Molecular Cell Biology (Lodish, Sixth Edition)

Citation preview

Iro ^/\aN

{ u e d u o l p u e u e u a o r J ' H' M

=t

erepnslel^|lned q6eoldopplH reLl)slorg {uoq}uv llo)s 'd ^^aqllen re6err)^luolA rosre)'v s!rq) )ra8 plourv qslpol{anre;1

NOIIIC] HIXIS

ADOtOtslll)

uvrn)llol l

ADOIOIIlll)

uvln)1101 1

o1{6olounuuur 1ln pue lilrsranrulprerupH1esluapnlselenpetbtepun saqleeiq6ao16lC ao] Llrorlpueul saqsrn6urlsrp uLelsr{s ounuJuirno s,iean aq} pue sasuodsel aunulLur lno ape^oo1{oldr-uasasntrn 6urpea;Epponn all] jo auO Ll).reasag lPq} s)tpP]snouP^aql setpnisq6ao14rC lorneqaquralslsaunuur ur sraq)rpasa.r lp)tpauJorB .lo,olntrlsulpeallellqMaql to roqurau e pue {bolouqrol}o a}n}rlsuls}lasnq)esspnJ eql le I6olor€lo rossa}oldsl Hg3gld ICOIH

{1rsranru6l o1,i6olorqllal soq)patlaq)s}argr6 lsea{ 6urppnq llourof }p s}uapnlsalenperO 'sr"uels{s ')1aue6'le)ruJeq)orq oq] pue s;1e> alPrqa].ia^ oM} ut seq>eordde slro;dxa {roletoqel letlaqllda lapoLlr lerrbolorq llal pue srq')JoMslql lol )lllell auerqujaur pue {1ue;odlla) o} uor}plelur sluauodtlotasoq}}o suot})un}1e)6o1orq pue aql oullept)nla uotalalsoy{rul}lP aql slueuodt-uor rrnau6urzuel:ereq)pue 6ur,!r1uep; lot uMou) sr fuo1ejoqe; slp 16o;orgJeln)alof\jpue ;o 11am sl UjH)SIIUS ANOHINV llaf +o aln]rlsulllaurof aq] ,torollalC oler)ossvpue Ilrsranru6l lleulof le {6o1org11a1 ,tolosserrord

pro}ue}S}e {1rsranru61 sluapnlsalenpel6o1 {6o1otqle1uau-rdolanep pup 's}uapn}sle)rpauro} srxsrueqleur espesrppue luauldolanap 'sluapnlsalenpet6tapun o1{6o;orq pue saq)pa}t}o)S rC ra)ue) ur srole;nbet salolaq} leluauLdolanep lla) leluau.rdolanap }o 'sl1aua6pue {6o1orq 6uttenotslp to; pue saua6xoqoaLuoq pue 6urleu6rs ut )lom lla)-llal]o spaJeur ,i;.re;ntr;red leluaudoqanap slq ro] uMou) st eg ,i6o1ot6lelueu.rdo1ena6 ro; IlaDo5 aql 1o luaprserdgsede pue sa)uaDspup s].rv1o ^LUeperyueltraLrlvaql pue sa)url)S ILuape:y aq] raquau]p sr prennog aH eln]rlsul saq6np pue aut)tpan aq] ]e lolebtlsa^Ul ;o leuorlpN ]o le)rpe/\ .d pue 's)r}auag[6o;o19;e1uaudo1ane6 lo looqls ,{ltstantulplo}ue}S1e buueeur6uaorg +orossalordsl I1"O)S M]HItVl l

rolda)arICH aq] 6urpnpur 'sllal aq1ro1luelrodurrsuralordrolderarpazuapeleqlpup pauol)seq pue snletedde ]o lno pue olut lololsaloq) ]o ]uaLUa^ouJ t61o9eqr q6notql6ur1:rger1 auerqLxotr 6urpuelsrapun Jno o] suorlnqrlluol epeLuseq,loietoqe;stH spleMesnoJeuJnu lo pa^la)alseqaq 'sasrnol{6o1otq-11ar alenper6se se r{6o;orslqd ueunq pup ,i6o;olqelenpetbtapun 6utq)ea}a^rlp^ouul lloM }o srqro1 {6o1ouq)e1 aq} }e ^6010rg peaqetrqM sl UlgllU) AINO6 }o aln}tlsuls}}asnqtessen 1oluauyeda6 aq} ur .rosso}old

srea{{ueul ro; selenpet6tepun o1s:r1eua6 lq6ne} spqoq olaqM1ll }e lo}p)npa alenpelblapun dot peztuOorar quaulppduol e se sr lasrp) 16 rie,rnqled fuo1ar>as aq] polols pue paplo+ aq] ut ete suralold +o fuoletlespue auPJqLuor.u pazrsaqlu{s ,{lrrneu ,rnoq sasserord )rspqaq} puelsrapuno} spoqtau letr6olorq11etpue >r1eua6 sasn 1o fuoleloqe1 sr;1,i6olouq:a1 oql le l6olorg1o lueuyeda6 oq++o peaHpue rossalordsl UISIV) .V SIUH) +o aln]rlsulsuosnq)essen

snal)nuaql 1o16olorqlla) ut aslno)pa)ue^pe ue pue ,{6olotrnpue ,i6olorqrelnlrlour ur sesrno)fuo1:nporlursaq)pa}aH slossatddns rouJnlpue seua6otuo{q pepotua slopel uotldtlrsuPl] uo relnttpedut 6utsn:o1'slla)uprlpuureur ur uorlpr]rur uorldu:sue;l a1e;n6et .reln)alouJ ]pLl]suotl)elalut alll salpnlsfuo1e:oqe; srH sosnJ|^ ut lorluo)aueblo; sr.usrupqfauj VNU]o srala^otsrp 1o pue 6ur>rlds ;eurbuoaq] ,o auo st aH sa)ual)SpuPsUV {uLapely ue)rlauv aq} Molla+ e osle sr .rC salabuy sol 'ptuJo}tlpJ eq} }e a}n}tlsul 1o }o )ra€ lo ^}tsla^tun ,i6o1org te;nralonaql +oraqureu.r e pue s)rlauogreln)alo/\pue ,{6o1ounu.rLll I6o;orqortr4}o lossa}oldst )Ulg olONuV {6o;ouqtalorqpue ,i6o1orq lla) ut saenot alenperbpue elenpel6lapunsaq)Pa}qstpol t6 uotleraylord raql suralotd pat+tluapt pue spq slla) ulels trlarodoleu-raq Uoddns serpnls 1eq1 lanou oslefuoleloqe;stg stolda:at pue urlarodorqytaoll] se q)ns 'suralotdtolda:et a)efns-lla) srslleueleuor]:un1pue B-lgl leJo^as lo 'ri6o1ors,{qd 6uruo;laql uo puP'suraloldete;rns-11er IueLu1o srsaqlulsorq aq] ^l.rpln)rupd auelqulaL!-llo) uo 1roMsrqro+umou} to1{1arto5ueluaurvaq} }o (t002)}uoprsard llaMsl oH {6o1otg11a1 spMpue se)uat)Spue suv ,roILlapety ueluourvaLi}pue p sa)ual)S {uLepe:y aq} raquraur qsrpol osle sr JC rl)reesau 1o leuotleN }o lp)tpaurot€.io}e}n}t}sulpeaqo}tqMoq} +o laqulorx e pue ribo;ouqlal]o aln]tlsuls]]osnq)esspl4 aq] ]e 6uuaaurbuaorg ]o Jossaloldpue ri6olor6]o lossaloldsl HSIOOI AI1UVH

SUOHINV]HI INOSV

PusLIsHgn: SaraTenney ExecurrvE Eonon: Katherine Ahr DpvEr-opneNrRI- Eorrons: Matthew Tontonoz, Erica PantagesFrost, Elizabeth Rice AssocIRrp Pnoyrcr MexecBR: Hannah Thonet AssrsreNr EorroR: Nick Tymoczko AsssocrerEDnacton op MeRrrrrNc: Debbie Clare SeNIoRPno;ecr Eonon: Mary Louise Byrd Tsxr DpsrcNBR: Marsha Cohen Pacp Mnreup: Aptara, Inc. CovpR Drsrcrs:BlakeLogan IlLuslReltoN CooRorNetoR: SusanTimmrns ILrustR,q.rroNs: Nerwork Graphics, Erica Beade,H. Adam Steinberg Puoro Eorron: Cecilia Varas PHoro RpseencHrn: Christina Micek PRooucnoN CooRoneton: Susan\7ein Mron RNo Suppr-rvENTSEDrroR: Hannah Thonet Mron DEvgLoplns: Biostudio, Inc., Sumanas,Inc. CotvtposrrloN: Aptara, Inc. MeNupRcruRrNG: RR Donnelley & SonsCompany

About the cover: Mitotic PtK2 cellsin late anaphasestainedblue for DNA and greenfor tubulin. Courtesyof Torsten\gittman. Library of CongressCataloging-in-PublicationData Molecular cell biology I Harvey Lodish . . . [et al.]. -6th ed. p. cm. Includesbibliographicalreferencesand index. 1. Cytology. 2. Molecular biology. I. Lodish, Harvey F. QH581.2.M6552007 57L5-dc22 2007006188 ISBN-13: 97 8-0-7167-7601-7 ISBN-10:0-7167-7 601-4 @ 1986,1.990,1995,2000,2004,2008 by tJf.H. Freemanand Company All rights reserved. Printed in the United Statesof America Secondprinting W. H. Freemanand Company 41 Madison Avenue,New York, NY 10010 Houndmills, Basingstoke RG21 6XS, England

www.whfreeman.com

To our studentsand to our teachers, from whom we continueto learn, and to our families,for their support, and love encouragement,

PREFACE

I n writing the sixth edition of Molecular Cell Biology I we have incorporated many of the spectacularadvances I made over the past four years in biomedical science,driven in part by new experimental technologiesthat have revolutionized many fields. High-velocity techniquesfor sequencing DNA, for example,have generatedthe completesequence of dozens of eukaryotic genomes;these in turn have led to important discoveriesabout the organization of the human genome and regulation of gene expression,as well as novel insights into the evolution of life-forms and the functions of individual members of multiprotein families. New imaging techniqueshave generated profound revelations about cell organization and movement, and new molecular structures have greatly increased our understanding of life processes such as cell-cell signaling, photosynthesis, gene transcription, and chromatin structure.

what we know. A number of experimental organisms,from yeaststo worms to mice, are used throughout so the student can seehow discoveriesmade with a "lower organism" can Iead directly to insights even about human biology and disease.This experimental approach, evident in the text itself, has also been thoroughly integrated into the pedagogical framework. For example:

New Author Team

r Updated Perspectives for the Future essays explore potential applications of future discoveriesand unanswered questions that lie ahead in research.

Two new authors have been instrumental in refocusing this book toward these exciting new developments. Anthony Bretscher of Cornell University is known for identifying and characterizing new components of the actin cytoskeleton and elucidating their biological functions in relation to cell polarity and membrane traffic. Hidde Ploegh, of the Massachusetts Institute of Technologg has made major contributions to our understanding of immune system behavior, particularly in regard to the various tactics that viruses employ to evade our immune responsesand the ways our immune systemresponds. Both authors are widely recognized for their researchas well as their classroomteachingabilities. 'We are grateful to Paul Matsudaira, Jim Darnell, Larry ZipurskS and David Baltimore for their exceptional contributions to the previous editions of Molecular Cell Biology. Much of their vision and insight is apparent at many places in this book.

Experimental Emphasis The hallmark of Molecular Cell Biology has always been the use of experiments to teach students how we have learned

r Experimental Figures lead students through important experimental results. r Classic Experiments essaysfocus on historically important and Nobel Prize-winningexperiments. r New and revised Analyze the Data problems at the end of each chapter require the student to synthesizereal experimental data to answer a seriesof questions.

showsthe locationof DNAand multiple microscopy Fluorescence et al, 2006,Science BNG Giepmans proteins withinthe samecell.lFrom 3'12:217 |

vtl

New Discoveries, New Methodologies

Coiled-coilstalk

Motor head Microtubule

Necklinkers

(+)

Methodological advances continue to expand and enrich our knowledge of molecular cell biology and lead to new understanding. Following are just a selection of the new experimental methodologiesand cutting-edgescienceintroduced in this edition: r Expanded coverage of proteomics, including organelle proteome profiling and advances in mass spectroscopy (Chapter 3)

I ForwardmotorbindsB-tubulin, ADP ^ releasing

p ForwardheadbindsATP

?

r Expanded coverageof RNAi, including the use of shRNAs to inhibit any gene of interest in a cultured cell or organism (Chapters5, 8) r Updated discussionsof chromatin, including structure and condensation(Chapter 6), control of geneexpressionby chromatin remodeling (Chapter 71, and chromatin-remodeling proteins and tumor development(Chapter 25) r Evolution (Chapter 6)

p

Conformationalchangein necklinkercausesrear head to swing forward

of chromosomes and the mitochondrion

r New molecular models, including pre-initiation complex and mediator complex (Chapter 7); annular phospholipids (Chapter 1,0);Caz* ATPase (Chapter 11); rhodopsin, transducin, and protein kinase A (Chapter 15); and myosin ATPase (Chapter 17) r Latest advancesin light and electron microscopy, including cryoelectron tomography (Chapter 9) r Reactive oxygen species(ROS) (Chapter 12)

ADB [ rue* forwardheadreleases ^ trailingheadhydrolyzesATP V and releases P.

r Role of supercomplexesin electron transport (Chapter 12) r Human epidermal growth factor receptors (HERs) and treatment of cancer (Chapter 16) r Myosin ATPasecycle (Chapter 17) r Kinesin-1MPase cycle (Chapter 18) r Use of retrovirus infection for tracing cell lineage (Chapter 21) r Axon guidance molecules (Chapter 23) r Somaticgenerearrangementin immune cells (Chapter 24) r Cancer stem cells (Chapter25) r Use of DNA (Chapter25)

Figure18-22 Kinesin-1 usesATPto "walk" downa microtubule

vill

PREFACE

microarray analysis in tumor typing

XI

l)vl tud

'JPalre^o PunoJ0q UEJuonrpo qxrs oql ur seldr.uExeIEJrpau arll Jo rsII aleldruoJ v .luau -uorr^uo rElnllotrErlxe rrar{l ur selErpdqoqfff, pup surelord ol puB sllor reqlo ol sllal lreuuof, pu? .usrloqElou alEurprooJ 'uorldtrcsuerl 'slueulelour VNC alelnSor 1ac ezdptec leql saxalduroc-sy1ao ur saxalduroc ulolo.rdrrpur Jo Surpuelsrapun polrerop e uo e8urq suonecqdde osaql Jo dueyrg .8urureo1 ere doql eruerrs f,rseq eql ;o suonecrldde leolurp oql roJ uorlerca.rddeup stuepnls errr8ol eleudordde a;aqm sreldeqr eqt lnoq8norql uo^ol\ e.reselduexe lelrperu esaql .sossasrp ueunq luecrlruSrsJoqro pu€ JafupJ Jo' sluauleeJl ,/\^.ou ol pel azreqlSolorq relntalol'u puE rEFIIel Jrs?qur secuelpu due141

'uaq) pue uosraur] Ie]nltlsulq)-teosau le)pauotguo]sog [ 3 1oIsaynoSl sllol al)snu'tolut slla) (lselqo^Lu)a]tllalesasnou.l fueurrd ]o arnllnl ur uotlerluola]]lC0€-6 eln611lelueuuedxS

a)uenelau le)tpaw -relotrr

'Iuolsds eunrurur olprgalra^ aqf or dSolorq IIar Jeln r ;o soldrcur.rdsarldde (y7 nlderla) .,d3oyounurur1,,

'uorldaf,ardrosuas pue Surpur; r{l?d ur sef,uE^pplso Ipuoxe -tel agr Surpnycur's11acpazrpnods aseql dSolorq ;o [e] aqr 1o aSerairocperJrunseprrrord(97 raldeq3) ..slle] e^raN,, r 'tuaurdole,rep uErI?luruEru uo snJoJpaseerf,urr{lr^,\(uorlptu.ro;u.rauedq8norql uorlezr -lrlroJ uro{ luerudola,rep sleluoruppunJor{l sruaso.rd(77 Jo ;erdBr{J) ,.luarudola,taq;o dSolorg IIaJ relnreloW ar{J" r

eerqt sepnlrur KSoTotgila) nlrcapw

:sraldeqr rvrau Jo uourpe qrxrs aql

sraldeq) /naN I p]eogprpMvuosltNUpuualaq-L]o Isa]Jnofl 'talrnuord eleuJa]pue a1eu.r 6ulnnoqs11ar66a ueunq paztltyal

'uorl€ruJoJur IEuorlrpPPr.lfnru ot srolfnJlsur pue sluapnls Darrp leql selr -ru? ^\erleJ e,Lrsueqa.rduroJ pu€ serpnls {reurpuel Jo sef,ue -raJeJ rsrl olep-ot-dn uE spr{ retdeqr r{leg 'lprralpru ^\au Jo .sretdeqc to uorrrppp aqt roJ uoor e>lerrrol sn Surlgeue ,tg scrdol Iueur ;o a8ereloc pasnf,ol Iereles SurzrueS.roa.r 'sesJnof, lsoru Jo uorlBzrue8ro Pu? peulluparls osle ql{r eql rorrru PUEIErraleu polplor alrun 01 slueluoJ Jo elqBl aql pasr^al a^pq o^\ 'drlunoo agl punoJ€ ruor; dSoyorq IIel to srossayord691 ue{r erotu r{tr^. uorlellnsuof, uI

uollezrue6lg nnel reldeqr qlee Jo derupeor peor-ol-dsea ue eprrrord mou sEurpeeqpatuauo-ldeJuoJ JEOIJ 'alqrssod Jaleregl\ uo8rel Sur;rourer's8urpvaqpue ]xel aqr par;qdurs osle e^Eq 3/N 'Oruof,01 sr lpq^{ Jot uorlEpunoJ pllos e epr^ -ord pue arnlrrd 8rq eqr stuapnls e,rr8deql teql aJnsSur4rur 'sarerlrJeao ralduqc aql parrordurr aAEq olt ,relncrlred u1 'olqrssalJearotu sluar.urradxa pue sldaruoc eqr qroq Sunleru ol luerullturuor Jno porJrsuoluralerl e.ry\pue 'sleluaurepun; eql Jo suorleueldxe reelJ roJ poau .sluepnls qlr^\ sluaurdo -la^ap lrJnuaps lselel eql;o a8eraloJ Jno arueleq ol peln o^prl o/N 'alqeqceordde aJoru sluopnls ro; Sullunep eg uEf, leqt Ierraleru o{pru ol op uer'sluepnls alenper8 pue olen -perS.rapunqloq paruarredxesE ,er\,r. req^ pe)se to srer.{Jeal pue IJEq dels e ualel a^Er{olr uorlrpo qlxrs eql 3ur1r.rmu1

f1r;rqrssa))V pue [1r.re11 poseat)ul

'd) dlr,rr1ce eseroruolel pue puotselqornaN OVtt sluaurt?eJlreJuef,ls?arfl ftttt'd) (Ogtt 'd) sarderaqlraruer raqlo pue le^ealC (Stf 'd) seuroqdrudl srsdleuederreonrly'q pue t 'd) sarderaqtref,uef,se suralord lernlBu 3_ursn

(gttf

(rrrro)

l)vl I ud

rc99'd) seleq?Ic (SSS'd) eur8ue pue uuac.{y3orlr51 'd) sutalo.rd.C pue sulxol IEIJelreg G* (tEg 'd) (ggE) .rolcel qr.to.r8 prlaqrrda PUPreruer lseorg .d) luetulEert Erullrsv GZg

(sog'o)

pue (g4) enualo.ralseloqrraddqIETTnUEJ sllol ruelsreJuef,d;pueprot posnore stsotuorletuBldsuerl Ioralsalor.{r'ICI'I 'd) aSerolsleurososdl ses€oslp (tOtt 'd) sourrre1 ftOg (SeS'd) srsorqr;rrrsd3 (f eOt 'd) uorssarddnsounuul ( S S S ' d )s e l l l l e l u r o u q e (geOt'd) ssouJEOC (ezord) IerretoruprJ ot peel uec dlquresseaurostxorede^Illoleq (SSS'd) 3urp1o;snuutalord pue eruas,{qdurg s.rat.rodsuerl Jret{lpup sJeDrrusuEJlornau taBrel s8n.rq 'd) sarpoular 'd) (eOS srsoldcoxa Jotlrusu?JtoJneu pue rusrlntog luqreq pue dlr,rtloerelrodtluB dCV/dIV kZOt (ZOS'd) rrxol dlq8rq eq uer (595) sercadsua8dxo a^Ilreeg (Sf Ot 'd) urladur leraqdrradlaS.relseseesrpeunuurolnv (Set 'd) uotlertdsar relnllol sllgil{ul aprued3 (ltOt 'd) srsorelf,sa1drrp7,q 'd) uorrerpdqappur Eraloq3 'd) snsrelul snlls pue dl4rualur a1ery fiw GSe 'd) sllar ruets slueldsupn ,,trrorreru ouog pue QgV'dl suollJeJluor elJsnlu uPOqPUEasEdIV *Y*3N 6Ze (SSl 'd) sISorqIJorlsdcpue suralord3gy rrlrrpua6l (Ste 'd) spuno^,\uDIs sllel J IEeq leturaprda 'dl sller ruots asn rnnederogl GVV'd) snpldrsursaloqelPpue'utsserdosezr'utrodenby ftte 1o 'dl (eeg 'd) reJu€r pue slurod>paqeapdc-1ya3 k* loretsalol{r PUEsrsorelrsorol{lv sqre5-de1 (te S 'd) surelord .rosserddns-rorunl 0n.' d) aseesrP (SSg 'd) sroruef,uelunq pue dezngredUOIUI (ZSS'd) sertrleurouqe OVt.'d) (ng) snrr,r'dcuarcrlepounulul uelunH leluaurdola,rappup JeJueJsosnpf uorlcunlsrp-uop 'd) eltuessel€ql (ZgS'd) euolselqourler.,(retrpa.ra11 \Vt. (OlE 'd) soseeslplectSolornaupue suot8ar salIIIel€sorJIW (qqS 'd) saseasrpaldrrynuresnef,suorlelnru VNW-I (ggg 'd) dqdorre elrsnlu leurds pue Surddrls uoxg ( ggS'd) .,(cuercr;ap uorsaqpe-eldrolne-I (SeZ' d) auro.rpudss,eu,,{e1co3 (ses'd) PuE ulnsoluou8rd euraporay ,(qdo.rrsdpJelnf,snu ouuoglnq pue 'uolela4sordc'p,q3g_eq1 6eZ'd) rarueJ sesnetsaua8ut &r.LrperossardarJo oluosqv ftgZ'd) refuef, leart ol srollqlqul oseJetuololueunq Surs;1 Uze'a) G.VZ' d) slsoleruorqlJornoN asnel VN(lluI uI suollelnry ftVZ'd) sasEOSIP 6VZ' d) souosoqlr orlosotdr uo slleJJeruereJllPe^EI{sJIloIqIluV puE lErJPuoqf,olltu 'd) dueru sasnecsluatualo11 ;o lrrrtqolN cueua8 sespeslp fttZ relnJsntuorneuosner selIIIelEsorf,I6 GZZ'dl seseoslp (SOZ'd) srsorqltcrlsdr dpnls ol POSnare erllu lno{rou) ueunq esnel salalp luelnry (Se 'd) seseaslp f 'd) sasncnnaderaqr qtt A Get suralord ueunq azrseqludssenbruqcalVNC lueulqluofeg sarqdonsdprplnf,sntulelrue8uoc osner suortetnu ua8e11o3 gZg 'd) stlaJapensslt-elllrouuoo pup d,Lrncg kZe'dl ernlreJ IPuer ot PEelspaJaPauErgrueu luouosEq rElnreuolc upunq rq8rarseelt? esner seue8utxauuo3 (etS 'd) sos€osrp (Sf S 'd) eseosrppue uodsuun relnllerered (gf S 'd) oseasrpurIS pue urelSoursapuIreI{p€J Ge t'd) saseosrpdueu asnecsuorletnur ururel y-add1 (OSZ'd) sreprosrpdrosuasesner slJOJeperlrc dreurtr4 Ogt'd) osposrpleel ot ullnqru roage s8nrq

(ee rd)

6w'a)

eunrperrl uI slool crtsou8erp p;raltod a.le sdel.leo.rc1uVNCI sarqtedodtuorprerrrqdorlraddq asnecsuoll€lnru ulelord Itgt'd) fitt' d) dqdorrsdp rplnf,snlu ouuel{f,nq Erreletupue EItuouEIIel-oPIlIs llotte solelleurqolSouleg (OgZ 'd) serrueuecrldroraqd5 (fSt 'd) sller rno IrElrE AdH pue AIH sE t{f,nssasnrl1 GOt'd) IorelsalollJpue slsoralf,soreqtv 'd) uDls raf,uef, PuE urelsdsrredal-uotstcxoaPuoollnN 9O t' a) aseesrps(rer.ureqzlv (Stt (Slt 'd) raf,ueruolo3 gOt'd) aspasrpueaq pue ref,uerlrotlp saseetordWVCy (ZS 'd) dde.raqloueqf,uI POSnare sJollqlqul auoseetord r{lrrq osnef,suolletntu Surleu8rsSoqe8pag (OOZ'd) sDOJOp (lS 'd) dllrtlce utalord llqgul uec s8n.rq (Ug 'd) rof,uet ueunq pue eua8NEId (tt'd) 8urp1o;sruutalord Jo soseeslq (SSS'd) rerupl upunq pue suralord se6 ueIIEIuLu€t{ 'dl (gg 'O) uorlef,Ipeu uI sretuosloorols eua8 pu€ reruer lseerg ftSg Tagg eql Io uorrprr;r1drue 'd) strods ecuernpuepup unarodorqldrg Gtg 'd) :erer.rdorddesE lxal oql ul dde.raqr lZtg 'xel eql readde suorleleJJof,IEJIurll JaJeIJqIEUoIIIpPV raf,uef,pup (JSI-C) rorce; Surrelnurrls-duolocailoolnuerg 'd) srorunt upunq pue uortrqlqur qrmor8 'gg1 ur uortecrldde I€f,IuIIr e Jo lrels eql sleu8rsuorl sIr{I fttg

sNorlv)llddv

1v)lNll)

MEDIA AND SUPPLEMENTS to-use format. Visit http://ebooks.bfwpub.com more.

For Students Companion Web Site www.whfreeman. com,/lodish5e NEW: Podcastsnarrated by the authors give students a deeperunderstanding of key figures in the text and a senseof the thrill of discovery. r NEW: Now available for your MP3 player or personal computer, more than 1,25animations and researchvideos show the dynamic nature of key cellular processesand important experimental techniques.The animations were storyboarded by the textbook authors in conjunction with BioStudio, Inc., and programmed by Sumanas,Inc. r Classic Experiment essaysfocus on classicgroundbreaking experiments and explore the investigativeprocess. r Online Quizzing is provided, including multiple-choice and short answer questions. -4), written Student SolutionsManual (ISBN:1-4292-01.27 'Wong, Richard Walker, Glenda by Brian Storrie, Eric A. Gillaspn and Jill Sible of Virginia Polytechnic Institute and State University and updated by Cindy Klevickis of James Madison University and Greg M. Kelly of the University of Western Ontario, contains complete workedout solutions to all the end-of-chapter problems in the textbook. NEW: eBook (ISBN: 1,-4292-0955-0)New to the sixth edition, this customizable eBook fully integrates the complete contents of the text and its interactive media in a format that features a variety of helpful study tools, including fulltext searching,note-taking, bookmarking, highlighting, and more. Easily accessibleon any Internet-connectedcomputer via a standard rWebbrowser, the eBook enablesstudents to take an active approach to their learning in an intuitive, easy-

to learn

For Instructors Companion Web Site www.whfreeman.com/lodish6e All the student resources,plus: r All figures and tables from the book in ipeg and layered PowerPoint formats, which instructors can edit or project section by section, allowing students to follow underlying concepts.Optimized for lecture-hall presentation' including enhancedcolors, enlarged labels, and boldface rype. r Test Bank in editable Microsoft'Word format now featuringnetu and reuisedquestionsfor every chapter.The test bank is written by Brian Storrie of the University of Arkansas for Medical Sciencesand Eric A. Wong, Richard Walker, Glenda GillaspS and Jill Sible of Virginia Polytechnic Institute and StateUniversity and revisedby Cindy Klevickis of JamesMadison University and Greg M. Kelly of the University of Ontario. r Additional Analyze the Data problems are available in PDF format. r NEW: Lecture-ready Personal ResponseSystem "clicker" questions are available as Microsoft'Word files and Microsoft PowerPoint slides. Instructor's Resource CD-ROM (ISBN: 1'-4292-0126-6) includesall the instructor's resourcesfrom the Web site' including all the illustrations from the text, animations, videos, test bank files, clicker questions, and the solutions manual files. 'l'-4292-0477-X) conOverhead tansparency Set (ISBN: for optimized text' the from key illustrations tains 250 presentation. lecture-hall

17 ,7 ,L Cell Mlgra:1jon Coordlnates Fore Gen.radon Adheslon and M€mbdne Rccvdlng

wlth Cell

II !

i{r

eBook bfwpu b.com http://ebooks.

Ela@'o@f'*dq

FlWre L7,38,tu9 4, 4. I

)a:99 -, w,P" : :l9Y t !i,'Pn.by yl:-'-'l].y?

.dh.t. b t 6. att lLrt t.6. l.^.lltpo4t. be foalrdh.tto^t (2) ftr 6. rlb,bdh ^ 6. att bodr 6. b dol d6. t..t d Aott

PREFACE

'

ACKNOWLEDGMENTS In updating, revising and rewriting this book, we were given 'We invaluable help by many colleagues. thank the following people who generouslygave of their time and expertiseby making contributions to specific chapters in their areas of interest, providing us with detailed information about their courses, or by reading and commenting on one or more chapters: Steven Ackerman, (Jniuersity of Massacbusetts,Boston Richard AdIe4 IJniuersity of Michigan, Dearborn Karen Aguirre, Coastal Carolina lJniuersity Jeff Bachant, Uniuersity of California, Riuerside Kenneth Balazovich, Uniuersity of Michigan Ben A. Barres, Stanford Uniuersity Karen K. Bernd, Dauidson College Sanford Bernstein, San Diego State (Jniuersity Doug Black, Howard Hughes Medical Institute and (Jniuersity of California, Los Angeles Richard L. Blanton, North Carolina State (Jniuersny Justin Blau, New York [Jniuersity Steven Block, Stanford IJniuersity Jonathan E. Boyson, Uniuersity of Vermont Janet Braam. Rice Uniuersity Roger Bradleg Montana State Uniuersity IilTilliam S. Bradshaq Brigham Young (Jniuersity Gregory G. Brown, McGill (Jniuersity \Tilliam J. Brown, Cornell IJniuersity Max M. Burger, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland David Burgess,Boston College Robin K. Cameron, McMaster [Jniuersity \7. Zacheus Cande, (Jniuersity of California, Berkeley Steven A. Carr, Broad lnstitute of Haruard (Jniuersity and Massacbusetts Institute of Technology Alice Y. Cheung, IJniuersity of Massacbusetts, Amberst Dennis O. Clegg, Uniuersity of California, Santa Barbara Paul Clifton, Utah State IJniuersity Randy \7. Cohen, California State [Jniuersity, Northridge Richard Dickerson, (Jniuersity of California, Los Angeles Patrick J. DiMario, Louisiana State Uniuersity Santosh R. D'Mello, [Jniuersity of Texas, Dallas Chris Doe, HHMI and [Jniuersity of Oregon Robert S. Dotson, Tulane [Jniuersity 'Sfilliam Dowhan, IJniuersity of Texas-Houston Medical School Gerald B. Downes, (Jniuersity of Massachusetts,Amherst Erastus C. Dudley, Huntingdon College Susan Dutcher, V/ashington (Jniuersity School of Medicine Matt Elrod-Erickson, Middle TennesseeState Uniuersity Susan Ely, Cornell Uniuersity Charles P. Emerson Jr., Boston Biomedical ResearchInstitute Irene M. Evans, Rochester Institute of Technology James G. Evans,.Whitehead Institute Bio Imaging Center, Massachusetts Institute of Technology Marilyn Gist Farquhar, Uniuersity of California, San Diego

X

PREFACE

Xavier Fernandez-Busquets, Bioengineering Instituteof Catalonia, Uniuersitatde Barcelona,Spain TerrenceG. Frey,San Diego State Uniuersity Margaret T. Fuller,Stanford UniuersitySchoolof Medicine KendraJ. Golden, WbitmanCollege David S. Goldfarb,RochesterUniuersity Martha J. Grossel,ConnecticutCollege LawrenceI. Grossman,WayneStateUniuersitySchoolof Medicine Michael Grunstein, Uniuersityof California, Los Angeles, Schoolof Medicine Barry M. Gumbiner, Uniuersityof Virginia 'Wei Guo, Uniuersityof Pennsyluarua Leah Haimo, Uniuersityof California, Riuerside Heidi E. Hamm, Vanderbilt(JniuersityMedicalSchool Craig M. Hart, Louisiana State Uniuersity Merill B. Hille, Uniuersityof Washington Jerry E. Honts, Drake Uniuersity H. Robert Horvitz, Massachusetts Institute of Technology Richard Hynes, Massachusetts lnstitute of Technologyand Howard HughesMedical Inshtute Harry Itagaki,Kenyon College ElizabethR. Jamieson,Smitb College Marie A. Janicke,State Uniuersityof New York, Buffalo Bradley'W.Jones,Uniuersityof Mississippi Mark Kainz, ColgateUniuersity Naohiro Kato, Louisiana State Uniuersity Amy E. Keating, Massachusetts lnstitute of Technology CharlesH. Keith, Uniuersityof Georgia Thomas C. S. Keller lll, Florida State Uniuersity 'V/estern Greg M. Kelly, Uniuersityof Ontario StephenKendall, California State Uniuersity,Fullerton FelipeKierszenbaum, MichiganStateUniuersity Cindy Klevickis,JamesMadison lJniuersity Brian Kobilka, StanfordUniuersityMedicalSchool. Martina Koniger, WellesleyUniuersfiy CatherineKoo, Caldwell College Keith G. Kozminski, Uniuersityof Virginia StevenI(. IJHernault, Emory Uruuerstty Douglas Lauffenburger, Massacbusetts Institute of Tecbnology RobertJ. Lefkowitz, HHMI and Duke UniuersityMedical School R. L. Levine,McGill Uniuersity FangJu Ltn, CoastalCarolina [Jniuersity ElizabethLord, Uniuersityof California, Riuerside Liqun Luo, StanfordUniuersity Grant MacGregor, Uniuersityof California, Iruine Jennifer O. Manilay, IJniuersityof California, Merced Barry Margul ies,Towson Uniuer stty C. William McCurdy, Uniuersityof California, Dauis, and LawrenceBerkeleyNational Laboratory Dennis \il/. McGee, State Uniuersityof New York, Binghamton JamesMcGrath, RochesterSchoolof Medicine David D. McKemy, Uniuersityof SouthernCalifornia Roderick MacKinnon, Rockefeller(Jniuersity JamesA. McNew, Rice Uniuersity

X

l)vl lud

'uoqey{ auuv eJr.^ A,tt (qBaop applfl:raqelN snel) pue reslp) elpq 'V sresl^pepue 'e1rrEpuerpraHs.rorq8nep pue acruef e;rrrr dru (nqtslatg ftuoqluy) :rnpred no1 .fue141 rasr^pe pue ';dolssralXer1Jsnq'11oo5re1a4'uor5 erlnf 'loc5 ulorurl te11ng (*rttnt) lare8rey,q(nocg '4 maqtlpw\ fre8err; enqsof pue 'reSorry ueqreuofte8err;4perpllry ta8arry ,(e['1 te8arry lcueg Qa8auy {,1uoyl :lltaN,O dqrel e;r.u f,ut (nsruy 'V sltqJ) :d;eq5 1q4 pue 'uorde13 pr,req 'grrur5 euqe8uy '1rag dalrrqg '>1rag.furaf t1e5 a;rrrrdru (4ng plouty) qooq sqt Jo suorlrpalsrrJ erll uo Surler -oqpllor ro; IIeureCurrf pue erourlleg pr^pq osle pue lreuuerg .,{aupd5 prre:epur.ZuolroN srolurur :l.poqea4trlor1 pup f,pEsIpu€ 'dpoqea4ernrg 'qsrpol arueqdar5 pue lqsrpol s€rqoJpue 'engsof 'erqdo5pue 'rpreqlSurlsrr)',{rpo'I uruery lgaurat5.4aerpuv pse ?ururgpue rrrg rpraH uerplrrllpuerSpue uarppqclur pup traurets 'e;r.trdu f :^\oul e.trterl^{ Jo qcnu sn 3ut e1aue4 (tls1pol Kan,wyfl -qJeatpue serpnlsrno ur sn SurSernocue roJ sresrlpepue sJolueu Jno ol pue looq E r{Jnsuo lJolr\ ol selet tr eurt eqt sn Surluer8 roJ pue sn SurrrdsurroJ sarTnupt rno ot slueql lercadsAlleurg ^rolEroqElIoJS aql to sreq 'qs1e16 'uostelN 'rg8ru; aqnf -ueru te11er1X blcmg pup qog qrr51 'srue8our41o;a8roeg 'sue,rgsauref'3uE 'uerrouoqauuy 1ore3 'qsngauerq 'unoJueurgd1e5:t1elsu,^,orno ot slueqf d.re141 'u^ers glnu PUPorroilr^rQ eruosAIIPIT -adsa luorlrpe srql ur af,uenltur ol enurluof, uonrpe eqt ot suon qry$ -nqrrluof, eIq erN esor{.4a esoqr a8pal,v'oulre ol pFo.{\ Ferrp 'ue.r8ordlre aql Surcuequa ur >lro^rrag pue 8urdo1e,r,ap scrqderg ro; (ruoc'srrqderC3flW'.{\^\rrr) f,{IW Jo epeogEruE pue slapou rplnrelou InJrrnpaq srq pue rq8rsurlerr ;o tuaudola,rep -So8epadsrqro; Sragurelsruepv 'H or palgepuroslpare elN 'uoItIPe slql uI euoP arr.no.,{ 1Iero; nod IUPqJ 'srotrpoolqeireuer aredaql 'a)r5 qtaqezrTg pue 'tsorg sa8etu€d eJuE 'zouoluol .e\aquew 'sJotrpalxel Jno to lueulruruof, eIII plno.4 a^4.'relnctlredu1 pu? tuelel egr a8pal-rnouTre.or ^e.t\ JeAeur slarxereqr {ooq 3 elnP -ord ot eruruelo lJo.ry\ol sseu8uqp.Lr Jrer{tJoJpue Joqel rreql JoJ urel[ uesns pue 'seren eqrca3 'o1zcouil1 1c151'suturutl uesn5 'tauoql ere5'>1aqcse1 uare;tr'udetg qrna'pa51.g qeuueH'lauuel 'IerlIN purlsrrrl3'ue3o1a>1e19 'uago3 Eqsrery'are13atqqeq IIIS 'prdg esrnol .fue1aq tqy ete) Tueqr e26 dueduro3 pup uerueerC 'H 'lA.tE sraulredSurqsqqnd rno Jo uolleroqellorpaDrluurof, pue IntereJaqt rnoqtr-a alqrssoduaeqa^Eqtou plno^\ uolllpasIqI 'stseJpodpue sluarurredxg Jrss?l3er{tto lueur -do1a.rap rar{ roJ euozrrv drrsra^runer{tJo apueze1psl.1or InJ Jo -ater8 euIIuOegt to uolsl^erreq osye ar€ elN'sualgord Surzzrn| erur8rrnto elqls illf .ro; dlrsralrun elels pue elnlrlsul cruqcat.(1o4 ot pue 'suorlsanb4ueg lsel pue sruelqo;dsldacuo3 erll -t\el^a1 ^\eu tuallaf,xe 1o drgsroqtne rrer{t JoJ oIJEtuO 1o llrsre.,rrun aql 1o 1.11a;'yq Sarg pue dtrsrerrrun uosrperyseuef ;o sDlrl^el) ,(pur3 or 'suralqord eteq eqt ezlleuy .treu }o tuaurdola.tap Jer{JoJ'aprsra,rrg'erurotrle3 ;o ,{trs:a,rruner{l Jo ourreH qee'I ol uorlercerddepue apnlrter8rno sserdxaol aIrTosle pFo.a.rel6 otuol.uvuos 1p nrua) qqpaH soxal Jo Kysnnrun aqa'neS aauaUA KSolouqcal]o ainfl$ul s$asnq)ossow'uJ" 'g IOeIITIIAJ hplnog 'opo.to!o)]o r("trsnmug'enySutq 'yq dreS Klrsnntup umotg'1assa15

Kltsnnru1 sur4dog suqo['pue1pue16.{lrerrag puu IWHH'uoury elqa8uy r(4o1ouqta1 Jo aluttsul silasnq)pssow erte13 Kltsn nruI auorpul'1ezr1e16 Knstanrunilaurc)'r3on 1 rello1 uoleuoq8utg'4to7 mayl]o K"ysnmu1ata75'>1rtlsngue1 'C uegod teuqsre4 qqetlurv puolCtoytg Jo K11stanr.u2 qlaq€zllg a,Lowqupms'ua11en a8a11o2 oluona Jo Klts.tantun'adadorl luorul1 trll'saqurol'W lreqoU 02 or.utB Klrs.tantun qqoamuoLuLu puvpaz may7'uo13u41a1yt 'uo13ur11ary tlrsnnrun or.to::r1'a1rrrd5-alepsaeI In€d Jo 'S oBarq uog 'otu.to]ttp) /o ftqstamu1 pup IWHH to1.{e1 uesns aurnt 1' orut oJt1o2 ] o Klrsn nr'un' e nda8eel aule3 autntl 'orutoJtlo2]o Kysnntu1 'ur1ten43euIlsIrLIJ K7ls n nr.u 2 auu)tpauag'qretu11el5ef,Iuore1 Knsnruu7 stnoj lutos'reruad5uesn5 autxpaytrJo poqzs i('1rsnntu1stno7 luros',{rl.us 'S dro8arg 'a1eu5'1 ueqder5 sala&uyso7'pxutoJllp3Jo t("xtstantu4 'W sef,urq aprshntd 'o1u.toJ11o3 Jo K|rstantu1'ItP"lS Krysnntu7uolSutqsoly uh$a6 tpun3-q8ut5 nuy qleqeztlg3ut1-ue1 qun Jo.Klrs'tantug'nrq5 lvtshntun anp.tnd'uerureqs'vslno'I ue8ro141 KSolouqtal]o anr$ul silasnq)ossory'3uag5 Kyuantug a1ol5uol8urqsrr11'uap1aq5'V llrg eualreyq n ntu2'zvret15 ouatpulutaqwo5 ]o r(1ts euetq [.to1,rtr ,g Luollllg Jo a8a11o3'sa1eq5 uospw a8nag'ore5 pleuoq K1ts.tanru1 'V pl^e( maN'ouellqf,f,If,S K4snnr.un 4rcI KTlsnntunptoJuolg'roPIeuqrS PI^€Q auotpayl pozps {4rsnntun uo1Sutqsaytte;aeqc5ueaf Jo s.ta ntun aw I g otuon1rtsuua7' AtueS'J eulerro'I K1r Kysnntun umola8toag'plemuasoa'C auuy 'Q silasnq)psspry'Srequesog rreqog KSolouqcalJo ag1t1su1 Kltsnnrun p.toJuolS'r8reqoa lelea auntpayy ]o loo.Ps sllodzuolPulJo Kltsnntug'a4rtg '; dre141 Kpsnntu2 ploJuolg'tlol€d retlreg I€I KqsnnruI ilautoJ'eudnr4 przreq K4snu.un axpisouptstnoTSaJFd uelsrl) xnaq'aru.toJlp) Jo r(puanr.u2{1s1e,rtr4uruetrJ aBa11o3 lsnqutv'etlJo4 f,IuIuoC Iooqzslwtpaw plPnrPH'tuou8t4EJsef,uerC '11uredrp Kala4ng'otutoJuo1]o KlrsnntuJlPuPIWHH'1ale4 Kltsnntun p.toJuolg'org duoqtuy 'W pnnw 'orutoJno3]o K'ltsnuun'snlclg P1r'eC Kystanr.u 2 p toluoqS'assn51 leog uosxpow'utsuocst6 Jo tbrs,lanrun'IqurelN'y'q sauref ouoztty Jo rtpstantun 'uroq8r51uely ( dt11tq4 uBtpdtupq) -oupqI n stoutll I o K1tst a,uu n'>1reur.la'a51 Kysnruug 3uno7 utaqSt.tg'leun1A1';tr uordg ueg uoualrytr 1at4snntu7 a1o1goru'tolr1o3'derrn141 tsure4 po q?SPzrPaWpn/t.tt2H'egloo141 eletued rtp.sn ntun ap 75 otuon1Ksuua4' 11eqtl1tl1 KTts n nrun ptqtunp) SellIWqFqEzIIE aunrpaYg Jo loops eue[1t[1 K1tst antu2 ptoJualSPuP IW H H'ctrro1ua1t141 oBatq uog 'otutoJllo) ]o Kttsnnrup Jeyq erueqdel5

CONTENTS IN BRIEF

Part I 1. 2. 3. Part ll 4. 5. 6. 7. 8. Part lll 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Chemical and MolecularFoundations1 L i f e B e g i n sw i th C e l l s 1 C h e mi caFl o u n d a ti o n s3 1 ProteinStructureand Function 63

Genetics and MolecularBiology 111 B a s i cM o l e c u l aG r e n e t i cM e c h a n i s m1s 1 1 M o l e cu l a Ge r n e ti cT e ch n i ques1G5 G e n e s,Ge n o mi cs, a n d C h rom osomes 215 Transcriptional Controlof Gene Expression269 Post-transcriptional GeneControl 323

CeflStructureand Function 371 V i su a l i zi n gF, ra cti o n a ti n g, and Cultur ingCells 371 BiomembraneStructure 409 Transmembrane Transportof lonsand small Molecures437 C e ll u l a rE n e rg e ti cs4 7 9 M o vi n gP ro te i n si n to Me mbr anes and Or ganelles533 VesicularTraffic,Secretion,and Endocytosis579 C e llS i g n a l i n gl : S i g n a T l ra nsduction and Shor t- Ter m CellularResponses 62 3 c e l l si g n a l i n gl l : si g n a l i n gPathways That contr ol GeneActivity 665 C e l lOrg a n i za ti o n a n d Mo v ementl: M icr ofilaments713 C e l lOrg a n i za ti o n a n d Mo v ementll: Micr otubules and Inter m ediate Filaments157 I n t e g ra ti n gC e l l si n to T i ssues801

Part lV CellGrowth and Development847 20. Regulatingthe EukaryoticCellCycle 847 21. 22. 23. 24. 25.

C e l lB i rth ,L i n e a g ea, n d D e ath 905 T h e Mo l e cu l a C r e l lB i o l o g yof Developm entg4g N e rveC e l l s 1 0 0 1 l m m u n o l o g y1 0 5 5 Cancer 1107

CONTENTS

PartI Chemical and MolecularFoundations

E

The Diversityand Commonality of Cells

18

C e l l sG r o w a n d D i v i d e

WITHCELLS 1 L I F EB E G I N S

CellsDie from AggravatedAssaultor an Internal Program 19

1

!!|

InvestigatingCellsand Their Parts 20

All CellsAre Prokaryoticor Eukaryotic

1

U n i c e l l u l aO r r g a n i s m sH e l p a n d H u r t U s

4

VirusesAre the Ultimate Parasites

6

Cell BiologyRevealsthe Size,Shape,Location, 20 and Movementsof Cell Components Biochemistryand BiophysicsRevealthe Molecular Structureand Chemistryof PurifiedCell Constituents 21

C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n

6

of DamagedGenes 22 GeneticsRevealsthe Consequences

EvenSingleCellsCan HaveSex

7

We Developfrom a SingleCell

8

GenomicsRevealsDifferencesin the Structure of EntireGenomes and Expression

23

DevelopmentalBiologyRevealsChangesin the Propertiesof Cellsas They Specialize

23

Stem Cells,Fundamentalto FormingTissues and Organs,Offer MedicalOpportunities

@The

Choosingthe Right ExperimentalOrganismfor the Job 25 BiologicalStudiesUse Multiple The Most Successful 27 Approaches

M o l e c u l eos f a c e l l

Small MoleculesCarryEnergy,TransmitSignals, a n d A r e L i n k e di n t o M a c r o m o l e c u l e s ProteinsGive CellsStructureand Perform Most CellularTasks

'

s|

on Evolution 28 A GenomePerspective

10

Metabolic Proteins,the GeneticCode,and Organelle StructuresAre NearlYUniversal

't1

The Genome ls Packagedinto Chromosomesand R e p l i c a t e dD u r i n gC e l lD i v i s i o n

Darwin'sldeasAbout the Evolutionof Whole Animals Are Relevantto Genes

12

Mutations May Be Good, Bad, or lndifferent

13

Many GenesControllingDevelopmentAre Remarkably 28 S i m i l a ri n H u m a n sa n d O t h e r A n i m a l s Human Medicinels Informed by Researchon Other 29 Organisms

NucleicAcidsCarryCoded Information for Making Proteinsat the Right Time and Place

E

Thework of cells

28

14

C e l l sB u i l da n d D e g r a d eN u m e r o u sM o l e c u l e s and Structures

15

Animal CellsProduceTheir Own External E n v i r o n m e nat n d G l u e s

16

CellsChangeShapeand Move

16

CellsSenseand Send Information

16

covalent Bondsand Noncovalent lnteractions

to CellsRegulateTheir Gene Expression M e e t C h a n g i n gN e e d s

17

the Structureof an Atom Determines TheElectronic of CovalentBondslt CanMake 33 Numberand Geometry

31

FOUNDATIONS 2 CHEMICAL ![

CONTENTS

.

32

E l e c t r o n sM a y B e S h a r e dE q u a l l yo r U n e q u a l l y i n C o v a l e n tB o n d s

34

L i f e D e p e n d so n t h e C o u p l i n go f U n f a v o r a b l eC h e m i c a l Reactionswith EnergeticallyFavorableReactions 57

CovalentBondsAre Much Strongerand More StableThan NoncovalentInteractions

Hydrolysisof ATPReleases SubstantialFreeEnergy and DrivesMany CellularProcesses

lonic InteractionsAre Attractionsbetween Oppositely C h a r g e dl o n s 36 HydrogenBondsDeterminethe Water Solubility o f U n c h a r g e dM o l e c u l e s 37

ATPls GeneratedDuring Photosynthesis and Respiration 59 N A D - a n d F A DC o u p l eM a n y B i o l o g i c aO l xidation and ReductionReactions

Van der Waals InteractionsAre Causedby T r a n s i e nD t iooles

37

The HydrophobicEffectCausesNonpolar Moleculesto Adhere to One Another

38

3 P R O T E ISNT R U C T U R ED AN FUNCTION

M o l e c u l a rC o m p l e m e n t a r i tM y e d i a t e dv i a NoncovalentInteractionspermitsTight, H i g h l yS p e c i f i cB i n d i n go f B i o m o l e c u l e s

3e

![

C h e m i c alB u i l d i n gB l o ckso f C e i l s

@

A m i n o A c i d sD i f f e r i n gO n l y i n T h e i rS i d eC h a i n s ComposeProteins

40 41

FiveDifferent NucleotidesAre Usedto Build N u c l e i cA c i d s Monosaccharides Joined by GlycosidicBonds Form Linearand Branchedpolysaccharides

44

PhospholipidsAssociateNoncovalentlyto Form the BasicBilayerStructureof Biomembranes

C h e m i c aEl q u i l i b ri u m

49

EquilibriumConstantsReflectthe Extentof a ChemicalReaction

50

![

Hierarchical Structureof proteins

SecondaryStructuresAre the Core Elements of ProteinArchitecture

66

Overall Foldingof a PolypeptideChainYields Its TertiaryStructure

67

Different Waysof Depictingthe Conformationof ProteinsConveyDifferent Typesof Information

68

StructuralMotifs Are RegularCombinationsof Secondaryand TertiaryStructures

68

S t r u c t u r aal n d F u n c t i o n aD l o m a i n sA r e M o d u l e s of TertiaryStructure

70

ProteinsAssociateinto Multimeric Structuresand M a c r o m o l e c u l aAr s s e m b l i e s

72

Membersof Protein FamiliesHavea Common EvolutionaryAncestor

72

![

BiologicalFluidsHaveCharacteristic pH Values

51

P l a n a rP e p t i d eB o n d sL i m i tt h e S h a p e si n t o Which ProteinsCan Fold

B i o c h e m i caEl n e rg e ti cs

@

SeveralFormsof EnergyAre lmportant in BiologicalSystems CellsCan TransformOne Typeof Energyinto Another The Changein Free EnergyDeterminesthe Direction of a ChemicalReaction The AG'' of a ReactionCan Be Calculated from lts K"o The Rateof a ReactionDependson the Activation EnergyNecessary to Energizethe Reactantsinto a TransitionState

xvi

.

coNTENTs

64 65

50

B u f f e r sM a i n t a i nt h e p H o f I n t r a c e l l u l aar n d E x t r a c e l l u l aFrl u i d s

63

The PrimaryStructureof a Protein ls lts Linear Arrangementof Amino Acids

ChemicalReactionsin CellsAre at SteadyState DissociationConstantsof Binding ReactionsReflect the Affinity of InteractingMolecules Hydrogenlons Are Releasedby Acidsand Taken Up by Bases

57

50

52 52

ProteinFolding

74 74

I n f o r m a t i o nD i r e c t i n ga P r o t e i n ' sF o l d i n gl s E n c o d e di n l t s A m i n o A c i d S e q u e n c e

74 Foldingof Proteinsin Vivo ls Promotedby Chaperones 7 5 AlternativelyFoldedProteinsAre lmplicatedin Diseases 77

54

ProteinFunction

78

S p e c i f i cB i n d i n go f L i g a n d sU n d e r l i e st h e Functionsof Most Proteins

7g

EnzymesAre Highly Efficientand SpecificCatalysts

79

An Enzyme'sActive Site BindsSubstratesand CarriesOut Catalysis

g0

SerineProteasesDemonstrateHow an Enzyme'sActive Site Works

g1

Enzymesin a Common PathwayAre Often physically Associatedwith One Another

g4

!f, 54 55 55 56

56

EnzymesCalledMolecularMotors ConvertEnergy into Motion

!E-

!l|

r.r RegulatingProteinFunctionl: ProteinDegradation

RegulatedSynthesisand Degradationof Proteins is a FundamentalPropertyof Cells

CentrifugationCan SeparateParticlesand MoleculesThat Differ in Massor Density

86 86

The Proteasomels a ComplexMolecularMachine Usedto DegradeProteins Ubiquitin Marks CytosolicProteinsfor Degradation in Proteasomes

RegulatingProteinFunctionll: Noncovalentand Covalent Modifications NoncovalentBinding PermitsAllosteric, or Cooperative,Regulationof Proteins

Purifying,Detecting,and Proteins Characterizing

85

SeparatesMoleculeson the Basis Electrophoresis Ratio of Their Charge-to-Mass Liquid ChromatographyResolvesProteinsby Mass,Charge,or BindingAffinitY

92 92 94 96

Highly SpecificEnzymeand Antibody AssaysCan Detect IndividualProteins Toolsfor Detecting Are Indispensable Radioisotopes M o l e c u l e s Biological MassSpectrometryCan Determinethe Mass and Sequenceof Proteins

101

88

Protein PrimaryStructureCan Be Determinedby ChemicalMethods and from Gene Sequences

103

89

Protein Conformationls Determinedby Sophisticated 103 PhysicalMethods

88

NoncovalentBindingof Calciumand GTPAre Widely UsedAs AllostericSwitchesto Control ProteinActivity 90 Phosphorylationand DephosphorylationCovalently RegulateProteinActivitY

91

ProteolyticCleavagelrreversiblyActivatesor lnactivatesSome Proteins

91

Higher-OrderRegulationIncludesControl of Protein Locationand Concentration

92

g

98 99

105

Proteomics

Proteomicsls the Studyof All or a LargeSubset of Proteinsin a BiologicalSYstem AdvancedTechniquesin MassSpectrometryAre Criticalto ProteomicAnalYsis

105 106

Partll Geneticsand MolecularBiologY 4 B A S I CM O L E C U L AGRE N E T I C MECHANISMS @

structureof NucleicAcids

111 113

Organizationof GenesDiffersin Prokaryoticand EukaryoticDNA to EukaryoticPrecursormRNAsAre Processed l RNAs F o r mF u n c t i o n am the Number of AlternativeRNA Splicinglncreases from a SingleEukaryoticGene ProteinsExpressed

A NucleicAcid Strand ls a LinearPolymerwith End-to-EndDirectionality

113

@

N a t i v eD N A l s a D o u b l eH e l i xo f C o m p l e m e n t a r y A n t i p a r a l l eS l trands

114

DNA Can Undergo ReversibleStrandSeparation

115

MessengerRNA CarriesInformation from DNA in a Three-LetterGeneticCode The FoldedStructureof IRNA Promoteslts Decoding

TorsionalStressin DNA ls Relievedby Enzymes

117

Different Typesof RNA ExhibitVarious ConformationsRelatedto Their Functions

118

@ T r a n s c r i p t i o n o f P ro te i n -C o d i ng Genesand Formationof Functional 120 MRNA A TemplateDNA Strand ls Transcribedinto a ComplementaryRNA Chain by RNA Polymerase

120

122 123 125

The Decodingof mRNAbY tRNAs 127

Functions NonstandardBasePairingOften OccursBetween C o d o n sa n d A n t i c o d o n s Amino Acids BecomeActivatedWhen Covalently L i n k e dt o t R N A s

@

StepwiseSynthesisof Proteins on Rabosomes

Machines RibosomesAre Protein-Synthesizing

CONTENTS

127 129 130 131

132 132

XVII

Methionyl-tRNA,tttRecognizes the AUG StartCodon T r a n s l a t i o nI n i t i a t i o nU s u a l l yO c c u r sa t t h e F i r s t A U G f r o m t h e 5 ' E n do f a n m R N A D u r i n gC h a i nE l o n g a t i o nE a c hl n c o m i n g Aminoacyl-tRNAMovesThrough Three RibosomalSites Translationls Terminatedby ReleaseFactors When a Stop Codon ls Reached Polysomesand RapidRibosomeRecyclingIncrease the Efficiencyof Translation

DNAReptication

!f|

133 133

E! 135

166

137

166

138

Segregationof Mutations in BreedingExperiments RevealsTheir Dominanceor Recessivity

167

ConditionalMutations Can Be Usedto Study EssentialGenesin Yeast

EO

139 140

Duplex DNA ls Unwound and Daughter StrandsAre Formedat the DNA ReplicationFork

't41 141 143

Recessive Lethal Mutations in DiploidsCan Be l d e n t i f i e db y I n b r e e d i n ga n d M a i n t a i n e di n Heterozygotes

171 ComplementationTestsDetermineWhether Different 't7 Recessive Mutations Are in the SameGene 1 Double Mutants Are Usefulin Assessing the Order in Which ProteinsFunction 171 GeneticSuppression and SyntheticLethalityCan RevealInteractingor Redundantproteins 173 GenesCan Be ldentified by Their Map position on the Chromosome 174

D N A R e p a i ra n d R e co mb i n a ti o n 145

!!|

p|

DNA Polymerases IntroduceCopyingErrors and Also CorrectThem

145

C h e m i c aal n d R a d i a t i o nD a m a g et o D N A C a n Leadto Mutations

145

H i g h - F i d e l i tD y N A E x c i s i o n - R e p aSiyr s t e m s Recognize a n d R e p a i rD a m a g e B a s eE x c i s i o n R e p a i r sT . G M i s m a t c h eas n d D a m a g e dB a s e s

147

MismatchExcisionRepairsOther Mismatchesand S m a l lI n s e r t i o n a s nd Deletions

147

NucleotideExcisionRepairsChemicalAdducts That Distort Normal DNA Shape Two SystemsUtilize Recombinationto Repair D o u b l e - S t r a nB d r e a k si n D N A H o m o l o g o u sR e c o m b i n a t i oC n a n R e o a i rD N A D a m a g ea n d G e n e r a t eG e n e t i cD i v e r s i t y

s f th e C e l l u l a r @ V i r u s e s : pa ra si te o GeneticSystem Most Viral Host RangesAre Narrow Viral CapsidsAre RegularArraysof One or a Few Typesof protein

147

148 149 150

154

'

CONTENTS

DNACloningand Char acter i z ati on176

RestrictionEnzymesand DNA LigasesAllow Insertionof DNA Fragmentsinto CloningVectors

176

E. coli PlasmidVectorsAre Suitablefor Cloning lsolatedDNA Fragments

178

cDNA LibrariesRepresentthe Sequences of Protein-CodingGenes

179 cDNAsPreparedby ReverseTranscriptionof Cellular mRNAsCan Be Clonedto GeneratecDNA Libraries 1 8 1 DNA LibrariesCan Be Screenedby Hybridization to an Oligonucleotidp erobe 181 YeastGenomicLibrariesCan Be Constructedwith ShuttleVectorsand Screenedby Functionar Complementation 182 Gel Electrophoresis Allows Separationof Vector DNA from Cloned Fragments 184 Cloned DNA MoleculesAre SequencedRapidly by the DideoxyChain-Termination Methoc 187 The PolymeraseChain ReactionAmplifiesa Specific DNA Sequencefrom a ComplexMixture 188

154 154

VirusesCan Be Clonedand Counted in plaqueAssays 1 5 5 LyticViral Growth CyclesLeadto the Death of Host Cells 1 5 5 Viral DNA ls Integrated into the Host-CellGenome in SomeNonlyticViral Growth Cycles 158

XVIII

GeneticAnalysisof Mutationsto ldentifyand StudyGenes

R e c e s s i vaen d D o m i n a n tM u t a n t A l l e l e sG e n e r a l l y HaveOppositeEffectson Gene Function

D N A P o l y m e r a s eR s e q u i r ea p r i m e r t o I n i t i a t e Replication

SeveralProteinsParticipatein DNA Replication D N A R e p l i c a t i o nU s u a l l yO c c u r sB i d i r e c t i o n a l lfyr o m E a c hO r i g i n

5 M O L E C U L AGRE N E T T C E C H N T Q U1E6Ss

ff|

UsingClonedDNA Fragments to Study GeneExpression

191

HybridizationTechniquespermit Detection of SpecificDNA Fragmentsand mRNAs

't91

DNA MicroarraysCan Be Usedto Evaluatethe Expression of Many Genesat One Time

192

ClusterAnalysisof Multiple ExpressionExperiments ldentifies Co-regulatedGenes

193

N o n p r o t e i n - C o d i nG g e n e sE n c o d eF u n c t i o n a l RNAs

SystemsCan ProduceLarge E. coli Expression Quantitiesof Proteinsfrom ClonedGenes VectorsCan Be Designedfor PlasmidExpression U s ei n A n i m a l C e l l s

Or ganization Chr om osomal of Genesand NoncodingDNA

196

222

223

s o n t a i nM u c h G e n o m e so f M a n y O r g a n i s m C N o n f u n c t i o n aD l NA

l d e n t i f y in ga n d L o ca ti n gH u ma n Disease Ge n e s

DNAsAre Concentrated Most Simple-Sequence in SpecificChromosomalLocations

224

199

DNA FingerprintingDependson Differences DNAs in Length of SimPle-Sequence

225

D N A P o l y m o r p h i s mAsr e U s e di n L i n k a g e - M a p p i n g H u m a nM u t a t i o n s

200

SpacerDNA Occupiesa Significant Unclassified Portion of the Genome

225

LinkageStudiesCan Map DiseaseGeneswith a Resolutionof About 1 Centimorgan

201

FurtherAnalysisls Neededto Locatea DiseaseGene i n C l o n e dD N A

202

f[

Show One of Three Major Many InheritedDiseases Patternsof Inheritance

198

]f|

M a n y I n h e r i t e dD i s e a s eRs e s u l ft r o m M u l t i p l eG e n e t i c 203 Defects

ffl

Inactivatingthe Functionof S p e c i f i cGe n e si n E u ka ryo te s

Normal YeastGenesCan Be Replacedwith Mutant A l l e l e sb y H o m o l o g o u sR e c o m b i n a t i o n

204 205

Transcriptionof GenesLigatedto a Regulated PromoterCan Be ControlledExperimentally SpecificGenesCan Be PermanentlyInactivatedin t h e G e r m L i n eo f M i c e

207

SomaticCell RecombinationCan InactivateGenes in SpecificTissues

208

D o m i n a n t - N e g a t i vAel l e l e sC a n F u n c t i o n a l l y l n h i b i t S o m eG e n e s RNA InterferenceCausesGene Inactivationby g RNA D e s t r o y i n gt h e C o r r e s p o n d i nm

6 G E N E SG , E N O M I C SA, N D CHROMOSOMES

][

EukaryoticGeneStructure

(Mobile)DNA Transposable Elem ents

Movement of Mobile ElementsInvolvesa D N A o r a n R N AI n t e r m e d i a t e

226

Are Presentin Prokaryotes DNA Transposons and Eukaryotes

227

BehaveLike Intracellular LTRRetrotransposons Retroviruses Transposeby a Distinct Non-LTRRetrotransposons Mechanism RNAsAre Found in Genomic Other Retrotransposed DNA M o b i l e D N A E l e m e n t sH a v eS i g n i f i c a n t l Iyn f l u e n c e d Evolution

@

215 217

Most EukaryoticGenesContain Intronsand Produce m R N A sE n c o d i n gS i n g l eP r o t e i n s

217

n nits S i m p l ea n d C o m p l e xT r a n s c r i p t i oU A r e F o u n di n E u k a r y o t i cG e n o m e s

217

P r o t e i n - C o d i nG g e n e sM a y B e S o l i t a r yo r B e l o n g t o a G e n eF a m i l y

219

HeavilyUsedGene ProductsAre Encodedby Multiple 221 Copiesof Genes

229 230 234 234

236

or ganelleDNAs

M i t o c h o n d r i aC o n t a i nM u l t i p l e m t D N A M o l e c u l e s

210

226

mtDNA ls InheritedCytoplasmically The Size,Structure,and Coding Capacityof mtDNA Vary ConsiderablyBetweenOrganisms Productsof MitochondrialGenesAre Not Exported M i t o c h o n d r i aE v o l v e df r o m a S i n g l eE n d o s y m b i o t i c Bacterium EventInvolvinga Rickettsia-like

237 237 238 240

240

MitochondrialGeneticCodesDiffer from the StandardNuclearCode

240

Mutations in MitochondrialDNA CauseSeveral G e n e t i cD i s e a s eisn H u m a n s ChloroplastsContain LargeDNAsOften Encoding M o r e T h a n a H u n d r e dP r o t e i n s

242

Anal Ys i s Genome- wide Genom ics: of GeneStructureand Expression 243 SuggestFunctionsof Newly StoredSequences ldentified Genesand Proteins

CONTENTS

243

xtx

Comparisonof RelatedSequences from Different SpeciesCan Give Cluesto Evolutionary Relationships Among proteins G e n e sC a n B e l d e n t i f i e dW i t h i n G e n o m i cD N A Sequences

244

S m a l lM o l e c u l e sR e g u l a t eE x p r e s s i oonf M a n y BacterialGenesvia DNA-BindingRepressors and Activators

273

244

TranscriptionInitiation from Some Promoters RequiresAlternativeSigmaFactors

273

Transcriptionby osa-RNAPolymerasels Controlled by ActivatorsThat Bind Farfrom the Promoter

274

Many BacterialResponses Are Controlledby Two-ComponentRegulatorySystems

275

T h e N u m b e ro f P r o t e i n - C o d i nG g e n e si n a n Organism'G s e n o m el s N o t D i r e c t l yR e l a t e d to lts BiologicalComplexity S i n g l eN u c l e o t i d eP o l y m o r p h i s masn d G e n eC o p y Number VariationAre lmportant Determinants of DifferencesBetween Individualsof a Species

246

@ St r u c t u r alOrg a n i za ti o n of EukaryoticChromosomes

][

ChromatinExistsin Extendedand CondensedForms Modificationsof HistoneTailsControl Chromatin Condensationand Function NonhistoneProteinsProvidea StructuralScaffold for Long ChromatinLoops A d d i t i o n a lN o n h i s t o n ep r o t e i n sR e g u l a t e Transcriptionand Replication

247 248

276

250

Three EukaryoticPolymerases CatalyzeFormation of Different RNAs

278

254

The LargestSubunit in RNA Polymerasell Hasan EssentialCarboxyl-Terminal Repeat

2s6

ChromosomeNumber;Size,and Shapeat Metaphase Are Species-Specific 257 During Metaphase,ChromosomesCan Be D i s t i n g u i s h ebdy B a n d i n gp a t t e r n sa n d C h r o m o s o m eP a i n t i n g 25g C h r o m o s o m eP a i n t i n ga n d D N A S e q u e n c i n g Reveal the Evolutionof Chromosomes 259 InterphasePolyteneChromosomesArise by DNA A m p l i fi c a t i o n 260 Three FunctionalElementsAre Requiredfor Replication a n d S t a b l eI n h e r i t a n c eo f C h r o m o s o m e s 261 CentromereSequences Vary Greatlyin Length 263 Addition of TelomericSequencesby Telomerase PreventsShorteningof Chromosomes

263

7 TRANSCRIPTIONALCONTROL O F G E N EE X P R E S S I O N 269 Controlof GeneExpression in Bacteria

W

271

TranscriptionInitiation by BacterialRNA polymerase RequiresAssociationwith a SigmaFactor 271 Initiation of /ac Operon TranscriptionCan Be Repressed and Activated 271

XX

O

CONTENTS

276

RegulatoryElementsin EukaryoticDNA Are Found Both Closeto and Many KilobasesAway from TranscriptionStart Sites

M o r p h o l og ya n d F u n cti o n aEl l e m ents o f E u k a r y o ti C c h ro mo so me s 257

@

overview of EukaryoticGene Controland RNAPolymerases

279 RNA Polymerasell InitiatesTranscriptionat DNA SequencesCorrespondingto the 5' Cap of mRNAs 280

E

Regulator ysequences in pr ote i nCodingGenes 282

The TATABox, Initiators,and CpG lslandsFunction as Promotersin EukaryoticDNA

282

Promoter-Proximal ElementsHelp Regulate EukaryoticGenes

282

Distant EnhancersOften Stimu late Transcription by RNA Polymerasell

284

Most EukaryoticGenesAre Regulatedby Multiple Transcription-Control Elements

s @Activator s and Repr essorof Transcription

286

Footprintingand Gel-ShiftAssaysDetect protein-DNA Interactions 286 ActivatorsAre Modular ProteinsComposed of DistinctFunctionalDomainsand promote Transcription 288 Repressors Inhibit Transcriptionand Are the FunctionalConverseof Activators

290

D N A - B i n d i n gD o m a i n sC a n B e C l a s s i f i eidn t o NumerousStructuralTypes

290

StructurallyDiverseActivation and Repression DomainsRegulateTranscription

293

TranscriptionFactorInteractionsIncrease Gene-ControlOptions

294

M u l t i p r o t e i nC o m p l e x e F s o r mo n E n h a n c e r s

295

TranscriptionInitiation by RNA Polymerasell GeneralTranscriptionFactorsPositionRNA Polymerasell at Start Sitesand Assistin Initiation

296

@

GENE 8 POST- TRANSCRIPTIONAL CONTROL

Formationof HeterochromatinSilencesGene at Telomeres,Near Centromeres, Expression and in Other Regions

299

Can Direct HistoneDeacetylationand Repressors Methylation at SpecificGenes

303

ActivatorsCan Direct HistoneAcetylationand Methylation at SpecificGenes

305

FactorsHelp Activate or Chromatin-Remodeling RepressTranscription

306

HistoneModificationsVary Greatlyin Their Stabilities 307 307

Transcriptionof Many GenesRequiresOrdered Binding 308 and Functionof Activatorsand Co-activators The YeastTwo-HybridSystemExploitsActivator Flexibility to DetectcDNAsThat EncodeInteractingProteins 310

E

312

ResponseElementsContain Inverted Nuclear-Receptor 313 or Direct Repeats Hormone Bindingto a NuclearReceptorRegulateslts 313 Activity as a TranscriptionFactor

![

RegulatedElongationand Terminationof Transcription

314

Transcriptionof the HIV Genome ls Regulatedby an Antitermination Mechanism

315

Pausingof RNA Polymerasell Promoter-Proximal Occursin Some RapidlyInducedGenes

316

@

f[

other EukaryoticTranscription Systems

TranscriptionInitiation by Pol I and Pol lll ls Analogousto That by Pol ll

315 316

of EukaryoticPre-mRNA325 Processing

The 5' Cap ls Added to NascentRNAsShortlyAfter TranscriptionInitiation

325

A DiverseSet of Proteinswith ConservedRNA-B|nding 326 DomainsAssociatewith Pre-mRNAs SplicingOccursat Short,ConservedSequencesin Pre-mRNAsvia Two TransesterificationReactions

329 330

with Pre-mRNA During Splicing,snRNAsBase-Pair Assembledfrom snRNPsand a Spliceosomes, Pre-mRNA,CarryOut SPlicing

330

C h a i nE l o n g a t i o nb y R N AP o l y m e r a slel l s C o u p l e d Factors to the Presenceof RNA-Processing

333

SRProteinsContributeto Exon Definition in Long Pre-mRNAs Group ll Introns ProvideCluesto the Self-Splicing Evolutionof snRNAs

334

3' Cleavageand Polyadenylationof Pre-mRNAs 335 Are Tightly CouPled DegradeRNAThat ls Processed NuclearExonucleases 336 Out of Pre-mRNAs

Regulationof Transcription-Factor 311 Activity

All NuclearReceptorsSharea Common Domain Structure

323

298

o f T ra n sc r iption M o l e c u l a Me r ch a n i sms 299 and Activation Repression

The Mediator ComplexFormsa MolecularBridge BetweenActivation Domainsand Pol ll

317

296

SequentialAssemblyof ProteinsFormsthe Pol ll TranscriptionPreinitiationComplexin Vitro In Vivo TranscriptionInitiation by Pol ll Requires Additional Proteins

Mitochondrialand ChloroplastDNAsAre RNA Transcribedby Organelle-Specific Polymerases

Regulation of Pre-mRNA Processing

337

AlternativeSplicingls the PrimaryMechanismfor RegulatingmRNA Processing

337

A Cascadeof RegulatedRNA SplicingControls DrosophilaSexualDifferentiation

338

and ActivatorsControl Splicing SplicingRepressors at AlternativeSites

339

of RNA Editing Alters the Sequences SomePre-mRNAs

340

]f|

Transportof mRNAAcrossthe NuclearEnveloPe

341

NuclearPore ComplexesControl lmport and Export 342 from the Nucleus Are Not Exportedfrom in Spliceosomes Pre-mRNAs 345 the Nucleus HIV Rev Protein Regulatesthe Transportof Unspliced 346 Viral mRNAs

CONTENTS

'

xxi

CytoplasmicMechanisms of posttranscriptionalControl 347

f!|

Micro RNAsRepressTranslationof SpecificmRNAs RNA InterferenceInducesDegradationof precisely ComplementarymRNAs CytoplasmicPolyadenylationpromotesTranslation of SomemRNAs

Localizationof mRNAsPermitsProductionof Proteinsat SpecificRegionsWithin the Cytoplasm

357

347

Processing of rRNAand IRNA

358

349

f[

351

Pre-rRNAGenesFunctionas NucleolarOrganizers a n d A r e S i m i l a ri n A l l E u k a r y o t e s

359

Small NucleolarRNAsAssistin Processing Pre-rRNAs

360 363

Degradationof mRNAsin the CytoplasmOccurs by SeveralMechanisms

352

ProteinSynthesisCan Be GloballyRegulated

353

Self-Splicing Group I IntronsWere the First Examplesof CatalyticRNA

356

Pre-tRNAsUndergo ExtensiveModification in the Nucleus

363

357

N u c l e a rB o d i e sA r e F u n c t i o n a l lS y p e c i a l i z eN d uclear Domains

3G4

Sequence-Specific RNA-BindingproteinsControl SpecificmRNATranslation SurveillanceMechanismspreventTranslationof lmproperlyProcessed mRNAs

Partlll CellStructureand Function 9 V I S U A L I Z IN G, F R A C T ION A T IN G, A N D C U L T U R I NC GE L L S 371 Organellesof the EukaryoticCell

lll

372

Phase-Contrast and DifferentiallnterferenceContrast MicroscopyVisualizeUnstainedLivingCells 381 Fluorescence MicroscopyCan Localizeand euantify S p e c i f i cM o l e c u l e si n L i v eC e l l s 382 l m a g i n gS u b c e l l u l aDr e t a i l sO f t e n R e q u i r e tsh a t t h e SamplesBe Fixed,Sectioned,and Stained

T h e P l a s m aM e m b r a n eH a sM a n y C o m m o nF u n c t i o n s in All Cells 372 EndosomesTake Up 5oluble Macromolecules from the Cell Exterior 372

lmmunofluorescence MicroscopyCan Detect Specific Proteinsin FixedCells

38s

Confocaland DeconvolutionMicroscopyEnable Visualizationof Three-Dimensional Objects

386

Lysosomes Are Acidic OrganellesThat Contain a Batteryof DegradativeEnzymes

Graphicsand lnformaticsHaveTransformed Modern Microscopy

373

Peroxisomes DegradeFattyAcidsand ToxicCompounds 374 The EndoplasmicReticulumls a Network of InterconnectedInternal Membranes 375 The Golgi ComplexProcesses and SortsSecreted a n d M e m b r a n ep r o t e i n s

376

P l a n tV a c u o l e sS t o r eS m a l lM o l e c u l e sa n d E n a b l ea C e l lt o E l o n g a t eR a p i d l y

Eil

ElectronMicroscopy: Methods and Applications

388

Resolutionof Transmission ElectronMicroscopyis VastlyGreaterThan That of Light Microscopy

3gg

377

T h e N u c l e u sC o n t a i n st h e D N A G e n o m e ,R N A SyntheticApparatus,and a FibrousMatrix

CryoelectronMicroscopyAllows Visualizationof ParticlesWithout Fixationor Staining

399

378

MitochondriaAre the PrincipalSitesof ATp Productionin Aerobic NonphotosyntheticCells

378

ElectronMicroscopyof Metal-CoatedSpecimens Can RevealSurfaceFeaturesof Cellsand Their Components

390

ChloroplastsContain Internal Compartmentsin Which Photosynthesis Takesplace

379

!!| g

L i g h t M i cro sco p y: vi su a l i zi n gC ell Structureand Localizingproteins W i t h i n C el l s 380

The Resolutionof the Light Microscopels About 0.2 pm

xxii

.

coNTENrs

381

Purificationof Cell Organelles

391

Disruptionof CellsReleases Their Organelles and Other Contents

391

CentrifugationCan SeparateMany Typesof Organelles

392

Organelle-Specific AntibodiesAre Usefulin P r e p a r i n gH i g h l yP u r i f i e dO r g a n e l l e s

393

l s o l a t i o nC , u l tu re a , n d Differentiation 394 of MetazoanCells Flow CytometrySeparatesDifferent CellTypes

394

C u l t u r eo f A n i m a l C e l l sR e q u i r e sN u t r i e n t - R i c h M e d i a a n d S p e c i aSl o l i dS u r f a c e s

395

P r i m a r yC e l lC u l t u r e sC a n B e U s e dt o S t u d yC e l l Differentiation

396

P r i m a r yC e l lC u l t u r e sa n d C e l lS t r a i n sH a v ea F i n i t e L i f eS p a n

396

TransformedCellsCan Grow Indefinitelyin Culture

397

SomeCell LinesUndergo Differentiationin Culture

398

H y b r i dC e l l sC a l l e dH y b r i d o m a sP r o d u c eA b u n d a n t M o n o c l o n aA l ntibodies

400

HATMedium ls CommonlvUsedto lsolate HybridCells

402

organelles 407 Cnsstc ExprRturrur9.1 separating

ET R U C T U R E 1 O B I O M E M B R A NS s[

409

B i o m e mb ra n e L s:i p i dC o mp o sition 411 a n i za ti o n a n d S t r u ctu raOrg l

P h o s p h o l i p i dSsp o n t a n e o u s lFyo r mB i l a y e r s

411

B i l a y e r sF o r ma S e a l e dC o m p a r t m e n t Phospholipid S u r r o u n d i n ga n I n t e r n a lA q u e o u sS p a c e

411

M o t i f s H e l pT a r g e tP e r i p h e r a l Lipid-Binding Proteinsto the Membrane

427

ProteinsCan Be Removedfrom Membranes by Detergentsor High-SaltSolutions

427

$f,

sphingolipids, Phospholipids, SYnthesis andCholesterol: Movement and Intracellular

429

Fatty AcidsSynthesisls Mediated by Several lmportant Enzymes

430

SmallCytosolicProteinsFacilitateMovement of FattyAcids Incorporationof Fatty Acids into Membrane Lipids TakesPlaceon OrganelleMembranes

430 431

F l i p p a s eM s o v e P h o s p h o l i p i df sr o m O n e M e m b r a n e Leafletto the OppositeLeaflet

431

Cholesterolls Synthesizedby Enzymesin the Cytosol and ERMembrane

432

Cholesterola nd Phospholi pids Are Transported BetweenOrganellesby SeveralMechanisms

433

1 1 T R A N S M E M B R A NTER A N S P O R T OF IONSAND SMALLM OLECU LES437

E

overview of Membrane Transport

l lasses B i o m e m b r a n eC s o n t a i nT h r e eP r i n c i p aC of Lipids

415

M o s t L i p i d sa n d M a n y P r o t e i n sA r e L a t e r a l l yM o b i l e in Biomembranes

416

O n l y S m a l lH y d r o p h o b i cM o l e c u l e sC r o s sM e m b r a n e s 438 b y S i m p l eD i f f u s i o n

L i p i dC o m p o s i t i o nI n f l u e n c etsh e P h y s i c a l Propertiesof Membranes

418

Membrane ProteinsMediate Transportof Most Moleculesand All lons Across Biomembranes

Lipid Compositionls Different in the Exoplasmic and CytosolicLeaflets

419

s l u s t e rw i t h S p e c i f i c C h o l e s t e r oal n d S p h i n g o l i p i dC P r o t e i n si n M e m b r a n eM i c r o d o m a i n s

420

ProteinComponents Biomembranes: 421 and BasicFunctions

@

438

439

uniport Transportof Glucose and Water

441

Most TransmembraneProteinsHave M e m b r a n e - S p a n n i nagH e l i c e s

SeveralFeaturesDistinguishUniport Transportfrom S i m p l eD i f f u s i o n GLUT1UniporterTransportsGlucoseinto Most M a m m a l i a nC e l l s s F a m i l yo f S u g a r T h e H u m a nG e n o m eE n c o d e a TransportingGLUTProtetns

M u l t i p l e B S t r a n d si n P o r i n sF o r m M e m b r a n e - S p a n n i n"gB a r r e l s "

424

TransportProteinsCan Be EnrichedWithin Artificial M e m b r a n e sa n d C e l l s

CovalentlyAttached HydrocarbonChainsAnchor Some Proteinsto Membranes

424

$[|

ProteinsInteractwith Membranesin Three Different Ways

s re A l l T r a n s m e m b r a nPer o t e i n sa n d G l y c o l i p i dA A s y m m e t r i c a l lO y r i e n t e di n t h e B i l a y e r

421

OsmoticPressureCausesWater to Move Across Membranes AquaporinsIncreasethe Water Permeabilityof Cell Membranes

CONTENTS

O

44'l 442 443 443 4M 4M

xxiii

pumpsand the ATP-powered I n t r a c el l u l al ro n i cE n vi ro n me nt 447

@

Different Classes of PumpsExhibitCharacteristic Structuraland Functionalproperties ATP-Poweredlon PumpsGenerateand M a i n t a i nl o n i cG r a d i e n t sA c r o s sC e l l u l a r Membranes MuscleRelaxationDependson Ca2*ATpases That Pump Ca" from the Cytosolinto the S a r c o p l a s mR i ce t i c u i u m CalmodulinRegulates the plasmaMembrane Ca'* PumpsThat Control CytosolicCa2+ Concentrations N a - / K - A T P a s eM a i n t a i n st h e I n t r a c e l l u l aN r a* a n d K * C o n c e n t r a t i o nisn A n i m a l C e l l s V-ClassH* ATPases Maintain the Acidity of Lysosomes and Vacuoles BacterialPermeases Are ABC proteinsThat lmport a Variety of Nutrientsfrom the Environment

N o n g a te dto n C h a n n e l a s n d th e potential RestingMembrane

SelectiveMovement of lons Createsa TransmembraneElectricpotential Difference T h e M e m b r a n eP o t e n t i a li n A n i m a l C e l l sD e p e n d s Largelyon Potassiumlon MovementsThrough O p e n R e s t i n gK + C h a n n e l s

467

Na*-LinkedCa2*Antiporter ExportsCa2* from C a r d i a cM u s c l eC e l l s 447

448

449

451 452 453

454

The Approximately50 MammalianABCTransporters PlayDiverseand lmportant Rolesin Cell and O r g a nP h y s i o l o g y 455 C e r t a i nA B CP r o t e i n s" F l i p ' ,p h o s p h o l i p i d s and Other Lipid-SolubleSubstratesfrom One Membrane Leafletto the Opposite Leaflet 456

El

BacterialSymporterStructureRevealsthe Mechanismof SubstrateBinding

SeveralCotransportersRegulate CytosolicpH

468

A PutativeCation ExchangeProtein Playsa K e y R o l ei n E v o l u t i o no f H u m a nS k i n Pigmentation

469

NumerousTransportProteinsEnablePlant Vacuolesto AccumulateMetabolitesand lons

469

ft|

Transeprrnerar Transporr

Multiple TransportProteinsAre Neededto Move Glucoseand Amino AcidsAcross Epithelia

471

SimpleRehydrationTherapyDependson the OsmoticGradientCreatedby Absorption o f G l u c o s ea n d N a +

411

ParietalCellsAcidify the StomachContentsWhile M a i n t a i n i n ga N e u t r a lC y t o s o l i cp H

472

Cusslc ExprntueruT 11.1 stumbting Upon ActiveTransport

477

12 CELLULAR ENERGETICS 458

@ 458

470

FirstStepsof Glucoseand Fatty Acid CatabolismGlycolysis : and the CitricAcid €ycle

479

480

(Stagel), CytosolicEnzymes During Glycolysis ConvertGlucoseto Pyruvate

481

460

lon ChannelsContain a SelectivityFilter Formed from ConservedTransmembraneSegments

The Rate of Glycolysis ls Adjustedto Meet the Cell'sNeed for ATP

483

461

Glucosels FermentedUnder AnaerobicConditions

PatchClampsPermit Measurementof lon M o v e m e n t sT h r o u g hS i n g l eC h a n n e l s

485

463

U n d e r A e r o b i cC o n d i t i o n s ,M i t o c h o n d r i a E f fi c i e n t l yO x i d i z eP y r u v a t ea n d G e n e r a t e ATP (Stagesll-lV)

485

464

MitochondriaAre DynamicOrganelleswith Two Structurallyand FunctionallyDistinctMembranes

485

464

In Stagell, Pyruvatels Oxidizedto CO2and HighEnergyElectronsStored in ReducedCoenzymes

487

Novel lon ChannelsCan Be Characterizedby a Combinationof Oocyte Expression and P a t c hC l a m p i n g N a - E n t r yi n t o M a m m a l i a nC e l l sH a sa N e g a t i v e Changein FreeEnergy(AG)

Cotransportby symportersand Antiporters

@|

N a * - L i n k e dS y m p o r t e r lsm p o r t A m i n o A c i d sa n d G l u c o s ei n t o A n i m a l C e l l sA g a i n s tH i g h ConcentrationGradients

xxiv

.

coNTENTs

465

466

Transportersin the Inner MitochondrialMembrane H e l p M a i n t a i nA p p r o p r i a t eC y t o s o l i a c nd Matrix Concentrationsof NAD* and NADH MitochondrialOxidation of Fatty AcidsGenerates ATP PeroxisomalOxidation of Fatty AcidsGenerates No ATP

491

@ Th e

El e ctro nT ra n sp o rtch a i n a n d Generationof the Proton-Motive 493 Force

the StepwiseElectronTransportEfficientlyReleases EnergyStored in NADH and FADH2

493

ElectronTransportin Mitochondria ls Coupledto P r o t o nP u m p i n g

493

ElectronsFlow from FADH2and NADHto 02 Through Four Multiprotein Complexes

PhotoelectronTransportfrom EnergizedReactionCenterChlorophylla Producesa Charge Separation

514

I n t e r n a lA n t e n n a a n d L i g h t - H a r v e s t i n g C o m p l e x e sI n c r e a s et h e E f f i c i e n c yo f Photosynthesis

515

494

MolecularAnalysisof Photosystems

517

ReductionPotentialsof ElectronCarriersFavor ElectronFlow from NADH to 02

499

The SinglePhotosystemof PurpleBacteria Generatesa Proton-MotiveForcebut No 02

517

ExperimentsUsing PurifiedComplexesEstablished the Stoichiometryof Proton Pumping

499

The Q CycleIncreases the Numberof Protons Translocated as ElectronsFlow Through C o m p l e xl l l

LinearElectronFlow Through Both Plant Photosystems, PSlland PSl,Generatesa Proton-MotiveForce, 519 02, and NADPH

500

A n O x y g e n - E v o l v i nC g o m p l e xl s L o c a t e do n t h e L u m i n a lS u r f a c eo f t h e P S l lR e a c t i o n Center

520

CellsUse Multiple Mechanismsto ProtectAgainst Damagefrom ReactiveOxygenSpeciesDuring PhotoelectronTransPort

521

CyclicElectronFlow Through PSIGeneratesa Proton-MotiveForcebut No NADPHor 02

522

I and ll Are RelativeActivitiesof Photosystems Regulated

523

The Proton-MotiveForcein Mitochondria ls Due Largelyto a Voltage GradientAcrossthe Inner Membrane ToxicBy-productsof ElectronTransportCan D a m a g eC e l l s

[[

Harnessingthe Proton-Motive F o r c ef o r E n e rg y-R e q u i ri n g Processes

502 502

503

[[l

The Mechanismof ATPSynthesisls Shared Among Bacteria,Mitochondria,and Chloroplasts

505

ATPSynthaseComprisesTwo Multiprotein ComplexesTermedFeand F1

505

During co2 Metabolism Photosynthesis

524

RubiscoFixesCO2in the ChloroplastStroma

525

Synthesisof SucroseUsing FixedCO2ls Completed in the Cytosol

525

506

Light and RubiscoActivaseStimulateCO2 Fixation

525

ATP-ADPExchangeAcrossthe Inner Mitochondrial Membrane ls Poweredby the Proton-Motive Force

s09

Which Competeswith Photorespiration, ls Reducedin PlantsThat Fix Photosynthesis, CO2by the C4PathwaY

527

R a t eo f M i t o c h o n d r i aO l x i d a t i o nN o r m a l l yD e p e n d s on ADP Levels

510

Brown-FatMitochondria Usethe Proton-Motive Forceto GenerateHeat

510

Rotation of the Ft 1 Subunit,Driven by Proton Movement Through F6,Powers ATPSynthesis

@

1 3 M O V I N GP R O T E I NISN T O A N D O R G A N E L L E Ss33 MEMBRANES

Ph o t o s yn th e siasn d L i g h t-A b sor bing Pigments

ThylakoidMembranesin ChloroplastsAre the Sites in Plants of Photosynthesis

511 511

Occur Three of the Four Stagesin Photosynthesis O n l y D u r i n gl l l u m i n a t i o n

511

EachPhoton of Light Hasa DefinedAmount of Energy

513

Comprisea ReactionCenterand Photosystems Complexes AssociatedLight-Harvesting

514

$[

of secretoryProteins Translocation 535 Acr ossthe ERMembr ane

A HydrophobicN-TerminalSignalSequenceTargets NascentSecretoryProteinsto the ER

536

CotranslationalTranslocationls lnitiated by Two Proteins GTP-Hydrolyzing

537

Passageof Growing PolypeptidesThrough the Transloconls Driven by EnergyReleasedDuring Translation

539

CONTENTS

.

XXV

ATPHydrolysisPowersPost-translational Translocationof SomeSecretoryProteinsin yeast

Insertionof proteinsinto the E RM e mb ra n e

$[

540

542

543 InternalStop-Transfer and Signal-AnchorSequences proteins DetermineTopologyof Single-Pass 544 M u l t i p a s sP r o t e i n sH a v eM u l t i p l e I n t e r n a l TopogenicSequences 546 A P h o s p h o l i p iA d n c h o rT e t h e r sS o m eC e l l - S u r f a c e Proteinsto the Membrane 547 The Topologyof a Membrane ProteinOften Can Be Deducedfrom lts Sequence 547

A Preformed/V-LinkedOligosaccharide ls Added to M a n y P r o t e i n si n t h e R o u g hE R O l i g o s a c c h a r i dSei d eC h a i n sM a y p r o m o t eF o l d i n g and Stabilityof Glycoproteins D i s u l f i d eB o n d sA r e F o r m e da n d R e a r r a n g e db y P r o t e i n si n t h e E RL u m e n C h a p e r o n eas n d O t h e r E Rp r o t e i n sF a c i l i t a t eF o l d i n g and Assemblyof Proteins lmproperlyFoldedProteinsin the ERlnduce Expression of Protein-FoldingCatalysts U n a s s e m b l eodr M i s f o l d e dP r o t e i n si n t h e E RA r e Often Transportedto the Cytosolfor Degradation

550

ss2

coNTENTs

569

Largeand Small MoleculesEnter and Leavethe Nucleusvia NuclearPoreComplexes

570

lmportinsTransportProteinsContainingNuclearL o c a l i z a t i oS n i g n a l si n t o t h e N u c l e u s

571

ExportinsTransportProteinsContainingNuclear-Export S i g n a l so u t o f t h e N u c l e u s 573 573

1 4 V E S I C U L ATRR A F F I CS, E C R E T I O N , AND ENDOCYTOSIS 579 Techniques for Studyingthe SecretoryPathway

s80

Transportof a protein Through the Secretory pathway Can Be Assayedin tiving Cells

5g2

YeastMutants Define Major Stagesand Many Componentsin VesicularTransport

584

Cell-FreeTransportAssaysAllow Dissectionof I n d i v i d u aS l t e p si n V e s i c u l aTr r a n s p o r t

585

![ ss2 555

556

and Chloroplasts

.

Transportinto and out of the Nucleus

s[

552

A m p h i p a t h i cN - T e r m i n aSl i g n a lS e q u e n c eD sirect Proteinsto the MitochondrialMatrix 558 M i t o c h o n d r i aP l r o t e i nl m p o r t R e q u i r e sO u t e r - M e m b r a n e R e c e p t o ras n d T r a n s l o c o ni sn B o t h M e m b r a n e s 559 Studieswith ChimericProteinsDemonstratelmportant Featuresof Mitochondriallmport 550 T h r e eE n e r g yI n p u t sA r e N e e d e dt o l m p o r t P r o t e i n s into Mitochondria 501 Multiple Signalsand PathwaysTarget Proteinsto SubmitochondrialCompartments 561 T a r g e t i n go f C h l o r o p l a sSt t r o m a lP r o t e i n sl s S i m i l a rt o lmport of MitochondrialMatrix Proteins 565 ProteinsAre Targetedto Thylakoidsby Mechanisms Relatedto TranslocationAcrossthe Bacterial I n n e rM e m b r a n e 565 xxvi

s68

549

Sortingof Proteinsto Mitochondria 557

E!|

P e r o x i s o m aM l e m b r a n ea n d M a t r i x P r o t e i n sA r e Incorporated by Different Pathways

Most mRNAsAre Exportedfrom the Nucleusby a R a n - l n d e p e n d e nMt e c h a n i s m

Pr o t e i nMo d i fi ca ti o n s, F o l d i n g, and Quality Control in the ER

Sortingof Peroxisomal Proteins sG7

CytosolicReceptorTargetsProteinswith an SKL Sequenceat the C-Terminusinto the Peroxisomal Matrix

SeveralTopologicalClasses of Integral Membrane ProteinsAre Synthesizedon the ER

s[

s[

@

M olecularMechanisms of Vesicular Traffic

Assembryof a protein coat DrivesVesicle F o r m a t i o na n d S e l e c t i o no f C a r g o Molecules A ConservedSet of GTPaseSwitch proteinsControls Assemblyof Different VesicleCoats T a r g e t i n gS e q u e n c e os n C a r g o p r o t e i n sM a k e S p e c i f i cM o l e c u l a rC o n t a c t sw i t h C o a t Proteins

585

5g6 5g7

588

Rab GTPases Control Dockingof Vesicleson Target Membranes PairedSetsof SNAREproteinsMediate Fusionof Vesicleswith Target Membranes

591

Dissociationof SNAREComplexesAfter Membrane F u s i o nl s D r i v e nb y A T PH y d r o l y s i s

5g1

589

@

EarlyStagesof the Secretory Pathway

592

COPIIVesiclesMediate Transportfrom the ER to the Golgi COPIVesiclesMediate RetrogradeTransportwithin t h e G o l g ia n d f r o m t h e G o l g i t o t h e E R

ss4

AnterogradeTransportThrough the Golgi Occurs by CisternalMaturation

595

@

Later stages of the secretory Pathway

597

VesiclesCoatedwith Clathrinand/or Adapter Proteins 598 Mediate SeveralTransportSteps

l: SIGNAL 1 5 C E L LS I G N A L I N G AND SHORT- T ER M TRANSDUCTION 623 RESPONSES CELLULAR [[

Signalto cellular FromExtracellular 625 Response

Signaling S i g n a l i n gC e l l sP r o d u c ea n d R e l e a s e Molecules

625

SignalingMoleculesCan Act Locallyor at a Distance

625

B i n d i n go f S i g n a l i n gM o l e c u l e sA c t i v a t e s Receptorson Target Cells

626

D y n a m i nl s R e q u i r e df o r P i n c h i n gO f f o f C l a t h r i n Vesicles

599

$[

Mannose6-PhosphateResiduesTargetSoluble Proteinsto Lysosomes

600

ReceptorProteinsBind LigandsSpecifically

627

RevealedKey Study of LysosomalStorageDiseases Componentsof the LysosomalSorting Pathway

602

The DissociationConstantls a Measureof the Affinity of a Receptorfor lts Ligand

628

ProteinAggregation in the trans-GolgiMay Function in Sorting Proteinsto RegulatedSecretoryVesicles 602

BindingAssaysAre Usedto Detect Receptorsand DetermineTheir Affinities for Ligands

628

Some ProteinsUndergo ProteolyticProcessing After Leavingthe trans-Golgi

603

SeveralPathwaysSort Membrane Proteinsto the Apical or BasolateralRegionof PolarizedCells

to a SignalingMolecule MaximalCellularResponse UsuallyDoesNot RequireActivationof All Receptors

629

604

Sensitivityof a Cellto ExternalSignalsls Determined by the Number of SurfaceReceptorsand Their 631 Affinity for Ligand

Receptor-Mediated Endocytosis 606

631 ReceptorsCan Be Purifiedby Affinity Techniques ReceptorsAre FrequentlyExpressedfrom Cloned Genes 6 3 1

!f|

CellsTake Up Lipidsfrom the Blood in the Form of Large,Well-Defined LipoproteinComplexes

608

The Acidic pH of Late EndosomesCausesMost Receptor-Ligand Complexesto Dissociate

610

[!|

DirectingMembraneProteins and CytosolicMaterialsto the Lysosome

MultivesicularEndosomesSegregateMembrane ProteinsDestinedfor the Lysosomal Membranefrom ProteinsDestinedfor LysosomalDegradation

627

606

Receptorsfor Low-DensityLipoproteinand Other LigandsContain Sorting SignalsThat Target Them for Endocytosis

The EndocyticPathwayDeliverslron to Cellswithout Complex Dissociationof the Receptor-Transferrin in Endosomes

studying cell-Surface Receptors

611

Highly ConservedComPonents Signal-Transduction of Intracellular 632 Pathways GTP-BindingProteinsAre FrequentlyUsedAs On/Off Switches are Employedin Protein Kinasesand Phosphatases Virtually All SignalingPathways Carryand Amplify Signalsfrom SecondMessengers Many Receptors

612

612

RetrovirusesBud from the PlasmaMembrane by a Process Endosomes 614 Similarto Formationof Multivesicular

14.1 Following a Protein Cnsstc ExprRturruT 621 out of thecell

s!|

633 634 634

of G ProteinGeneralElements 535 SYstems RecePtor Coupled

G Protein-CoupledReceptorsAre a Largeand Diverse 635 Familywith a Common Structureand Function G Protein-CoupledReceptorsActivate Exchangeof G T Pf o r G D Po n t h e a S u b u n i to f a T r i m e r i cG 637 Protein Different G ProteinsAre Activated by Different GPCRs 539 and ln Turn RegulateDifferent EffectorProteins

CONTENTS

.

xxvii

G Protein-Coupled Receptors T h a t R eg u l a tel o n C h a n n e l s

640

AcetylcholineReceptorsin the Heart Muscle Activate a G ProteinThat OpensK+ Channels

641

Light ActivatesG..-CoupledRhodopsins

641

Activationof RhodopsinInducesClosingof cGMP-GatedCation Channels

642

[f|

Rod CellsAdapt to Varying Levelsof Ambient Light Becauseof Opsin Phosphorylationand Binding of Arrestin

644

G Protein-GoupledReceptorsThat Activateor Inhibit Adenylyl Cyclase 646

$!|

Adenylyl Cyclasels Stimulatedand Inhibited by Different Receptor-Ligand Complexes

646

StructuralStudiesEstablishedHow G,,.GTpBinds to and ActivatesAdenylyl Cyclase

646

cAMP ActivatesProtein KinaseA by Releasing CatalyticSubunits

547

GlycogenMetabolismls Regulatedby Hormone-lnducedActivation of Protein KinaseA

648

cAMP-MediatedActivation of Protein KinaseA ProducesDiverseResponses in Different Cell Types

649

S i g n a lA m p l i f i c a t i o nC o m m o n l yO c c u r si n M a n y SignalingPathways

550

S e v e r aM l e c h a n i s mD s o w n - R e g u l a tS e ignaling from G Protein-CoupledReceptors

651

Anchoring ProteinsLocalizeEffectsof cAMp to SpecificRegionsof the Cell

ActivatePhospholipase C

PhosphorylatedDerivativesof InositolAre lmportant SecondMessengers C a l c i u ml o n R e l e a s e from the Endoplasmic Reticulumis Triggeredby lp3 The Ca2*/Calmodulin ComplexMediatesMany CellularResponses to ExternalSignals Diacylglycerol(DAG)Activatesprotein KinaseC, Which RegulatesMany Other proteins Signal-lnducedRelaxationof VascularSmooth Musclels Mediated by cGMp-Activated Protein KinaseG

Clnsstc ExprnlveruT 15.1 TheInfancy of signal Transduction-GTPStimulationof cAMP Synthesis 663

15 CELL-SIGNALIN l lG : SIGNALING PATHWAYSTHAT C O N T R O L GENE ACTIVITY 66s

sl|

IntegratingResponses of cells t o En v i ro n me n taInl fl u e n ce s

'

CONTENTS

668 668

RadioactiveTaggingWas Usedto ldentify TGFp Receptors

669

ActivatedTGFBReceptorsPhosphorylateSmad TranscriptionFactors

670

NegativeFeedbackLoopsRegulateTGFB/Smad Signaling

671

Lossof TGFBSignalingPlaysa Key Role in Cancer

671

sf|

cytokineReceptors andthe JAK/STAT Pathway

672 672

CytokineReceptorsHaveSimilarStructuresand Activate SimilarSignalingPathways

673

JAK KinasesActivate STATTranscriptionFactors

674

654

ComplementationGeneticsRevealedThat JAK and STATProteinsTransduceCytokine Signals

677

654

Signalingfrom CytokineReceptorsls Regulated by NegativeSignals

678

655

Mutant ErythropoietinReceptorThat Cannot Be TurnedOff Leadsto IncreasedNumbersof Erythrocytes

679

553

6s6

656

657

Integrationof Multiple SecondMessengersRegulates Glycogenolysis 557

XXVIII

Activation of Smads

A TGFBSignalingMoleculels Formedby Cleavage of an InactivePrecursor

ReceprorTyrosrneKrnases

679

L i g a n dB i n d i n gL e a d st o P h o s p h o r y l a t i oann d Activation of IntrinsicKinasein RTKs

680

Overexpression of HER2,a Receptor TyrosineKinase,Occursin Some Breast Cancers

680

s[ $f|

TGFFReceptors and the Direct

CytokinesInfluenceDevelopmentof Many Cell Types

G Protein-coupled Receptors That

sfl

I n s u l i na n d G l u c a g o nW o r k T o g e t h e rt o M a i n t a i na StableBlood GlucoseLevel

HedgehogSignalingRelievesRepressionof Target Genes

D o m a i n sA r e l m p o r t a n tf o r B i n d i n gS i g n a l Conserved TransductionProteinsto ActivatedReceptors Down-regulationof RTKSignalingOccursby Endocytosis and LysosomalDegradation

683

@

700

PathwaysThat InvolveSignal - l nduc ed 703 ProteinCleavage

o f R a sa n d MA P K i n ase @ Act i v a t i o n 584 Pathways

Degradationof an Inhibitor ProteinActivatesthe NF-rBTranscriptionFactors

703

Ras,a GTPaseSwitch Protein,CyclesBetweenActive and InactiveStates

68s

Ligand-ActivatedNotch ls CleavedTwice,Releasing a TranscriptionFactor

705

ReceptorTyrosineKinasesAre Linkedto Rasby Adapter Proteins

685

GeneticStudiesin Drosophilaldentified Key Proteinsin the Ras/MAP Signal-Transducing KinasePathway

685

Binding of SosProteinto InactiveRasCausesa ConformationalChangeThat ActivatesRas

587

SignalsPassfrom Activated Rasto a Cascadeof Protein Kinases

688

MAP KinaseRegulatesthe Activity of Many Transcription 690 Genes FactorsControlling Early-Response G Protein-CoupledReceptorsTransmitSignalsto MAP Kinasein YeastMating Pathways

691

ScaffoldProteinsSeparateMultiple MAP Kinase Pathwaysin EukaryoticCells

692

The Ras/MAPKinasePathwayCan InduceDiverse C e l l u l a rR e s p o n s e s

693

sf|

Phosphoinositides as signal Transducers

Phospholipase C" ls Activatedby SomeRTKsand CytokineReceptors

694

6e4

Recruitmentof Pl-3 Kinaseto Hormone-Stimulated ReceptorsLeadsto Synthesisof Phosphorylated Phosphatidyl inositols in the Plasma Accumulationof Pl 3-Phosphates Membrane Leadsto Activation of SeveralKinases 695 Activated Protein KinaseB InducesMany CellularResponses

696

The Pl-3KinasePathwayls NegativelyRegulated by PTENPhosphatase

s!|

698

Arrestin ActivatesSeveralKinaseCascades 698 GPCR-Bound Wnt SignalsTrigger Releaseof a Transcription Factorfrom CytosolicProteinComplex

RegulatedIntramembraneProteolysisof SREBP a TranscriptionFactorThat Acts to Releases Maintain Phospholipidand CholesterolLevels

1 7 C E L LO R G A N I Z A T I OANN D 713 M O V E M E N Tl : M I C R O F I L A M E N T S

@

and Actin Microfilaments Structures

715

Actin ls Ancient,Abundant, and Highly Conserved G-ActinMonomersAssembleinto Long, Helical F-ActinPolymers

717

F-ActinHasStructuraland FunctionalPolarity

718

@

Dynamicsof Actin Filaments

717

718

Actin Polymerizationin Vitro Proceedsin Three Steps 7 1 9 Actin FilamentsGrow Fasterat (+) EndsThan at 720 (-) Ends Actin FilamentTreadmillingls Acceleratedby P r o f i l i na n d C o f i l i n Providesa Reservoirof Actin for Thymosin-B4 Polymerization CappingProteinsBlockAssemblyand Disassembly a t A c t i n F i l a m e n tE n d s

Activationof GeneTranscription Cell-Surface by Seven-spanning 697 Receptors

CREBLinkscAMP and Protein KinaseA to Activation of GeneTranscription

CatalyzeCleavageof Many Matrix Metalloproteases 706 S i g n a l i n gP r o t e i n sf r o m t h e C e l lS u r f a c e InappropriateCleavageof Amyloid PrecursorProtein Can Leadto Alzheimer'sDisease

@

of Actin Filament Mechanisms AssemblY

F o r m i n sA s s e m b l eU n b r a n c h e dF i l a m e n t s The Arp2/3 ComplexNucleatesBranchedFilament Assembly lntracellularMovementsCan Be Poweredby Actin Polymerization

CONTENTS

721 722 722

723 723 724 726 xxix

ToxinsThat Perturbthe Pool of Actin Monomers Are Usefulfor StudyingActin Dynamics

726

ChemotacticGradientsInduceAltered Phosphoinositide LevelsBetweenthe Front and Backof a Cell 750

Cnsstc ExpenlvlrruT 17.1 Looking at Muscle

o r g a n i z a ti o no f A cti n -B a secellular d Structures 728

@

C r o s s - L i n k i nPgr o t e i n sO r g a n i z eA c t i n F i l a m e n t si n t o Bundlesor Networks

728

Adaptor ProteinsLink Actin Filamentsto Membranes

728

Myosins: Actin-Based Motor Proteins

fifl

Contraction

755

1 8 C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E S AND INTERMEDIATE FILAM EN T S 757 [[

731

Micr otubuleStr uctur eand Or ganization

758

732

MicrotubuleWalls Are PolarizedStructuresBuilt f r o m a B - T u b u l i nD i m e r s

758

M y o s i n sM a k e U p a L a r g eF a m i l yo f MechanochemicalMotor proteins

733

MicrotubulesAre Assembledfrom MTOCsto GenerateDiverseOrganizations

760

C o n f o r m a t i o n aCl h a n g e si n t h e M y o s i nH e a d CoupleATPHydrolysisto Movement

736

Myosin HeadsTake DiscreteStepsAlong Actin Filaments

736

MyosinV Walks Hand Over Hand Down an Actin Filament

MicrotubulesAre DynamicStructuresDue to Kinetic Differencesat Their Ends

763

737

I n d i v i d u aM l i c r o t u b u l e sE x h i b i tD y n a m i cI n s t a bIi t y

763

LocaIized A s s e m b l ya n d " S e a r c h - a n d - C a p t u r e " H e l p O r g a n i z eM i c r o t u b u l e s

766

DrugsAffecting Tubulin PolymerizationAre Useful Experimentallyand to Treat Diseases

766

M y o s i n sH a v eH e a d ,N e c k ,a n d T a i l D o m a i n sw i t h DistinctFunctions

Myosin-poweredMovements

@

M y o s i nT h i c kF i l a m e n t sa n d A c t i n T h i n F i l a m e n t s i n S k e l e t aM l u s c l eS l i d eP a s tO n e A n o t h e r D u r i n gC o n t r a c t i o n

738

ftf|

738

[f!

S k e l e t aM l u s c l el s S t r u c t u r e db y S t a b i l i z i n ga n d ScaffoldingProteins

740

Contractionof SkeletalMusclels Regulatedby Ca2* a n d A c t i n - B i n d i n gP r o t e i n s

740

Actin and Myosin ll Form ContractileBundlesin N o n m u s c l eC e l l s

741 Myosin-DependentMechanismsRegulate C o n t r a c t i o ni n S m o o t hM u s c l ea n d N o n m u s c l eC e l l s 742 Myosin-V-Bound VesiclesAre CarriedAlong Actin Filaments 743

Microtubule Dynamics

762

Regulationof MicrotubuleStructure and Dynam ics 767

MicrotubulesAre Stabilizedby Side-and End-Binding Proteins

767

M i c r o t u b u l e sA r e D i s a s s e m b l ebdy E n d B i n d i n g and SeveringProteins

768

@

Kinesinsand Dyneins:Micr otubul eBasedMotor Proteins 769

Organellesin AxonsAre TransportedAlong Microtubulesin Both Directions

769

Kinesin-1PowersAnterogradeTransportof VesiclesDown Axons Toward the (+) End of Microtubules

770

745

KinesinsForm a LargeProtein Familywith Diverse Functions

771

747

Kinesin-l ls a Highly Processive Motor

772

C e l lM i g r a t i o nI n v o l v e st h e C o o r d i n a t eR e g u l a t i o n of Cdc42,Rac,and Rho

748

Dynein Motors TransportOrganellesTowardthe (-) E n do f M i c r o t u b u l e s

774

Migrating CellsAre Steeredby Chemotactic Molecules

750

Kinesinsand DyneinsCooperatein the Transport o f O r g a n e l l eT s hroughout he Cell

715

c e l l M i g r a t i o ns: i g n a l i n ga n d Chemotaxis

@

C e l lM i g r a t i o nC o o r d i n a t e F s o r c eG e n e r a t i o nw i t h C e l lA d h e s i o na n d M e m b r a n eR e c y c l i n g The SmallGTP-BindingProteinsCdc42,Rac,and Rho Control Actin Organization

XXX

.

CONTENTS

745

fifl

Ciliaand Flagella: Microtubule777 BasedSurfaceStructures

E u k a r y o t i cC i l i aa n d F l a g e l l aC o n t a i nL o n g D o u b l e t MicrotubulesBridged by Dynein Motors

777

r e a t i n gA r e P r o d u c e db y C i l i a r ya n d F l a g e l l a B C o n t r o l l e dS l i d i n go f O u t e r D o u b l e t Microtubules

778

IntraflagellarTransportMoves Material Up and D o w n C i l i aa n d F l a g e l l a

779

Defectsin IntraflagellarTransportCauseDisease by Affecting SensoryPrimaryCilia

780

Microfilamentsand MicrotubulesCooperateto TransportMelanosomes Cdc42Coordi nates M icrotubules and M icrofi laments D u r i n g C e l lM i g r a t i o n

1 9 I N T E G R A T I NCGE L L SI N T O TISSUES

$[

797

801

cell- celland cell- Matr ixAdhes i on: 803 An Overview

C e l l - A d h e s i oM n o l e c u l e sB i n dt o O n e A n o t h e r a n d t o I n t r a c e l l u l aPr r o t e i n s

803

782

805

C e n t r o s o m eD s u p l i c a t eE a r l yi n t h e C e l lC y c l ei n Preparationfor Mitosis

The ExtracellularMatrix Participatesin Adhesion, S i g n a l i n ga, n d O t h e r F u n c t i o n s

783

807

of The Mitotic SpindleContainsThree Classes Microtubules

The Evolutionof MultifacetedAdhesionMolecules Enabledthe Evolutionof DiverseAnimal Tissues

784

M i c r o t u b u l eD y n a m i c sI n c r e a s eDs r a m a t i c a l l y in Mitosis

784

$!|

Mitosis

MitosisCan Be Dividedinto Six Phases

781

l u r i n gM i t o s i s M i c r o t u b u l e sT r e a d m i l D

785

The KinetochoreCapturesand HelpsTransport Chromosomes

786

DuplicatedChromosomesAre Aligned by Motors a n d T r e a d m i l l i n gM i c r o t u b u l e s

788

to Polesby AnaphaseA MovesChromosomes Microtubule Shortening

789

AnaphaseB SeparatesPolesby the Combined A c t i o n o f K i n e s i n sa n d D y n e i n

789

s o n t r i b u t et o S D i n d l e A d d i t i o n a lM e c h a n i s mC Formation

789

CytokinesisSplitsthe DuplicatedCell in Two

789

e h e i r M i c r o t u b u l e sa n d P l a n tC e l l sR e o r g a n i z T B u i l da N e w C e l l W a l li n M i t o s i s

790

$fl

IntermediateFilaments

791

@

Junctio ns cell- celland celI- ECM and TheirAdhesionMolecules 808

, nd t p i c a l ,L a t e r a l a E p i t h e l i aC l e l l sH a v eD i s t i n c A BasalSurfaces

808

ThreeTypesof JunctionsMediate Many Cell-Cell and Cell-ECMInteractions

809

l l d h e s i o n si n A d h e r e n s C a d h e r i n sM e d i a t eC e l l - C e A J u n c t i o n sa n d D e s m o s o m e s

810

Tight JunctionsSealOff Body Cavitiesand Restrict D i f f u s i o no f M e m b r a n eC o m p o n e n t s

814

Adhesionsin EpithelialCells 816 IntegrinsMediateCelI-ECM s llow Small G a p J u n c t i o n sC o m p o s e do f C o n n e x i n A Moleculesto PassDirectlyBetweenAdjacent Cells 817

M atr ix l: The Extr acellular The BasalLamina

820

l a m i n aP r o v i d e sa F o u n d a t i o nf o r The BasaL Assemblyof Cellsinto Tissues

820 821

IntermediateFilamentsAre Assembledfrom S u b u n i tD i m e r s

792

M a t r i x P r o t e i n ,H e l p s L a m i n i n ,a M u l t i a d h e s i v e Cross-linkComponentsof the BasalLamina

Intermediate FilamentsProteins Are Expressed Manner in a Tissue-Specific

792

Type lV Collagenls a Major Structural Sheet-Forming 821 l amina C o m p o n e n to f t h e B a s a L

I n t e r m e d i a t eF i l a m e n t sA r e D y n a m i c

795

Defectsin Laminsand KeratinsCauseManv Diseases 795

[[

coordinationand cooperation between CytoskeletalElements 796

Proteins lntermediateFilament-Associated C o n t r i b u t et o C e l l u l a rO r g a n i z a t i o n

796

Components Perlecan,a Proteoglycan,Cross-links Receptors of the BasalLaminaand Cell-Surface

@The

Mra t r i xl l : Extracellula Connectiveand Other Tissues

s r e t h e M a j o r F i b r o u sP r o t e i n s F i b r i l l a rC o l l a g e n A in the ECMof ConnectiveTissues

CONTENTS

824

825 825

XXXI

F i b r i l l a rC o l l a g e nl s S e c r e t e da n d A s s e m b l e di n t o FibrilsOutsideof the Cell

926

ConnectionsBetweenthe ECMand Cytoskeleton Are Defectivein MuscularDystrophy

835

T y p eI a n d l l C o l l a g e n A s ssociate with Nonfibrillar Collagensto Form DiverseStructures

826

l g C A M sM e d i a t eC e l l - C e l l A d h e s i oi n N e u r o n a l and Other Tissues

836

LeukocyteMovement into Tissuesls Orchestrated by a Precisely Timed Sequenceof Adhesive Interactions

837

Proteoglycans and Their ConstituentGAGsPlay DiverseRolesin the ECM

827

HyaluronanResists Compression, Facilitates Cell Migration, and GivesCartilagelts Gel-likeProperties 829 FibronectinsInterconnectCellsand Matrix, Influencing Cell Shape,Differentiation,and Movement 830

AdhesiveInteractionsin Motile a n d N o n mo ti l eC e l l s

[[

I n t e g r i n sR e l a yS i g n a l sB e t w e e nC e l l sa n d T h e i r T h r e e - D i m e n s i o nE an l vironment

p[

PtantTissues

839

T h e P l a n tC e l lW a l l l s a L a m i n a t eo f C e l l u l o s eF i b r i l s in a Matrix of Glycoproteins

833 833

Regulationof Integrin-MediatedAdhesionand S i g n a l i n gC o n t r o l sC e l l M o v e m e n t

Looseningof the Cell Wall PermitsPlant Cell Growth

840

Plasmodesmata DirectlyConnectthe Cytosolsof A d j a c e n tC e l l si n H i g h e rP l a n t s Only a Few AdhesiveMoleculesHave Been ldentified in Plants

841

PartlV CellGrowth and Development 20 R E G U L AT IN T GH E E U K A R Y OT IC C E L LC Y C L E 847 Overviewof the CellCycle and lts Control

E[

849

The Cell Cyclels an Ordered Seriesof Events L e a d i n gt o C e l lR e p l i c a t i o n

849

RegulatedProtein Phosphorylationand DegradationControl Passage Through the Cell Cycle

849

DiverseExperimentalSystemsHave Been Usedto ldentify and lsolateCell-Cycle Control proteins

851

MPF Activity

853

Maturation-PromotingFactor(MpF)Stimulates Meiotic Maturation in Oocytesand Mitosis i n S o m a t i cC e l l s

854

Mitotic CyclinWas Firstldentified in EarlvSea U r c h i nE m b r y o s

856

CyclinB Levelsand KinaseActivity of MitosisPromoting Factor(MPF)ChangeTogetherin CyclingXenopusEgg Extracts Anaphase-Promoting Complex(APC/C)Controls Degradationof Mitotic Cyclinsand Exit from Mitosis

.

CONTENTS

8s6

8s8

Cyclin-Dependent KinaseRegulation Dur ingMitosis 8s9

MPFComponentsAre ConservedBetween Lower and Higher Eukaryotes

860

Phosphorylationof the CDKSubunit Regulatesthe KinaseActivity of MPF

851

ConformationalChangesInducedby Cyclin Binding and PhosphorylationIncrease MPFActivity

862

@

Controlof Mitosisby Cyclinsand

@

![fl

MolecularMechanisms for Regulating Mitotic Events

864

Phosphorylationof NuclearLaminsand Other ProteinsPromotesEarlyMitotic Events

864

U n l i n k i n go f S i s t e rC h r o m a t i d sI n i t i a t e sA n a p h a s e

867

ChromosomeDecondensationand Reassembly of the NuclearEnvelopeDependon Dephosphorylation of MPFSubstrates

870

!!f|

Cyclin-cDK and Ubiquitin-protein Ligase Control of S phase

872

A Cyclin-Dependent Kinase(CDK)ls Criticalfor S-PhaseEntry in S. cerevisiae

872

Three G1CyclinsAssociatewith 5. cerevrsrae CDK to Form S-Phase-Promoting Factors

874

Degradationof the S-Phase Inhibitor TriggersDNA Replication Multiple CyclinsRegulatethe KinaseActivity of 5. cerevisiaeCDK During Different Cell-CyclePhases 877 Replication a t E a c hO r i g i n l s I n i t i a t e dO n l y O n c e During the Cell Cycle

!fil

877 |ffirhe

cell-cyclecontrolin Mammalian Cells

2 1 C E L LB I R T H L, I N E A G EA, N D DEATH

879

90s

Birthof cells:Stemcells, 905

N i c h e s ,a n d L i n e a g e

Stem CellsGive Riseto Both Stem Cellsand DifferentiatingCells

906

RestrictedDuring Cell FatesAre Progressively Development

907

The CompleteCell Lineageof C. elegansls Known

908

M a m m a l i a nR e s t r i c t i o n P o i n t l s A n a l o g o u st o STARTin Yeast Cells

880

Multiple CDKsand CyclinsRegulatePassage of M a m m a l i a nC e l l sT h r o u g ht h e C e l lC y c l e

881

HeterochronicMutants ProvideCluesAbout Control of Cell Lineage

909

RegulatedExpression of Two Classes of Genes R e t u r n sG eM a m m a l i a nC e l l st o t h e C e l lC y c l e

881

Cultured EmbryonicStem CellsCan Differentiateinto V a r i o u sC e l lT y p e s

911

Passage Through the RestrictionPoint Dependson Phosphorylationof the Tumor-Suppressor Rb Protein 882

Adult Stem Cellsfor Different Animal TissuesOccupy 912 S u s t a i n i n gN i c h e s

C y c l i nA l s R e q u i r e df o r D N A S y n t h e s iasn d C D K 1 for Entry into Mitosis

883

MeristemsAre Nichesfor Stem Cellsin Postnatal Plants

Two Typesof Cyclin-CDKInhibitorsContributeto Cell-Cycle Control in Mammals

883

@

!![

in cell-Cycle Checkpoints Regulation

The Presenceof UnreplicatedDNA PreventsEntry into Mitosis lmproper Assemblyof the Mitotic SpindlePrevents the Initiation of Anaphase ProperSegregationof Daughter Chromosomesls Monitored by the Mitotic Exit Network

884 888

92O

cell-Typespecificationin Yeast

921

Mati ng-TypeTranscription FactorsSpecify CellTypes

922

MCMl and a1-MCM1ComplexesActivate Gene Transcription

923

a2-MCMI and cr2-a1ComplexesRepressTranscription 923 888

PheromonesInduceMating of ct and a Cellsto G e n e r a t ea T h i r d C e l l T Y P e

923

889

Arrest of Cellswith DamagedDNA Depends Cell-Cycle 891 on Tumor Suppressors

!!|

and Differentiation Specification 924 of Muscle

EmbryonicSomitesGive Riseto Myoblasts

925

MyogenicGenesWere Firstldentified in Studieswith Cultured Fibroblasts

925

Two Classesof Regulatory FactorsAct in Concert to Guide Productionof MuscleCells

926

CohesinSubunit Recombinationand a Meiosis-Specific for the SpecializedChromosome Are Necessary 895 Segregationin Meiosis|

Differentiationof Myoblastsls Under Positiveand NegativeControl

927

Cell-CellSignalsAre Crucialfor Determinationand Migration of Myoblasts

928

SpecialPropertiesof Rec8Regulatelts Cleavagein M e i o s i sI a n d l l

896

bHLH RegulatoryProteinsFunctionin Creationof Other Tissues

929

The Monopolin ComplexCo-orientsSister Kinetochoresin Meiosis|

898

Tensionon SpindleMicrotubulesContributesto ProperSpindleAttachment

898

!![

Meiosis:A specialTypeof cell Division

Key FeaturesDistinguishMeiosisfrom Mitosis

892 892

Protein of G1Cyclinsand a Meiosis-Specific Repression 895 KinasePromote Premeiotic5 Phase

Emerging 20.1 cellBiology Cnsstc ExpenlueruT from the Sea:The Discoveryof Cyclins

903

@

Regulationof Asymmetric CellDivision

930

YeastMating-TypeSwitchingDependsupon AsymmetricCell Division

CONTENTS

930 '

xxxiii

ProteinsThat RegulateAsymmetryAre Localizedat OppositeEndsof Dividing Neuroblastsin Drosophila 9 3 1

cell Deathand tts Regulation

!@

936

ProgrammedCell Death OccursThrough Apoptosis Neurotrophinspromote Survivalof Neurons

937

A Cascadeof CaspaseProteinsFunctionsin One Apoptotic Pathway

938

Pro-ApoptoticRegulatorsPermit CaspaseActivation in the Absenceof TrophicFactors

941

SomeTrophicFactorsInduceInactivationof a Pro-ApoptoticRegulator

942

Tumor NecrosisFactorand RelatedDeath Signals PromoteCell Murder by Activating Caspases

943

937

2 2 T H EM O L E C U L ACRE L LB I O L O G Y O FD E V E L O P M E N T 949

Controlof Body Segmentation: Themesand Variationsin Insects and Vertebrates 969 Early Drosophila Development ls an Exercisein Speed 970 TranscriptionalControl Specifiesthe Embryo's Anterior and Posterior

971

TranslationInhibitorsReinforceAnterior-Posterior Patterning

973

InsectSegmentationls Controlledby a Cascade of Transcription Factors

974

VertebrateSegmentationls Controlledby Cyclical Expression of RegulatoryGenes

977

DifferencesBetweenSegmentsAre Controlledby Hox Genes

978

Hox-GeneExpressionls Maintained by a Variety of Mechanisms

982

Flower DevelopmentRequiresSpatiallyRegulated Productionof TranscriptionFactors

983

!![ H i g h l i g h tso f D e ve l o p me n t

@

DevelopmentProgresses from Egg and Sperm to an EarlyEmbryo

As the EmbryoDevelops,Cell LayersBecomeTissues and Organs 951 GenesThat RegulateDevelopmentAre at the Heart of Evolution

Neural Development

985

N e u r u l a t i o nB e g i n sF o r m a t i o no f t h e B r a i na n d SpinalCord

986

950 9s0

952

cell-TypeSpecification in Early

SignalGradientsand TranscriptionFactorsSpecifyCell Typesin the NeuralTube and Somites 987 Most Neuronsin the BrainArise in the Innermost NeuralTube and Migrate Outward

988

LateralInhibition Mediated by Notch Signaling CausesEarlyNeural Cellsto BecomeDifferent

988

Gametogenesis and Fertilization g53

@

Growth and Patterningof Limbs 990

G e r m - l i n eC e l l sA r e A l l T h a t W e I n h e r i t

953

!![

FertilizationUnifiesthe Genome

955

Hox GenesDeterminethe Right Placesfor Limbsto Grow

990

958

Limb DevelopmentDependson Integration o f M u l t i p l e E x t r a c e l l u l aSri g n a lG r a d i e n t s

991

958

Hox GenesAlso Control Fine Patterning of Limb Structures

992

So Fa[ So Good

994

Genomiclmprinting ControlsGene Activation Accordingto Maternal or PaternalChromosome Origin Too Much of a Good Thing: The X Chromosomels Regulatedby DosageCompensation

cell Diversityand patterning

!f,

in EarlyVertebrateEmbryos

CleavageLeadsto the FirstDifferentiationEvents The Genomesof Most SomaticCellsAre Complete

Cussrc ExprRlueruT 22.1 UsingLethal

961 961

SignalGradientsMay InduceDifferent Cell Fates

963

SignalAntagonistsInfluenceCell Fatesand Tissue Induction

965

A Cascadeof SignalsDistinguishes Left from Right

956

.

coNTENTs

999

960

GastrulationCreatesMultiple TissueLayers,Which BecomePolarized

xxxiv

Mutationsto Study Development

959

23 NERVE CELLS !fl

Neurons andGlia:Building Blocksof the NervousSystem

1001

lOOz

Information FlowsThrough Neuronsfrom Dendrites to Axons 1003

NXXX

.

slNliNo)

I1r:r;rrad5sllqlqxl z90L 'asualaq aur-lpJtqI aql ,{lrunurur;anrldepy +o Iyunr.uur;anrldepy pue aleuul qlog sesseduo:u3 1eq1funtul o1 esuodsagxa;duol e sl uotleuuel+ul tgot

possoJ)eJV siaureg le)ruaq) pue le)tueq)a[ Jauv asue+ac]o aurl puo)es e sapt^oJd{lrunulur; eleuul

650I

sua6oqle6}sure6y esualac ,rolar{e1p.rr3e r u r o l s a u e p u n o gl e ) t u a q ) p u p l e ) t u e q ) a l sapol qdu{1 pue sonssrlut a)uaprseud1 a1e1 pue r{pogaq} }noq6nolql a}eln)lt) say{:o1na1

650I LS1L

sosualaolsoH lo /na!rua^o @

LSOI

saltsluaJa#tq 1e a1e:r;dagpue selnou luala#tC q6notql {pog eqr la1u3 sue6oq1e4

LSgl

vzol EZol EZOL €20r ZZ0l

ZZOL 0z0l 6t0l

ss0r

A9o]oNnwNtvz

8r0t 8t0t

suoxv aprng os;y uo1e1n6ey ;elueuldo;ana6 saln)alonj e)ueprn9 uoxv j|'osatltuel Jnol elv aJeql

gVOL

urnl ol euo) qlmorg eql asne) saln)alonja)ueptng uoxv

LVOL

EtOI

zvll

suotpauuo) uoxv +o uep{5 paJeprg ue polee^audeyl ;epaloulleu aql o r n p n J l sa ) u e p l n g {.rosua5pazuo}on e sl euo) qwoJg oql

0t0r

0t0r

vt0L €t0t €t 0 t Et0t t t0t

6ur1e6le1pue qlmoJg uoxv

6ur;;o.r1uo) :ssa))n5ol qled aql ]!!|

600r 600t

slopo pola6 slolderag +o eJoqlold V png elsel q)el ul slla) slasqns r{q pesua5eJV sa}sel ,{leuur.16 anrl +o u o t l o w p u e p u n o sp e l a o s l l a ) J e l J a u u l

9€0r vgol Z€OI

plro4 aql ;o sa6eur;sturoJslla) uor;6u e9 a;dr11 n yr1uloJ+uotleu.ro;uI palet6a1u1

6Z0L

a J n s s e Jpdu e ' q ) n o l 'plo) 'leaH 'uted palaq s1;a1fuosuesoueq)al4

t€0r

820r

fuo1sr11 fueuorln;on3pa;rragsa{3 slla) a^JeNanrlrsue5-1q6tl saJnleal a{3 aq1

LZOI

600r /00 t

900t

900t

leuueq) uol eLll ol alnqtlluol loyderag a u r ; o q : ; { 1 a r y) t u t } o ) t Na q } u ! s } ! u n q n So ^ t J l l V uotpeJluo) al)snlAol speel slauu eq) uorle) paleg-auIlor1tl{1e:yrro6u ruadg sol)rsan:r1deu{5 epl(rag louue) urueu{q 6urpel s1ue1ny1 {;3 sJellil.usueJloJneN +o a)eldnou Jo uorlepel6eq {q paleurulal s; sasdeu{51e 6ur;eu6r5 auerqruolAeu,tseld aql qllm sal)tsenrgtdeu{5 y + o u o t s n l s a l e ; n 6 a gu t a l o l d 6 u r p u r g - u n r : ; e 1 sJellil,usueJloJnoN

,to esealags.ra66rl1+ze) +o xnl+ul auelqual^ eruseldoql Jeeu peztle)o'lalv lalll,usuetlolnaN qllm pepeo'l sal)tsanrrldeu{5 sutelold 1.rodr1uy pelu!'l-+H {q sa;:rsel rrldeu{5 olur papodsue4 aJV sJe}}l,usuel}olnaN sornllnJls :r1deu{s1so6 pue trldeu{set6 1o {lquassy selrnbegsasdeu{5}o uotleruJoJ

sosdeu^S le uollet!unuuo) fl!| sasdeu{5pue sq}eaqsur;aIy1e)npold etlg suoxv paleur;a{y1ur opoN ol apoN LlJol+,,dr.un;,, sletlualoduotpv uotpnpuo) as;ndul ;o fir:o;a4 eql saseaJ)u;uorleur;a{61 rlrtolJuol el)olg elod uedg oql olul 1uaur6a56urlenrpeu;-leuueq)aql 'to luaue^ol u o r l e z u e ; o d e qa u e l q u a n j o t esuodsegur o^ol4 se)tleHn y5 6ursua5-a6e11o4 sernpnJls

Jelrulse^eHslauueLl) uol poleg-e6e11on 1;y dIV +oa)uesqvaql u! sletlualoduotpv {ue61pnpuo) ue) slle) o^JaN u o r l n u r u r cl n o q l l ^ {;;euorpelrprul palebedoJde.tv slet}ue}oduotpv sletluelod uorpv alelouag slouueq) *) pue +eN peleg - a 6 e 1 ; o 4 ; o6 u r s o 1 1 p u e 6 u r u e d g; e r l u e n b e 5 "tJ ol asol) sl lerlualod uorl)V aql +o apnlru6eyl aql

slla) o^raN ut sletlualod uotl)v lo uolle6edol6 eql pue slouueq) uot paleg-a6ellon

6ut;;eru5pue ,6utlsel ,6u!lpaH

LZOI

'6ur;aa3'6u;aa5 :slle)leuotlesuas ]ftfl

SZgL ole)tunwuo) ol suoJnaNlvtollVoslv suorpunpde9 SZOI

lerlualod uorl)V ue eleJaueD o] uotst)ec ouoN-lo-llv ue e)ey! sllo) o^JeN

s00I s00r €00r

@

suoJneNa;dr1;n61 ;o pasodtuol slrn)r) 6ur;eu6r5sesl urals{5sno^JoNeql sasdeu{5 er^ suolnaN uaa/V\lags/v\olJuotletuJolul slerlueloduotl)v polle) ^/\oll uol +o soslndse sa^olNuotleLuro+ul

peard5ue) pue a^rse^ulaJV slla) Jorunl lrlelselal4

G0t t

s;;a1burlera+rlordur aleur6r.rgfi;;ens1sJe)ue)

0l I I

slassanpool8 MaN +o uorleruJoJsal|nbeuqwoJg rotunl uorlelndod{1r.roury1; e ag ue) slle) uie}SJe)ue)

tttt

tltr

6 0 tt

Ja)ue) lo

lasuo aql pueslla) lounl Ell

LOLL

ulfNv) sz

)rleuos S0lt sauegurlnqo;6ounururl ;o luaura6uereau :auoauoraSsaua9o/nl u'?z lNlwtuldxf )tss\n) sua6oqled 1o I1er.rel e pure6y {ilunuu; o^rpa}ord lr)rll seur))en

l0 t t

slle) I pue I uaa/l^laSuolleroqello) serrnbagsarpoqrluvfirurgy-qbrp +o uorpnpord sg;a36urluasel6-ue61luy +o uotle^rpv ol speal s.rolda)aua)r'l-llol rro1uer.ua6e6u3 surauedreln)eloulor)et4pa^ueq-ue6oq1e6rto {1aue1 e a^ralrad slolderag a)ll-llol

660t 660t L6Ol

L6OL esuodseue^rldepv aql ur slla) uals^s-eunuul lo uolleroqellof @

?80r 280t z80t

t80t

sen) )rpeloueq)

960r s60t s60I v60l t50t

saurlouraq) {q peprnor4 ol asuodsegur a^ol4 say{ro>;na1

slle)8pueI+o

6801,

ur;nqolbounuurlue +o uorpod (qe)l aqf selqueseu.roldaraulla)-I aql lo alnpnrls aql

880r

SINIINO)

.

Inxxx

^emqled rrYbopu3 aql ol pale^tloq sua6rluy sluasard^eMqled )HlA ll ssel) suabgluv rr;osoy{1s}uesaJdAennqted)HtA I ssel) a)e+rnslla) eql ol palsod pue sllnpoJd )HlA r.llltvrpexa;duo3 e:y sluaulbeJl ulelord q)lrl6 {q ssa:o.r6oql sl uo!}eluesald uabtluy loldarag lle)-I eql qltm pelalul pue sua6gluveprlde6 purg seln)elon )Hu\l

6LOL salnrolol4)HtA +o sassel)putlstq onnl {q peplnD aly saryadold luale#tG qllm slla) I leuorpunJ papu5eu )Ht Pue r11;rad5 8/0 t ua6gluv sl slla) r rxoloil),to firnrpy 6ut11t;aq1 s4e.r9 pelag to ]derrv o] LLOI sat:ed5aues aql lo slenPl^lPul palelerun orvtl+o {rtttqv eq} seulura}a6 )HW eql 9L0l,

sL0l vL0l

ELOI ELOL

960r

sJeryel4a)eynS ,ro uorsse:dx3pue uolpnpo.rdautloy{3 lteLl1uo paseg sessel).rofe61aarql olur pepr^rqarv slla) I tG) s l l a ) a u n u u l r e q l o o 1s ; e u 6 1 5 apr^ord1eq1seurlop{1,ro{er.ry ue a)npord slle) I 5ty pue 6 u t t 1 >l o + p e z t l e t ) a d a toldarel-o3 gC) eql fure1 s;1a3I rrxolol{) uorle^rpv llnl ro+ ;eu6r5;o sed{.1onnl elrnbag slla) I uorpelas anrlebappue enrlrsod+o ssa)oJde q6norql do;anaq saln)elot 1961 6urzru6o)ag+o alqedel slla) I

t60t

uorlerluoJa#!cpue uorleJo+rloJd sre66u1s.roldareurtltrad5-ua6rluvet^ 6ur1eu615 s1uer.u6e5 auag f pue 'C h uaeMlaq suotpunf eql u! pepo)ul sanplsauelqerrenllaql ]|.o ^uen qllM esle^tc fua4 ery s-roldarag1;a3-1 s a u a gu r l n q o l 6 o u n u l u t ; ol Jelrursieuuel4 e ur pa6uerreagaJV saueg U)I

880r

tueudola^ao lle)-I pue ' sr ol da) au l la) -I's lto) L @

880t

LLOJ

690t

uo!leluaseJd

uabrtuvpue)HW.qL'ljEE eleyrl laql ur;nqo;bounuurl;o adllosl aql q)llms ue) slla) I 61 pelana5 6ur1e61o1 61punog-auelqulal46ut1e61uto.t; q)llrvrsslla) I 'esuodsegenrldepy ue 6utlnq rolda:eg lla) E-erd e ruoJ+lndul salrnbagluetudo;aneolla)-g salllul#v panordutlqltm serpoqlluv 'to uolpoles pue >t1euo5 uorleJauogaql sMollV uorlelnur.rad{;1 s1uau6a5aue9 f Pue 'q h sa^lo^ulsn)ol uteq)-^^eeHaqt ;o luaula6uelleag

auag f Puen lo s1uar.u6a5 {;qrrassysaltnbagauag uleq)-}q6llleuol}lunJV

luaudola^ec lle)-g pue 69Ot flrsre^rc fpoqrluv lo uollereuag|fff| saryedol6 leuorpunl 890r sll sauruJelaq uot6ag luelsuo) s,urlnqol6ounululuV l{1r:r;rrad5 LgOL altsgnbx3Jteql lol sluno)lv seln)eloy! r{poqtluy }o alnpnJ}S leuolsuaulO-eaJqIeql puog opr+lnslqe ^q LgOL pazrlrqelsslaaqs $ orv'r1 'to pasodu.tol plol )llslrepereq) e e^eH suleuoq urlnqo;6ounuul

990r s90I €90t

€90t

ut;nqo16ounuu1 'anbrul e se)npord petnqtJlstq{11euo13r llo) I q)el suotpunl luaJe#tc qll/v\qlel 'prx3 sad{1os;ur;nqo;6ounuull a;dt11nyr1 sureL{)rq6lt pue {aeag ;o 6ut}stsuo) arnpnJls paruasuo)e e^eH sut;nqo;6ounuu.t1

u o ll) u n l p u e a r n lr n r ls :su lln q o l6 0 u nuul

SpecificMutations TransformCulturedCells into Tumor Cells

1113

A Multi-hit Model of CancerInduction ls Supported by SeveralLinesof Evidence

1114

Successive OncogenicMutations Can Be Traced in Colon Cancers

1116

PatternsCan DNA MicroarrayAnalysisof Expression RevealSubtle DifferencesBetweenTumor Cells

1116

@ Th e

G e ne ti cB a si so f ca n ce r

1119

Gai n-of-FunctionM utationsConvertProto-oncogenes 1119 into Oncogenes VirusesContainOncogenesor Cancer-Causing Activate CellularProto-oncogenes

1121

Mutations in Tumor-Suppressor Loss-of-Function GenesAre Oncogenic

1123

Genes Inherited Mutations in Tumor-Suppressor lncreaseCancerRisk

1123

Aberrationsin SignalingPathwaysThat Control DevelopmentAre Associatedwith Many Cancers 1124

!fl

oncogenicMutationsin GrowthPromoting Proteins

1127

OncogenicReceptorsCan Promote Proliferationin the Absenceof External Growth Factors

1127

Viral Activators of Growth-Factor ReceptorsAct as Oncoproteins

1128

Many OncogenesEncodeConstitutivelyActive Proteins Sig nal-Transduction

1129

InappropriateProductionof NuclearTranscription FactorsCan InduceTransformation

11 3 0

M o l e c u l a rC e l lB i o l o g yl s C h a n g i n gH o w C a n c e r ls Treated

1132

MutationsCausingLossof and Gr owth- lnhibiting Controls Cell-Cycle

1134

from MutationsThat Promote UnregulatedPassage Gr to 5 PhaseAre Oncogenic

1134

MutationsAffecting ChromatinLoss-of-Function RemodelingProteinsContributeto Tumors

1135

Lossof p53 Abolishesthe DNA-DamageCheckpoint 1136 Apoptotic GenesCan Functionas Proto-oncogenes 1137 Genes or Tumor-Suppressor CheckpointsOften Leadsto Failureof Cell-Cycle T u m o rC e l l s i n Aneuploidy

1138

and CaretakerGenes Carcinogens 11 3 9 in Cancer CarcinogensInduceCancerby DamagingDNA

11 3 9

SomeCarcinogensHave Been Linkedto Specific Cancers

1139

Lossof DNA-RepairSystemsCan Leadto Cancer

1141

Contributesto TelomeraseExpression lmmortalizationof CancerCells

1143

GLOSSARY

G-1 l-1

INDEX

CONTENTS

.

xxxvtl

uorlnlo^l uo a^tlledsJadatuouag v

s u e d J r a q l p u e s l l a ) 6 u r 1 e 6 r 1 s a n u ; v't

0z

s't

8Z

vl o

I

slla) +o )ro^ aqf lla) e j|.oseln)alon aql slla) +o Alleuourruol pue Ilrstenrq eq1

E't

z'r t'L

I Nn r n o

:suropSur>1 rnot apnlrur selodre{ng .rrlodre4ne pue rr}o -dre4ord-s11acyo sad& o^\t stsrsuof,asrelrun pcrSolorq eq1 Jo

)llo^relnl ro )rlo^Jplord otv sllo) llv 'sllaf snorrB^ Jo uorlf,unt puE ernlrnrls Oqt ur serlrrpFurs saJuereurp puE 0r{l .>1ooqsrqt tno r{toq srseqrplnlolotu aqt uo snrot IIr^ e_ry\ Jo -q8norqr sploJunsllar drols aqr sy de.u atuesoqt dllecrseq to ur sassecordperetlldtuol dueur tno drrec pue sorntpal Iernl -rnrls urPlref,Orpqssllof, 'seruera;;rprJqto snorerunupue 11e asaqralrdsaq 'poo; rse8rpsn dyeqpue seurtsolurrno ur e^rl lerlt prretrpq eql ro rre eqt ruorJ uaSo.ltrutrerlxe ol srueld dlaq reqr Euatr€q oql op se 'sursrue8royo saddl reqto qlr.41r uorlerfosse asolf, ur aArTJo seruolof,tuJot sJar.{lo.uorlelosr ur elrl stusrue8rorelnllarrun oruosq8noqrly .sllel reqlo qtr^{ pa^lo^ur dyateurrturare srusrueS.ro relnllarrllnu ur sllar lsory 'sraqlo roJ luourarrnba.ralnlosqe ue sr lnq sllal atuos sllr>l ua8dxg 'elgers,{llernlcnrtspue dreuouersdla8rel arp srer{tO

'sJaJrloJpue seqeue 'so;n1lnl1s to sarloru ur eesuef a.rl sE Sur8ueqc-lseJe^eq pue dlprder e^ou aruos '(1-1 arn8r4) sadeqs pup sezrs lo f,lBrrcr, Surzeute ue ur otuoJ sllaJ

slla) Io f1r;euourruo)pue Alrsla^lqaql lU 's11arSurdpnts sdp.^a snorrel ar{l ruo{ urpel uef, to aA\ let{,ryrpup (suorlf,unJIeJrtrrr pup sluenlrlsuoJ Jrsgq Jroql 's11ec dlrs.rallp aqt Surcnporrurdq rerdeqr anSolord srql ;o ur truls elrN'slleJ rltpJp pue 'ayr1'qrrrg aqr 1o ,{lors petJceJ Jo -ulnru oqt 3ur,teo,ndgenper8 'splaryasegt to IIE r.uoryu,ry\pJp saqreo.rddelpluorurrodxapue slq8rsureqrrJsep11r,ue,tt 'sral -deqc SurrnolloJer{r uI 'uorletuour.radxa;o a1d1spue srseqd ',(8o1orq -rua u^\o slr spq splort eseql Jo qre1 leluarudolo,r -ep pue'eruerf,s relndruor tSolorsdqd'slrlaue8 1(docsonrur iiSoyorqrplnf,oloru'sorsdqdorq'dtlsruaqcorqreqtoSol sBur.rq (qcr.r sr lSolorq r lerll erualrs a,rrlBr8elut [e] JEInJOI6W 'puodsa; o1 sdem eleudordde tsotu oqt uo aprrep daqr moq pue sllor q8no.rqrs^{olJuorler.uro;ur^/rl,oq lnoqe dlrelnrrued 'peureal eg ol sureruoJlunotup asuerururue '11115 'Joqlo qJEe aJuenlturpu€ qrnot dagr ,vroq'urEluoJ daqt sernlcnrls tEr{na 's11ac sluouodruor ar{t tnoqp Etep.ry\au;ouorsoldxaue aJe! 1o o.tr 'suado d.rntuec tsrrJ-.{tue,v\teqr sV 'aJII Jo lrun Ietueruep 'aJIIyo sartradord>lreurller{ -unt oql sr IIar eqr tpr{t SuneJrpul aqt IIe trqrqxo susrueS.rorulnlloJrun aldurrsua,lg '1ac epurs e Jo lsrsuof, sulsrue8rodueru 1nq 'salnlf,n.rlsxalduror olur pezrve8rc sllel Jo suor1lrrtro suorllq ureluor srusrueSrore1 -NIIE]qFI.U

JOTIIO PUP EII

'OJII

OUIJEP SEIIITIqE OSAqI 'SUOIIJE

-eJ lef,rtuor1J lo AeJreSur.zetueue lno ,llrct pue'rlnurrls 1 ol puodsar 'uorleru.ro;urssecord 'aonpo.rder?rro-r8uer I sarpoq rno urot teql sllel lenpr^rpur eql 'sa,rlesrnoa>1r I 'sraLtr)easau oioq6l{poq ueurnqp }o sllo)uotllu}0L aq}astJe []ul utads pue 66a up jo uotuneql uJoJjs1leta16urs oslea.teLl)tqM 1;rnn 'uradsqlrnn'66a ueunq aq] 'llo)(urrl)Jaloulol)tLU 667- al6ursy

Sll:l) HIIM SNID] B ]J II

U] IdVH)

uEeJoeqt w deaPselru uoeq o^ErIsllel f,nodrElord / Puno} '3rl 'eggl X S' sr rltrea zr0l lnoqr to lelol e Surq8ra.n uo ErratJeq Jo regrunu pelerurlse eqJ 'srusrueS.rolenplrr 'lq8ram s(uprunq -lpul /r0I x t ueql ororu sluoserda.rsrql e8erat'eaqr 1o 3>1S'I-I pareurnseuE JoJ tunof,Je euetJpq led '3 or_0l x SZ rnoqe ;o rq8rerrtd;p e seq vJnr.nl)eq tpz olq)tnq?sg el8ursy 'sseruorqs.r{trpaaql ;o ued a8nq e dn a>1eru deqr tlenprzrrpur llptus erE salo,fue>1ord qSnoqlly 'uonezrueB 'losoldr lo 'lorrolur leuJalur 1o ecuese.rdaql Surlerrpur -Jo

s ' r ] l ) H r - r As N r 9 3 8l l l ' l

I

t urravul

ere suralo.rdduetu tsluatu snoenbBJreql ur pazllelol dyasrcerd -lredtuor papunog-euprgueu e^Eq tou oP sllal Ielrelleq q8noqrly 'sller Jo suleql snotueru€llJeq ro relnllorlun 0q ro'etrelcegouedo eql lsursrue8ropa11ac urc'ae31e uaarS-en1q -apurs are setodre{ord;o adrb snoJetunue'eualteg 'adfi sqr Jo slleJJolslsuof,salofrelord IV'FZ-I ern8rg) uooeztue8to Ieuralur aldulrs dlalueler e seq PUE'snalcnuPouIleP€ S>IJEI 'euelqtuatu euseld aqr dq Papunorrns sI letll luolulJedluol crtodre>1ord'EorqtrrePUEElrel pasolJa18urse Jo lslsuoJ s11ac -l€q apnllu satodru>1or4'ststlord pue'r8un;'sleuttue'slueld

'pa]rutlun nno11e{ q)ee'satuolo) talq6nepaleoplsulsasseu-r pleqlru(q)Uedaur)rpay\ slenst^,/qrqs {ueu ;o dn apeLu ) puelassa) [ ]ul ]o looqls 'sllo) 'slop 'nelg riuetu dn apeu.t alqtst^ pio]uets to anlq se {1rlenru61 uolourqseg'uoplog (6)Ued[]rstanrun uaar6 aql 1o n lenpt^tpur JJa[ xo^loA'e61e uaet6pa11el-e;6uts (+)]Jed)ul'pa]rulrlul-1 (a)uedlul 'pa]rurlun aresaraqds a6relaqt 'snarne ualaH s1ensrl7;e:r6o10rg eurlore) leluolo) 'slaq)reasau punoMe le ]ol) (p) paztltsso+ :s11ar a;6uts e6lel slensrlTueqburuunl o]oqd/HlN ()) tul Ued C uqo[(p)Uedrul V (a) s60arnesoutp 'pa]rullun olesllaluaal6eql eprnotd qdleu(q)Uedlul 'slaqlleasau poolqa)eu o] sa)uplsqns slensrn oloqd7ra16ne9 leq] slalaleld /uosurqou aq};o ped ale (sa}l{ro)nal) aunLuu.tl pue 'u..orpa+ur tueD(e)Uedlpoo] puetaleM+oUodsuet]lo] saqnlolur paurof lq6rl pueuuals{s ole slla) slla)poolqaltqMaql 'seyilolqyire0utleaq-uao^xo pr6ue Iq peyoddns oreslla)aql uoaMlaq sered5uo1ela)s asolnllel 's1ue;d 'dol poolq par ut uMoqs'slla)poolg()) ^ep6e6oueqlaul rolo) oslej oqf paxr} re;nrsen ur ,{1turr; (q) areld ur ore slle) oq} iueld ant6oltle)+ouaulnJaql ut antlleql solfads uror+por..ls oresllo)plopue'rllr^aLl]JosasPq aql reau,i;snonurluo: Ogt< o] asr.i ]o sJa]rl pueaptxotp uoqlel6utpenuol o1se6ue6orp{q aurosaueqlaLu r!ro] slla)nnel ,ipoqaq];o spedraqlool uodsue.rl ro} poolq euapeqaeq)le {q ,i6raualraq}a)npoldleq} (pul)/esoueqlanl) pailalsuer] aql ol ]aaqsletlaqltde eq1qonotqtpoolpa5e6tpuloJ+ }o 'sn;1n sseLu (q) uaquaure]pue'all8'po;enbogseq)nsesaaq> a>npold alesluaulnN]aaqssnonurluol e urslla)IueLusureluor v 's;;a> Outptntp q)el 'a.loquMoqsaurlsalur ol pasnereq)tqM 's1)elsrn)QoDe7alPasaqf q6norqlarr;s e 'sllo)+oremole1r1-ra6urJ snslan )tloqelaupue'(sllallr1ofue1na alou:euol)eqnl(e) satlt^t])e aql urse'leaqsler;eqlrda ue urol ue)slla)(6) uro[oqeql ]e qlnq 'o^oLll ltaqtul le]]lp o] ^lt1lqe rrlo,telord)uotleztue6lo {q a;qrsrn eq1sr{poq llo)oql lurolord e }o uorl)npo.r}ur lPulalul luerseronl; asaqlul pa}el}snlll u1'sqdelooloqd o} uol}lppe sllar,{6oloqdroLu apeurseMllo)aq1'salrrpuap aq1qbnorqls1lar ]o )roMlaupaq)uerq oq] jo auos 'sazlspue sadeqs srsllo)]o ,ileuen;e:roo;otldloul e6re1 raqloqllMsuot])auuo) ueqlarourulro]uel leql ooo'00 t ;1at y (+)slla) ,{;qrperrur ue 'urnllaqara) aLl}}o uornauelurlrn4el6urs lo luauruosse6ulpunolseue ul auo) slla) l-l lunglJ V

(b) Eukaryoticcell

( a ) P r o k a r y o t icce l l P e r i p l a s m iscp a c e a n d c e l lw a l l

Golgi vesicles

Lysosome

O u t e rm e m b r a n e

I n n e r( p l a s m a ) membrane

Nucleoid lu'btrm I

Nucleoid

N u c l e a rm e m b r a n e P l a s m am e m b r a n e Golgi vesicles Mitochondrion Peroxisome Lysosome

I n n e r( p l a s m a )m e m b r a n e

Periplasmicspace Outer membrane R o u g he n d o p l a s m i c reticulum

1-2 Prokaryotic cellshavea simplerinternal A FIGURE micrograph of a organizationthan eukaryoticcells.(a)Electron coli a commonintestinal bacteriumThe thin sectionof Escherichia withina of the bacterial DNA,is notenclosed nucleoid, consisting by two membraneE coli andsomeotherbacterra aresurrounded spaceThethincellwallis membranes separated bythe periplasmic (b)Electron mrcrograph of a plasma adjacent to the innermembrane antibodies Onlya single cell,a typeof whitebloodcellthatsecretes (theplasma membrane membrane) surrounds thecell,butthe manymembrane-limited compartments, or interior contains Thedefining of eukaryotic cellsis organelles. characteristic

whichis DNAwithina definednucleus, of thecellular segregation is membrane Theouternuclear by a doublemembrane bounded for factory a reticulum, withthe roughendoplasmic continuous process andmodifyproteins, proteinsGolgivesicles assembling to digestcellmaterials generate lysosomes energy, mitochondria and process usingoxygen, molecules them,peroxisomes recycle them to the surfaceto release carrycellmaterials vesicles secretory E M u r r a y . P a r t ( b ) fromPC G B u r d e t t a n d R D J lPart(a)courtesyofl lJltrastructure: A Functional 1993,CellandTissue Cross andK L Mercer, andCompanyl W H Freeman Perspective,

and 40 miles up in the atmosphere;they are quite adaptable! The carbon stored in bacteria is nearly as much as the carbon stored in plants. Eukaryotic cells, unlike prokaryotic cells, contain a defined membrane-bound nucleus and extensive internal membranes that enclose other compartments called organelles (Figure 1-2b). The region of the cell lying betweenthe plasma membrane and the nucleus is the cytoplasm, comprising the cytosol (aqueousphase)and the organelles.Eukaryotescompriseall membersof the plant and animal kingdoms,including

the fungi, which exist in both multicellular forms (molds) and unicellular forms (yeasts),and the protozoans (proto, primitive zoan, animal), which are exclusively unicellular. Eukaryotic cells are commonly about 10-100 pm across' generally much larger than bacteria. A typical human fibroblast, a connective tissuecell, might be about 15 p,m acrosswith a volume and dry weight some thousandsof times those of an E. coli bacterial cell. An ameba, a single-celledprotozoan, can be more than 0.5 mm long. An ostrich egg beginsas a singlecell that is evenlarger and is easilyvisible to the naked eye. THE DIVERSITA Y N D C O M M O N A L I T YO F C E L L S

All cells are thought to have evolved from a common progenitor becausethe structures and moleculesin all cells have so many similarities. In recent years, detailed analysis of the DNA sequencesfrom a variety of prokaryotic organisms has revealed two distinct types: the bacteria and the archaea. Working on rhe assumption that organisms with more similar genesevolvedfrom a common progenitor more recently than those with more dissimilar genes,researchers have developed the evolutionary lineage tree shown in Figure 1-3. According to this tree, the archaeaand the eukaryotes diverged from bacteria billions of years ago before they diverged from each other. In addition to DNA sequence distinctions that define the three groups of organisms,

Animals

Plants Fungi

Ciliates

Euglena

Microsporidia

EUKARYOTA S I i m em o l d s Diplomonads (Giardia lamblia)

EUBACTERIA E. coli

Sulfolobus ARCHAEA B. subtilus

Thermococcus

Thermotoga

Methanobacteriu m Halococcus

Flavobacteria G r e e ns u l f u r bacteria

Halobacterium Methanococcus jannaschii

Borrelia burgdorferi

P r e s u m e dc o m m o n p r o g e n i t o r of all extant organisms P r e s u m e dc o m m o n p r o g e n i t o r of archaebacteria and eukaryotes

A FIGURE 1-3 All organismsfrom simplebacteriato complex mammalsprobablyevolvedfrom a common,single-celled progenitor.Thisfamilytreedepicts theevolutionary relations among thethreemajorlineages of organisms. Thestructure of thetreewas initially ascertained frommorphological criteria: creatures that look alikewereput closetogetherMorerecently the sequences of DNA andproteins havebeenexamined asa moreinformation-rich criterion for assigning relationships Thegreater thesimilarities in thesemacromolecular sequences, the moreclosely related organisms arethoughtto be.Thetreesbasedon morphological comparisons andthefossilrecordgenerally agreewellwith thosebasedon molecular data.Althoughallorganisms in the eubacterial and archaean lineages areprokaryotes, archaea aremoresimilar to eukaryotes ("true"bacteria) thanto eubacteria in somerespects For instance, archaean andeukaryotic genomes encodehomologous histone proteins, whichassociate with DNA;in contrast, bacteria lack histonesLikewise, the RNAandproteincomponents of archaean ribosomes aremorelikethosein eukaryotes thanthosein bacteria. C H A P T E RI

I

L I F EB E G I N SW I T H C E L L S

archaeacell membraneshave chemical properties that differ dramatically from those of bacteria and eukaryotes. Many archaeansgrow in unusual,often extreme,environments that may resemblethe ancient conditions that existed when life first appeared on earth. For instance, halophiles ("salt loving") require high concentrationsof salt to survive, and thermoacidophiles("heat and acid loving") grow in hot (80' C) sulfur springs,where a pH of lessthan 2 is common. Still other archaeanslive in oxygen-free milieus and generate methane(CH+) by combining water with carbon dioxide.

U n i c e l l u l aO r r g a n i s m sH e l p a n d H u r t U s Bacteria and archaea, the most abundant single-celledorganisms,are commonly 1-2 pm in size. Despite their small size and simple architecture,they are remarkable biochemical factories,converting simple chemicalsinto complex biological molecules.Bacteria are critical to the earth's ecology, but some cause major diseases:bubonic plague (Black Death) from Yersiniapestis,strep throat from Streptomyces, tuberculosis from Mycobacterium tubercwlosis, anthrax from Bacillus anthracis, cholera from Vibrio cholerae, and food poisoning from certain types of E. coli and Salmonella. Humans are walking repositories of bacteria, as are all plants and animals.'We provide food and shelter for a staggering number of "bugs," with the greatestconcentration in our intestines.In return for the food and shelter that allow them to reproduce, bacteria help us digest our food. One common gut bacterium,E. coli, is also a favorite experimental organism. In responseto signalsfrom bacteria such as E. coli, the intestinal cells form appropriate shapesto provide a niche where bacteria can live, thus facilitating proper digestion by the combined efforts of the bacterial and the intestinal cells. Conversely,exposure to intestinal cells changes the properties of the bacteria so that they participare more effectivelyin human digestion. Such communication and responseis a common feature of cells. The normal, peacefulmutualism of humans and bacteria is sometimesviolated by one or both parties. When bacteria begin to grow where they are dangerousto us (e.g.,in the bloodstream or in a wound), the cells of our immune system fight back, neutralizingor devouring the intruders. Powerful antibiotic medicines,which selectivelypoison prokaryotic cells, provide rapid assistanceto our relatively slow-developingimmune response.Understanding the molecular biology of bacterial cells leads to an understanding of how bacteria are normally poisonedby antibiotics,how they becomeresistantto antibiotics, and what processesor structurespresent in bacterial but not human cells might be usefully targeted by new drugs. Like bacteria) protozoa are usually beneficial members of the food chain. They play key roles in the fertility of soil, controlling bacterial populations and excreting nitrogenous and phosphatecompounds,and are key playersin wastetreatment systems-both natural and man-made.These unicellular eukaryotesare also critical parts of marine ecosystems, consuming large quantities of phytoplankton and harboring photosynthetic algae, which use sunlight to produce biologically usefulenergyforms and small fuel molecules.

Enteringand Exitinga LiverCell Sporozoite S' vid"o: Plasmodium (b)

(a)

Redbloodcel ,I SPorulation

E *

,,

"F1 ,H;,",

\

Merozoites-

,11 )A

|,Ervuy rEo Gametocytes uo,

Huum maann F=>^

Mosquito

\ -\ / . / - \ - / /\ l,r'

+/L

I

+

( 0;r"""Y"!

FfGURE 1-4 Plasmodiumorganisms,the parasitesthat cause malaria,are single-celledprotozoanswith a remarkablelife cycle.ManyPlasmodium species areknown,andtheycaninfecta The hosts. varrety of animals, cycling betweeninsect andvertebrate fourspecies thatcausemalaria in humansundergo several dramatic (a)Diagram withintheirhumanandmosquito hosts. transformations of the lifecycleSporozoites entera humanhostwhenan infected Anopheles mosquitobitesa personfl Theymigrateto the liver, intothe wheretheydevelop intomerozoites, whicharereleased fromsporozoites, sothis bloodE. Merozortes differsubstantially "to transform" (Greek, isa metamorphosis or "many transformation merozoites invaderedbloodcells(RBCs) and shapes")Circulating producedby somePlasmodium reproduce withinthem B Proteins RBCs, causing the cellsto species moveto the surface of infected infected RBCs adhere to thewallsof bloodvessels. Thrsprevents fromcirculating to thespleen, wherecellsof the immunesystem theyharbor woulddestroy the RBCs andthe Plasmodium organisms in RBCs for a periodof time Aftergrowingandreproducing suddenly characteristic of eachPlasmodium species, the merozoites

from largenumbersof infectedcells4. lt burstforth in synchrony chillsthatare isthiseventthatbringson thefeversandshaking of malariaSomeof the released symptoms thewell-known a cycleof production creating RBCs, infectadditional merozoites intomaleand develop somemerozoites Eventually, andinfection. cells, These 5, anothermetamorphosis. femalegametocytes cannot whichcontainhalftheusualnumberof chromosomes, in bloodIo an Anopheles theyaretransferred for longunless survive gametocytes are the stomach, mosquito s In the mosquito. yetanother intospermor eggs(gametes), transformed flagellaon of longhairlike markedby development metamorphosis zygotesZ, of spermandeggsgenerates the sperm6 Fusion wallandgrowinto intothecellsof thestomach whichimplant of Rupture sporozoites for producing factories essentially oocysts, of sporozoites E; thesemigrateto the thousands an oocystreleases glands, settingthe stagefor infectionof anotherhuman salivary and of matureoocysts micrograph electron host.(b)Scanning stomach of sudace the external abut Oocysts sporozoites. emerging them that protects withina membrane wallcellsandareencased (b)courtesy of R E Sinden ] fromthe hostimmunesystem. lPart

However, some protozoa do give us grief: Entamoeba histolytica causesdysentery;Trichomonas uaginalis,vaginitis; and Trypanosoma brucei, sleeping sickness.Each year the deadliest of the protozoa, Plasmodium falciparum and related species,is the cause of more than 300 million new casesof malaria, a diseasethat kills 1.5 to 3 million people annually. These protozoans inhabit mammals and mosquitoes alternately,changing their morphology and behavior in responseto signalsin each of theseenvironments.They also recognize receptors on the surfacesof the cells they infect. The complex life cycle of Plasmodium dramatically illustrates how a singlecell can adapt to each new challenge it encounters(Figure 1-4). All of the transformations in cell type that occur during the Plasmodium Iife cycle are gov-

erned by instructions encodedin the geneticmaterial of this parasite and triggered by environmental inputs. The other group of single-celledeukaryotes, the yeasts, also have their good and bad points with respect to humans. as do their multicellular cousins, the molds. Yeasts and molds, which collectively constitute the fungi, have an important ecological role in breaking down plant and animal remains for reuse.They also make numerous antibiotics and are used in the manufacture of bread, beer,wine, and cheese.Not so pleasant are fungal diseases'which range from relatively innocuous skin infections such as jock itch and athlete's foot to life-threatening Pneumocystis carinii pneumonia, a common cause of death among AIDS patients. A N D C O M M O N A L I T YO F C E L L S THE DIVERSITY

VirusesAre the Ultimate Parasites

opportunity. Viral infections can be devastatingly destructive, causing cells to break open and tissuesto fall apart. However, many methods for manipulating cells depend on using virusesto convey geneticmaterial into cells.To do this, the portion of the viral genetic material that is potentially harmful is replaced with other genetic material, including human genes.The altered viruses, or vectors, still can enter cellstoting the introduced geneswith them (Chapter 9). One day, diseasescausedby defectivegenesmay be treated by using viral vectors to introduce a normal copy of a defective gene into patients. Current research is dedicated to overcoming the considerableobstaclesto this approach, such as getting the introduced genesto work at the right placesand trmes.

Not all microscopic pathogensare cells. The other most familiar disease-causingorganisms are the viruses, which make use of the machinery inside the cellsthey infect to copy themselves.Virus-causeddiseasesare numerous and all too familiar: chickenpox, influenza, some types of pneumonia, polio, measles,rabies,hepatitis,the common cold, and many others. Smallpox, once a worldwide scourge,was eradicated by a decade-longglobal immunization effort beginning in the mid-1960s. Mral infections in plants (e.g.,dwarf mosaic virus in corn) have a major economic impact on crop production. Planting of virus-resistantvarieties, developed by traditional breeding methods and more recently by geneticengineeringtechniques,can reduce crop lossessignificantly. Most viruses have a rather limited host range, infecting certain bacteria,plants, or animals(Figure1-5). Becausevirusescannot grow or reproduce on their own, they are in this sensenot consideredto be alive. To survive. a virus must infect a host cell and take over its internal machinery to synthesizeviral proteins and in some casesreplicate the viral geneticmaterial. When newly made virusesare releasedby budding from the cell membrane or when the infected cell bursts, the cycle starts anew. Viruses are much smaller than cells,on the order of 100 nanometer (nm) in diameter; in comparison, bacterial cells are usually >1000 nm (1 nm : 10-e meters).A virus is typically composedof a protein coat that enclosesa core containing the genetic material, which carries the information for producing more viruses (Chapter 4). The coat protects a virus from the environment and allows it to stick to, or enter,specifichost cells. In some viruses, the protein coat is surrounded by an outer membrane-likeenvelope. The ability of viruses to rransport genetic material into cells and tissuesrepresentsa medical menaceand a medical

The most remarkable feature of organismsis their ability to reproduce.Biological reproduction, combined with continuing evolutionary selectionfor a highly functional body plan, is why today's horseshoecrabs look much as they did 300 million years ago, a time span during which entire mountain ranges have risen or fallen. The Teton Mountains in S7yoming,now about 14,000 feet high and still growing, did not exist a mere 10 million years ago. Yet horseshoecrabs, with a life span of about 19 years,have faithfully reproduced their ancient selvesmore than half a million times during that period. The common impression that biological structure is transient and geologicalstructure is stableis the exact opposite of the truth. Despite the limited duration of our individual lives, reproduction gives us a potential for immortality that a mountain or a rock does not have. Whereas some species have changed little over great periods of time, other organismshave changeddramatically

(a)T4 bacteriophage

(b)Tobaccomosaicvirus

C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n

,

50nm

,

(c) Adenovirus

A FIGURE 1-5 Virusesmust infect a host cell to grow and reproduce.Theseelectronmicrographs illustrate someof the structural variety (a)T4 bacteriophage exhibited byviruses. (bracket) attaches to a bacterial cellviaa tailstructureViruses that infect bacteria arecalledbacteriophages, (b)Tobacco or simplyphages. mosarc viruscauses a mottlingof the leaves of infected tobacco C H A P T E R1

I

L I F EB E G I N SW I T H C E L L S

plantsandstuntstherrgrowth.(c)Adenovirus causes eyeand respiratory tractrnfections in humans. Thisvirushasan outer membranous envelope fromwhichlongglycoprotein spikesprotrude [Part(a) from A Levine,1991, Viruses,ScientificAmericanLibrary,p 20 Part(b) courtesyof R C Valentine Part(c) courtesyof RobleyC Williams, Universityof Californial

during the same period. The changescame in responseto pressuresfrom the environment that causedincreasedsurvival of variant individuals. Both stasis and change are possible becausethe machinery of cells does an amazingly precisejob of copying geneticmaterial, yet its rare errors introduce some variation. If environmental conditions continue to select more or less the existing form, as in the caseof horseshoecrabs, the specieswill changelittle. If a new variant has a survival advantage, perhaps because conditions have changed, it may persist and replace the old form. Populations of bacteria exposedto antibiotics, for example, changetheir properties dramatically to escapeand live. They do this becauserare mutations, changesin the genetic material that allow antibiotic resistance,keep some cells alive while the cells without those mutations die. Most populations of any single specieshave a large repertoire of genetic alterations becausethere is a low but significant error rate in copying the genes.That error rate increasesin the presenceof radiation such as sunlight or certain chemical poisons. Current genomeproiects are exploring geneticvariation among humans. "The" human genome sequencethat has been determined already is just one version among billions. Understanding variation is essentialto learn how we respond differently to certain infections or drugs and to exploring how our geneticheritage combines with our experienceand learning to make each of us unique. Underlying the reproduction of organismsis the copying of cells, something that must be precise in order to control the size, shape, and organization of animals and to prevent unwanted growth, such as cancer.The cell is a machine that can copy itself, unlike viruses,which cannot do so on their own. As we will seein Chapters 20 and 21',the cell cyclefrom a single cell copying its own contents through division into two cells-is controlled by a seriesof elegant switches and cross-checking mechanisms. Reproducing cells accurately is a matter of life and death.

EvenSingleCellsCan Have Sex If genetic material was never shared or exchanged,each individual would be the beginning of a new clone of individuals, and the members of a clone would share most of the same genetic strengths and weaknesses.Sex is a processof mingling genetic variation from two individuals, creating new individuals with a combination of properties unlike either parent and that may be beneficial for survival and reproduction. Each chromosome except the sex chromosomes is representedtwice, one copy from the father and one from the mother. Since each pair of chromosomes trades piecesduring the formation of eggs and sperm, new combinations of genesare created and inherited together in the next generation-variation is accelerated. The other benefit of having two copiesof each chromosome is that a poorly functioning gene is backed up by the other copy. The common yeast used to make bread and beer, Saccharomycescereuisiae,appearsfairly frequently in this

Budding lS. cerevisi ael

1-6 The yeast Saccharomycescerevisraereproduces A FfGURE sexuallyand asexually'(a)Twocellsthat differin matingtype' calleda andcr,canmateto forman a/a cellIl. Thea anda cellsare theycontaina singlecopyof eachyeast meaning haploid, halfthe usualnumber'Matingyieldsa diploida/ctcell chromosome, Duringvegetative two containing copiesof eachchromosome. process an asexual by mitoticbudding, growth,diploidcellsmultiply a diploidcellsundergomeiosis, conditions, E Understarvation B. Rupture to formhaploidascospores typeof celldivision, special into whichcangerminate fourhaploidspores, releases of an ascus asexually E. (b)Scanning alsocanmultiply cells4. These haploid Aftereachbudbreaks of buddingyeastcells. micrograph electron buds free,a scaris left at the buddingsite,so the numberof previous (b) AbbeyA/isuals M bacteria are cells [Part canbe counted.Theorange lncl Unlimited, book becauseit has proven to be a great experimental organism. Like many other unicellular organisms' yeasts h"u. t*o mating types that are conceptually like the male and female gametes(eggsand sperm) of higher organisms'

ubiquitous. A N D C O M M O N A L I T YO F C E L L S THE DIVERSITY

.

7

We Developfrom a SingleCell In 1827, German physician Karl von Baer discovered that mammals grow from eggs that come from the mother's ovary. Fertilization of an egg by a sperm cell yields a zygote, a visually unimpressive cell 200 pm in diameter. Every human being begins as a zygote,which housesall the necessaryinstructions for building a human body containing about 100 trillion (1014)cells, an amazing feat. Development begins with the fertrlizedegg cell dividing into two, four, then eight cells, forming the very early embryo (Figure 1-7). Continued cell proliferation and then differentiation

Chapters 16 and 22. Making different kinds of cells-muscle, skin, bone, neuron, blood cells-is not enough to produce the human body. The cells must be properly arranged and organized into tissues,organs, and appendages.Our two hands have the same kinds of cells, yet their different arrangements-in a mirror image-are critical for function. In addition, many cells exhibit distinct functional and/or structural asymmetries, a property often called polarity. From such polarized cells arise asymmetric, polarized tissuessuch as the lining of the intestinesand structures such as hands and hearts. The features that make some cellspolarized and how they arise also are coveredin later chapters,including Chapter 21.

Video:EarlyEmbryonicDevelopment < FIGURE 1-7 The first few cell divisionsof a fertilized egg set the stagefor all subsequentdevelopment.A developing mouseembryois shownat (a)the two-cell,(b) four-cell, and(c)eight-cell stages. Theembryoissurrounded by supporting membranes. The corresponding stepsin human development occurduringthe firstfew daysafterfertilization. Researchers. IClaudeEdelmann/Photo I n cl

C H A P T E R1

I

L I F EB E G I N SW I T H C E L L S

Stem Cells,Fundamentalto FormingTissues and Organs,Offer MedicalOpportunities The biology of stem cells, cells that can give rise to specific cell types and tissues,has generated, great interest. 'We can 'Sfhen contrast stem cells to the simpler types of bacteria. a bacterial E. coli cell divides, both daughter cells are pretty much equivalent in content, size, and shape. In some other bacteria and in many casesof eukaryotic cell division, the two daughters differ in important ways. Although both will have the same genetic material, the cells may differ in size, shape, and contents. The cells may have different fates, that is, they may become different types of differentiated cell. A division that produces two different daughter cells is sometimes described as an asymmetric cell division. Stem-celldivisions are a specialcaseof asymmetric division. One of the two daughter cells is identical to the parent cell; the other follows a path of differentiation, such as becoming a blood cell. The parent cell, called a stem cell, can go on reproducing itself at every division, at each division also producing anorher blood cell. Mosr tissuesin our bodies form from stem cells. Blood, for example, is produced from stem cells that residein the bone marrow and continue to produce new blood cellsfor our entire lives. This is the basis of the often successfulbone marrow transplants that are used to treat cancer patients who have had their blood stem cells damaged by cancer treatments: what is being transplanted is stem cells.However, blood stem cells produce onlv more of themselvesand blood cells, not othir cell types. Thus eachtissuemust have its own stem cells,at leastduring the period of development when the tissue is formed. Stem cells for each tissue arise from even more capable stem cells that have the ability to form multiple stem cell types. The first stem cells are found in early embryos,where all the cells are capable of producing all cell types. In mammals the ultimate stem cell is the fertilized egg, which produces early embryo cells capable of forming all the tissuesof the body. This capability is illustrated by the formation of identical twins, which occur naturally when the mass of cells composing an early embryo divides into two parts, each of which develops and grows into an individual animal. This means that the cells cannor have divided up their embryo-forming duties prior to the time of embryo division. Each cell in an eight-cell-stagemouse embryo has the potential to give rise to any part of the entire animal. Cells with this capability are referred to as embryonic stem (ES)cells. As we will learn in Chapter 22, ES cells can be grown in the laboratory (cultured) and will develop into various types of differentiated cells under appropriate conditions. The ability to make and manipulate mammalian embryos in the laboratory has led to new medical opportunities as well as various social and ethical concerns.In vitro fertilization, for instance, has allowed many otherwise infertile couples to have children. One technique involves extractron of nuclei from defective sperm incapable of normally fertilizing an egg,injection of the nuclei into eggs,and implantation of the resulting fertilized eggsinto the mother.

In recent years, nuclei taken from cells of adult animals have been used to produce new animals. In this procedure' the nucleus is removed from a body cell (e.g., skin or blood cell) of a donor animal and introduced into an unfertilized mammalian egg that has beendeprived of its own nucleus.In a step that has now been done with mice, cows, sheep, mules, and some other animals, the egg with its donor nucleus is implanted into a foster mother. The ability of such a donor nucleusto direct the developmentof an entire animal shows that all the information required for life is retained in the nuclei of some adult cells. Sinceall the cells in an animal produced in this way have the genesof the single original donor cell, the new animal is a genetic clone of the donor (Figure 1-8), though the animals may differ anyway due to their distinct environments and experiences.Repeating the processcan give rise to many clones. Nuclei taken from ES cellswork especiallywell, whereasnuclei from other parts of the body at later times in life work far lesswell. The majority of embryos produced by this technique do not survive due to birth defects,so the donor nuclei may not have all the needed information or the nuclei may be damaged by the cloning process.Even those animals that are born alive have abnormalities,including acceleratedaging. The "rooting" of plants, in contrast, is a type of cloning that is readily accomplished by gardeners,farmers, and laboratory technicians. Scientific interest in the cloning of humans is very limited. Virtually all scientistsoppose it becauseof its high risk to the embryo (also, most people don't believe there is a

critical shortage of twins and triplets). Of much gteater scientific and medical interest is the ability to generatespecific cell types starting from embryonic or adult stem cells' This procedure, somatic cell nuclear transfer (SCNT)' produces cells that are grown in culture and never turned into

If the cells are produced using a donor nucleus from a patient, the properties of the cells may allow them to escaperejection by the patient's immune system,opening new possibilities for cell-transplanttherapies.

E

The Moleculesof a Cell

Molecular cell biologists explore how all the remarkable

ture and function.

Small MoleculesCarryEnergy,TransmitSignals, and Are Linkedinto Macromolecules Much of the cell's content is a watery soup flavored with small molecules (e.g., simple sugars' amino acids, vitamins) and ions (e.g.,sodium, chloride, calcium ions)' The locations conce.tirations of small molecules and ions within the "nd controlled by numerous proteins inserted in cellular are cell membranes. These pumps' transporters, and ion channels move nearly all small moleculesand ions into or out of the cell and its organelles(Chapter 11). One of the best-known small molecules is adenosine triphosphate (ATP), which storesreadily available chemical 'When ..r"rgy itt two of its chemical bonds (seeFigure 2-31)' cells"splitapart theseenergy-richbonds in AIP' the released ..r.rgy ."n b. harnessed to power an energy-requiring pro..tt such as muscle contraction or protein biosynthesis' 1-8 Fivegeneticallyidenticalclonedsheep.An early To obtain energy for making ATR cells break down food a FIGURE intofivegroupsof cellsandeachwas sheepembryowasdivided molecules.For instance, when sugar is degraded to carbon natural like the much mother, intoa surrogate implanted separately dioxide and water, the energy stored in the original chemical process of twinningAt an earlystagethecellsareableto adjustand bonds is releasedand much of it can be "captured" in ATP the cellsbecome laterin development forman entireanimal; (Chapter 72).Bacterial,plant, and animal cells can all make way andcanno longerdo so.An alternative progressively restricted ATP ty this process.In addition, plants and a few other orsingle-celled the nucleiof multiple isto replace to cloneanimals ganisms can harvest energy from sunlight to form ATP in with donornucleifromcellsof an adultsheep'Eachembryo embryos photosynthesis. ' was to the adultfromwhichthe nucleus identical will be genetically Other small moleculesact as signalsboth within and beto theseprocedures survive of embryos obtained.Lowpercentages tween cells; such signals direct numerous cellular activities on the givehealthy andthefull impactof the techniques animals, effect on our bodies of Library/Photo (Chapters iS uttd f e ;. fne powerful Photo Tompkinson/Science animalsis not yet known.lGeoff flooding of instantaneous the from comes a frightening event Incl Researchers, S F A CELL T H E M O L E C U L EO

O

9

the body with epinephrine, a small-moleculehormone that mobilizes the "fight-or-flight" response.The movements neededto fight or flee are triggered by nerve impulses that flow from the brain to our muscleswith the aid of neurotransmitters, another type of small-moleculesignal that we discussin Chapter23.

acids. Sugars,for example, are the monomers used to form polysaccharides. These macromolecules are critical structural components of plant cell walls and insect skeletons.A typical polysaccharideis a linear or branched chain of repeating identical sugar units. Such a chain carries information: the number of units. However, if the units arenot identical, then the order and type of units carry additional information. As we will seein Chapter 6, some polysaccharides exhibit the greaterinformational complexity associated with a linear code made up of different units assembledin a particular order. But this property is most typical of the two other types of biological macromolecules-proteins and nucleic acids.

ProteinsGive CellsStructureand perform Most CellularTasks The varied, intricate structures of proteins enable them to carry out numerous functions. Cells string together Z0 dif_ ferent amino acids in a linear chain to foim a protein (see Figure 2-14).Proteins commonly range in length from 100

to 1000 amino acids, but some are much shorter and others longer. We obtain amino acids either by synthesizingthem from other moleculesor by breaking down proteins that we eat. The "essential" amino acids, from a dietary standpoint, are the eight that we cannot synthesizeand must obtain from food. Beans and corn together have all eight, making their combination particularly nutritious. Once a chain of amino acids is formed, it folds into a complex shape,conferring a distinctive three-dimensionalstructure and function on each protein (Figure1-9). Some proteins are similar to one another and therefore can be consideredmembers of a protein family. A few hundred such families have been identified. Most proreins are designedto work in particular places within a cell or to be releasedinto the extracellular (extra, "outside") space.Elaborate cellular pathways ensurethat proteins are transported to their proper intracellular (intra, "within") locations or secreted (Chapters 13 and14). Proteins can serveas structural componentsof a cell. for example,by forming an internal skeleton(Chapters tO, tZ, and 18). They can be sensorsthat changeshapeas remperature, ion concentrations, or other properties of the cell change. They can import and export substancesacross the plasmamembrane(Chapter11). They can be enzymes,causing chemicalreactionsto occur much more rapidly than they would without the aid of theseprotein catalysts(Chapter 3). They can bind to a specificgene,turning it on or off (Chapter 7). They can be extracellular signals,releasedfrom one cell to communicatewith other cells, or intracellular signals, carrying information within the cell (Chapters 15 and 16). They can be motors that move other moleculesaround, burning chemicalenergy (ATP) to do so (Chapters17 and 1g).

Insulin

Glutaminesynthetase Lrturamtne synthetase

Hemoglobin

FIGURE 1-9 Proteinsvary greatly in size,shape,and function.Thesemodels of thewater-accessible surface of some representative proteins aredrawnto a commonscaleandreveal the numerous projections andcrevices on thesurface. Eachprotern hasa definedthree-dimensional shape(conformation) thatisstabilized by numerous chemrcal interactions discussed in Chapters 2 and3. The illustrated proteins include (glutamine enzymes synthetase and

10

C H A P T E R1

|

L | F EB E G T N W S |TH CELLS

DNA molecule

lmmunoglobulin

Adenylate K tn a s e

L i p i db i l a y e r

adenylatekinase),an antibody(immunoglobuiln), a hormone (insulin), and the bloods oxygencarrier(hemoglobin)Modelsof a segmentof the nucleicacid DNA and a smallregionof the lipid bilayerthat formscellularmembranes(seeSection1.3)demonstrate the relativewidth of thesestructures comparedwith that of tvpical proteins.[Courtesy of GarethWhite]

LI

'l'll)

v lo slln)r'lot^ tH_

puerls(^reluaLualduol) lolqOnep^^ououo 'xrlaqa;qnop pue spuerlsleur6uoor..l1 leurbrLo +o ouo 6urureluorqleo arnpold eq11o serdo>oMl sr aLr-ro)]no oLlf spuerlsAreluaueldLuor 'uorlerrldar ol saleldual se pesnpue punoMUnare spuerlsoM] aql

6utn6 Qq6ry)seseqg pue I aql uoaMlaqpuesaseqf pue slxtloqalqnop spuoqua6otp,iq V oql uaoMloq )eoM{q paztltqels slqnop e urol ol raqlo q)ea punole punoM aqlQlal,'xrlor.l spuerlsfuelueurelduo) orv\llo slslsuo)VNC 0t-l lunglJ V

spuerls ra1q6ne6

'g rardeq3 ur lretepur uortElsuertpuB uorldrrrsuPrl lno d.rrel leqt stuauodruor IIer eql euruexe al1 'opof, f,rteu -aE les.ra.trundlreeu aql ot Surprorre aruanbasVNUIU oqr dq parelrrp rap.ro asne;d eqt ur sprre ourrue raglaSol s>1ur1 (uorlelsuB-rl SurrnC 'uorlelsueJl pue selqtuesseatuosoqrrar{l pell€r 1na u1 vNd IBrlrur aqr vNC Sursnureqr VNU p otur saprloe]f,nuJo a8e>1ur1 aql sez.{1etec 'au.,(zua 'VNC 'aserorudlod y51g e8rel pepuens-alqnop V eql Jo uorsre^ (VNU) prJp Jrelf,nuoqrrpepuerts-a13urs e otur pardor sr aua8 e yo uor8er Surpor eqt 'uorldrrcsuprl pellel 'rs-rr; '(II-I aqt uI arn8lg) suretordorur VNC ur uorlerurot -ur pepol aqt ol sorrasur sessecordo.la.lesn slla] lJeluor 'opuru sr uratord Jr{t sllal qlrr{.a uI puE uel{,{\ slorluof, teqt uo8al Ktolap8at E pue urelord e ;o acuanb -es prf,p ounue aqt sarpads rcq7 uot8at Surpoc e :strud orrrl ureluoJ dluorutuoc suralord 3ur1eu Jot suorlfnJlsur d.r.rer teqr saue8eql'000'SZ-000'02 lnoqe'suerunq lsaue8pues -noqt .,taale e^eq errolf,pq tso14 '3uo1 seprloallnu 000'00I or 000S a.re dlyecrddrqlrq^\ 'souo8 eql (strun leuorlrunt elerrslP olul poPI^lPsl VNq Jo uorlrod Surreaq-uotlEturolul oql 'puerts e 3uo1esoprloalJnuJo rapro reaurl Jrll 'aluenb -esslr ur seprser VNC dq perrrpr uorteruroturlrleueg or{J. 'gfleru lou oP ]Pql sPuprls ror{rodueu sureluotroslEuorl vNC -nlos oqt ue^a 'Surraddrzot peal 'r llr.{\ puerts dretuauralduroc rel{lo eqt Surureluocuortnlos e ur raded aql Suopos taded 'alduruxerod ;o aoarde o] Paqleue pue par;rrndsI puerls auo;t reqlo aql Sursnpue.rtseuo Surlcalaprot Intesndlauarlxa sruorl -enpuq;1.i:rl prf,e f,rolf,nur.lrns 'suorlrpuoo arnle.radtuetpue tles drz dlsnoauetuods11,udaqt 'pale.r rq8rr eqr ur raqtaSor 4ceq -edes a.le spuprts .,(reluerualduol teqt Suols os sr spuerls Jr o.,rueqr;o Surqrreru,{-ruluaualduol srr{J '(91-1 arn8r4)3 e qtr.&\peqlteru sr J qrea pue 1 e ser1puerls reqlo eqt 'y ue seq pupJls ouo Jeleror{,la.:uouJnrlsuof aydrursp spr{ x[eq elqnop 'puens rqr VNC r{leg to auoqlleq lerqeq eqr tuo4 lno Sunoal 'puerts -ord stred eseq eqt qtrlr E ur puo ol pua paurol VNCI are '9 pue 'J t'V peter^argqe'seprtoalcnuluora11rp rnoC

'(y .rardeq3)saseqctue8ro rtlcdc ureluor sernlrnrts rrar.{lasnEreqsaspqse ol perraJererP uelto Oseql:sJPlloelJnu pellel sreuouoru Jo pasoduor are spupns VNC 'x1eq elqnop e Sururol 'srxe uoruruor e punore polrol ere lpqt spuerts IeJrlor{3uo1om.lJo stsrsuoJVNC Jo arnlfnrls IeuoIS -uaurp-oorr{l eql '(VNC) prf,p f,Ielf,nuoqrrfxooppaller retu -d1od 'lerroleru aqt uI peur€r sr utelord Jo pul>ltllee e crlaua8 acnpord ot aJor{1\l. pue 'uaqrrt hrroq rnoge uoIleIUJotuI oql

a)eld pue ourrl lq6tu aql le surelord6ur4ey1ro; uolleturo+ulpapo) f.r.re1sp!)v )rel)nN '(solnralou 80I x S) utlle utotord IErnltnDS lu€punqE eqr or (salncalotu000'02) utalo.rdroldoc 'dleprm -a.r sutetord alrnb eqt uo.r; sarrB,r Surpurq-urlnsur erpr 'e8e.ra,reuo urolord tuere;trp Jo eruepunqe aql 'lce1 u1 1o addr qree Jo selnrelouruorlpu e ot osolf,suleluol IIOJE snqr isurelord ruere;Jrp000'0I rnoqe suleluor IIat re^II p leq] raprsuoJ taqrrny dels auo uollelnf,lef, srqt drrec oI '(Ez0I x ZO'9) punoduor lerlueqr due 1o ayourrad salnrelotu Jo requnu eql laqunu s,orpeSorrypue lq8ra,t uratord Ielol eqt trrorl sp IIer re^II .radsalncalou utarord 60I x 6'L lnoqe 'suratord f,rlofre>lna to reqrunu letor oqt elelnllel uer e,l.r '(1ou73) 00L'TS yo rq8rorr ;o lerrddr sr enlE^ srql Surunssy relnrelorrr p seq uratord lseef e8erarreeql '3 or_0I x L er{r 'tq8ta,trs(ller e to tuer sr ureto.rdrelnller rq8rarvr ;o I€tol -nd 97,,(laleturxo.rddpro; slunoJJe uralord erur5 '3 r_0I x g'g q8ra.uplno.vi aql'1uF E0'I Jo &lsuep 11ec e Sururns IIer -sy '(s;arqqlur ro) etunlol e seg 'aprse e*l o_0I x V't p srql '(1ac re,rr1) uo (tuc rurl eqnr E ,{1q3nor'11ac SI SI00'0) 'req ardcoledeqe se r1rns'11accrlodre>1ne lerrd& e a4et s.ta1 -unu srqt eterurtseoJ 'lasrr urelureru pue alerado ol spJJu IIar e selnrelotu urarord dueur .uoq >1serq8nu a^.\txaN 'dtlulJul 3uo1e pre,&ol 1a,'nsr sutetord algrssodIo reqrunu .ro'alqetsun'luele.trnba llleuorr eqt'elqElunorsrpasr,&\rar.{lo 'seruanb -lunJ dueru aq asaql Surunsse ua,tg p1no.l.r leqt Jo '3uo1 sprre -as uralord luereJJrp alqrssod oov17ere araqt ounue 00t rnoqe sr ureto.rd..1errddl,,E Jr lng 'ecue13lsrr; re alqrssodrursrueestI is>lserperre^ eseqlurotred or papaeu surolord ruereJtrpoqt IIE ruroJ splre ouIIuE 0Z uel ,{\oH

ur pegrrtsap are aruosrldarer{r Jo sluauodruol raqto dueru aql lpue;rs VNC € otur seprtoepnu Surlury ro; alqrsuodsa_r sr eserau,(1odVNq 'leur8r.roeql ot Iefrluepr ql€e ,serrlaq elqnop 1o ;red e sr eruortno eqI .(0I-I a.rn8rgaas)pue_us dreruetualduoJ 1y\eue olur saprloopnu elquesse ol aleldual e se qrea sasnpup seuosoruorql eqr ul vNC Ielrleq PuErls -elqnop spuertso.,vrtrqt seteredas,aruosrlde.r eqt (eurqrpru Jo uorlerrlda.ruralordrllnru a8rel e ,saprlrp 11ace arurl d.ra,rg 'pec ol 'JlpLlreqlo eq.l 'asaql !tuoy41o1 IlEq peJen og UEJ'seue8eqt;o JIeq snLlrpue '(71-1 ern8r4)sauosoruorqr seq uerunq Jo 'lpH 9t IIal lpur -rou dre,te 'rulads pue s33a uondarxe egr qtrl1 .VNC 1o to luerualduror eJrluastr sasr.rduorrusrue8roue ;o auroua8eq1 '(e7-1 a-rn8r1 aes) aql yo uor8arI€rtueJaqt ur ,saurt duetu 11ac 'sat1alncalorusrqr lq13ua1ur ralaurllru Jlesrluo >lrEqPePIoJ B tnoqE alnrelotu VNC relnrrrr a13ursB ur sJprseruorterurot 's11arrrtodrelord u1 'suretord -ut cttaue83qt to IIe ro tsoru urEtJaJqtr.4apelerJosseelnfelou VNC Jeeurl a13urse surel -uor aruosoruonll qleg '(9 ;erdeq3) saruosoruorrlf,se.{\ou{ J^\ soJnlJnrls JprlrrueJor.[l olur peplot dle,rrsuelxa ,snap -nu agl ur petef,ol sr sllJJ lrodrelna ul VNC er.[]Jo rsory

u o t s r ^ t ol l o ) 6 u u n c p o l e ) r l d o up u e saurosotuotq)olur pa6e{)ed sl oruouagoql 'paroldxa Surag ere uorteln8e.r 11rrs ,saue8 1o ed& srql Jo &rnbrqn pue sursrupq)au eql qSnoql II€ ro tsoru alelnSer deu sygg llerus seteturlsaaruos dg 'sldrrcsuerlauaS;o drrpqerspue uortrnpord ,uors Surtuln8er -sardxa aue8 uo tleJJef,rt€uerp p e^eq uel selnrelour VNU 'serurtrq8u aqr sllet le rq8rr aqr ur saua8tq8u or1tsale^ IIpruS -ItrE legt ualsds IortuoJ alrsrnbxaup ruroJ ol padoldruaa.re srolreJ uotldtnsue;l lueretlrp Jo sperpunq 'susruefiro xald -ruoJ ul 'seua8pereln8er aqr Suqcalasol dlpr;rceds Surpurq -VNC u,4o str Surtnquluoc uralord auo ueql JJou qlr.^A 'saxaldruor uleloJdrtlnu sE lJo,ln uouo sJolfet uortdr.rrsuerl ']as eqt ur aueSqrea rEau lsrxo rotret teqr roJ satrsSurpurq ;r seua8Jo las E eteln8er dlateurproor uer rotf,€J uortdr.rcs 'uorssarda;pue uortelrl -uert Jo addl auo 1o serdocaldrrlnyq '11ar -re eua8 e Jo drlclJlradseqt Suunsue ;o VNC aql ur rnl -lo e dlug .seprtoaltnu IIr.^aacuanbesrJf,nsqrpe 1o sardor.ra.a1 rnoJ Jo due aq uec uortrsodr1f,Ee erurs (9tS'gV1't) saruanb -as alqrssod o^eq uec s.rred espq 0I Sururetuor ylrlq 0rt '3uo1srred aseq y51q ;o ruaru8asy ZI-9 $oqe seouanbes t.roqs azruSoJerllr,l.r'uralord Surpurg-y51q e d1lecrdd1 'VNC s,ller e ut luaserdspuesnoqlJrll Jo rno seue8,ua1e tsnl 1o suor8a.rdroleln8ar aqr o1 dlyerluare;ardpurq ot elqp ere .{eql reql dlasnard os pedeqs are srotreJ uortdrrrsue{ '(1 retdeq3) saue8 relncrtred ;o uortdursuerl Surssard 'saqrlr,rs sE tJE pue -ar ;o SurtB,r.rlJe JerJlre VNC ot purq (srolreJuorldrrrsuprl qcrq,rrr pallEt suralordSurpurq-ypq uo spuadapdtr,rrlceaua8yo lortuoJ r{tns 'speeuluorrnr }eetu ol 'go surato.rd;o arrolrada.r rragl Surldepe ,(qareqr ro uo saue8 rrPeds Suturnt dq suortrpuof, ur se8ueqc.ro sleu8rs leuJetxe 'esrol aJrApue IeuJetxeot puodsa;ue) sllJJlueru taaoerory s11ecdaupr>1,{q peenpord tou er€ teqt suralord oruos ernp -ord s1larra,tq dq,rrr. s(teqJ 'sureto-rdaleu ot pesn pup (uo paurnt lo 'a,ttlf,eare saua8esoqtJo eruosdluo ad.{rIIac r{rea

s r r r ) H r M s N r g r sl j r ' r I

t u:ravul

zl

ur tnq 'seua3ueunq to tes IInJ aql uretuor serpogrno ur sllar eL[] IIE dl.reau'aruetsur roC 'paquf,suurl aq UEJ seua8rraql eJeq.4 pue uoq,lt IoJluoc ol sde,,vreleq s{usrup8ro 11y ' u r a t o r do t u l s J l n f , 'salncalotuy51a re8rel -elou VNUIU to uouplsuerr eqt pue (seLuosoluorqr Jo drlllqers eql Jo uollrunJ Pue erntf,nrts '3uo1 sepnoepnu 'sVNU aqr aleln8a.rd11err;roads 002-02 'dr{lqers pue Surssecord VNU pue ornl Ilprus sas€f,dueru u1 -fnrts eruosoruoJqlSurpnycurtlrrrrlre eua8 slf,odseduetu 1o Suneln8e.rur alor tuet.rodurr flqelreua; e Aeld ol puno' ueaq osle ser{VNU 'flruareg razrsaqludsuratord pue repear VNUIU luarrr]'a pue asrcard dlqe>1reua.rp a{pru ol surel -ord 69 ueqt eroril qlrrr,rdn rueat leqt sureqf,VNU JnoJ seq euosoqrr agt (oldurexa roC 'aurLlleru rplnlelou e Surplrnq ro; {ro^\euer; E se enres uer y51g 'useldoldc ol snolf,nu ruoJJ uorleruro;ur Surr.ra;supJlur aloJ slr ol uortrppe uI

ureqlrpourle olurspr)eouru.re 6ur1ur1 {1;erruaqr {q ureloLd e puearuenbes slrpear]eql sourosoqu {q punoqsr}r oloqm alqrlosse 'useldoyb y11y (VNUul) eq] o1 sa^oLr; rebuesseu ernleur oql 'llo) p ul :E de15saruanbas 6urporuou oAoLUar o1pessa:ord rrloi{re>1ne sr aq1: E dals eleldua]e sespuer]s ldursuerl VNCaq],o auo pepuerls-a16urs 6ursn]drnsuert e olurseprloapnu 6ur>1ur1 VNUU-ard esereuri;od aq_La]rsuelsoq] 'uorle)ol>rloeds VNc oq] 6uo;esenou.r 'VNC ue,Louorldursuerl sur6aq asereu{1od e ]e auebpale^r])e VNU aq]o] punoqxelduoruorlpr]rur ure1o.rdr1lnu e 1o{lquasse6urnnollo3 : g da15uJeq]ole^r]lepuelorluo),{eq}seue6:r;r:edsaq11o ,lo1eln6er aql o1purqsrolreluorldursue{:It da15'ssarord suor6el delsrllnu e {q surelold;o saruenbaspr)eourue oq} o}ul pouo^uo)s! vNc u! uollewrolu!papo)aqM-! lun9lJ v ureq3prce ouil..uv I 1 I I

v l g 1 o u o r o e ro u r p o c u o N V r u U ; ou o t 6 a l6 u t P o c - u t a 1 o l 3 vNc Jo uorOorpaqrrcsuerluoN -n2q2q2qa V N C + o u o r 0 o rp a q u 3 s u e {

uollolJcsue{

d uorleAllcv

can be "painted" for easy 1-12 Chromosomes FIGURE A normalhumanhas23 pairsof morphologically identification. fromthe onememberof eachpairis inherited chromosomes; distinct motherandthe othermemberfromthefather(Left)A chromosome whenthe froma humanbodycellmidwaythroughmitosis, spread wastreatedwith Thispreparation arefullycondensed chromosomes reaqents thatalloweachof the 22 pairs staininq fluorescent-labeled

Chapter 4. The molecular design of DNA and the remarkable properties of the replisome ensure rapid, highly accurate copying. Many DNA polymerasemoleculeswork in concert, each one copying part of a chromosome.The entire genome of fruit flies, about 1.2 x 10o nucleotideslong, can be copied in three minutes! Becauseof the accuracyof DNA replication, nearly all the cells in our bodies carry the same genetic instructions, and we can inherit Mom's brown hair and Dad's blue eyes. A rather dramatic example of genecontrol involves inac'Women tivation of an entire chromosomein human females. have two X chromosomes,whereas men have one X chromosome and one Y chromosome, which has different genes than the X chromosome. Yet the genes on the X chromosome must, for the most part, be equally active in female cells(XX) and male cells(XY). To achievethis balance'one of the X chromosomesin female cells is chemically modified and condensedinto a very small mass called a Barr body' which is inactive and never transcribed. Surprisingly,we inherit a small amount of geneticmaterial entirely and uniquely from our mothers. This is the circular DNA present in mitochondria' the organelles in eukaryotic cells that synthesizeAIP using the energy released by the breakdown of nutrients. Mitochondria contain multiple copies of their own DNA genomes,which code for some of the mitochondrialproteins(Chapter6). Becauseeachhuman inherits mitochondrial DNA only from his or her mother (it comes with the egg but not the sperm), the

colorwhen to appearin a different andtheX andY chromosomes of multiplex Thistechnique microscope viewedin a fluorescence (M-FISH) iscalled sometimes in situhybridization fluorescence (RiSht) fromthe (Chapter Chromosomes 6) painting chromosome orderof size, in pairsrndescending preparation on the leftarranged of X andY chromosomes Thepresence an arraycalleda karyotype. of M R Speicher] asmale [Courtesy the sexof the individual identifies

distinctive features of a particular mitochondrial DNA can be used to trace maternal history. Chloroplasts, the organelles that carry out photosynthesisin plants' also have Ih.i, o*tt circular genomes'Both mitochondria and chloroolasts are believedto be derived from endosymbionts,bacteii" th"t took up residenceinside eukaryotic cells in a mutually beneficialpartnership.The mitochondrial and chloroplast circular DNAs appeatto have originated as bacterial genomes' which also are usually circular,though the organellegenomes have lost most of the bacterialgenes.

Mutations May Be Good, Bad,or Indifferent Mistakes occasionallydo occur spontaneouslyduring DNA replication, causing changesin the sequenceof nucleotides' Soch ch"ng.s, or mutations, also can arise from radiation that causesdamageto the nucleotidechain or from chemical poisons, such as those in cigarette smoke, that lead to errors iuring the DNA-copying process (Chapter 25)' Mutations .o-.1., various forms: a simple swap of one nucleotide for another; the deletion, insertion, or inversion of one to millions of nucleotides in the DNA of one chromosome; and translocation of a stretch of DNA from one chromosome to another. In sexually reproducing animals such as ourselves,mutations can be inherited only if they are presentin cellsthat potentially contribute to the formation of offspring' Such germJine cells include eggs'sperm' and their precursor cells' T H E M O L E C U L EOSF A C E L L

O

13

Body cells that do not contribute to offspring are called somatic cells. Mutations that occur in these cells never are inherited, although they may contribute to the onset of cancer. Plants have a less distinct division between somatic and germ-line cells, since many plant cells can function in both capacltres. Mutated genesthat encode altered proteins or that cannot be controlled properly ."u.. .rurn..ous inherited diseases.For example, sickle-celldiseaseis atributable to a single nucleotide substitution in the hemoglobin gene, which encodesthe protein that carries oxygen in red blood cells. The single amino acid changecausedby the sickle cell muta-

Sequencingof the human genome has shown that a

transcription factors typically are only 70-12 nucleotides long, a few single-nucleotidemutations might convert a nonfunctional bit of DNA into a functional protein-binding regulatory site. Much of the nonessentialDNA in both eukaryotesand prokaryotes consistsof highly repeatedsequencesthat can move from one place in the genome to another. These mobile DNA elementscan jump (transpose)inro genes,most commonly damaging but sometimes activating them. Jumping generallyoccursrarely enoughto avoid endangering the host organism. Mobile elements,which *... di.coveredfirst in plants, are responsiblefor leaf color variegation and the diverse beautiful color patterns of Indian corn kernels.By jumping in and out of ge.resthat control prgmentationas plant developmentprogresses,the mobile elemenrsgive rise to elaboratecolorid putt...rr. Mobile elements were later found in bacteria, in which they often carry and, unfortunatelS disseminategenesfor antibiotic resrstance. Now we understand that mobile elements have multi_ plied and slowly accumulatedin genomesover evolutionary time, becoming a universal property of genomesin preseni_ day_organisms. They account for an astounding45 percent of the human genome. Some of our own mobile DNA ele_ ments are copies-often highly mutated and damaged_of genomesfrom viruses that spend part of their life iycle as DNA segmentsinserted into host-cell DNA. Thus we carry in our chromosomes the genetic residues of infections ac_ quired by our ancestors.Once viewedonly as molecularpar_ asites,mobile DNA elementsare now thoueht to have ion_ tnbuted significantly to the evolution of higher organisms (Chapter6). '14

.

cHAprE1 R | L | F EB E G t Nwsl r H c E L L s

IE

TheWorkof Cetts

In essence,any cell is simply a compartment with a watery interior that is separatedfrom the external environment by a surfacemembrane (the plasma membrane) that preventsthe free flow of moleculesin and out. In addition, as we've noted, eukaryotic cells have extensive internal membranes that further subdivide the cell into various comparrmenrs, the organelles.Each compartment has contents and properties, such as specializedproteins or a certain pH, suited to its job. The plasma membrane and other cellular membranes are composedprimarily of two layers of phospholipid molecules. These bipartite moleculeshave a "water-loving" (hydrophilic) end and a "water-hating" (hydrophobic) end. The two phospholipid layersof a membrane are orienred with all the hydrophilic ends directed toward the inner and outer surfacesand the hydrophobic ends buried within the interior (Figure1-13). Smalleramounts of other lipids, such as cholesterol, and many kinds of proteins are inserted into the phospholipid framework. The lipid moleculesand someproteins can float sidewisein the plane of the membrane,giving membranes a fluid character. This fluidity allows cells to change shape and even move. However, the attachment of some membrane proteins to other molecules inside or outside the cell restricts their lateral movement. We will learn more about membranes and how molecules cross them in Chapters10 and 11. The cytosol and the internal spacesof organellesdiffer from each other and from the cell exterior in terms of aciditg ionic composition, and protein contents. For example, the composition of saltsinside the cell is often drastically different from what is outside. Becauseof these different ,,microclimates," each cell compartment has its own assigned

Cholesterol

Water-seeking h e a dg r o u p

Water

FIGURE 1-13Thewatery interiorof cellsis surroundedby the plasmamembrane,a two-layeredshell of phospholipids. Thephospholipid molecules areoriented with theirfattyacylchains (blacksquiggly lines) facinginwardandtheirwater-seeking head groups(whitespheres) facingoutward.Thusbothsidesof the membrane arelinedby headgroups, mainlycharged phosphates, adjacent to thewateryspaces tnside andoutside thecell.All biological membranes havethesamebasicphospholipid bilayer structureCholesterol (red)andvarious (notshown)are proteins embedded in the bilayer. Theinteriorspaceisactually muchlarger relative to thevolumeof the plasma membrane depicted here.

tasks in the overall work of the cell (Chapters 10, L2, and 13). The unique functions and microclimates of the various cell compartm€nts are due largely to the proteins that reside in their membranesor interior. 'We can think of the entire cell compartment as a factory dedicatedto sustainingthe well-being of the cell. Much cellular work is performed by molecular machines, some housed in the cytosol, some attached to the cytoskeleton' and some in various organelles.Here we quickly review the major tasks that cells carry out in their pursuit of the good life.

Cells needto break down worn-out or obsoleteparts into small molecules that can be discarded or recycled' This housekeepingtask is assignedlargely to lysosomes,organelles crammedwith degradativeenzymes.The interior of a lysosome has a pH of about 5.0' roughly 100 times more acidic than that of the surrounding cytosol. This aids in the breakdown of materials by lysosomal enzymes' which are specially

CellsBuild and DegradeNumerous Moleculesand Structures As chemical factories,cells produce an enormous number of complex molecules from simple chemical building blocks. All of this syntheticwork is powered by chemical energy extracted primarily from sugarsand fats or sunlight, in the case of plant cells, and stored primarily in ATP, the universal "currency" of chemical energy (Figure 1-14). In animal and plant cells, most ATP is produced by large molecular machines located in two organelles,mitochondria and chloroplasts. Similar machines for generating ATP are located in the plasma membrane of bacterial cells. Both mitochondria and chloroplasts are thought to have originated as bacteria that took up residenceinside eukaryotic cells and then became welcome collaborators (Chapter 12). Directly or indirectlS all of our food is created by plant cells using sunlight to build complex macromolecules during photosynthesis. Even underground oil suppliesare derived from the decay of plant material.

llll+ overviewAnimation:BiologicalE

Most of the structural and functional properties of cells de-

cell and most membrane proteins, however' are made on ribosomes associatedwith the endoplasmic reticulum (ER)' This organelleproduces,processes'and ships out both proteins and lipids. Prolein chains produced on the ER move to the Golgi comple*, where they are further modified before being for*ari.d to their final destinations. Proteins that travel in this way contain short sequencesof amino acids or attached sugar ch"itts (oligosaccharides)that serve as addressesfor directing them to their correct destinations. These addresseswork becausethey arerecognizedand bound by other proteins that do the sorting and shipping in various cell compartments'

lnterconversions or Light (photosynthesis) compoundswith high potentialenergY(resPiration )

Synthesisof cellularmacromolecules(DNA, RNA, proteins, polysaccharides)

Synthesisof other cellularconstituents (suchas membrane phospholipidsand certainrequired metabolites)

Cellularmovements, includingmusclecontraction,crawling movements of entire cells, and movement of chromosomesduring mitosis

A FIGURE1-14 ATP is the most common molecule used by cells to capture and transfer energy. ATPis formed from ADP and in plantsand by the inorganicphosphate(Pi)by photosynthesis

Transportof moleculesagainst a concentration

gradient

Generationof an electricpotential acrossa memDrane

Heat

for nerve 1fi..:.,;t;,",

by andfatsin mostcells.Theenergyreleased of sugars breakdown processes (hydrolysis) of PifromATPdrivesmanycellular thesplitting

THEWORK OF CELLS

15

lntermediate f ilaments

Microtubules

FIGURE 1-15 The three types of cytoskeletalfilamentshave characteristic distributionswithin cells.Threeviewsof the same cell.A cultured fibroblast wastreatedwith threedifferent antibodv preparations. Eachantibody bindsspecifically to the protein monomers formingonetypeof filamentandischemically linkedto a differently colored fluorescent dye(green, blue,or red).Visualization

A n i m a l C e l l sP r o d u c eT h e i r O w n E x t e r n a l E n v i r o n m e nat n d G l u e s The simplest multicellular animals are sinsle cells embedded in a jelly of proteins and polysaccharides."ll.d th. extracellular matrix. Cells themselvesproduce and secretethesematerials, thus creating their own immediate environment (Chapter 19). Collagen, the single most abundant protein in the animal kingdom, is a major component of the extracellular matrix in most tissues.In animals, the extracellularmatrix cushions and lubricates cells. A specialized,especially tough matrix, the basal lamina, forms a supporting layer un_ derlying sheetlikecell layers and helps pr.u"rrt the cells from rlppmg apart. The cells in animal tissuesare ..glued" togerher by cell_ adhesionmolecules(CAMs) embeddedin their surfacemem_

Microfilaments

of the stainedcellin a fluorescence microscope reveals the location of filaments boundto a particular preparation dye-antibody In this case,intermediate filaments green;microtubules, arestained blue; and microfilaments, red.All threefibersystems contributeto the shapeandmovements of cells.lcourtesy of V Small ]

animal cells (Figure 1-15). The cytoskeleton prevents the plasma membrane of animal cells from relaxing into a sphere(Chapter 10); it also functions in cell locomotion and the intracellular transport of vesicles,chromosomes, and macromolecules(Chapters 17 and 18). The cytoskeletoncan be linked through the cell surfaceto the extracellular matrix or to the cytoskeletonof other cells,thus helping to form tissues(Chapter 19). All cytoskeletal filaments are long polymers of protein subunits. Elaborate systemsregulatethe assembly dirursembly of the cytoskeleton,thereby controlling cell".rd shape.In some cells the cytoskeletonis relatively stable, but in others it changesshapecontinuously. Shrinkageof the cytoskeleton in some parts of the cell and its growrh in other parts can produce coordinated changesin shape that result in cell locomotion. For instance,a cell can send out an extensionthat attachesto a surfaceor to other cellsand then retract the cell body from the other end. As this processcontinues due to coordinated changesin the cytoskeleton,the cell moves forward. Cells can move at rates on the order of 20 pm./second.

C e l l sC h a n g eS h a p ea n d M o v e

largement but not movement of cells from one position to another.

Cells changeshapeand move becausetheir internal skeleton. the cytoskeleton,exerts forces on the rest of the cell and its

CellsSenseand SendInformation

attachments- Three types of protein filaments, organized into networks and bundles, form the cytoskeleton within

A living cell continuously monitors its surroundings and adjusts its own activities and composition accordingly. Cells also communicate by deliberatelysendingsignalsthat can be receivedand interpreted by other cells. Suchsignalsare com_ mon not only within an individual organism but also be_ tween organisms.For instance,the odor of a pear signals a food source to us and other animals; consumption of the

16

.

c H A p r E1R | L I F E B E G t Nwst r H c E L L s

pear by an animal aids in distributing the pear'sseeds.Everyone benefits! The signals employed by cells include simple small chemicals, gases, proteins, light, and mechanical movements. Cells possessnumerous receptor proteins for detecting signals and elaborate pathways for transmitting them within the cell to evoke a response.At any time, a cell may be able to senseonly some of the signalsaround it, and how a cell responds to a signal may change with time. In some cases,receivingone signal primes a cell to respond to a subsequentdifferent signal in a particular way. Both changesin the environment (e.g.,an increaseor decreasein a particular nutrient or the light level) and signals receivedfrom other cells representexternal information that cells must process.The most rapid responsesto such signals generally involve changesin the location or activity of preexisting proteins. For instance, soon after you eat a carbohydrate-rich meal, glucosepours into your bloodstream.The rise in blood glucose is sensedby B cells in the pancreas, which respond by releasingtheir stored supply of the protein hormone insulin. The circulating insulin signal causesglucose transporters in the cytoplasm of fat and muscle cells to move to the cell surface,where they begin importing glucose. Meanwhile, liver cellsalso are furiously taking in glucosevia a different glucosetransporter.In both liver and musclecells, an intracellular signalingpathway triggeredby binding of insulin to cell-surfacereceptorsactivatesa key enzymeneeded to make glycogen,a large glucosepolymer (Figure 1-16a). The net result of thesecell responsesis that your blood glucoselevel falls and extra glucoseis stored as glycogen,which your cells can use as a glucosesource when you skip a meal to cram for a test. The ability of cells to send and respond to signalsis crucial to development. Many developmentallyimportant signals are secretedproteins produced by specific cells at specific times and places in a developing organism. Often a receiving cell integratesmultiple signals in deciding how to behave,for example, to differentiate into a particular tissue type, to extend a process,to die, to send back a confirming s i g n a l( y e s ,I ' m h e r e ! ) ,o r t o m i g r a t e . The functions of about half the proteins in humans, roundworms, yeast, and severalother eukaryotic organisms have beenpredicted basedon analysesof genomic sequences (Chapter6). Suchanalyseshave revealedthat at least10-15 percent of the proteins in eukaryotesfunction as secretedextracellular signals, signal receptors, or intracellular signaltransduction proteins, which pass along a signal through a seriesof steps culminating in a particular cellular response (e.g., increased glycogen synthesis).Clearly, signaling and signal transduction are maior activities of cells.

CellsRegulateTheir Gene Expressionto Meet C h a n g i n gN e e d s In addition to modulating the activities of existing proteins, cells often respond to changing circumstancesand to signals from other cells by altering the amount or types of proteins they contain. Gene expression,the overall processof selectively reading and using genetic information, is commonly

(a)

B o u n ds i g n a l

Surfacereceptors

)?'.. OO o o

6

s

I

Active enzyme

enzyme (b) Cytosolic Receptor-hormone

tm

m R N A+ + j----\

o oo Protein

Oc

#

ot

^

I mRNA

Nucleus

lncreasedtranscription of specificgenes

1-16 Externalsignalscommonlycausea changein FIGURE the activity of preexistingproteinsor in the amountsand or of a hormone typesof proteinsthat cellsproduce.(a)Binding cantriggeran receptors to itsspecific molecule othersignaling of a the activity or decreases pathway that increases intracellular in to receptors bindingof insulin protein. Forexample, preexisting of cellsleadsto activation of liverandmuscle membrane the plasma from of glycogen in thesynthesis a keyenzyme glycogen synthase, within arelocated for steroidhormones glucose(b)Thereceptors complexes hormone-receptor The surface the cell cells,noton to increased tarqetgenes,leading of specific transcription activate that bindto proteins. Manysignals production of the encoded pathways, to alsoact,by morecomplex on the cellsurface receptors geneexPression modulate

controlled at the level of transcription, the first step in the production of proteins. In this way cells can produce a pariicular mRNA only when the encoded protein is needed' thus minimizing wasted energy. Producing an mRNA is, however,only the first in a chain of regulatedeventsthat together determine whether an active protein product is produced from a particular gene. Transcriptional control of geneexpressionwas first decisively demonstratedin the responseof the gut bacterium E. coli to different sugar sources.E. coli cells prefer glucose as a sugar source,but they can survive on lactosein a pinch' These bacteria use both a DNA-binding repressor protein and a DNA-binding actiuator protein to change the rate of transcription of three genesneededto metabolize lactosedepending on the relative amounts of glucoseand lactosepresint (Chapter 4). Such dual positiveinegativecontrol of gene exoression fine-tunes the bacterial cell's enzymatic equip-..rt fo. the job at hand. Like bacterial cells, unicellular eukaryotes may be subjected to widely varying environmental conditions that require extensivechangesin cellular structuresand function' T H EW O R K O F C E L L S

17

For instance,in starvation conditions yeastcellsstop growing and form dormant spores (seeFigure 1-6). In multicellular organisms, however, the environment around most cells is relatively constant. The major purpose of gene control in us and in other complex organismsis to tailor the properties of various cell types to the benefit of the entire animal or plant. Control of gene activity in eukaryotic cells usually involves a balance between the actions of transcriptional activators and repressors.Binding of activators to specificDNA regulatory sequencescalled enhancers turns on transcription, and binding of repressorsto other regulatory sequences called silencersturns off transcription. In Chapters 7 and 8, we take a closelook at transcriptional activators and repressors and how they operate, as well as other mechanismsfor controlling gene expression.In an extreme case,expression of a particular gene could occur only in part of the brain, only during eveninghours, only during a certain stageof development, only after a large meal, and so forth. Many external signals modify the activity of transcriptional activators and repressorsthat control specific genes. For example, lipid-soluble steroid hormones, such as estrogen and testosterone,can diffuse across the plasma membrane and bind to their specific receptors located in the cytoplasm or nucleus(Figure 1-16b).Hormone binding changes

Overview Animation: Life Cycle of a Cell {lltt Nondividing cells

o

Resting c el l s

R N Aa n d protein synthesis G2

A FIGURE 1-17 Duringgrowth, eukaryoticcellscontinually progressthrough the four stagesof the cell cycle, generatingnew daughtercells.In mostproliferating cells,the four phases of the cellcycleproceedsuccessively, takingfrom 10-20 hoursdepending on celltypeanddevelopmental state Duringinterphase, whichconsists of the G,,,S,andG2phases, the cellroughly doubles itsmassReplication of DNAdurinqthe S phaseleaves the cellwith four copiesof eachtypeof chromosome Inthe mitotic(M)phase, thechromosomes are evenlypartitioned to two daughter cells,andthecytoplasm divides roughlyin halfin mostcases. Undercertainconditions suchasstarvation or whena tissuehasreached itsfinalsize,cells willstopcycling andremainin a waitingstatecalledGs Mostcells in Gscanreenter thecycleif conditions chanqe.

18

C H A P T E R1

|

L t F EB E G T N W S |TH CELLS

the shape of the receptor so that it can bind to specific enhancer sequencesin the DNA, thus turning the receptor into a transcriptional activator. By this rather simple signaltransduction pathway, steroid hormones cause cells to change which genes they transcribe (Chapter 7). Since steroid hormones can circulate in the bloodstream, they can affect the properties of many or all cells in a temporally coordinated manner. Binding of many other hormones and of growth factors to receptors on the cell surface triggers different signal-transduction pathways that also lead to changesin the transcription of specific genes (Chapters 15 and 16). Although thesepathways involve multiple components and are more complicated than those transducing steroid hormone signals,the generalidea is the same.

CellsGrow and Divide As we have discussed,reproduction is at the heart of biology; rocks don't do it. The reproduction of organisms depends on the reproduction of cells. The simplest type of reproduction entails the division of a "parent" cell into two "daughter" cells.This occurs as part of the cell cycle,a series of eventsthat preparesa cell to divide followed by the actual division process, called mitosis. The eukaryotic cell cycle commonly is representedas four stages(Figure 1-17). The chromosomes and the DNA they carry are copied during the S (synthesis)phase.The replicatedchromosomesseparare during the M (mitotic) phase,with each daughter cell getting a copy of each chromosome during cell division. The M and S phasesare separatedby two gap stages,the G1 phase and G2 phase, during which mRNAs and proteins are made. In single-celledorganisms,both daughtercellsoften (though not always) resemblethe parent cell. In multicellular organisms, stem cells can give rise to two different cells, one that resembles the parent cell and one that does not. Such asymmetric cell division is critical to the generationof different cell types in the body (Chapter21). During growth the cell cycle operatescontinuously with newly formed daughter cells immediately embarking on their own path ro mitosis. Under optimal conditions bacteria can divide to form two daughter cells once every 30 minutes. At this rate, in an hour one cell becomesfour: in a day one cell becomesmore than 101a, which if dried would weigh about 25 grams. Under normal circumstances, however, growth cannot continue at this rate becausethe food supply becomeslimiting. Most eukaryotic cells take considerably longer than bacterial cells to grow and divide. Moreover, the cell cycle in adult plants and animalsnormally is highly regulated(Chapter20). This tight control prevents imbalanced, excessivegrowth of tissueswhile ensuring that worn-out or damaged cells are replaced and that additional cells are formed in responseto new circumstancesor developmental needs.For instance, the proliferation of red blood cellsincreasessubstantiallywhen a person ascendsto a higher altitude and needs more capacity to capture oxygen. Somehighly specializedcells in adult animals, such as nervecellsand striatedmusclecells,rarely divide, if at all. The fundamental defect in cancer is loss of the ability to

control the growth and division of cells.In Chapter 25, we examine the molecular and cellular eventsthat lead to inappropriate, uncontrolled proliferation of cells. Mitosis is an asexual process since the daughter cells carry the exact samegeneticinformation as the parental cell. ln sexwalreproduction, fusion of two cells produces a third cell that contains genetic information from each parental cell. Sincesuch fusions would causean ever-increasingnumber of chromosomes, sexual reproductive cycles employ a special type of cell division, called meiosis,that reducesthe number of chromosomesin preparation for fusion (seeFigure 5-3). Cells with a full set of chromosomes are called diploid cells. During meiosis, a diploid cell replicates its chromosomes as usual for mitosis but then divides twice without copying the chromosomesin between. Each of the resulting four daughter cells, which have only half the full number of chromosomes,is said to be haploid. Sexual reproduction occurs in animals and plants and even in unicellular organismssuch as yeasts(seeFigure 1-5). Animals spendconsiderabletime and energygeneratingeggs and sperm, the haploid cells, called gametes,which are used for sexualreproduction. A human femalewill produce about half a million eggsin a lifetime, all of thesecells forming before she is born; a young human male produces about 100 million sperm each day. Gametes are formed from diploid precursor germ-line cells,which in humans contain 46 chromosomes.In humans the X and Y chromosomesare called sex chromosomes becausethey determine whether an individual is male or female. In human diploid cells, the 44 remaining chromosomes,called autosomes,occur as pairs of 22 different kinds. Through meiosis,a man produces sperm that have 22 chromosomes plus either an X or a Y, and a woman produces ova (unfertilized eggs) with 22 chromosomesplus an X. Fusion of an egg and sperm (fertilization) yields a fertilized egg,the zygote,with46 chromosomes,one pair of each of the 22 kinds and a pair of Xs in femalesor an X and a Y in males(Figure1-18). Errors during meiosiscan lead to disorders resulting from an abnormal number of chromosomes.These include Down's syndrome, caused by an extra chromosome 21, and Klinefelter's syndrome, causedby an extra X chromosome.

Diploid (2n)

Haploid(1nl

(",,o )

One type of female gamete

Two types of male gamete

Diploid (2n) Female zygote

Male zygote

meiosis 1-18 Dad madeyou a boy or girl. In animals, FIGURE Themale cellsformseggsandsperm(gametes). of diploidprecursor the sexof the parentproduces two typesof spermanddetermines sex chromosomes; Y are the here, X and asshown zygoteIn humans, fromthe maleparentto a Y chromosome the zygotemustreceive (non-sex chromosomes). developintoa male A : autosomes bing" between the fingers; the cells in the webbing subsequently die in an orderly and precisepattern that leavesthe fingers and thumb free to play the piano. Nerve cells in the brain soon die if they do not make proper or useful electrical

CellsDie from AggravatedAssaultor an lnternal Program 'lfhen

cells in multicellular organismsare badly damagedor infectedwith a virus, they die. Cell death resulting from such a traumatic event is messy and often releasespotentially toxic cell constituents that can damage surrounding cells. Cells also may die when they fail to receivea life-maintaining signal or when they receive a death signal. In this type of programmed cell death, called apoptosis, a dying cell actually produces proteins necessaryfor self-destruction.Death by apoptosis avoids the releaseof potentially toxic cell constituents(Figure1-19). Programmed cell death is critical to the proper development and functioning of our bodies (Chapter 21). During fetal life, for instance,our hands initially developwith "web-

1-19ApoPtoticcellsbreakapartwithout spewing FIGURE forth cell constituentsthat might harm neighboringcells. looklikethe cellon the left Cells Whitebloodcellsnormally likethe cellon the programmed celldeath(apoptosis), undergoing The arereleased blebsthateventually surface right,formnumerous is growth Apoptosis signals. it lackscertain cellisdyingbecause cellswherethey cells,to remove virus-infected to eliminate important (likethewebbingthatdisappears asfingersdevelop), arenot needed cellsthatwouldreactwith ourown immunesystem andto destroy Inc] Unlimited, Murtil/isuals bodies[Gopal THE WORK OF CELLS

19

connectionswith other cells. Some developinglymphocytes, the immune-systemcells intended to recognizeforeign proteins and polysaccharides,have the ability to reac against our own tissues.Such self-reactivelymphocytes becomeprogrammed to die before they fully marure. If these cells are not weededout before reaching maturity, they can causeautoimmune diseases,in which our immune system destroys the very tissuesit is meant to protect.

IE

InvestigatingCellsand TheirParts

To build an integrared understanding of how the various molecular components that underlie cellular functions work together in a living cell, we must draw on various perspectives. Here, we look at how five disciplines-cell biology, biochemistry and biophysics,genetics,genomics,and developmental biology-can conrribure to our knowledge of cell structure and function. The experimental approachesof each field probe the cell's inner workings in different ways, allowing us to ask different types of questions about cells and what they do. Cell division provides a good example to illustrate the role of different perspectivesin analyzing a complex cellular process.Although we discussthe different disciplinesseparatelyfor clarity, in practice most biologists use multiple approachesin concert. This is part of the fun of

Nanometers

The goal of cell biologists is to understandhow a cell is able to control its own shape and surface properties, transport materials to the right locations, copy itself, and receiveand send signals.Cell biologists use severaltypes of microscopy to observecells, while at the same time labeling specificcell components and altering them to seewhat happens.Analysesare generally done at the micrometer scale. Actual observation of cells awaited development of the first, crude microscopes in the early 1600s. A compound

Meters

Assemblies Macromolecules

Glucose

C-C bond

Cell Biology Revealsthe Size,Shape,Location, and Movementsof Cell Components

Millimeters

Micrometers

Small molecules Atoms

cell biologg putting together genetics with microscopy or enzymology with development. The realm of biology rangesin scalemore than a billionfold (Figure 1-20). Beyond that, it's ecology and earth science at the "macro" end, chemistry and physics at the "micro" end. The visible plants and animals that surround us are measuredin meters (100-102 m). By looking closely,we can seea biologicalworld of millimeters(1 mm : 10-3 m) and even tenths of millimeters (10-a m). Setting aside oddities like chicken eggs, most cells are 1-100 micrometers (1 pm : 10-5 m) long and thus clearlyvisibleonly when magnified. To seethe structureswithin cells, we must go farther down the sizescaleto 10-100 nanometers(1 nm : 10 v m).

Hemoglobin

M u l t i c e l l u l aor r g a n i s m s

Cells

Ribosome

C. elegans

Bacterium

Mitochondrion

Newborn human

Bumblebee

I

J 1010m

10sm

1 0 ' 8m

'1 0-7m

0.1nm

1 nm

10nm

1 0 0n m

10'6m 1 pm

1 0 - 5m 10pm

FIGURE 1-20 Biologistsare interestedin objectsrangingin sizefrom smallmolecules to the tallesttrees.A sampling of biological objects aligned on a logarithmic scale(a)TheDNAdouble helixhasa diameter of about2 nm (b)Eight-cell-stage numan embryothreedaysafterfertilization, about200 rr"macross(c)A wolf

20

CHAPTER 1

I

L I F EB E G I N S WITHCELLS

1 0 - am 1 0 0u m

1 0 - 3m

1 0 2m

1 0 ' 1m

1 mm

1 0m m

100mm

1 0 0m 1m

spider, about15 mm across(d)Emperor penguins areabout1 m tall. [Part(a)Will andDeniMclntyrePart(b)YorgasNikas/photo Researchers, Inc Part(c)GaryGauglerl/isuals Unlimited, Inc Part(d)HughS Rosefuisuals Unlimited. lncl

microscope,the most useful type of light microscope,has two lenses.The total magnifying power is the product of the magnificationby each lens. As better lenseswere invented, the magnifying power and the ability to distinguish closely spacedobjects, the resolution, increasedgreatly. Modern cclmpoundmicroscopesmagnify the view about a thousandfold, so that a bacterium 1 micrometer (1 p-) long looks like it's a millimeterlong. Objectsabout 0.2 pm apart can be discernedin theseinstruments. Microscopy is most powerful when particular components of the cell are stained or labeled specifically,enabling them to be easilyseenand locatedwithin the cell. A simple exampleis stainingwith dyesthat bind specificallyto DNA to visualize the chromosomes. Specific proteins can be detected by harnessingthe binding specificityof antibodies,the proteins whose normal task is to help defend animals against infection and foreign substances.In general,eachtype of antibody binds to one protein or large polysaccharideand no other (Chapter 3). Purified antibodies can be chemically linked to a fluorescent molecule, which permits their detection in a specialfluorescencemicroscope(Chapter 3). lf a cell or tissueis treated with a detergentthat partially dissolvescell membranes,fluorescentantibodiescan drift in and bind to the specific protein they recognize. When the sample is viewed in the microscope,the bound fluorescent antibodies identify the location of the target protein (see F i g u r e1 - 1 5 ) . Betrer still is pinpointing proteins in living cells with intact membranes. One way of doing this is to introduce an engineeredgenethat codesfor a hybrid protein: part of the hybrid protein is the cellular protein of interest;the other part is a protein that fluoresceswhen struck by ultraviolet light. A common fluorescentprotein used for this purpose is green fluorescent protein (GFP), a natural protein that makes some iellyfish colorful and fluorescent. GFP "tagging" could reveal,for instance,that a particular protein is first made on the endoplasmic reticulum and then is moved by the cell into the lysosomes.In this case,first the endoplasmicreticulum and later the lysosomeswould glow in the dark. Chromosomesare visible in the light microscopeonly during mitosis,when they becomehighly condensed.The extraordinary behavior of chromosomesduring mitosis first was discoveredusing the improved compound microscopes of the late 1800s.About halfway through mitosis,the replicatedchromosomesbeginto move apart. Microtubules,one of the three types of cytoskeletalfilaments, participate in this movementof chromosomesduring mitosis.Fluorescenttagging of tubulin, the protein subunitthat polymerizesto form microtubules, reveals structural details of cell division that otherwisecould not be seenand allows observationof chromosomemovement(Figure1-21). Electron miqroscopesuse a focusedbeam of electronsinstead of a beam of light. In transmission electron microscopy, specimensare cut into very thin sections and placedunder a high vacuum,precludingexaminationof living cells. The resolution of transmission electron micro-

llll|

5e6nsAnimation:Mitosis

A FIGURE1-21 During the later stages of mitosis, microtubules (red) pull the replicated chromosomes(black) toward the ends of a dividing cell. Thisplantcell is stained and with a DNA-bindingdye (ethidium)to revealchromosomes antibodiesspecificfor tubulinto reveal with f luorescent-tagged microtubulesAt this stagein mitosis,the two copiesof each replicatedchromosome(calledchromatids)haveseparatedand are of AndrewBajer ] movinqawavf rom eachother [Courtesy

scopes,about 0.1 nm, permits fine structural details to be distinguished,and their powerful magnification would make a 1-pm-long bacterialcell look like a soccerball. Most of the organelles in eukaryotic cells and the double-layered structure of the plasma membrane were first observedwith electron microscopes(Chapter 9). \fith new specializedelectron microscopy techniques,three-dimensionalmodels of organellesand large protein complexes can be constructed from multiple images.But to obtain a more detailed look at the individual macromoleculeswithin cells, we must turn to techniqueswithin the purview of biochemistry and biophysics.

s e v e a tl h e B i o c h e m i s t r ay n d B i o p h y s i c R M o l e c u l a rS t r u c t u r ea n d C h e m i s t r yo f P u r i f i e d Cell Constituents Biochemistsextract the contentsof cellsand separatethe constituents based on differencesin their chemical or physical properties,a processcalledfractionation. The attention to individual moleculesmeans operating at the nanometer scale. Of particular interest are proteins, the workhorses of many A typical fractionation schemeinvolvesuse cellular processes. of various separationtechniquesin a sequentialfashion.These separationtechniquescommonly are basedon differencesin the size of moleculesor the electric charge on their surface (Chapter 3). To purify a particular protein of interest, a purification scheme is designed so that each step yields a

I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

2'l

J

^c

*o

;s'-s'

4"8" 2

1

-

3

-

4

-

5

-

I

FIGURE 1-22 Biochemicalpurificationof a protein from a cell extract often requiresseveralseparationtechniques.The purificationcanbe followedby gelelectrophoresis of thestarting proteinmixture andthefractions obtained fromeachpurification step Inthisprocedure, a sample isapplied to wellsin thetop of a gelatin-like slabandan electric fieldisappliedInthe presence of appropriate saltanddetergent concentrations, the proteins move throughthefibersof the geltowardtheanode,with largerproteins movingmoreslowlythroughthe gelthansmaller ones(seeFigure 3-35)Whenthegelisstained, proteins separated arevisible as distinctbandswhoseintensities areroughlyproportional to the proteinconcentration Shownhereareschematic depictions of oels for thestarting (lane1)andsamples mixture of proteins takenaiter eachof several purification stepsIn the firststep,saltf ractionation, proteinsthat precipitated with a certainamountof saltwereredissolved; electrophoresis (lane2) showsthatit of thissample contains fewerproteins thantheoriginal mixtureThesample then wassubjected in succession to threetypesof column chromatography proteins thatseparate by electrical charge, size,or bindingaffinityfor a particular (seeFigure smallmolecule 3-37).The finalpreparation isquitepure,ascanbe seenfromtheappearance of justoneproteinbandin lane5 [AfterJBergetal,2o02,Biochemistry W H Freeman andCompany, p 87l preparation with fewer and fewer contaminating proteins, until finally only the protein of interestremains (Figure 1-22). The initial purification of a protein of interest from a cell extract often is a tedious, time-consumingtask. Once a small amount of purified protein is obtained, antibodies to it can be produced by methods discussedin Chapter 19. For a biochemist, antibodies are near-perfecttools for isolating larger amounts of a protein of interest for further analysis.In effect, antibodies can "pluck out" the protein they specifically recognizeand bind from a semipure sample containing numerous different proteins. An increasinglycommon alterna-

22

C H A P T E R1

I

L I F EB E G I N SW I T H C E L L S

tive is to engineera gene that encodesa protein of interest with a small attached protein "tag," which can be used to pull out the protein from whole cell extracts. Purification of a protein is a necessaryprelude to studies on how it catalyzesa chemical reaction or carries out other functions and how its activiry is regulated. Some enzymesare made of multiple protein chains (subunits) with one chain catalyzing a chemical reaction and other chains regulating when and where that reaction occurs. The molecular machines that perform many critical cell processesconstitute even larger assembliesof proteins. By separating the individual proteins composing such assemblies,their individual catalytic or other activitiescan be assessed. For example,purification and study of the activity of the individual proteins composing the DNA replication machine provided clues about how they work together to replicate DNA during cell division (Chapter 4). The folded, three-dimensionalstructure, or conformation, of a protein is vital to its function. To understand the relation between the function of a protein and its form, we need to know both what it does and its detailed structure. The most widely used method for determining the complex structures of proteins, DNA, and RNA is x-ray crystallography, one of the powerful tools of biophysics.Computer-assistedanalysisof the data often permits the location of every atom in a large, complex molecule to be determined. The double-helix structure of DNA, which is key to its role in herediry was first proposed basedon x-ray crystallographic studies.Throughout this book you will encounter numerous examplesof protein structuresas we zero in on how proteins work.

GeneticsRevealsthe Consequences of DamagedGenes Biochemical and crystallographic studies can tell us much about an individual protein, but they cannot prove that it is required for cell division or any other cell process.The importance of a protein is demonstratedmost firmly if a mutation that prevents its synthesis or makes it nonfunctional adverselyaffects the processunder study. !(/e define the genotype of an organism as its composition of genes;the term also is commonly used in referenceto different versionsof a singlegeneor a small number of genes of interest in an individual organism. A diploid organism generally carries two versions (alleles)of each gene, one derived from each parent. There are important exceptions, such as the geneson the X and Y chromosomesin males of some species,including our own. The phenotype is the visible outcome of a gene's action, such as blue eyes versus brown eyesor the shapesof peas.In the early days of genetics, the location and chemical identity of genes were unknown; only the observablecharacteristics,the phenotypes, could be followed. The concept that genesare like "beads" on a long "string," the chromosome,was p,roposedearly in the 1900s basedon geneticwork with the fruit fly Drosophila. In the classicalgeneticsapproach, mutants are isolated that lack the ability to do something a normal organism can do. Often large genetic "screens" are done to look for many different mutant individuals (e.g., fruit flies, yeast cells) that are

unable to complete a certain process,such as cell division or muscle formation. In experimental organismsor cultured cells, mutations usually are produced by treatment with a mutagen, a chemical or physical agent that promotes mutations in a largely random fashion.But how can we isolateand maintain mutant organismsor cellsthat are defectivein some process, such as cell division, that is necessaryfor survival?One way is to look for organisms with a temperature-sensitivemutation. Thesemutants are able to grow at one temperature,the permissiue temperature, but not at anothe5 usually higher temperature, the nonpermissiuetemperature.Normal cells can grow at either temperature. In most cases,a temperature-sensitivemutant produces an altered protein that works at the permissive temperaturebut unfolds and is nonfunctional at the nonpermissive temperature. Temperature-sensitive screensare readily done with viruses,bacteria, yeast,roundworms, and fruit flies. By analyzing the effectsof numerous different temperaturesensitivemutations that altered cell division, geneticistsdiscoveredall the genesnecessaryfor cell division without knowing anything, initially, about which proteins they encode or how theseproteinsparticipatein the process.The greatpower of geneticsis to reveal the existenceand relevanceof proteins without prior knowledge of their biochemicalidentity or molecular function. Eventually these "mutation-defined" genes were isolatedand replicated(cloned)with recombinantDNA techniquesdiscussedin Chapter 5. \fith the isolatedgenesin hand, the encodedproteins could be produced in the test tube or in engineeredbacteria or cultured cells. Then the biochemistscould investigatewhether the proteins associatewith other proteins or DNA or catalyze particular chemical reactions during cell division (Chapter20). The analysis of genome sequencesfrom various organisms during the past decadehas identified many previously unknown DNA regions that are likely to encode proteins (i.e., protein-coding genes).The generalfunction of the protein encoded by a sequence-identifiedgene may be deduced by analogy with known proteins of similar sequence.Rather than randomly isolating mutations in novel genes, several techniquesare now available for inactivating specific genes by engineering mutations into them or destroying their mRNA with interfering RNA molecules(Chapter 5). The effects of such deliberategene-specificinactivation procedures provide information about the role of the encoded proteins in living organisms. This application of genetic techniques starts with a gene/proteinsequenceand ends up with a mutant phenotype; traditional genetics starts with a mutant phenotype and ends up with a gene/proteinsequence.

GenomicsRevealsDifferencesin the Structure and Expressionof EntireGenomes Biochemistry and geneticsgenerally focus on one gene and '$flhile powerful, these tradiits encoded protein at a time. tional approachesdo not give a comprehensiveview of the structure and activity of an organism'sgenome,its entire set of genes.The field of genomicsdoes just that, encompassing the molecular characterizationof whole genomesand the determination of global patterns of geneexpression.The recent

completion of the genome sequencesfor more than 100 speciesof bacteria and severaleukaryotesnow permits comparisons of entire genomes from different species.The results provide overwhelming evidenceof the molecular unity of life and the evolutionary processesthat made us what we are (seeSection 1.5). Genomics-basedmethods for comparing thousands of piecesof DNA from different individuals all at the same time are proving useful in tracing the history and migrations of plants and animals and in following the inheritance of diseasesin human families. DNA microarrays can simultaneously detect all the mRNAs presentin a cell, thereby indicating which genesare being transcribed. Such global patterns of gene expression clearly show that liver cells transcribe a quite different set of genesthan do white blood cellsor skin cells. Changesin gene expressionalso can be monitored during a diseaseprocess' in responseto drugs or other external signals'and during development. For instance,the identification of all the mRNAs presentin cultured fibroblasts before, during' and after they divide has given us an overall view of transcriptional changesthat occur during cell division (Figure 1-23). Cancet diagnosis is being transformed becausepreviously indistinguishablecancer cells have distinct gene expressionpatterns and prognoses (Chapter 25). Similar studies with different organisms and cell types are revealing what is universal about the genesinvolved in cell division and what is specific to particular organisms.To find out which genesare directly regulatedby a transcription factor, chromatin containing the protein of interest can be purified with an antibody and the associated DNA analyzed on microarrays, a procedure called chromatin immunopreclpltatlon. The entire complement of proteins in a cell, its proteome' is controlled in part by changesin genetranscription.The regulated synthesis,processing,localization' and degradationof specific proteins also play roles in determining the proteome of a particular cell. Learning how proteins bind to other proteins, often in large, multiprotein complexes, is providing a comprehensiveview of the molecular machines important for cell functioning. The field of proteomics will advance dramatically once high-throughput x-ray crystallography, currently under development,permits researchersto rapidly determine the structuresof hundredsor thousandsof proteins.

DevelopmentalBiology RevealsChangesin the Propertiesof Cellsas They Specialize Another approach to viewing cells comes from studying how they changeduring development of a complex organism. Bacteria, algae, and unicellular eukaryotes (protozoans, yeasts) often, but by no means always, can work solo. The concerted actions of the trillions of cells that compose our bodies require an enormous amount of communication and division of labor. During the development of multicellular organisms, differentiation processesform hundreds of cell types, each specialized for a particular task: transmission of electric signals by neurons, transport of oxygen by red blood cells, destruction of infecting bacteria by macrophages' contraction by muscle cells, chemicalprocessingby liver cells,and so on. I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

23

EXPERIMENTAL FIGURE 1-23 Microarrayanalysisof normal growing braincellsand braintumor cells.An experiment likethis isa starting pointfor learning howtumorcellsdifferfromnormal cellsRNAwasextracted fromnormalgrowingmousebraincells fromthecerebellum andfroma tumorof thecerebellum TheRNA fromthe tumorwaslabeled with a reddye,andRNAfromthe normal,non-tumorous cerebellum waslabeled with a greendye The two RNApreparations weremixedandhybridized to a microarray containing thousands of spotsof DNA Eachspotcontains the DNA sequence of onegene Unbound RNAwaswashedawayandthe microarray wasexposed to UVlight,whichcauses the dyesto fluoresce Spotsthataregreenhaveboundmostlynormalcerebellum RNA,spotsthatareredhaveboundmostlytumorRNA,andspots thatareyellowhaveboundroughly equalamounts of each.The faintlystained spotsrepresent genesfor whichthereislittleRNAin eithersampleThedataindicate whichgeneshavebeentranscribed in tumors,normalcerebellum, or both Onlypartof the datais shownhereTheentiredatasetrequires analyzing the colorsof more than25,000spots,allof whichcanbefittedontoonemicroscope slide.Precise measurements of colorintensity areactually madeby a spectrophotometer, but lookingby eyeshowsthat manygenesare morehighlyexpressed in normalor tumorcells.Someof these differences arethe consequence of the changeintotumorcells,but somemayreveal geneexpression changes thatcausethetumorsto form In addition, proteins madeexclusively in tumors,andperhaps necessary for uncontrolled growth,maybe candidate targets for discovering anti-cancer drugs [Courtesy of TalRaveh andl\y'atthew Scott, StanfordUniversitySchoolof Medicinel

Many of the differences among differentiated cells are due to production of specificsetsof proteins neededto carry out the unique functions of each cell type; that is, only a subsetof an organism'sgenesis transcribedat any given time or in any given cell. Such differential gene expressionat different times or in different cell types occurs in bacteria, fungi, plants, animals, and even viruses. Differential gene

24

CHAPTER 1

I

L I F EB E G I N S WITHCELLS

expression is readily apparent in an early fly embryo, in which all the cells look alike until they are stained to detect the proteins encoded by particular genes (Figure 1-24). Transcription can changewithin one cell type in responseto an external signal or in accordancewith a biological clock; some genes,for instance,undergo a dalIy cycle between low and high transcription rates. Producing different kinds of cells is not enough to make an organism, any more than collecting all the parts of a truck in one pile gives you a truck. The various cell types must be organized and assembled into all the tissues and organs. Even more remarkable, these body parts must work almost immediately after their formation and continue working during the growth process. For instance, the human heart begins to beat when it is lessthan 3 mm long, when we are mere 23-day-old embryos, and continues beating as it grows into a fist-sizemuscle. From a few hundred cells to billions, and still ticking. In the developing organism, cells grow and divide at some times and not others, they assembleand communicate, they prevent or repair errors in the developmentalprocess, and they coordinate each tissuewith others. In the adult organism,cell division largely stops in most organs.If part of an organ such as the liver is damaged or removed, cell division resumesuntil the organ is regenerated.The legend goes that Zets punished Prometheus for giving humans fire by

FIGURE 1-24 Differentialgene expressioncan be detectedin early fly embryosbefore cellsare morphologicallydifferent. An earlyDrosophila embryohasabout6000cellscoveringitssurface, mostof whichareindistinguishable by simplelightmicroscopy. lf the embryois madepermeable to antibodies with a detergent that partially dissolves membranes, theantibodies canfindandbindto the proteins theyrecognize Inthisembryowe seeantibodies tagged with a fluorescent labelboundto proteins thatarein the nuclei; each smallsphere corresponds to onenucleus. Threedifferent antibodies wereused,eachspecific proteinandeachgivinga for a different distinct green,or blue)in a fluorescence color(yellow, microscope. Theredcolorisaddedto highlight overlaps between theyellowand bluestainsThelocations proteins of thedifferent showthatthe cells arein factdifferent at thisearlystage,with particular genesturned on in specific stripes of cells. Thesegenescontrolthesubdivision of the bodyintorepeating segments, likethe blackandyellowstripes of a hornet.[Courtesy of Sean Carroll, University of Wisconsin ]

chaining him to a rock and having an eagleeat his liver. The punishment was eternal because,as the Greeks evidently knew, the liver regenerates. Developmental studies involve watching where, when, and how different kinds of cells form, discovering which signals trigger and coordinate developmentalevents,and understandingthe differential gene action that underliesdifferentiation (Chapters 16 and 21). During development we can see cells change in their normal context of other cells. Cell biology, biochemistry, cell biology, genetics, and genomics approachesare all employed in studying cells during development.

C h o o s i n gt h e R i g h t E x p e r i m e n t aOl r g a n i s m for the Job Our current understanding of the molecular functioning of cells rests on studieswith viruses,bacteria, yeast, protozoa, slime molds, plants, frogs, sea urchins, worms, insects,fish, chickens, mice, and humans. For various reasons, some organisms are more appropriate than others for answering particular questions.Becauseof the evolutionary conservation of genes,proteins, organelles,cell types, and so forth, discoveriesabout biological structuresand functions obtained with one experimental organism often apply to others. Thus researchersgenerallyconduct studieswith the organism that is most suitable for rapidly and completely answering the question being posed, knowing that the results obtained in one organismare likely to be broadly applicable.Figure1-25 summarizesthe typical experimental usesof various organisms whose genomeshave been sequencedcompletely or nearly so. The availability of the genomesequencesfor these organisms makes them particularly useful for geneticsand genomicsstudies. Bacteria have several advantagesas experimental organisms: they grow rapidlS possesselegant mechanisms for controlling gene activity, and have powerful genetics. This latter property relates to the small size of bacterial genomes,the easeof obtaining mutants, the availability of techniquesfor transferring genes into bacteria, an enormous wealth of knowledge about bacterial gene control and protein functions, and the relative simplicity of mapping genesrelative to one another in the genome. Singlecelledyeastsnot only have some of the sameadvantagesas bacteria but also possessthe cell organization,marked by the presenceof a nucleusand organelles,that is characteristic of all eukaryotes. Studiesof cellsin specializedtissuesmake use of animal and plant "models," that is, experimentalorganismswith attributes typical of many others. Nerve cells and muscle cells, for instance,traditionally were studied in mammals cells,such or in creatureswith especiallylarge or accessible as the giant neural cells of the squid and sea hare or the flight musclesof birds. More recently,muscle and nerve development have been extensively studied in fruit flies (Drosophila melanogaster),roundworms (Caenorhabditis elegans),and zebrafish(Danio rerio), in which mutants can be readily isolated. Organisms with large-celledembryos

that develop outside the mother (e.g.' frogs, sea urchins, fish, and chickens) are extremely useful for tracing the fates of cells as they form different tissues and for making extracts for biochemicalstudies.For instance,a key protein in regulating mitosis was first identified in studies with frog and sea urchin embryos and subsequentlypurified from extracts (Chapter 20lt. Using recombinant DNA techniques,researcherscan engineer specificgenesto contain mutations that inactivate or increaseproduction of their encodedproteins. Suchgenescan be introduced into the embryos of worms, flies, frogs, sea urchins, chickens,mice, a variety of plants, and other organisms,permitting the effectsof activating a geneabnormally or inhibiting a normal gene function to be assessed.This approach is being used extensivelyto produce mouse versions of human genetic diseases.Inactivating particular genes by introducing short piecesof interfering RNA is allowing quick tests of gene functions possiblein many organisms.The expansion of genomeproiectsto critically important diseaseorganisms,such as malaria, and to creaturesthat span the evolutionary tree is bringing new options for medicine and new insights into how living organisms have diversified to take advantageof every possibleecologicalniche. Mice have one enormous advantage over other experimental organisms:they are the closestto humans of any animal for which powerful geneticapproachesare feasible.Engineered mouse genes carrying mutations similar to those associatedwith a particular inherited diseasein humans can be introduced into mouse embryonic stem (ES) cells. These cells can be injected into an early embryo, which is then implanted into a pseudopregnantfemale mouse (a mouse treated with hormones to trigger physiological changes neededfor pregnancy) (Chapter 5). If the mice that develop from the injected ES cells exhibit diseasessimilar to the human disease,then the link between the diseaseand mutations in a particular geneor genesis supported. Once mouse models of a human diseaseare available, further studies on the molecular defectscausing the diseasecan be done and new treatmentscan be tested,thereby minimizing human exposure to untested treatments. Large-scalegenetic screens are being done that take advantageof newly designedmutagenic transposons.The transposons allow efficient generation of mouse mutants and rapid identification of the gene that has been hit in each one. A continuous unplanned genetic screen has been per\What we mean formed on human populations for millennia. is that all sorts of human variations have arisen and have been noticed, since they affect visible or noticeable human characteristics.Thousandsof inherited traits have beenidentified and, more recently,mapped to locations on the chromosomes.Some of these traits are inherited propensitiesto get a disease;others are eye color or other minor characteristics. Geneticvariations in virtually every aspectof cell biology can be found in human populations, allowing studiesof normal and diseasestatesand of variant cells in culture. Less-commonexperimental organisms offer possibilities for exploring unique or exotic properties of cells and for studying standard properties of cells that are exaggeratedin I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

25

Podcast:Common ExperimentalOrganisms

Viruses

Bacteria

Proteinsinvolvedin DNA, RNA, protein synthesis Gene regulation Cancerand control of cell oroliferation Transportof proteinsand organellesinsidecells Infectionand immunity Possiblegene therapy approaches

Proteinsinvolvedin DNA, RNA, protein synthesis, metabolism Gene regulation Targetsfor new antibiotics Cell cycle Signaling

Yeast (Saccharo myce s cerev is i ael

Roundworm I Caenorh abd itis elegansl

Controlof cell cycle and cell division Proteinsecretionand membrane biogenesis Functionof the cytoskeleton Cell differentiation Aging Gene regulationand chromosome structure

26

C H A P T E R1

I

Developmentof the body plan C e l ll i n e a g e Formationand function of the nervous system Controlof programmedcell death Cell proliferationand cancergenes Aging Behavior Gene regulationand chromosome structure

Fruit ffy (Drosophi Ia mel anoga sterl

Zebrafish

Developmentof the body plan Generationof differentiatedcell lineages Formationof the nervoussystem, heart,and musculature Programmedcell death Geneticcontrol of behavior Cancergenesand control of cell proliferation Controlof cell polarization Effectsof drugs, alcohol,pesticides

Developmentof vertebratebody tissues Formationand function of brain and nervous syslem Birth defects Cancer

Mice, includingculturedcells

Plant (A ra b i d ops is th aIi anal

Developmentof body tissues F u n c t i o no f m a m m a l i a ni m m u n e system Formationand function of brain and nervoussystem Models of cancersand other human diseases Gene regulationand inheritance Infectiousdisease

Developmentand patterningof tissues Geneticsof cell biology Agricultural applications Physiology Gene regulation lmmunity Infectiousdisease

L I F EB E G I N SW I T H C E L L S

< FIGURE 1-25 Eachexperimentalorganismusedin cell (a) biology has advantagesfor certaintypes of studies.Viruses (b)havesmallgenomes andbacteria amenable to genetic dissection Manyinsights intogenecontrolinitially camefromstudies with these (c)hasthe cellular organismsTheyeastSaccharomyces cerevisiae organization of a eukaryote but isa relatively simplesingle-celled genetically. organism thatiseasyto growandto manipulate Inthe (d),whichhasa small nematode worm Caenorhabditis elegans numberof cellsarranged in a nearlyidentical wayin everyworm,the formation of eachindividual cellcanbe tracedThefruitfly (e),firstusedto discover Drosophila melanogaster the properties of genesthat chromosomes, hasbeenespecially valuable in identifying controlembryonic development Manyof thesegenesare evolutionarily conserved in humansThezebraf ishDaniorerio(f)is genesthatcontrol usedfor rapidgenetic screens to identify development andorganogenesis. Of theexperimental animal (g)areevolutionarily systems, mice(Musmusculus) the closestto humans andhaveprovided models for studying numerous human genetic andinfectious diseases. Themustard-family weed Arabidopsis thaliana,sometimes described asthe Drosophila of the plantkingdom, genes hasbeenusedfor genetic screens to identify involved in nearlyeveryaspect of plantlife Genome sequencing is completed for manyviruses andbacterial species, theyeast Saccharomyces cerevisiae, the roundwormC. elegans,the f ruit fly D. melanogaste4 humans,andthe plantArabidopsis thalianallis mostlycompleted for miceandin progress for zebrafish Other particularly organisms, frogs,seaurchins, chickens, andslimemolds, to be immensely continue valuable for cellbiologyresearch Increasingly, a widevariety of otherspecies for areused,especially (a)Visuals studies of evolution of cellsandmechanisms Unlimited, [Part Inc Part(b)KariLountmaa/Science Photo Library/Photo Researchers, Inc Part (c)ScimavPhoto Researchers, Inc Part(d)Photo Researchers, IncPart(e) Darwin Dale/Photo Researchers, Inc,Part(f)IngeSpencel'/isuals Inc Unlimited, Part(g)J M Labavjancanatuisuals Unlimited, Inc Part(h)Darwin Dale/Photo Researchers, Inc]

a useful fashion in a particular animal. For example, the ends of chromosomes,the telomeres,are extremely dilute in most cells.Human cells typically contain 92 telomeres(46 chromosomesX 2 endsper chromosome).In contrast,some protozoawith unusual "fragmented" chromosomescontain millions of telomeres per cell. Taking advantage of the unique properties of this well-chosen experimental organism has led to important recent discoveriesabout telomere structure.

The Most SuccessfulBiologicalStudiesUse M u l t i p l eA p p r o a c h e s We have discussedfive classesof approachesto biological problems: cell biology biochemistry and biophysics, genetics, genomics,and developmentalbiology. Each has its own types of experiments,and most biological problems require more than one approach in order to reach a satisfying understanding of mechanism. Now we will survey how these approacheshave been applied to the study of cell division to emphasizehow important it is to use multiple types of experlments.

Cell division was viewed, and indeed discovered,by some of the earliest usersof microscopes.More recently a variety of kinds of microscopy, including confocal and electron microscopy and time-lapseimaging (Chapter 9), have been used to characterizethe stepsof the cell cycle. Most biology begins with this sort of observation, defining the mysteries that must be tackled experimentally.Then manipulations begin. Antibodies were made againstproteins that play critical roles in cell division both to detect proteins and in some casesto interfere with the functions of those proteins. Key proteins were fused to fluorescentproteins, starting with the iellyfish greenfluorescentprotein (GFP),so that key proteins could be followed in living cells. Questions about when and where proteins work could be addressedand their functions defined to an extent. The apparatusof cell division, such as the mitotic spindle and other protein complexes,was purified and analyzed using the approaches of biochemistry and biophysics. Each protein had to be purified to find out whether it is part of a complex of proteins bound together as a machine, and the structuresof key proteins were determined using x-ray crystallography and other methods (Chapter 3). Previously unknown enzymatic activities were detected in extracts using assays such as measuring the attachment of phosphate groups to cell division regulatory proteins by kinases, and then the relevant kinasescould be purified. Finding a novel protein in a complex of proteins involved in cell division makes it a good bet that the protein does something important, a sort of "guilt by association," but it doesnot provide proof that the protein matters. For that one must turn to genetics. Genetics can be used to identify mutants in the newly found protein. If cell division fails in a living organism when the protein is not working, you know the protein matters. Geneticsis also a way to identify previously unknown genesand proteins sincescreenscan be done (especiallyin bacteria and yeast, but also in more complex Iab organisms)to look for all the genesthat are neededfor cell division. The newly discoveredproteins can be incorporated into a complete picture of the mechanicsof cell division machinery. Genomics provides another way to look for working parts of the cell-division machine. Since it is often true that mRNAs and proteins are produced only when they are needed, using microarrays to look for all geneswhose expressionvaries with the cell cycle is a powerful approach to identify candidatesfor cell division regulators. Having identified new genesthat are required for cell division, one must find out how thesegenes'protein products work. Simply knowing that a protein matters to cell division is not enough to understand the mechanism.Thus it is necessaryto return to biochemical and biophysical approaches to work out the molecular biology and to cell biology to monitor protein locations and movements. Finally, cell division does not happen in a vacuum; it happens in the context of the life cycle of the organism. To fully appreciatehow the regulation works and is used, it is important to use the approachesof developmental biology

I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T s

27

to revealwhen and where cell division normally happensand why. In theseexperimentsthe times and placesof cell division during the developmentof the organism are monitored, and then the signals that stimulate or suppresscell division are identified and studied. Errors in developmental control of cell division, revealedby studying mutants, can causeorgans and tissuesto be the wrong sizeor cancer. The samekinds of approachesthat work for cell division can be applied to many other biological challenges,such as Iearning how muscles form or how they work or how the brain functions. Often it's good to use every tool in the toolkit.

tf,|

A GenomePerspective

on Evolution Comprehensivestudies of genesand proteins from many organismsare giving us an extraordinary documentationof the history of life. Nature is a laboratory that has been conducting experimentsfor billions of years, and some of the most successfulgenomesthat emergedare still with us. 'We share with other eukaryotes thousands of individual proteins, hundredsof macromolecularmachines,and most of our organelles,all as a result of our sharedevolutionary history. New insights into molecular cell biology arising from genomicsare leadingto a fuller appreciationof rhe elegant molecular machines that arose during billions of yearsof genetictinkering and evolutionaryselectionfor the most efficient, precise designs. Due ro alternative RNA splicing,the number of proteins vastly exceedsthe number of genes,and the functions of many variant proteins and assembliesof proteins remain to be discovered. Once a more complete description of cells is in hand, we will be ready to fully investigate the rippling, flowing dynamics of living systems.

MetabolicProteins,the GeneticCode, a n d O r g a n e l l eS t r u c t u r e sA r e N e a r l yU n i v e r s a l Even organisms that look incredibly different share many biochemical properties. For instance, the enzymesthat catalyze degradation of sugars and many other simple chemical reactions in cells have similar structures and mechanisms in most living things. The genetic code whereby the nucleotide sequencesof mRNA specifiesthe amino acid sequencesof proteins can be read equally well by a bacterial cell and a human cell. Becauseof the universalnature of the genetic code, bacterial "factories" can be designedto manufacture growth factors, insulin, clotting factors, and other human proteins with therapeutic uses. The biochemical similarities among organisms also extend to the organelles found in eukaryotic cells.The basic structuresand functions of these subcellular components are largely conservedin all eukaryotes.

28

Computer analysisof DNA sequencedata, now available for numerousbacterialspeciesand severaleukaryotes, can locate protein-coding geneswithin genomes.\fith the aid of the geneticcode, the amino acid sequencesof proteins can be deduced from the corresponding gene sequences.Although simple conceptually, "finding" genes and deducing the amino acid sequencesof their encoded proteins is complicated in practice becauseof the many noncodingregionsin eukaryoticDNA (Chapter5). Despite the difficulties and occasional ambiguities in analyzing DNA sequences, comparisonsof the genomesfrom a wide range of organisms provide stunning, compelling evidence for the conservation of the molecular mechanisms that build and change organisms and for the common evolut i o n a r y h i s t o r yo f a l l s p e c i e s .

CHAPTER 1

I

L I F EB E G I N S WITHCELLS

Darwin'sldeasAbout the Evolutionof Whole A n i m a l sA r e R e l e v a n t o G e n e s Darwin did not know that genesexist or how they change, but we do: the DNA replication machine makes an error, or a mutagen causesreplacement of one nucleotide with another or breakage of a chromosome. Some changesin the genomeare innocuous, some mildly harmful, some deadly; a very few are beneficial. Mutations can change the sequence of a gene in a way that modifies the activity of the encoded protein or alters when, where, and in what amounts the protein is produced in the body. Gene-sequencechangesthat are harmful will be lost from a population of organisms becausethe affected individuals cannot survive as well as their relatives. This selection process is exactly what Darwin described without knowing the underlying mechanismsthat cause organisms to vary. Thus the selectionof whole organisms for survival is really a selectionof genes,or more accurately sets of genes.A population of organismsoften contains many variants that are all roughly equally well-suited to the prevailing conditions. When conditions change-a fire, a flood, loss of preferred food supply, climate shift-variants that are better able to adapt will survive, and those less suited to the new conditions will begin to die out. In this way, rhe genetic composition of a population of organisms can change over tlme.

M a n y G e n e sC o n t r o l l i n gD e v e l o p m e n t A r e R e m a r k a b l yS i m i l a ri n H u m a n s a n d O t h e rA n i m a l s As humans, we probably have a biasedand somewhat exaggerated view of our status in the animal kingdom. Pride in our swollen forebrain and its associatedmental capabilities may blind us to the remarkably sophisticated abilities of other species:navigation by birds, the sonar systemof bats, homing by salmon, or the flight of a fIy. Despite all the evidencefor evolutionary unity at the cellular and physiological levels,everyoneexpectedthat genes

(a)

Genes

Flv

(d)

Mammal

(e)

< FIGURE 1-26 Similargenes,conservedduringevolution, processes in diverseanimals. regulatemanydevelopmental to havehada commonancestor areestimated Insects andmammals abouthalfa billionyearsago.Theysharegenesthatcontrolsimilar of the processes, suchasgrowthof heartandeyesandorganization times. conservation of functionfromancient bodyplan,indicating (a)Hoxgenesarefoundin clusters of mostor on thechromosomes proteins thatcontrolthe allanimalsHoxgenesencoderelated of activities of othergenesHoxgenesdirectthedevelopment as axisof manyanimals, alongthe head-to-tail different segments Eachgeneisactivated colors. indicated by corresponding (transcriptionally) axisand regionalongthe head-to-tail in a specific in micethe Hox there Forexample, controls the growthof tissues of vertebrae shapes genesareresponsible for thedistinctive Hoxgenesin fliescausebodypartsto formin affecting Mutations on the head. suchaslegsin lieuof antennae thewronglocations, genesprovide address andserveto direct a head-to-tail These in the rightplaces(b)Development of the rightstructures formation a genecalled eyesin fruitfliesrequires of the largecompound (c)Flieswith inactivated (namedfor the mutantphenotype). eyeless the human geneslackeyes(d)Normalhumaneyesrequire eyeless (e)Peoplelacking to eyeiess. gene,calledPax6,that corresponds anrrdia,a lackof adequatePax6functionhavethe geneticdisease encodehighlyrelatedproteins irisesin the eyesPax6andeyeless from of othergenesandaredescended theactivities that regulate (b)and(c)Andreas gene lParts Hefti, Interdepartmental the sameancestral (lEM) of BaselPart(d) of theUniversity Biocenter Microscopy Electron Unlimitedl Inc Part(e)Visuals Researchers. Fraser/Photo @Simon

that, as far as we can tell, are utterly absentfrom certain lineagesof animals. Plants, not surprisingly,exhibit many such differencesfrom animals after a billion-year separation in their evolution. Yet certain DNA-binding proteins differ between peasand cows at only two amino acids out of 1'021

regulating animal development would differ greatly from one phylum to the next. After all, insectsand seaurchins and mammals look so different. \(e must have many unique proteins to createa brain like ours . . . or must we? The fruits of research in developmental genetics during the past two decadesrevealthat insectsand mammals, which have a common ancestor about half a billion years ago, possessmany genes(Figure 1-26). Indeed, similar development-regulating a large number of these genes appear to be conserved in many and perhaps all animals. Remarkably, the developmental functions of the proteins encoded by thesegenesare also often preserved.For instance,certain proteins involved in eye development in insects are related to protein regulators of eye developmentin mammals. Samefor development of the heart, gut, lungs, and capillaries and for placementof body parts along the head-to-tail and back-to-front body axes (Chapter19). This is not to say that all genesor proteins are evolutionarily conserved. Many striking examples exist of proteins

H u m a nM e d i c i n el s I n f o r m e db y R e s e a r c h on OtherOrganisms Mutations that occur in certain genesduring the course of our lives contribute to formation of various human cancers. The normal, wild-type forms of such "cancer-causing"genes generallyencodeproteins that help regulatecell proliferation or death (Chapter 21').'Wealso can inherit from our parents mutant copies of genesthat causeall manner of genetic diseases,such as cystic fibrosis, muscular dystrophy' sickle cell anemia, and Huntington's disease.Happily we can also inherit genesthat make us robustly resist disease.A remarkable number of genesassociatedwith cancer and other human diseasesare present in evolutionarily distant animals. For example, a recent study shows that more than threequarters of the known human diseasegenes are related to genesfound in the fruit fIy Drosophila. Vith the identification of human diseasegenesin other organisms,experimental studies in experimentally tractable organisms should lead to rapid progress in understanding

OEN E V O L U T I O N A G E N O M EP E R S P E C T I V

29

the normal functions of the disease-relatedgenes and what occurs when things go awry. ConverselS the disease states themselvesconstitute a genetic analysis with well-studied phenotypes. All the genesthat can be altered to cause a certain diseasemay encode a group of functionally related proteins. Thus clues about the normal cellular functions of pro-

30

.

cHAPTERI I

L I F EB E G I N SW I T H C E L L S

teins come from human diseasesand can be used to guide initial research into mechanism. For instance, genesinitially identified becauseof their link to cancer in humans can be studied in the context of normal development in various model organisms, providing further insight about the functions of their protein products.

CHAPTER

CHEMICAL FOUNDATIONS Polarized light microscopic imageof crystalsof ATBwhose hydrolysis isa primarysourceof energythat drivesmanycellular chemical reactions[Dr.ArthurM SiegelmaMr'isuals Unlimited ]

he life of a cell dependson thousands of chemical interactions and reactions exquisitely coordinated with one another in time and sDaceand under the influence of the cell's genetic instructions and its environment. By understandingat a molecular level theseinteractions and reactions, we can begin to answer fundamental questionsabout cellular life: How doesa cell extract critical nutrients and information from its environment? How does a cell convert the energy stored in nutrients into work (movement,synthesis of critical components)?How does a cell transform nutrients into the fundamental structuresrequired for its survival (cell wall, nucleus, nucleic acids, proteins, cytoskeleton)? How does a cell link itself to other cells to form a tissue? How do cells communicate with one another so that a complex, efficiently functioning organism can develop and thrive? One of the goals of Molecular Cell Biology is to provide answers to these and other questions about the structure and function of cells and organisms in terms of the properties of individual moleculesand ions. For example, the properties of one such molecule,water, have controlled and continue to control the evolution, structure, and function of cells. You cannot understand biology without appreciating how the properties of water control the chemistry of life. Life first arose in a watery environment. Constituting 70-80 percent by weight of most cells, water is the most abundant molecule in biological systems. It is within this aqueous milieu that small molecules and ions, which make up about 7 percent of the weight of living matter, assembleinto the larger macromoleculesand macromolecular aggregatesthat make up a cell's machinery and architecture and so the remaining mass of organisms.

These small molecules include amino acids (the building blocks of proteins), nucleotides (the building blocks of DNA and RNA), lipids (the building blocks of biomembranes), and sugars (the building blocks of starchesand cellulose). Many biomolecules(e.g.,sugars)readily dissolvein water; these molecules are called hydrophilic (water liking). Others (e.g., cholesterol)are oilS fatlike substancesthat shun water; these are said to be hydrophobic (water fearing). Still other biomolecules(e.g.,phospholipids)are a bit schizophrenic, containing both hydrophilic and hydrophobic regions; these molecule are said to be amphipathic. Phospholipids are used to build the flexible membranes that form the wall-like boundaries of cells and their internal organelles.The smooth functioning of cells, tissues' and organisms depends on all these molecules, from the smallest to the largest. Indeed, the chemistry of the simple proton (H*) can be as important to the survival of a human cell as that of each gigantic, genetic-code-carrying DNA molecule (the mass of the DNA molecule in human

OUTLINE 2.1

CovalentBondsand NoncovalentInteractions 32

2.2

of Cells BuildingBlocks Chemical

40

2.3

Equilibrium Chemical

49

2.4

Energetics Biochemical

54

31

(a) Molecularcomplementarity

(bl Chemicalbuilding blocks

ProteinA

Polymerization p&. d'

",llrill t

1

ProteinB

S m a l lm o l e c u l e subunits (dl Chemicalbond energy

{c} Chemicalequilibrium

e#

Macromolecule

"High-energy" phosphoa n h y dr i d e bonds

kr Kl

K^^_

K1

Adenosine triphosphate

k,

A FIGURE2-1 Chemistry of life: four key concepts.(a) Molecular complementarity liesat the heartof all biomolecular interactions, as when two proteinswith complementary shapesand chemical propertiescometogetherto form a tightlybound complex (b) Small moleculesserveas buildingblocksfor IargerstructuresForexample, to generatethe information-carrying macromolecule DNA,four small nucleotidebuildingblocksare covalentlylinkedinto long strings (polymers), which then wrap aroundeachother to form the double helix (c) Chemicalreactionsare reversible, and the distributionof the chemicalsbetweenstartingreagents(/eft)and the productsof the

(nght)depends reactions on the rateconstants of the forward(k1, (k,,lowerarrow)reactions upperarrow)andreverse Theratioof these,K"o,provides an informative measure of the relative amounts (d)In of products andreactants thatwill be present at equilibrium manycases, the source of energyfor chemical reactions in cellsisthe hydrolysis of the molecule ATPThisenergyisreleased whena highenergyphosphoanhydride bondlinkingthe B andy phosphates in (red)isbrokenby the addition theATPmolecule of a watermolecule,

c h r o m o s o m e1 i s 8 . 6 x 1 0 r 0 t i m e s t h a t o f a p r o t o n ! ) . T h e chemical interactions of all of these molecules,large and small, with water and with one anothet define the nature of life. Luckily, although many types of biomolecules interacr and react in numerous and complex pathways to form functional cells and organisms, a relatively small number of chemical principles are necessaryto understand cellular processes at the molecularlevel (Figure 2-l).ln this chapter we review these key principles, some of which you already know well. Sfe begin with the covalent bonds that connect atoms into a molecule and the noncovalent forcesthat stabilize groups of atoms into functional structures within and between molecules. We then consider the key properties of the basic chemical building blocks of macromoleculesand macromolecular assemblies.After reviewing those aspectsof chemical equilibrium that are most relevant to biological systems,we end the chapter with basic principles of bio-

chemical energetics,including the central role of ATP (adenosinetriphosphate) in capturing and transferring energy in cellular metabolism.

32

cHAPTER2 |

CHEMICALFOUNDATTONS

TOrmlnOAUI'311O l',.

A

CovalentBondsand

NoncovalentInteractions Strong and weak attractive forces between atoms are the "glue" that holds them together in individual moleculesand permits interactions between different biomolecules.Strong forces form a covalent bond when two atoms share one pair of electrons("single" bond) or multiple pairs of electrons ("double" bond, "triple" bond, etc.). The weak attractive forces of noncovalent interactions are equally important in determining the properties and functions of biomolecules such as proteins, nucleic acids, carbohydrates,and lipids. .We will first review covalent bonds and then discussthe four

Electrons

Covalentbond H

H

H

H

A FIGURE 2-2 Covalentbondsform by the sharingof electrons. Covalent bonds,the strongforcesthat holdatomstogetherinto molecules, formwhenatomsshareelectrons fromtherroutermost electron orbitals. Eachatomformsa definednumberandqeometrv of covalent bonds

major types of noncovalent interactions:ionic bonds, hydrogen bonds, van der Waals interactions,and the hydrophobic effect.

The ElectronicStructureof an Atom D e t e r m i n e st h e N u m b e ra n d G e o m e t r y of CovalentBondslt Can Make Hydrogen, oxygen, carbon, nitrogen,phosphorus,and sulfur are the most abundant elementsin biological molecules. Theseatoms, which rarely exist as isolatedentities,readily form covalent bonds, using electronsin the outermost electron orbitals surrounding their nuclei (Figure 2-2\. As a rule, each type of atom forms a characteristicnumber of covalent bonds with other atoms, with a well-defined geometry determined by the atom's size and by both the distribution of electrons around the nucleus and the number of electronsthat it can share.In some cases(e.g.,carbon), the number of stable covalent bonds formed is fixed; in other cases (e.g., sulfur), different numbers of stable covalent bonds are possible. All the biological building blocks are organizedaround the carbon atom, which normally forms four covalent bonds with three or four other atoms. As illustrated in Figure 2-3a for formaldehyde, carbon can bond to three atoms, all in a common plane. The carbon atom forms two typical single bonds with two atoms and a double bond (two shared electron pairs) with the third atom. In the absenceof other constraints, atoms joined by a single bond generally can rotate freely about the bond axis, whereas those connected by a double bond cannot. The rigid planarity imposed by double bonds has enormous significancefor the shapesand flexibility of biomoleculessuch as phospholipids, proteins, and nucleic acids. Carbon can also bond to four rather than three atoms. As illustrated by the methane (CHa) molecule,when carbon is bonded to four other atoms, the angle between any two bonds is 109.5' and the positions of bonded atoms define the four points of a tetrahedron(Figure2-3b). This geome-

try defines the structures of many biomolecules.A carbon (or any other) atom bonded to four dissimilar atoms or groups in a nonplanar configuration is said to be asymmetric. The tetrahedral orientation of bonds formed by an asymmetric carbon atom can be arranged in three-dimensional space in two different ways, producing molecules that are mirror imagesof eachother, a property calledchirality (from the Greek word cheir,meaning "hand") (Figure2-4). Such moleculesare called optical isomers,or stereoisomers.Many molecules in cells contain at least one asymmetric carbon atom, often called a chiral carbon atom. The different stereoisomersof a molecule usually have completely different biological activities becausethe arrangement of atoms within their structures differs, yielding their unique abilities to interact and chemically react with other molecules. Some drugs are mixtures of the stereoisomersof small moleculesin which only one stereoisomerhas the biological activity of interest.The use of a pure single stereoisomer of the chemical in place of the mixture can result in a more potent drug with reduced sideeffects.For example,one stereoisomerof the antidepressantdrug citalopram (Celexa) is

(al Formaldehyde

H

c:o H

(b) Methane H

I

H-C-H

I

H Chemical structure

Ball-and-stick model

Space-filling model

2-3 Geometryof bondswhen carbonis covalently A FIGURE linked to three or four other atoms.(a)A carbonatomcanbe (CHzO). Inthiscase,the bondedto threeatoms,asin formaldehyde participate in two singlebondsandone electrons carbon-bonding etoms , h i c ha l ll i ei n t h es a m ep l a n eU. n l i k a d o u b l eb o n dw canrotatef reelyaboutthe by a singlebond,whichusually connected by a doublebondcannot(b)Whena bondaxis,thoseconnected (CH+), the carbonatomformsfoursinglebonds,asin methane (all in the formof in space oriented case) are H in this atoms bonded indicates on the leftclearly Theletterrepresentation a tetrahedron. andthe bondingpattern of the molecule theatomiccomposition the geometric modelin the centerillustrates Theball-and-stick of the balls the diameters bonds, but and of the atoms arrangement are electrons the atomsandtheirnonbonding representing with the bondlengthsThesizesof the smallcompared unrealistically modelon the rightmore cloudsin the space-filling electron in threedimensions thestructure represent accuratelv C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S

33

FIGURE 2-4 Stereoisomers. Manymolecules in cellscontain at leastoneasymmetric carbonatom Thetetrahedral orientation of bondsformedbyan asymmetric carbonatomcanbearranged in three-dimensional spacein two different ways,producing molecules that aremirrorimages, or stereoisomers, of eachother.Shownhereis thecommonstructure of an aminoacid,with itscentral asymmetric carbonandfourattached groups, including the Rgroup,discussed in Section 2 2. Aminoacidscanexistin two mirror-image forms, designated r ando. Although properties thechemical of such stereoisomers areidentical, theirbiological activities aredistinctOnly r aminoacidsarefoundin oroterns

that can participate in noncovalent interactions. Sulfur forms two covalent bonds in hydrogen sulfide (H2S)but also can accommodate six covalent bonds, as in sulfuric acid (H2SO4)and its sulfate derivatives.Nitrogen and phosphorus each have five electronsto share.In ammonia (NH3), the nitrogen atom forms three covalent bonds; the pair of electrons around the atom not involved in a covalent bond can take part in noncovalent interactions. In the ammonium ion (NH+*), nitrogen forms four covalent bonds, which have a tetrahedral geometry. Phosphoruscommonly forms five covalent bonds, as in phosphoric acid (H3POa) and its phosphate derivatives,which form the backbone of nucleic acids. Phosphategroups covalently attachedto proteins play a key role in regulating the activity of many proteins, and the central moleculein cellular energetics,ATP, contains three phosphate groups (seeSection 2.4). A summary of common covalent linkagesand functional groups (portions of molecules that confer distinctive chemical properties) is provided in Table 2-2.

ElectronsMay Be SharedEqually o r U n e q u a l l yi n C o v a l e n tB o n d s

The extent of an atom's ability to attract an electron is called its electronegatiuity. ln a bond between atoms with identical or similar electronegativities,the bonding electrons are es170 times more potent than the other. Somestereoisomershave sentially sharedequally betweenthe two atoms, as is the case very different activities. Darvon is a pain reliever,whereas its for most C-C and C-H bonds. Such bonds are called nonstereoisomer,Novrad (Daruon spelled backward), is a cough polar. In many molecules,the bonded atoms have different suppressant. One stereoisomer of ketamine is an anesthetic, electronegativities,resulting in unequal sharing of the elecwhereas the other causeshallucinations. I trons. The bond betweenthem is said to be polar. One end of a polar bond has a partial negativecharge The number of covalent bonds formed by other common (6-), and the other end has a partial positive charge ( 6+). atoms is shown in Table 2-1,.A hydrogen atom forms only In an O-H bond, for example, the greater electronegativone covalent bond. An atom of oxygen usually forms only ity of the oxygen atom relative to hydrogen results in the two covalent bonds but has two additional pairs of electrons electrons spending more time around the oxygen atom than the hydrogen. Thus the O-H bond possessesan electric dipole, a positive charge separated from an equal but opposite negative charge. The amount of 6- charge on the oxygen atom of a O-H dipole is approximately 25 percent of that of an electron, with an equivalent 6+ charge on the H atom. Becauseof its two O-H bonds that are nor AT()M AIIO USUAI. I,IUMBTB TYPICAL I)UTER TI.ICIROI'IS {]FCl)VATElrlT BI]NDS Bt]I'IIl GIOMETBY on exact opposite sides of the O atom, water molecules (HzO) are dipoles (Figure 2-5) that form electrostatic,noncovalent interactions with one another and with other molH I H ecules. These interactions play a critical role in almost o z every biochemical interaction and so are fundamental to ,ror cell biology. S 214,or5 St The polarity of the O:P double bond in H3PO4 results ,' in a resonancehybrid, a structure between the two forms N shown below in which nonbonding electrons are shown as 3or4 pairs of dots:

-T-

5

P

4

-'rP

I

-?-

H

CHAPTER2 I

CHEMICALFOUNDATIONS

I

:o

o I

H-O-P-O-H il"

o.

34

H

I

e

l*

H-O-P-O-H

I

o

FUNCTIONAL GROUPS

o -c-

o -c-o-

Carbonyl

Carboxyl

(ketone)

( c a r b o x y l i ca c i d )

o -o-P-oI

oPhosphate

oo

iltl -o-P-o-P-

o-

o-

Pyrophosphate (phosphorylated molecule)

(diphosphate)

LINKAGES

o til -c-o-cI

o -N-C-

ll

I Amide

In the resonancehybrid on the right, one of the electrons from the P:O double bond has accumulatedaround the O atom, giving it a negativecharge and leaving the P atom with a positive charge. These chargesare important in noncovalent interactions.

C o v a l e n tB o n d sA r e M u c h S t r o n g e r and More StableThan NoncovalentInteractions Covalentbonds are very stable(i.e.,consideredto be strong) becausethe energies required to break them are much greater than the thermal energy available at room temperature (25 "C) or body temperature(37'C). For example,the

ol

H

2-5 The dipolenatureof a water molecule.The A FIGURE a partialcharge(aweakerchargethantheone symbolE represents proton) in the a Because of thedifference on an electron or of H andO, eachof the polarH-O bondsin electronegativities of eachof anddirections of the dipoles waterisa dipoleThesizes the netdrstance andamountof charqe the bondsdetermine separation, or dipolemoment,of the molecule

thermal energy at25 "C is approximately 0.6 kilocalorie per mole (kcal/mol), whereas the energy required to break the carbon-carbonsingle bond (C-C) in ethane is about 140 times larger (Figure 2-6). Consequently' at room temperature (25 "C), fewer than f. in 1012ethanemoleculesis broken into a pair of 'CH3 molecules,each containing an unpaired, nonbondingelectron(calleda radical). Covalent single bonds in biological moleculeshave energies similar to the energy of the C-C bond in ethane. Because more electrons are shared between atoms in double bonds, they require more energy to break than single bonds. For instance, it takes 84 kcal/mol to break a single C-O bond but 170 kcal/mol to break a C:O double bond' The most common double bonds in biological molecules are C:O, C:N, C:C, and P:O. In contrast, the energy required to break noncovalent interactions is only 1-5 kcalimol, much less than the bond energiesof covalent bonds (seeFigure 2-5). Indeed' noncovalent interactions are weak enough that they are constantly being formed and broken at room temperature' Although these interactions are weak and have a transient existenceat physiologicaltemperatures(25-37 "C)' multiple noncovalent interactions can, as we will see' act together to produce highly stable and specific associations between different parts of alatge molecule or between different macromolecules. Below, we review the four main types of noncovalent interactions and then consider their roles in the binding of biomoleculesto one another and to other molecules. C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S

35

> FIGURE 2-6 Relativeenergiesof covalent Noncovalent interactions bondsand noncovalentinteractions. Bond lonic energies aredefinedastheenergyrequired to breaka particular typeof linkageCovalent Hydrogen b o n d si ,n c l u d i nt g bonds h o s ef o r s i n g l (eC - C ) a n d double(C:C) carbon-carbon bonds.areoneto Thermal two powersof 10stronger thannoncovalent energy interactions Thelatteraresomewhat greater thanthethermalenergyof theenvironment at (25"C) Many normalroomtemperature processes biological arecoupled to the energy 0.24 released duringhydrolysis of a phosphoanhydride bondin ATP

Covalentbonds

Hydrolysisof ATP p h o s p h o a n h y d r i dbeo n d

c-c

C=C

240 kcal/mol

Increasingbond strength

lonic InteractionsAre Attractions between OppositelyChargedlons Ionic interactions result from the attraction of a positively charged ion-a cation-for a negatively charged ion-an anion. In sodium chloride (NaCl), for example, the bonding electron contributed by the sodium atom is completely transferred to the chlorine arom. (Figure 2-7a). Unlike covalent bonds, ionic interactionsdo not have fixed or specificgeometric orientations becausethe electrostatic field around an ion-its attraction for an opposite charge-is uniform in all directions. In solid NaCl, many ions pack tightly together in an alternating pattern to permit opposite charges to align and thus form a highly orderedcrystallinearray (saltcrystals)(Figure2-7b). tVhen solid saltsdissolvein water, the ions separatefrom one another and are stabilized by their interactionswith water molecules.In aqueoussolutions,simpleions of biological

(al

significance,suchas Na*, K*, Ca2*,Mg2* rand Cl-, are hydrated, surrounded by a stable shell of water moleculesheld in place by ionic interactions betweenthe central ion and the oppositely charged end of the water dipole (Figure 2-7c). Most ionic compounds dissolvereadily in water becausethe energy of hydration, the energy releasedwhen ions tightly bind water molecules,is greater than the lattice energy that stabilizesthe crystal structure. Parts or all of the aqueousbydration shell must be removed from ions when they directly interact with proteins. For example, water of hydration is lost when ions pass through protein pores in the cell membrane during nerve conduction. The relative strength of the interaction betweentwo ions, A- and C-, dependson the concentration of other ions in a solution. The higher the concentration of other ions (e.g., Na* and Cl-), the more opportunities A and C* have to

(b)

1,' -> ---.+

cl Cl

Donationof electron

.-#

s

-d q{-

+ Hro dissolving +-Crystallizing

-d

U A FIGURE 2-7 Electrostaticinteractionsof oppositelycharged dissolved in water,the ionsseparate andtheircharges, no longer ionsof salt(NaCl)in crystalsand in aqueoussolution.(a)In balanced by immediately adjacent ionsof opposite charge, are crystalline tablesalt,sodiumatomsarepositively charged stabilized ions(Na+) by interactions with polarwaterWatermolecules andthe dueto the lossof oneelectron each,whereas chloride ionsareheldtogetherby electrostatic atomsare interactions between the correspondingly (Ct I 5Ugainingoneelectron negatively charged charges on the ionandthe partialcharges on thewater'soxygenand each(b)Insolidform,ioniccompounds formneatlyordered hydrogen atomsIn aqueous arrays, solutions, all ionsaresurrounded by a or crystals, of tightlypackedionsin whichthe positive andnegatively hydration shellof watermolecules charged ionscounterbalance eachother.(c)Whenthe crvstals are

36

CHAPTER2 I

CHEMICALFOUNDATIONS

(c)

(b)

(a)

:O-H

I

H H

iuH tll O-H

I o-H

H

I :O-H

:O-H

:O-H

H

ll

H-O:

I :O-H

HH H-O

Water-water

: O-H

H-O: I

I

H HH

:o -i-rtr-

:O- CHs

Alcohol-water

I

:N-CHs H

Amine-water

H

H

o -i - o-

.H-O: I H

Peptide group-water

Ester group-water

2-8 Hydrogenbondingof water with itself and with A FIGURE in an outerelectrons Eachpairof nonbonding other compounds. atomin a hydrogen atomcanaccepta hydrogen oxygen or a nitrogen andtheaminogroupscanalsoformhydrogen bond Thehydroxyl forms bondswithwater.(a)In liquidwater,eachwatermolecule others, creating a dynamic hydrogen bondswithseveral transient

(b)Wateralsocanform molecules networkof hydrogen-bonded for the high accounting andamines, bondswith alcohols hydrogen (c)Thepeptide groupandestergroup, of thesecompounds solubility participate in commonly in manybiomolecules, whicharepresent bondswithwateror polargroupsin othermolecules. hydrogen

interact ionically with these other ions and thus the lower the energyrequired to break the interaction betweenA- and C*. As a result, increasingthe concentrationsof saltssuch as NaCl in a solution of biological moleculescan weaken and even disrupt the ionic interactions holding the biomolecules together.

O-H bonds within a singlewater molecule (Figure2-8a). The strengthof a hydrogen bond betweenwater molecules (approximately 5 kcal/mol) is much weaker than a covalent O-H bond (roughly 110 kcal/mol), although it is greater than that for many other hydrogen bonds in biological molecules(t-2 kcall mol). The extensivehydrogen bonding between water molecules accounts for many of the key properties of this compound' including its unusually high melting and boiling points and its ability to interact with (e.g.,dissolve)many other molecules. The solubility of unchargedsubstancesin an aqueousenvironment dependslargely on their ability to form hydrogen bondswith water.For instance,the hydroxyl group (-OH) in an alcohol (XCH2OH) and the amino group (-NH2) in amines (XCH2NH2) can form severalhydrogen bonds with water, enabling these moleculesto dissolvein water to high concentrations(Figure2-8b). In general,moleculeswith polar bonds that easilyform hydrogen bonds with water' as well as charged moleculesand ions that interact with the dipole in water, can readily dissolve in water; that is' they are hydrophilic (water liking). Many biological moleculescontain, in addition to hydroxyl and amino groups' peptide and ester groups, which form hydrogen bonds with water via otherwisenonbondedelectronson their carbonyl oxygens(Figure 2-8c). X-ray crystallography combined with computational analysispermits an accuratedepiction of the distribution of the outermostunbondedelectronsof atoms as well as the electrons in covalent bonds, as illustrated in Figure 2-9.

HydrogenBondsDeterminethe Water S o l u b i l i t yo f U n c h a r g e dM o l e c u l e s A hydrogen bond is the interaction of a partially positively charged hydrogen atom in a molecular dipole (e.9., water) with unpaired electronsfrom another atom, either in the same (intramolecular\or different Untermolecular)molecule.Normall5 a hydrogen atom forms a covalent bond with only one other atom. However, a hydrogen atom covalentlybonded to an electronegativedonor atom D may form an additional weak association,the hydrogen bond, with an acceptoratom A, which must have a nonbonding pair of electronsavailable for the interaction: D6--H6+

+ : 46- i-

D6--H6*:.....:: 46-

uyarolln uona The length of the covalent D-H bond is a bit longer than it would be if there were no hydrogen bond becausethe acceptor "pulls" the hydrogen away from the donor. An important feature of all hydrogen bonds is directionality. In the strongest hydrogen bonds, the donor atom, the hydrogen atom, and the acceptor atom all lie in a straight line. Nonlinear hydrogen bonds are weaker than linear ones; still, multiple nonlinear hydrogen bonds help to stabilize the three-dimensionalstructuresof many proteins. Hydrogen bonds are both longer and weaker than covalent bonds betweenthe same atoms. In water, for example, the distance between the nuclei of the hydrogen and oxygen atoms of adjacent, hydrogen-bondedmoleculesis about 0.27 nm, about twice the length of the covalent

Van der WaalsInteractions Are Causedby TransientDiPoles When any two atoms approach each other closel5 they create a weak, nonspecific attractive force called a van der Waals interaction. Thesenonspecificinteractionsresult from the momentary random fluctuations in the distribution of the electrons of any atom' which give rise to a transtent C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S

37

clouds, the atoms are said to be in van der $7aalscontact. The strength of the van der rWaalsinteraction is about 1 kcal/mol. weaker than typical hydrogen bonds and only slightly higher than the averagethermal energy of moleculesat 25 "C. Thus multiple van der Waals interactions,a van der'Waalsinteraction in conjunction with other noncovalent interactions, or both are required to significantly influencethe stability of inter- and intramolecular conracrs.

The HydrophobicEffectCausesNonpolar Moleculesto Adhere to One Another Becausenonpolar molecules do not contain charged groups, possessa dipole moment, or becomehydrated,they are insoluble or almost insolublein water; that is, they are hydrophobic (water fearing).The covalentbonds betweentwo carbon atoms and between carbon and hydrogen atoms are the most common nonpolar bonds in biological systems.Hydrocarbons-molecules made up only of carbon and hydrogen-are virtually insoluble in water. Large triacylglycerols(or triglycerides),which make up A FIGURE 2-9 Distributionof bondingand outer nonbonding animal fats and vegetableoils, also are insolublein water. As we electronsin the peptidegroup.Shownhereisa peptidebond will see later, the major portion of these moleculesconsistsof linkingtwo aminoacidswithina proteincalledcrambinTheblack long hydrocarbon chains. After being shaken in water, triacyllinesrepresent thecovalent bondsbetween atomsThered(negative) glycerols form a separatephase.A familiar example is the sepaandblue(positive) linesrepresent contours of chargedetermined ration of oil from the water-basedvinegar in an oil-and-vinegar usingx-raycrystallography andcomputational methods. Thegreater salad dressing. the numberof contourlines,the higherthe charge. Thehighdensity Nonpolar moleculesor nonpolar portions of molecules of redcontourlinesbetweenatomsrepresents the covalent bonds (shared tend to aggregatein water owing to a phenomenoncalled pairs). electron Thetwo setsof redcontourlinesemanating fromtheoxygen(O)andnotfallingon a covalent the hydrophobic effect. Becausewater molecules cannot (black bond line) represent thetwo pairsof nonbonded form hydrogen bonds with nonpolar substances,they tend electrons on the oxygen that areavailable to participate in hydrogen bondingThehighdensity to form "cages" of relatiuely rigid hydrogen-bonded of bluecontourlinesnearthe hydrogen (H)bondedto nitrogen (N) pentagons and hexagons around nonpolar molecules represents a partialpositive charge, indicating thatthisH canactasa donorin hydrogen bonding. C Jelsch etal, 2000,procNat,t. [From Acad SciUSA97.3171 CourtesyofM M Teeterl

unequal distribution of electrons. If two noncovalently bonded atoms are close enough together, electrons of one atom will perturb the electronsof the other. This perturbation generatesa transient dipole in the secondatom, and the two dipoles will attract each other weakly (Figure 2-10). Similarly, a polar covalent bond in one molecule will attract an oppositely oriented dipole in another. Van der'Waals interactions, involving either transiently induced or permanent electric dipoles, occur in all types of molecules, both polar and nonpolar. In particular, van der \Waalsinteractions are responsible for the cohesion between nonpolar moleculessuch as heptane,CH3-(CH2)s-CH:, that cannot form hydrogen bonds or ionic interactionswith 'Waals other molecules.The strength of van der interactions decreasesrapidly with increasingdistance;thus thesenoncovalent bonds can form only when atoms are quite closeto one another. However, if atoms get too close together,they become repelled by the negative charges of their electrons. \fhen the van der Waals attraction between rwo aroms exactly balances the repulsion between their two electron

38

CHAPTER2 |

cHEM|CALFOUNDAT|ONS

ii

k----t

Covalent radius ( 0 . 0 6n 2m )

van derWaals radius ( 0 . 1 4n m )

A FIGURE 2-10 Two oxygenmoleculesin van der Waals contact.ln thismodel,redindicates negative chargeandblue positive indicates chargeTransient dipoles in theelectron clouds of all atomsgiveriseto weakattractive forces,calledvander Waals interactions. Eachtypeof atomhasa characteristic vanderWaals radius at whichvanderWaalsinteractions withotheratomsare optimal. Because atomsrepeloneanotherif theyarecloseenough together for theirouterelectrons to overlap withoutbeingshared in a covalent bond,thevanderWaalsradiusisa measure of thesizeof theelectron cloudsurrounding an atom Thecovalent radrus indicated hereisfor thedoublebondof O:O; thesinqle-bond covalent radius of oxygenisslightly longer.

Nonpolar substance

Watersreleasedinto bulk solution H i g h l yo r d e r e d w a t e rm o l e c u l e s

\\ o o'o \.t

o

o Hydrophobic aggregation

@o Lowerentropy

Higherentropy

2-11 Schematic depictionof the hydrophobiceffect' FIGURE in molecules thatformaroundnonpolar Cagesof watermolecules in thesurrounding thanwatermolecules aremoreordered solution r esd u c et h s en u m b e r b u l kl i q u i dA g g r e g a t i o nf n o n p o l amr o l e c u l e resulting in a in highlyordered cages, involved of watermolecules compared state(nErht) moreenergetical lyf avorable higher-entropy, state(/eft) with the unaggregated

(Figure 2-1I, Ieft). This state is energeticallyunfavorable becauseit decreasesthe randomness(entropy) of the population of water molecules.(The role of entropy in chemic a l s y s t e m si s d i s c u s s e di n a l a t e r s e c t i o n . )I f n o n p o l a r moleculesin an aqueousenvironment aggregatewith their hydrophobic surfacesfacing each other, the hydrophobic s u r f a c e a r e a e x p o s e dt o w a t e r i s r e d u c e d ( F i g u r e 2 - 1 1 , right). As a consequence,lesswater is neededto form the cages surrounding the nonpolar molecules, and entropy increases(an energeticallymore favorable state)relative to the unaggregatedstate. In a sense,then, water squeezes the nonpolar moleculesinto spontaneouslyforming aggregates.Rather than constituting an attractive force such as in hydrogen bonds, the hydrophobic effect resultsfrom an a v o i d a n c e o f a n u n s t a b l e s t a t e ( e x t e n s i v ew a t e r c a g e s a r o u n d i n d i v i d u a ln o n p o l a r m o l e c u l e s ) . Nonpolar moleculescan also associate,albeit weakly' through van der Vaals interactions.The net result of the hydrophobic and van der Waals interactionsis a very powerful tendencyfor hydrophobic moleculesto interact with one another,not with water. Simply put, like dissolueslike' Polar moleculesdissolve in polar solvents such as water; nonpolar moleculesdissolvein nonpolar solventssuch as hexane.

y ediated M o l e c u l a rC o m p l e m e n t a r i t M PermitsTight, Interactions Noncovalent via H i g h l yS p e c i f i cB i n d i n go f B i o m o l e c u l e s Both inside and outside cells, ions and molecuiesare constantly bumping into one another.The greaterthe number of copiesof any two types of moleculesper unit volume (i'e.,

the higher their concentration)' the more likely they are to encounter one another.Vhen two moleculesencounter each other, they most likely will simply bounce apart becausethe noncovalent interactions that would bind them together are weak and have a transient existenceat physiological temperatures. However, moleculesthat exhibit molecular complementarity, a lock-and-key kind of fit between their shapes, form multiple noncharges,or other physical properties,can '$7hen two such struccovalent interactions at close range. turally complementarymoleculesbump into each other' they can bind (stick) together. Figure 2-12 tllustrates how multiple, different weak bonds can bind two proteins together. Almost any other arrangement of the same groups on the two surfaceswould not allow the molecules to bind so tightly. Such multiple, specificinteractions betweencomplementary regions within a orotein molecule allow it to fold into a unique threedimensionalshape(Chapter 3) and hold the two chains of DNA togetherin a double helix (Chapter 4)' Similar interactionsunderliethe associationof groups of more than two molecules into multimolecular complexes, leading to formation of muscle fibers' to the gluelike associationsbetween cells in solid tissues,and to numerous other cellular structures. Depending on the number and strength of the noncovalent interactions betweenthe two moleculesand on their environment, their binding may be tight (strong) or loose (weak) and, as a consequence,either long lasting or transient.The higher the affinity of two moleculesfor each other, the better the molecular "fit" between them, the more noncovalent interactionscan form, and the tighter they can bind

-cH3 | -CH3 H3( ,

_cH3 H3C_

ProteinB ProteinA Stable complex

ProteinC ProteinA Less stable comPlex

andthe bindingof complementarity 2-12 Molecular FIGURE Thecomplementary proteinsvia multiplenoncovalentinteractions. of two proteinsurfaces polarity, andhydrophobicity charges, shapes, produce a whichin combination weakinteractions, permrt multiple from molecular deviations Because binding. tight and stronginteraction surface a parlicular binding, weaken substantially complementarity canbindtightlyto onlyoneor a usually of anygivenbiomolecule region of the Thecomplementarity of othermolecules verylimitednumber much more bind permits to them left on the two proteinmolecules proteins on therighi tightlythanthetwo noncomplementary INTERACTIONS T C O V A L E N TB O N D SA N D N O N C O V A L E N T

39

together.An important quantitative measureof affinity is the binding dissociationconstant K6, describedlater. As we discussin Chapter 3, nearly all the chemicalreactionsthat occur in cellsalso dependon the binding propertiesof enzymes.Theseproteins not only speedup, or cataIyze, reactions but also do so with a high degree of specificity, a reflection of their ability to bind tightly to only one or a few related molecules. The specificity of intermolecularinteractionsand reactions,which dependson molecular complementarity,is essentialfor many processes critical to life.

Covalent Bonds and Noncovalent Interactions Covalent bonds, which bind the atoms composing a olecule in a fixed orientation, consist of pairs of electrons shared by two atoms. They are stable in biological systems because the relatively high energies required to break them (50-200 kcal/mol) are much larger than the thermal kinetic energyavailableat room (25 .C) or body (37 "C) temperatures. r Many moleculesin cells contain at least one asymmetric carbon atom, which is bonded to four dissimilar atoms. Such moleculescan exist as optical isomers (mirror images),designatedn and r (seeFigure 2-4), which have different biological activities.In biological systems, nearly all sugarsare D isomers,whereasnearly all amino acids are L isomers. trons may be sharedequally or unequally in covalent Atoms that differ in electronegativityform polar cobonds in which the bonding electronsare distributed unequally. One end of a polar bond has a partial positive charge and the other end has a partial negativechaige (see Figure2-5). r Noncovalent interactions between atoms are considerably weaker than covalent bonds, with bond energlesranging from about 1-5 kcal/mol (seeFigure 2-6). r Four main types of noncovalent interactions occur in biological systems:ionic bonds, hydrogen bonds, van der \faals interactions, and interactions due to the hvdroohobic effect. r Ionic bonds result from the electrostatic attraction between the positive and negativechargesof ions. In aqueous solutions, all cations and anions are surrounded by a shell of bound water molecules(seeFigure Z-7c).Increasingthe salt (e.g.,NaCl) concentrarionof a solutioncan weakenthe relative strength of and even break the ionic bonds between biomolecules. hydrogen bond, a hydrogen atom covalently bonded electronegativeatom associateswith an accepror whose nonbonding electrons attract the hydrogen gure 2-8).

40

CHAPTER2 |

cHEMTCALFOUNDATTONS

r Weak and relatively nonspecificvan der'Sfaalsinteractions are createdwheneverany two atoms approach each other closely. They result from the attraction between transient dipoles associatedwith all molecules (seeFigure2-10). r In an aqueousenvironment, nonpolar moleculesor nonpolar portions of larger molecules are driven together by the hydrophobic effect, thereby reducing the exrent of their direct contactwith water molecules(seeFigure2-11). r Molecular complementarity is the lock-and-key fit between moleculeswhose shapes,charges,and other physical properties are complementary.Multiple noncovalent interactions can form betweencomplementary molecules,causing them to bind tightly (seeFigure2-12),but not between moleculesthat are not complementary. r The high degreeof binding specificity that results from molecular complementarity is one of the features that underlies intermolecular interactions and thus is essentialfor many processescritical to life.

E

Chemical BuildingBlocksof Cells

A common theme in biology is the construction of large molecules (macromolecules)and structures by the covalent or noncovalent associationof many similar or identical smaller molecules.The three most abundant classesof the critically important biological macromolecules-proteins, nucleic acids, and polysaccharides-are all polymers composed of multiple covalently linked building block small molecules,or monomers (Figure 2-13). Proteins are linear polymers containing 10 to severalthousand amino acids linked by peptide bonds. Nucleic acids are linear polymers containing hundreds to millions of nucleotides linked by phosphodiester bonds. Polysaccharidesare linear or branched polymers of monosaccharides(sugars)such as glucose linked by glycosidic bonds. Although the actual mechanismsby which covalent bonds betweenmonomers form are complex and will be discussedlater, the formation of a covalent bond between two monomer molecules usually involves the net loss of a hydrogen (H) from one monomer and a hydroxyl (OH) from the other monomer-or the net loss of one water-and can therefore be thought of as a debydration reaction. These bonds are stable under normal biological conditions (e.g., 37"C, neutral pH), and so thesebiopolymers are stable and can perform a wide variety of jobs in cells (store information, catalyze chemical reactions, serve as structural elements in defining cell shapeand movement, etc.). Macromolecular structures can also be assembledusing noncovalent interactions. The macromolecular two-layered (bilayer) structure of cellular membranesis built uo bv the noncovalent assemblyof many thousandsof small molecules called phospholipids (seeFigure 2-13).In this chapter, we will focus on the characteristicsof the monomeric chemical

POLYMERS

MONOMERS

ttl

H2N-C -C-OH I R

HHOHHOHHOHHO

HO

HO +

ttl

|

I R

I

l

ll,l

L ll

N-C-CrN-C-C-OH I I | R* R3 I

peptidebond

Amino acid

o tl

| ll I

| ll |

H-N-C-ciN-C-C | I lt R2 R, l"

oH

H-N-C-C-

PolYPePtide

B L

Base

?1,

HO-P-O

I

HO-P-OJs,

.OH

6Nucleic acid

Nucleotide

glYcosidicbond

Polysaccharide

Monosaccharide

I Hyoropr'irt. h e a dg r o u p J

Phospholipid

2-13 Overviewof the cell'sprincipalchemical FIGURE threemajortypesof biological buifdingblocks.(Top)The of bythe polymerization assembled are each macromolecules (monomers) type:proteins of a particular smallmolecules multiple acidsfromnucleotides 3),nucleic fromaminoacids(Chapter building blocks-amino acids,nucleotides,sugars'and phospholipids. The structure, function, and assemblyof proteins, nucleic acids, polysaccharides,and biomembranes are discussedin subsequentchapters.

A m i n o A c i d sD i f f e r i n gO n l y i n T h e i r SideChainsComposeProteins The monomeric building blocks of proteins ate 20 amino acids, which when incorporated into a protein polymer are sometimescalled residues.All amino acidshave a characteristic structureconsistingof a central alpha (cr)carbon atom (C') bonded to four different chemical groups: an amino (NHz)

(sugars)Each from monosaccharides (Chapter 4), andpolysaccharides whose reaction polymer by a linkedintothe iscovalently monomer (Boftom) In (dehydration) molecule net resultis lossof a water into a bilayer assemble noncovalently monomers phospholipid contrast, (chapter10) membranes of allcellular whichformsthebasis structure, group, a carboxylic acid or carboxyl (COOH) group (hence amino acid),a hydrogen (H) atom, and one variable Ih. *-. group, called a side chain or R group' Becausethe ct carbon i.-nali amino acids except glycine is asymmetric,thesemolecules can exist in two mirror-image forms called by convention the I (dextro) and the r- (levo) isomers (seeFigure 2-4)' The two isomerscannot be interconverted(one made identical with the other) without breaking and then re-forming a chemical bond in one of them. !7ith rare exceptions, only the I- forms of amino acids are found in proteins' To understand the three-dimensional structures and functions of proteins' discussedin detail in Chapter 3, you must be familiar with some of the distinctive properties of

B U I L D I N GB L O C K SO F C E L L S CHEMICAL

41

H Y D R O P H O BA I CM I N OA C I D S

cooI

*H3N-C-H I cHs

coo-

+ H . N - c I- H

"l

CH HsC

cooI

+H3N-c-H

CHs

cooI

cooI

+H3N-c-H

cooI

cooI

'H"N-C-H

-l

cooI

*H.N-C-H

-l

H-C - CH.

CH,

CH,

CH,

CH

C:CH

t"

lcHs

t-

HaC

t-

CHs

Lr*t OH

\/ Alanine ( A l ao r A l

Valine (Val orVl

lsoleucine ( l l eo r l l

Leucine (Leu or Ll

H Y D R O P H I LA I CM I N OA C I D S

Methionine (Met or M)

Acidic amino acids

cooI

coo-

I +H3N-c-H

I CHt

CH,

l-

NHs*

Lysine (Lys or Kl

CH,

t-

CH, lCH,

tCH, t-

Glycine (Gty or G)

.

H-C-OH

coo-

oH

cHs

Aspartate (Asp or Dl

Serine (Ser or Sl

Threonine (Thr orT)

I

'H"N-C-H

-l

CH,

IcH, )' cooGlutamate ( G l uo r E )

proline (pro or p)

amino acids, which are determined in part by their side chains. You need not memorize the detailed structure of each type of side chain to understandhow proteins work because amino acids can be classified into several broad categories basedon the size,shape,charge,hydrophobicity (a measure of water solubility), and chemical reactivity of the side chains (Figure 2-74). However, you should be familiar with the generalproperties of each category. Amino acids with nonpolar side chains are hydrophobic and so poorly soluble in water. The larger the nonpolar side chain, the more hydrophobic-less water soluble-the amino acid. The noncyclic side chains of alanine, ualine, leucine, and. isoleucine(calledaliphatic),as well as methionine,consisren_ tirely of hydrocarbons, except for the one sulfur arom in me_ thionine, and all are nonpolar. phenylalanine, tyrosine, and. 42

r"l t-i

coo+H3N-C-H

coo-

l-

NH i C: NHr+ lNHz Arginine (Argor Rl

SPECIALAMINO ACIDS

Cysteine (Cys or C)

cootl

CH,

CH,

Tryptophan (TrporW)

Polar amino acids with uncharged R groups

*H3N-C-H

I 'H"N-C-H -l

lCH,

Tyrosine (TyrorYl

+H.N-c-H

-l

coo-

Phenylalanine (Phe or Fl

c H A p r E R 2| c H E M t c A L F o u N D A T t o N s

FIGURE 2-14 The 20 commonaminoacidsusedto build proteins.Thesidechain(Rgroup;red)determines thecharacteristic properties of eachaminoacidandisthe basisfor grouping amino acidsintothreemaincategories: hydrophobic, hydrophilic, and special. Shownarethe ionized formsthatexistat the pH(=7)of the cytosolIn parentheses arethethree-letter andone-letter abbreviations for eachaminoacid.

tryptophan have large, bulky aromatic side chains. In later chapters, we will see in detail how these hydrophobic side chains under the influence of the hydrophobic effeci often pack in the interior of proteins or line the surfacesof proteins that are embeddedwithin hydrophobic regions of biomembranes. Amino acids with polar side chains are hydrophilic; the most hydrophilic of theseamino acids is the subsetwith side chains that are charged (ionized) at the pH typical of biological fluids (=7)-both inside and outside the cell (seeSectio-n 2.3). Arginine and lysine have positively charged side chains and are called basic amino acids;aspartic acid and glutamic acid have negatively charged side chains due to the carboxylic acid groups in their side chains (their charged forms are called aspartate and glutamate) and are called acidic. A fifth amino acid, histidine, has a side chain containing a ring

with two nitrogens, called imidazole, which can shift from being positively charged to uncharged depending on small changesin the acidity of its environment:

I

A c e t y ll y s i n e

C H 3 - C - N - C H 2 - C H 2 - C H 2 - C H " - C H - C-Ol O -

NH.-

o - o - Ptl o - c H 2 - c H - c o o -

ll Phosphoserine

ll O

NH"OH

pH 5.8

PH 7.8

The activities of many proteins are modulated by shifts in environmental acidity through protonation or deprotonation of histidine side chains. Asparagine and glutamine are uncharged but have polar side chains containing amide groups with extensivehydrogen-bondingcapacities'Similarl5 serine and threonine are unchargedbut have polar hydroxyl groups, which also participate in hydrogen bonds with other polar molecules. Lastl5 cysteine,glycine, and proline exhibit specialroles in proteins becauseof the unique properties of their side chains. The side chain of cysteine contains a reactive sulfhydryl group (-SH), which can oxidize to form a covato a secondcysteine: lent disulfide bond (-S-S-/

I I- H

I

I H,9-9H

3-Hydroxyproline

II H2C\ * N //CH-COOH, HC:C-CH2-CH-COOtrl NH.H3C-N-C-.N

3-Methylhistidine

H

-ooc y-Carboxyglutamate

-OOC

c H - c H' "l - c H - c o o ilH.*

of aminoacidside 2-15 €ommonmodifications A FIGURE others andnumerous residues chainsin proteins.Thesemodified groups(red)to theamino chemical of various areformedby addition chain of a polypeptide acidsidechainsduringor aftersynthesis

N-H

N-H

I

H-C-CH2-

SH+ HS-CH2q

ll

C:O

C:O

reveals that they contain upward of 100 different amino acids.Chemical modifications of the amino acids account for

ll

tl H-N

r''l ll

H-C-

ll O:C ll

C H 2- S - S -

N-H C H 2- c - H

C:O

Regionswithin a singleprotein chain (intramolecular) or in separatechains (intermolecular) sometimesare crosslinked through disulfide bonds. Disulfide bonds stabilizethe folded structure of some proteins. The smallestamino acid, glycine, has a singlehydrogen atom as its R group. Its small sizeallows it to fit into tight spaces.Unlike the other common amino acids, the side chain of proline bends around to form a ring by covalently bonding to the nitrogen atom (amino group) attached to the Co. As a result' proline is very rigid and createsa fixed kink in a protein chain, limiting how a protein can fold in the region of proline residues. Some amino acids are more abundant in proteins than others. Cysteine,tryptophan, and methionine are rare amino acids: together they constitute approximately 5 percent of the amino acids in a protein. Four amino acids-leucine, serine, lysine, and glutamic acid-are the most abundant amino acids, totaling 32 percent of all the amino acid residuesin a typical protein. However, the amino acid composition of proteins can vary widely from thesevalues. Although cellsusethe 20 amino acidsshown in Figure2-14 inthe initial synthesisof proteins' analysisof cellular proteins

and plants but less studied-perhaps becauseof the relative insta-bilityof phosphorylatedhistidine-and apparently rare in mammals.ltt. iia. chains of asparagine,serine,and threonine are sitesfor glycosylation,the attachmentof linear and branched carbohydrate chains. Many secretedproteins and membrane proteins contain glycosylated residues' Other amino acid modifications found in selectedproteins include the hydroxylation of proline and lysine residuesin collagen (Chapter 19), the methylation of histidine residuesin memb."ne rec.ptors' and the 7 carboxylation of glutamate in blood-clotting factors such as prothrombin' Acetylation, addition of an acetyl group' to the amino gro,rp oi the N-terminal residue, is the most common form acid chemical modification, affecting an estimated 6f "-ino 80 percentof all proteins:

o lllll

RO

cH3-c- r y- g- cll HH Acetylated N-terminus OF CELLS B U I L D I N GB L O C K S CHEMICAL

O

43

This modification may play an important role in controlling the life span of proteins within cells becausenonacetylateJ proteins are rapidly degraded.

PURINES NH,

O

ltl

Five Different Nucleotides A r e U s e dt o B u i l d N u c l e i cA c i d s Two types of chemically similar nucleic acids, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). are the principal genetic-information-carryingmoleculesof the cell. The monomers from which DNA and RNA polymers are built, called nucleotides,all have a common structure: a phosphategroup linked by a phosphoesterbond to a pentose (a five-carbon sugar molecule) that in turn is linked io a nitrogen- and carbon-containing ring structure commonly referred to as a base (Figure 2-16a).In RNA, the pentoseis ribose; in DNA, it is deoxyribose that at posirion 2, has a proton rather than the hydroxyl group at that site in ribose

HH Adenine (A)

(Gl Guanine

PYRIMIDINES NH,

il

tru/?--cH

r"^tl

.r'"'ii" vl H Cytosine (C)

A FIGURE 2-17 Chemical structuresof the principalbasesin nucleicacids.ln nucleic acidsandnucleotides, nitrogen 9 of purines andnitrogen1 of pyrimidines (red)arebondedto the 1, carbonof riboseor deoxyribose U isonlyin RNA,andT isonlyin DNA Both R N Aa n dD N Ac o n t a iA n , G ,a n dC .

position 1 of a pyrimidine (N1). The acidic character of

which form ionic interactions with the negatively charged phosphates. Cells and extracellular fluids in organismscontain small concentrationsof nucleosides,combinations of a baseand a sugar without a phosphate.Nucleotides are nucleosidesthat have one, two, or three phosphate groups esterified at the 5' hydroxyl. Nucleoside monophosphateshave a single esterified phosphate (seeFigure 2-16a); nucleoside diphosphates contain a pyrophosphategroup:

oo

-o-l-o-A-o-

(a) Adenine

Nhz l

(b)

'-c'

5',

N ; , 6 ; -c

\

HOCH2

daH HC.1 : 1c s,/ -'N' N

o - o - P - O - c H5',

o-

5',

HOCH2

Phosphate 2'

OH

H

Ribose Adenosine 5'-monophosphate (AMP}

2-Deoxyribose

FIGURE 2-15 Commonstructureof nucleotides. (a)Adenosine 5' -monophosphate (AMp),a nucleotide present in RNAByconvention, thecarbonatomsof the pentose sugarin nucleotides arenumbered with primes. In naturalnucleotides, the 1, carbonisjoinedby a B linkage to the base(inthiscaseadenine); boththe base(blue)and the phosphate on the 5, hydroxyl (red)extendabovethe planeof the sugarring.(b)Ribose anddeoxyribose, the pentoses in RNAand DNA,respectively 44

.

c H A p r E R 2| c H E M t c A L F o u N D A T t o N S

o-

o-

Pyrophosphate

and nucleosidetriphosphateshave a third phosphate.Table 2-3 lists the names of the nucleosidesand nucleotidesin nucleic acids and the various forms of nucleosidephosphates.The nucleosidetriphosphatesare used in the synthesisof nucleic acids,which we cover in Chapter 4. Among their other functions in the cell, GTP participares in intracellular signaling a_ndacts as an energy reservoir,particularly in protein synthesis, and ATP, discussedlater in this chapter, is the most widely usedbiologicalenergycarrier.

M o n o s a c c h a r i d eJso i n e db y G l y c o s i d i B c onds Form Linearand Branchedpolysaccharides The building blocks of the polysaccharidesare rhe simple sugars, or monosaccharides.Monosaccharides are carbohy_ drates,which are literally covalently bonded combinationsof carbon and water in a one-to-one ratio (CH2O),, where n e q u a l s3 , 4 , 5 , 6 , o r 7 . H e x o s e s( n : 6 ) a n d p e n t o s e (sn : 5 ) are the most common monosaccharides. All monosaccharides

GUANINT(G}

ADENINE(A)

nNe Jin

t,,'o^o

innNe f Iino*e

uBACrr(lJ) THYMINE(I)

cYT0srNt(q

Adenosine

Guanosine

Cytidine

Uridine

Deoxyadenosine

Deoxyguanosine

Deoxycytidine

DeoxYthYmidine

Adenylate

Guanylate

Cytidylate

UridYlate

Deoxycytidylate

Deoxythymidylate

Deoxyadenylate

DeoxyguanYlate

Nucleoside monophosphates

AMP

GMP

CMP

UMP

Nucieosidediphosphates

ADP

GDP

CDP

UDP

Nucleoside triphosphates

ATP

GTP

CTP

UTP

Deoxynucleoside mono-, di-, and triphosphates

dAMP,etc.

dGMP,etc'

dCMP, etc

dTMP' etc.

contain hydroxyl (-OH) a keto group:

oo

- c t- icl - H lll

Aldehyde

groups and either an aldehyde or

Htt,

6

(-rQ

n-J'-oH

H

- c -Lcf - c - l

1

0) occurs spontaneously(AG < 0). An endothermic reaction (AH > 0) will occur spontaneouslyif AS increasesenough so that the T AS term can overcome the positive AH. Many biological reactions lead to an increase in order and thus a decreasein entropy (AS < 0). An obvious example is the reaction that links amino acids to form a protein. A solution of protein molecules has a lower entropy than does a solution of the same amino acids unlinked because the free movementof any amino acid in a protein is restricted when it is bound into a long chain. Often cells compensate for decreases in entropy by "coupling" suchsynthetic,entropylowering reactions with independent reactions that have a very highly negariveAG (seebelow). In this way cells can convert sources of energy in their environment into the building of highly organized structures and metabolic pathways that are essentialfor life. The actual changein free energy AG during a reacrion is influenced by temperature, pressure,and the initial concentrations of reactants and products and usually differs from AG''. Most biologicalreactions-like othersthat take place in aqueoussolutions-also are affected by the pH of the so'We lurion. can estimate free-energy changes for different temperaturesand initial concentrationsusing the equation AG:

AC.'-r RTln Q:

AC"'+ RTln

fproducts] Q-7) I reacrantsl

where R is the gas constanrof 1.987 call(degree.mol), T is the temperature (in degreesKelvin), and Q is the initial rctio of products to reactants. For a reaction A + B == C. in which two molecules combine to form a third, e in Equation2-7 equalstcl/lAltBl.In this case,an increasein the initial concentration of either [A] or [B] will result in a larger negative value for AG and thus drive the reaction toward more formation of C. Regardlessof the AG"' for a particular biochemicalreaction, it will proceed spontaneouslywithin cells only if AG is negative, given the intracellular concentrations of reactants and products. For example, the conversion of glyceraldehyde 3-phosphate (G3P) to dihydroxyacetonephosphate (DHAP), two inrermediatesin the breakdownof glucose, .^DHAP G3P . has a AGo' of -1840 caVmol.If the initial concentrationsof G3P and DHAP are equal, then AG : AGo' becauseRT ln 1 : 0; in this situation, the reversiblereaction G3P : DHAP will proceedspontaneouslyin the direction of DHAp formation until equilibrium is reached. However, if the initial 56

CHAPTER2 I

CHEMICALFOUNDATIONS

[DHAP] is 0.1 M and the initial [G3P] is 0.001 M, with other conditions standard,then Q in Equation 2-7 equals0.1/0.001 : 100, giving a AG of *887 caVmol.Under theseconditions, the reaction will proceed in the direction of formation of G3P. The AG for a reaction is independent of the reaction rare. Indeed, under usual physiological conditions, few if any of the biochemical reactions neededto sustain life would occur without some mechanism for increasing reaction rates. As we describe below and in more detail in Chapter 3, the rates of reactions in biological systemsare usually determinedby the activity of enzymes,the protein catalyststhat acceleratethe formation of products from reactantswithout altering the value of AG.

The Ad' of a ReactionCan Be Galculatedfrom lts K.o A chemical mixture at equilibrium is in a stable state of minimal free energy.For a system at equilibrium (AG : 0, Q : K"o), we can write AGo': -2.3RTlogK.o: -1362logK.o

Q-8)

under standard conditions (note the changeto base 10 logarithms). Thus if we determinethe concentrationsof reactants and products at equilibrium (i.e., the K.o), we can calculate the value of AGo'. For example,the K.o for the inrerconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate (G3P : DHAP) is 22.2 under standard conditions. Substitutingthis value into Equation 2-8, we can easily calculatethe AGo' for this reaction as - 1840 callmol. By rearranging Equation 2-8 and taking the antilogarithm, we obtain Keq :

10-(aG"'/2'3RT)

(2-e)

From this expression,it is clear that if AGo' is negative,the exponent will be positive and henceK.o will be greater than 1. Therefore at equilibrium rhere will be more products than reactants; in other words, the formation of products from reactants is favored. Conversely,if AG'' is positive, the exponent will be negativeand K.o will be lessthan 1.

The Rateof a ReactionDependson the Activation EnergyNecessary to Energize the Reactantsinto a TransitionState As a chemical reaction proceeds, reactants approach each otherl some bonds begin to form while others begin to break. One way to think of the state of the moleculesduring this transition is that there are strains in the electronic configurations of the atoms and their bonds. In order for the collection of atoms to move from the relatively stable state of the reactants to this intermediate state during the reaction, an introduction of energy is necessary.This is illustrated in the reaction energy diagram in Figure 2-30. Thus the collection of atoms is transiently in a higher-energysrare at some point during the course of the reaction. The state during a chemicalreaction at which the systemis at its highest energy level is called the transition state or transition-

+

II

Transitionstate (uncatalyzed)

Transitionstate (catalyzed) 6 0) 0) 0) LI

Products

Progressof reaction----> 2-30 Activationenergy of uncatalyzedand A FIGURE reactionpathway catalyzedchemicalreactions.Thishypothetical (blue)depicts proceeds A thechanges in freeenergyG asa reaction reaction willtakeplacespontaneously if thefreeenergy(G)of the (AG< 0). However, products all islessthanthatof the reactants proceed reactions throughone(shownhere)or morehighchemical isinversely states, andthe rateof a reaction energytransition proportional in to theactivation energy(AG*),whichisthe difference the reactants andthetransition stateIn a freeenergybetween (red),thefreeenergies of the reactants and reaction catalyzed products stateis areunchanged but thefreeenergyof thetransition lowered, thusincreasing thevelocity of the reaction

state intermediate.The energy neededto excite the reactants to this higher-energystate is called the activation energy of the reaction. The activation energy is usually representedby AGt, analogousto the representationof the changein Gibbs free energy (AG) already discussed.From the transition state, the collection of atoms can either releaseenergy as the reaction products are formed or releaseenergy as the atoms go "backward" and re-form the original reactants.The velocity (V) at which products are generated from reactants during the reaction under a given set of conditions (temperature, pressure,reactant concentrations)will depend on the concentration of material in the transition state, which in turn will depend on the activation energy and the characteristic rate constant (z) at which the transition state ls converted to products. The higher the activation energy, the lower the fraction of reactantsthat reach the transition state and the slower the overall rate of the reaction. The relationship between the concentration of reactants,z and V is (AG+/2 3RT) v : u lreactants]x 10 From this equation, we can seethat lowering the activation energy-that is, decreasingthe free energy of the transition stateAG+-leads to an accelerationof the overall reactionrate V. A reduction in AGt of 1.36 kcal/mol leads to a tenfold increasein the rate of the reaction, whereas a 2.72 kcal/mol reduction increasesthe rate 100-fold. Thus relatively small changesin AGt can lead to large changesin the overall rate of the reaction.

Catalystssuch as enzymes(Chapter 3) acceleratereaction rates by lowering the relative energy of the transition state and so the activation energy (seeFigure 2-30). The relative energiesof reactantsand products will determineif a reaction is thermodynamically favorable (negativeAG), whereas the activation energy will determine how rapidly products form (reaction kinetics). Thermodynamically favorable reactions will not occur if the activation energiesare too high.

L i f e D e p e n d so n t h e C o u p l i n go f U n f a v o r a b l e s ith Energetically C h e m i c aR l e a c t i o nw FavorableReactions unfavorable(AG > 0) Many processesin cellsareenergetically include the synExamples and will not proceed spontaneously. of a substance transport nucleotides and thesis of DNA from a higher concenlower to from a plasma membrane acrossthe or endergonic, energy-requiring, out an can carry tration. Cells reaction (AGr > 0) by coupling it to an energy-releasing,or exergonic, reaction (AGz < 0) if the sum of the two reactionshas an overall net negativeAG. Suppose,for example,that the reactionA = B + X has a AG of + 5 kcaUmoland that the reactionX = Y + Zhas a L,G of -10 kcal/mol:

(1) A

B+X

AG:+Skcal/mol

( 2\ x

Y+Z

AG:-10kcal/mol

Sum: A.

^B+Y+Z

AG'' :-5kcal/mol

In the absenceof the secondreaction' there would be much more A than B at equilibrium. However, becausethe conversion of X to Y + Z is such a favorable reaction, it will pull the first process toward the formation of B and the consumption of A. Energetically unfavorable reactions in cells often are coupled to the energy-releasinghydrolysis of ATP' as we discussnext.

Hydrolysisof ATPReleasesSubstantialFree E n e r g ya n d D r i v e sM a n y C e l l u l a rP r o c e s s e s In almost all organisms, adenosinetriphosphate, or AIP, is the most important molecule for capturing' transiently storing, and subsequentlytransferring energy to perform work (e.g.,biosynthesis,mechanicalmotion). The useful energyin an ATP molecule is contained in phosphoanhydride bonds, which are covalent bonds formed from the condensationof two moleculesof phosphateby the loss of water: OO

ti o- -i-on + Ho-P-o- . * ll

o-

o-

o

llll o--P-O-?-OI I o o-

+ H2O

B I O C H E M I C AELN E R G E T I C S

57

pH. During synthesis of ATP, a large input of energy is required to force the negative charges in ADP and P1 together.ConverselSmuch energy is releasedwhen ATP is hy*zc'-a-N rtl \u Phosphoanhydride bonds drolyzed to ADP and P;. In comparison, formation of the HC__*,rC_;1 phosphoester o bond between an unchargedhydroxyl in glycto to - o - P -i ol |- lL|Pl - O _ | . P - O erol and P1requires less energy, and less energy is released cH2-owhen this bond is hydrolyzed. tll oo-o H Cells have evolved protein-mediated mechanismsfor transferringthe free energyreleasedby hydrolysis of phosphoanhydride bonds to other molecules, thereby driving HO OH reactions that would otherwise be energeticallyunfavorAdenosine triphosphate able. For example, if the AG for the reaction B + C -+ D is positive but less than the AG for hydrolysis of ATP, the reA FIGURE 2-31 Adenosinetriphosphate(ATP).Thetwo phosphoanhydride bonds(red)in ATP, whichlinkthethreephosphate action can be driven to the right by coupling it to hydrolygroups, eachhasa AG'of about-7 3 kcal/mol for hydrolysis sis of the terminal phosphoanhydride bond in ATP. In one Hydrolysis of thesebonds,especially theterminal one,rsrnesource common mechanism of such energy coupling, some of the of energythatdrivesmanyenergy-requiring reactions in biological energy stored in this phosphoanhydride bond is transferred systems. to one of the reactants by breaking the bond in ATP and forming a covalent bond between the releasedphosphate group and one of the reactants.The phosphorylatedinterAn ATP molecule has two key phosphoanhydride (also mediate generatedin this way can then react with C to calledphosphodiester)bonds (Figure 2-31). Hydrolysis of a form D * P; in a reaction that has a negativeAG: phosphoanhydride bond (-) in each of the following reactions has a highly negativeAG'' of about -7.3 kcal/mol: NH"

B+ATPTB-p+ADP Ap-p-p + H2O ----+ Ap-p + Pi + H+ (ATP) Ap-p-p+H2O(ATP)

B-p+C-+D+P;

(ADP) Ap+PPi+H+ (AMP)

The overallreaction B+C+ATP:-D+ADP+P.

Ap-p + H2O ----+ Ap + P; * H+ (ADP)

(AMP)

In thesereactions,Pi standsfor inorganic phosphate(pO+, ) and PP;for inorganic pyrophosphate,two phosphategroups linked by a phosphoanhydride bond. As the top two reactions show, the removal of a phosphate or a pyrophosphate group from ATP leaves adenosinediphosphate (ADp) or adenosinemonophosphate (AMP), respecively. A phosphoanhydride bond or other high-energy bond (commonly denoted by -) is not intrinsically different from other covalent bonds. High-energy bonds simply releaseespecially large amounts of energywhen broken by addition of water (hydrolyzed). For instance,the AG"' for hydrolysis of a phosphoanhydride bond in ATP (-7.3 kcal/mol) is more than three times the AGo' for hydrolysis of the phosphoester bond (red) in glycerol3-phosphate(-Z.2kcallmol):

ooH Ho-p-o-cHr-lH - cH2oH I

U Glycerol3-phosphate

A principal reason for this differenceis that ATp and its hydrolysis products ADP and P1are highly charged at neutral

58

CHAPTER2 I

CHEMICALFOUNDATIONS

is energeticallyfavorable (AG < 0). An alternative mechanism of energy coupling is to use the energy releasedby ATP hydrolysis to changethe conformation of the molecule to an "energy-rich" stressedstate. In turn, the energy stored as conformational stresscan be released as the molecule "relaxes" back into its unstressed conformation. If this relaxation processcan be mechanistically coupled to another reaction, the releasedenergycan be harnessedto drive important cellular processes. As with many biosynthetic reactions,transport of molecules into or out of the cell often has a positive AG and thus requires an input of energy to proceed. Such simple transport reactions do not directly involve the making or breaking of covalent bonds; thus the AGo' is 0. In the case of a substancemoving into a cell, Equation 2-7 becomes

AG: Rrt"+."9*

(2-10)

where [C1"] is the initial concentration of the substanceinside the cell and [Co",] is its concentration outside the cell. 'We can see from Equation 2-10 that AG is positive for transport of a substanceinto a cell against its concentration gradient (when [Cr"] > [C"",]); the energy to drive such "uphill" transport often is supplied by the hydrolysis of

ATP. Conversely,when a substancemoves down its concentration gradient ([C",,] > [C,"] ), AG is negative. Such "downhill" transport releasesenergythat can be coupledto an energy-requiringreaction, say, the movement of another substanceuphill acrossa membrane or the synthesisof ATP itself (seeChapters 1I and 12).

ATPls GeneratedDuring Photosynthesis and Respiration Clearlg to continue functioning, cells must constantly replenish their ATP supply. In nearly all cells,the initial energysource whose energyis ultimately transformed into the phosphoanhydride bondsof ATP and bonds in other compoundsis sunlight. In photosynthesis,plants and certain microorganismscan trap the energy in light and use it to synthesizeAIP from ADP and P1.Much of the ATP produced in photosynthesisis hydrolyzed to provide energyfor the conversionof carbon dioxide to sixcarbon sugars,a processcalledcarbon fixation: ATP ADP + P. 6co2 + 6 Hro V c 6 H 1 2 o+6 o 0 2 In animals, the free energy in sugars and other molecules derived from food is releasedin the processof respiration. All synthesisof ATP in animal cells and in nonphotosynthetic microorganismsresultsfrom the chemicaltransformation of energy-richcompounds in the diet (e.g., glucose, starch). We discussthe mechanismsof photosynthesisand cellular respiration in Chapter 12. The completeoxidation of glucoseto yield carbon dioxide, c6Flpo6 + 6 02 --->6 co2 + 6 H2o has a AG'' of -686 kcal/mol and is the reverseof photosynthetic carbon fixation. Cells employ an elaborate set of protein-mediated reactions to couple the oxidation of 1 molecule of glucoseto the synthesisof as many as 30 moleculesof ATP from 30 molecules of ADP. This oxygen-dependent (aerobic) degradation (catabolism) of glucose is the major pathway for generating ATP in all animal cells, nonphotosyntheticplant cells, and many bacterial cells. Catabolism of fatty acids can also be an important source of ATP. Light energy captured in photosynthesisis not the only source of chemical energy for all cells. Certain microorganisms that live in or around deepoceanvents,where adequate sunlight is unavailable,derive the energyfor converting ADP and Pi into ATP from the oxidation of reduced inorganic compounds.Thesereducedcompounds originate deepin the earth and are releasedat the vents.

not accompanythe formation of new chemical bonds or the releaseof energythat can be coupled to other reactions.The loss of electronsfrom an atom or a molecule is called oxidation, and the gain of electrons by an atom or a molecule is called reduction. Becauseelectrons are neither created nor destroyedin a chemical reaction, if one atom or molecule is oxidized, another must be reduced. For example' oxygen draws electronsfrom ps2+ (ferrous) ions to form Fe3* (ferric) ions, a reaction that occurs as part of the process by which carbohydrates are degraded in mitochondria. Each oxygen atom receivestwo electrons, one from each of two Fe"- ions: 2 Fe2* + Yz oz -->2 Fe3* + 02Thus Fe2*is oxidized, and 02 is reduced.Suchreactionsin which one molecule is reduced and another oxidized often are referred to as redox reactions. Oxygen is an electron acceptor in many redox reactions in cells under aerobic conditions. Many biologically important oxidation and reduction reactions involve the removal or the addition of hydrogen atoms (protons plus electrons) rather than the transfer of isolated electronson their own. The oxidation of succinate to fumarate, which also occurs in mitochondria, is an example (Figure 2-32). Protons are soluble in aqueous solutions (as H3O+), but electrons are not and must be transferred directly from one atom or molecule to another without a water-dissolvedintermediate.In this rype of oxidation reaction' electrons often are transferred to small electron-carrying molecules, sometimes referred to as coenzymes.The most common of theseelectron carriers are NAD* (nicotinamide adeninedinucleotide),which is reducedto NADH' and FAD (flavin adenine dinucleotide), which is reduced to FADH2 (Figure 2-33). The reduced forms of these coenzymescan transfer protons and electrons to other molecules, thereby reducing them.

c-oI

H-C-H | H-C-H I

c- oo

Succinate

N A D * a n d F A DC o u p l eM a n y B i o l o g i c a l Oxidation and ReductionReactions In many chemical reactions, electrons are transferred from one atom or molecule to another; this transfer may or may

o c-o

o

---\----\---2 v v ze 2H*

I c-H

c-H I

c-o o

Fumarate

to fumarate.Inthis of succinate 2-32 Conversion A FIGURE aspartof the citric in mitochondria occurs which reaction, oxidation andtwo protonsTheseare losestwo electrons acidcycle,succinate it to FADH2. to FAD,reducing transferred

B I O C H E M I C AELN E R G E T I C S

59

(b) Reduced: FADH2 Reduced: NADH

o c-NH2

+ le

I

Hll

I

Ribose I 2P

I Ribitol I 2P

Ribose I 2P

I

..1

A O e n o sn re

Adenosine

NAD++H++2e-.-

NADH

Ribitol I 2P

I

I

Adenosine

Adenosine FAD+2H- +2e-

i-

FADH2

FIGURE 2-33 Theelectron-carrying coenzymesNAD+and FAD. (a)NAD*(nicotinamide adenine dinucleotide) isreduced to NADHby the addition of two electrons andoneprotonsimultaneously In manybiological redoxreactions, a pairof hydrogen atoms(two protons andtwo electrons) areremoved froma moleculeIn some cases, oneof the protons andbothelectrons aretransferred to NAD-;the otherprotonisreleased intosolution(b)FAD(flavin

adenine dinucleotide) isreduced to FADH2 bythe additionof two electrons andtwo protons, whensuccinate asoccurs isconverted to (seeFigure fumarate 2-32)ln thistwo-stepreaction, addition of one electron together with oneprotonfirstgenerates a short-lived (notshown), semiquinone intermediate whichthenaccepts a second electron andproton.

To describeredox reactions, such as the reaction of ferrous ion (Fe2+)and oxygen (O2), it is easiestto divide them into two half-reactions:

reduce)a compound with a more positivereduction potential. In this type of reaction, the changein electricpotential AE is the sum of the reduction and oxidation potentialsfor the two half-reactions.The AE for a redox reaction is related to the changein free energyAG by the following expression:

Oxidation of Fe2n: Reduction of 02:

2 Fez* --+ 2 Fe3* -t 2 e2 e * 1/z02 -- 02-

In this case,the reducedoxygen (O2-) readily reactswith two protons to form one water molecule(HzO). The readinesswith which an atom or a molecule gains an electron is its reduction potential E. The tendencyto lose electrons,the oxidation potential, has rhe same magnitude but opposite sign as the reduction potential for the reversereaction. Reduction porentials are measuredin volts (V) from an arbitrary zero point set ar the reduction potential of the following half-reactionunder standard conditions (25 "C. 1 atm, and reactantsat 1 M):

H+ + e- IY'Z

*t,

oxidation

The value of E for a molecule or an atom under standard conditions is its standardreduction potential, E'6 A molecule or an ion with a positive E'e has a higher affinity for electronsthan the H* ion does under standardconditions. Conversely,a molecule or ion with a negative E'6 has a lower affinity for electronsthan the H* ion does under standard conditions.Like the valuesof AGo', standardreduction potentials may differ somewhat from those found under the conditions in a cell becausethe concentrations of reactants in a cell are not 1 M. In a redox reaction,electronsmove spontaneouslytoward atoms or moleculeshaving more positiuereductionporentials. In other words, a compound having a more negativereduction potential can transfer electrons spontaneouslyto (i.e.,

60

CHAPTER2 I

CHEMICALFOUNDATIONS

AG (callmol) : -n (23,064)AE (volts)

(2-11)

wheren is the number of electronstransferred.Note that a redox reactionwith a positiveAE value will have a negativeAG and thus will tend to proceedspontaneouslyfrom left to right.

BiochemicalEnergetics r The changein free energy AG is the most useful measure for predicting the direction of chemicalreactionsin biological systems.Chemical reactions tend to proceed spontaneouslyin the direction for which AG is negative.The magnitude of AG is independentof the reaction rate. r The chemicalfree-energychangeAGo' equals -2.3 RT log K.o. Thus the value of AGo' can be calculatedfrom the experimentally determinedconcentrationsof reactantsand products at equilibrium. r The rate of a reaction dependson the activation energy neededto energizereactantsto a rransition state. Catalysts such as enzymesspeedup reactions by lowering the activation energyof the transitionstate. r A chemical reaction having a positive AG can proceed if it is coupled with a reaction having a negariveAG of larger magnitude. r Many otherwise energetically unfavorable cellular processesare driven by the hydrolysis of phosphoanhydride bonds in ATP (seeFigure2-31).

r Directly or indirectlS light energycaptured by photosynthesisin plants and photosynthetic bacteria is the ultimate source of chemical energy for almost all cells. r An oxidation reaction (loss of electrons)is always coupled with a reduction reaction (gain of electrons). r Biological oxidation and reduction reactions often are coupled by electron-carryingcoenzymessuch as NADand FAD (seeFigure2-33). r Oxidation-reduction reactionswith a positive AE have a negativeAG and thus tend to proceed spontaneously.

KeyTerms acid 52

hydrophobic 3 1

a carbon atom (C*) 41

hydrophobic effect 38

amino acids41

ionic interactions 36

amphipathic 31

molecular complementarity 39

base52 buffers 52 chemicalpotential energy54

monosaccharides44

covalentbond 32

nucleotides44

dehydration r eaction 4 0 AG (free-energy change)55

oxidation 59 pH 51

disulfide bond 43

p h o s p h o a n h y d r i dbeo n d s5 7

endergonic55

phospholipid btlayers41

endothermic 55

polar 34

energy coupling 58 enthalpy (H) 55

polymer 40 redox reaction 59

entropy (S)55

reduction 59

equilibrium constant49

saturated 47

exergonic 55

steadystate50

exothermic56

stereoisomers 33

fatty acids 47

unsaturated 47

hydrogen bond 37

van der $0aals interactions37

hydrophilic 31

44 nucleosides

Review the Concepts 1,. The gecko is a reptile with an amazing ability to climb smooth surfaces,including glass.Recentdiscoveriesindicate that geckosstick to smooth surfacesvia van der'lfaals interactions between septaeon their feet and the smooth surface. How is this method of stickinessadvantageousover covalent interactions?Given that van der Sfaalsforces are among the weakest molecular interactions, how can the gecko's feet stick so effectively? 2. The K* channel is an example of a transmembraneprotein (a protein that spans the phospholipid bilayer of the plasmamembrane).What typesof amino acidsare likely to be found (a) lining the channel through which K* passes,(b) in contact with the hydrophobic core of the phospholipid bilayer

containing fatty acylgroups, (c) in the cytosolic domain of the protein, and (d) in the extracellulardomain of the protein? 3. V-M-Y-F-E-N: This is the single-letteramino acid abbreviation for a peptide. What is the net charge of this peptide can at pH 7.0? An enzyme called a protein tyrosine kinase 'Sfhat attach phosphatesto the hydroxyl groups of tyrosine. is the net charge of the peptide at pH 7.0 after it has been phosphorylated by a tyrosine kinase? \What is the likely source of phosphateutilized by the kinase for this reaction? 4. Disulfide bonds help to stabilize the three-dimensional structure of proteins. What amino acids are involved in the formation of disulfide bonds?Does the formation of a disulfide bond increaseor decreaseentropy (AS)? 5. In the 1960s, the drug thalidomide was prescribed to pregnant women to treat morning sickness. However' thalidomide caused severe limb defects in the children of some women who took the drug, and its use for morning sicknesswas discontinued.It is now known that thalidomide was administered as a mixture of two stereoisomericcomthe pounds, one of which relieved morning sicknessand 'Sfhat defects' birth for the other of which was responsible Why might two such closely related comare stereoisomers? pounds have such different physiologic effects? 6. Name the compound shown below.

HrrriS)c-)x ll :,

se C i H

;; ll ll

Hrl',t-c\fi)c-i/ o -o- P-o-o-P-o-o-?-o-9Hz I o-

I

o-

I o

5',

-o 2',

OH OH Is this nucleotide a component of DNA, RNA, or both? Name one other function of this compound. 7. The chemical basis of blood-group specificity residesin the carbohydratesdisplayedon the surfaceof red blood cells. Carbohydrateshave the potential for great structural diversity. Indeed,the structural complexity of the oligosaccharides that can be formed from four sugarsis greater than that for oligopeptidesfrom four amino acids. Sfhat propertiesof carbohydratesmake this great structural diversity possible? 8. Ammonia (NH:) is a weak basethat under acidic conditions becomesprotonated to the ammonium ion in the following reaction: NH, + H- -+ NH+NH3 freely permeates biological membranes, including those of lysosomes.The lysosomeis a subcellular organelle with a pH of about 4.5-5.0; the pH of cytoplasmis -7'0. lWhat is the effect on the pH of the fluid content of lysosomes R E V I E WT H E C O N C E P T S

61

when cells are exposedto ammonia? No/e: Protonated ammonia does not diffuse freely acrossmembranes. 9. Consider the binding reaction L + R -+ LR, where L is 'S7hen a ligand and R is its receptor. 1 x 10 3 M L is added to a solution containing5 x 10-2 M R, 90% of the L binds to form LR. rVhat is the K.o of this reaction? How will the K.o be affected by the addition of a protein that catalyzes this binding reaction?V/hat is the K6? 10. Vhat is the ionization state of phosphoric acid in the cytoplasm? Vhy is phosphoric acid such a physiologically important compound? 1 1 . T h e A G o ' f o r t h e r e a c t i o nX + Y - + X Y i s - 1 0 0 0 callmol. What is the AG at 25 'C (298 Kelvin) starting with 0.01 M each X, I and XY? Suggesttwo ways one could make this reaction energeticallyfavorable. 12. According to health experts,saturatedfatty acids,which come from animal fats, are a major factor contributing to coronary heart disease.What distinguishesa saturated fatty acid from an unsaturated fatty acid, and to what does the term saturated refer?RecentlS trans unsaturatedfatty acids, or trans fats, which raise total cholesterollevelsin the bodv. have also been implicated in heart disease.How does the cis stereoisomerdiffer from the trans configuration, and what effect does the cis configuration have on the structure of the fatty acid chain? 13. Chemicalmodifications to amino acidscontribute to the diversity and function of proteins. For instance,7-carboxylation of specific amino acids is required to make some proteins biologically active. What particular amino acid undergoes this modification, and what is the biological relevance?Warfarin, a derivative of coumarin, which is present in many plants, inhibits 7-carboxylation of this amino acid and was used in the past as a rat poison. At present, it is also used clinically in humans. What patients might be prescribedwarfarin and whv?

62

CHAPTER2 I

CHEMICALFOUNDATIONS

References Albertg R. A., and R. J. Silbey.2005. PhysicalChemistry, 4th ed. Wiley. Atkins, P.,and J. de Paula. 2005. The Elementsof Physical Chemistry,4th ed. \7. H. Freemanand Company. Berg,J. M., J. L. Tymoczko, and L. Stryer.2007. Biochemistry, 5th ed. W. H. Freemanand Company. Cantor, P. R., and C. R. Schimmel.1980. BiophysicalChemistry.V. H. Freemanand Company. Davenport,H.W. 1974. ABC of Acid-BaseChemistry,6rh ed.. University of ChicagoPress. Eisenberg,D., and D. Crothers.1,979.PhysicalChemistryuith Applications to the Life Sciences.Benjamin-Cummings. Guyton, A. C., and J. E. Hall. 2000. Textbook of Medical Physiology, 10th ed. Saunders. Hill, T. J. 1977. FreeEnergy Transductionin Biology. Academic Press. Klotz, I. M. 1978. Energy Changesin BiochemicalReactions. AcademicPress. Murray, R. K., et al. 1999.Harper'sBiocbemistry,25thed. Lange. Nicholls, D. G., and S.J. Ferguson.1992. Bioenergetics2. Academic Press. Oxtoby, D., H. Gillis, and N. Nachtrieb. 2003. Principlesof Modern Chemistry,Sth ed. Saunders. Sharon,N. 1980. Carbohydrates. Sci.Am.243(5):90-116. Tanford, C. 1980. The Hydropl;obic Effect: Formation of Micellesand Biological Membranes,2d ed. \filey. Tinoco, I., K. Sauer,and J. Wang. 200L. PhysicalChemistryPrinciplesand Applications in Biological Sciences,4thed. Prentice Hall. Van Holde, K., W. Johnson,and P.Ho. 1998. Principlesof PhysicalBiochemistry.PrenticeHall. Voet, D., and J. Voet. 2004. Biochemistry,3d,ed. \7iley. Wood, !(. B., et al. 1981,.Biochemistry:A ProblemsApproach, 2d ed. Benjamin-Cummings.

CHAPTER

STRUCTURE PROTEIN AND FUNCTION

Ribbondiagramof a betapropeller domainfrom the humansignaling (spheres) proteinKeaplTenwatermolecules areboundto eachof lvlanyproteinsarebuiltfrom multiple, the sixbladesof the propeller. X Li,C A independently stableproteindomains[FromL J Beamet Bottoms, andM Hannink,2005,ActaCrystallogr D: Biol.Crystallogr of Robert Hube[Martinsried 51(10):1335-1342I Credit.Courtesy

roteins, which are polymers of amino acids, come in many sizesand shapes.Their three-dimensionaldiversity reflects underlying structural differences: principally variations in their lengths and amino acid sequences,and in some cases,differencesalso in the number of disulfide bonds or the attachment of small moleculesor ions to their amino acid side chains. In general,the linear, unbranched polymer of amino acidscomposing any protein will fold into only one or a few closely related three-dimensional shapes-called conformations. The conformation of a protein together with the distinctive chemical properties of its amino acid side chains determines its function. As a consequence,proteins can perform a dazzling array of distinct functions inside and outside of cells that either are essentialfor life or provide selective evolutionary advantageto the cell or organism that contains them. It is, therefore, not surprising that characterizing the structuresand activities of proteins is a fundamental prerequisitefor understanding how cells work. Much of this textbook is devoted to examining how proteins act together to enablecells to live and function properly. Many proteins can be grouped into just a few broad func. Structural proteins, for example,determinethe tional classes shapesof cellsand their extracellularenvironments,and serve as guide wires or rails to direct the intracellularmovement of moleculesand organelles.They usually are formed by the assemblyof multiple protein subunitsinto very large,long strucnres. Scaffold proteins bring other proteins together into

ordered arrays to perform specific functions more efficiently than if those proteins were not assembledtogether. Enzymes are proteins that catalyze chemical reactions. Membrane transport proteins permit the flow of ions and molecules acrosscellular membranes. Regulatoryproteins act as siSnals' sensors,and switches to control the activities of cells by altering the functions of other proteins and genes.These include signaling proteins, such as hormones and cell-surfacereceptors that transmit extracellular signals to the cell interior'

OUTLIN E 3.1

HierarchicalStructureof Proteins

64

3.2

P r o t e i nF o l d i n g

74

3.3

ProteinFunction

78

3.4

RegulatingProteinFunction ProteinDegradation

3.5

R e g u l a t i n gP r o t e i nF u n c t i o nl l : Noncovalentand CovalentModifications

3.6

Purifying,Detecting,and CharacterizingProteins

3.7

Proteomics

92 105

63

Motor proteins are responsiblefor moving other proteins, organelles,cells-even whole organisms.Any one protein can be a member of more than one protein class,as is the caseof some cell-surfacesignaling receptorsthat are both enzymes and regulator proteins becausethey transmit signals from outside to inside cells by catalyztngchemical reactions. To accomplishefficientlytheir diversemissionssomeproreinsassembleinto largecomplexes,often calledmolecularmachines. How do proteinsmediateso many diversefunctions?They do this by exploiting a few simple activiries.Most fundamentally, proteins bind-to one anorher,to other macromolecules, suchas DNA, and to smallmoleculesand ions.In many cases such binding can induce a conformational changein the protein and thus influenceits activity. Binding is basedon molecular complementaritybetweena protein and its binding partner, as describedin Chapter 2. A second key activity is enzymatic catalysis.Appropriate folding of a protein will place some amino acid side chainsand carboxyl and amino groups of the backboneinto positions that permit the catalysis of covalent bond rearrangements.A third activity involves folding into a channel or pore within a membrane through which moleculesand ions flow. Although theseare especially crucial protein activities,they are not the only ones. For example,fish that live in frigid waters-rhe Antarctic borchsand Arctic cods-have antifreezeproteins in their circulatory systems to preventwater crystallizationat subzerotemperatures. A completeunderstandingof how proteins permit cells to live and thrive requiresthe identificationand characterization of all the proteins used by a cell. In a sense,molecular cell biologistswant to compile a completeprotein 'parts list' and consrructan all-inclusive"users manual" that describeshow theseprclteinswork. Compiling a comprehensive protein parts list has become feasiblein recent years with the sequencingof entire genomes-complete sets of g e n e s - o f m o r e a n d m o r e o r g a n i s m s .F r o m a c o m p u r e r analysisof genome sequences,researcherscan deducethe number of amino acidsand their sequenceof most of the encoded proteins (Chapter5). The rerrn proreomewas coined to refer to the entire protein complementof an organism.The human genome conrains 20,000-25,000 genes(only four times that of the single-cellyeast Saccharomycescereuisiae). However, it encodesabout 33.000 different protein because of variation in mRNA producrion (e.g..alteinativesplicing (Chapter8)). Even more variation is generaredby 100 types of protein modification that can produce hundreds of thousandsof distinct human proteins.By comparingprotein sequencesand structures of proteins of unknown function to those of known function, scientistscan often deduce much about their functions. In the past, characterizationof protein function by genetic, biochemical, or physiological methods often precededthe identification of particular proteins. In the moderngenomicand proteomicera,a protein is usuallyidentified prior to determining its function. In this chapter,we begin our study of how the structureof a protein givesriseto its function, a themethat recursthroughout this book (Figure 3-1). The first secrion examineshow chains of amino acid building blocks are arrangedin a threedimensional structural hierarchy.The next section discusses 64

CHAPTER 3

|

PROTEIN S T R U C T U RAEN D F U N C T I O t T

MOLECULAR STRUCTURE Primary (sequence)

S e c o n d a r y( l o c a lf o l d i n g )

Tertiary ( l o n g - r a n gf e olding)

Supramolecular (large-scale assembly)

Ouaternary (multimericstructure)

(b)

Regulation

@ FUNCTION Transport i

C a t a l y s i s4

FIGURE 3-1 Overviewof proteinstructureand function. (a)Proteins areassembled according to a hierarchy of structures A polypeptide l i 'nse asr e q u e n coef a m i n oa c i d sl i n k e db y p e p t i d e bonds(primary structure) foldsinto localhelices or sheets (secondary structure) thatpackrntolarge(longer-range) complex (tertiary three-dimensional structures structu re) Someindividual polypeptides (quaternary associate intomultichain complexes structure), whichin somecases canbeverylarge,consisting of tensto (supramolecular (b)Protein hundreds of subunits assemblies) function includes organization of thegenome, otherproteins, lipidbilayer (structure); membranes, andcytoplasm controlof proteinactivity (regulation), monitoring of theenvironment andtransmitting resultant (signaling), information flowof smallmolecules andionsacross (transport); membranes (viaenzymes); catalysis of chemical reactions (viamotorproteins) andgeneration of forcefor movement These functions andothersarisefromspecifrc bindinginteractions and conformatronal changes in thestructure of a properly foldedprotein how proteins fold into thesestructures.\(e then rurn to protein function, focusingon enzymes,the specialclassof proteinsthat catalyzechemicalreactions.Variousmechanismsthat cellsuse to control the activitiesand life spansof proteinsare coveredin the next two sections.Next comesa sectionon commonly used techniquesin the biologist'stool kit for isolating proteins and characterizingtheir properties.The chapter concludeswith a discussionof the burgeoningfield of proteomics.

ff,t

Hierarchical Structureof Proteins

A protein chain folds into a distinct three-dimensionalshape that is stabilizedby noncovalent interacrionsberween regions in the linear sequenceof amino acids.A kcy concept rn

(a) Primarystructure -Ala -Glu -Val-Thr-Asp- Pro-Gly-

(b) Secondarystructure s heli

(c) Tertiarystructure

Domain

bond formation between the amino group of one amino acid and the carboxyl group of another results in the net releaseof a water molecule (dehydration) (Figure 3-3a). The repeated amide N, cr carbon (C*), carbonyl C and oxygen atoms of each amino acid residue form the backbone of a protein molecule from which the various side-chaingroups project (Figure 3-3b, c). As a consequenceof the peptide linkage, the backbone exhibits directionality becauseall the amino groups are located on the same side of the Co atoms. Thus one end of a protein has a free (unlinked) amino group (the N-terminus), and the other end has a free carboxyl group (the C-terminus). The sequenceof a protein chain is conventionally written with its

(d) Ouaternarystructure (a)

HO tll

HO

ttl

*HsN- Cd-CR1

O- + *H3N- Co-C-

3l

ll lN t,o \7

O-

n'

HOHO

riltll

(a)Thelinear FIGURE 3-2 Fourlevelsof proteinhierarchy. peptide bondsisthe acids linked together by of amino sequence (b)Folding primary chainintolocala structure. of the polypeptide (c)Secondary represents secondary structure. helices or B sheets loopsandturnsin a single together with various structural elements polypeptide stablestructure, chainpackintoa largerindependently distinct domains; thisistertiarystructure(d) whichmayinclude polypeptides can with theirown tertiary structures Someindividual complex. a multichain intoa quaternary structure defining associate understandinghow proteins work is that function is deriued from three-dimensional structure, and three-dimensional structure, which is determined primarily by noncoualent interactions betueen regions in the linear sequenceof amino acids, is specifiedby amino acid seqwence.Indeed,principles relating biologicalstructureand function initially were formulated by the biologistsJohannvon Goethe(1'749-1,832),Ernst Haeckel (1834-1.91.9),and D'Arcy Thompson \1'860-1'948). They greatly influencedthe school of "organic" architecture pioneeredin the early rwentieth century that is epitomizedby the dicta "form follows function" (Louis Sullivan)and "form is function" (Frank Lloyd'Wright). Here, we considerthe architectureof proteins at four levelsof organization: primar5 secondary,tertiary, and quaternary (Figure 3-2).

The PrimaryStructureof a Proteinls lts Linear Arrangemeno t f AminoAcids As discussedin Chapter 2, proteins are constructed by the polymerizationof 20 differenttypesof amino acids.Individual amino acids are linked together in linear, unbranched chains by covalent amide bonds, called peptide bonds, with occasional disulfide bonds covalently linking side chains together.Peptide

PePtide bond (b)

(C-terminus)

(N-terminus) (c)

amino 3-3 Structureof a polypeptide.(a)Individual A FIGURE via reactions form which peptide bonds, by together linked are acids the R1,R2,etc.,represent thatresultin a lossof water(dehydration). polymers of ("Rgroups")of aminoacids.(b)Linear sidechains whichhave aminoacidsarecalledpolypeptides, oeptidebond-linked (C-terminus) (N-terminus) end carboxyl free and a a freeaminoend linkingthe (c)A ball-and-stick modelshowspeptidebonds(yellow) atom(blue)of oneaminoacid(aa)with thecarbonyl aminonitrogen onein thechain.TheR groups of an adjacent carbonatom(gray) (black) of theaminoacids atoms (green) cr carbon extendfromthe properties of distinct the determine largely chains side These individual oroteins EF P R O T E I N S H I E R A R C H I C ASLT R U C T U RO

65

N-terminal amino acid on the left and its C-terminal amino acid on the right, and the amino acids are numbered sequentially starting from the amino terminus (number 1). The primary structure of a protein is simply the linear arrangement, or sequence,of the amino acid residuesthat compose it. Many terms are used to denote the chains formed by the polymerization of amino acids. A short chain of amino acids linked by peptide bonds and having a defined sequenceis called an oligopeptide, or just peptide; longer chains are referred to as polypeptides. peptides generally contain fewer than 20-30 amino acid residues, whereas polypeptides are often 200-500 residueslong. The longest protein described to date is the muscle protein titin with 25,926 residues.We generally reservethe term protein for a polypeptide (or complex of polypeptides) that has a welldefined three-dimensionalstructure. It is implied that proteins and peptides are the natural products of a cell. The size of a protein or a polypeptide is reported as its massin daltons (a dalton is 1 atomic massunit) or as its molecular weight (M\7), which is a dimensionlessnumber. For example,a 10,000-MI7 protein has a mass of 10,000 daltons (Da), or 10 kilodaltons (kDa). In the penultimatesection of this chapter, we will consider different methods for measuringthe sizesand other physical characteristicsof proteins. The known and predicted proteins encoded by the yeast genome have an averagemolecular weight of 52,728 and contain, on average,466 amino acid residues.The average molecular weight of amino acids in proteins is 113, taking into account their averagerelative abundances.This value can be used to estimate the number of residuesin a protein from its molecular weight or, conversely,its molecular weight from the number of residues.

SecondaryStructuresAre the Core Elements of ProteinArchitecture The secondlevel in the hierarchy of protein strucrure is secondary structure. Secondary structures are stable spatial arrangementsof segmentsof a polypeptide chain held together by hydrogen bonds between backbone amide and carbonyl groups and often involving repeatingstructural patterns. A singlepolypeptide may contain multiple types of secondary structure in various portions of the chain, depending on its sequence.The principal secondarystructures are the alpha (c) helix, the beta (p) sheet,and a short U-shapedbeta (F) turn. Portions of the polypeptide that don't form these structures,but neverthelesshave a well-defined,stableshape, are said to have anirregular structure.The term random ioil appliesto highly flexible portions of a polypeptide chain that have no fixed three-dimensionalstructure.In an averageprotein, 50 percent of the polypeptide chain exists as ct helices and B sheets;the remainder of the molecule is in coils and turns. Thus, ct helicesand B sheetsare the major internal supportive elementsin most proteins. In this section,we exploie the shapesof secondarystructuresand the forces that iavor their formation. In later sections,we examine how linear arrays of secondary structure fold together into larger, more complex arrangementscalled tertiary structure. 66

.

c H A p r E 3R |

pRorEtN s r R u c r u RA EN DF U N c l o N

A m i n ot e r m i n u s

Carboxylterminus

A FIGURE 3-4 The ct helix, a commonsecondarystructurein proteins.Thepolypeptide (seenasa ribbon)isfoldedinto backbone a spiralthat isheldin placeby hydrogen bondsbetweenbackbone oxygenandhydrogen atomsOnlyhydrogens involved in bondingare shown.Theoutersurface of the helixiscovered bv theside-chain R groups(green)

The a Helix In a polypeptide segmentfolded into an o helix, the backbone forms a spiral structure in which the carbonyl oxygen atom of eachpeptide bond is hydrogen-bonded to the amide hydrogen atom of the amino acid four residues farther along the chain (in the direction of the C-terminus) (Figure 3-4). Within an a helix, all the backbone amino and carboxyl groups are hydrogen-bondedto one another,exceptat the very beginning and end of the helix. This periodic arrangementof bonds confersan amino-to-carboxy-terminal directionality on the helix becauseall the hydrogen bond acceptors (e.g.,the carbonyl groups) have the same orientation (pointing in the downward direction in Figure 3-4) and results in a structure in which there is a complete turn of the spiral every 3.6 residues.An ct helix 36 amino acids long has 10 turns of the helix and is 5.4 nm long (0.54 nm/turn). The stable arrangement of hydrogen-bonded amino acids in the a helix holds the bar:kbone in a straight, rodlike cylinder from which the side chains point outward. The relative hydrophobic or hydrophilic quality of a particular helix within a protein is determined entirely by the

characteristicsof the side chains, becauseall the polar amino and carboxyl groups of the peptide backboneare engagedin hydrogen bonding with one another in the helix. In watersoluble proteins, the hydrophilic helicestend to be found on the outside surfaces,where they can interact with the aqueous environment, whereas hydrophobic helices tend to be buried within the core of the folded protein. The amino acid proline is usually not found in cr helices,becausethe covalent bonding of its amino group with a carbon in the side chain preventsits participation in stabilizingthe backbonethrough normal hydrogen bonding. While the classic ct helix is the most intrinsically stable, and most common helical form in proteins, there are variations, such as more tightly or loosely twisted helices.For example, in a specializedhelix called a coiled coil (describedseveralsectionsfarther on), the helix is more tightly wound (3.5 residuesand 0.51 nm per turn). The p Sheet Another type of secondarystructure, the B sheet,consistsof laterally packed B strands.Each B strand is a short (5- to 8-residue),nearly fully extended polypeptide segment. Unlike in the ct helix (where hydrogen bonding between the amino and carboxyl groups in the backbone occursbetlveen nearly adjacentresidues),hydrogenbonding in the B sheetoccurs between backbone atoms in separate,but adjacent, B strands (Figure 3-5a). Thesedistinct B strandsmay be either within a single polypeptide chain, with short or long loops be-

(a) Topview

'lo e

"{r ...)

Amino termrnus

Carboxyl termrnus

(b) Side

A FIGURE 3-5 The p sheet,anothercommonsecondary structurein proteins.(a)Topviewof a simplethree-stranded B p strandsThestabilizing rrydrogen bonds sheetwith antiparallel lines(b)Side by greendashed areindicated the B strands between (green) projection groups aboveand R of the viewof a B sheetThe in thisview Thefixedbond belowthe planeof thesheetisobvious produce contour a pleated backbone anglesin the polypeptide

of four residues, 3-6 Structureof a p turn' Composed A FIGURE chain(=180"U-turn)The of a polypeptide thedirection B turnsreverse 50 percent exact matches,or "identities") and related functions or structureswere defined as an evolutionarily related family, while a swperfamilyencompassedtwo or more families in which the interfamily sequencesmatched lesswell (=30-40 percent identities)than within one family. It is generally thought that proteins with 30 percent sequenceidentity are likely to have similar three-dimensionalstructures; however, proteins with far less sequencematching can have very similar structures.Recently,revised definitions of family and superfamily have been proposed, in which a family comprisesproteins with a clear evolutionary relationship (>30 percentidentity or additional structural and functional information showing common descent but FIGURE 3-25 Substratebindingin the activesite of typsin_ N like serineproteases. (a)Theactivesiteof trypsin(bluemolecule) H wrtha boundsubstrate (blackmolecule) Thesubstrate formsa two_ B i n d i n gs i t e stranded B sheetwith the bindingsite,andthe sidechainof an (Rr)in thesubstrate arginine is boundin the side-chain-specificitv binding p o c k e tl .t sp o s i t i v ecl h y a r g egdu a n i d i n i ugmr o u pi s stabilized by the negative chargeon the sidechainof the enzyme,s A s p - 1 8 9T h i sb i n d i n g a l i g n tsh ep e p t i d b e o n do f t h ea r g i n i n e appropriately (b) for hydrolysis catalyzed by theenzyme,s active_site c a t a l y tti rci a d( s i d ec h a i nos f S e r - 1 9 5H,i s - 5 a7 n dA s p - 1 0 2()b. )T h e a m i n oa c i d sl i n i n gt h es i d e - c h a i n - s p e c ibf i n c idt iyn g pocket d e t e r m i ni e t ss h a p ea n dc h a r g ea,n dt h u si t sb i n d i n g properties Trypsin accommodates the positively charged sidechainsof arginine and lysine; chymotrypsin, large,hydrophobic sidechainssuchas p h e n y l a l a n ianned; e l a s t a ssem , a lsl r d ec h a i nssu c ha sg l y c i naen d

alanine.[Part(a)modified from.J.J. perona andC. S. Craik,1991. J. Biol. Chen 272(48).29987-29990 I

Peptidebond to be cleaved

Side-chainspecificity binding pocket Asp-189

Chymotrypsin

Elastase

enzyme'sAsp-189. Trypsin has a marked preferencefor hydrolyzing proteins (black in Figure 3-25a) at the carboxyl side of a residue with a long positively charged side chain (arginine or lysine), becausethe side chain is stabilizedin the specificity binding pocket by the negativeAsp-189. Slight differencesin the structures of otherwise similar specificity pockets help explain the differing substratespecificities of the two related serine proteases:chymotrypsin prefers large aromatic groups (asin Phe,Tyr, Trp), and elastaseprefers the small side chains of Gly and Ala (Figure 3-25b1.The unchargedSer-189in chymotrypsinallows large, uncharged,hydrophobic side chains to bind stably in the pocket. The branchedaliphatic side chainsof valine and threoninein elastase replace glycines in the sides of the pocket in trypsin and thus prevent large side chains in substratesfrom binding, but allow stablebinding of the short alanineor glycinesidechain. In the catalytic site, all three enzymesuse the hydroxyl group on the side chain of a serine in position 195 to catalyzethe hydrolysis ofpeptide bonds in protein substrates.A catalytic triad formed by the three side chains of Ser-195, His-57, Asp-102 participates in what is essentiallya twostep reaction. Figure 3-26 shows how the catalytic triad co-

operates in breaking the peptide bond, with Asp-102 and His-57 supporting the attack of the hydroxyl oxygen of Ser-195 on the carbonyl carbon in the substrate.This attack initially forms an unstable transition state with four groups attached to this carbon (tetrahedral intermediate). Breaking of the C-N peptide bond then releasesone part of the protein (NH3-P2), while the other part remains covalently attached to the enzymevia an ester bond to the serine'soxygen, forming a relatively stable intermediate (the acyl enzyme). The subsequentreplacementof this oxygen by one from water, in a reaction involving another unstable tetrahedral intermediate, leadsto releaseof the final product (P1-COOH). The tetrahedral intermediates are partially stabilized by hydrogen bonding from the enzyme'sbackbone amino groups in what is called the oxyanion hole.The large family of serine proteasesand related enzymeswith an active-siteserine illustrates how an efficient reaction mechanism is used over and over by distinct enzymesto catalyzesimilar reactions. The serineproteasemechanismpoints out severalgeneral key featuresof enzymaticcatalysis:(1) enzymecatalytic sites are designedto stabilizethe binding of a transition state' thus lowering the activation energy and acceleratingthe overall

(c) Acyl enzyme (ES'complex)

(b) Tetrahedralintermediate (transitionstate)

( a ) E Sc o m p l e x

o H 'r'r.,..-N

D , rH; N

"c

/ Oxyanion hole

P.,/

P,,, H

T,o (f) EP complex

(d) Acyl enzyme (ES'complex)

(e) Tetrahedralintermediate (transitionstate)

(b)

pH 4.0

lsoelectric f o c u s i n g( l E F )

ET

.66

I

.2 o o, o

X

pH10.0 Apply first gel to top of second

34r

z

6 f OJU

pH 4.0

pH 10.0 16

0

I o o o q)

v

Separate

s lil,i??"?l" by size FIGURE3-36 Two-dimensionalgel A EXPERIMENTAL proteins on the basis of charge and separates electrophoresis mass.(a) In this technique,proteinsarefirst separatedinto bandson focusing(step[) The the basisof their chargesby isoelectric resultinggel strip is appliedto an SD5-polyacrylamide 9el (stepZ), (step B) (b) In this mass and the proteinsare separatedinto spotsby

cells,each fromcultured gelof a proteinextract two-dimensional by canbe detected Polypeptides a singlepolypeptide spotrepresents Each as autoradiography such dyes,ashere,or by othertechniques point(pl)andmolecular by itsisoelectric polypeptide ischaracterized (b)courtesy of J Celis ] weiqht IPart

PG ROTEINS AN , D CHARACTERIZIN P U R I F Y I N GD,E T E C T I N G

95

opposite end. A charged protein will migrate through the gradient until ir reachesits isoelectricpoint (pI), the pH at which the net charge of the protein is zero. This technique, called isoelectric focusing (IEF), can resolve proteins that differ by only one charge unit. Proteins that have been separated on an IEF gel can then be separatedin a second dimension on the basis of their molecular weights. To accomplish this separation,the IEF gel strip is placed lengthwiseon one outside edge of a sheetlike (two-dimensional, or slab) polyacrylamide gel, this time saturated with SDS. When an electric field is imposed, the proteins will migrate from the IEF gel into the SDS slab gel and then separateaccording to their masses. The sequential resolution of proteins by charge and mass can achieve excellent separation of cellular proteins (Figure 3-35b). For example, two-dimensional gels have been very useful in comparing the proteomes in undifferentiated and differentiated cells or in normal and cancer cells becauseas many as 1000 proteins can be resolvedas individual spots simultaneously.Sophisticatedmethods have been developed to permit the comparison of complex patterns of proteins in two-dimensional gels from related, but distinct,samples(e.g.,tissuefrom a normal versusa mutant individual) to permir identification of differencesin rhe types or amounts of proteins in the samples (seesection on proteomics,below).

Liquid ChromatographyResolvesproteins b y M a s s ,C h a r g e o , r B i n d i n gA f f i n i t y A third common technique for separating mixtures of proteins or fragments of proteins, as well as other molecules,is basedon the principle that moleculesdissolvedin a solution can differentially interact (bind and dissociate)with a particular solid surface, depending on the physical and chemical properties of the molecule and the surface.If the solution is allowed to flow across the surface, then molecules that interact frequently with the surface will spend more time bound to the surface and thus flow past the ,,rrfac. -or. slowly than molecules that interact infrequently with it. In this technique, called liquid chromatography (LC), the sample is placed on rop of a tightly packed column of spherical beadsheld within a glassor plastic cylinder.The samplethen flows down the column, usually driven by gravitational or hydrostatic forces alone or with the assistanceof a pump, and small aliquots of fluid flowing out of the column, called fractions, are collected sequentially for subsequentanalysis for the presenceof the proteins of interest.The nature of the beads in the column determineswhether the separation of proteins dependson differencesin mass, charge, or binding affinity. Gel Filtration Chromatography proteins that differ in masscan be separatedon a column composedof porous beads made from polyacrylamide, dextran (a bacterial polysaccharide), or agarose(a seaweedderivative)-a technique called gel filtration chromatography. Although proteins flow around the spherical beads in gel filtration chromatography they spend 96

o

c H A p r E 3R |

pRorEtN s r R u c r u RA EN DF U N c l o N

some time within the large depressionsthat cover a bead'ssurface. Becausesmaller proteins can penetrate into thesedepressions more readily than larger proteins can, they travel through a gel filtration column more slowly than larger proteins (Figure 3-37a). (In contrast, proteins migrate throwgh the pores in an electrophoreticgel; thus smaller proteins move faster than larger ones.) The total volume of liquid required to elute (or separateand remove) a protein from a gel filtration column depends on its mass: the smaller the mass, the more time it is trapped on the beads,the greater the elution volume. By use of proteins of known mass as standards to calibrate the column, the elution volume can be usedto estimatethe massof a protein in a mixture. A protein's shapeas well as its masscan influence the elution volume. lon-Exchange Chromatography In ion-exchangechromatographg a secondtype of liquid chromatography proteins are separatedon the basis of differencesin their charges.This techniquemakesuseof speciallymodified beadswhose surfaces are coveredby amino groups or carboxyl groups and thus carry either a positive charge (NHr*) or a negativecharge(COO ) at neutral pH. The proteins in a mixture carry various net charges at any given pH. When a solution of a protein mixture flows through a column of positively charged beads,only proteins with a net negative charge (acidic proteins) adhere to the beads; neutral and positively charged (basic) proteins flow unimpeded through the column (Figure 3-37b). The acidic proteins are then eluted selectivelyfrom the column by passing a solution of increasingconcentrationsof salt (a salt gradient) through the column. At low sah concentratrons, protein moleculesand beads are attracred by their opposite charges. At higher salt concentrations, negative salt ions bind to the positively charged beads, displacing the negatively charged proteins. In a gradient of increasing salt concentration, weakly bound proteins, those with relatively low charge, are eluted first and highly charged proteins are eluted last. Similarly, a negatively charged column can be used to retain and fractionate basic (positively charged) protelns. Affinity Chromatography The ability of proteins to bind specifically to other molecules is the basis of affinity chromatography. In this technique, ligand or orher molecules that bind to the protein of interest are covalently attached to the beads used to form the column. Ligands can be enzyme substrates,inhibitors or their analogues,or other small moleculesthat bind to specificproteins. In a widely usedform of this techniqu e-anti b o dy -aff in ity, or immu n oaffinity, ch r o matography-the attached molecule is an antibody specific for the desiredprotein (Figure 3-37c). (\7e discussantibodies as tools to study proteins next). An affinity column in principle will retain only those proteins that bind the molecule attached to the beads; the remaining proteins, regardlessof their charges or masses, will pass through the column because they do not bind. However, if a retained protein is in turn bound to other molecules, forming a complex, then the entire complex is

(c)Antibody-affi nity chromatography

(a) Gel filtrationchromatography

Load in pH 7 buffer

Largeprotein Small protein Layer sample on column

Add buffer to wash

O Protein recognized by antibody

proteins through column

o Proteinnot recognized by antibody

Polymergel bead

2

1

Elute with + pH3 buffer

EIuted fractions Antibody 1

2 Eluted f ractions

(b) lon-exchange chromatography Negativelycharged proteino Anions retained by beads

Positivelycharged proternO Layer sample on column

Elute negatively charged protein with salt solution

(Nacl)fl fl

Eluted fractions

Positively charged gel bead

3

2

1

fractions

FTGURE 3-37 Threecommonlyusedliquid EXPERIMENTAL techniquesseparateproteinson the basisof chromatographic mass,charge,or affinity for a specificbinding partner.(a)Gel proteins thatdifferin sizeA separates chromatography filtration layered on thetop of a cylinder iscarefully mixture of proteins proteins packed travelthroughthe with porousbeadsSmaller proteins columnmoreslowlythanlargerproteinsThusdifferent in theeluateflowingout of the bottomof thecolumnat emerging in canbe collected elutionvolumes) times(different different chromatography tubes,calledfractions(b)lon-exchange separate packed with proteins thatdifferin netchargein columns separates (shown positive or a here) charge carry either a beads that special havingthe samenetchargeasthe beads charge. Proteins negative proteins having whereas andflow throughthe column, arerepelled

chargebindto the beadsmoreor lesstightly, the opposite Boundproteins-inthiscase, on theirstructures. depending a salt elutedby passing subsequently charged-are negatively As the ions the column (usually of NaClor KCI)through gradient (more bound proteins tightly the displace they bindto the beads, in orderto be released). highersaltconcentration proteins require is of proteins a mixture (c)In antibody-affinity chromatography, with beadsto whicha specific passed througha columnpacked attachedOnlyproteinwith highaffinityfor the iscovalently antibody proteins flow by thecolumn;allthe nonbinding isretained antibody the boundproteiniseluted through.Afterthe columniswashed, the with an acidicsolutionor someothersolutionthat disrupts protein flows out of then released the complexes; antigen-antibody is collected and thecolumn

retained on the column. The proteins bound to the affinity column are then eluted by adding an excessof a soluble form of the ligand or by changing the salt concentration or pH such that the binding to the moleculeon the column is

disrupted. The ability of this technique to separatepartrcular proteins dependson the selectionof appropriate binding partners that bind more tightly to the protein of interest than to other proteins. . P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S

97

H i g h l y S p e c i f i cE n z y m ea n d A n t i b o d yA s s a y s Can DetectIndividualProteins The purification of a protein, or any other molecule, requires a specific assay that can detect the presenceof the molecule of interest as it is separatedfrom other molecules (e.g.,in column or density-gradientfractions or gel bands or spots).An assaycapitalizeson somehighly distinctivecharacteristicof a protein: the ability to bind a particular ligand, to catalyze a particular reaction, or to be recognized by a specific antibody. An assaymust also be simple and fast to minimize errors and the possibilitythat the protein of interest becomesdenaturedor degradedwhile the assayis performed. The goal of any purification scheme is to isolate sufficient amounts of a given protein for study; thus a useful assaymust also be sensitiveenough that only a small proportion of the available material is consumed by it. Many common protein assaysrequire just 10 e to 1.0 12g of material. Chromogenic and Light-Emitting Enzyme Reactions Many assaysare tailored to detect some functional aspectof a protein.For example,enzymaticactivity assaysare basedon the ability to detect the loss of substrateor the formation of product. Some enzymatic assaysutilize chromogenic substrates, which changecolor in the courseof the reaction. (Somesubstrates are naturally chromogenic; if they are not, they can be linked to a chromogenic molecule.)Becauseof the specificiryof an enzyme for its substrate,only samples that contain the enzyme will change color in the presenceof a chromogenic substrate; the rate of the reaction provides a measure of the quantity of enzymepresent.Enzymesthat catalyzechromogenic reactionscan also be fused or chemically linked to an antibody and used to "report" the presenceor location of an antigen to which the antibody binds (seebelow). Antibody Assays As noted earlier, antibodies have the distinctive characteristicof binding tightly and specificallyto antigens.As a consequence,preparations of antibodies that recognizea protein antigen of interest can be generatedand used to detect the presenceof the protein, either in a complex mixture of other proteins (finding a needle in a haystack, as it were) or in a partially purified preparation of a particular protein. The tight binding of the antibody to its antigen, and thus the presenceof the antigen, can be visualized by labeling the antibody with an enzyme)a fluorescent molecule, or radioactive isotopes. For example, luciferase, an enzyme present in fireflies and some bacteria, can be linked to an anribody. In the presenceof ATp and the substrate luciferin, luciferase catalyzesa light-emitting reaction. In either case,after the antibody binds to the protein of in-

98

C H A P T E R3

|

p R o T E t NS T R U C T U RAEN D F U N C T T O N

naturally fluorescent protein found in jellyfish (seeFigure 9-12). Alternatively after the first antibody binds to its target protein, a second, labeled antibody is used to bind to the complex of the first antibody and its target. This combination of two antibodiespermits very high sensitivity in the detection of a targetprotein. To generatethe antibodies, the intact protein or a fragment of the protein is injected into an animal (usually a rabbit, mouse, or goat). Sometimesa short synthetic peptide of 10-15 residuesbasedon the sequenceof the protein is used as the antigen to induce antibody formation. A synthetic peptide, when coupled to a large protein carrier, can induce an animal to produce antibodies that bind to that portion (the epitope) of the full-sized, natural protein. Biosynthetically or chemically attaching the epitope to an unrelatedprotein is called epitope tagging. As we'll see throughout this book, antibodies generatedusing either peptide epitopes or intact proteins are extremely versatilereagentsfor isolating, detecting,and characterizingprotelns. Detecting Proteins in Gels Proteins embedded within a one- or two-dimensionalgel usually are not visible. The two general approachesfor detecting proteins in gels are either to Iabel or stain the proteins while they are still within the gel or to electrophoreticallytransfer the proteins to a membrane made of nitrocellulose or polyvinylidene difluoride and then detect them. Proteinswithin gels are usually stainedwith an organic dye or a silver-basedstain, both detectedwith normal visible light, or with a fluorescent dye that requires specialized detection equipment. Coomassieblue is the most commonly usedorganic dye, typically usedto detect=1000 ng of protein, with a lower limit of detectionof =4-1,0 ng. Silver stainingor fluorescencestainingare more sensitive(lower limit of =1 ng). Coomassie and other stains can also be used to visualize proteins after transfer to membranes;however.the most common method to visualize proteins in these membranes is immunoblotting, commonly called'Westernblotting. Western blotting combines the resolving power of gel electrophoresisand the specificityof antibodies.This multistep procedure is commonly used to separateproteins and then identify a specificprotein of interest. As shown in Figure 3-38, two different antibodies are used in this method, one that is specific for the desiredprotein and a secondthat binds to the first and is linked to an enzymeor other molecule that permits detectionof the first antibody (and thus the protein of interesr to which it binds). Enzymesto which the secondantibody is attachedcan either generatea visible colored product or, by a processcalled chemiluminescence,produce light that can readily be recorded by film or a sensitive detector. The two different antibodies, sometimescalled a "sandwich," are used to amplify the signalsand improve sensitivity.If an antibody is not available,but the geneencodingthe protein is available and can be used to expressthe protein, recombinant DNA methods (Chapter 5) can irrcorporatea small peptide epitope (epitope tagging) into the normal sequenceof the protein that can be detected by a commercially available antibody to that epitope.

Illl+ Technique Animation:lmmunoblotting I

Antibody detection

Electrophoresisand transfer

!

Chromogenic detection

E

S D S - p o l y a c r y l a m i d e g e lM e m b r a n e

with lncubate A b ,( J ) ; washexcess

Incubatewith enzYmelinkedAb2 (Y); wash excess

Reactwith substrate for Ab2-linkedenzyme

FIGURE 3-38 Westernblotting A EXPERIMENTAL (immunoblotting) combinesseveraltechniquesto resolveand detect a specificprotein. StepE: Aftera proteinmixturehasbeen bands(orspots, throughan SDSgel,theseparated electrophoresed (blotted) fromthe gelonto gel)aretransferred for a two-dimensional removedStepZ: whichit isnot readily a porousmembraneJrom (Abr)specific of antibody isfloodedwith a solution Themembrane for a while.Onlythe protern andallowedto incubate for the desired thisproteinbindsthe antibody, bandcontaining membrane-bound (whoseposition cannotbe molecules forminga layerof antibody point) iswashedto remove Thenthe membrane seenat this

u n b o u nA d b ' . S t e pE : T h e m e m b r a ni sei n c u b a t ewdi t ha s e c o n d andbindsto thefirstAb1 (Ab2)thatspecifically recognizes antibody (e.9., to linked eitheran enzyme iscovalently antibody Thissecond reaction), a chromogenic can catalyze which phosphatase, alkaline can whosepresence or someothersubstance isotope, radioactive and the location Step4: Finally, with greatsensitivity be detected (e.g, by a deep-purple amountof boundAb2aredetected permitting the reaction), fromchromogenic precipitate (and the destred of the mass) therefore mobility electrophoretic (based on band aswellasitsquantity oroteinto be determined,

RadioisotopesAre lndispensableTools l olecules f o r D e t e c t i n gB i o l o g i c aM

Radioisotopes Useful in Biological Research Hundreds of biological compounds (e.g.,amino acids,nucleosides,and numerous metabolic intermediates)labeled with various radioisotopes are commercially available. These preparations vary considerably in their specific actiuity, which is the amount of radioactivity per unit of material, measured in

A sensitivemethod for tracking a protein or other biological molecule is by detecting the radioactivity emitted from radioisotopesintroduced into the molecule. At least one atom in a radiolabeled molecule is present in a radioactive form, called a radioisotope.

ISOTOPE

intensrtv).

}|ALF-tIIE

Phosphorus-32

1'4.3days

Iodine-125

60.4 days

Sulfur-35

87.5days

Tritium (hydrogen-3)

12.4 years

Carbon-14

5730.4years

dom/mmol) are available.Likewise' commercialpreparations tH-l"b.l.d nucleic acid precursorshave much ligher speoi 1aC-labeled cific activities than those of the corresponding preparations.In most experiments'the former are preferable t".uot. they allow RNA or DNA to be adequatelylabeled alter a shorter time of incorporation or require a smaller cell sample.Various phosphate-containingcompounds in which every phosphorus atom is the radioisotope phosphorus-32 P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S

99

a^rereadily available. Becauseof their high specific activity, 32P-labeled nucleotides are routinely ur.d to label nucleic acids in cell-freesystems. Labeled compounds in which a radioisotope replaces atoms normally present in the molecule have virtually the same chemical properties as the corresponding nonlabeled compounds. Enzymes,for instance,generally cannot distinguish betweensubstrateslabeledin this way and their nonlabeled substrates.The presenceof such radioactive atoms is indicatedwith the isotopein brackets(no hyphen) as a prefix (e.g., [3H]leucine).In .ontrurt, labeling almost all biomolecules (e.g., protein or nucleic acid) with the radioisotope iodine-125 (12sI)requires the covalent addition of 12sIto a molecule that normally does nor have iodine as part of its structure.Becausethis labelingproceduremodifies the chemical structure, the biological activity of the labeled molecule may differ somewhat from that of the nonlabeledform. The presenceof such radioactive atoms is indicated with the isotope as a prefix with a hyphen (no bracket) (e.g.,125l-trypsin). Standardmethods for labeling proteins with 12sIresult in covalent attachmentof the 125Iprimarily to the aromatic rings of tyrosine side chains (mono- and diiodotyrosine).

disintegrations per minute per small pixel of surface area. These instruments, which can be thought of as a kind of reusable electronic film, are commonly used to quantitate radioactive molecules separated by gel electrophoresisand are replacingphotographic emulsionsfor this purpose. A combination of labeling and biochemical techniques and of visual and quantitative detectionmethods is often employed in labeling experiments.For instance,to identify the major proteins synthesizedby a particular cell type, a sample of the cells is incubated with a radioactive amino acid (e.g., [35S]methionine)for a few minutes, during which time the Lbeled amino acid mixes with the cellular pool of unlabeled amino acids and some of the labeled amino acid is biosynthetically incorporated into newly synthesizedprotein. Subsequently unincorporatedradioactive amino acid is washed away from the cells. The cells are harvested,the mixture of cellular proteins is extractedfrom the cells (for example, by a detergentsolution), and then separatedby gel electrophoresis; and the gel is subjectedto autoradiography or phosphorimager analysis.The radioactive bands correspondto newly synthesizedproteins, which have incorporatei the radiolabeled amino acid. Alternatively, the proteins can be resolved by liquid chromatographS and the radioactivity in the eluted Labeling Experiments and Detection of Radiolabeled fractions can be determinedquantitatively with a counter.To Molecules Whether labeledcompoundsare detectedby audetect only one specificprotein, rather than all the proteins toradiography, a semiquantitativevisual assay or their radioacbiosyntheticallylabeled this way, a specific antibody to the tivity is measuredin an appropriate ',counter," a highly quantiprotein of interestcan be usedto precipitatethe protein away tative assaythat can determine the amount of a radiolabeled from the other proteins in the sample (immunoprecipitation). compound in a sample, depends on the nature of the experiThe precipitate is then solubilized in a detergenr,separating ment. In some experiments,both types of detection are used. the antibody from the protein, and the sampleis subjectedto In one use of autoradiographS a tissue,cell, or cell conSDS-PAGEfollowed by autoradiography.In this type of exstituent is labeledwith a radioactivecompound, unassociated periment, a fluorescent compound that is activated by the radioactivematerial is washed away, and the structure of the radiation is often infusedinto the gel so that the light emitted sample is stabilized either by chemically cross-linking the can be used to detect the presenceof the labeled protein, macromolecules('fixation') or by freezingit. The sample is either using film or a two-dimensional electronicdetector. then overlaid with a photographic emulsion sensitiveto radiPulse-chase experimentsare particularly usefulfor tracing ation. Developmentof the emulsion yields small silver grains changesin the intracellular location of proreins or the modiwhose distribution correspondsto that of the radioactivemafication of a protein or metabolit. ou.i time. In this experiterial and is usuallydetectedby microscopy.Autoradiographic mental protocol, a cell sample is exposed to a radiolabeled studiesof whole cellswere crucial in determiningthe intracelcompound that can be incorporated or otherwiseattachedto lular siteswhere various macromoleculesare synthesizedand a cellular molecule of interest-the "pulse"-for a brief pethe subsequentmovements of these macromoleculeswithin riod of time, then washedwith buffer to remove the unincorporated label, and finally incubated with an unlabeled form of the compound-the "chase" (Figure 3-39). Samplestaken periodically during the chaseperiod are assayedto determine the location or chemicalform of the radiolabel as a function of time. Often, pulse-chaseexperiments,in which the protein Quantitative measurementsof the amount of radioactivis detected by autoradiography after immunoprecipitation ity in a labeledmarerial are performed with severaldifferent and SDS-PAGE,are usedto follow the rate of synthesis,modification, and degradation of proteins by adding radioactive amino acid precursorsduring the pulse and then detectingthe amounts and characteristicsof the radioactiveprotein during the chase.One can thus observepostsyntheticmodifications of the protein that changeits electrophoreticmobility and the rate of degradation of a specificprotein. A classicuse of the pulse-chasetechniquewas in studiesto elucidatethe pathwav traversedby secretedproteins from their site of synihesisin the endoplasmicreticulum to the cell surface(Chapter 14). 100

.

c H A p r E3R | p R o r E t N s r R U c r u RAEN DF U N c l o N

Normal protein

p-e.

(b) Mutant 'Drotein

m '.,& o-

"'

key component, which provides a measure of the relative abundancesof each of the ions in the sample.The fourth essential component is a computerized data systemthat is used to calibrate the instrument; acquire,store, and processthe resulting data; and often direct the instrument automatically to collect additional specific types of data from the sample' based on the initial observations. This type of automated feedback is used for the tandem MS (MS/MS) peptidesequencingmethods describedbelow. The two most frequently usedmethods of generatingions of proteins and protein fragmentsare (1) matrix-assistedlaser desorption/ionization(MALDI) and (2) electrospray(ES)' In MALbI (Figure 3-40) the peptide or protein sampleis mixed with a low-molecular-weight,W-absorbing organic acid (the matrix) and then dried on a metal tatget. Energy from a laser ionizes and vaporizes the sample producing singly charged molecular ions from the constituentmolecules'In ES (Figure 3-41.a),the sample of peptidesor proteins in solution is converted into a fine mist of tiny droplets by spraying through a

l"j,\'J," ;:::,'J."'Ll3,' "".' il?HT":',i"H experimentscan FIGURE 3-39 Pulse-chase A EXPERTMENTAL track the pathway of protein modificationor movement newlysynthesized within cells.(a)Tofollowthefateof a specific proteinin a cell,cellswereincubated for 0 5 hr with [3sS]methionine proteins, (thepulse) andthe radioactive to labelallnewlysynthesized intothe cellswasthenwashedaway aminoacidnot incorporated (thechase) for varying timesup to Thecellswerefurtherrncubated to weresubjected from each timeof chase 24 hours,andsamples protein(here,the lowto isolate onespecific immunoprecipitation of the immunoprecipitates receptor) SDS-PAGE lipoprotein density permitted visualization of the one followedby autoradiography (p) protein, asa smallprecursor synthesized whichis initially specific (m) of addition form by larger mature modified to a rapidly andthen from proteinwasconverted Abouthalfof the labeled carbohydrates after0.5 hourof p to m duringthe pulse,the restwasconverted to stablefor 6-8 hoursbeforeit begins chaseTheproteinremains (b) (indicated The same intensity). band by reduced be degraded in cellsin whicha mutantformof the wasperformed experiment to converted proteinismadeThemutantp formcannotbe properly thanthe normalprotein the m form,andit is morequicklydegraded 1986, J CellEloi H A Brush, andM Krieger, fromK F Kozarsky, lAdapted 1567-1s7s 102(5) I Roshanl(eab 021-66950639

arated by the mass analyzeron the basis of their mlz. The two most frequently used mass analyzersare timeof-flight (TOF) instruments and ion traps. TOF instruments e*ploit the fact that the time it takes an ion to passthrough the length of the analyzer before reaching the detector is

Metal target

I

lonization +++ O@o

MassSpectrometryCan Determinethe Mass and Sequenceof Proteins Mass spectrometry (MS) is a powerful technique for characthe tertzingproteins.MS is particularly useful in determining 'With such inmass of a protein or fragments of a protein. formation in hand, it is also possibleto determinepart of or all the protein's sequence.This method permits the very highly accuratedirect determination of the ratio of the mass (m) of a charged molecule (ion) to its charge (z), ot mlz. Techniquesare then used to deducethe absolutemass of the ion. There are four key features of all mass spectrometers. The first is an ion source,from which charge, usually in the form of protons, is transferred to the peptide or protein molecules.The formation of these ions occurs in the presenceof a high electric field that then directs the chargedmolecular ions into the second key component' the mass analyzer. The mass analyzer,which is always in a high vacuum chamber, physically separatesthe ions on the basis of their differing mass-to-charge(mlz) ratios. The mass-separated ions are subsequentlydirected to strike a detector,the third

Sample

@

t

q)

c

Lightestions arrive at detector first

Time 3-40 Molecularmasscan be FIGURE a EXPERIMENTAL laserdesorption/ionization determinedby matrix-assisted massspectrometry'ln a MALDI-TOF time-of-flight (MALDI-TOF) pulses of lightfroma laserionizea proteinor massspectrometer, on a metaltarget(stepE) An that isabsorbed peptidemixture towardthe detector the ionsin the sample fieldaccelerates electric to the isproportional (steosEl andEl).Thetimeto the detector the having (mlz) ions For ratio. mass-to-charge the squarerootof timeto the ionsmovefaster(shorter the smaller samecharge, usingthetimeof flight weightiscalculated Themolecular detector). of a standard PG ROTEINS O AN , D CHARACTERIZIN P U R I F Y I N GD,E T E C T I N G

101

(a)

Electrospray needle

Atmosphere Vacuum

Mass Detector analyzer

----_Y--------Y-

Droplets containing solvatedions

rons

Mass spectrometer

Electrosprayionization (b)

568.65

100 3go ;80 o.^ /u o o

F60 Ebo €40 930 620 #10 0

MS/MS of mlz 836.47

o

0)

q) CC

100 90 FIIVGYVDDTOFVR 80 70 60 693.26 50 706.62 40 30 765.40 421.33 20 473.15 549.46 261.30 10 0 300 400 500 600 700 800

1142.53

1298.60 1497.46

1251 1124.44

1398.48 1536.14

900

1000

1100

1200

1300

14oO 15oO 1600

m/z EXPERIMENTAL FIGURE 3-41 Molecularmassof proteins and peptidescan be determinedby electrosprayionization ion-trap massspectrometry.(a)Electrospray (ES)ionization proteins converts andpeptides in a solution intohighlycharged gaseous ionsby passing the solution througha needle(forming the droplets) thathasa highvoltageacross it (charging the droplets). Evaporation of thesolvent produces gaseous ionstharenrera mass spectrometer. Theionsareanalyzed by an ion-trapmassanalyzer that thendirectsionsto the detector. (b) Toppanet:Massspectrumof a mixture of threemajorandseveral minorpeptides ispresented asthe relative abundance of the ionsstriking (y axis)asa the detector functionof the mass-to-charge (m/z)ralio(x axis).Bottornpanel:ln an MS/MSinstrument (suchasthe iontrapshownin part(a)),a

102

C H A P T E R3

|

p R o T E t NS T R U C T U RAEN D F U N C T T O N

peptideioncanbe selected specific for fragmentatron intosmaller ionsthatarethenanalyzed anddetected. (also TheMS/MS spectrum calledthe product-ion spectrum) provides detailed structural information aboutthe parention,including sequence information for peptidesHere,the ionwith a mlzof 836.47wasselected, fragmented andthe m/z massspectrumof the productionswas measured Notethereis'nolongeran ionwith an m/zoI g36.47 present because it wasfragmented. Fromthevarying sizes of the productions,the understanding that peptidebondsareoftenbroken in suchexperiments, the knownmlzvalues for individual aminoacid fragments, anddatabase information, the sequence of the peptide, FIIVGYVDDTQFVR, canbe deduced. ona fiourefrom [part(a)based part(b),unpublished 5 Carr; datafromS Carr I

proportional to the square root of mlz (smaller ions move fasterthan larger oneswith the samecharge,seeFigure 3-40). In ion-trap analyzers,tunable electric fields are used to cap'trap,' ions with a specific mlz and to sequentially ture, or pass the trapped ions out of the analyzer onto the detector (seeFigure 3-4ta). By varying the electric fields, ions with a wide range of mlz values can be examined one by one, producing a massspectrum,which is a graph of mlz (x axis) versus relative abundance(y axis) (Figure 3-41'b,top panel). In tandem, or MS/MS, instruments, any given parent ion in the original mass spectrum (Figure 3-41b, top panel) can be broken into smallerions by collisionwith an inmass-selected, ert gas, and then the mlz and relative abundancesof the resulting fragment ions measured (Figure 3-41b, bottom panel), all within the samemachinein about 0.1 s per selectedparent ion. This secondround of fragmentationand analysispermits the sequencesof short peptides ( FIGURE to each areaddedby a DNApolymerase Nucleotides synthesis. (indicated by growingdaughter strandin the 5'-+3' direction f roma continuously strandissynthesized Theleading arrowheads) (red) is strand lagging end. The primer at its 5' singleRNA thatare frommultipleRNAprimers discontinuously synthesized duplexis aseachnewregionof the parental formedperiodically produces Okazaki initially of theseprimers unwoundElongation prevtous the growing approaches fragment As each fragments, areligated andthefragments primer, the primerisremoved of the entire in synthesis results eventually of thisprocess Repetition l a g g i nsgt r a n d .

5', P o i n to f j o i n i n g L a g g i n gs t r a n d Okazakifragment P a r e n t aD l NAduPlex

S h o r tR N A p r i m e r

5', Leadingstrand

DNA REPLICATION

14'l

FocusAnimation:Coordinationof Leading- and

flltt strandsynthesis

(a) SV40DNA replicationfork

E::i1,""';

31

5',

Primase

L a g g i n gs t r a n d

E

pPol E Rfc PCNA

Primer

(b}PCNA RPA

a

Doublestranded DNA

Leadingstrand

(c)RPA

FIGURE 4-31 Model of an SV40DNA replicationfork. (a)A hexamer of largeT-antigen ([), a viralprotein. functions asa helicase to unwindthe parental DNAstrands. Single-strand regions of the parental template unwoundby largeT-antigen areboundby multiple copies of the heterotrimeric proteinRpA(Z). Theleading strandissynthesized by a complex of DNApolymerase S (polS),

(E). (b)Thethreesubunits fragment of pCNA,shownin different colors, forma circular structure with a centralholethroughwhich double-stranded DNApasses. A diagram of DNAisshownjn the centerof a ribbonmodelof the pCNAtrimer.Thediaqram at the

pCNAboundto DNAin upperleftshowsthe iconrepresenting parta. (c)Thelargesubunitof RPAcontains two domains thatbind single-stranded DNA.Onthe left,thestructure determined for the two DNA-binding domains of the largesubunitboundto single_ stranded DNAisshownwith the DNAbackbone (whitebackbone with bluebases) parallel to the planeof the page Notethatthe singleDNAstrandisextended withthe bases exposed, an optimal conformation for replication by a DNApolymerase Onthe right,the viewisdownthe lengthof thesingleDNAstrand,revealing how RpA wraparoundthe DNA.Thediagram B strands at bottomcenter showsthe iconrepresenting heterotrimeric RpAboundto singlestranded DNAin part(a).[part (a)adapted fromS J Flint etal, 2000, pathogenesis, Virology: Molecular Biology, andControl, ASMpress; part(b) afterJ M Gulbis et al, 1996,Cetl87:297; andpart(c)afterA Bochkarev etal, 1997, Nature 385:176 l

Figure 4-31 depicts the multiple proteins that coordinate copying of SV40 DNA at a replication fork. The assembled proteins at a replication fork further illustrate the concept of molecular machines introduced in Chapter 3. These multi_ component complexes permit the cell to carry out an ordered sequenceof eventsthat accomplishessentialcell functions. The molecular machine that replicatesSV40 DNA con_ tains only one viral protein. All other proteins involved in 142

o

c H A p r E R4

I

B A s t cM o L E c u L A R G E N E T tM c ECHANtsMs

llll+ FocusAnimation:Bidirectional of DNA Reptication EcoRl

SV40 DNA replication are provided by the host cell. This viral protein, large T-antigen, forms a hexamer that unwinds the parental strandsat a replication fork. Primers for leading and lagging daughter-strandDNA are synthesizedby a complex of primase, which synthesizesa short RNA primer, and DNA polymerased (Pol a), which extendsthe RNA primer with deoxynucleotides,forming a mixed RNA-DNA primer' The primer is extended into daughter-strand DNA by DNA polymerase6 (Pol 6), which is lesslikely to make errors during copying of the templatestrand than is Pol ct becauseof its proofreading mechanism (see Section 4.6 below). Pol E forms a complex with R/c (replication factor C) and PCNA (proliferating cell ntclear antigen), which displaces the primase-Pol crcomplex following primer synthesis.As illustrated in Figure 4-31,b,PCNA is a homotrimeric protein that has a central hole through which the daughterduplex DNA passes, thereby preventing the PCNA-Rfc-Pol 6 complex from dissociating from the template.Pol E is the main polymeraseused by eukaryotes for elongating DNA strands during replication. After parental DNA is separatedinto single-strandedtemplatesat the replication fork, it is bound by multiple copiesof RPA (replicationprotein A), a heterotrimericprotein (Figure 4-31,c).Binding of RPA maintains the template in a uniform conformation optimal for copying by DNA polymerases. Bound RPA proteins are dislodgedfrom the parental strands by Pol cr and Pol 6 as they synthesizethe complementary strandsbase-pairedwith the parental strands. Severaleukaryotic proteins that function in DNA replication are not depictedin Figure 4-31. DNA polymerasee also contributes to the synthesisof cellular chromosomal DNA, though its exact role is uncertain. Still other specializedDNA polymerasesare involved in repair of mismatchesand damagedlesionsin DNA (seeSection4.6). A topoisomeraseassociateswith the parental DNA aheadof the helicaseto remove torsional stressintroduced by the unwinding of the parental strands. Ribonuclease H and FEN I remove the ribonucleotidesat the 5' ends of Okazaki fragments;these are replacedby deoxynucleotidesadded by DNA polymerase6 as it Okazaki extendsthe upstream Okazaki fragment. Successive fragments are coupled by DNA ligase through standard 5'-+3' phosphoesterbonds.Replicationof a linear DNA molecule presentsa specialproblem at the ends of the molecule sincethe 5'-most RNA primers of the lagging strandscannot be replacedby DNA by this mechanism.In most eukaryotes' this problem is solved by the RNA-protein complex called telomerasethat carriesits own templateas discussedin Chapter 6, Genes,Genomics,and Chromosomes.

D N A R e p l i c a t i o nU s u a l l yO c c u r sB i d i r e c t i o n a l l y f r o m E a c hO r i g i n As indicatedin Figures4-30 and 4-31, both parental DNA strands that are exposedby local unwinding at a replication fork are copied into a daughter strand. In theory, DNA replication from a singleorigin could involve one replication fork

C i r c u l a vr i r a l cnromosome

o

q)

o)

E

.,*.,."0, o g;-ri.r-" o";"; r;,;*; a EXPERTME;I of microscopy bidirectionalreplicationof SV40DNA.Electron growthof DNAstrands bidirectional SV40DNAindicates replicating cells viralDNAfromSV4O-infected froman origin.Thereplicating one site recognizes which EcoRl, enzyme restriction wascut by the for a a landmark DNA.Thiswasdoneto provide in thecircular recognition the FcoRl in the 5V40genome: sequence specific asthe endsof linearDNA recognized isnoweasily sequence micrographs Electron microscopy. by electron vrsualized molecules a collection showed molecules DNA SV40 replicating of EcoRl-cut "bubbles," longerreplication with increasingly of cut molecules from eachendof the cut whosecentersarea constantdistance with Thisfindingisconsistent chaingrowthin two molecules at thecenterof a bubble, froma commonoriginlocated directions etal, G C Fareed diagrams' [See in thecorresponding asillustrated of N P Salzman l photographs courtesy J Virol10:484; 1972, that moves in one direction. Alternatively, two replication forks might assembleat a single origin and then move in opposite directions, leading to bidirectional growth of both i",rght., strands.Severaltypes of experiments,including the orr. sho*tt in Figure 4-32, provided early evidencein support of bidirectional strand growth. The general consensusis that all prokaryotic and eukaryoticlels employ a bidirectional mechanism of DNA DNA REpL1CAT;ON o

143

FocusAnimation:Coordination of Leading-and Lagging-strand flltt Synthesis Helicases

< FIGURE 4-33 Bidirectional mechanism of DNAreplication. Theleftreplication fork hereiscomparable to the replication fork diagrammed in Figure 4-31, whichalsoshowsproteins otherthan largeT-antigen lop:TwolargeT-antigen hexameric helicases first EJrn*'no'nn bindat the replication originin opposite orientations. Step[: Using energyprovided fromATPhydrolysis, the helicases movein opposite directions, unwinding the parental DNAandgenerating single-strand templates thatareboundby RPAproteinsStepE: primase_pol o complexes synthesize (red)base-paired shortprimers to eachof the parental primersynthesis separated strands. Stepg: pCNA-Rfc-pol 6 complexes E t".o,nn-.trand replace the primase-Pol crcomplexes J andextendtheshortprimers, generating (darkgreen)at eachreplication the leading strands fork Step@: Thehelicases furtherunwindthe parental strands, andRpA proteins bindto the newlyexposed single-strand regions. Stepg: I PCNA-Rfc-Pol 6 complexes extend the leading strands further Step6: extension ! | Leading-strand Primase-Pol ctcomplexes primers synthesize for lagging-strand t synthesis at eachreplication fork Stepfl: pCNA-Rfc-pol 6 complexes displace the primase-Pol o complexes andextendthe lagging-strand (lightgreen), Okazaki fragments whicheventually areligatedto the 5' endsof the leading strandsTheposition whereligation occursis represented by a circleReplication continues byfurtherunwinding of theparental strands andsynthesis of leading andlagging strands asin Steps4-Z Althoughdepicted asindividual stepsfor clarity, unwinding andsynthesis of leading andlaggingstrands occurconcunentlv I

Unlike SV40 DNA, eukaryotic chromosomal DNA moleculescontain multiple replication origins separatedby tens to hundreds of kilobases.A six-subunit protein called ORC, for origin recognition complex, binds to each origin and associateswith other proteins required to load cellular hexameric helicasescomposed of six homologous MCM proteins (for minichromosome maintenance. the genetic screen initially_used to identify the genes encoding them). Two opt.nn,"n-strand primersynrhesis posed MCM helicases separatethe parental strands at an

extension El I Leaoing-strand

+

E J

extension Z I a.nn,nn-strand

+

transcription of most genes,control of the initiation step is the primary mechanismfor regulatingcellular DNA replication. Ac_ S t r a n dl i g a t i o n

replication. In the caseof SV40 DNA, replication is initiated by binding of two large T-antigen hexamiric helicasesto the

into two daughtercells.We discussthe various regulatory mech_ anismsthat determinethe rate of cell division in Chapter 20.

DNA Replication Each strand in a parental duplex DNA acts as a template r synthesisof a daughter strand and remains base-paired the new strand, forming a daughter duplex (using a 144

C H A P T E R4

|

B A S T CM O L E C U L A RG E N E T T M C ECHANTSMS

semiconservativemechanism).New strands are formed the 5'--+3'direction. r Replication begins at a sequencecalled an origin. Each eukaryotic chromosomal DNA molecule contains multiple replication origins. r DNA polymerases,unlike RNA polymerases,cannot unwind the strandsof duplex DNA and cannot initiate synthesis of new strandscomplementaryto the templatestrands. r At a replication fork, one daughter strand (the leading strand) is elongated continuously. The other daughter strand (the lagging strand) is formed as a seriesof discontinuous Okazaki fragments from primers synthesizedevery few hundred nucleotides(Figure 4-30). r The ribonucleotidesat the 5' end of each Okazaki fragment are removed and replacedby elongation of the 3' end of the next Okazaki fragment. Finally adjacent Okazaki fragments are joined by DNA ligase. r Helicasesuse energyfrom ATP hydrolysis to separatethe parental (template)DNA strands. Primase synthesizesa short RNA primer, which remains base-pairedto the template DNA. This initially is extendedat the 3' end by DNA polymerase a (Pol c), resulting in a short (S')RNA(3' )DNA daughterstrand. r Most of the DNA in eukaryotic cells is synthesizedby Pol 6, which takes over from Pol ct and continues elongation of the daughterstrandin the 5'+3' direction.Pol 6 remains stably associatedwith the template by binding to Rfc protein, which in turn binds to PCNA, a trimeric protein that encirclesthe daughter duplex DNA (seeFigure 4-31). r DNA replication generally occurs by a bidirectional mechanism in which two replication forks form at an origin and move in opposite directions, with both template strandsbeing copied at eachfork (seeFigure4-33)' r Synthesisof eukaryotic DNA in vivo is regulatedby controlling the activity of the MCM helicasesthat initiate DNA replication at multiple origins spacedalong chromosomal DNA.

![

DNARepairand Recombination

Damage to DNA is unavoidable and arises in many ways. DNA damage can be caused by spontaneouscleavageof chemicalbonds in DNA, by environmental agentssuch as ultraviolet and ionizing radiation, and by reaction with genotoxic chemicalsthat are by-products of normal cellular metabolism or occur in the environment' A mutation in the normal DNA sequencecan occur during replication when a DNA polymerase inserts the wrong nucleotide as it reads a damagedtemplate. Mutations also occur at a low frequency as the result of copying errors introduced by DNA polymeraseswhen they replicate an undamagedtemplate. If such mutations were left uncorrected, cells might accumulate so many mutations that they could no longer function properly, In addition, the DNA in germ cells might incur too many

mutations for viable offspring to be formed' Thus the prevention of DNA sequenceerrors in all types of cellsis important for survival, and several cellular mechanismsfor repairing damagedDNA and correcting sequenceerrors have evolved' One mechanismfor repairing double-strandedDNA breaks' by a processcalled recombination, is also used by eukaryotic cells to generatenew combinations of maternal and paternal geneson each chromosome through the exchangeof segments of the chromosomesduring the production of germ cells (e'g', sperm and eggs). Significantln defects in DNA repair mechanisms and cancerare closelyrelated. !7hen repair mechanismsare compromised, mutations accumulate in the cell's DNA. If these mutations affect genes that are normally involved in the careful regulation of cell division, cells can begin to divide uncontrollably, leading to tumor formation, and cancer' Chapter 25 outlines in detail how cancer arisesfrom defects in DNA repair.We will encountera few examplesin this section, as well, as we first consider the ways in which DNA integrity can be compromised' and then discussthe repair mechanismsthat cells have evolved to ensurethe fidelity of this very important molecule'

IntroduceCopyingErrors DNA Polymerases and Also CorrectThem The first line of defensein preventingmutations is DNA polymerase itself. Occasionally, when replicative DNA polymerasesprogressalong the template DNA, an incorrect nuto the growing 3' end of the daughterstrand cleotideit for instance,in(seeFigure"da.a 4-31,).E. coll DNA polymerases., troduce about 1 incorrect nucleotideper 104 polymerizednucleotides.Yet the measuredmutation rate in bacterial cells is much lower: about 1 mistake in 10e nucleotidesincorporated into a growing strand. This remarkable accuracy is largely due to proofreading by E. coli DNA polymerases' Pro-ofreadingdependson a 3'-+5' exonucleaseactiuity of some DNA polymerases.Sfhen an incorrect baseis incorporated during DNA synthesis,base-pairingbetweenthe 3' nucleotideof the nascentstrand and the templatestrand doesnot occur.As a result,the polymerasepauses'then transfersthe 3' end of the growing chain to its exonucleasesite, where the incorrectmispairedbaseis removed (Figure4-34)' Then the 3' end is transferredback to the polymerasesite' where this region is copied correctly. Like the E. coli DNA polymerases' I*o enkaryotic DNA polymerases,6 and e, used for replication of most chromosomal DNA in animal cells' also have proofreading activity. It seemslikely that proofreading is indispensablefor all cellsto avoid excessivemutatlons'

C h e m i c aal n d R a d i a t i o nD a m a g et o D N A Can Leadto Mutations DNA is continually subiectedto a baffage of damaging chemical reactions; estimatesof the number of DNA damin a singlehuman cell range from 10a to 105 per "g..,r..t,, day! Even if DNA were not exposedto damaging chemicals, ..it"in aspectsof DNA structure are inherently unstable' D N A R E P A I RA N D R E C O M B I N A T I O N O

145

Frngers

Thumb

t^

Thumb

Fingers urowtng strand

Pol

Template strand

Exo

FIGURE 4-34 Proofreading by DNApolymerase. All DNA polymerases havea similar three-dimensional structure, which resembles a half-opened righthandThe,,fingers,, bindthesinglestranded segment of thetemplatestrand,andthe polymerase catalytic (Pol)liesin thejunctionbetween activity thefingers andpalm.As long asthecorrect nucleotides areaddedto the3, endof thegrowing strand,it remains in the polymerase site Incorporation of an incorrect

baseat the3' endcauses melting of thenewlyformedendof the duplexAsa result, pauses, thepolymerase andthe3,endof the growingstrandistransferred to the 3,-+5,exonuclease site(Exo) about3 nm away,wherethe mtspaired baseandprobably otherbases areremoved. Subsequently, the 3' endflipsbackintothe polymerase siteandelongation resumes. fromC M Joyce andT.T.Steltz, [Adapted 1995, 1 Bacteriol 177:6321,and S Bellandl Baker, 1998, Ceil92:2951

For example, the bond connecting a purine baseto deoxyribose is prone ro hydrolysis at low rate under physiological conditions, leaving a sugar without an attached base.Thus coding information is lost, and this can lead to a mutarion during DNA replication. Normal cellular reacrions,including the movement of electronsalong the electron-transportchain in mitochondria and lipid oxidation in peroxisomei, produce severalchemicalsthat react with and damage DNA, including hydroxyl radicals and superoxide (O2 ). These too can causemutations, including those that lead to cancers. Many spontaneous mutations are point mutations, which involve a changein a single basepair in the DNA se-

quence. One of the most frequent point mutations comes ftom deamination of a cytosine (C) base,which converts it into a uracil (U) base. In addition, the common modified baseS-methylcytosineforms thymine when it is deaminated. If thesealterationsare not correctedbefore the DNA is replicated,the cell will usethe strandcontainingU or T as template to form a U.A or T.A basepair, thus creating a permanent changeto the DNA sequence(Figure 4-35). Radiation from the environment can also have dramatic consequences for DNA. High-energy ionizing radiation such as x-rays and gamma rays causedouble-strandedbreaks in DNA. Uy radiation found in sunlight causesdistortions in

NH.

U

l' N'c-c-cr. ttl ozc'-r-cH I

Deamination

(.tl HNz"\c-cH"

trl

o-rctN.c

2-Deoxyribose

2-Deoxyribose

5-Methylcytosine

Thymine

5',

?21

Deamination

--+

5',

J

Replication

z

E .* Base-excision repatr 3',

3',

Witd-type DNA FIGURE 4-35 Deamination leadsto point mutations.A spontaneous pointmutation canformbydeamination of 5-methylcytosine (C)to formthymine(T).lf the resulting T.Gbasepair isnot restored to the normalC.Gbasepairby baseexcision_repair 146

.

Mutant DNA

5',

Wild-type DNA

(step[), it willleadto a permanent mechanisms changeIn sequence (r.e., a mutation) following (stepZ). Afteroneround DNAreplication of replication, onedaughter DNAmolecule willhavethe mutantT.A basepairandtheotherwillhavethewild-type C.Gbasepair.

c H A p r E4R | B A s t cM o L E c u L AGRE N E TM t cE c H A N t s M s

the DNA double helix that interfere with proper replication and transcription.

High-FidelitD y N A E x c i s i o n - R e p aSi ry s t e m s a n d R e p a i rD a m a g e Recognize In addition to proofreading,cellshaveother repair systemsfor preventingmutations due to copying errors, spontaneousmutation, and exposureto chemicalsand radiation. SeveralDNA excision-repair systemsthat normally operate with a high degree of accvracy have been well studied. These systemswere first elucidated through a combination of genetic and biochemicalstudiesin E. coli. Homologs of the key bacterialproteins exist in eukaryotes from yeast to humans, indicating that theseerror-freemechanismsaroseearly in evolution to protect DNA integrity. Each of these systems functions in a similar manner-a segmentof the damagedDNA strand is excised, and the gap is filled by DNA polymeraseand ligaseusing the complementary DNA strand as template. I7e will now turn to a closer look at some of the mechanisms of DNA repair, ranging from repair of single basemutations to repair of DNA broken acrossboth strands. Some of theseaccomplish their repairs with great accuracy;others are lessprecise.

BaseExcisionRepairsT.G Mismatchesand DamagedBases In humans, the most common type of point mutation is a C to T, which is causedby deamination of S-methyl C to T (see Figure 4-35). The conceptual problem with base excision repair in this case is determining which is the normal and which is the mutant DNA strand, and repairing the latter so that it is properly base-pairedwith the normal strand. Sincea G.T mismatch is almost invariably causedby chemical conversion of C to U or S-methyl C to T, the repair system evolvedto removethe T and replaceit with a C (Figure4-36). The G'T mismatch is recognized by a DNA glycosylase that flips the thymine base out of the helix and then hydrolyzes the bond that connects it to the sugar-phosphate DNA backbone. Following this initial incision, an apurinic (AP) endonucleasecuts the DNA strand near the abasicsite. The deoxyribose phosphate lacking the base is then removed and replaced with a C by a specializedrepair DNA polymerasethat reads the G in the template strand. As mentioned earlier, this repair must take place prior to DNA replication becausethe incorrect base in this pair, T, occurs naturally in normal DNA. Consequently,it would be able to engagein normal \Tatson-Crick basepairing during replication, generatinga stablepoint mutation that is now unable to be recognizedby repair mechanisms(seeFigure 4-35, s t e pZ ) . Human cells contain a battery of glycosylases'each of which is specific for a different set of chemically modified DNA bases.For example,one removesS-oxyguanine,an oxidized form of guanine, allowing its replacementby an undamagedG, and others remove basesmodified by alkylating agents.The resultingnucleotidelacking a baseis then replaced

3',

5' .*"'J.-r1.!!?sif..'-"1*.r

i{:iT,iil

-L*l-i-J_|j*l-_l-e 5' '' ,)a ' -lDNAglycosYlase

T*ril*rTl*r

-ji*j*|J*t-Li*L

ls A P EeI n d o n u c l e a s e

I t

* *T-r -i*rj""r I

!-r-.{e.+

liG:!"; fJ-

-^. )-

rr*

ls AP lyase I

(partof DNAPol0)

::*T-l- Tl*l]i-J-l-Lgl-l-i-J"-

PorF, DNA risase DNA

E

4-36 Baseexcisionrepair of a T'G mismatch.A DNA A FIGURE formedby usually for G'Tmismatches, glycosylase specific (see flipsthethymine 4-35), Figure residues of 5-mC deamination baseout of the helixandthencutsit awayfromthesugar-phosphate (blackdot).An justthe deoxyribose (stepn), leaving DNAbackbone site[apurinic baseless icfor the resultant specif endonuclease (stepE), and thencutsthe DNAbackbone | (APE1)l endonuclease apurinic endonuclease, by an phosphate is removed thedeoxyribose with DNApolymerase associated B, a specialized lyase(APlyase), usedin repair(stepB). Thegapisthenfilledin by DNApolymerase (stepZl),restoring theoriginal by DNAligase DNAPolB andsealed 42:2946] Chemie Angewandte pair 2003, O Schdrer, G.Cbase [After

by the repair mechanismjust discussed.A similar mechanism functions in the repair of lesions resulting from depwrination, the loss of a guanine or adenine base from DNA resulting from hydrolysis of the glycosylic bond between deoxyribose and the base.Depurination occursspontaneouslyand is fairly common in mammals. The resulting abasicsites,if left unrepaired, generatemutations during DNA replication because they cannot specifythe appropriatepaired base.

MismatchExcisionRepairsOther Mismatches a n d S m a l lI n s e r t i o n sa n d D e l e t i o n s Another process,also conservedfrom bacteria to man' principally eliminates base-pair mismatches and insertions or deletionsof one or a few nucleotidesthat are accidentallyintroduced by DNA polymerasesduring replication. As with base excision repair of a T in a T'G mismatch, the conceptual problem with mismatch excision repair is determining whic^his the normal and which is the mutant DNA strand, and repairing the latter. How this happensin human cellsis not known with certainty. It is thought that the proteins that bind to the mismatchedsegmentof DNA distinguishthe template AND RECOMBINATION . DNA REPAIR

147

z'lttsuz

Templatestrand

+ A

t:l 1ii

c

Newly synthesized d a u g h t e rs t r a n d

Cells use nucleotide excision repair to fix DNA regions containing chemically modified bases,often called chemical addwcts,that distort the normal shapeof DNA locally. A key to this type of repair is the ability of certain proteins to slide along the surfaceof a double-strandedDNA molecule looking for bulgesor other irregularities in the shapeof the double helix. For example, this mechanism repairs thyminethymine dimers, a common type of damage caused by UV light (Figure 4-38); thesedimers interfere with both replication and transcription of DNA.

lvtsxo\

-iil

!ll;l iilli

N u c l e o t i d eE x c i s i o nR e p a i r sC h e m i c aAl d d u c t s T h a t D i s t o r tN o r m a lD N A S h a p e

\

MLHl endonuclease, PMS2

a DNAhelicase DNAexonuclease

j

3' .-i- .,',,,"*.],--r,-'--;-"i,- 5,

3'd.

**5'

G a p r e p a i rb y D N A p o l y m e r a s ea n d l i g a s e

5',

l!. iri

3,h

3',

:liilai

ill||-fr

lllti:

Figure 4-39 illustrates how the nucleotide excisionrepair system repairs damaged DNA. Some 30 proteins are involved in this repair process,the first of which were identified through a study of the defectsin DNA repair in cultured cellsfrom individuals with xeroderma pigmentosum,a hereditarydiseaseassociatedwith a predisposition to cancer.Individuals with this diseasefrequently develop the skin cancers called melanomas and squdmous cell carcinomasif their skin is exposedto the UV rays in sunlight. Cells of affected patients lack a functional nucleotide excision-repair system. Mutations in any of at least seven different genes, called XP-A through Xp-G,

FIGURE 4-37 Mismatchexcisionrepairin humancells.The mismatch excision-repair pathway corrects errorsintroduced during replication A complex of the MSH2andMSH6proteins (bacterial MutShomologs 2 and6) bindsto a mispaired segment of DNAin sucha wayasto distinguish between thetemplate anonewty synthesized (steptr) Thistriggers daughter strands bindingof MLH.j andPMS2(bothhomologs of bacterial MutL)Theresulting DNAproteincomplex thenbindsan endonuclease thatcutsthe newly synthesized daughter strandNexta DNAhelicase unwinds the helix, andan exonuclease removes several nucleotides fromthecutendof the daughter strand,including the mismatched base(stepf,l) Finally, aswith baseexcision repair, thegapisthenfilledin by a DNA (Pol6, in thiscase) polymerase andsealed (stepS) by DNAligase

",x"

Deo

@ Oeo"yriU\e

Two thymine residues

and daughter strands; then the mispaired segmentof the daughter strand-the one with the replication error-is excisedand repaired to becomean exact;omplement of the templatestrand (Figure4-37).In contrastto baseexcisionrepair, mismatch excision repair occurs after DNA replication.

J,""Y,1,,"" o..

ouo*u.x"

Predispositionto a colon cancerknown as bereditary nonpolyposiscolorectalcancerresultsfrom an inherited loss-of-function mutation in one copy of either the MLHL or the MSH2 gene. The MSH2 and MLH1 proteins are essentialfor DNA mismatch repair (seeFigure 4-37). Cells with ar least one functional copy of each of these

H

@

o"o,.rx" 1..t

"

CH.

Thymine-thyminedimer residue

mon in noninherited forms of colon cancer.I 148

CHAPTER 4

|

FIGURE4-38 Formation of thymine-thymine dimers. The most commontype of DNA damagecausedby UV irradiation, thymine-thymine drmerscan be repairedby an excision-repair mechanism

BASTC M O L E C U L AG R E N E T TM CE C H A N T S M S

6VL

N O U _ V N l S n o f l uC N V U r v d l u V N C

'uorlJaslxeu (uoqrzutquo)at 'utsru oql snoSoloruoq tred sE possnJsrps Jo 'suorlElnrualnporlur uer rSELuEp -Eqreru yNC PuoreseqJ Io rreder ,\\oLIJo rldurexe euo sr uorto]oplrua8€tnru dlqrssod E r{f,nsJo uoueuroC '(69-g ernSrg)rurod SururoIeqr re srred eseq ]EJeAesJo ssol ur stlnser dlqerre,Lurssarord -rredaraql 'aruosouorqf, erues oql urory euoc stueru8eJ; VNC paurol eqt Jr ue^e fauord-rorre sr ssaco.rdsrql 'salnJaloruVNC o,ll.l 'Sururol-pua to spua snoSolotuoquou agl Sururoforso^lo^ur sno8o1outoquou'stJJsrLreS.ro relnllarrllnu ur s>leeJgpueJts -alqnop Sur.rrederroJ rusrueqreu tueurruope.rdoq1 'sIEOJq puErls-a]qnop rredar ot pe^lo^a aaeg suats.,(so,4AI 'passardxa sr eua8 reqt qrrq,^ ur addl 'oue8 reqtoue uor8a.rSurpooeql IIar ro le^el eqt Sur8ueqo 1o ot .{trurxord esolr otur aua8auo;o :atouo;d aqr Surrqplnoc luaua8ue.r.reeJ IeuosoruoJqf, € ro leruenbes pr)E ourrue luore;Jrp dlareldruoc e yo uorlrod lpurrural-J eqt ol pesnt ecuanbasprlp ourue auo ]o uoruod leuru.ret-51eqr ro; sepol legl aua8..ppqdq,,e elearf,plnoc Sururo[]cerrocur'aldruexa .rog 'saue8;o Suruorlcunt oqt tJeJJEuEtr terlt stuaua8uerre -er Ieruosoruorqcsso.r8ot peal uer VNC Jo spuerts elqnop dlJeln]rued ;o Sururo[a.rlJarrofur osnef,oqsuorsol aJOAas erp asaqJ 'VNC ul slearq puerts-elqnopesners8nrp raruer -rtue eurospue (uorterpe-r-l{" pue -x '3'e) uorlerpprSurzruol

v Nc u ! sle er gp ue rls- elqnoo redeu ol uorleurquo)auoz!lllnsuols^So^,\l '6g-7 ern8rg ur palcrdap g q8norqr sdalsJo suorrreer aqt pue 'HIIJJ ! 'turod xllaq VNC orlt Jo SuruodosreS teqt te Jo tueurlrnner -3rn srql iaserarudlod eqr 01 pelrufar sl 'gSJ 'uratord VNU '(raurp aunudql-eurudqlr ''3'a) vNC uo uorsele le ]lerusE pelletssauoreq aseraudlodVNU up;r 'uatsds srql u1 'suor8 -er leJrtrrf tsotu aql ot suoJto rreda.rstre.rrp.{1ruenr1;arred -a.rpaldnoc-uortdrrcsuert'selod.re>1ne raq8rq ur IIer auo due ur paqrrJsue.rlsr etuoua8 erlt Jo uortrer; Iletus e dluo acur5 ',updat palrlnot-uotlir.tcsua4 pollel-os-suorBa.r peqrrcs -uerluou ur uerll dle.trtoe Suraq euouaS eql ;o peqrrrsuert .raq8rq suor8a.rur el€J JetsEJqlnru e le parredarsr salodJ€>lne ur a8eruepVNC leql uortelrasqoeqt urpldxedleq deu rred

_er puB uorldr'su€' ur slrunqns vNC orr",Ir1:,ii; rrrH,,ru a;rnbar teqt sesserordreln1lecfuareJJrpoznl ul dlquresseural -ord e repurs seq ernleu pasn leql sreedde\'(I€-L a.rn8rg 'etrs aas)alertrurot ese;atu^,(1od VXU 8ur,rno11e trels aql te xllaq VNC eqt spur,\\unHII{J.;o dlrarrreaserrloqaqt 'uon -drrrsuul q'6€-V a;n8rg ur u.^oqs se'saserrlaqor ^(8o1our -orl aAEqslrunqns oser{r o./![ 'sllal crtodrelna ur .rredar Jo uorsrrxeeprtoepnu ro; parrnbaros]Eore'(19-1 ern8rl aas)

'nAZ lareqls awaq) alpueMabuV O lgV6ZtZn petdepVJ(tr do]s) pue'99t: ! l,t arnleN' IOOZ'sraleLrlreop 1u.ror1 u u t u t e u t aarq t p u e ' u o r l e l t ; d eVr N C u l s e e s e 6 ry1p 6 { q p a l e e s t > 1 t t 6 y56 {q petttJst de6 eq} ril;eut3saptloal)nuouoLx eseraur[1od l{1]>exe 'seseqpebeL-uep oq] qtlM luauOetl o1 peper6apsr q)rqM slqf (E dels)uorsalaq],to oplsqlee uo Uede VNC oq] sosealar puo)os oq] ]n) 'aseal)nuopuo saseqZ€-vZ slurod1epuetlspe6euLep ue se 6ut1leMou) D-dX uaql (E dols) e l-dx pue (aseol)nuopuo pue pulMUn potLlro+ sl soseqEZ=+o0lqqnqe lllun xll0qoqi 0z!llqelsap roqunj pue xelduor oql ol purq uaq] suralordVdUpue g-dx xllaq 'sltunqns 'srs{lorp{q olqnopaql putMUn,411eryed 61y Iq patannod 'HllJI lo1te1uotldtllsuei]s]lnllal ueqi xaldLlol ase)rloqosoqM slql (U dals)suralotdB€Zpue (urelord3 Lunsoluaubtderutepotax) eutulql I;1er1rur sl 'roLulp )-dX aql 1o xeldulote {q paztubo>er 'xrloqolqnopaq] uoruolslpsasne) e se q)ns leql uolsalvNC +o V'slle) ueunq ul ltedal uo!s!)xe ePlloal)nN 5€'t lun9lJ V yp6 edAl-p1t6

j,., j.,ILj-,11-LLL; ,,.-1,-.,

t

**t"-,n

VN8 ltr ".,,";1:3J I

.9

.€

VV

, .i'**.,n

,r"-;,-i'- ,s ln3 g-dx

lnc J-dx

9-dx

t

buruaoO x ! l o qatqnop VNo Jo lI

seua811e;o uorldrrcsueJtrot perrnberroloe; uortdrJ)suerl 'HIIJI, 'dlqelreruaA le.raue8e Jo strunqns aprrdeddlodaar; a ' G t - V e . r n 8 r ga e s )p o o l s -Jepun lle,t^.lAouare rreder uorsrJxeaprloelrnu ur surelord 'seruanbrsuoJaIUES eql o^Eq dX esoqrJo tsoru Jo solor aq1 pue edltouaqd erueseqt ernpo.rd11elunsoluau8rd erurep -oJax esnel pue ruelsls rredar srqt to uonplrtJeur ol peal

Double-strandbreak

vast majority of women with inherited susceptibility to breast cancer have a mutation in one allele of either the rttttttl BRCA-1 or the BRCA-2 genesrhat encodeproteins participating in this repair process.Loss or inactivation of the second allele inhibits the homologous recombination repair pathway and thus tends to induce cancerin mammary or ovarian epithelialcells,although at presentit is not clear why these estrogen-responsive tissuesare favored sites of carcinogenesis. Yeastscan repair double-strandbreaks induced by 1-irradiation. Isolation and analysisof radiationsensitive(RAD) mutants that are deficient in this reoair system facilitated study of the process. Virtually all the proteins a yeast Rad proteins have homologs in the human genome, Jo,n". and the human and yeastproteins function in an essentially identical fashion. A variety of DNA lesions not repaired by mechanisms discussedearlier can be repaired by mechanismsin which the damagedsequenceis replaced by a segmentcopied from the same or a highly homologous DNA sequenceon the homologous chromosome of diploid organisms,or the sister chromosome following DNA replication in all organisms.These mechanismsinvolve an exchange of strands between separate DNA moleculesand henceare collectivelyreferred to as FIGURE 4-40 Nonhomologous end-joining.Whensister D N A r ecombination mechanisms. chromatids arenot available to helprepairdouble-strand breaks, In addition to providing a mechanism for DNA repair, nucleotide sequences arebuttedtogetherthatwerenot apposed in similar recombination mechanismsgenerategeneticdiversity the unbroken DNA TheseDNAendsareusually fromthesame among the individuals of a speciesby causing the exchange chromosome locus,andwhenlinkedtogether, several basepairsare of large regions of chromosomesbetween the maternal and lost Occasionally, endsfromdifferent chromosomes areaccidentally paternal pair of homologous chromosomesduring the spejoinedtogetherA complex of two proteins, KuandDNA-dependent cial type of cellular division that generatesgerm cells (sperm protern kinase, bindsto theendsof a double-strand break(steptr) and eggs),meiosis (Figure 5-3). In fact the exchange of reAfterformation of a synapse, the endsarefurtherprocessed by gions of homologous chromosomes,called crossing over, is nucleases, resulting in removal (stepE), andthetwo of a few bases required for proper segregationof chromosomesduring the double-stranded molecules areligatedtogether(stepB) Asa result, first the double-strand meiotic cell division. Meiosis and the consequencesof breakis repaired, but several basepairsat thesite of the breakareremoved[Adapted generating new combinations of maternal and paternal fromG Chu,1997, J Biot Chem 272:24097; M Lieber et al, 1997,Curr. geneson one chromosome by recombination are discussed OpinGenet. Devel. T:99;andD van G a n t e t a l , 2 O O 1 ,N a t u r eR e v .G e n e t 2 : 1 9 6 I further in Chapter 5. The mechanismsleading to proper segregation of chromosomes during meiosis are discussedin Chapter 20. Here we will focus on the molecular mechanisms of DNA recombination, highlighting the exchangeof DNA strands betweentwo recombining DNA molecules.

TTTTTTTTTT

TTTTTTTTTT

chromosome to another. Such translocations may generare chimeric genesthat can have drastic effects on normal cell function, such as uncontrollable cell growth, which is the hallmark of cancer.The devastatingeffectsof double-strand breaks make this the "most unkindest cut of all," to borrow a phrase from Shakespeare's Julius Caesar.

H o m o l o g o u sR e c o m b i n a t i oC n a n R e p a i rD N A Damageand GenerateGeneticDiversity

150

.

cHAprER 4

|

Repair of a Collapsed Replication Fork An exampleof recombinationalDNA repair is the repair of a "collapsed,'replication fork. If a break in the phosphodiesterbackboneof one DNA strand is not repaired before a replication fork passes, the replicatedportions of the daughterchromosomesbecome separatedwhen the replication helicasereachesthe ,,nick,, in the parental DNA strand becausethere are no covalent bonds between the two fragments of the parental strand on either side of the nick. This processis called replication fork collapse (Figure4-41, stepn). If it is not repaired,it is generallylethal to at leastone daughtercell following cell division becauseof the loss of genetic information between the nick and the end of the chromosome.The recombination processthat repairs the resultingdouble-strandedbreak and regenerat.s r.pli."" tion fork involves multiple enzymesand other proteins, only some of which are mentionedhere.

B A s t cM o L E c u L A G R E N E T tMc E C H A N t s M s

DNAnick

5', 3',

I

I ll I Y

s'-e*onrcleaseacts on brokenend. Other daughter strand (pink)ligatedto repairedparentalstrand ( l i g h tb l u e )i n u n b r o k e n chromosome. 5', 3',

Jo

RecA-or Rad51-mediated strand invasion

Hollidaystructure

\--*

3' Ligateends

J

R e b u i l dr e p l i c a t i o fno r k a n d c o n t i n u er e p l i c a t i o n

(Figure 4-41.,steptr ). The lagging nascentstrand (pink) created on the unnicked, homologous' parent strand is ligated to the unreplicated portion of the parent chromosome, as shown in Figure 4-41, step Z). A critical protein required for the next step is RecA in bacteria, or the homologous Rad51 in S. cereuisiae and other eukaryotes. Multiple RecA/RadS1 molecules bind to the single-stranded DNA (now considered the invading strand) and catalyze its hybridization to a perfectly or nearly perfectly complementary sequencein another, homologous, double-strandedDNA molecule, either the ligated molecule created after fork collapse (as shown in the figure) or the other homologous chromosome in diploid organisms.The other strand (dark blue) of this target double-stranded DNA (the strand not basepairing with the invading strand) is displaced as a singlestranded loop of DNA along the region of hybridization between its complement and the invading strand (refer to Figure 4-4L, step B). The RecA/RadS1-catalyzedinvasion of a duplex DNA by a single-strandedcomplement of one of the strands is key to the recombination process. Since no basepairs are lost or gained in this process,calledstrand inuasion,it doesnot requirean input of energy. Next, the hybrid region between target DNA (pink) and the invading strand (dark red) is extended in the direction away from the break by proteins that use energy from ATP hydrolysis. This process is called branch migration (Ftgure 4-41,, step 4) becausethe point at which the target DNA strand (pink) crossesfrom one complementary strand (dark blue) to its complement in the broken DNA molecule (dark red), is called a branch in the DNA structure. In this diagram, the diagonal lines represent only one phosphodiesterbond. Molecular modeling and other studiesshow that the first baseon either side of the branch is base-pairedto a complementary nucleotide. As this branch miSrates to the Ieft, the number of base pairs remains constant; one new base pair formed with the (red) invading strand is matched by the loss of one base pair with the parental (dark blue)

strand. When the region of hybridization extends beyond the 5' 3', end of the broken strand (light blue), this broken parental DNA strand becomes increasingly single-stranded, as its repairof a collapsed A FIGURE 4-41 Recombinational complement, the invading (dark red) strand' base-pairsinfork. Parental strands arelightanddarkblueTheleading replication steadwith the target (pink) DNA strand. This single-stranded pink. red, lagging daughter strand is dark and the daughter strand (light blue) parcntal strand then base-pairswith the complephosphodiester mentary region of the other parental strand (dark blue) that represent a single linesin stepB andbeyond Diagonal colorSmallblack bondfromtheDNAstrandof theconesponding has likewise become single-strandedas the branch migrates of the phosphodiester cleavage arrowsfollowingstep@ represent to the left (Figure 4-41., step 4). The resulting structure is See in the Holliday structure. of DNAstrands bondsat the crossover called a Hotliday strwcture, after the geneticist who first prornalVm bb/ruva. htmI f or an animationof sdscedu/jou http://www. posed it as an intermediatein Seneticrecombination. Again, RuvAandRuvB. See catalyzed by E coliproteins branchmigration the diagonal lines in the diagram following step 4 represent genetics.wisc.edu/Holliday/holliday3D htmlfor an http://engels single phosphodiesterbonds (not a stretch of DNA), and all Seethetextfor a of theHolliday structure anditsresolution animation basesin the Holliday structure are base-pairedto compleLehninger fromD L Nelson andM M Cox,2005, discussion. [Adapted mentary basesin the parental strands. Cleavageof the phos4thed, W H Freeman andCompany.l of Biochemistry Principles phodiesterbonds that crossover from one parental strand to ' the other and ligation of the 5' and 3 ends base-pairedto the The first step in the repair of the double-strand break is same parental strands (stepsE and 6) result in the generaexonucleolytic digestionof the strand (light blue) that has its (dark tion of a structure similar to a replication fork. Rebinding of red) 5' end at the break, leaving a portion of the other replication fork proteins results in extension of the leading (the single-stranded one with the 3' end at the break) strand D N A R E P A I RA N D R E C O M B I N A T I O N

151

Jn

Ends digestedby exonucleases,leaving 3 ' s i n g l e - s t r a n d eedn d s

RecA-or Rads1-mediated strand invasion

!

3 ' e n d o f i n v a d i n gs t r a n di s e x t e n d e db y D N A p o l y m e r a s eu n t i lt h e d i s p l a c e d single-strand(dark blue) base-pairswith the other 3' single strand generated i n i t i a l l y( p i n k )

A a e n di s e x t e n d ebdy D N Ap o l y m e r a s e J

Ends are ligated

l I

Jo

I

Cleavephosphodiesterbonds indicated with arrows and ligatealternativeends

A FIGURE 4-42 Double-strand DNAbreakrepairby homologousrecombination. Forsimplicity, eachDNAdoublehelix is represented by two parallel lineswith the polarities of the strands indicated by arrowheads at their3, endsTheuppermolecule hasa double-strand breakNotethat in the diagram of the upperDNA strand past the point of the original strand break and reinitiation of lagging-strandsynthesis(step Z), thus regenerating a replication fork. The overall processallows the undamagedupper strand in the lower molecule following step E (pink/light blue) to serveas template for extensionof the leading (dark red) strand in step Z. Double-Stranded DNA Break Repair by Homologous Recombination A similar mechanism called bomolopous 152

o

c H A p r E4R |

molecule the strandwith its3' endat the rightison thetop,whilein thediagram of the lowerDNAmolecule thisstrandisdrawnon the bottom Seethetextfordiscussion. L Orr-WeaverandJ W [AfterT. Szostak, 1985,MicrobiolRev. 49:33.1 recombination can repafua double-strand break in a chromosome and can also exchangelarge segmentsof two double-strandedDNA molecules (Figure 4-42). Homologous recombination is also dependenton strand invasion catalyzed principally by RecA in bacteriaand Rad51 in eukaryotes(steps Il and tr). The 3' end of the invading DNA strand is then extended by a DNA polymerase,displacingthe parental strand as an enlargingsingle-stranded loop of DNA (dark blue, step E). Vhen DNA synthesis extends sufficiently far, the displaced

B A s t cM o L E c u L AGRE N E TM t cE c H A N t s M s

parentalstrand (dark blue)that is complementaryto the 3' single-strandedregion generatedat the other broken end of DNA (the pink single-stranded region on the left following step n), base-pair(step B). This 3' end the complementarysequences (pink) is then extendedby a DNA polymerase,using the displaced single-strandedloop of parental DNA (dark blue) as template(step4). Next, the two 3' ends generatedby DNA synthesisare ligated(stepE)to the 5'ends generatedin step I by 5'-exonucleasedigestion of the broken ends. This generatestwo Holliday structuresin the paired molecules(stepE).Branch migration of theseHolliday structurescan occur in either direction (not diagrammed).Finally, cleavageof the strands at the positions shown by the arrows, and ligation of the alternative 5' and 3' ends at each cleavedHolliday structure generatestwo recombinant chromosomesthat each contain the DNA of one parental DNA molecule (pink and red strands) on one side of the break point and the DNA of the other parental DNA molecule (light and dark blue) on the other sideof the break point (step6). Eachchromosomecontains a third region, located in the immediate vicinity of the initial break point, that forms a heterodwplex;here one strand from one parent is base-pairedto the complementary strand of the other parent (pink or red base-pairedto dark or light blue). Base-pair mismatches between the two parental strandsare usually repairedby repair mechanismsdiscussed above to generatea complementary basepair. In the process, sequencedifferencesbetween the two parents are lost, a processreferredto as geneconuersion. Figure 4-43 diagrams how cleavageof one or the other pair of strandsat the four-way strand junction in the Holliday structure generatesparental or recombinant molecules.This process,calledresolution of the Holliday structure,separates DNA molecules initially joined by RecA/RadS1-catalyzed strand invasion. Each Holliday structure in the intermediate following step El of Figure 4-42 can be cleavedand religated in the two possibleways shown by the two setsof small black arrows. Consequently,there are four possibleproducts of the

recombination process.Two of theseregeneratethe parental chromosomes(with the exceptionof the heteroduplexregion at the break point that is repaired into the sequenceof one parent or the other (geneconversion),and t'wo generaterecombinant chromosomesas shown inFigure 4-42. Meiotic Recombination Meiosis is the specializedform of cell division in eukaryotesthat generateshaploid germ cells (e.g.,sperm and eggs)from a diploid cell (Figure 20-38). At least one recombination occurs between the paternal and maternal homologous chromosomesbefore the first meiotic cell division. Recombination is initiated by an enzyme that makes a double-strandedbreak in the DNA of one chromosome at any one of a very large number of sites.The process diagrammed in Figure 4-42 is then followed. The entire process from cleavageof the DNA of one chromosome through resolution of the Holliday structuresis repeateduntil at least one recombination, also called a crossouer'occurs between one pair of each of the homologous chromosomes. As mentioned earlier,the resulting link betweenhomologous chromosomesis required for their proper segregationduring the first meiotic cell division (seeChapter 20). As a consequence,every germ cell contains multiple recombinant chromosomes made of large segmentsof either the maternal or the oaternal chromosome.

DNA Repair and Recombination Changesin the DNA sequenceresult from copying errs and the effects of various physical and chemical agents. r Many copying errors that occur during DNA replication are corrected by the proofreading function of DNA polymerasesthat can recognize incorrect (mispaired) basesat the 3' end of the growing strand and then remove them by activity (seeFigure4-34). an inherent3'-+5' exonuclease

E

3'

5',

5',

--.: |

5',

3',

I

t AI 4-43 Alternativeresolutionof a Hollidaystru€ture. FIGURE bond.lt linesrepresent a singlephosphodiester Diagonal andvertical the diagram of the by rotating to diagram the process issimplest have 180'sothatthetop andbottommolecules bottommolecule Cuttinqthe bondsasshownin Il and the samestrandorientations

chromosomes theoriginal regenerates theendsasindicated ligating asshownat the asshownin E andreligating Cuttingthe strands Seehttp://engels chromosomes recombtnant bottomgenerates for a three-dimensional genetics.wisc.edu/Holliday/holliday3D.html anditsresolution structure of the Holliday animation AND RECOMBINATION O DNA REPAIR

153

r Eukaryotic cells have three excision-repair systemsfor correcting mispaired basesand for removing W-induced thymine-thymine dimers or large chemical adducts from DNA. Base excision repair, mismatch repair, and nucleotide excision repair operate with high accuracy and generally do not introduce errors. r Repair of double-strand breaks by the nonhomologous end-joining pathway can link segmentsof DNA from different chromosomes, possibly forming an oncogenic translocation. The repair mechanismalso producesa small deletion, even when segmentsfrom the same chromosome are joined. r Inherited defectsin the nucleotideexcision-repairpathway, as in individuals with xeroderma pigmentosum, predisposethem to skin cancer.Inherited colon cancer frequently is associatedwith mutant forms of proteins essentialfor the mismatch repair pathway. Defects in repair by homologous recombination are associatedwith inheritanceof one murant alleleof the BRCA-1 or BRCA2 gene and result in predispositionto breast and uterine cancer. r Error-free repair of double-strand breaks in DNA is accomplished by homologous recombination using the undamaged sister chromatid as a template. This processcan lead to recombination of parental chromosomesand is exploited by eukaryotes to generate genetic diversity by recombination of paternal and maternal chromosomes in developinggerm cells.

of the Cellutar Wl Viruses:Parasites GeneticSystem Virusesare obligate,intracellularparasites.They cannot reproduce by themselvesand must commandeera host cell's machineryto synthesizeviral proteins and in some casesto replicate the viral genome. RNA viruses, which usually replicate in the host-cell cytoplasm, have an RNA genome, and DNA viruses,which commonly replicate in the hostcell nucleus, have a DNA genome (seeFigure 4-1). Viral genomesmay be single-or double-stranded,dependingon the specific type of virus. The entire infectious virus particle, called a virion, consistsof the nucleicacid and an outer shell of protein that both protects the viral nucleic acid and functions in the processof host-cell infection. The simplest viruses contain only enough RNA or DNA to encode four proteins; the most complex can encode=200 proteins. In addition to their obvious importanceas causesof disease,viruses are extremely useful as researchtools in the study of basic biological processes,such as thosediscussedin this chaoter.

Most Viral Host RangesAre Narrow The surfaceof a virion contains many copies of one type of protein that binds specificallyto multiple copies of a receptor protein on a host cell. This interaction determines the

154

CHAPTER 4

|

host range-the group of cell types that a virus can infectand beginsthe infection process.Most viruses have a rather limited host range. A virus that infects only bacteria is called a bacteriophage, or simply a phage. Viruses that infect animal or plant cells are referred to generally as animal uiruses or plant uiruses.A few viruses can grow in both plants or animals and the insects that feed on rhem. The highly mobile insects serve as vectors for transferring such viruses between susceptibleplant or animal hosts. Wide host ranges are also characteristicof some strictly animal viruses,such as vesicularstomatitis virus, which grows in insect vectors and in many different types of mammals. Most animal viruses,however, do not cross phyla, and some (e.g.,poliovirus) infect only closely related speciessuch as primates. The host-cell range of some animal viruses is further restricted to a limited number of cell types because only these cells have appropriare surface receptors to which the virions can attach.

V i r a l C a p s i d sA r e R e g u l a rA r r a y so f O n e o r a Few Typesof Protein The nucleic acid of a virion is enclosedwithin a protein coat, or capsid, composed of multiple copies of one prorein or a few different proteins, each of which is encoded by a single viral gene.Becauseof this structure, a virus is able to encode all the information for making a relatively large capsid in a small number of genes.This efficient use of geneticinformation is important, since only a limited amount of DNA or RNA, and therefore a limited number of genes,can fit into a virion capsid. A capsid plus the enclosednucleic acid is called a nucleocapsid. Nature has found two basic ways of arranging the multiple capsid protein subunits and the viral genome into a nucleocapsid.In some viruses,multiple copies of a single coat protein form a helical structure that enclosesand protects the viral RNA or DNA, which runs in a helical groove within the protein tube. Viruses with such a helical nucleocapsid, such as tobacco mosaic virus, have a rodlike shape (Figure 4-44a). The other major structural type is based on the icosahedron, a solid, approximately spherical object built of 20 identical faces,each of which is an equilateral triangle (Figure 4-44b). During infection, some icosahedral viruses interact with host cell-surfacereceptors via clefts in between the capsid subunits; others interact via long fiberlike proteins extending from the nucleocapsid. In many DNA bacteriophages,the viral DNA is located within an icosahedral "head" that is attached to a rodlike "tail." During infection, viral proteins at the tip of the tail bind to host-cellreceptors,and then the viral DNA passesdown the tail into the cytoplasm of the host cell (Figure 4-44c). In someviruses,the symmetricallyarrangednucleocapsid is coveredby an external membrane,or envelope,which consistsmainly of a phospholipid bilayer but also conrains one or two types of virus-encodedglycoproteins (Figure 4-44d).

BASTC M O L E C U L AG R E N E T TM CE C H A N T S M S

Poliovirus

Tobaccomosaicvirus (c)

(d)

50 nm

50 nm

A v i a n i n f l u e n z av i r u s

(a)Helical vlrus tobaccomosaic 4-44 Virionstructures. < FIGURE ( b )S m a li lc o s a h e dvr a i rlu sT h ee x a m p lseh o w ni sp o l i o v i r u s . of an enveloped virus,an example (c)Bacteriophage Ta (d)Influenza part(b) Library; Photo (a)O Bradfute, Arnold/Science Peter virus[Part Biozentrum/ (c) of Microbiology, part Department of T S Baker; courtesy t oe s e a r c h e rl sn,c l S c l e n c eP h o t oL i b r a r yp; a r t ( d ) J a m e sC a v a l l i n i / P h o R

T4 Bacteriophage

The phospholipidsin the viral envelopeare similar to thosein the plasmamembraneof an infectedhost cell.The virai envelope is, in fact, derivedby budding from that membrane,but contains mainly viral glycoproteins,as we discussshortly.

V i r u s e sC a n B e C l o n e da n d C o u n t e d i n P l a q u eA s s a y s The number of infectiousviral particlesin a samplecan be quantifiedby a plaque assay.This assayis performedby culturing a dilute sample of viral particleson a plate covered with host cells and then counting the number of local Iesions,called plaques,that develop (Figure 4-45). A plaque developson the plate wherevera singlevirion initially infects

a singlecell. The virus replicatesin this initial host cell and then lyses(ruptures)the cell, releasingmany progenyvirions that infect the neighboring cells on the plate. After a few such cyclesof infection, enough cells are lysed to produce a visibleclear area,the plaque,in the layer of remaininguninfectedcells. Sinceall the progeny virions in a plaque are derived from a single parental virus, they constitute a virus clone. This type of plaque assayis in standardusefor bacterialand animal viruses.Plant virusescan be assayedsimilarly by counting local lesions on plant leavesinoculated with viruses. Analysis of viral mutants' which are commonly isolated by plaque assays,has contributedextensivelyto current understandingof molecularcellularprocesses.

SYSTEM RENETIC V I R U 5 E SP: A R A S I T EOSF T H E C E L L U L A G

155

(a)

Confluentlayer of susceptiblehost cells growing on surfaceof a plate

A d d d i l u t es u s p e n s i o nc o n t a i n i n gv i r u s ; after infection,cover layer of cells with agar; incubate Plaoue

3. Replication-Yiral mRNAs are produced with the aid of the host-cell transcription machinery (DNA viruses)or by viral enzymes(RNA viruses).For both types of viruses, viral mRNAs are translated by the host-cell translation machinery. Production of multiple copies of the viral genome is carried out either by viral proteins alone or with the help of host-cell proteins. 4. Assembly-Yiral proteins and replicated genomesassociate to form progeny virions. 5. Release-Infected cell either ruptures suddenly (lysis), releasingall the newly formed virions at once, or disintegratesgraduallS with slow releaseof virions. Both cases lead to the death of the infected cell.

Eachplaque representscell lysis initiatedby one viral particle(agar restrictsmovement so that virus can infectonly contiguouscells)

Figure 4-46 rllustratesthe lytic cycle forT4 bacteriophage,a nonenveloped DNA virus that infects E. coli. Viral capsid proteins generally are made in large amounts becausemany

Freevirion

E. coli

chromosome

Xffi;ffi["-E a nn d d iinn i aj ne f ic n nt i o n a

EsE *?

,

/

/.. ,, 'll

l

l.: .T4DNA

Plaque

A EXPERIMENTAL FTGURE 4-45 The plaqueassaydetermines the numberof infectiousparticlesin a viral suspension. (a)Each lesion, or plaque, whichdevelops wherea singlevirioninitially infected a singlecell,constitutes a pureviralclone.(b)plaques on a lawnof Pseudomonasfluorescens bacteriamadeby bacteriophage polytechnique of Dr Pierre Rossi, $S1 [Part(b)Courtesy Ecole F6d€rale de (LBE-EPFL) Lausanne l Replication of viralDNA Expression of virallateproteins

LyticViral Growth CyclesLeadto the Death of Host Cells Although details vary among different types of viruses,those that exhibit a lytic cycle of growth proceed through the following general stages: l, Adsorption-Virion interacts with a host cell by binding of multiple copies of capsid protein to specificreceptors on the cell surface. 2. Penetration-Yiral genome crossesthe plasma membrane. For some viruses,viral proteins packagedinside the capsid also enter the host cell.

155

C H A P T E R4

|

A FIGURE 4-46 tytic replicationcycleof a nonenveloped, bacterialvirus.E.colibacteriophage T4 hasa double-stranded DNA genomeandlacksa membrane envelope Afterviralcoatproteins at thetip of thetailin T4 interact with specific receptor proteins on the exterior of the hostcell,theviralgenomeisinjected intothe host (stepIl). Host-cell enzymes thentranscribe viral"early"genesinto mRNAs andsubsequently translate theseintoviral"early"proteins (stepZ) Theearlyproteins replicate theviralDNAandinduce expression of viral"late"proteins (stepS) The by host-cell enzymes virallateproteins include capsid andassembly proteins andenzymes thatdegrade the host-cell DNA,supplying nucleotides for synthesis of moreviralDNA Progeny virions areassembled in thecell(stepZl) (step5) whenviralproteins andreleased lysethecell.Newly liberated viruses initiateanothercycleof infection in otherhost ceils

B A S t CM O L E C U L A RG E N E T T M C ECHANTSMS

Rabiesvirus *

N u c l e o c a p s i dp r o t e i n Matrixprotein

't

R e c e p t o r - b i n d i nggl y c o p r o t e i n

o V i r a lR N A p o l y m e r a s e

arOOinS $ V i r u s r e c e p t o r C e l lm e m b r a n e

;{b :'

I'

i

':

..,/ Cytosol

\enoo"vtori.

I Transnort fl

M a t r i xa n d n u c l e o c pa s i d synthesrs

A { Replication h' l.

t ER

.{

Transcription Nucleus

E# Release

FIGURE4-47 Lytic replication cycle of an enveloped, animal e dN A v i r u s . R a b i evsi r u si s a n e n v e l o p evdi r u sw i t h a s i n g l e - s t r a n d R genome The structuralcomponentsof this virusare depictedat the top After a virionadsorbsto multiplecopiesof a specifichost membraneprotein(steptr), the cellengulfsit in an endosome ( s t e pE ) A c e l l u l apr r o t e i ni n t h e e n d o s o m em e m b r a n ep u m p s H* ionsfrom the cytosolinto the endosomeinterior.The resulting s c o n f o r m a t i o n cahl a n g ei n t h e d e c r e a sien e n d o s o m apl H i n d u c e a leadingto fusionof the viralenvelopewith the viralglycoprotein, r e m b r a n ea n d r e l e a soef t h e n u c l e o c a p s i d e n d o s o m al il p i db i l a y em uses into the cytosol(stepsB and 4) ViralRNApolymerase in the cytosolto replicatethe viralRNA ribonucleoside triphosphates genome(stepE) and to synthesize viralmRNAs(step6) One of the glycoprotein, which is viralmRNAsencodesthe viraltransmembrane ( E R a) s i t i s r n s e r t e idn t o t h e m e m b r a n eo f t h e e n d o p l a s m ri ce t i c u l u m

(stepZ). Carbohydrate isadded ribosomes on ER-bound synthesized t h e i n s i d e E Rl u m e na n di sm o d i f i eads t o t h el a r g ef o l d e dd o m a i n passthroughthe glycoprotein andtheassociated the membrane fuse (stepE) Vesicles with matureglycoprotern Golgiapparatus on the viralglycoprotein depositing membrane, with the hostplasma the cell domainoutside withthe largereceptor-binding cellsurface on host-cell aretranslated (step9) Meanwhile, otherviralmRNAs andviralRNA protein, matrixprotein, intonucleocapsid ribosomes with replicated areassembled polymerase GtepIE). Theseproteins nucleocapsids GtepI[), RNA(brightred)intoprogeny viralgenomic domainof viraltransmembrane withthecytosolic whichthenassociate (stepIE) Theplasma membrane in the plasma glycoproteins t h en u c l e o c a p sf oi dr ,m i n ga " b u d "t h a t m e m b r a ni sef o l d e da r o u n d (stePIE) isreleased eventually

copiesof them are requiredfor the assemblyof eachprogeny virion. In eachinfectedcell, about 100-200 T4 progenyvirions are producedand releasedby lysis. The lytic cycle is somewhatmore complicatedfor DNA virusesthat infect eukaryoticcells.In most suchviruses,the DNA genome is transported (with some associated proteins)into the cell nucleus.Once inside the nucleus,the viral DNA is transcribedinto RNA by the host'stranscription machinery.Processingof the viral RNA primary transcript by host-cell enzymesyields viral mRNA, which is transportedto the cytoplasmand translatedinto viral pro-

teins by host-cellribosomes,tRNA, and translationfactors. The viral proteins are then transported back into the nucleus,where some of them either replicate the viral DNA directly or direct cellular proteinsto replicatethe viral DNA' as in the case of SV40 discussedearlier.Assembly of the capsid proteins with the newly replicated viral DNA occurs in the nucleus,yieldingthousandsto hundredsof thousands of progenyvirions. Most plant and animal viruseswith an RNA genome do not require nuclear functions for lytic replication. In some of these viruses, a virus-encoded enzyme that enters the host

G E N E T I CS Y S T E M SF T H E C E L L U L A R V I R U S E SP: A R A S I T EO

157

d u r i n g p e n e t r a t i o n t r a n s c r i b e st h e g e n o m i c R N A i n t o mRNAs in the cell cytoplasm. The mRNA is directly translated into viral proteins by the host-cell translation machinery. One or more of theseproteins then produces additional copies of the viral RNA genome. Finally, progeny genomes are assembledwith newly synthesizedcapsid proteins into p r o g e n yv i r i o n si n t h e c y t o p l a s m . After the synthesisof hundreds to hundreds of thousandsof new virions has beencompleted,dependingon the type of virus and host cell, most infecredbacterialcellsand some infectedplant and animal cellsare lysed,releasingall the virions at once. In many plant and animal viral infections, however, no discretelytic event occurs; rather, the dead host cell releasesthe virions as it gradually disintegrates. As noted previously,envelopedanimal virusesare surrounded by an outer phospholipid layer derived from the plasma membrane of host cells and containing abundant viral glycoproteins. The processesof adsorption and release of envelopedviruses differ substantially from these p r o c e s s e sf o r n o n e n v e l o p e dv i r u s e s . T o i l l u s t r a t e l y t i c replication of enveloped viruses, we consider the rabies v i r u s , w h o s e n u c l e o c a p s i dc o n s i s t so f a s i n g l e - s t r a n d e d RNA genome surrounded by multiple copies of nucleocapsid protein. Like most other lytic RNA viruses,rabies virions are replicated in the cytoplasm and do not require host-cell nuclear enzymes.As shown in Figure 4-47, a rabies virion is adsorbedto a host cell by binding to a specific cell-surfacereceptor moleculeand then entersthe cell by endocytosis.Progeny virions are releasedfrom a host cell by budding from the host-cellplasma membrane.Budding virions are clearly visible in electron micrographs of infected cells, as illustrated in Figure 4-48. Many tens of thousands of progeny virions bud from an infected host cell before it dies.

V i r a l D N A l s I n t e g r a t e di n t o t h e H o s t - C e l l G e n o m ei n S o m eN o n l y t i cV i r a l G r o w t h C y c l e s Some bacterial viruses, called temperate phages,can establish a nonlytic associationwith their host cellsthat doesnot kill the cell. For example,when bacteriophageinfectsE. coli, the viral DNA may be integrated into the host-cell chromosome rather than being replicated. The integrated viral DNA, called a prophage, is replicated as part of the cell's DNA from one host-cell generation to the next. This phenomenon is referred to as lysogeny. Under certain conditions, the prophage DNA is activated,leading to its excision from the host-cell chromosome and entrance into the lytic cycle, with subsequentproduction and releaseof progeny vlrtons. The genomesof a number of animal viruses also can rntegrateinto the host-cell genome. One of the most important are the retroviruses,which are envelopedviruses with a genomeconsistingof two identical srrandsof RNA. These viruses are known as retrouiruses because their RNA genome acts as a template for formation of a DNA

158

CHAPTER 4

I

A EXPERIMENTAL FIGURE 4-48 Progenyvirionsare released by budding.Progeny virions of enveloped viruses arereleased by buddingfrominfected cellsIn thistransmission electron micrograph of a cellinfected with measles virus,virionbudsareclearly visible protruding fromthe cellsurfaceMeasles virusisan enveloped RNA viruswith a helical nucleocapsid, likerabies virus,andreplicates as illustrated in Figure4-47 [From A Levine. 1991 Scientific , Viruses, p 221 American tibrary,

molecule-the opposite flow of geneticinformation compared with the more common transcription of DNA into RNA. In the retroviral life cycle (Figure 4-49), a viral enzyme called reversetranscriptaseinitially copies the viral RNA genome into single-strandedDNA complementary to the virion RNA; the same enzyme then catalyzessynthesisof a complementaryDNA strand. (This complex reaction is detailed in Chapter 6 when we consider closely r e l a t e d i n t r a c e l l u l a r p a r a s i t e sc a l l e d r e t r o t r a n s p o s o n s . ) The resulting double-strandedDNA is integrated into the chromosomal DNA of the infected cell. Finally, the integrated DNA, called a provirus, is transcribed by the cell's own machinery into RNA, which either is translated into viral proteins or is packagedwithin virion coat proteins to form progeny virions that are releasedby budding from the host-cell membrane. Becausemost retrovirusesdo not kill their host cells, infected cells can replicate,producing daughter cells with integrated proviral DNA. These daughter cells continue to rranscribe the proviral DNA and bud progeny virions. Some retrovirusescontain cancer-causing genes (oncogenes),and cellsinfectedby suchretrovirusesare oncogenically transformedinto tumor cells.Studiesof oncogenicretroviruses (mostly virusesof birds and mice) have revealeda great deal about the processes that leadto transformationof a normal cell into a cancercell (Chapter25).

BASTC MOLECULAG R E N E T TM CE C H A N T S M S

llll|

overview Animation: Life Cycle of a Retrovirus Genomic ssRNA Retrovirus Host-cell

Reverse transcription

Transportto n u c l e u sa n d integration

I \

*Y

havea genome FIGURE 4-49 Retrovirallife cycle.Retroviruses RNA and an outer copies of single-stranded of two identical interact in the envelope envelopeStep[: Afterviralglycoproteins protein, envelope host-cell membrane the retroviral with a specific membrane, allowing entryof the with the plasma fusesdirectly intothe cytoplasm of the cell StepE: Viralreverse nucleocapsid genomeintoa andotherproteins copytheviralssRNA transcriptase intothe DNA StepB: TheviraldsDNAis imported double-stranded

Among the known human retrovirusesare human Tc e l l l y m p h o t r o p h i c v i r u s ( H T L V ) , w h i c h c a u s e sa form of leukemia, and human immunodeficiency virus ( H I V ) , w h i c h c a u s e sa c q u i r e d i m m u n e d e f i c i e n c y s y n drome (AIDS). Both of these viruses can infect only specific cell types, primarily certain cells of the immune system and, in the caseof HIV, some central nervous system neurons and glial cells. Only these cells have cell-surface receptors that interact with viral envelope proteins, accounting for the host-cell specificity of these viruses.Unlike most other retroviruses,HIV eventually kills its host cells. The eventual death of large numbers of immunesystem cells results in the defectiveimmune response characteristicof AIDS. Some DNA viruses also can integrate into a host-cell chromosome. One example is the human papillomaviruses (HPVs), which most commonly cause warts and other benign skin lesions. The genomes of certain HPV serotypes, however,occasionallyintegrate into the chromosomal DNA of infected cervical epithelial cells, initiating developmentof cervical cancer. Routine Pap smears can detect cells in the early stagesof the transformation processinitiated by HPV integration, permitting effectivetreatment. I

sitesin the hostintooneof manypossible andintegrated nucleus chromosome onlyonehost-cell DNA.Forsimplicity, cellchromosomal istranscribed viralDNA(provirus) isdepictedStepZl: Theintegrated (darkred)and generating mRNAs RNApolymerase, bythe host-cell (brightred).Thehost-cell machinery genomicRNAmolecules andnucleocapsid intoglycoproteins theviralmRNAs translates by andarereleased virions thenassemble proteins. StepEt: Progeny 4-47. in Figure buddingasillustrated

Viruses: Parasitesof the Cellular Genetic System r Viruses are small parasitesthat can replicate only in host cells. Viral genomes may be either DNA (DNA viruses)or RNA (RNA viruses)and either single-or doublestranded. r The capsid, which surrounds the viral genome, is composed of multiple copiesof one or a small number of virusencodedproteins. Somevirusesalso have an outer envelope' which is similar to the plasma membrane but contains viral transmembraneproteins. r Most animal and plant DNA viruses require host-cell nuclear enzymesto carry out transcription of the viral genome into mRNA and production of progeny genomes' In contrast, most RNA viruses encode enzymesthat can transcribe the RNA genomeinto viral mRNA and produce new copies of the RNA genome. r Host-cell ribosomes, tRNAs, and translation factors are used in the synthesisof all viral proteins in infected cells.

G E N E T I CS Y S T E M SF T H E C E L L U L A R V I R U S E SP: A R A S I T EO

159

r Lytic viral infection entails adsorption, penetration, synthesisof viral proteins and progeny genomes(replication), assemblyof progeny virions, and releaseof hundreds to thousandsof virions, leadingto death of the host cell (seeFigure 4-46).Releaseofenveloped vrrusesoccurs by budding through the host-cell plasma membrane (see Figure 4-471. r Nonlytic infection occurs when the viral genome is integrated into the host-cell DNA and generally does not lead to cell death. r Retroviruses are enveloped animal viruses containing a single-stranded RNA genome. After a host cell is penetrated, reversetranscriptase,a viral enzymecarried in the virion, converts the viral RNA genome into doublestranded DNA, which integratesinto chromosomal DNA (seeFigure4-49). r Unlike infection by other retroviruses,HIV infection eventually kills host cells, causing the defects in the immune responsecharacteristicof AIDS. r Tumor viruses, which contain oncogenes,may have an RNA genome (e.g.,human T:cell lymphotrophic virus) or a DNA genome (e.g., human papillomaviruses).In the case of theseviruses,integration of the viral genomeinto a hostcell chromosome can causetransformation of the cell into a tumor cell.

In this chapter we first reviewed the basic structure of DNA and RNA and then describedfundamental aspectsof the transcription of DNA by RNA polym.rur.t. RNA polymerases are discussed in greater detail in Chapter 7, along with additional factors required for transcriotion i n i t i a t i o n i n e u k a r y o t i cc e l l sa n d i n t e r a c t i o n sw i t h . e g u l a tory transcription factors that control transcription initiation in both bacterial and eukaryotic cells. Next, we discussedthe geneticcode and the participation of IRNA and the protein-synthesizingmachine, the ribosome, in decoding the information in mRNA to allow accurateassembly of protein chains. Mechanisms that regulate protein synthesis are consideredfurther in Chapter 8. Then, we consideredthe molecular detailsunderlying the accuratereplication of DNA required for cell division. Chapter 20 coversthe mechanismsthat regulatewhen a cell replicates its DNA and that coordinate DNA replication with the complex process of mitosis that distributes the daughter DNA molecules equally to each daughter cell. The next section addressedmechanisms for repairing damage to DNA, including recombination mechanismsthat also lead to the generationof geneticdiversity among individuals of a species.This genetic recombination contributes to the diversity of traits subjectedro natural selectionduring the evolution of contemporary species.In Chapter 20, we discuss the mechanisms that segregatechromosomes into haploid germ cells, a processthat requires recombination 160

.

c H A p r E4R |

between maternal and paternal chromosomes.Finally, we discussed viruses, parasites of the cellular molecular genetic system and important model systemsand useful tools for studying multiple aspects of molecular cell biology. The basicmolecular geneticprocessesdiscussedin this chapter form the foundation of contemporary molecular cell biology. Our current understandingof theseprocesses is grounded in a wealth of experimentalresults and is not likely to change.However, the depth of our understanding will continue to increaseas additional details of the structures and interactionsof the macromolecularmachinesinvolved are uncovered. The determination in recent years of the three-dimensionalstructures of RNA polymerases, ribosomal subunits, and DNA replication proteins has allowed researchersto design ever more penerraung experimental approachesfor revealinghow thesemacromoleculesoperate at the molecular level. The detailed level of understanding currently being developed may allow the design of new and more effective drugs for treating illnessesof humans, crops, and livestock.For example,the recent high-resolutionstructuresof ribosomesare providing insights into the mechanism by which antibiotics inhibit bacterial protein synthesiswithout affecting the function of mammalian ribosomes. This new knowledge may allow the design of even more effective antibiotics. Similarln detailed understanding of the mechanisms regulating transcription of specifichuman genesmay lead to therapeutic strategies that can reduce or prevent inappropriate immune responsesthat lead to multiple sclerosisand arthritis, the inappropriate cell division that is the hallmark of cancer, and other pathological processes. Much of current biological researchis focusedon discovering how molecular interactions endow cells with decision-making capacity and their special properties. For this reason severalof the following chapters describecurrent knowledge about how such interactions regulate transcription and protein synthesisin multicellular organisms and how such regulation endows cells with the capacity to become specializedand grow into complicated organs. Other chapters deal with how protein-protein interactions underlie the construction of specialized organellesin cells, and how they determine cell shapeand movement. The rapid advancesin molecular cell biology in recent years hold promise that in the not too distant future we will understand how the regulation of specialized cell function, shape,and mobility coupled wirh regulared c e l l r e p l i c a t i o n a n d c e l l d e a t h ( a p o p t o s i s )l e a d t o t h e growth of complex organisms like flowering plants and human beings.

KeyTerms anticodon127 codons127 complementary L14

B A S tM c o L E c u L AGRE N E TM t cE c H A N t s M s

crossingover 150 deamination 146 depurination 147

DNA end-joining 149

polyribosomes138

DNA polymerases141

primary transcript 121

double helix 114 envelope(viral) 154

primer 141

excision-repair systems147

reading frame 1.28

exons 123 geneconversion153

recombination112

genetic code 127

retroviruses152

Holliday structure154

reversetranscriptase152

homologousrecombination repair L50

ribosomal RNA (rRNA) 112

introns 123

ribosomes127

lagging strand 141

RNA polymerase120

leading strand 141

thymine-thymine dimers 148

messengerRNA (mRNA) 112

promoter 1-21-

replicationfork 141

mutation 138

transcription1 12 transferRNA (IRNA) 112

Okazaki fragments 141 phosphodiesterbond 1 14

translation 112 'Watson-Crick basepairs 114

Review the Concepts l. \fhat are'Watson-Crickbasepairs? lWhy are they important? 2, TAIA box-binding protein binds to the minor groove of DNA, resulting in the bending of the DNA helix (seeFigure 4-5). Vhat property of DNA allows the TAIA box-binding protein to recognizethe DNA helix? 3. Preparing plasmid (double-stranded,circular) DNA for sequencinginvolves annealing a complementary,short, single-strandedoligonucleotide DNA primer to one strand of the plasmid template. This is routinely accomplishedby heating the plasmid DNA and primer to 90 "C and then 'C. \ilhy does slowly bringing the temperature down to 25 this protocol work? 4. \fhat difference between RNA and DNA helps to explain the greater stability of DNA? What implications does this have for the function of DNA? 5. What are the major differencesin the synthesisand structure of prokaryotic and eukaryotic mRNAs? 6, 'While investigatingthe function of a specificgrowth factor receptor gene from humans, researchersfound that two types of proteins are synthesizedfrom this gene. A larger protein containing a membrane-spanningdomain functions to recognizegrowth factors at the cell surface,stimulating a specific downstream signaling pathway. In contrast, a related, smaller protein is secretedfrom the cell and functions to bind availablegrowth factor circulating in the blood, thus inhibiting the downstream signaling pathway. Speculateon how the cell synthesizesthesedisparateproteins. 7. The transcription of many bacterial genesrelieson functional groups called operons,such as the tryptophan operon

'$7hat advantagesare (Figure 4-1.3a).'What is an operon? there to having genesarrangedin an operon' compared with the arrangementin eukaryotes? 8. Contrast how selectionof the translational start site occurs on bacterial,eukaryotic, and poliovirus mRNAs. 9. \fhat is the evidence that the 23S rRNA in the large rRNA subunit has a peptidyltransferaseactivity? 10. How would a mutation in the poly(A)-binding protein I gene affect translation? How would an electron micrograph of polyribosomesfrom such a mutant differ from the normal pattern? L1. What characteristic of DNA results in the requirement that some DNA synthesis is discontinuous? How are Okazaki fragments and DNA ligase utilized by the cell? L2. Eukaryotes have repair systems that prevent mutations due to copying errors and exposure to mutagens. 'What are the three excision-repair systemsfound in eukaryotes, and which one is responsible for correcting thymine-thymine dimers that form as a result of UV light damageto DNA? 13. DNA-repair systemsare responsiblefor maintaining genomic fidelity in normal cellsdespitethe high frequencywith which mutational eventsoccur.'Sfhattype of DNA mutation is generatedby (a) UV irradiation and (b) ionizing radiation? Describe the system responsiblefor repairing each of these types of mutations in mammalian cells. Postulatewhy a loss of function in one or more DNA-repair systems typifies many cancers. 14. lVhat is the name given to the processthat can repair DNA damage and generate genetic diversity? Briefly describe the similarities and differences of the two processes. 15. The genome of a retrovirus can integrate into the host-cellgenome.What geneis unique to retroviruses,and why is the protein encoded by this gene absolutely necessary for maintaining the retroviral life cycle?A number of retroviruses can infect certain human cells. List two of them, briefly describe the medical implications resulting from these infections, and describewhy only certain cells are infected.

Analyze the Data Protein synthesisin eukaryotes normally begins at the first AUG codon in the mRNA. Sometimes,however' the ribosomes do not begin protein synthesisat this first AUG but scan past it (leaky scanning),and protein synthesisbegins instead at an internal AUG. In order to understandwhat features of an mRNA affect efficiency of initiation at the first AUG, studies have been undertaken in which the synwas examined. thesisof chloramphenicolacetyltransferase protein referred a give rise to Translation of its messagecan protein, CAT smaller a slightly give rise to to as preCM or (see M. Kozak. 2005. Gene 367:1'3). The two proteins differ in that CAT lacks several amino acids found at the A N A L Y Z ET H E D A T A

T

161

N-terminus of preCAT. CAT is not derived by cleavageof preCAT but, instead, by initiation of translation of the mRNA at an internal AUG:

precAT Start

CAT Start

Stop

I

ll

vt m 7 c p p p " , ' ' , ", A U G 12

,:r [J[[*;,[[[[n

AUG

a. Resultsfrom a number of studieshave given rise to the hypothesisthat the sequence(-3)ACCAUGG(+4), in which the start codon AUG is shown in boldface,provides an optimal context for initiation of protein synthesisand ensures that ribosomesdo not scanpast this first AUG to begin initiation insteadat a downstreamAUG. In the numbering scheme used here, the A of the AUG initiation is designated(*1); bases5' of this are given negarivenumbers [so that the first b a s eo f t h i s s e q u e n cies ( - 3 ) ] , a n d b a s e s3 ' t o t h e ( + 1 ) A a r e given positive numbers [so that the last baseof this sequence is (+4)]. To test the hypothesisthat the start sire sequence (-3)ACCAUGG(+4) preventsleaky scanning,the chloramphenicol acetyltransferase mRNA sequencewas modified and the resulting effectson translation assessed. In the following figure, the sequence(red) surrounding the first AUG codon (black) of the mRNA that gives rise to the synthesisof preCAT is shown above lane 3. Modification of this messageis shown above the other gel lanes (altered nucleotides are in blue), and the completedproteins generatedfrom eachmodified messageappear as bands on the SDS-polyacrylamide gel below. The intensity of each band is an indication of the amount of that protein synthesized.Analyzethe alterations to the wild-type sequence,and describe how they affect translation. Are the positions of some nucleotidesmore important than others? Do the data shown in this figure provide support for the hypothesisthat the context in which the first AUG is presentaffectsefficiencyof translation from this site? Is ACCAUGG an optimal contexr for initiation from the first AUG?

CCCCA -3UUAAC UUCCC UUCCA (t

AUG1 I A A A A A U U U U ^-^^"-1U

PrtrLAr

G G IC +4UGGAA

preCAT+ * CAT+*

***{} s

G

G

b. What are some additional alterations to this message, other than those shown in the figure, that would further elucidate the importance of the ACCAUGG .

c H A p r E R4

|

c. A mutation causing a severe blood diseasehas been found in a single family (seeT. Matthes et a1.,2004, Blood 104:2181). The mutation, shown in red in the fig, ure below, has been mapped to the 5' untranslatedregion of the gene encoding hepcidin and has been found to alter the gene'smRNA. The shadedregions indicate the coding sequenceof the normal and mutant genes.No hepcidin is produced from the altered mRNA, and lack of hepcidin resultsin the disease.Can you provide a reasonableexplanation for the lack of synthesisof hepcidin in the family memberswho have inherited this mutation? $fhat can you deduce about the importance of the context in which the start site for initiation of protein synthesisoccurs in this c a s e?

Starthepcidin .....cCAGUGGGACAGCCAGACAGACGGcncCnricccncUG..... Norma

I

Y .....GCAAUGGGACAGCCAGACAGACGGCACGAUGGCACU........ Mutant

References Structure of Nucleic Acids Arnott, S. 2006. Historical article: DNA polymorphism and the earfy history of the double helix. TrendsBiochem. Sci.3l:349-354. Berger,J. M., and J. C. Wang. 1996. Recentdevelopmentsin DNA topoisomeraseII structureand mechanism.Curr. Opin. Struc. Biol. 6:84-90. Dickerson,R. E. 1983. The DNA helix and how it is read. Sci. Am. 249:94-1.'1.1.. Dickerson,R. E., and H. L. Ng. 2001. DNA srructurefrom A to B. Proc. Nat'l Acad. Sci.USA.98:6986-69888. Doudna, J. A., and T. R. Cech.2002.The chemicalrepertoireof natural ribozymes.N ature 418:222-228. Kornberg, A., and T. A. Baker.2005. DNA Replication.University Science,chap. 1. A good summary of the principlesof DNA structure. Lilley, D. M. 2005. Structure,folding and mechanismsof ribozymes.Curr. Opin. Struc.Biol. t5:313-323. Vicens,Q., and T. R. Cech.2005. Atomic level architectureof group I introns revealed.TrendsBiochem. Sci.3l:41-51. 'Wang, J. C. 1980. SuperhelicalDNA. TrendsBiochem. Sci. 5:279-221. Wigley,D.B. 1995. Srructureand mechanismof DNA topoisomerases.Ann. Reu.Biophys. Biomol. Struc.24:L85-208.

+

Lane12345

'162

sequence as an optimal context for synthesis of preCAT rather than CAT? How would you further examine w h e t h e rA a t t h e ( - 3 ) p o s i t i o n a n d G a t t h e ( + 4 ) p o s i t i o n are the most important nucleotidesto provide context for the AUG start?

Transcription of Protein-Coding Genes and Formation of Functional mRNA Brenner,S., F. Jacob,and M. Meselson.1,96L.An unstableintermediatecarrying information from genesto ribosomesfor protein synthesis.Natur e 190:576- 5 8'1,. Murakami, K. S., and S. A. Darst. 2003. BacterialRNA polvmerases:the whole story.Curr. Opin. Strwc.Biol. t3:31-39.

B A S t cM o L E c u L A R G E N E I cM E c H A N t s M s

Okamoto K., Y. Sugino,and M. Nomura. 1962. Synthesisand turnover of phagemessengerRNA in E. coli infectedwith bacteriophageT4 in the presenceof chloromycetin.J. Mol. Biol. 5:527-534. Steitz,T. A. 2006. Visualizingpolynucleotidepolymerase machinesat work. EMBO J.25:3458-3468. The Decoding of mRNA by tRNAs Alexander,R. !(., and P. Schimmel.2001. Domain-domain comProg. Nucl. Acid Res. munication in aminoacyl-tRNA synthetases. Mol. Biol. 69:31,7-349. Hatfield, D. L., and V. N. Gladyshev.2002.How seleniumhas altered our understanding of the genetic code.Mol. Cell Biol. 22:3565-3576. Hoagland, M. B., et al. 1958. A solubleribonucleicacid intermediatein protein synthesis./. Biol. Chem.23l:241-257. Ibba, M., and D. Soll. 2004. Aminoacyl-tRNAs: settingthe limits of the geneticcode.GenesDeu. 18:731-738. Khorana, G. H., et al. 1966. Polynucleotidesynthesisand the geneticcode. Cold Spring Harbor Symp. Quant. Biol.3l:3949. Nakanishi, K., and O. Nureki. 2005. Recentprogressof structural biology of IRNA processingand modification. MoL Cells l91.57-166. Nirenberg, M., et al. 1966.The RNA code in protein synthesis. Cold Spring Harbor Symp. Quant. Biol. 3t:ll-24. Rich. A.. and S.-H. Kim. 1978. The three-dimensionalstructure of transfer RNA. Scl.Am.240(1,1:52-62(offprint 1.377). Stepwise Synthesis of Proteins on Ribosomes Abbott, C. M., and C. G. Proud. 2004. Translationfactors:in sicknessand in health. TrendsBiocbem.Sci.2925-131. Auerbach,T., A. Bashan,and A. Yonath. 2004. Ribosomal antibiotics:structural basisfor resistance,synergismand selectivity. TrendsB iotech nol. 22:570- 576. Frank, J., et al. 2005. The role of IRNA as a molecularspring in decoding,accommodation,and peptidyl transfer.FEBSLett. 579:959-962. Ganoza,M. C., M. C. Kiel, and H. Aoki. 2002. Evolutionary conservationof reactionsin translation.Microbiol. Mol. Biol. Reu. 66:460485. Gualerzi,C. O., et al. 2001. Initiation factorsin the early events of mRNA translation in bacteria.Cold Spring Harbor Symp. Quant. Biol. 66:363-376. Hellen, C. U., and P. Sarnow.2001. Internal ribosomeentry sitesin eukaryotic mRNA molecules.Genet.Deuel. 15:1593-161'2. Kahveiian,A., G. Roy, and N. Sonenbery.200l. The mRNA closed-loopmodel: the function of PABPand PABP-interactingproteins in mRNA translation.Cold Spring Harbor Symp. Quant. Biol. 66:293-300. Kapp, L. D., and J. R. Lorsch. 2004. The molecularmechanics of eukaryotictranslation.Ann. Reu.Biochem. 73:657-704. Mitra, K., and J. Frank. 2006. Ribosomedynamics:insights from atomic structuremodeling into cryo-electronmicroscopy maps.Ann. Reu.Biophys.Biomol. Struc.35:299-3t7. Noller, H. F. 2005. RNA structure:readingthe ribosome. Science309:1508-15 14. Noller, H. F., et al. 2002. Translocationof IRNA during protein synthesis.FEBSLett. 514i1.1.-16. Polacek,N., and A. S. Mankin. 2005. The ribosomal peptidyl transferasecenter:structure,function, evolution, inhibition' Cdr. Reu.Biocbem.Mol. Biol. 4O:285-31.1. Preiss,T., and M. W. Hentze.2003. Startingthe protein synthesis machine:eukaryotic translation initiation. BioEssays 25:1201-1.211'. Richter,J. D., and N. Sonenberg.2005. Regulationof capdependenttranslation by eIF4E inhibitory proteins.Nature 433:477480.

Scheper,G. C., C. G. Proud, and M. S' van der Knaap. 2005' Defectivetranslation initiation causesvanishingof cerebralwhite matter.TrendsMol. Med. t2:1'59-1'66. Sonenberg,N. 2006. TranslationalControl in Biology and Medicine (CSH monograph).Cold SpringHarbor Press. Sonenberg,N., and T. E. Dever.2003. Eukaryotic translation initiation factors and regulators.Curr. Opin. Struc.Biol' 13:56-63' Steitz,T. A. 2005. On the structural basisof peptide-bondformation and antibiotic resistancefrom atomic structuresof the large ribosomal subunit. FEBSLett. 579:955-958' Taylor, S. S., N. M. Haste' and G. Ghosh. 2005. PKR and eIF2ct:integration of kinasedimerization,activation' and substrate docking. Cell 122:823-82 5. DNA Replication Brautigam, C. A, and T. A. Steitz. 1998' Structural and functional insights provided by crystal structures of DNA polymerases and their snbtti"t. complexes.Curr. Opin. Struc. Biol. 8:54-63. Bullock, P.A. 1997. The initiation of simian virus 40 DNA replication in vitro. Crit. Reu.Biochem.Mol. Biol' 32:503'568. DePamphilis,M. L., ed. 2006 DNA Replicationand Human Disease.Cold SpringHarbor Laboratory Press. Kornberg. A., and T. A. Baker.2005. DNA Replication' University Science. Langston,L. D., and M. O'Donnell. 2005. DNA replication: keep moving and don't mind the gap.Mol. Cells23:155-160' Mendez,J., and B. Stillman.2003. Perpetuatingthe double helix: molecularmachinesat eukaryotic DNA replication origins' 25:11'58-I1'67. BioEssays O'Donnell, M.2006. Replisomearchitectureand dynamicsin Eschericbia coli. l. B iol. Cbem. 281:106 5 3-106 56. Sclafani,R. A., R. J. Fletcher,and X. S' Chen' 2004' Two heads are better than one: regulation of DNA replication by hexameric helicases.GenesDeu. 18:2039-2045. DNA Repair and Recombination Andressoo,J. O., and J. H. Hoeijmakers'2005. Transcriptioncoupled repair and premature aging.Mutat. Res.577:179-1'94' Barnes,D. 8., and T. Lindahl. 2004. Repair and geneticconsequencesof endogenousDNA basedamagein mammalian cells'Ann' Reu.Genet.38:445476. Bell, C. E. 2005. Structureand mechanismoI Escherichiacoli RecA ATPase.Mol. Microbiol. 58:358-366. Friedberg,E. C., et al' 2006. DNA repair: from molecularmechanism to human disease.DNA Repair 5:986-996. Haber,J. E. 2000. Partnersand pathwaysrepairing a doublestrand break. Trends Genet. 16:259-264. system'Na/' Jiricny,1.2006. The multifacetedmismatch-repair Reu.Mol. Cell Biol.7:335-346. Khuu. P.A.. et al. 2005. The stacked-XDNA Holliday iunction and protein recognition.l. Mol. Recog.192234-242. Lilley, D. M., and R. M. Clegg' t993-The structureof the four--ay jonction in DNA. Ann. Reu-Biophys. Biomol' Struc' 22:299-328. Mirchandani, K. D., and A. D. D'Andrea- 2006. The Fanconi anemia/BRCAp"lh*"yt a coordinator of cross-link repail Exp' Cell Res.372:2647-2653' Mitchell, J. R., J. H. Hoeijmakers,and L. J. Niedernhofer'2003' Divide and.*qu.t, nucleotideexcisionrepair battlescancerand aging.Curr. Opin. Cell Biol.l5:232-240. Orr-Weaver,T. L., and J. I7. Szostak.1985. Fungal recombination. Microbiol. Reu. 49:33-58. Shin, D. S., et al. 2004. Structureand function of the double strand break repair machinery.DNA Repair 32863-873' '!food, R. D., M. Mitchell, and T. Lindahl. Human DNA repair Res.577:275-283. Mutat. senes. R E F E R E N C E 5o

16 3

Yoshida,K., and Y. Miki. 2004. Role of BRCA1 and BRCA2 as re-gulatorsof DNA repair, transcription, and cell cycle in responseto DNA damage.CancerSci. 95,:865-87t.

Viruses:Parasites of the CellularGeneticSystem Flint,S.J.,et al. 2000.Principles of Virology: Molecular Biology, Pathogenesis,and Control. ASM press. Hull, R. 2002. Matheuts' Plant Virology. Academic press.

164

C H A P T E R4

I

B A S T CM O L E C U L A RG E N E T T M C ECHANTSMS

Klug, A. 1999.The tobacco mosaic virus particle: structure and assembly.Phil. Trans, R. Soc.Lond. B Biol. SZi.354:531-535. Knipe, D. M., and P. M. Howley eds. 2001. Fields Virology. Lippincott Villiams & Ifilkins. Kornberg, A., and T. A. Baker. 1992. DNA Replication,2d, ed,. W. H. Freeman and Company. Good summary of bacteriophagemolecular biology.

CHAPTER

GENETIC MOLECULAR TECHNIQUES (RNA|) mostgenesin the canbe usedto silence RNAinterference worm on the right (markedby C. elegansgenome Thetransgenic expresses dsRNAto the muscle a GFPreporterin headneurons) geneunc-\5, resultingin the potentdegradationof the unc-/5 of the worm ln contrast, mRNAand leadingto completeparalysis body the typicalsinusoidal the wild-typeworm on the leftexhibits of JohnKim] movement[Courtesy

I n previous chapters, we were introduced to the variety of I tasks that proteins perform in biological systems.Indeed' I the very field of molecular cell biology seeksto understand the molecular mechanismsof individual proteins and how groups of proteins work together to perform their biological functions. In studying a newly discoveredprotein' cell biologistsusually begin by asking three questionsabout it: what is its function, where is it located, and what is its structure?To answer thesequestions,investigatorsemploy three tools: the genethat encodesthe protein, a mutant cell line or organism that lacks the function of the protein, and a sourceof the purified protein for biochemicalstudies.In this chapter we consider various aspectsof two basic experimentalstrategiesfor obtaining all three tools (Figure5-1). The first strategy,often referred to as classicalgenetics, beginswith isolation of a mutant that appearsto be defective in some process of interest. Genetic methods then are used to identify and isolate the affected gene. The isolated genecan be manipulatedto produce large quantitiesof the protein for biochemical experiments and to design probes for studies of where and when the encoded protein is expressedin an organism.The secondstrategyfollows essentially the same stepsas the classicalapproach but in reverse order, beginning with isolation of an interesting protein or its identification based on analysis of an organism's genomic sequence.Once the corresponding gene has been isolated, the gene can be altered and then reinsertedinto an organism. In both strategies,by examining the phenoof mutations that inactivate a particutypic consequences lar gene, geneticistsare able to connect knowledge about the sequence,structure, and biochemical activity of the

encoded protein to its function in the context of a living cell or multicellular organism. An important component in both strategiesfor studying a protein and its biological function is isolation of the corresponding gene. Thus we discussvarious techniques by which res.archits can isolate, sequence'and manipulate specific re-

we discusstechniquesthat abolish normal protein function in order to analyzethe role of the protein in the cell.

OUTLIN 5.1

GeneticAnalysisof Mutationsto ldentify and StudYGenes

166

5.2

DNA Cloningand Characterization

176

5,3

UsingClonedDNA Fragmentsto StudyGene ExPression

5.4

ldentifying and LocatingHuman DiseaseGenes

5.5

Inactivatingthe Functionof Specific Genesin EukarYotes

165

> FIGURE 5-1 Overviewof two strategies for relatingthe function,location,and structureof gene products,A mutant organism isthestarting pointfor the classical geneticstrategy(greenarrows)Thereverse (orange strategy arrows) usually beginswith identification of a protein-codrng sequence by analysis of genomesequence databases In bothstrategies, the actualgeneisisolated eitherfroma DNAlibrary or by specific amplification of the genesequence from genomicDNA Oncea clonedgeneis isolated, it canbe usedto produce theencoded protein in bacterial or eukaryotic expression systems Alternatively, a clonedgenecanbe rnactivated by oneof various techniques andusedto generate mutantcellsor orqanisms

Mutant organism/cell Comparisonof mutant and wild-type function G e n e t i ca n a l y s i s Screeningof DNA library

Clonedgene DNA sequencing

to ldentifyand StudyGenes As describedin Chapter 4, the information encoded in the DNA sequenceof genesspecifiesthe sequence-and therefore the structure and function-of every protein moleculein a cell. The power of geneticsas a tool foi studying cells and organismslies in the ability of researchersto selectivelyalter every copy of just one type of protein in a cell by making a change in the gene for that protein. Genetic analysesof mutants defectivein a particular processcan reveal (a) new genes required for the process to occur, (b) the order in which gene products act in the process,and (c) whether the proteins encoded by different genes interact with one another. Before seeinghow geneticstudiesof this rype can provide insights into the mechanism of complicated cellular or developmental process,we first explain some basic genetic terms used throughout our discussion. The different forms, or variants, of a geneare referred to as alleles.Geneticistscommonly refer to the numerous naturally occurring genetic variants that exist in populations, particularly human populations, as alleles.The term mutation usually is reserved for instances in which an allele is known to have been newly formed, such as after treatment of an experimental organism with a muragen, an agent that causesa heritable changein the DNA sequence. Stricdyspeaking,the particular serof alielesfor all the genes carried by an individual is its genotype.However, this term also is usedin a more restrictedsenseto denoteiust the allelesof the

type usually denoresan allele that is present at a much higher frequency than any of the other possible alternatives. 166

.

cHAprER 5

|

Databasesearchto identify protein-codingsequence PCRisolationof corresponding gene

Expression in cultured cells

Genetic Analysis of Mutations

ff,t

Gene inactivation

M o L E c u L AG R E N E T tr cE c H N t e u E s

Protein Localization Biochemical studies Determ i nati o n of structu re

Geneticistsdraw an important distinction between the genotype and the phenotype of an organism. The phenotype refers to all the physical attributes or traits of an individual that are the consequenceof a given genotype. In practice, however, the term phenotype often is used to denote the physical consequencesthat result from just the alleles that are under experimental study. Readily observable phenotypic characteristicsare critical in the geneticanalysisof mutations.

R e c e s s i vaen d D o m i n a n tM u t a n t A l l e l e s GenerallyHave OppositeEffectson G e n eF u n c t i o n A fundamental genetic difference between experimental organismsis whether their cells ca:i::ya single set of chromosomes or two copies of each chromosome. The former are referred to as haploid; the latter, as diploid. Complex multicellular organisms (e.g., fruit flies, mice, humans) are diploid, whereasmany simple unicellular organismsare haploid. Someorganisms,notably the yeastSaccharomycescereuisiae, can exist in either haploid or diploid states. Many cancer cells and rhe normal cells of some organisms, both plants and animals) caffy more than two copies of each chromosome.However, our discussionof genetictechniques and analysis relates to diploid organisms, including dipioid yeasts. Although many different allelesof a gene might occur in different organisms in a population, any individual diploid organism will carry two copiesof eachgeneand thus at most can have two different alleles.An individual with two different allelesis heterozygousfor a gene, whereas an individual that carriestwo identical allelesis homozygousfor a gene.A recessivemutant allele is defined as one in which both alleles must be mutant in order for the mutant phenotype to be observed;that is, the individual must be homozygous for the mutant allele to show the mutant phenotype.In contrast, the phenotypic consequences of a dominant mutant allelecan be

;;-;-'*i

=

GENOTYPE I l-----t DIPLOID PHENOTYPE

Wild type

Dominant =

i M uta nt

mutant alleles 5-2 Effectsof dominantand recessive FIGURE on phenotypein diploidorganisms.A singlecopyof a dominant bothcopies whereas a mutantphenotype, to produce alleleissufficient observedin a heterozygousindividual carrying one mutant and one wild-type allele(Figure5-2). 'Whether a mutant allele is recessiveor dominant provides valuable information about the function of the affectedgene and the nature of the causativemutation' Recessivealleles usually result from a mutation that inactivates the affected gene,leadingto a partial or completelossof fwnctioz. Suchrecessivemutations may remove part of the gene or the entire genefrom the chromosome,disrupt expressionof the gene,or alter the structure of the encoded protein, thereby altering its function. Conversely,dominant alleles are often the consequenceof a mutation that causessome kind of gain of function. Such dominant mutations may increase the activity of the encodedprotein, confer a new function on it, or lead to its inappropriate spatial or temporal pattern of expression. Dominant mutations in certain genes,however,are associated with a lossof function. For instance,somegenesatehaploinswfficient, meaning that both allelesare required for normal function. Removing or inactivating a singleallele in such a gene leadsto a mutant phenotype.In other rare instancesa dominant mutation in one allelemay lead to a structuralchangein the protein that interferes with the function of the wild-rype protein encoded by the other allele. This type of mutation, referred to as a dominant-negatiue,producesa phenotype similar to that obtainedfrom a loss-of-functionmutation. Someallelescan exhibit both recessiveand dominant properties.In such cases,statementsabout whether is dominant or recessivemust specify the phenoallele an type. For example, the allele of the hemoglobin gene in humans designatedHb'has more than one phenotypicconsequence.Individuals who are homozygousfor this allele (Hb'/Hb') have the debilitating diseasesickle-cellanemia, but heterozygousindividuals (Hb'/Hb") do not have the disease.Therefore, Hb' is recessiuefor the trait of sicklecell disease.On the other hand, heterozygous(Hb'/Hb') individuals are more resistantto malaria than homozygous (Hb"/Hb") individuals, revealing that Hb' is dominant for the trait of malaria resistance.I

to causea mutantphenotype allelemustbe present of a recessive causea lossof function;dominant usually mutations Recessive causea gainof functionor an alteredfunction usually mutations

mutationsthan dominant mutations.

Segregationof Mutations in Breeding ExperimentsRevealsTheir Dominance or RecessivitY Geneticistsexploit the normal life cycle of an organism to test for the dominance or recessivityof alleles' To see how this is done, we need first to review the type of cell division that gives rise to gametes (sperm and egg cells in higher plants and animals). l7hereas the body (somatic)cells of -ort *.tl,i.ellular organisms divide by mitosis, the germ

their genesmay exist in different allelic forms' Figure 5-3 depicts the major eventsin mitotic and meiotic cell division' InLitosis DNA replication is always followed by cell division, yielding two diploid daughter cells' In meiosis oze round'of DNe replication is followed by two separatecell divisions, yieldingfour haploid (12) cells that contain only one chromosome of each homologous pair' The apportion-

A commonly used agent for inducing mutations (mutagenesis) in experimental organisms is ethylmethane sulfonate (EMS). Although this mutagen can alter DNA sequencesin severalways, one of its most common effectsis to chemically modify guanine basesin DNA, ultimately leading to the conversionof a G'C basepair into an A T basepair. Such an alteration in the sequenceof a gene,which involves only a singlebasepair, is known as a point mutation. A silent A N D s T U D YG E N E S TO IDENTIFY OF MUTATIONS ANALYSIS GENETIC

167

Focus Animation: Mitosisftttt Focus

Meiosis

MEIOTIC CELL DIVISION

Paternal homolog Maternal homolog

S o m a t i cc e l l( 2 n )

Premeioticcell l2nl

I DNA reptication

vI

v

Replicated cnromosomes (4nl

DNA reptication

Replicated cnromosomes

I

I Homologous chromosomes + align; synapsisand crossing over

v

Mitotic apparatus

Mitotic appara

;

MetaphaseI

o o o

I

I

v

v

/\

/ Ceil division \

++

D a u g h t e cr e l l s( 2 n )

o o o o

E

FIGURE 5-3 Comparison of mitosisand meiosis.Both somatrc cellsandpremeiotic germcellshavetwo copies of each (2n),onematernal chromosome andonepaternal. In mitosis, the replicated chromosomes, eachcomposed of two sister chromatids, alignat the cellcenterin sucha waythatboth daughter cellsreceive a maternal andpaternal fromolog of each morphological typeof chromosome. Durinqthefirstmerctrc division, however, eachreplicated chromosome pairswith its nomotogous partnerat the cellcenter; thispairingoff isreferred Io assynapsis, andcrossing overbetweenhomoloqous

168

CHAPTER 5

|

M O L E C U L AG R E N E T TTCE C H N T O U E S

chromosomes is evidentat this stage One replicatedchromosomeof eachmorphological type then goesinto eachdaughtercell The resultingcellsundergoa seconddivisionwithout interveningDNA replication, with the srsterchromatidsof eachmorphological type beingapportionedto the daughtercells.In the secondmeiotic divisionthe alignmentof chromatidsand their equalsegregation into daughtercellsis the sameas in mttoticdivision.The alignmentof pairsof homologouschromosomein metaphaseI is randomwith respectto other chromosomepairs,resultingln a mix of paternally and maternallyderivedchromosomes in eachdaughtercell.

(a) Segregationof dominant mutation Mutant

Wild-tYPe

Gametes

F i r s tf i l i a l g e n e r a t i o nF, . : all offspringhave mutant phenotype

Gametes S e c o n df i l i a l g e n e r a t i o nF, r : 3/aof offspringhave mutant phenotype Normal

(b) Segregationof recessivemutation Mutanl

(Figure 5-4). If the F1 progeny exhibit the mutant trait' then ih."r.r.rr"n, allele is dominant; if the F1 progeny exhibit the wild-type trait, then the mutant is recessive'Further crossing b"t*.Ln F1 individualswill also revealdifferent patternsof inheritanceaccording to whether the mutation is dominant or recessive.When F1 individuals that are heterozygousfor a dominant allele are crossedamong themselves,three-fourths of the resulting F2 progeny will exhibit the mutant trait' In contrast, when F1 individuals that are heterozygous for a recessiveallele are crossedamong themselves,only one-fourth of the resultingF2 progeny will exhibit the mutant tratt' As noted earlier,the yeast S. cereuisiae,an lmportant experimental organism' can exist in either a haploid or a iiotoid state.1n these unicellular eukaryotes' crossesbetween haploid cells can determinewhether a mutant allele is dominani or recessive.Haploid yeast cells' which carry one copy of each chromosome' can be of two different mating types k.town as a and ct. Haploid cells of opposite mating type can mate to produce a/ct diploids, which carry two of each chromosome. If a new mutation with an ob.*i., servablephenotypeis isolatedin a haploid strain, the mutant strain can be mated to a wild-type strain of the opposite mating type to produce a/o diploids that are heterozygousfor thl mutant ull.t.. ff these diploids exhibit the mutant trait, then the mutant allele is dominant, but if the diploids appear as wild-type, then the mutant allele is recessive'I7hen a/ct diploids aie placed under starvation conditions, the cells undergo -eiosfu, giving rise to a tetrad of four haploid spores' t*Jof type a and two of type ct. Sporulation of a heterozygo.rsdipiiid cell yields two sporescalryils the mutant allele ina t*o carrying the wild-type allele (Figure 5-5)' Under Wild type (type a)

Gametes

Mutant (type o)

F i r s tf i l i a l g e n e r a t i o nF, r . no offspringhave mutant phenotype Diploid cells: w i l l n o t e x h i b i tm u t a n t phenotypeif mutation is recessive

Gametes S e c o n df i l i a l g e n e r a t i o nF, r : 1/+of offspringhave mutant phenotype

Haploid spores in tetrad: 2 will be mutant 2 will be wild tYPe

5-4 Segregationpatternsof dominantand A FIGURE recessivemutationsin crossesbetweentrue-breedingstrains o f d i p l o i do r g a n i s m sA. l l t h e o f f s p r i nign t h e f i r s t( F r ) the F1 lf the mutantalleleisdominant, generation areheterozygous. ( a ) p a r t l f t he i n p h e n o t y p a e s , m u t a n t t h e e x h i b i t o f f s p r i nw g ill g i l le x h i b itth e ee , F 1o f f s p r i nw m u t a na t l l e l ei s r e c e s s i vt h a ,si n p a r t( b ) C r o s s i nogf t h e F 1 w i l d - t y pp eh e n o t y p e different alsoproduces amongthemselves heterozygotes m u t a na t lleles r e c e s s i v e a n d f d o m i n a n t s e g r e g a t i or ant i o so r intheF,qeneration

of allelesin yeast'Haploid 5-5 Segregation FIGURE cellsof oppositematingtype(i e , oneof matingtype Saccharomyces an a/ctdiploidlf ctandoneof matingtypea) canmateto produce theothercarries and allele wild-type a dominant carries onehaploid gene, resulting the same of the allele mutant a recessive trait Undercertaln the dominant diploidwillexpress heterozygous haploidspores' four of tetrad a diploidcellwillforma conditions, trait and recessive the express will tetrad the Twoof the sporesin tratt dominant the express two will

A N D S T U D YG E N E S G E N E T I CA N A L Y S I SO F M U T A T I O N ST O I D E N T I F Y

169

appropriate conditions, yeastsporeswill germinate,produc_ rng vegetativehaploid srrains of both mating rypes.

(a)

p Incubate at 23 .C for 5 n

-+

C o n d i t i o n aM l u t a t i o n sC a n B e U s e dt o S t u d y E s s e n t i aGl e n e si n y e a s t

E

Add mutagen; distributeinto s m a l l e ra l i q u o t s

00001000 [000t[00

VVVV

The proceduresusedto identify and isolate mutants, referred to as genettc screens,depend on whether the experimental organism is haploid or diploid and, if the latter, whether the mutation is recessiveor dominant. Genes that encode proteins essentialfor life are among the most interestingand important ones ro study. Since phenotypic expressionof mutationsin essentialgenesleadsto death of the individual, ingeniousgeneticscreensare neededto isolateand maintain organismswith a lethal mutation.

rI V V V

Colonies

Incubate at23'C

ff5fi:JJ:ru

Temperature-sensitive for growth; growth at 23., no growth at 36.

(b) Wild type

cdc28 mutants

t

t

tagenizedyeastcellsthat could grow normally at23 "C but that could not form a colony when placedat 36;C (Figure5_6a). Once temperature-sensitive murants were isolated,fu.th., analysisrevealedthat some indeed were defectivein cell divi-

l

o cdcZmutants

Cg

6 ,G

l{ t.. v

o' t-

170

o

c H A p r E5R |

MoLEcuLA GRE N E TrtEcc H N t e u E S

ao

FIGURE 5-6 Haploidyeastscarrying < EXPERIMENTAL lethalmutationsare maintainedat temperature-sensitive permissive temperatureand analyzedat nonpermissive cellfor temperature-sensttive screen temperature.(a)Genetic grow form (cdc) and yeast that in Yeasts mutants divisioncycle but not at 36 "C temperature) at 23 'C (permissive colonres thatblocks (nonpermissive maycarrya lethalmutation temperature) for blocksat (b)Assay colonies of temperature-sensitive celldivision. of wildin thecellcycle.Shownherearemicrographs stages specific after mutants different temperature-sensitive yeast two and type cells, for 6 h Wild-type temperature at the nonpermissive incubation sizes of buds, to grow,canbe seenwith alldifferent whichcontinue cellsin the of the cellcycleIn contrast, stages different reflecting in thecell stage at a specific a block exhibit lowertwo micrographs of a cycleThecdc28mutantsarrestat a pointbeforeemergence cellsThecdcZmutants, appearasunbudded newbudandtherefore justbeforeseparation of the mothercellandbud whicharrest (a)see (emerging cell),appearascellswith largebudslPart daughter L H H a r t w e l l ,1 9 6 -,l J B a c t e r i o9l 3 j 6 6 2 ; p a r t ( b ) f r o m L M H e r e f o r d a n d L H Hartwell.1914,J Mol Biol 84.4451

ComplementationTestsDetermineWhether Mutations Are in the Different Recessive S a m eG e n e In the genetic approach to studying a particular cellular process, researchersoften isolate multiple recessivemutaiion, that produce the same phenotype. A common test for

ganism heterozygousfor both mutations (i'e', carrying one a one b allele)will exhibit the mutant phenotype beIll.le "nd causeneither allele provides a functional copy of the gene' In contrast, if mutation a and b arein separategenes,then heterozygotescarrying a single copy of each mutant allele will not;;hibit the mutant phenotype becausea wild-type allele

theseyeastmutants did not simply fail to grow, as they might if they carried a mutation affecting generalcellular metabolism. Rather,at the nonpermissivetemperature,the mutants of interest grew normally for part of the cell cycle but then arrestedat a particular stageof the cell cycle,so that many cells at this stagewere seen(Figure 5-6b). Most cdc mutations in that is, when haploid cdc strainsare mated yeastare recessive; to wild-type haploids,the resulting heterozygousdiploids are nor defectivein cell division. neither temperature-sensitive

R e c e s s i vLee t h a lM u t a t i o n si n D i p l o i d sC a n B e l d e n t i f i e db y I n b r e e d i n ga n d M a i n t a i n e d in Heterozygotes In diploid organisms,phenotypesresulting from recessive mutations can be observedonly in individuals homozygous for the mutant alleles.Sincemutagenesisin a diploid organism typically changesonly one allele of a gene' yielding heterozygousmutants, geneticscreensmust include inbreeding stepsto generateprogeny that are homozygous for the mutanl alleles. The geneticist H. Muller developed a general and efficient procedure for carrying out such inbreeding experiments in the fruit fly Drosophila. Recessivelethal mutaiions in Drosophila and other diploid organisms can be maintained in heterozygousindividuals and their phenotypic consequencesanalyzedin homozygotes. The Muller approach was used to great effect by C' Niisslein-Volhard and E. \Tieschaus, who systematically lethal mutations affectingembryogenesis screenedfor recessive in Drosophila. Dead homozygousembryos carrying recessive lethal mutations identifiedby this screenwere examinedunder the microscope for specific morphological defects in the embryos.Current understandingof the molecularmechanisms underlyingdevelopmentof multicellularorganismsis based,in large part, on the detailedpicture of embryonic development rWe revealedby characterizationof theseDrosoprila mutants. will discusssome of the fundamental discoveriesbased on thesegeneticstudies\n Chapter 22.

lar characterization of the CDC genes and their encoded proteins, as describedin detail in Chapter 20, has provided a ?r"-.*ork for understanding how cell division is regulated in organismsranging from yeast to humans'

Double Mutants Are Useful in Assessing t h e O r d e ri n W h i c h P r o t e i n sF u n c t i o n Based on careful analysis of mutant phenotypes associated with a particular cellular process' researchersoften can deduce the order in which a set of genesand their protein products function. Two general types of processesare amenable to such analysis: (a) biosynthetic pathways in which a precursor material is convertedvia one or more intermediatesto a final product and (b) signaling pathways that regulate other processesand involve the flow of information rather than chemical intermediates.

A N D S T U D YG E N E S TO IDENTIFY OF MUTATIONS ANALYSIS GENETIC

171

> EXPERIMENTA FLI G U R E5 - 7 Complementation analysisdetermines whether recessivemutations are in the same or different genes. Complementation testsin yeastare performedby matinghaploida and o cellscarryingdifferentrecessrve mutations to producediploidcells In the analysis of cdc mutations,pairsof differenthaploid temperature-sensitive cdc strainswere systematically matedand the resultingdiploids testedfor growth at the permissive and nonpermrsstve temperaturesIn this hypothetical example,the cdcX and cdcy mutants c o m p l e m e net a c ho t h e ra n d t h u s h a v e mutationstn differentgenes,whereasthe cdcX and cdcZmutantshavemutationsin the s a m eg e n e

M a t e h a p l o i d so f oppositemating types and carryingdifferent recessivetemperaturesensitivecdc mutations

Mutant (type a)

Mutant (type cr)

(cdcX)

(cdcY\

\_./

\_-/ \/

Mutant (typea)

Mutant (typeo)

\cdcx)

(cdcz)

\_-./

\_-,i

\/

\r'

cdcXlcdcY (type a/o)

\r'

cdcXlcdcZ (type a/o) P l a t ea n d i n c u b a t e at permissive temperature

T e s tr e s u l t i n gd i p l o i d s for a temperaturesensitivecdc phenotype

2C

aa

Growth

No growth

6A

PHENOTYPE: Wild type INTERPRETATION:

36'C

Growth indicatesthat mutations cdcX and cdcY are in differentgenes

Y9

M utant Absenceof growth indicatesthat mutations c d c Xa n d c d c Z a r e i n t h e s a m eg e n e -Ir-V----l...t-^_ia"'-l___LF

Respectivewild-typealleles p r o v i d en o r m a lf u n c t i o n

next. In E. coLi,the genesencodingtheseenzymeslie adja_ cent to one another in the genome, constituting the trp operon (seeFigure 4-73a). The order of action of the differ_ ent genesfor theseenzymes,hencethe order of the biochemi_ cal reactions in the pathway, initially was deducedfrom the types of intermediatecompoundsthat accumulatedin each

In Chapter 14 we discussthe classicuse of the double_ mutant.strategyto help elucidatethe secretorypathway. In this pathway proteins to be secreredfrom the cell rnou. i.otheir site of synthesison the rough endoplasmicreticulum (ER) to the Golgi complex. then to ,..r.toiy vesicles, and fi_ n a l l y t o r h ec e l l s u r f a c e . Ordering of Signaling pathways As we learn in later chapters,expressionof many eukaryoticgenesis regulated by signaling pathways that are initiateJ by extracellular 172

.

c H A p r E5R | M o L E c u L AGRE N E Tr tEcc H N t o u E S

B o t h a l l e l e sn o n f u n c t i o n a l

hormones, growth factors, or other signals. Such signaling pathways may include numerouscomponents,and doublemutant analysisofren can provide insight into the functions and interactionsof thesecompon.nrr.th. only prerequisite for obtaining useful information from this type of analysisis that the two mutations must have opposite effects on the output of the sameregulatedpathway. Most commonl5 one mutatio_nrepressesexpressionof a particular reporrer gene even when the signal is present, while another mutation resultsin reporter geneexpressionevenwhen the signalis absent(i.e.,constirutiveexpression).As illustratedin Figure5_gb, two simple regulatory mechanismsare consistentwith such

Note that this technique differs from complementation analysisjust describedin that when testingtwo recessive mu_ tations, the double mutant created is homozygolzsfor both mutations.Furthermore,dominant mutantscan be subiected t o d o u b l e - m u t a natn a l v s i s .

(a)Analysisof a biosyntheticpathway 1. A m u t a t i o ni n A a c c u m u l a t e isn t e r m e d i a t e e. A m u t a t i o ni n B a c c u m u l a t e isn t e r m e d i a t 2 OF PHENOTYPE D O U B L EM U T A N T

A d o u b l em u t a t i o ni n A a n d B a c c u m u l a t e s 1. intermediate INTERPRETATION: The reaction catalYzedby A precedes the reaction catalYzedbY B. I

: '

Eo'Et'E (b)Analysisof a signalingpathway

mutant allelewould have a mutant phenotype(Figure5-9a)' The observation of genetic suppressionin yeast strains carrying a mutant actin allele (act1-1) and a secondmutation (sic5) in another gene provided early evidence for a

A mutation in A gives repressedreporterexpresslon. A mutation in B gives constitutivereporterexpression. PHENOTYPE OF : D O U B L EM U T A N T : A d o u b l em u t a t i o ni n A a n d B g i v e s repressedreporterexPression. I , INTERPRETATION: A positively regulates repofter expression and is negativelYregulated bY B' .

(a)Suppression GenotyPe AB Phenotype Wildtype

aB Mutant

ab Ab Mutant SuPPressed

rNrERPRErAloP H E N O T Y PO EF D O U B L EM U T A N T

INr ERPRErArIoN:

/ed\

t I i/t l

A d o u b l em u t a t i o ni n A a n d B g i v e s constitutivereporterexpression.

\UU,/

pression i7 f .x 7:; i:';:;':, t""1 i' fJ3i,,1,133 A--€--l

B

(b) Synthetic lethality 1 Genotype

AB Phenotype Wild type

5-8 Analysisof doublemutantsoften canorderthe FIGURE in stepsin biosyntheticor signalingpathways'Whenmutations process but have genesaffectthe samecellular two different of the doublemutant phenotypes, the phenotype different distinctly genes mustfunction(a) which the two in the order canoftenreveal pathway, a thataffectthe samebiosynthetic Inthe caseof mutations t em e d t a t e l y t i l la c c u m u l attheei n t e r m e d i ai m d o u b l em u t a nw in the by the proteinthatactsearlier preceding the stepcatalyzed of a signaling analysis organism(b)Double-mutant wild-type on effects haveopposite if two mutations pathwayispossible phenotype gene In thiscase,the observed of a reporter expression aboutthe orderin which information of the doublemutantprovides regulators or negative actandwhethertheyarepositive the proteins

G e n e t i cS u p p r e s s i o an n d S y n t h e t i cL e t h a l i t y t roteins C a n R e v e a Il n t e r a c t i n go r R e d u n d a n P Two other types of genetic analysis can provide additional clues about how proteinsthat function in the samecellular processmay interactwith one anotherin the living cell. Both of thesemethods,which are applicablein many experimental organisms,involve the use of double mutants in which the phenotypic effects of one mutation are changed by the presenceof a secondmutation. Suppressor Mutations The first type of analysisis basedon geneticsuppression.To understandthis phenomenon,suppose

aB Partial defect

ab

Ab

Severe defect

Partial defect

rNrERPRErArto-

,fii\

( | 11I ) U,/

\U

i

(cl Synthetic lethality 2 Genotype

AB

Phenotype Wildtype

aB Wild type

ab

Ab Wild tYPe

or 5-9 Mutationsthat resultin geneticsuppression A FIGURE proteins' redundant or interacting reveal syntheticlethality proteins with two defective (a)Observation thatdoublemutants phenotype butthatsinglemutantsgive (A andB)havea wild-type thatthefunctionof eachprotein indicates a mutantphenotype thatdouble other.(b)Observation the with on interaction depends mutants single than defect phenotypic severe more a have mutants (e g , subunits of a heterodimer) thattwo proteins alsoisevidence (c)Observation thata double to functionnormally. mustinteract mUtantisnonviab|ebutthatthecorrespondingsing|emutants f unctionin thattwo proteins indicates phenotype thewild-type product an essential to produce pathways redundant

A N D S T U D YG E N E S O F M U T A T I O N sT O I D E N T I F Y ANALYSIS GENETIC

.

173

direct interaction in vivo between the proteins encoded by the two genes.Later biochemical studies showed that these two proteins-Act1 and Sac6-do indeed inreract in the construction of functional actin structureswithin the cell. Synthetic Lethal Mutations Another phenomenon,called synthetic lethality, produces a phenotypic effect opposrte to that of suppression.In this case,the deleteriouseffectof one mutation is greatly exacerbated(rather than suppressed)by a secondmutation in a relatedgene.One situation in which such synthetic lethal mutations can occur is illustrated in Figure 5-9b. In this example,a heterodimericprotein is partiall5 but not completely,inactivatedby mutations in eiiher one of the nonidentical subunits.However, in double mutants carrying specificmurationsin the genesencodingboth subunits,little interaction betweensubunits occurs,iesulting in severephenotypic effects. Synthetic lethal mutations

product cannor be synthesizedand the double mutanrs will be nonviable.

(a)

G e n e sC a nB e l d e n t i f i e db y T h e i r M a p p o s i t i o n on the Chromosome The preceding discussionof genetic analysisillustrates how a geneticistcan gain insight into gene function by observing the phenotypic effectsproduced by joining together different combinations of mutant allelesin the samecell or organism. For example, combinations of different alleles of the same gene in a diploid can be used to determine whether a mutation is dominant or recessiveor whether two different recessive mutations are in the same gene.Furthermore, combinations of mutations in different genes can be used to determine the order of gene function in a pathway or to identify functional relationships between genessuch as suppressionand synthetic enhancement.Generally speaking,all these methods can be viewed as analytical tests baseJ on gene functioz. \7e will now consider a fundamentally different type of genetic analysis based on gene positioz. Studies designedto determine the position of a gene on a chromosome, often referred to as genetic mapping studies, can be usedto identify the geneaffected by a particular mutation or to determine whether two mutations are in the samegene. In many organisms generic mapping studies rely on exchangesof genetic information that occur during meiosis. As discussedin Chapter 4 and as shown in Figure 5-10a,

(b)Consider two linkedgenesA and Bwith recessive allelesa and b.

-'-lf m2

Replicated chromosomes (4nl

Crossof two mutantsto construct a doublyheterozygous strain:

and over

AlAblb

x alaBlB

I

MetaphaseI

A i

A n a p h a s eI

Crossof double heterozygoteto test strain: A b aBab

..

a

b

I

*\ /

";

I

I

A n a p h a s el l

v

I

v AbaBABab abuA"brb \___________-Y_

Parentaltypes

_________YJ

Recombinanttypes

m1 1n

1n

Parental gamete

tn

Recombinantgametes

tn

parental gamete

A FIGURE5-10 Recombinationduring meiosis can be used to map the position of genes. (a)Shown is an individualthat carries two mutations,designatedm/ (yellow)andm2 (green),that are on the maternaland paternalversionsof the samechromosomelf crosslngoveroccursat an intervalbetweenml andm2 beforethe first meloticdivision,then two recombinantgametesare produced; one carriesboth m / and m2, whereasthe other carriesneither mutation The longerthe distancebetweentwo mutationson a 174

.

cHAprER s

I

M o l E c u L A RG E N E T tTcE C H N t o u E S

GeneticdistancebetweenA and B can be determinedfrom frequencyof parentaland recombinantgametes: gametes Geneticdistancein cM = 100, Itotbi.nant total gametes chromatid,the more likelythey are to be separatedby recombination and the greaterthe proportionof recombinantgametesproduced (b) In a typicalmappingexperiment,a strainthat is heterozygous for two differentgenesis constructedThe frequencyof parentalor recombinantgametesproducedby thls straincan be determjned from the phenotypesof the progenyin a testcross to a homozygous recessive strain The geneticmap distancein centimorgans (cM) is givenas the percentof the gametesthat are recombinant

genetic recombination takes place before the first meiotic cell division in germ cells,when the replicatedchromosomes of each homologous pair align with each other. At this time, homologous DNA sequenceson maternally and paternally derived chromatids can exchangewith each other, a process 'We now know that the resulting known as crossing over. crossovers between homologous chromosomes provide structural links that are important for the proper segregation of pairs of homologous chromatids to opposite poles during the first meiotic cell division (for discussionseeChapter 20). Considertwo different mutations,one inheritedfrom each parent, that arc located close to one another on the same chromosome.Two different typesof gametescan be produced according to whether a crossoveroccurs betweenthe mutations during meiosis. If no crossoveroccurs between them, gametesknown as parental types,which contain either one or the other mutation, will be produced. In contrast' if a crossoveroccurs betweenthe two mutations' gametesknown as recombinant types will be produced. In this example recombinant chromosomeswould contain either both mutations, or neither of them. The sites of recombination occur more or lessat random along the length of chromosomes;thus the closertogethertwo genesare, the lesslikely that recombination will occur between them during meiosis. In other words, the lessfrequently recombination occursbetweentwo genes on the same chromosome, the more tightly they are Iinked and tbe closer together they are. Two genesthat are sufficiently close together such that there are significantly fewer recombinant gametes produced than parental gametes are consideredto be geneticallylinked. The technique of recombinational mapping was devised in 1911 by A. Sturtevantwhile he was an undergraduate working in the laboratory of T. H. Morgan at Columbia University. Originally used in studies on Drosophila, this technique is still used today to assessthe distance between two genetic loci on the same chromosome in many experlmental organisms. A typical experiment designedto determine the map distancebetweentwo geneticpositions would involve two steps. In the first step' a strain is constructed that carriesa different mutation at eachposition, or locus. In to dethe secondstep, the progeny of this strain are assessed termine the relative frequency of inheritance of parental or recombinant types.A typical way to determinethe frequency of recombination between two genesis to cross one diploid parent heterozygousat each of the genetic loci to another parent homozygousfor each gene.For such a cross,the proportion of recombinant progeny is readily determined becauserecombinant phenotypeswill differ from the parental phenotypes.By convention, one genetic map unit is defined as the distance between two positions along a chromosome that results in one recombinant individual in 100 total progeny. The distance corresponding to this 1 percent recombination frequency is called a centimorgan (cM) in honor of Sturtevant'smentor,Morgan (seeFigure 5-10b). A complete discussion of the methods of genetic mapping experiments is beyond the scope of this introductory discussion;however,two featuresof measuringdistancesby recombination mapping need particular emphasis.First, the

frequency of genetic exchange between two loci is strictly proportional to the physical distancein basepairs separating ih.- o.tly for loci that are relatively close together (sa5 less than about 10 cM). For loci that are farther apart than this' a distance measured by the frequency of genetic exchange tends to underestimatethe physical distance becauseof the possibility of two or more crossoversoccurring within an inie.ual. In the limiting casein which the number of recombinant types will equal the number of parental types, the two loci under considerationcould be far apart on the same chromosome or they could be on different chromosomes, and in such casesthe loci are consideredto be unlinked' A secondimportant concept neededfor interpretation of

recombination frequency (i.e.' a genetic distance of 1 cM) representsa physical distanceof about 2.8 kilobasesin yeast distance of about 400 kilobases in compared *lth " Drosophila and about 780 kilobasesin humans' One of the chief uses of genetic mapping studies is to

human diseasescan be identified using such methods' A second general use of mapping experiments is to determine *hethe. two different mutations are in the samegene' If two mutations are in the same gene, they will exhibit tight linkage in mapping experiments,but if they are in different g..r.{ they will usually be unlinked or exhibit weak linkage'

Genetic Analysis of Mutations to ldentify and Study Genes r Diploid organisms carry two copies (alleles)of each gene,whereashaploid organismscarry only one copy' r Recessivemutations lead to a loss of function, which is masked if a normal allele of the gene is present' For the mutant phenotype to occur' both alleles must carry the mutatl0n. r Dominant mutations lead to a mutant phenotype in the presenceof a normal allele of the gene.The phenotypesassociatedwith dominant mutations often representa gain of function but in the caseof some genesresult from a loss of function. r In meiosis,a diploid cell undergoesone DNA replication and two cell divisions, yielding four haploid cells in which maternal and paternal alleles are randomly assorted (see Figure5-3).

GENES G E N E T I CA N A L Y S I SO F M U T A T I O N ST O I D E N T I F YA N D S T U D Y

175

r Dominant and recessivemutations exhibit characteristic segregatlonpatternsin geneticcrosses(seeFigure5_4). r I n h a p l o i d y e a s t .t e m p e r a t u r e - s e n s i t i vmeu t a t i o n s a r e particularly useful for identifying and studying genesessential to survival. r The number of functionally related genesinvolved in a process can be defined by complemenration analysis (see Figure-5-7). r The order in which genesfunction in a signaling pathway can be deducedfrom the phenotype of double mutants defectivein two stepsin the affectedprocess. r Functionally significant interactions between Drorelns can be deduced from the phenotypic effects o? allelespecificsuppressormutations or syntheticlethal mutations. r Genetic mapping experimentsmake use of crossingover between homologous chromosomes during meiosis to measurethe genetic distance between two different muta_ tions on the samechromosome.

DNACloningand Characterization

f[

is simply any DNA molecule composed of sequencesde_ rived from different sources. The key to cloning a DNA fragment of interest is to link it to a vector DNA molecule that can replicate within a host cell. After a singlerecombinant DNA molecule,composedof a vector plus an insertedDNA fragmenr, is introdu.id irrto " host cell, the inserted DNA is replicated along with the vec_ tor, generating a large number of identical DNA molecules. The basic schemecan be summarizedas follows:

vectorsexist, our discussionwill mainly focus on plasmid vectors in E. coli host cells,which are commonly used. Various techniquescan then be employed to identify the sequenceo{ interest from this collection of DNA fragments,known as a DNA library. Once a specificDNA fragment is isolated,it is typically characterizedby determining the exact sequenceof nucleotidesin the molecule. We end with a discussionof the polymerasechain reaction (PCR).This powerful and versatile techniquecan be usedin many ways to generatelarge quantities of a specificsequenceand otherwisemanipulate ONR m the laboratory.The various usesof cloned DNA frasmentsare discussed in subsequent sections.

RestrictionEnzymesand DNA Ligases A l l o w I n s e r t i o no f D N A F r a g m e n t si n t o CloningVectors A major objective of DNA cloning is to obtain discrete, small regions of an organism'sDNA that constitute specific genes.In addition, only relatively small DNA moleculescan be cloned in any of the availablevectors.For thesereasons, the very long DNA molecules that compose an organism's genome must be cleavedinto fragments that can be inserted into the vector DNA. Two types of enzymes-restriction enzymes and DNA ligases-facilitate production of such recombinantDNA molecules. Cutting DNA Molecules into Small Fragments Restric_ tion enzymesare endonucleasesproduced by bacteria that typically recognizespecific 4- to 8-bp sequences,calledrestrictin sites, and then cleaveboth DNA strandsat this site. Restriction sites commonly are short palindromic sequences;that is, the restriction-sitesequenceis the sameon each DNA strand when read in the 5'-+3' direction (Figure5-11). For each restriction enzyme) bacteria also produce a modification enzyme, which protects a bacterium's own DNA from cleavageby modifying it at or near eachpotenrial cleavagesite. The modification enzymeadds a methyl group to one or two bases,usually within the restriction site. When

Vector + DNA fragment EcoRl

J

+

RecombinantDNA

J

J'

r(-

|

J

Cleavage EcoRl I

Isolation, sequencing,and manipulation of purified DNA fragment Although investigatorshave devisednumerous experimental variations, this flow diagram indicatesthe essentialsteDsin DNA cloning. In this section,we first describemethods for isolating a specificsequenceof DNA from a seaof other DNA sequences. This processoften involvescutting the genomernto fragmentsand then placing each fragment ii a vector so that the entire collection can be propagatedas recombinantmole_ cules in separatehost cells. While many different types of '176 .

c H A p r E5R I M o L E c u L AGRE N E Tr tEcc H N t e u E s

|

ti +

Replication of recombinant DNA within host cells

Stickyends

s',.),

-------r

G . TTAA

c ----r

3'

\l

5

FIGURE 5-11 Cleavage of DNAby the restrictionenzyme EcoRf.Thisrestriction enzymefromE colimakesstaqqered cutsat the specific 6-bppalindromic sequence shown,yielding fragments withsingle-stranded, "sticky,, complementary ends.Manyother restriction enzymes alsoproduce fragments with stickyends

a methyl group is presentthere, the restriction endonuclease is prevented from cutting the DNA. Together with the restriction endonuclease,the methylating enzyme forms a restriction-modification system that protects the host DNA while it destroysincomingforeign DNA (e.g.,bacteriophage DNA or DNA taken up during transformation) by cleaving it at all the restriction sitesin the DNA. Many restriction enzymesmake staggeredcuts in the two DNA strands at their recognition site, generatingfragments that have a single-stranded"tail" at both ends,sticky ends (seeFigure 5-11). The tails on the fragmentsgeneratedat a given restriction site are complementaryto those on all other fragments generated by the same restriction enzyme. At room temperature, these single-strandedregions can transiently base-pairwith thoseon other DNA fragmentsgenerated with the same restriction enzyme. A few restriction enzymes,such as Alul and SmaI, cleaveboth DNA strandsat the same point within the restriction site, generating fragmentswith "blunt" (flush)endsin which all the nucleotides at the fragment ends are base-pairedto nucleotides in the complementarystrand.

The DNA isolated from an individual organism has a specificsequence,which purely by chancewill contain a specific set of restriction sites' Thus a given restriction enzyme will cut the DNA from a particular source into a reproducible set of fragmentscalled restriction fragments.The frequency with which a restriction enzymecuts DNA, and thus the average size of the resulting restriction fragments, depends largely on the length of the recognition site. For example, a restriction enzyme that recognizesa 4-bp site will cleaveDNA an averageof once every4", or 256, basepairs, whereas an enzyme that recognizesan 8-bp sequencewill cleaveDNA an averageof onceevery4E basepairs (=65 kb)' Restriction enzymes have been purified from several hundred different speciesof bacteria, allowing DNA molecules to be cut at a large number of different sequencescorresponding to the recognitionsitesof theseenzymes(seeTable5-1)' Inserting DNA Fragments into Vectors DNA fragments with either sticky endsor blunt endscan be insertedinto vector DNA with the aid of DNA ligases.During normal DNA replication, DNA ligase catalyzestheend-to-endioining (ligation) of

ENZYME

MICRO()RGANISM SOURCE

SITERECOGNITION

BamHl

B a cillu s amy lo liq u efaciens

-G-G-A-T-C.C. -C-C-T-A-G-G.

PBODUCED ENOS

J Sticky

1 J Sau3A

aureus Staphylococcus

-G-A-T-C.C-T-A-G-

Sticky

t EcoRl

Escherichia coli

Hindlll

Haemophilus influenzae

J -G-A-A-T-T-C-C.T-T-A-A-G t J -A,A-G.C-T-T.

Sticky

Sticky

-T-T-C-G-A-4.

T Smal

Serratia mdrcescens

J -C-C-C-G-G-G-G-G-G-C-C-C-

Blunt

1

Notl

No cardia otiti di s-cauiarum

-G-C_G-G.C-C-G-C-C-G-C-C-G-G-C-G-

Sticky

t Many of these recognition sequencesare included in a common polylinker sequence(seeFigure 5-13). D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

177

short fragments of DNA called Okazaki fragments. For purposesof DNA cloning, purified DNA ligaseis usedto covalently join the ends of a restriction fragment and vector DNA that have complementaryends (Figure5-12). The vector DNA and restriction fragment are covalently ligated rogetherthrough the standard3'-+5' phosphodiesterbonds of DNA. In addition to Iigating complementary sticky ends, the DNA ligasefrom bacteriophage T4 can ligate any two blunt DNA ends. However, blunt-end ligation is inherently inefficient and requires a higher concentration of both DNA and DNA ligasethan doesligation of sticky ends.

E. coliPlasmidVectorsAre Suitablefor Cloning lsolatedDNA Fragments Plasmidsare circular, double-strandedDNA (dsDNA) molecules that are separatefrom a cell's chromosomal DNA. These extrachromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g.,yeast),exist in a

Genomic DNA fragments (a)

3' P-AATT oH__r-----::-.---r 5,

VectorDNA (a')

(b)

s', oH 3'r---------r-TTAA-p

+

P-C GHO-:5'

3' (c)

P-A G CT HOj-:-:r

3' 5,

I

I Complementary I endsbase-pair I

v

OHP

lltr 3'r------]-TTAA

/\ PHO 2 ATP T4 DNA ligase 2AMP+2PPi (a')

5'I-AATT

(a)

3',---------F++ii

3'

s,

FIGURE 5-12 Ligationof restrictionfragmentswith complementary stickyends.In thisexample, vectorDNAcutwith EcoRl ismixedwith a sample containing restriction fragments produced by cleaving genomicDNAwith several different restriction enzymes Theshortbasesequences composing the stickyendsof eachfragment typeareshownThestickyendon the cutvectorDNA (a')base-pairs onlywith thecomplementary stickyendson theEcoR/ fragment(a)in the genomic sample. Theadjacent 3, hydroxyl and5, phosphate groups(red)on the base-paired fragments thenare joined(ligated) covalently byT4 DNAligase. 178

.

c H A p r E R5

|

MoLEcuLAR G E N E T trcE c H N t o u E s

Polylinker

Plasmid cloning vector

FIGURE 5-13 Basiccomponentsof a plasmidcloningvector that can replicatewithin an E. coli cell. plasmid vectorscontaina genesuchasamp',whichencodes selectable the enzyme andconfers resistance to ampicillin. Exogenous P-lactamase DNAcan be inserted intothe bracketed regionwithoutdisturbing theabilityof the plasmidto replicate or express theamp,gene plasmid vectors alsocontaina replication origin(ORl) sequence whereDNA replication isinitiated by host-cell enzymes. Inclusion of a synthetic polylinker containing the recognition sequences for several different restriction enzymes increases the versatility of a plasmidvectorThe vectorisdesigned sothateachsitein the polvlinker is unioueon t h ep l a s m i d .

parasitic or symbiotic relationship with their host cell. Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division. During cell division, copies of the plasmid DNA segregateto each daughtercell, assuring continued propagation of the plasmid through successive generationsof the host cell. The plasmidsmost commonly usedin recombinant DNA technology are those that replicate in E. col/. Investigators have engineeredtheseplasmids to optimize their use as vectors in DNA cloning. For instance, removal of unneeded portions from naturally occurring E. coli plasmids yields plasmid vectors, (=1.2-3 kb in circumferential length, that contain three regions essentialfor DNA cloning: a replication origin; a marker that permits selection,usually a drugresistancegene;and a region in which exogenousDNA fragments can be inserted (Figure 5-13). Host-cell enzymes replicate a plasmid beginning at the replication origin (ORI), a specific DNA sequenceof 50-100 basepairs. Once DNA replication is initiated at the ORI, it continues around the circular plasmid regardlessof its nucleotide sequence.Thus any DNA sequenceinsertedinto such a plasmid is replicated along with the rest of the plasmid DNA. Figure 5-14 outlines the general procedure for cloning a DNA fragment using E. coli plasmid vectors. \lhen E. coli cells are mixed with recombinant vector DNA under certain conditions, a small fraction of the cells will take up the plasmid DNA, a process known as transformation. Typically, 1, cell in about 10,000 incorporates a single plasmld DNA molecule and thus becomes transformed. After plasmid vectors are incubated with E. coli, those cells that take up the plasmid can be easily selectedfrom the much larger number of cells. For instance,if the plasmid carries a gene that confers resistance to the antibiotic ampicillin,

> EXPERIMENTAL FIGURE 5-14 DNAcloningin a plasmid vector permitsamplificationof a DNAfragment.A fragmentof an intoa plasmid vectorcontaining DNAto be clonedisfirstinserted gene(amp'),suchasthatshownin Figure 5-13 ampicillin-resistance molecule by incorporation of a plasmid Onlythefew cellstransformed cells, the mediumIntransformed willsurvive on ampicillin-containing plasmid in intodaughter cells,resulting DNAreplicates andsegregates formation of an ampicillin-resistant colonv

transformed cells can be selected by growing them in an ampicillin-containing medium. DNA fragments from a few base pairs up to =10 kb commonly are insertedinto plasmid vectors. When a recombinant plasmid with an inserted DNA fragment transforms an E. coli cell, all the antibiotic-resistantprogeny cells that arise from the initial transformed cell will contain plasmids with the sameinsertedDNA. The insertedDNA is replicated along with the rest of the plasmid DNA and segregatesto daughter cells as the colony grows. In this way, the initial fragment of DNA is replicated in the colony of cells into a large number of identical copies. Since all the cells in a colony arise from a single transformed parental cell, they constitute a clone of cells, and the initial fragment of DNA inserted into the parental plasmid is referred to as cloned DNA or a DNA clone. The versatility of an E. coli plasmid vector is increased by the addition of a polylinker, a synthetically generated sequencecontaining one copy of severaldifferent restriction sitesthat are not presentelsewherein the plasmid sequence (seeFigure 5-13). \fhen such a vector is treated with a restriction enzyme that recognizesa restriction site in the polylinker, the vector is cut only once within the polylinker. Subsequentlyany DNA fragment of appropriate length produced with the same restriction enzymecan be insertedinto the cut plasmid with DNA ligase.Plasmids containing a polylinker permit a researcher to use the same plasmid vector when cloning DNA fragmentsgenerated with different restriction enzymes,which simplifies experimentalprocedures. For some purposes,such as the isolation and manipulation of large segmentsof the human genome, it is desirable to clone DNA segments as large as several megabases [L megabase(Mb) : 1 million nucleotides).For this purpose specializedplasmid vectors known as BACs (bacterialartificial chromosomes)have been developed.One type of BAC uses a replication origin derived from an endogenousplasmid of E. coli known as the F factor. The F factor and cloning vectors derived from it can be stably maintained at a single copy per E. coli cell even when they contain inserted sequencesof up to about 2 Mb. Production of BAC libraries requires special methods for the isolation, ligation, and transformation of large segmentsof DNA becausesegments of DNA larger than about 20 kb are highly vulnerable to mechanical breakage by even standard manipulations such as prpetung.

DNA fragment to be cloned Enzymaticallyinsert DNA into plasmid vector

Recombinant plasmid

Mix E. coliwith olasmids in presenceol CaCl2;heat-pulse C u l t u r eo n n u t r i e n ta g a r p l a t e sc o n t a i n i n ga m p i c i l l i n

E. coli cnromosome

Transformedcell SUTVIVES

Cellsthat do not t a k e u p p l a s m i dd i e o n a m p i c i l l i np l a t e s

I P l a s m i replication d I

+

certmurtiotication I

g Colony of cells,each containingcopies of the same recombinantPlasmid

GDNALibrariesRepresentthe Sequences of Protein-CodingGenes A collection of DNA molecules each cloned into a vector molecule is known as a DNA library. !7hen genomic DNA from a particular organism is the source of the starting DNA, the set of clones that collectively represent all the DNA sequencesin the genome is known as a genomic D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N .

179

I lJ

+F*ry65' TranscriUe RNAintocDNA

< FIGURE 5-15 A cDNAlibrary containsrepresentative copiesof cellularmRNAsequences. A mixtureof mRNAsis pointfor preparing thestarting plasmid recombinant clones eachcontaining a cDNA.Transforming E coliwith the plasmids recombinant generates a setof cDNAclones representing allthecellular mRNAs. Seethetextfor a step-bysteodiscussion.

g I n " . o u " R N Aw i t ha t k a t i E J A d dp o t y ( d c ) t a i l Single-stranded 3' G GGGT-___--I cDNA

T T T T 5' t,,,.,.

EI lil$:fl6"#lli",

Y 5'I 3'GGGGI_---__--lTTTT5' -

complementary | Synttresize

sJ strand Double-stranded cDNA r (Jbbb a

-

i ^ F

3', - - r

I fTT5

oRl sites 5', 3'GGGG

E

I

C T T A A G E G G G G I _ _ _ _ _ _ - _ _T_ IT T T E C

lf Gf]GGGGI-.-_lT Stickyend

.

.,*"" withEcoRl T T TECTTAA

I Individual clones

180

T TAAGE

c H A p r E Rs

I

rJl:ru:HT:;,0?,,

MoLEcuLAR G E N E I Cr E c H N t e u E s

nJ.,,with EcoRl

library. Such genomic libraries are ideal for representingthe geneticcontent of relatively simple organismssuch as bacteria or yeast, but presentcertain experimental difficulties for higher eukaryotes.First, the genesfrom such organismsusually contain extensiveintron sequencesand therefore can be too large to be inserted intact into plasmid vectors. As a result, the sequencesof individual genesare broken apart and carried in more than one clone. Moreover, the presenceof introns and long intergenic regions in genomic DNA often makes it difficult to identify the important parts of a gene that actually encode protein sequences.For example, only about 1.5 percentof the hunran genomeactually represents protein-coding genesequences. Thus for many studies,cellular mRNAs, which lack the noncoding regionspresentin genomic DNA, are a more useful starting material for generating a DNA library. In this approach, DNA copies of mRNAs, called complementaryDNAs (cDNAs), are synthesized and cloned into plasmid vectors. A large collection of the resulting cDNA clones, representingall the mRNAs expressedin a cell type, is called a cDNA library.

cDNAsPreparedby ReverseTranscription o f C e l l u l a rm R N A sC a n B e C l o n e dt o G e n e r a t ec D N AL i b r a r i e s The first stepin preparing a cDNA library is to isolatethe total mRNA from the cell type or tissueof interest.Becauseof their poly(A) tails, mRNAs are easily separated from the much more prevalent rRNAs and tRNAs presentin a cell extract by use of a column to which short strings of thymidylate (oligo-dTs) are linked to the matrix. The generalprocedure for preparing a cDNA library from a mixture of cellular mRNAs is outlined in Figure 5-15. The enzymereversetranscriptase,which is found in retroviruses,is usedto synthesize a strand of DNA complementary to each mRNA molecule, starting from an oligo-dT primer (steps1 and2). The resulting cDNA-mRNA hybrid moleculesare converted in several stepsto double-strandedcDNA moleculescorrespondingto all the mRNA molecules in the original preparation (steps 3-5). Each double-stranded cDNA contains an oligodC.oligo-dG double-strandedregion at one end and an oligo-dT.oligo-dA double-strandedregion at the other end. Methylation of the cDNA protects it from subsequent restrictionenzymecleavage(step6). To prepare double-stranded cDNAs for cloning, short double-strandedDNA moleculescontaining the recognition site for a particular restriction enzymeare ligatedto both ends of the cDNAs using DNA ligasefrom bacteriophageT4 (Figure 5-15, step 7). As noted earlier,this ligasecan join "bluntended" double-strandedDNA moleculeslacking sticky ends. The resulting moleculesare then treated with the restriction enzymespecificfor the attachedlinker, generatingcDNA moleculeswith sticky endsat eachend (step8a). In a separateprocedure,plasmid DNA first is treatedwith the samerestriction enzymeto produce the appropriate sticky ends (step8b). The vector and the collection of cDNAs, all containing complementary sticky ends, then are mixed and joined covalently by DNA ligase(Figure 5-15, step 9). The resulting

DNA moleculesare transformed into E. coli cellsto generate individual clones;each clone carrying a cDNA derived from a singlemRNA. Becausedifferent genesare transcribed at very different rates,cDNA clonescorrespondingto abundantly transcribed genes will be representedmany times in a cDNA library, whereas cDNAs corresponding to infrequently transcribed geneswill be extremely rare or not presentat all. This property is advantageousif an investigatoris interestedin a gene that is transcribedat a high rate in a particular cell type' In this case,a cDNA library preparedfrom mRNAs expressedin that cell type will be enriched in the cDNA of interest, facilitating isolation of clonescarrying that cDNA from the library. Howeve! to have a reasonablechance of including clones corresponding to slowly transcribedgenes'mammalian cDNA libiaries must contain 1.06-107individual recombinantclones.

D N A L i b r a r i e sC a n B e S c r e e n e db y H y b r i d i z a t i o n e robe to an OligonucleotidP Both genomic and cDNA libraries of various organisms contain hundredsof thousandsto upwards of a million individual clones in the case of higher eukaryotes. Two general approaches are available for screening libraries to identify clonescarrying a geneor other DNA region of interest: (1) detectionwith oligonucleotideprobes that bind to the clone of interest and (2) detection basedon expression of the encoded protein. Here we describe the first method; an example of the secondmethod is presentedin the next section. The basis for screeningwith oligonucleotideprobes is hybridization, the ability of complementary singlestrandedDNA or RNA moleculesto associate(hybridize) specificallywith each other via basepairing. As discussed in Chapter 4, double-stranded(duplex) DNA can be denatured (melted) into single strands by heating in a dilute salt solution. If the temperature then is lowered and the ion concentration raised, complementary single strands will reassociate(hybridize) into duplexes.In a mixture of nucleic acids, only complementary single strands (or strands containing complementary regions) will reassociate; moreover,the extent of their reassociationis virtually unaffectedby the presenceof noncomplementarystrands. As we will seelater in this chapter,the ability to identify a particular DNA or RNA sequencewithin a highly complex mixture of moleculesthrough nucleic acid hybridization is the basis for many techniquesemployed to study gene expresslon. The stepsinvolved in screeningan E. coli plasmid cDNA library are depicted in Figure 5-16. First' the DNA to be screenedmust be attachedto a solid support. A replica of the petri dish containing a large number of individual E. coli clones is reproduced on the surfaceof a nitrocellulose membrane. The DNA on the membrane is denatured, and the membrane is then incubated in a solution containing a radioactively labeled probe specificfor the recombinant DNA containing the fragment of interest. Under hybridization conditions (near neutral pH, 40-65 "C' 0.3-0'6 M NaCl), D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N .

181

*'**

5eu

I n d i v i d u acl o l o n i e s

B o u n ds i n g l e - s t r a n d eDdN A

Master plate of E. coli colonies

Filter

P l a c en i t r o c e l l u l o sfei l t e ro n D l a t e t o p i c ku p c e l l sf r o m e a c hc o l o n y Hvbridized c o m p l e m e n t a rD y NAs

N i t r o c e l l u l o sfei l t e r

Wash away labeledDNA that does not hybridizeto DNA bound to filter

r"uorr autoradiography f

I

Performaut o r a d i o g r a p h y

o)

S i g n a la p p e a r so v e r p l a s m i dD N A t h a t i s complementary to probe

A EXPERIMENTAL FIGURE 5-16 cDNAlibrariescan be screened with a radiolabeledprobeto identifya cloneof interest.The a p p e a r a noc fea s p o to n t h ea u t o r a d i o g r ai nmd i c a t et hs ep r e s e n c e o f a r e c o m b i n acnl to n ec o n t a i n i nDgN Ac o m p l e m e n t a t or yt h e probeTheposition of the spoton the autoradioqram isthe mirror

i m a g eo f t h ep o s i t i oonf t h a tp a r t i c u l a c lro n eo n t h eo r i g i n aple t r i dish(although for easeof comparison, it isnot shownreversed h e r e )A. l i g n i ntgh ea u t o r a d i o g r awm i i s hw i l l i t ht h eo r i g i n aple t r d locatethe corresponding clonefromwhichE colicellscanbe recovered

this labeledprobe hybridizesro any complemenrarynucleic acid strandsbound to the membrane.Any excessprobe that doesnot hybridize is washedaway, and the labeledhybrids are detectedby autoradiography of the filter. This technique can be usedto screenboth genomicand cDNA libraries,but is most commonly usedto isolatespecificcDNAs. ClearlS identification of specific clones by the membrane-hybridizationtechnique depends on the availa b i l i t y o f c o m p l e m e n r a r yr a d i o l a b e l e d p r o b e s . F o r a n oligonucleotideto be useful as a probe, ir must be long enough for its sequenceto occur uniquely in the clone of interest and not in any other clones. For most purposes, this condition is satisfied by oligonucleotidescontaining a b o u t 2 0 n u c l e o t i d e s .T h i s i s b e c a u s ea s p e c i f i c2 0 - n u c l e o t i d e s e q u e n c eo c c u r s o n c e i n e v e r y 4 2 0 ( = 1 0 1 2 )n u cleotides.Since all genomesare much smaller (=3 x t0e nucleotidesfor humans),a specific20-nucleotidesequence in a genomeusually occurs only once. With automated ins t r u m e n t s n o w a v a i l a b l e , r e s e a r c h e r sc a n p r o g r a m t h e chemicalsynthesisof oligonucleotidesof specificsequence up to about 100 nucleotideslong. Longer probes can be preparedby the polymerasechain reaction (pCR), a widely

usedtechniquefor amplifying specificDNA sequences rhat is describedlater. How might an investigator design an oligonucleotide probe to identify a clone encoding a particular protein? It helps if all or a portion of the amino acid sequenceof the protein is known. Thanks to the availability of the complete genomic sequencesfor humans and some important model organisms such as the mouse, Drosophila, and the roundworm Caenorbabditis elegans,a researchercan use an appropriate computer program to searchthe genomic sequence database for the coding sequencethat corresponds to the amino acid sequenceof the protein under study. If a match is found, then a single, unique DNA probe based on this known genomic sequencewill hybridize perfectly with the clone encoding the protein of interest.

182

.

cHAprER s

I

M o l E c u L A RG E N E T rI cE c H N t e u E S

YeastGenomicLibrariesCan Be Constructed with Shuttle Vectorsand Screenedby F u n c t i o n aC l omplementation In somecasesa DNA library can be screenedfor the ability to express a functional protein that complements a recessive

Polylinker

(a)

Shuttlevector

CEN (b)

l^

dNos mil$

mmrs

Yeastgenomic DNA

Transform E. coli Screenfor amoicillinresistance 'as\ +

*

***

I lsolateandpoolrecombinant from 105transformed I plasmids { F. colicolonies complementation Assayyeastgenomiclibraryby functional

5-17 A yeastgenomiclibrarycan be < EXPERIMENTAL FIGURE plasmid vector that can replicatein shuttle in a constructed shuttlevector of a typicalplasmid yeastand E.coli.(a)Components genesThepresence of a yeastoriginof for cloningSaccharomyces (CEN) (ARS) allowsstable anda yeastcentromere DNAreplication isa yeast yeast included Also in andsegregation replication markersuchasURA3,whichallowsa ura3 mutantto selectable sequences thevectorcontains growon mediumlackinguracilFinally, in E.coli(ORlandamp')anda polylinker andselection for replication (b)Typical protocol for of yeastDNAfragments. for easyinsertion yeast of total digestion genomic Partial yeast library a constructing with an fragments to generate genomic DNAwith5au3Aisadjusted to acceptthe sizeof about10 kb Thevectorisprepared average the whichproduces with BamHl, genomic by digestion fragments E. coli that clone of transformed samestickyendsas5au3A.Each a singletype contains resistance for ampicillin growsafterselection of yeastDNAfragment in a genomic DNA fragment inserted into a plasmid vector. To construct a plasmid genomic library that is to be screenedby functional complementationin yeast cells,the plasmid vector must be capable of replication in both E. coli cells and yeast cells. This type of vector' capable of propagation in two different hosts, is called a shuttle vector. The structure of a typical yeast shuttle vector is shown in Figure 5-L7a. This vector contains the basic elements that permit cloning of DNA fragmentsin E- coli.In addition, the shuttle vector contains an autonomouslyreplicating sequence(ARS), which functions as an origin for DNA replication in yeast; a yeast centromere (called CEN), which allows faithful segregationof the plasmid during yeastcell division; and a yeastgeneencodingan enzymefor uracil synthesis (URA3), which serves as a selectable marker in an approprlate yeastmutant. To increasethe probability that all regions of the yeast genome are successfullycloned and represented in the plasmid library, the genomic DNA usually is only partially digest.d to yield overlapping restriction fragments of =tb t U. These fragments are then ligated into the shuttle vector in which the polylinker has been cleavedwith a restriction enzymethat produces sticky ends complementary to those on the yeast DNA fragments (Figure 5-17b). Becausethe 10-kb restriction fragmentsof yeastDNA are incorporated into the shuttle u.ito., randomly, at least 10s E. coli colonies,each containing a particular recombinant shuttle vector, are necessaryto assurethat each region of yeast DNA has a high probability of being representedin

the library at least once. mutation. Sucha screeningstrategywould be an efficientway Figure 5-18 outlines how such a yeast genomic library to isolate a cloned genethat correspondsto an interestingrecan be screenedto isolate the wild-type gene corresponding cessivemutation identified in an experimental organism. To one of the temperature-sensitivecdc mutations mentioned to illustrate this method, referredto as functional complementain this chapter. The starting yeast strain is a double earlier E. coli tion, we describehow yeast genescloned in special that requires uracil for growth due to a ura3 mttamutant identify yeast cells to plasmidscan be introduced into mutant is temperature-sensitivedue to a cdc28 mutation and tion strain. in the mutant the wild-type genethat is defective by its phenotype (see Figure 5-6). Recombinant identified purpose of screening for the Libraries constructed from the yeast genomic library are mixed isolated plasmids from constructed are among yeast gene sequencesusually genomic DNA rather than cDNA. BecauseSaccharomyces with yeast cells under conditions that promote transformation of the cells with foreign DNA. Sincetransformed yeast genesdo not contain multiple introns, they are sufficiently cells carry a plasmid-borne copy of the wild-type URA3 compact that the entire sequenceof a genecan be included D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

183

Libraryof yeast genomic DNA carrying URA3selectivemarker

Temperature-sensitive cdc-mutant yeast; ura3 (requiresuracil)

Transformyeast by treatmentwith LiOAC,PEG.and heat shocr Plateand incubateat permissivetemperature o n m e d i u m l a c k i n gu r a c i l

Only colonies carrYtng a URA3 marker are able to grow

23'C

R e p l i c a - p l a taen d incubateat nonpermissive te m perature

Only colonies carrying a wild-type CDC gene are able to grow

36'C

EXPERIMENTAL FIGURE 5-18 Screening of a yeastgenomic libraryby functionalcomplementation can identifyclones carryingthe normalform of a mutantyeastgene.Inthis example, a wild-type CDCgeneis isolated by complementation of a cdcyeastmutant TheSaccharomyces strainusedfor screentng rne yeastlibrary carries ura3 anda temperature-sensitive cdcmutation Thismutantstrainisgrownandmaintained at a permisstve (23"C) Pooled temperature plasmids prepared recombinant as

shownin Figure 5-17arerncubated with the mutantyeastcellsunder conditions thatpromotetransformation Therelatively few transformed yeastcells, plasmid whichcontainrecombinant DNA. cangrowin theabsence of uracilat 23 'C Whentransformed yeast colonies arereplica-plated andplacedat 36'C (a nonpermissrve temperature), onlyclones carrying a libraryplasmid thatcontains the wild-type copyof the CDCgenewillsurviveL|OAC= lithiumacetate; PEG= polyethylene glycol

gene,they can be selectedby their ability to grow in the absenceof uracil. Typically, about 20 petri dishes, each containing about 500 yeast transformants, are sufficient to represent the entire yeast genome. This collection of yeast transformants can be maintained at 23"C, a temperature permissivefor growth of the cdc28 mutant. The entire colIection on 20 plates is then transferredto replica plates, which are placed at 36 "C, a nonpermissivetemperature for cdc mutants. Yeastcoloniesthat carry recombinant plasmids expressinga wild-type copy of the CDC28 genewill be able to grow at 36'C. Once temperature-resistant yeastcolonies have been identified, plasmid DNA can be extracted from the cultured yeast cells and analyzed by subcloning and DNA sequencing,topics we take up next.

the well into which the original DNA mixture was placed at the start of the electrophoreticrun. Smaller moleculesmove through the gel matrix more readily than larger molecules, so that molecules of different length migrate as distinct bands. Smaller DNA moleculesfrom about 10 to 2000 nucleotides can be separatedelectrophoretically on polyacrylamide gels, and larger molecules from about 200 nucleotidesto more than 20 kb on agarose gels. A common method for visualizingseparatedDNA bands on a gel is to incubatethe gel in a solution containing the fluorescentdye ethidium bromide. This planar moleculebinds to DNA by intercalating berweenthe base pairs. Binding concentratesethidium in the DNA and also increasesits intrinsic fluorescence.As a result, when the gel is illuminated with ultraviolet light, the regions of the gel containing DNA fluoresce much more brightly than the regionsof the gel without DNA. Once a cloned DNA fragment, especiallya long one, has beenseparatedfrom vector DNA, it often is treated with various restriction enzymesto yield smallerfragments.After separation by gel electrophoresis,all or some of these smaller fragments can be ligated individually into a plasmid vector and cloned in E. coli by the usual procedure. This process, known as subcloning,is an important step in rearranging parts of genesinto useful new configurations. For instance, an investigator who wants to change the conditions under which a geneis expressedmight usesubcloningto replacethe normal promoter associatedwith a cloned genewith a DNA segmentcontaining a different promoter. Subcloningalso can be used to obtain cloned DNA fragments that are of an appropriate length for determining the nucleotidesequence.

Gel Electrophoresis Allows Separationof Vector DNA from ClonedFragments In order to manipulate or sequencea cloned DNA fragment, it sometimesmust first be separatedfrom the vector DNA. This can be accomplished by cutting the recombinant DNA clone with the samerestriction enzymeusedto produce the recombinant vectors originally. The cloned DNA and vector DNA then are subjected to gel electrophoresis, a powerful method for separatingDNA moleculesof different size(Figure5-19). Near neutral pH, DNA molecules carry a large negative charge and therefore move toward the positive electrode during gel electrophoresis.Becausethe gel marrix restricrs random diffusion of the molecules, molecules of the same length migrate together as a band whose width equalsthat of '184 .

c H A p r E5 R | M o L E c u L AGRE N E TrtEc c H N t e u E s

(a)

DNA restrictionfragments

dKs

I n , u . " m i x t u r ei n t h ew e l lo f I a n a g a r o soer p o l y a c r y l a m i d e A n n l ve l e c t r ifci e l d I sel.

v Well

__l

- (Negativeelectrode)

Gelparticle

Pores

+ (Positive lectrode) M o l e c u l e sm o v e t h r o u g hp o r e s in gel at a rate inversely p r o p o r t i o n atlo t h e i r c h a i nl e n g t h

separates 5-19 Gel electrophoresis FIGURE < EXPERIMENTAL by DNA moleculesof different lengths.(a)A gel is prepared p o u r i n ga l i q u i dc o n t a i n i negi t h e rm e l t e da g a r o soer t w o g l a s sp l a t e sa f e w u n p o l y m e r i zaecdr y l a m i dbee t w e e n m i l l i m e t ear sp a r t A s t h e a g a r o sseo l i d i f i eosr t h e a c r y l a m i d e e v a l sf)o r m s p o l y m e r i z ienst op o l y a c r y l a m i ad eg ,e lm a t r i x( o r a n g o p o l y m e r s T h e d i m e n s i o nosf o f c h a i n s c o n s i s t i nogf l o n g ,t a n g l e d s ,r p o r e sd, e p e n do n t h e t h e i n t e r c o n n e c t icnhga n n e l o f t h e a g a r o soer a c r y l a m i dues e dt o f o r mt h e g e l concentration T h es e p a r a t ebda n d sc a nb e v i s u a l i z ebdy a u t o r a d i o g r a p(hi fyt h e n yt e f r a g m e n tasr er a d i o l a b e l eodr)b y a d d i t i o no f a f l u o r e s c e d ( e g , e t h i d i u mb r o m i d et )h a t b i n d st o D N A ( b )A p h o t o g r a pohf a ( E t B r )E t B rb i n d st o D N Aa n d g e ls t a i n e w d i t h e t h i d i u mb r o m i d e f l u o r e s c eusn d e rU Vl i g h t ,T h eb a n d si n t h ef a r l e f ta n df a r r i g h t f r a g m e n tosf k n o w ns i z e l a n e sa r ek n o w na s D N Al a d d e r s - D N A h e l e n g t ho f t h e D N A f o r d e t e r m i n i nt g t h a ts e r v ea sa r e f e r e n c e f r a g m e n tisn t h e e x p e r i m e n tsaal m p l eI P a r(tb )S c i e n cPeh o t o L i b r alr y

o- o - PI : o

-O-P:O

I o

I

o

- O - P I: O

- O - P I: O

I o Subjectto autoradiography or incubatewith fluorescentdye

I

-O-P:O

l o

ase

- O - PI : O I O

I

CH,

QH,

HH Signals corresponding to DNA bands

Deoxyribonucleoside triphosPhate (dNTP)

Dideoxyribonucleoside triphosPhate (ddNTP)

of deoxyribonucleoside 5-20 Structures FIGURE triphosphate (dNTP) dideoxyribonucleoside and triphosphate intoa growingDNA residue (ddNTP). of a ddNTP Incorporation at thatpoint elongation strandterminates D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

185

Technique Animation:DideoxySequencing of DNA{tttt (b)

P r i m e r5 ' T e m p l a t e3 ' -

5'

(a) 5'TAGCTGACTC3' 3' A T C G A C T G A G T C A A G A A C T A T T G G G C T T A A

I DNApolymerase

+ ddGTP

I | + dATB dGTB dCTB dTTp | + ddGTPin low concentration I

v 5'TAGCTGACTCAG3' 3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

-A

-G

5 ' T A G C T G A CT C A GT T C + T T G 3 ' 3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

_A

EG

-G

5' TAGCTGACTCAGTTJTTCNTNACCCG3' 3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

-T

T

etc.

I

I

etc.

I

Denatureand separatedaughterstrandsby electrophoresis I N N N N A A T G A A TA G A T A T A T A G G G G A A T T G A G T 20 30 40

AAATAG 110

TTGG GTAA 120

ATGGT 130

GGTA

ATAG

GGGGAT

TGTTT 140

a EXPERIMENTAL FTGURE 5-21 ClonedDNAscan be sequencedby the Sangermethod, usingfluorescent-tagged dideoxyribonucleoside (ddNTps). triphosphates (a)A single (template) strandof the DNAto be sequenced (blueletters) is hybridized to a synthetic primer(blackletters) deoxyribonucleotide Theprimeriselongated in a reaction mixture containing thefour normaldeoxyribonucleoside triphosphates plusa relatively small amountof oneof thefourdideoxyribonucleoside triphosphates. ln thisexample, (yellow) ddGTP ispresentBecause of the relatively low concentration of ddGTP, incorporation of a ddGTBandthus chaintermination, occurs at a givenposition in the sequence only about1 percent of thetime Eventually the reaction mixture will containa mixture of prematurely (truncated) terminated dauqhter

186

CHAPTER 5

I

MOLECULAG RENETIC TECHNIQUES

T

TAGAGT

GA

TG

AGG

GTGAAATTGTTATGTAAA 150 160

AIG 80

AAG TTGAGTATT 90-

170

A

A

AA '180

I

AT

fragments endingat everyoccurrence of ddGTp(b)Toobtainthe complete sequence of a templateDNA,four separate reactions are performed, eachwith a different dideoxyribonucleoside triphosphate (ddNTP) TheddNTP thatterminates eachtruncated fragment canbe identified by useof ddNTPs taggedwith four differentfluorescent dyes(indicated (c)In an automated bycolored highlights). sequencing machine, thefourreaction mixtures aresubjected to gel electrophoresis, andtheorderof appearance of eachof thefour drfferent fluorescent dyesat the endof the gelis recordedShown hereisa sample printoutfroman automated sequencer fromwhich thesequence of theoriginal template DNAcanbe deduced fromthe sequence of thesynthesized strandN = nucleotide thatcannotbe (c)fromGriffiths assigned[Part et al, Figure 14-27 I

lsolation of a genomic library spanning the genome of interest A l i g n i n gl i b r a r yc l o n e s by hybridizationor restriction-site mapping

Ordered set of clones spanningthe genome

Sequencing of orderedclones

Genomicsequence > FIGURE 5-22 Two Strategies for Assembling WholeGenome Sequences. Onemethoddepends on isolating andassembling a set thatspanthe genomeThiscanbe doneby of clonedDNAsegments or by alignment of matching clonedsegments by hybridization restriction sitemapsTheDNAsequence of the ordered clones can genomic intoa complete The thenbe assembled sequence

of Sequencing r a n d o ml i b r a r yc l o n e s

Sequenceof unordered fragments, for about lO-fold coverageof eachgenomic segment

Aligning sequenced clonesby comPuter Assembled genomlcsequence DNA easeof automated on the relative methoddepends alternative the library stepof ordering the laborrous andbypasses sequencing sothateachsegment clones enoughrandomlibrary Bysequencing to it ispossible to 10 times genome from 3 is represented of the of the alignment by computer sequence thegenomrc reconstruct fragments verylargenumberof sequence

guished by their corresponding fluorescent label (Figure 521b). For example,all truncated fragmentsthat end with a G would fluoresce one color (e.g., yellow), and those ending with an A would fluoresceanother color (e.g',red), regardless The complete characterizationof any cloned DNA fragment requires determination of its nucleotide sequence.F. Sanger of their lengths. The mixtures of truncated daughter fragments from each of the four reactionsare subjectedto elecand his colleaguesdeveloped the method now most comtrophoresison specialpolyacrylamidegels that can separate monly used to determine the exact nucleotide sequenceof single-strandedDNA moleculesdiffering in length by only 1 DNA fragmentsup to =500 nucleotideslong. The basic idea nucleotide.A fluorescencedetector that can distinguish the behindthis method is to synthesizefromthe DNA fragmentto four fluorescenttags is located at the end of the gel. The sebe sequenceda set of daughterstrandsthat are labeledat one quence of the original DNA template strand can be deterend and differ in length by one nucleotide.Separationof the mined from the order in which different labeledfragmentsmitruncated daughterstrandsby gel electrophoresiscan then esgrate past the fluorescencedetector (Figure 5-21'c). tablish the nucleotidesequenceof the original DNA fragment. In order to sequencea long continuousregion of genomic Synthesisof truncated daughterstandsis accomplishedby triphosphates(ddNTPs). DNA or even the entire genomeof an organism, researchers use of 2',3'-dideoxyribonucleoside These molecules,in contrast to normal deoxyribonucleotides usually employ one of the strategiesoutlined in Figure 5-22. (dNTPs), lack a 3' hydroxyl group (Figure 5-20). Although The first method requiresthe isolation of a collection of cloned overlap. Once the sequence DNA fragmentswhose sequences ddNTPs can be incorporated into a growing DNA chain by oligonucleotidesbased is determined, fragments of these of one DNA polymerase,once incorporated they cannot form a for use as synthesized can be chemically sequence on that phosphodiesterbond with the next incoming nucleotide In fragments. overlapping the adjacent in sequencing primers triphosphate.Thus incorporation of a ddNTP terminates is determined DNA of long stretch of a sequence way, the this resulting at in a daughterstrand truncated chain synthesis, incrementally by sequencingof the overlapping cloned DNA specific positions correspondingto the basecomplementary fragments that compose it. A second method, which is called added ddNTP on the template strand. to the the time-consumbypasses whole genomeshotgunseqwencing, Sequencingusing the Sangerdideoxy chain-termination ing step of isolating an ordered collection of DNA segments method is usually carried out using an automated DNA sethat span the genome.This method involves simply sequencing quencing machine. The reaction begins by denaturing a random clones from a genomic library. A total number of double-strandedDNA fragment to generatetemplate strands for in vitro DNA synthesis.A syntheticoligodeoxynucleotide clonesare chosenfor sequencingso that on averageeach segabout 10 times.This degreeof ment of the genomeis sequenced is usedas the primer for the polymerizationreactionthat concoverageensuresthat eachsegmentof the genomeis sequenced tains a low concentrationof eachof the four ddNTPs in addimore than once.The entiregenomicsequenceis then assembled tion to higher concentrationsof the normal dNTPs. The using using a computeralgorithm that alignsall the sequences, ddNTPs are randomly incorporated at the positions of the their regionsof overlap.\7hole genomeshotgunsequencingis correspondingdNTP, causingtermination of polymerization of the fastestand most cost-effectivemethod for sequencinglong at thosepositionsin the sequence(Figure5-21,a).Inclusion stretchesof DNA, and most genomes'including the human fluorescenttagsof different colors on eachof the four ddNTPs genome,have beensequencedby this method. allows each set of truncated daughterfragmentsto be distin-

Rapidly C l o n e dD N A M o l e c u l e sA r e S e q u e n c e d b y t h e D i d e o x yC h a i n - T e r m i n a t i oMne t h o d

D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

187

TechniqueAnimation: PolymeraseChain Reaction{tttt

c Y c l e1

, Denaturationof DNA I A n n e a l i n go f p r i m e r s

E l o n g a t i o no f p r i m e r s

I

c Y c l e2

, Denaturationof DNA I A n n e a l i n go f p r i m e r s

Elongationof primers

{

G Y c l e3

< EXPERIMENTAL FIGURE 5-23 The polymerase chainreaction(PCR) is widely used to amplifyDNAregionsof known sequences. Toamplifya specific regionof DNA,an investigator willchemically primers synthesize two different oligonucleotrde complementary to (designated sequences of approximately 18 bases flankingtheregionof interest aslightblue anddarkbluebars)Thecomplete reaction iscomposed of a complex mixture of doublegenomic stranded DNA(usually DNAcontaining thetargetsequence of interest), a stoichiometric excess of bothprimers, thefourdeoxynucleoside triphosphates, anda heatstableDNApolymerase knownasTaqpolymerase. DuringeachPCRcycle, the reaction mixture isfirstheatedto separate thestrands andthencooledto allowtheprimers to bindto complementary sequences flanking the regionto beamplified. Iaq polymerase thenextends eachprimerfromits3' end,generating newlysynthesized strands thatextendin the3' direction to the 5' endof thetemplate strand.Duringthethirdcycle, two double-stranded DNAmolecules aregenerated equalin lengthto thesequence of theregionto beamplified Ineachsuccessive cyclethetargetsegment, whichwillannealto theprimers, isduplicated, andwilleventually vastlyoutnumber allotherDNAsegments in thereaction mixture Successive PCRcycles canbeautomated bycycling the reaction for timedintervals at high temperature for DNAmeltingandat a definedlowertemperature for theannealing and portions elongation of thecycleA reaction thatcycles 20 timeswillamplifythespecific target sequence 1-million-fold

, Denaturationof DNA I n n n e a t i n so f p r i m e r s

E l o n g a t i o no f p r i m e r s

I

L

Cycles4, 5, 6, etc.

T h e P o l y m e r a sC e h a i nR e a c t i o nA m p l i f i e sa SpecificDNA Sequencefrom a ComplexMixture If the nucleotide sequencesat the ends of a parricular DNA region are known, the intervening fragment can be amplified directly by the polymerase chain reaction (PCR). Here we describethe basic PCR technique and three situations in which it is used. 188

.

cHAprER s

I

M o L E c u L AG R E N E I cr E c H N t e u E s

The PCR dependson the ability to alternately denature (melt) double-strandedDNA moleculesand hybridizecomplementary singlestrandsin a controlled fashion. As outlined in Figure5-23, a typical PCR procedurebeginsby heat-denaturation of a DNA sampleinto singlestrands.Next, two synthetic oligonucleotidescomplementaryto the 3' ends of the target DNA segmentof interestare addedin greatexcessto the denatured DNA, and the temperarure is lowered to 50-60 'C. Thesespecificoligonucleotides,which are at a very high concentration,will hybridizewith their complementarysequences in the DNA sample, whereas the long strands of the sample DNA remain apart becauseof their low concentration.The hybridized oligonucleotidesthen serveas primers for DNA chain synthesisin the presenceof deoxynucleotides(dNTPs) and a temperature-resistantDNA polymerase such as that from Thermusaquaticus(a bacteriumthat livesin hot springs).This enzymq called Taq polymerase, can remain active even after being heatedto 95 oC and can extend the primers at temperaturesup to72"C. When synthesisis complete,the whole mixture is then heatedto 95 "C to denaturethe newly formed DNA duplexes.After the temperatureis lowered again, another cycle of synthesistakes place becauseexcessprimer is still present. Repeated cycles of denaturation (heating) followed by hybridization and synthesis(cooling) quickly amplify the sequence of interest. At each cycle, the number of copies of the sequencebetweenthe primer sitesis doubled; therefore, the desired sequenceincreasesexponentially-about a million-fold after 20 cycles-whereas all other sequencesin the original DNA sampleremain unamplified. Direct lsolation of a Specific Segment of Genomic DNA For organismsin which all or most of the genome has been sequenced,PCR amplification starting with the total genomic DNA often is the easiestway to obtain a specificDNA region of interest for cloning. In this application, the two oligonucleotideprimers are designedto hybridize to sequences flanking the genomic region of interest and to include sequences

Regionto be amplified 5'

3'

P r i m e r1

D N As y n t h e s i s

s'

5'

P r i m e r2

5-24 A specifictarget region in total FIGURE < EXPERIMENTAL genomicDNAcan be amplifiedby PCRfor usein cloning.Each to oneendof thetargetsequence primerfor PCRiscomplementary that enzyme for a restriction sequence the recognition andincludes primer doesnot havea sitewithinthetargetregion.In thisexample, primer2 contains a Hindlll whereas a BamHl sequence, 1 contains (Notethatfor clarity, icationof only in anyround,amplif sequence isshown,the onein brackets oneof thetwo strands ) After aretreatedwith appropriate thetargetsegments amplification, generating with stickyendsThese fragments restriction enzymes, plasmid vectors andcloned into complementary canbe incorporated (seeFigure 5-13). in E colibythe usualprocedure

c

Prime1 r

C o n t i n u ef o r = 2 0 PCRcycles Cut with restriction enzymes

Stickyend

-Stickyend Ligatewith plasmidvector with stickyends

that are recognizedby specificrestriction enzymes(Figure 524). After amplification of the desired target sequencefor about 20 PCR cycles,cleavagewith the appropriate restriction enzymesproduces sticky ends that allow efficient ligation of the fragment into a plasmid vector cleaved by the same restriction enzymesin the polylinker. The resulting recombinant plasmids,all carrying the identical genomic DNA segment,can then be cloned in E. coli cells.\fith certain refinements of the PCR, even DNA segmentsgreater than 10 kb in length can be amplified and cloned in this way. Note that this method does not involve cloning of large numbers of restriction fragments derived from genomic DNA and their subsequentscreeningto identify the specificfragment of interest. In effect, the PCR method inverts this traditional approach and thus avoids its most tedious aspects.The PCR method is useful for isolating genesequencesto be manipulated in a variety of useful ways describedlater. In addition the PCR method can be used to isolate gene sequencesfrom mutant organismsto determinehow they differ from the wild type. A variation on the PCR method allows PCR amplification of a specificcDNA sequencefrom cellular mRNAs. This

method, known as reuersetranscriptase-PcR /R?PCR/, begins with the same procedure describedpreviously for isolation of cDNA from a collection of cellular mRNAs' Typically' an oligo-dT primer, which will hybridize to the 3' poly(A) tail of the mRNA, is used as the primer for the first strand of cDNA synthesisby reversetranscriptase.A specific cDNA can then be isolatedfrom this complex mixture of cDNAs by PCR amplification using two oligonucleotide primers designedto match sequencesat the 5' and 3' ends of the corresponding mRNA. As described previously, these primers could be designedto include restriction sitesto facilitate the insertion of amplified cDNA into a suitableplasmid vector. Preparation of Probes Earlier we discussedhow oligonucleotide probes for hybridization assayscan be chemically synthesized.Preparation of such probes by PCR amplification requires chemical synthesisof only two relatively short primers corresponding to the two ends of the target sequence. The starting sample for PCR amplification of the target sequencecan be a preparation of genomic DNA, or a preparation of cDNA synthesizedfrom the total cellular 32PmRNA. To generatea radiolabeled product from PCR, labeled dNTPs are included during the last several amplification cycles.Becauseprobes prepared by PCR are relatively 3'P atoms incorporated into long and have many radioactive them, theseprobes usually give a stronger and more specific signal than chemically synthesizedprobes. Tagging of Genes by Insertion Mutations Another useful application of the PCR is to amplify a "tagged" gene from the genomic DNA of a mutant strain. This approach is a simpler method for identifying genesassociatedwith a particular mutant phenotype than screening of a library by functionalcomplementation(seeFigure5-18). The key to this use of the PCR is the ability to produce mutations by insertion of a known DNA sequenceinto the genome of an experimental organism. Such insertion mutations can be generated by use of mobile DNA elements, which can move (or transpose)from one chromosomal site to another. As discussedin more detail in Chapter 5, these DNA sequencesoccur naturally in the genomesof most organisms and may give rise to loss-of-function mutations if they transposeinto a protein-coding region. For example, researchershave modified a Drosophila mobile DNA element, known as the P element, to optimize D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

r89

> EXPERIMENTAL FIGURE 5-25 The genomic sequenceat the insertionsite of a transposon is revealedby PCRamplificationand Restriction sites:t sequencing. Toobtainthe DNAsequence of the insertion siteof a P-element transooson it is necessary to PCR-amplify thejunctionbetween knowntransposon sequences andunknown flankingchromosomal sequences Onemethodto achieve thisisto cleave genomic DNAwith a restriction enzyme thatcleaves oncewithinthe transposon sequence Ligation of the resulting restriction fragments willgenerate circular DNA molecules. Byusingappropriately designed DNA primers thatmatchtransposon sequences it is possible junction to PCR-amplify the desired fragmentFinally, (see a DNAsequencing reaction Figure 5-21)is performed usrngthe PCR-amplified fragmentasa template andan oligonucleotide primerthat matches sequences neartheendof the transposon, to obtainthesequence of thejunction between thetransposon andchromosome

I ransDoson

I cr, *it, I

restriction enzyme

Ligate to circularize

PCRprimers

Sequencing pflmer

I PCRamplification with Orimers to transposon I

-+

its use in the experimental generation of insertron mutations. Once it has been demonstratedthat insertion of a P element causesa mutation with an interestingphenotype, the genomic sequencesadjacentto the insertion site can be amplified by a variation of the standardPCR protocol that usessyntheticprimers complementaryto the known P-element sequencebut that allows unknown neighboring sequencesto be amplified. One such method, depicted in Figure 5-25, beginsby cleavingDrosophila genomic DNA containing a P-elementinsertion with a restriction enzyme that cleavesonce within the P-elementDNA. The collection of cleaved DNA fragmenrs treated with DNA ligase yields circular molecules,some of which will contain P-element DNA. The chromosomal region flanking the P element can then be amplified by PCR using primers that match P-elementsequencesand are elongatedin opposite directions. The sequenceof the resulting amplified fragment can then be determined using a third DNA primer. The crucial sequencefor identifying the site of P-element insertion is the junction between the end of the P-element and genomic sequences.Overall, this approach avoids the cloning of large numbers of DNA fragments and their screeningto detect a cloned DNA correspondingto a mutated geneof interest. Similar methods have been applied to other organrsms for which insertion mutations can be generatedusing either mobile DNA elements or viruses with sequencedgenomes that can insertrandomly into the genome. 190

CHAPTER 5

|

MOLECULAG R E N E T TTCE C H N T Q U E S

DNA Cloning and Characterization r In DNA cloning, recombinant DNA molecules are formed in vitro by inserting DNA fragments into vecror DNA molecules.The recombinant DNA molecules are then introduced into host cells, where they replicate, producing large numbers of recombinant DNA molecules. r Restriction enzymes (endonucleases)typically cut DNA at specific4- to 8-bp palindromic sequences,producing defined fragments that often have self-complementarysinglestrandedtails (stickyends). I Two restriction fragments with complementary ends can be joined with DNA ligase to form a recombinant DNA molecule(seeFigure 5-12). t E. coli cloning vectors are small circular DNA molecules (plasmids)that include three functional regions:an origin of replication, a drug-resistancegene, and a site where a DNA fragmentcan be inserted.Transformedcellscarrying a vector grow into colonieson the selectionmedium (seeFigure5-13). r A cDNA library is a set of cDNA clones prepared from the mRNAs isolated from a particular type of tissue. A genomic library is a set of clones carrying restriction fragments produced by cleavageof the entire genome. r In cDNA cloning, expressed mRNAs are reversetranscribedinto complementaryDNAs, or cDNAs. By a series of reactions,single-strandedcDNAs are converted into

double-strandedDNAs, which can then be ligated into a plasmidvector (seeFigure5-15). r A particular cloned DNA fragment within a library can be detected by hybridization to a radiolabeledoligonucleotidewhose sequenceis complementaryto a portion of the fragment(seeFigure5-16). r Shuttle vectors that replicate in both yeast and E. coli can be used to construct a yeastgenomic library. Specificgenes can be isolated by their ability to complementthe correspondingmutant genesin yeastcells(seeFigure5-17). r Long cloned DNA fragments often are cleavedwith restriction enzymes,producing smallerfragmentsthat are then separatedby gel electrophoresisand subclonedin plasmid vectorsprior to sequencingor experimentalmanipulation. r DNA fragmentsup to about 500 nucleotideslong are sequenced in automated instruments based on the Sanger (dideoxychain-termination)method (seeFigure5-21). r lil/hole genome sequencescan be assembledfrom the sequencesof a large number of overlappingclones from a genomiclibrary (seeFigure 5-22). r The polymerasechain reaction(PCR)permitsexponential amplification of a specificsegmentof DNA from just a single initial template DNA molecule if the sequenceflanking the DNA regionto be amplifiedis known (seeFigure5-23). r PCR is a highly versatilemethod that can be programmed to amplify a specificgenomicDNA sequence,a cDNA, or a sequenceat the junction betweena transposableelement and flanking chromosomalsequences.

EE UsingClonedDNAFragments to StudyGeneExpression In the last sectionwe describedthe basictechniquesfor using recombinantDNA technologyto isolatespecificDNA clones, and ways in which the clones can be further characterized.

Now we considerhow an isolatedDNA clone can be usedto study geneexpression.We discussseveralwidely usedgeneral techniquesthat rely on nucleic acid hybridization to elucidate when and where genesare expressed,as well as methods for generatinglarge quantitiesof protein and otherwisemaniputo determinetheir expressionpatlating amino acid sequences terns,structure,and function. More specificapplicationsof all thesebasictechniquesare examinedin the following sections.

s e r m i tD e t e c t i o no f H y b r i d i z a t i o nT e c h n i q u eP F r a g m e n t s a n d mRNAs S p e c i f i cD N A Two very sensitivemethodsfor detectinga particular DNA or RNA sequencewithin a complex mixture combine separation and hybridizationwith a complementary by gel electrophoresis probe. A third method involveshybridizing DNA radiolabeled labeledprobesdirectly onto a preparedtissuesample.\Wewill encounter referencesto all three of these techniques,which have numerousapplications,in other chapters. Southern Blotting The first hybridizationtechniqueto detect DNA fragments of a specific sequenceis known as Southern blotting after its originator E. M. Southern.This techniqueis capable of detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavageof the entire human genomewith a restrictionenzyme.When sucha complex mixture is subjectedto gel electrophoresis,so many different fragmentsof nearly the same length are presentit is not possibleto resolveany particular DNA fragmentsas a discrete band on the gel. Neverthelessit is possibleto identify a particular fragmentmigrating as a band on the gel by its ability to hybridize to a specificDNA probe. To accomplish this, the restriction fragments present in the gel are denatured with alkali and transferredonto a nitrocellulosefilter or nylon membraneby blotting (Figure 5-26). This procedurepreservesthe distribution of the fragmentsin the gel, creatinga replica of the gel on the filter. (The blot is usedbecauseprobesdo not readily diffuseinto the original gel.)The filter then is incubatedunder hybridization conditions with a specificradiolabeledDNA probe, which usually is

DNA I I Cleavewith restriction enzymes I Gel V

Autoradiogram

Ni t r o c e l l luo s e Ni t r o c e l l luo s e Gel ----.f----

Hybridizewith l a b e l e dD N A o r R N Ap r o b e

---r---T---T

A l k a l i n es o l u t i o n C a p i l l a r ya c t i o nt r a n s f e r s DNAfrom gelto nitrocellulose

FIGURE A EXPERIMENTAL 5-25 Southernblot techniquecan detecta specificDNAfragmentin a complexmixtureof threedifferent restrictionfragments.Thediagram depicts to fragments in the gel,butthe procedure canbe applied restriction

that of DNAfragmentsOnlyfragments of millions a mixture probewillgivea signalon an autoradiogram to a labeled hybridize mRNAs specifrc blottingdetects calledtVorthern technique A similar J Mol Blol98:508 1975, l withina mixturelseeE M Southern,

U S I N GC L O N E DD N A F R A G M E N TTSO S T U D YG E N EE X P R E S S I O N

191

generatedfrom a cloned restriction fragment. The DNA restriction fragmentthat is complementaryto the probe hybridizes,and its location on the filter can be revealedby autoradiography. Northern Blotting One of the most basicways to characterize a cloned geneis to determinewhen and where rn an organism the geneis expressed.Expressionof a particular genecan be followed by assayingfor the corresponding mRNA by Northern blotting, named, in a play on words, after the related method of Southern blotting. An RNA sample, often the total cellular RNA, is denatured by treatment with an agent such as formaldehyde that disrupts the hydrogen bonds between base pairs, ensuring that all the RNA moleculeshave an unfolded, linear conformation. The individual RNAs are separated according to size by gel electrophoresisand transferred to a nitrocellulose filter to which the extendeddenatured RNAs adhere.As in Southernbloning, the filter then is exposedto a labeled DNA probe that is complementaryto the geneof interest; finally, the labeled filter is subjected to autoradiography. Becausethe amount of a specificRNA in a samplecan be estimated from a Norrhern blot, the procedureis widely used ro compare the amounts of a particular mRNA in cells under differentconditions(Figure5-27). In Situ Hybridization Northern blotting requiresextracring the mRNA from a cell or mixture of cells,which meansthat the cellsare removedfrom their normal location within an orsan-

UN

{k

5kb-

2kb-

ism or tissue.As a result, the location of a cell and its relation to its neighbors is lost. To retain such positional information in precisestudiesof geneexpression,a whole or sectionedtissueor evena whole permeabilizedembryo may be subjectedto in situ hybridization to detect the mRNA encoded by a particular gene.This technique allows genetranscription to be monitored in both time and space(Figure5-28).

D N A M i c r o a r r a y sC a nB e U s e dt o E v a l u a t et h e E x p r e s s i o on f M a n y G e n e sa t O n e T i m e Monitoring the expressionof thousandsof genessimultaneously is possiblewith DNA microarray analysis,another technique based on the concept of nucleic acid hybridization. A DNA microarray consists of an organized array of thousands of individual, closely packed gene-specific sequencesattached to the surfaceof a glassmicroscopeslide. By coupling microarray analysis with the results from genome sequencing projects, researchers can analyze the global patterns of gene expression of an organism during specificphysiological responsesor developmentalprocesses. Preparation of DNA Microarrays In one methodfor preparing microarrays,an =1-kb portion of the coding region of each gene analyzedis individually amplified by the PCR. A robotic deviceis used to apply each amplified DNA sample to the surface of a glass microscope slide, which then is chemically processedto permanently attach the DNA sequencesto the glasssurfaceand to denature them. A typical array might contain =6000 spotsof DNA in a2 x 2- cm grid. In an alternative merhod, multiple DNA oligonucleotides, usually at least 20 nucleotides in length, are synthesizedfrom an initial nucleotide that is covalently bound to the surface of a glass slide. The synthesisof an oligonucleotide of specific sequencecan be programmed in a small region on the surfaceof the slide. Severaloligonucleotidesequencesfrom a singlegeneare thus synthesizedin neighboring regions of the slide to analyzeexpressionof that gene. Vith this method, oligonucleotidesrepresentingthousands of genescan be produced on a singleglassslide. Becausethe methods for constructing these arrays of synthetic oligonucleotideswere adapted from methods for manufacturing microscopic integrated circuits used in computers, these types of oligonucleotidemicroarrays are often called DNA chips.

1kb-0.6s kb -

w

t-flHl,.'l'"

A EXPERIMENTAL FIcURE5-27 Northernblot analysisreveals increased expression of p-globinmRNAin differentiated erythroleukemia cells.ThetotalmRNAin extracts of erythroleukemia cellsthatweregrowingbut uninduced andin cells induced to stopgrowingandallowedto differentiate for 4g hoursor 96 hourswasanalyzed by Northern blottingfor B-globin mRNAThe density of a bandisproportional to theamountof mRNApresent TheB-globin mRNAisbarelydetectable in uninduced cells(UNlane) but increases morethan1000-fold by 96 hoursafterdifferentiation is induced[Courtesy of L Kole] 192

.

cHAprER 5

|

M o L E c u L AG R E N E T tr cE c H N t e u E s

Using Microarrays to Compare Gene Expression under Different Conditions The initial stepin a microarrayexpression study is to preparefluorescentlylabeledcDNAs corresponding to the mRNAs expressedby the cellsunder study.When the cDNA preparation is applied to a microarray,sporsrepresenting genesthat are expressedwill hybridize under appropriate conditions to their complementarycDNAs in the labeledprobe mix, and can subsequentlybe detectedin a scanninglasermicroscope. Figure 5-29 depicts how this method can be applied to examine the changes in gene expression observed after starved human fibroblasts are transferred to a rich, serumcontaining, growth medium. In this type of experiment, the separatecDNA preparations from starvedand serum-grown

(c)

DorsaI Head

NT

Notochord

A EXPERIMENTAL FIGURE 5-28 In situ hybridizationcandetect activityof specificgenesin whole and sectionedembryos.The s p e c i m ei n s p e r m e a b i l i zbeydt r e a t m e nwt i t h d e t e r g e natn da protease to expose the mRNAto the probeA DNAor RNAprobe, s p e c i f ifco r t h e m R N Ao f i n t e r e sits, m a d ew i t h n u c l e o t i daen a l o g s c o n t a i n i ncgh e m i c aglr o u p tsh a tc a nb e r e c o g n i z ebdy a n t i b o d i e s A f t e rt h e p e r m e a b i l i zsepde c i m ehna sb e e ni n c u b a t ewdi t ht h e p r o b eu n d e cr o n d i t i o nt h s a tp r o m o t e hybrid z a t i o nt,h ee x c e s s p r o b ei s r e m o v ew d i t ha s e r i eosf w a s h e sT h es p e c i m ei n st h e n i n c u b a t ei d n a s o l u t i ocno n t a i n i nagn a n t i b o dtyh a tb i n d st o t h e p r o b eT hs a n t i b o diysc o v a i e n tl loyi n e dt o a r e p o r t eern z y m (ee g , se h o r s e r asdh p e r o x i d a o r a l k a l i npeh o s p h a t a st hea) tp r o d u c eas productAfterexcess coloredreaction antibodyhasbeenremoved,

drecipitate o r t h e r e p o r t eern z y m iesa d d e dA c o l o r e p s u b s t r a tf e f o r m sw h e r et h e p r o b eh a sh y b r i d i z et od t h e m R N Ab e i n g d e t e c t e d( a )A w h o l em o u s e m b r y oa t a b o u t1 0 d a y so f probedfor Sonichedgehog mRNAThestainmarks development ( r e d a l o n gt h e m e s o d e r rmu n n i n g a r o d o f a r r o w ) , t h en o t o c h o r d f u t u r es p i n acl o r d ( b )A s e c t i oonf a m o u s e m b r y os i m i l atro t h a t i n o a r t( a ) T h ed o r s a l / v e n tar xailso f t h e n e u r atlu b e( N T c) a nb e (redarrow) notochord seen,with the Sonichedgehog-expressing ( b l u e v e n t r a (l c )A s t i l f l a r t h e r a r r o w ) e n d o d e r m b e l o wi t a n dt h e during embryoprobedfor an mRNAproduced wholeDrosophila is patternof bodysegments Therepeating tracheadevelopment of L visibleAnterior(head)is up,ventralisto the left Icourtesy andN/ P Scott Milenkovic l

fibroblastsare labeledwith differently colored fluorescent dyes.A DNA array comprising8600 mammaliangenesthen is incubatedwith a mixture containingequal amountsof the t w o c D N A p r e p a r a t i o n su n d e r h y b r i d i z a t i o nc o n d i t i o n s . After unhybridizedcDNA is washed away, the intensity of green and red fluorescenceat each DNA spot is measured microscopeand storedin computerfiles using a fluorescence under the name of eachgeneaccordingto its known position on the slide.The relativeintensitiesof red and greenfluorescencesignalsat eachspot are a ffreasureof the relativelevel of expressionof that genein resp()nseto serum.Genesthat are not transcribedunder thesegrowth conditions give no detectablesignal. Genesthat are transcribed at the same level under both conditions will hybridize equally to both r e d a n d g r e e n - l a b e l e dc D N A p r e p a r a t i o n s .M i c r o a r r a y analysisof geneexpressionin fibroblastsshowedthat transcriptionof about 500 of the 8600 genesexaminedchanged substantiallyafter addition of serum.

the many different changesin cell physiology that occur when cells are transferred from one medium to another. I n o t h e r w o r d s , g e n e st h a t a p p e a rt o b e c o - r e g u l a t e di n a s i n g l e m i c r o a r r a y e x p r e s s i o ne x p e r i m e n t m a y u n d e r g o changesin expressionfor very different reasonsand may a c t u a l l y h a v e v e r y d i f f e r e n t b i o l o g i c a l f u n c t i o n s .A s o l u t i o n t o t h i s p r o b l e m i s t o c o m b i n et h e i n f o r m a t i o n f r o m a set of expressionarray experimentsto find genesthat are similarly regulated under a variety of conditions or over a oeriod of time. This more informative use of multiple expressionarray experiments is illustrated by examining the relative expression of the 8600 genesat different times after serum addition, generatingmore than 104 individual piecesof data. A computer program, relatedto the one usedto determinethe can organizethese of differentprotein sequences' relatedness over the expression genes show similar that data and cluster cluster such Remarkably, serum addition. after time course particproteins genes whose encoded groups sets of analysis ipate in a common cellular process, such as cholesterol biosynthesisor the cell cycle (Figure5-30).

C l u s t e rA n a l y s i so f M u l t i p l e E x p r e s s i o n E x p e r i m e n t sl d e n t i f i e sC o - r e g u l a t e G d enes F i r m c o n c l u s i c l n sr a r e l y c a n b e d r a w n f r o m a s i n g l e m i c r o a r r a y e x p e r i m e n t a b o u t w h e t h e r g e n e st h a t e x h i b i t s i m i l a r c h a n g e si n e x p r e s s i o na r e c o - r e g u l a t e da n d h e n c e likely to be closely related functionally. For example, m a n y o f t h e o b s e r v e dd i f f e r e n c e si n g e n e e x p r e s s i o nj u s t e fs d e s c r i b e di n f i b r o b l a s t sc o u l d b e i n d i r e c t c o n s e < r u e n c o

analysiswill be a powerful F.i In the future, microarray di"enustic tool in medicine. For instance,particular fiil setsof .RXRt have beenfound to distinguishtumors with a poor prognosisfrom those with a good prognosis.Previously indistinguishablediseasevariations are now detectable' Analysisof tumor biopsiesfor thesedistinguishingmRNAs

U S I N GC L O N E DD N A F R A G M E N TTSO S T U D YG E N EE X P R E S S I O N .

193

Technique Animation:Synthesizing an Oligonucleotide Array flllt TechniqueAnimation: Screeningfor Patternsof Gene Expression Fibroblasts w i t h o u ts e r u m

I

t s o t a tteo t a tm R N A I

+

Reverse-transcribe I to cDNA labeledwith lr/ a fluorescentdye {

Green dye--J

cDNAs hvbridized to DNAs for a single gene

J I I

Hybridizeto DNA mrcroarray

< EXPERIMENTAL FIGURE 5-29 DNAmicroarrayanalysiscan revealdifferencesin gene expressionin fibroblastsunder differentexperimentalconditions.(a)In thisexample, cDNA prepared frommRNAisolated fromfibroblasts eitherstarved for serumor afterserumadditionislabeled with different fluorescent dyesA microarray composed of DNAspotsrepresenting 8600 genesisexposed mammalian to an equalmixture of thetwo cDNA preparations underhybridization conditions Theratioof the intensities of redandgreenfluorescence overeachspot,detected with a scanning confocal lasermicroscope, indicates the relative expression of eachgenein response to serum.(b)A micrograph of a smallsegment of an actualDNAmicroarray. Eachspotin this 16 x genehybridized 16arraycontains DNAfroma different to control andexperimental cDNAsamples labeled with redandgreen fluorescent dyes(A yellowspotindicates equalhybridization of greenandredfluorescence, indicating no changein gene (b)Alfred expression) Pasieka/Photo Researchers, Inc] [Part

Wash Measuregreen and red fluorescenceover eachspot

will help physiciansto selectthe most appropriate treatment. As more patternsof geneexpressioncharacteristicof various diseasedtissuesare recognized,the diagnostic use of DNA microarrays will be extendedto other conditions.I

E. coliExpressionSystemsCan ProduceLarge Quantitiesof Proteinsfrom ClonedGenes

A lf a spot is green,expressionof that gene decreasesin cells after serum addition

.E l f a s p o t i s r e d ,e x p r e s s i o no f t h a t g e n e i n c r e a s e isn c e l l s after,seru m,addition

Many protein hormones and other signalingor regulatory proteins are normally expressedat very low concentrations,precluding their isolation and purification in large quantities by standard biochemical techniques. Widespread therapeutic use of such proteins, as well as basicresearchon their structureand functions, dependson efficient proceduresfor producing them in large amounts at reasonablecost. RecombinantDNA techniquesthat turn E. coli cells into factories for synthesizinglow-abundance proteins now are used to commercially produce granulocyte colony-stimulating factor (G-CSF), insulin, growth hormone, and other human proteins with therapeuticuses. For example, G-CSF stimulatesthe production of granulocytes, the phagocytic white blood cells critical to defense against bacterial infections. Administration of G-CSF to cancer patients helps offset the reduction in granulocyte production caused by chemotherapeuticagents, thereby protecting patients againstseriousinfection while they are receiving chemotherapy.I The first step in producing large amounts of a lowabundanceprotein is to obtain a cDNA clone encoding the full-length protein by methods discussedpreviously.The second step is to engineerplasmid vectorsthat will expresslarge amounts of the encoded protein when it is inserted into E. coli cells.The key to designingsuch expressionvectors is inclusion of a promoter, a DNA sequencefrom which transcription of the cDNA can begin. Consider,for example, the

194

c H A P T E R5

|

M O L E C U L AG R E N E T TTCE C H N T Q U E S

Eachcolumn representsa differentgene at times after addition of serum

0)

E -

-r-

BCD

A A EXPERIMENTAL FIGURE 5-30 Clusteranalysisof data from multiple microarrayexpressionexperimentscan identify cogeneswas of 8600mammalian regulatedgenes.Theexpression at timeintervals overa 24-hour detected by microarray analysis periodafterserum-starved wereprovided with serum.The fibroblasts that algorithm diagram shownhereisbasedon a computer cluster groupsgenesshowingsimilar with a compared changes in expression overtime.Eachcolumnof colored serum-starved controlsample a time boxesrepresents a singlegene,andeachrow represents point.A redboxindicates relative to the in expression an increase in expression; anda blackbox,no control;a greenbox,a decrease

relatively simple system for expressing G-CSF shown in Figure 5-31. In this case,G-CSF is expressedin E. coli transformed with plasmid vectors that contain the lac promoter adjacentto the cloned cDNA encoding G-CSF.Transcription from the lac promoter occurs at high rates only when lactose, or a lactose analog such as isopropylthiogalactoside (IPTG), is added to the culture medium. Even larger quantities of a desired protein can be produced in more complicated E. coli expressionsystems. To aid in purification of a eukaryotic protein produced in an E. coli expression system, researchersoften modify the cDNA encodingthe recombinant protein to facilitate its separation from endogenousE. coli proteins. A commonly used modification of this type is to add a short nucleotide sequenceto the end of the cDNA, so that the expressedprotein will have six histidine residuesat the C-terminus. Proteins modified in this way bind tightly to an affinity matrix > EXPERIMENTAL FIGURE 5-31 Someeukaryoticproteinscan be producedin E coli cellsfrom plasmidvectorscontainingthe vectorcontainsa fragment lac promoter.(a)Theplasmidexpression the /acpromoterandthe containing of the E colichromosome analogIPTG, of the lactose lacZgene.Inthe presence neighboring /acZ normallytranscribes the iacZgene,producing RNApolymerase protein, intotheencoded mRNA,whichistranslated B-galactosidase (b)The/acZgenecanbe cut out of the expression vectorwith andreplaced by a clonedcDNA,in thiscaseone restriction enzymes granulocyte Whenthe factor(G-CSF). colony-stimulating encoding plasmid intoE.collcells,additionof IPTG istransformed resulting produce G-CSF fromthe/acpromoter transcription andsubsequent protein intoG-CSF mRNA.whichistranslated

at thetop The"tree"diagram icantchangein expression. signif genescanbe patterns for individual showshowthe expression to grouptogetherthe geneswith fashion in a hierarchical organized overtime'Five of expression in theirpatterns similarity the greatest geneswereidentified in this regulated of coordinately clusters by the barsat the bottom.Eachcluster asindicated experiment, proteins functionin a geneswhoseencoded multiple contains (A),thecellcycle process: biosynthesis cholesterol particular cellular (C),signaling andangiogenesis (B),the immediate-early response (E).[Courtesy of Michael (D),andwoundhealing andtissueremodeling Laboratoryl National Berkeley Lawrence B Eisen,

(- -*R^*fu \/

,

p-galactosidase.

- IPTG (b)

Transform E. coli

- IPTG

+ IPTG

G E N EE X P R E S S I O N TO STUDY U S I N GC L O N E DD N A F R A G M E N T S

195

that contains chelated nickel atoms, whereas most E. coli proteins will not bind to such a matrix. The bound proteins can be releasedfrom the nickel atoms by decreasingthe pH of the surrounding medium. In most cases,this procedure yields a pure recombinant protein that is functional, since addition of short amino acid sequencesto either the Cterminusor the N-terminus of a protein usually doesnot interfere with the protein's biochemicalactivity.

{a) Transient transfection cDNA

I transtectcultured I c e l l sb y l i p i dt r e a t m e n t or electroporation J

PlasmidExpressionVectorsCan Be Designedfor U s ei n A n i m a lC e l l s \Vhile bacterial expressionsystemscan be used successfully to createlarge quantitiesof someproteins, bacteriacannot be used in all cases.Many experimenrsto examine the function of a protein in an appropriate cellular context requlre expression of a geneticallymodified protein in cultured animal cells. Genesare cloned into specializedeukaryotic expressionvec, tors and are introduced into cultured animal cells by a processcalled transfection.Two common methods for transfecting animal cells differ in whether the recombinant vecor DNA is or is not integratedinto the host-cellgenomic DNA. In both methods,culturedanimal cellsmust be treatedto facilitatetheir initial uptake of the recombinantplasmidvector. This can be done by exposingcells to a preparationof lipids that penetrarethe plasma membrane, increasingits permeability to DNA. Alternatively, subjecting cells to a brief electric shock of severalthousand volts, a technique known as electroporation, makes them transiently permeableto DNA. Usuallythe plasmidDNA is addedin sufficient concentrationto ensurethat a large proportion of the cultured cellswill receiveat leastone copy of the plasmidDNA. Researchers have also harnessedvirusesfor their use in the laboratory; virusescan be modified ro contain DNA of interest, which is then introduced into host cells by simply infecting them with the recombinant virus. Transient Transfection The simplestof the two expression methods, calledtransient transfectioz, employs a vector similar to the yeast shuttle vectors describedpreviously. For use in mammalian cells,plasmid vectorsare engineeredalso to carry an origin of replicationderivedfrom a virus that infecrsmammalian cells, a strong promoter recognized by mammalian RNA polymerase,and the cloned cDNA encodingthe protein to be expressedadjacentto the promoter (Figure5-32a). Once such a plasmid vector entersa mammalian cell, the viral origin of replication allows it to replicate efficientl5 generating numerousplasmidsfrom which the protein is expressed.However,duringcell divisionsuchplasmidsare nor faithfullysegregatedinto both daughtercellsand in time a substantialfraction of the cells in a culture will not contain a plasmid, hencethe name t ransi ent t ransfecti on. Stable Transfection (Transformation) If an introduced vector integratesinto the genomeof the host cell, the genomeis permanentlyalteredand the cell is said to be transformed.lntegration most likely is accomplishedby mammalian enzymes that normally function in DNA repair and recombination. 196

c H A P T E R5

|

MOLECULAG R E N E T TTCE C H N T Q U E S

P r o t e i ni s e x p r e s s e d f r o m c D N A i n p l a s m i dD N A (b) Stable transfection (transformation) Promoter

cDNA

cultured I Transfect I c e l l sb y l i p i dt r e a t m e n t or electroporation {

Proteinis expressedfrom cDNA integrated i n t o h o s tc h r o m o s o m e A EXPERIMENTAL FIGURE5-32 Transientand stable transfection with specially designed plasmid vectors permit expression of cloned genes in cultured animal cells.Bothmethodsemployplasmid vectorsthat containthe usualelements-ORl,selectablemarker(e.g , amp'), and polylinker-that permitpropagationin E. coli and inserlionof a clonedcDNAwith an adjacentanimalpromoter.Forsimplicity, these elementsarenot depicted(a)In transient transfection, the plasmid vectorcontainsan originof replication for a virusthat can replicatern the culturedanimalcells Sincethe vectoris not incorporated into the genome of the culturedcells,production of the cDNA-encoded proteincontinues onlyfor a limitedtime (b) In stabletransfection, the vectorcarries a selectable markersuchas neo',whichconfersresistance to G-418,The relatively few transfectedanimalcellsthat integratethe exogenousDNA into theirgenomesareselected on mediumcontainingG-418 Because the vectoris integratedinto the genome,thesestablytransfected,or transformed, cellswill continueto producethe cDNA-encoded protein as longasthe cultureis maintainedSeethe textfor discussion

L6l

N O T S S I U d XrTN r 9 A O n I S O l _ S I N l U \ l 9 V U l V N O q t N O ] )

'dlr,nrlrsues pue uortnlosar ratear8 qtrlr ueuo lnq'd1snor.r.e.rdpeqrrlsep stuorurredxeuorlezrprrq -d,{ trlt uI ol erep relrtursseprlord poqrau srql 'pesserdxa sr aua8 p uerJ,l.rpup eJeq,4^aurruJolopot pesn eq uef ural -ord relrodar eqr uorsserdxoeqr 'S'S uortres ur paqrrrsep Jo sE lerurup lrue8sue.rlp to uortearr dg ro aua8 perlrpou oqr Surureluoc Jottol uorsse.rdxaprurseld € qlr.4auorlf,oJsupJt dq .roqrrosller olur petnporluror sr eua8 plrqdq eqr uer{l1 'tseJolur urato.rder{l ot uretord ralrodar aql 1o ;o uorssard -xe treuuof, leqr eue8 plJqlq E atEJrJ ol sde,r,ro,AalaqrJJsap e,r\ areH 'sller ur potlotap eq dlluarua.tuor uer rep et)) uta7otQluacsatonlf uaaB se qtns urato.rd.rel.rodere Sursnuo serloruaryopoqteu srql 'surJtordrrro.,{.re4na Jo uorlezr]pto] relnlleJertur pue uorssardxa aql dpnls ot ^,(errre apr,r -ord uer srotf,e^uorssardxE 6ur66e1 utaloJd pue auag

DNtSn

'8It-C ot aruelsrsJr,{q :o1 percelasaq uer ra>lrer.u pau pue aua8 pauolc paler8alur dlqers e rJtr^\ sllal 'uortra; 'er-uoue8tsoq -suelt Jlqetslo1 Jspl Jr..lt ul se',{.ressarau;1 Jqt otul paterSelurueql pup sna]f,nueqt olur peuodsuerlsrqcrq,u'y51q otur paqrrrsuert-esrelarsr sacuanbasUII IErr^ ,(q palueg aua8pauolc erJtuortralur IIer raqv 'sllaf,re^rl pue apsntu 'suor -nau 's11aruals rrtarodoreruaqSurpnlcur'sadlt IIeJ uer1erurueur ;o ^,berre,topr.^ e ttoJur ol salcrtred snrr,t SurllnseJor{l .{\olle 'selcrued snrr.. ererrns palaldtuot jo oql uo uralord il^\ pue adole.,ruosnrllrluel aqr areldar dppea.r uuc qrrq,ra.'(urel Ieurrou -ord g-n5n) snrrl srJrteurotsrelnJrsa^aql uralotdor.{p aqr sr 1o txeluor srql ur posnuratord adola,ruauoruruol V 'addrilar p3.rer perrsepe lreJur ot salcruedsnrr,rprrgdq Surtlnsareql .ln.ollpilr,r\ snrrlrluel lueurqruof,eJe oluJ peleJodrocur uaq,rt teqt uralord odolr^ueIErr^E Jo uorssardxes.^Aolle pruseld leur1aq1 .alcrrred snrr^rlual e olur Surureluoc-a11 3ur3e ]Euorllunj vNu 1err,t 'uratord edole,rua ->1cedro; d.rBssarau rofeu aq] ro] ldecxo 1e-rr.r 'saua8 eqr IIe sorrrel pruseld Sur8e4cedeqt sE u^\ou{ 1e.rr,r

prtuseld puoras V 'VNC leruosoruorqr otur paterSaturaq ueql pue uorldrnsuerl esraler dq VUC olq perdol aq uer IIaJ E olur uorpnporlur uo reqr aFlelotu VNU te8rpl pelceJurdlye.rr,r '9 Ierr^ e ;o srsaqtudslrorrp soJuenbes911 1e4tt rerdeq3 ur poquJsopsV 'soruenbosUII snrr^uua1dq palueg pau se q)ns Je)JeruelqerrelasE ot txou lseJJlur;o eua8 peuop p sur€luoo ptutspli .topan eql se u,{\ou{ 'ptuseld tsrr} ar{J 'sllaf, Ierurue togr€t otul eua8 pauop e Jo uorpnportur luerlrlle rot elqEtrns salcrlred snJr^rlual lueurqruof,eJacnpo.rdol posn a-re'uorlla; -suert tuersuerrdq qlac otur pof,nporlur'sprruseldluaraJJrpeer{l 'gg-g ern8rg ur u,{\oqs sV 'sasnilnuual sp u.ln.oul sasnrrlorler Jo ssEIJe uro{ pelrJep sr uorssa.rdxa lerrl qfns euo yo eldruexe uV 'sllel Ieurue ur pessardxadlqers eq uef, oue8 parJrporu E r{lrq^\ dq dcuercgla eql aseeJrurdlreer8 ol VNC leurosoru -orr{J olur uoruasur luenbasqnspuP s[e] Ieurup otul lErrotEru rrleuoSJo uortfnPoJlur .ro1sasnrrrrdq Pesnsusrusr{JeruJrsEqar{] perroldxae^eq sreqrreeseUsuels^S uolsseldxl lerlnoJlau 's1ana1 aqr rsaqSrq tp tserelurIo uralord eql aonpordlerp asoqtdynueprot paueorJs a.redyyensnstuptre;SueJtelq€ts eqr 'erolaraql 'VNCI poues -ur eqt Surqr.rrsue.rl saler rrer{l ur re}tp il1.^a8It-C or luetsrs Jo 'aruoua8eql ul selrstuopueJ -eJ sauolJ paruro;sueJl Ienpr^pw le srnJf,o uorler8atut asnprefl '8It-C Jo uorterluoJuooq8rq e ;o ecuesardeql ur euolJ e ol esrJarrr8pue aAr Jns IIr,r\ aruos -oruoJr{Jtsoq er{l olur JolJeA uorssardxaaql peteJSolura^pq lerlt sller esoqr,{1ug 'qZt-S arnSrgur pourltno s uorltaJsua,4 alqals tq VNICII pauoll e Sursse.rdxe ro; arnparord Jrseq eql '8It-g sE u.,taou>l un,{uroeu ol peleler dlecrueqt punoduroc rrxol E ot oJu€tsrsersJeJuof,r4tw,n'(,oau pareu8rsep)esera; -suertoqdsoqd urcdruoeuroJ eue8aql sr re>lreruaIqEDOIOS pesn lluoruruoo y 'VNCIT pelresur eql Surquf,suprtJo seter Jreqt ur JaJtIPuerJoIIyl' 8It-C ol luelslsersauolJpaurotsuel IEnpI^P -ur tetuoua8aql ur selrsuopueJ le sJnJrouorte.r8aluresnetofl

uorlecrloor ;o ur6r.ro lerln u reloro leocleltA r o Jo u o 9

pouolS

vNU ler^orlou

u o t s s n ) s t pl o J Ll x a l

aq] aos 'auoue6 uetleuueur eql olu! seue6 pauo;>1o uogle.rEalu!luat)t#a JOI pasn eq ue) slopa^ leJt^oJlau €E-Slun9tJ lVINlNtUldXl
leJe uorlf,unJ pue ef,uonbososorl^, sureloJd pue seue8 osogf qtr^/\lrnpoJd str pue aua8aseosrpe to seJuenb to 'uretord papolue str pup aueSpellelJp -es eql Jo uosrredr.uoJ oqt dJuueprot sr eseasrpuewnq patrrequr.(ue roy osnec3urd1 -rapun aqt Surraqdrcepur dels rsry yecrddraqt 'sdepe.trop 'suorlpJaua8^(ueruJoJlxeu eql ol uorleJoua8euo ruor; I passedueoqo^Erlreqr salelletuetntu Surlsrxaarddq pesnerare seseesrppelrJer{urto sosef lsoru 'uorleraua8Surpara.rdaql ur esore uorlelnru.1\.euE r.uo{ lynsarderu aue8 ,,eseesrp,, e leql q8noqlly 'seseesrp paluor{urSur.rrnrcodluounuoc lsour or{l}o

[:il'j:i:lfffi:"*;"f"' x":T:Ht",,j;i H i:i,l:'.i;

53nOlNH)lI )l1lNl9

UV'ln):l'lol^l I

S UlldvH)

85t

E sepof,ueleql ecuanbesvNC lrol{s P qlrrrr lr 3ur r{lloJls uorls -sry dq lseralul aue8eqt d;1pourol sr utalord Ero uoneJol rel Jo -nllef,Elur aqr Surrerap ro; 3ur33el dCC ol e^Ileuratle uV 'pelelol dlleurrou st utalord roldoJor oql qlII{^{ tp etIS aql'suorneu drosuasur Erlrf,Iprslp eqr lp eouetsoronltdCC dq pazrlecol oq ueJ uotsnJ-unxo,ld Suulnsar aql loldef,er eqt Jo aruonbas Surpoc aqr ot d{C 3ur4uq dq pelcn.llsuoc 'lserluoc uI 'suorneu osoqryo ruseld sr aua8plrqdq eqr uoq.,ra 'uorsn} 'suo.rnaucgtoadsut pessatdxasl -o{c eql 3ur11r1 dJC -npruotd e sp u.^aou>ldllensn uorlern8r;uoJ P uI dCD to ocuanbasSurpocaqt ot dpce.rrppe{ull sI roldef,erlu€roPo aqt .ro; ralourord eqt ueqlN 'suo8ala'3 ur uralord .roldoco.rtue -ropo ue;o uotsserdxeeqr dpnls ol sluaurtradxa3ur33er-449 yo sedlr tueroJ;Ip o^\l Jo asn aqt sot€rlsnll yg-g arn8tg

seua9aseasrc

puebut^llluaplEE uerunH6ur1ero1 'uorlJeloP^PoqrluB roJ eootroaue ro ddc qlr-tr suratord;o 3ur33eroqr sr spoqlau aseqlto uorlecrlddB luel.rodur uV 'slloJ uerleurueru ro lseod ur soua8peuoll sserdxa 01 pesn ag uEf, sJolrel uorsserdxa rrtofre4ng r 'aua8pauolc e ruo{ uralord E slunoue tuepunqe uorl to Jo -cnpord ol{t ^ olle sprruselduroq po^uop sJotf,o^uorssardxgr 'suorlJunJ Pelel -o.r dlerrSolorg e^eq leqt surotord epoJuelluouruoc seua8 'suorlrpuor snorre^ rapun pareln8a.r Petpln8ar-ocgtns dl.relrtursore lpql saua8d;rluepr upr sluourrradxauorssard -xa de.rrBorcnualdrllnur ruou EtEpoql 1o srsdleuerolsnl3 r '(67-9 a;n8rgaas) suorlrpuor tuaraJlrprepun sllel eruesoql ur ro sllar;o saddr tuareJtrpur seua8Jo spuesnorJl;o uorsserdxrJo le^el e^rle -lar erlr slletap dlsnoauetlnurs srsdleuederrBo.rcrurVNC r 'uouezrprrqdgnlls ut dq s11ar8ural1ur perlorep

' pa^uap) uo.r1 2] 668:t8//;p'96611eia e1dn6ue5 [ (Spuet sarn6tl suolnourrloedsururalo.rd 0irpo jo uorle)olleurouoql ])allal pa^lesqo oq]+opuo aq1 elll)rilosuas oq ue) uorlnqt.llslp 01po.ualur fiosuesaq11odt1aq11e {;uosrpuesuolnau lelstpoql ]e luetedde uotsn]dlD-0[lpOaq]ase)srq] eq1o1pele6te]stutalo.rd auerqtrlor.rJ u 1a r u a n b absu r p o6t l r p g q t 6 u e l - ; 1onq1]+ op u ae q 1o 16 1 96 u t 1 u t 1 rilluetsatonll ate (q)'paleqel seMuorsn}-ura}ord {q palrnrlsuo: V Apoqllo)aq}}eq} prlos)solupuap{losuespue(Molrepaqsep) (n,lo.r.ie a1o5 sue6aya) jo peaqaql ursuolnau{rosuestl,ttrads;ourse;doyi: stutalotdslql 0l.lpo]o suopo)pt)eoulue eq1urpassardxe ,{qpaleraua6 senn olll o] dl9 6ur>1ur1 lno+]sll+oq] pueleloLuo.rd ueel6ro; aluenbes uorsn]-ralourord V (e) (dlg) utelordlua>selon11 sue6eyaf Jo '0 tlpo lolda:el ]uelopo eue6aq1o] pesn]sean slqlul 'seua6pauol) eue6eqi 'lueutuedxa e 6urporue le)ruraLll uo.r; passerdxesu1e1o.td lo uotleztte)olrelnlla) alellll)el 6ur66elutelordpue auag t€-S lgn9lJ'lVINlt tluldxl V Lurl0 L

oq uel sVNUru rr;rcadsJo uortnqrJtslppue aruasardaq1 r 'erntxrru E urqtr^\ y11g cr;rcads E slf,elapSurrrolq uraquoN 'OT-S arnSrg aas) eqord ylqq ;o anbruqoarrelrturs eq1palagelolper .{retuaruelduoJ E rltr.{\ uouezrpr.rqfq pue tarlry 'srsa.roqdol p or spueq paleredasaqr ;o (8uruolq) ra;sue.rr -ce1e 1eBSururquor ,tq arnlxrur xalduoc e urqlr^ luatu -8er; ygq rr;rcads 'a13urse pelop uEf, Surnolq urorllnos r uolssardxf auag Apnls ol sluau6erl VNC pauol) 6ursn ulelord uolsnl dJ0-0l,uoo :uolsnl-ulolord (ql

'lpuorlrunJ urerual llrrs pue eleJelotuer aua8pouoll E uorlpf,rtrpotu 1o soddr lpq.ry\uo spuadap uaryo uralord ua,tr8 e 3et ot dCC ro adotrde trorls E esn ol rer.{laq^\Jo arroql eqJ 'adolrda eqr ro; cr;rcads ,(poqrrue Ipuolrouour eql qlr^.\ sllel dq peioerepog upf, uJelord eql 1o Surlaqel-aJuef,soronlJounrurur oqt Jo ruroJ pe33er-adorrdapasserdxeeql'aue8 perJrpouroLIl Sururetuor Jolf,el uorsserdxapruseld e qtr.r uortf,eJsueJlJal -1y '3u83al aio4Qa sp u.,raou>l $ por{terusrql'acuaq ladolrda ue peller srdpoqrrueue dq punog aq upl lpql epudedtror{se r{rns dpoqrrue leuolJouoru u.taou>le dq pazruSof,erspne ourlue Jo

dl9 ol posnl relourord 0luoo :uolsnl-rolourord (e)

AUTOSOMAL RECESSIVE Sickle-cellanemia

Abnormal hemoelobin causesdeformation of red blood cells, which can become lodged in capillaries; also confers resistanceto malaria'

11625of sub-SaharanAfrican origin

Cysric fibrosis

Defective chloride channel (CFTR) in epithelial cells leads to excessivemucus in lungs.

1'12500of European origin

Phenylketonuria (PKU)

Defective enzyme in phenylalanine metabolism (tyrosine hydroxylase) results in excessphenylalanine, leading to mental retardation, unless restricted by diet.

1/10,000 of European origin

Tay-Sachsdisease

Defective hexosaminidaseenzyme leads to accumulation of excesssphingolipids in the lysosomesof neurons, impairing neural development.

1/1000 easternEuropean Jews

Huntington's disease

Defective neural protein (huntingtin) may assembleinto aggregatescausing damage to neural ttssue.

1/10,000of Europeanorigin

Hypercholesterolemia

Defective LDL receptor leads to excessivecholesterol in blood and early heart attacks.

tlI22French Canadians

Duchenne muscular dystrophy (DMD)

Defective cytoskeletal protein dystrophin leads to impaired muscle function.

1/3500 males

Hemophilia A

Defective blood clotting factor VIII leads to uncontrolled bleedine.

1-2110,000 males

AUTOSOMAL DOMINANT

X-LINKED RECESSIVE

provide clues to the molecular and cellular causeof the disease.Historically, researchershave usedwhatever phenotypic clues might be relevant to make guessesabout the molecular basis of inherited diseases.An early example of successful guessworkwas the hypothesisthat sickle-cellanemia,known to be a diseaseof blood cells,might be causedby defectivehemoglobin. This idea led to identification of a specificamino acid substitution in hemoglobin that causespolymerization of the defectivehemoglobin molecules,causingthe sickle-like deformation of red blood cellsin individuals who have inherited two copiesof the Hb' allele for sickle-cellhemoglobin. Most often, however,the genesresponsiblefor inherited diseasesmust be found without any prior knowledge or reasonablehypothesesabout the nature of the affectedgeneor its encodedprotein. In this section,we will seehow human geneticistscan find the generesponsiblefor an inherited disease by following the segregationof the diseasein families' The segregationof the diseasecan be correlatedwith the segregation of many other genetic markers, eventually leading to identification of the chromosomalposition of the affected

gene.This information, along with knowledgeof the sequence of th. hn-".t genome,can ultimately allow the affectedgene mutations to be pinpointed. and the disease-causing

s howOneof Three M a n y I n h e r i t e dD i s e a s e S Major Patternsof Inheritance Human genetic diseasesthat result from mutation in one specific gene exhibit severalinheritance patterns depending on the nature and chromosomal location of the alleles that causethem. One characteristicpattern is that exhibited by a dominant allele in an autosome (that is, one of the 22 human chromosomes that is not a sex chromosome). Becausean autosomal dominant allele is expressed in the heterozygote,usually at least one of the parents of an affected individual will also have the disease'It is often the case that the diseasescaused by dominant alleles appear later in life after the reproductive age. If this were n o i t h e c a s e , n a t u r a l s e l e c t i o nw o u l d h a v e e l i m i n a t e d the allele during human evolution. An example of an EENES H U M A N D I S E A SG I D E N T I F Y I NAGN D L O C A T I N G

.

199

(al Autosomal dominant: Huntington's disease

d

AHD/A* Affected

x

A+/A+ Q

I

Males and females

A-/ANot affected

AHD/A+ Affected (b) Autosomal recessive: Cystic fibrosis

d AcFrR/A+ Carrier

x , I

AcFrR/A+ ? Carrier

* Males and females

ACFTR /ACFTR

ACFTR/A+

Affected (c) X-finked recessive: Duchennemuscular dystrophy

d X* ly

Carrier x

XDMD/X+ Q Carrier

I

v

Males PMDIY Affected

A-/ANoncarrier

A+/AcFrR

Carrier

x+ly Unaffected

Females YDMD/x+ Carrier

x+/x+ Noncarrier

FIGURE 5-35 Threecommoninheritancepatternsfor human geneticdiseases. Wild-type (A)andsexchromosomes autosomal (X andY)areindicated by superscript plussigns(a)In an autosomal d o m i n a ndti s o r d es ru c ha sH u n t i n g t osnd i s e a soen, l yo n em u t a n t alleleisneeded to conferthe diseaself eitherparentis heterozygous for the mutantHDallele,hisor herchildren havea 50 percent c h a n coef i n h e r i t i nt h g em u t a nat l l e l e a n dg e t t i n g t h ed i s e a s(eb )l n an autosomal recessive disorder suchascystic fibrosis, two mutant alleles mustbe present to conferthe diseaseBothparents mustbe heterozygous carriers of the mutantCFIRgenefor theirchildren to be at riskof beingaffected or beingcarriers(c)An X-linked recessive disease suchasDuchenne muscular dystrophy iscauseo oy a recessive mutation on theX chromosome andexhibits thetypicalsex_ linkedsegregatron patternMalesbornto mothers heterozygous for a mutantDMDallelehavea 50 percent chance of inheriting the mutantalleleandbeingaffectedFemales bornto heterozvqous mothers havea 50 percent chance of beinqcarriers

a u t o s o m a l d o m i n a n t d i s e a s ei s H u n t i n g t o n ' s d i s e a s e ,a neural degenerativediseasethat generally strikes in midto late life. If either parenr carries a murant HD allele, e a c h o f h i s o r h e r c h i l d r e n ( r e g a r d l e s so f s e x ) h a s a 5 0 percent chance of inheriting rhe mutanr allele and being a f f e c t e d( F i g u r e5 - 3 5 a ) . A recessiveallelein an autosomeexhibits a quite different segregatronpattern. For an autosomal recessiueallele, both parentsmust be hererozygouscarriersof the allelein order for their children to be at risk of being affectedwith the disease. Each child of heterozygousparenrshas a 25 percentchanceof receiving both recessiveallelesand thus being affected,a 50 percentchanceof receivingone normal and one mutant allele and thus being a carrier, and a 25 percentchanceof receiving two normal alleles.A clear exampleof an autosomalrecessive diseaseis cystic fibrosis, which results from a defectivechloride-channelgeneknown as CFTR (Figure5-35b). Relatedindividuals (e.g.,first or secondcousins)have a relatively high 200

.

c H A p r EsR I

probability of being carriers for the same recessivealleles. Thus children born to related parents are much more likely than those born to unrelated parents to be homozygousfor, and thereforeaffectedby, an autosomalrecessivedisorder. The third common pattern of inheritanceis that of an XLinked recessiueallele. A recessiveallele on the X chromosome will most often be expressedin males,who receiveonly one X chromosome from their mother, but not in females, who receivean X chromosome from both their mother and their father. This leadsto a distinctive sex-linked segregation pattern where the diseaseis exhibited much more frequently in males than in females.For example, Duchenne muscular dystrophy(DMD), a muscledegenerative diseasethat specifically affects males, is causedby a recessiveallele on the X chromosome. DMD exhibits the typical sex-linked segregation pattern in which mothers who are heterozygous and therefore phenotypically normal can act as carriers, rransmitting the DMD allele, and therefore the disease,to 50 percent of their male progeny (Figure5-35c).

M o L E c u L AGRE N E Tr tEcc H N l o u E s

D N A P o l y m o r p h i s mAs r e U s e di n L i n k a g e M a p p i n gH u m a nM u t a t i o n s Once the mode of inheritancehas beendetermined,the next step in determining the position of a diseaseallele is to genetically map its position with respect to known genetic markers using the basic principle of genetic linkage as described in Section5.1. The presenceof many different already mapped genetic rraits, or markers, distributed along the length of a chromosome facilitatesthe mapping of a new mutation by assessingits possiblelinkage to these marker genesln appropriate crosses.The more markers that are available,the more preciselya mutarion can be mapped.The density of genetic markers neededfor a high-resolution human genetic map is about one marker every 5 centimorgans (cM) (as discussedpreviously,one geneticmap unir, or centimorgan, is defined as the distance between two positions along a chromosome that results in one recombinant individual in 100 progeny).Thus a high-resolutiongenericmap requires25 or so geneticmarkersof known position spread along the length of each human chromosome. In the experimentalorganismscommonly used in genetic studies,numerous markers with easily detectablephenotypes are readily availablefor geneticmapping of mutations.This is not the casefor mapping geneswhose mutant allelesare associated with inherited diseasesin humans. However" recombinant DNA technologyhas made availablea wealth of useful DNA-based molecular markers. Becausemost of the human genomedoesnot code for protein, alarge amount of sequence variation exists betweenindividuals. Indeed, it has been estimated that nucleotidedifferencesbetweenunrelatedindividuals can be detectedon an averageof every 103 nucleotides.If thesevariations in DNA sequence,referredto as DNA polymorphisms,can be followed from one generationto the next, they can serve as genetic markers for linkage studies. Currently, a panel of as many as 104 different known polymorphisms whose locations have been mapped in the human genomeis usedfor geneticlinkage studiesin humans.

Restriction fragment length polymorphisms (RFLPs) were the first type of molecular markers used in linkage studies.RFLPsarisebecausemutationscan createor destroy the sitesrecognizedby specificrestriction enzymesthat happen to lie in human DNA, leadingto variationsbetweenindividuals in the length of restriction fragments produced from identical regionsof the genome.Differencesin the sizes of restriction fragments betweenindividuals can be detected by Southern blotting with a probe specific for a region of DNA known to contain an RFLP (Figure5-36a).The segregation and meiotic recombination of such DNA polymorphisms can be followed like typical genetic markers. Figure 5-36b illustrates how RFLP analysis of a family can detect the segregaticlnof an RFLP that can be used to test for statistically significant linkage to the allele for an inherited diseaseor some other human trait of interest. The amassedgenomic sequenceinformation from different humans has led to identification of other useful DNA polymorphisms in recent years. Single-nwcleotidepolymorphisms (SNPs) constitute the most abundant type and are therefore useful for constructing geneticmaps of maximum resolution. Another useful type of DNA polymorphism consistsof a variable number of repetitionsof a one- two-, or Suchpolymorphisms,known as simple three-basesequence. sequencerepeats (SSRs)or microsatellites, presumably are formed by recombinationor a slippagemechanismof either the template or newly synthesizedstrands during DNA Hybridization banding pattern from individual with both allele 1 and allele2

lat

Chromosomalarrangement

't'iri

; g

'ti

t

I

:

i't

:

't

replication. A useful property of SSRsis that different individuals will often have different numbers of repeats.The existenceof multiple versionsof an SSRmakes it more likely to produce an informative segregationpattern in a given pedigreeand therefore be of more generalusein mapping the positionsof diseasegenes.If an SNP or SSRaltersa restriction site, it can be detectedby RFLP analysis. More commonly' however, these polymorphisms do not alter restriction fragments and must be detectedby PCR amplification and DNA sequenclng.

Geneswith a LinkageStudiesCanMap Disease Resolutionof About 1 Centimorgan lVithout going into all the technical considerations,let's see how the allele conferring a particular dominant trait (e.9., familial hypercholesterolemia)might be mapped. The first step is to obtain DNA samplesfrom all the members of a family containing individuals that exhibit the disease.The DNA from each affected and unaffected individual then is analyzed to determine the identity of a large number of known DNA polymorphisms (either SSR or SNP markers can be used). The segregationpattern of each DNA polymorphism within the family is then compared with the segrigation of the diseaseunder study to find those polymorphisms that tend to segregatealong with the disease' Finaily, computer analysisof the segregationdata is used to (b) Grandparents

G randparents

Enzyme Enzynre B

Mutation at site a2 Preventscleavage

i+

,f

e R e s t r i c t i o ne n d o n u c l e a s A

j

B R e s t r i c t i o ne n d o n u c l e a s e

.1,

Fragment lengths

tr

F-okb-l

As

v

[ - - - l P r o b e ds i n g l e - c o p yr e g i o n

Alleles

E

FIGURE5-36 Restrictionfragment length A EXPERIMENTAL can be followed like genetic markers. polymorphisms (RFLPs) (a) In the two homologouschromosomes shown,DNA is treatedwith enzymes(A and B),which cut DNA at two differentrestriction (a and b) The resultingf ragmentsare subjected differentsequences probe (seeFigure5-26)with a radioactive to Southernblot analysis that bindsto the indicatedDNA region(green)to detectthe betweenthe two homologous fragments Sinceno differences by the B enzyme, recoqnized occurin the sequences chromosomes by the probe,as indicatedby a only one fragmentis recognized band However,treatmentwith enzymeA singlehybridization producesradiographically distinctf ragmentsof two differentlengths

tr

r,,*, .!!,

Ilikb-l Fs kb-l

(a1-a2anda1-a3),and two bandsare seen,indicatingthat a mutationhascausedthe lossof one of the a sitesin one of the two of the DNA from a analysis chromosomes(b) Pedigreebasedon RFLP regionknown to be presenton chromosome5. The DNA samples enzymeIaql and analyzedby Southern were cut with the restriction of the genomeexistsin threeallelic region this family, blotting In this by Iaql sitesspaced10, 7 '7, or 6 5 kb apart forms characterized Eachindividualhastwo alleles;somecontainallele2 (7 7 kb) on both at this site Circles and othersare heterozygous chromosomes, gel lanesare in the The males indicate indicatefemales;squares and are generally pedigree above in the the subjects as order same , e l5l 1 : 3 1 ]9 l l tearl , 1 9 8 7C r D o n i s - K ee a l i g n e db e l o wt h e m l A f t e H . I D E N T I F Y IA NN GD L O C A T I NHGU M A ND I S E A SGEE N E S

201

calculatethe likelihood of linkage between each DNA polymorphism and the disease-causing allele. In practice, segregation data are collected from different families exhibiting the same diseaseand pooled. The more families exhibiting a parricular diseasethaican be examined, the greater the statistical significance of evidence for linkage that can be obtained and the grearerthe precisionwith which the distancecan be measuredbetween a linked DNA oolvmorphism and a diseaseallele.Most family studieshave a maximum of about 100 individualsin which linkage betweena diseasegene and a panel of DNA polymorphismscan be tested. This number of individuals setsthe practical upper limit on the resolutionof sucha mapping study to about 1 cenrimorgan,or a physicaldistanceof about 7.5 x 10s basepairs. A phenomenoncalledlinkagedisequilibriumisthe basisfor an alternative strategy,which in some casescan afford a higher degreeof resolution in mapping studies.This approach dependson the particular circumstancein which a genericdisease commonly found in a particular population resultsfrom a single mutation that occurredmany generationsin the past. The DNA polymorphismscarriedby this ancestralchromosomeare collectively known as the haplotype of that chromosome. As the diseasealleleis passedfrom one generationto the next, only the polymorphisms that are closestto the diseasegenewill not be separatedfrom it by recombination. After many generarlons the region that contains the diseasegenewill be evident because this will be the only region of the chromosome thar will carry the haplotype of the ancestralchromosome conservedthrough many generations(Figure5-37).By assessing the distributionof specificmarkersin all the affectedindividualsin a population, geneticistscan identify DNA markers tightly associatedwith

appearedon the ancestralchromosome-in somecasesthis can amount to finding markersthat are so closelylinked to the diseasegenethat even after hundredsof meiosesthey have never beenseparatedby recombinarion.

FurtherAnalysisls Neededto Locatea Disease G e n ei n C l o n e dD N A Although linkage mapping can usually locare a human diseasegene to a region containing about 105 base pairs, as many as 10 different genesmay be located in a region of this size. The ultimate objective of a mapping study is to locate the genewithin a cloned segmenrof DNA and then to determine the nucleotide sequenceof this fragment. The relative scalesof a chromosomalgenetic-ap anJphysicalmaps corresponding to ordered sers of plasmid clones and the nucleotidesequenceare shown in Figure5-38. One strategyfor further localizinga diseasegenewithin the genome is to identify mRNA encoded by DNA in the region of the geneunder study. Comparison of geneexpressionin tissues 2O2

.

c H A p r E 5R |

M o l E c u L AG R E N E TrtEc c H N t e u E s

Generation

1

Newmu+6+:^h

f f l

I Generation 2

cr"ototot"

inp.,.ti",l?i'.": | | F Yitl dirrerent \ huptotvp?'' mf I J nantotvoe fVeioti"recombination

ilI tl ff

I I

'""tt''"" ll iltl ililililt|

A FIGURE 5-37 Linkagedisequilibrium studiesof human populationscan be usedto map genesat high resolution.A newdisease mutation willarisein the contextof an ancestral chromosome amonga setof polymorphisms known asthehaplotype (indicated by pinkshading). Aftermanygenerations, chromosomes thatcarrythe disease mutation willalsocarrysegments of the ancestral haplotype thathavenot beenseparated fromthe disease mutationby recombination Thebluesegments of these chromosomes general represent haplotypes derived fromthegeneral population andnotfromtheancestral haplotype in whichthe mutatron originally aroseThisphenomenon is knownaslinkage disequilibrium Thepositionof the disease mutationcanbe located by scanning chromosomes containing thedisease mutation for highly polymorphisms conserved corresponding to theancestral haplotype from normal and affected individuals may suggesttissuesin which a particular diseasegenenormally is expressed.For instance,a mutation that phenotypically affects muscle, but no other tissue,might be in a genethat is expressedonly in muscle tissue. The expressionof mRNA in both normal and affected individuals generally is determined by Northern blotting or in situ hybridization of labeledDNA or RNA to tissuesections. Northern blots, in situ hybridization, or microarray experiments permit comparison of both the level of expression and the size of mRNAs in mutant and wild-type tissues.Although the sensitivity of in situ hybridization is lower than that of Northern blot analysis,it can be very helpful in identifying an mRNA that is expressedat low levels in a given tissue but at very high levels in a subclassof cells within that tissue.An mRNA that is altered or missing in various individuals affected with a diseasecomparedwith wild-type individualswould be an excellent candidate for encoding the protein whose disrupted function causesthat disease. In many cases,point mutations that give rise to diseasecausing allelesmay result in no detectablechange in the level of expression or electrophoretic mobility of mRNAs. Thus if comparison of the mRNAs expressedin normal and affected individuals reveals no detectabledifferencesin the candidate mRNAs, a search for point mutarions in the

Polymorphic or BAC (l

A T U

T T A tr

A A T (A 750 kb

T A

c

u

'l

T A T T a

u

T A u U

\T LEVELOF RESOLUTION:Cytogenetic map

Physical map

Linkage map

M E T H O DO F Linkageto restriction DETECTION:Chromosome b a n d i n gp a t t e r n f r a g m e n tl e n g t hp o l y morPhismsRFLPS, in Fluorescence s i t u h y b r i d i z a t i o n s i n g l en u c l e o t i d ep o l y (FISH) m o r P h i s m sS N P s ,a n d s l m p l es e q u e n c e repeatsSSRs

5-38 The relationshipbetweenthe geneticand A FIGURE a deptcts Thediagram physicalmapsof a humanchromosome' of detailThe levels at different analyzed humanchromosome when asa wholecanbeviewedin the lightmicroscope chromosome andthe at metaphase, statethatoccurs it isin a condensed by canbe determined ic sequences locationof specif approximate (FISH) At the nextlevelof detail, in sltuhybridization fluorescence

DNA regions encoding the mRNAs is undertaken. Now that highly efficientmethodsfor sequencingDNA are availfrequently determinethe sequenceof canable, researchers DNA isolatedfrom affectedindividualsto of regions didate identify point mutations. The overall strategyis to search for a coding sequencethat consistentlyshowspossiblydelereriousalterationsin DNA from individualsthat exhibit the disease.A limitation of this approachis that the region near the affectedgene may carry naturally occurring polymorphisms unrelated to the gene of interest. Such polymorphisms,not functionally related to the disease,can lead to misidentificationof the DNA fragmentcarrying the geneof interest.For this reason,the more mutant allelesavailable for analysis,the more likely that a gene will be correctly identified.

Sequence map

Hybridization Sanger (dideoxY) to plasmid sequenclng clones

2d to Genomes' FromGenes et al, 2003,Genetics: fromL Hartwell lAdapted Hill ed, McGraw I

s e s u l tf r o m M u l t i p l e M a n y I n h e r i t e dD i s e a s e R GeneticDefects Most of the inherited human diseasesthat are now understood at the molecular level are monogenetic traits; that is, a clearly discerniblediseasestate is produced by a defect in a single gene.Monogenic diseasescausedby mutation in one specific gene exhibit one of the characteristic inheritance patterns shown in Figure 5-35' The genes associatedwith most of the common monogenic diseases have already been mapped using DNA-based markers as describedpreviouslY. However, many other inherited diseasesshow more complicatedpatterns of inheritance,making the identification of the underlying genetic causemuch more difficult' EENES H U M A N D I S E A SG I D E N T I F Y I NAGN D L O C A T I N G

O

203

One type of added complexity that is frequently encountered is genetic heterogeneity.In such cases,mutations in any one of multiple different genes can cause the same disease.For example, retinitis pigmentosa,which is characterizedby degenerationof the retina usually leading to blindness,can be causedby mutations in any one of more than 60 different genes. In human linkage studies, data from multiple families usually must be combined to deter_ mine whether a statisricallysignificant linkage exists be_ t w e e n a d i s e a s eg e n e a n d k n o w n m o l e c u l a r m a r k e r s . Genetic heterogeneitysuch as rhar exhibited by retinitis pigmentosa can confound such an approach becauseany statisticaltrend in the mapping data from one family tends to be canceledout by the data obtained from another fam_ i l y w i t h a n u n r e l a t e dc a u s a t i v eg e n e . Human geneticisrsused two different approachesto _ identify the many genesassociatedwith retinitii pigmenrosa. The first approach relied on mapping studies in &ception_ ally large single families rhar contained a sufficient number of affectedindividuals to provide statisticallysignificant evidencefor linkage betweenknown DNA polymolphisms and a single causativegene. The genesidentified in such studies

retinitis pigmentosa.This approach of using additional in_ formation to focus screeningefforts on a s.rbiet of candidate genesled to identificationof additional rare causativemura_

ldentifying and Locating Human DiseaseGenes r Inherited diseasesand other traits in humans show t h r e e m a j o r p a t t e r n s o f i n h e r i t a n c e :a u t o s o m a l d o m i nant, autosomal recessive,and X-linked recessive(see F i g u r e5 - 3 5 ) . r Genesfor human diseasesand other trairs can be mapped by determining their cosegregation during meiosis with markers whose locations in the genome are known. The closer a geneis to a particular marker, the more likelv thev are to cosegregate, of human geneswith great precision requires of molecular markers distributed along the es. The most useful markers are differencesin the DNA sequence(polymorphisms) between individuals in noncoding regions of the genome. r DNA polymorphisms useful in mapping human genesinclude restriction fragment length polymorphisms (RFLps), single-nucleotidepolymorphisms (SNps), and simple sequencerepeats(SSRs). inkage mapping often can locate a human diseasegene a chromosomal region that includes as many as l0 genes.To identify the geneof interestwithin this candidate region_typicallyrequires expressionanalysis and compari_ son of DNA sequencesbetween wild-type and disiaseaffectedindividuals. r Some inherited diseasescan result from mutations in dif_ ferent genesin different individuals (geneticheterogeneity). The occurrence and severity of other diseasesdepend on the presenceof mutant allelesof multiple genesin the same individuals (polygenictraits). Mapping of the genesassociated with such diseasesis particularly difficult becausethe occurrenceof the diseasecannot readily be correlated to a singlechromosomallocus.

ff,l Inactivatingthe Functionof SpecificGenesin Eukaryotes The elucidation of DNA and protein sequencesrn recent y e a r s h a s l e d t o i d e n t i f i c a t i o no f m a n y g e n e s .u s i n g s e quencepatternsin genomicDNA and the sequencesimilar_ ity of the encodedproteins with proteins oi known function. As discussedin Chapter 6, the general functions of proteins identified by sequencesearchesmay be predicted by analogy with known proteins. However, the precisein vivo roles of such "new" proteins may be lrnclearin the ab_ senceof mutant forms of the correspondinggenes.In this

CHAPTER 5

J

MOLECULAG RENETIC TECHNIQUES

Three basic approachesunderlie these gene-inactivation techniques:(1) replacinga normal genewith other sequences, (2) introducing an allelewhose encodedprotein inhibits functioning of the expressednormal protein, and (3) promoting destructionof the mRNA expressedfrom a gene.The normal endogenousgeneis modified in techniquesbasedon the first approach but is not modified in the other approaches'

(a)

20-nt flanking sequence

.-f-hi"_l

s y n r h sr i s DNJA

-

NormalYeastGenesCan Be Replacedwith M u t a n t A l l e l e sb y H o m o l o g o u sR e c o m b i n a t i o n Modifying the genomeof the yeastS. cereuisiaeis particularly easy for two reasons:yeast cells readily take up exogenous DNA under certain conditions, and the introduced DNA is efficiently exchangedfor the homologous chromosomal site in the recipient cell. This specific,targeted recombination of identical stretchesof DNA allows any genein yeastchromosomesto be replacedwith a mutant allele. (As we discussin Section5.1, recombination between homologous chromosomesalso occurs naturally during meiosis.) In one popular method for disrupting yeast genesin this fashion, the PCR is used to generatea disruption construct containing a selectablemarker that subsequentlyis transfectedinto yeastcells.As shown in Figure 5-39a,primersfor PCR amplification of the selectablemarker are designedto flankinclude about 20 nucleotidesidenticalwith sequences amplified resulting The replaced. ing the yeast gene to be constructcomprisesthe selectablemarker (e.g.,the kdnMX gene,which llke neo' confers resistanceto G-41 8 ) flanked by about 20 basepairs that match the ends of the target yeast gene. Transformeddiploid yeast cells in which one of the two copiesof the target endogenousgenehas beenreplaced by the disruption construct are identified by their resistance phenotype.Theseheterozygous to G-418 or other selectable diploid yeastcellsgenerallygrow normally regardlessof the function of the target gene,but half the haploid sporesderived from thesecellswill carry only the disruptedallele(Figure 5-39b). If a geneis essentialfor viability,then sporescarrying a disruptedallelewill not survive. Disruption of yeastgenesby this method is proving parthe role of proteinsidentifiedby ticularly usefulin assessing analysisof the entire genomic DNA sequence(seeChapter 6). A large consortiumof scientistshas replacedeach of the approximately 6000 genesidentified by this analysiswith the kanMX disruption construct and determinedwhich gene disruptionslead to nonviablehaploid spores.Theseanalyses have shown that about 4500 of the 6000 yeastgenesare not

gene may be viable becauseof operation of backup or compensatorypathways.To investigatethis possibility,yeast geneticistscurrently are searchingfor syntheticlethal mutations that might reveal nonessentialgeneswith redundant functions(seeFigure 5-9c).

*Fri-"|.

-

Z

Disruption construct

(b)

Four haploid spores

FLI G U R E5 - 3 9 H o m o l o g o u s r e c o m b i n a t i o n a EXPERIMENTA constructs can inactivate specific disruption with transfected ( a ) y e a s t . A s u i t a b l ec o n s t r u cfto r d i s r u p t i n ga g e n e s i n target . h et w o p r i m e r s t a r g e tg e n ec a n b e p r e p a r e db y t h e P C R T d e s i g n e df o r t h i s p u r p o s ee a c hc o n t a i na s e q u e n c eo f a b o u t 2 0 yeast n u c l e o t i d e(sn t )t h a t i s h o m o l o g o u st o o n e e n d o f t h e t a r g e t o f D NA a s e g m e n t a m p l i f y t o n e e d e d s e q u e n c e s a s g e n ea s w e l l carryinga selectablemarkergene such as kanMX, which confers to G-418. (b) When recipientdiploid Saccharomyces resistance c e l l sa r et r a n s f o r m e dw i t h t h e g e n e d i s r u p t i o nc o n s t r u c t , h o m o l o g o u sr e c o m b i n a t i obne t w e e nt h e e n d so f t h e c o n s t r u c t s i l l i n t e g r a t et h e a n d t h e c o r r e s p o n d i ncgh r o m o s o m asl e q u e n c ew

nonvrable

O F S P E C I F IGCE N E SI N E U K A R Y O T E S I N A C T I V A T I NTGH E F U N C T I O N

205

(a) Formation of ES cells carrying a knockout mutation neor

fuHSV

G e n eX r e p l a c e m e nct o n s t r u c t

Homologous ,/ r. recombination,? "il" ./

r'

ES-ceilDNA

;

G e n eX

O t h e rg e n e s

I I Gene-targeted linsertion

I II R a n d o m ltnserilon

v

v

M u t a t i o ni n g e n eX

N o m u t a t i o ni n g e n eX

Cellsare resistantto G - 4 1 8a n d g a n c i c l o v i r

C e l l sa r e r e s i s t a ntto G - 4 1 8b u t s e n s i t i v e to ganciclovir

(b) Positive and negative selection of recombinant ES cells O OO 6+

N o n r e c o m b i n a ncte l l s Recombinantswith gene-targetedinsertion

| - r " . , w i t hG - 4 1 8 ( p o s i t i vsee l e c t i o n ) J

o.',oo o-oo Ag

I fr"u, with ganciclovir ( n e g a t i v es e l e c t i o n ) |

*

o, -o rro

< EXPERIMENTAL FIGURE 5-40 lsolationof mouseEScellswith a gene-targeteddisruption is the first stage in productionof knockoutmice.(a)Whenexogenous DNAisintroduced rnto embryonic stem(ES) cells,randominsertion vianonhomologous recombrnation occursmuchmorefrequently thangene-targeted insertion viahomologous recombination Recombinant cellsin which onealleleof geneX (orange andwhite)isdisrupted canbe obtained by usinga recombinant vectorthatcarries geneX disrupted with neo'(green), whichconfers resistance to G-418,and,outside the regionof homology, tkHsv(yellow), thethymidine genefrom kinase herpes simplex vrrusTheviralthymidlne kinase, unlikethe endogenous mouseenzyme, canconvert the nucleotide analog ganciclovir intothe monophosphate form;thisisthenmodified to t h et r i p h o s p h af o t er m ,w h i c hi n h i b i tcse l l u l aDrN Ar e p l i c a t i o i nnE S cellsThusganciclovir iscytotoxic for recombinant EScellscarrying the tkHsv gene.Nonhomologous insertion includes the tkHsv gene, whereas homologous insertion doesnot;therefore, onlycellswith nonhomologous insertion aresensitive to ganciclovir (b) Recombinant cellsareselected by treatment with G-41g,since cellsthatfailto pickup DNAor integrate it intotheirgenomeare sensitive to thiscytotoxic compoundThesurvivrng recombinant cells aretreatedwith ganciclovir Onlycellswith a targeted disruption in geneX, andtherefore lacklng geneanditsaccompanying the fkHsv cytotoxicity, willsurvive[See S L Mansour etal, 1988,Nature336.348l

A useful promoter for this purpose is the yeast GAL1 promoter, which is active in cells grown on galactose but completely inactive in cells grown on glucose. In this approach, the coding sequenceof an essentialgene (X) ligated to the GALl promoter is inserted into a yeast shuttle u..to. (seeFigure 5-17a).The recombinant vector then is introduced into haploid yeastcellsin which geneX has beendisrupted. Haploid cells that are transformed will grow on galactosemedium, since the normal copy of gene X on the vector is expressedin the presenceof galactose.\fhen the cells are transferredto a glucose-containingmedium, geneX no longer is transcribed;as the cells divide, the amount of

c'l

o o-

E S c e l l sw i t h t a r g e t e dd i s r u p t i o ni n g e n eX

Transcriptionof GenesLigatedto a Regulated P r o m o t e rC a n B e C o n t r o l l e dE x p e r i m e n t a l l y Although disruption of an essenrialgene required for cell g r o w t h w i l l y i e l dn o n v i a h l es p o r e sr.h i i m e r h o dp r o v i d e sl i m l e infclrmation about what the encodedprotein actually does in cells.To learn more about how a specificgenecontributesto cell growth and viability investigarorsmust be able to selec_ tively inactivatethe genein a population of growing cells.One method for doing rhis empkrysa regularedpro-oi.. to selec_ tively shut off transcription of an essentialgene. 206

o

c H A p r E5R I

M o L E c u L AGRE N E Tr tEcc H N t o u E S

In an early application of this method, researchersex, plored the function of cytosolic Hsc70 genes ln yeasr. Haploid cells with a disruption in all four redundant Hsc70 geneswere nonviable, unlessthe cells carried a vector containing a copy of the Hsc70 gene that could be ex_ pressedfrom the GALI promoter on galactosemedium. On transfer to glucose,the vector-carryingcells eventually stopped growing becauseof insufficient Hsc70 acivity. C a r e f u l e x a m i n a r i o n o f t h e s e d y i n g c e l l s r e v e a l e dt h a t their secretory proteins could no longer enter the endoplasmic rericulum (ER). This study provided the first evi_ dencefor the unexpectedrole of Hsc70 protein in translocation of secretory proteins into the ER, a process e x a m i n e di n d e t a i l i n C h a p t e r 1 3 .

#

uia"o: Microinjectionof ESCellsinto a Blastocyst

for a FIGURE 5-41 EScellsheterozygous > EXPERIMENTAL disruptedgene are usedto producegene-targetedknockout for a knockout cellsheterozygous stem(ES) mice.Step1: Embryonic (X andhomozygous for a dominant mutationin a geneof interest alleleof a markergene(here,browncoatcolor,A) aretransplanted thatarehomozygous cavityof 4 5-dayembryos intothe blastocoel alleleof the marker(here,blackcoatcolor,a) Step2: for a recessive female intoa pseudopregnant thenareimplanted Theearlyembryos by indicated cellsarechimeras, ES-derived containing Thoseprogeny micethenare theirmixedblackandbrowncoats.Step3: Chimeric fromthismatinghaveE5to blackmice;brownprogeny backcrossed of DNAisolated 4-6: Analysis derived cellsin theirgermline Steps brownmice canidentify froma smallamountof tailtissue of thesemice alleleIntercrossing for the knockout heterozygous that allele, produces for the disrupted homozygous someindividuals 1989,Trends fromM R.Capecchi, mice.lAdapted is,knockout Genet5:70.1

E gffl'"? ::l[T;"0?]X'J"'""' Brown mouse (NA, X-lx+) B l a c km o u s e (ala, X+lX+l 4.5-dayblastocyst

I

EIl"[';:Xl'.ffi ['Ji:;i]:il:; * A F o s t e rm o t h e r

SpecificGenesCan Be PermanentlyInactivated i n t h e G e r m L i n eo f M i c e Many of the methods for disrupting genesin yeast can be applied to genesof higher eukaryotes.Thesealteredgenescan be introduced into the germ line via homologous recombination to produce animals with a gene knockout, or simply "knockout." Knockout mice in which a specificgeneis disrupted are a powerful experimental systemfor studying mammalian development, behavior, and physiology. They also are useful in studying the molecular basisof certain human geneticdiseases. Gene-targetedknockout mice are generatedby a two-stage procedure.In the first stage,a DNA construct containing a disrupted allele of a particular target gene is introduced into embryonic stem (ES)cells.Thesecells,which are derived from the blastocyst,can be grown in culture through many generations (seeFigure 21'-7).In a small fraction of transfectedcells, the introduced DNA undergoeshomologousrecombinationwith the target gene, although recombination at nonhomologous chromosomal sitesoccurs much more frequently' To selectfor cells in which homologous gene-targetedinsertion occurs' the recombinant DNA construct introduced into ES cells needsto include two selectablemarker genes (Figure 5-40). One of thesegenes(neo'),which confersG-418 resistance,is inserted within the target gene (X), thereby disrupting it. The other selectable gene, the thymidine kinase gene from herpes simplex virus (lAHsv),confers sensitivity to ganciclovir, a cytotoxic nucleotide analog; it is inserted into the construct outside the target-genesequence.Only ES cells that undergo homologous reIo-bitr"tiott, and therefore do not incorporate tkHsv, can survivein the presenceof both G-418 and ganciclovir.In these cellsone alleleof geneX will be disrupted. In the second stage in production of knockout mice' ES cellsheterozygousfor a knockout mutation in geneX are injected into a recipient wild-type mouse blastocyst,which subsequentlyis transferred into a surrogatepseudopregnant female mouse (Figure 5-41). The resultingprogeny will be chimeras, containing tissues derived from both the transplantedES cellsand the host cells.If the ES cellsalso are ho-

Black I S.t""t chimericmicefor to wild-typeblackmice I crosses

v

germcells: All germcells: Possible a/X+ A/X+tA/X-: a/X* =r I tt cell-deriveo ProgenY s I will be brown

v

a/a, X+/X* Progenyfrom ES cell-derived germcells !| | s.re"n brown ProgenYDNA -* to identify X'lX+ heterozygotes al EI

Mate X-lX* heterozYgotes

Screen Progeny DNA to identifY lil - I l, X tx- homozYgotes

Knockoutmouse

mozygous for a visible marker trait (e.g., coat color)' then chimeric progeny in which the ES cells survived and proliferated can te identified easily.Chimeric mice are then mated with mice homozygous for another allele of the marker trait to determine if the knockout mutation is incorporated into the germ line. Finally, mating of mice' each heterozygous for the knockout allele, will produce progeny homozygous for the knockout mutation.

C E N E SI N E U K A R Y O T E S T H E F U N C T I O NO F S P E C I F I G INACTIVATING

207

loxP mouse

Cre mouse

A l l c e l l sc a r r ye n d o g e n o u sg e n e Xwith /oxPsitesflankingexon 2

Heterozygousfor gene X knockout; all cellscarry cre gene

Cell{ype-specif ic promoter

Cellsnot expressingCre

CellsexpressingCre

-4lJ@

G e n ef u n c t i o ni s n o r m a r

IoxP-Cre mouse All cells carry one copy of loxpmodified gene X, one copy of g e n eX k n o c k o u ta, n d c r e g e n e s

EXPERIMENTAL FTGURE 5-42 The loxp-Crerecombination system can knock out genes in specificcell types. A /oxpsjte is insertedon eachsideof the essential exon2 of the targetgeneX (blue)by homologousrecombination, producinga /oxpmouse.Since the /oxPsitesare in introns,they do not disruptthe functionof X T h e C r e m o u s ec a r r i e o s n e g e n eX k n o c k o u at l l e l ea n d a n i n t r o d u c e d p'l linkedto a cell-type_specific cre gene (orange)from bacteriophage promoter(yellow)The cre gene is incorporatedinto the mouse genomeby nonhomologousrecombination and doesnot affectthe

Development of knockout mice that mimic certain human diseasescan be illustrated by cystic fibrosis. By methodsdiscussedin Section5.4, the recessrve murarion that causesthis diseaseeventuallywas shown to be located in a geneknown as CFTR, which encodesa chloride chan_

rnice are currently being used as a model systemfor stud ing this geneticdiseaseand developingeffectivetherapies

S o m a t i cC e l lR e c o m b i n a t i o C n a nI n a c t i v a t e G e n e si n S p e c i f i cT i s s u e s Investigatorsoften are interestedin examining the effects o f k n o c k o u t m u t a t i o n si n a p a r t i c u l a rt i s s u eo i t h e m o u s e , at a specificstagein development,or both. However, mice carrying a germ-line knockout may have defectsin numer_ ous tissuesor die before the developmentalstageof inter_ est. To addressthis problem, mouse geneticistshave de_ v i s e d a c l e v e r t e c h n i q u et o i n a c t i v a l er a r g e r g e n e s l n 208

.

c H A p r E5R | M o L E c u L AGRE N E Tr tEcc H N t o u E S

-

FE G e n ef u n c t i o n isdisrupted

functionof othergenesln the/oxp-Cre mtcethat resultfrom crossing, Creproteinisproduced onlyin thosecellsin whichthe promoter isactiveThusthesearethe onlycellsin which recombrnation between the/oxPsites catalyzed by Creoccurs, leading to deletion of exon2 Since the otheralleleisa constitutive geneX knockout, deletion between the/oxpsitesresults in complete l o s so f f u n c t i o on f g e n e X i na l lc e l l se x p r e s s iC ng r e B yu s i n g promoters, different researchers canstudytheeffects of knockinq out geneX in various typesof cells specifictypes of somatic cells or at particular times during development This techniqueemploys site-specificDNA recombination sites(calledloxP sires)and the enzymeCre that catalyzesrecombination between them. The loxP-Cre recombination system is derived from bacteriophageP1, but this sitespecificrecombination sysremalso functions when placed in mouse cells. An essentialfeature of this technique is that expression of Cre is controlled by a cell-type-specificpromoter. In loxP-Cre mice generatedby the procedure depicted in Figure 5-42,inactivation of the geneof interest (X) occurs only in cells in which the promoter controlling the cre gene is active. An early application of this techniqueprovided strong evidencethat a particular neurotransmitterreceptor is important for learning and memory. Previouspharmacological and physiologicalstudieshad indicated that normal learning requiresthe NMDA classof glutamatereceptorsin the hippocampus,a region of the brain. But mice in which the gene encoding an NMDA receptor subunit was knocked out died neonatally,precluding analysis of the receptor's role in learning. Following the protocol in Figure 5-42, researchersgeneratedmice in which the receptorsubunit gene was inactivatedin the hippocampusbut expressedin other tissues. These mice survived to adulthood and showed

llll+ Technique Mouse Animation:creatinga Transgenic z/ar\ \\"2

DNA injected into a Pronucleusof a Mouse

miceare produced FIGURE 5-43 Transgenic > EXPERIMENTAL by randomintegrationof a foreigngeneinto the mousegerm (themale intooneof thetwo pronuclei DNAinjected line.Foreign hasa good bythe parents) andfemalehaploidnucleicontributed of the intothe chromosomes integrated of beingrandomly chance intothe recipient is integrated a transgene diploidzygoteBecause it doesnot disrupt recombination, genomeby nonhomologous l l7 . 2 7 3 1 L B r i n s t e r e, t1a9l 8 1C e n d o g e n o u s g e n[ S e se e R , e2

Injectforeign DNA intooneofthe pronuclei

Pronuclei

Fertilizedmouse egg Prlor t o f u s i o no f m a l e a n d f e m a l ep r o n u c l e i

II Transferinjectedeggs I into foster mother

learning and memory defects,confirming a role for thesereceptors in the ability of mice to encode their experiences rnto memory.

I

+

D o m i n a n t - N e g a t i vAel l e l e sC a n F u n c t i o n a l l y l n h i b i t S o m eG e n e s In diploid organisms,as noted in Section5.1, the phenotypic effect of a recessiveallele is expressedonly in homozygous individuals,whereasdominant allelesare expressedin heterozygotes.Thus an individual must carry two copies of a recessiveallele but only one copy of a dominant allele to exhibit the corresponding phenotypes. Sfe have seen how strains of mice that are homozygous for a given recessive knockout mutation can be produced by crossingindividuals that are heterozygousfor the same knockout mutation (see Figure 5-41). For experimentswith cultured animal cells, however, it is usually difficult to disrupt both copies of a genein order to produce a mutant phenotype.Moreover, the difficulty in producing strainswith both copiesof a genemutated is often compounded by the presenceof related genes of similar function that must also be inactivated in order to reveal an observablephenotype. For certain genes,the difficulties in producing homozygous knockout mutants can be avoided by use of an allele carrying a dominant-negativemutation. Theseallelesare genetically dominant; that is, they produce a mutant phenotype even in cells carrying a wild-type copy of the gene. However, unlike other types of dominant alleles,dominantnegativeallelesproduce a phenotypeequivalentto that of a loss-of-function mutatton. Useful dominant-negativealleleshave been identified for a variety of genesand can be introduced into cultured cells by transfection or into the germ line of mice or other organisms. In both cases,the introduced geneis integratedinto the genome by nonhomologous recombination. Such randomly inserted genesare called transgenes;the cells or organisms carrying them are referred to as transgenic.Transgenescarrying a dominant-negative allele usually are engineeredso that the allele is controlled by a regulatedpromoter, allowing expressionof the mutant protein in different tissuesat different times. As noted above, the random integration of exogenousDNA via nonhomologousrecombinationoccursat

o About 10-30% of offspringwill contain foreign DNA in chromosomesof a l l t h e i r t i s s u e sa n d g e r m l i n e Breed mice expressing foreign DNA to proPagate DNA in germline

@

@

a much higher frequency than insertion via homologous recombination. Becauseof this phenomenon' the production of transgenicmice is an efficient and straightforward process

GTPasesfrom an inactive GDP-bound state to an active GTP-bound state dependson their interacting with a corresponding guanine nucleotide exchangefactor (GEF)' A ,rr,.riu.r,small GTPase that permanently binds to the GEF orotein will block conversion of endogenous wild-type imall GTPasesto the active GTP-bound state' thereby inhibiting them from performing their switching function ( F i g u r e5 - 4 4 ) .

C E N E SI N E U K A R Y O T E S T H E F U N C T I O NO F S P E C I F I G INACTIVATING

(a) Cellsexpressingonly wild-type allelesof a small GTPase

Inactive

j

fGo' \-/ Wildtype

(b) Cellsexpressingboth wild-type allelesand a dominant-negativeallele

D o m i n a n t - n e guve a mutant

FIGURE 5-44 Inactivationof the functionof a wild-type GTPase by the actionof a dominant-negative mutantallele. (a)Small(monomeric) (purple) GTPases areactivated by their interaction with a guaninenucleotide exchange factor(GEF), which catalyzes the exchange of GDpfor GTp(b)Introduction of a dominant-negative alleleof a smallGTpase geneintocultured cellsor transgenic animals leadsto expression of a mutantGTpase that binds to andinactivates the GEFAsa result, endogenous wild-type copies of thesamesmallGTPase aretrappedin the inactive GDp-bound stateA singledominant-negative allelethuscauses a loss-offunctionphenotype in heterozygotes similar to thatseenin homozygotes carrying two recessive loss-of-f unctionalleles

R N AI n t e r f e r e n c e C a u s e sG e n eI n a c t i v a t i o nb y Destroyingthe CorrespondingmRNA A recently discoveredphenomenon known as RNA interference (RNAi) is perhaps the mosr straightforward method to inhibir the function of specificgenes.This approach is technically simpler than the methods described above for disrupting genes.First observed in the roundworm C. elegans,RNAi refers to rhe ability of doublestranded RNA to block expression of its corresponding single-strandedmRNA but not that of mRNAs with a difl ferent sequence. As describedin Chapter 8, the phenomenon of RNAi rests on the general ability of eukaryotic cells to cleave double-strandedRNA inro short (23-nt) double-stranded segmentsknown as small inhibitory RNA (siRNA). The RNA endonuclease that catalyzesthis reaction, known as

tween one strand of the siRNA and its complementaryse_ quenceon the target nRNA; subsequently,specificnucle_ asesin the RISC complex then cleave the mRNA/siRNA hybrid. This model accounts for the specificity of RNAi, since it dependson basepairing, and for its potency in si_ lencing gene function, since the complem entary mRNA is 21O

.

cHAprER 5

|

M o L E c u L AG R E N E T tr cE c H N t e u E s

permanently destroyed by nucleolytic degradation. The normal function of both Dicer and RISC is to allow for gene regulation by small endogenous RNA molecules known as micro RNAs (miRNAs). Researchersexploit the micro RNA pathway for intentional silencing of a gene of interest by using either of two generalmethods for generatingsiRNAs of defined sequence. In the first method a double-strandedRNA corresponding to the target genesequenceis produced by in vitro transcription of both senseand antisensecopiesofthis sequence(Figure5-45a). This dsRNA is injected into the gonad of an adult worm. where it is converted to siRNA by Dicer in the developing embryos. In conjunction with the RISC complex, the siRNA molecules causethe corresponding mRNA molecules to be destroyedrapidly. The resulting worms display a phenotype similar to the one that would result from disruption of the corresponding gene itself. In some cases,entry of just a few moleculesof a particular dsRNA into a cell is sufficient to inactivate many copies of the corresponding mRNA. Figure 5-45b illustrates the ability of an injected dsRNA to interfere with production of the corresponding endogenousmRNA in C. elegansembryos.In this experiment, the mRNA levels in embryos were determined by incubating the embryos with a fluorescently labeled probe specific for the mRNA of interest. This technique,in situ hybridization, is useful in assayingexpressionof a particular mRNA in cells and tissuesections. The second method is to produce a specific doublestranded RNA in vivo. An efficient way ro do this is to expressa synthetic gene thar is designedto contain tandem segmentsof both senseand anti-sensesequencescorresDonding to the target gene (Figure 5-45c). \X/henthis gene is transcribed, a double-strandedRNA "hairpin" srructure forms, known as small hairpin RNA, or shRNA. The shRNA will then be cleaved by Dicer to form siRNA molecules. The lentiviral expressionvectors are particularly useful for introducing synrhericgenes for the expressionof shRNA constructsinto animal cells. Both RNAi methods lend themselves to systematic studiesto inactivateeach of the known genesin an organism and to observewhat goeswrong. For example, in initial studies with C. elegans,RNA interferencewith 16,700 genes(about 86 percent of the genome)yielded 1722 visibly abnormal phenotypes.The geneswhose functional inactivation causesparticular abnormal phenotypescan be grouped into sets;each member of a set presumably controls the same signals or events.The regulatory relations between the genes in the set-for example, the genes that control muscledevelopment-can then be worked out. Other organismsin which RNAi-mediated geneinactivation has been successfulinclude Drosophila, many kinds of plants, zebra fish, the frog Xenopus, and mice, and are now the subjects of large-scaleRNAi screens.For example, lentiviral vectors have been designedto inactivate by RNAi more than 10,000 different genes expressedin cultured mammalian cells. The function of the inactivated genescan be inferred from defects in growth or morphology of cell clones transfectedwith lentiviral vectors.

fi| ,oo."rt: RNAInterference (a) In vitro production of double-stranded RNA Antisensetranscript

--\(-

Injected

Noninjected

(c) In vivo production of double-stranded RNA

(RNA|)can 5-45 RNAinterference FIGURE < EXPERIMENTAL functionallyinactivategenesin C.elegansand other for RNA(dsRNA) of double-stranded organisms.(a)Invitroproduction gene, the gene of sequence The coding target RNA|of a specific DNA,is of genomic fromeithera cDNAcloneor a segment derived to a strong vectoradjacent in a plasmid placedin two orientations in vitrousingRNA of bothconstructs promoterTranscription in yields manyRNAcoptes triphosphates polymerase andribonucleoside (identical or sequence) mRNA with the orientation thesense these conditions, orientationUndersuitable antisense complementary to formdsRNAWhenthe willhybridize RNAmolecules complementary by DicerintosiRNAs(b) intocells,it iscleaved isinjected dsRNA by RNA|(seethe worm embryos in of mex3RNAexpression lnhibition was (Left) RNA in embryos mex3 of Expression mechanism) for the text for thismRNA,that with a probespecific by in situhybridization assayed product(Rtgtht) a colored(purple) thatproduces is,linkedto an enzyme mex3 double-stranded with froma worminjected Theembryoderived indicated by mRNA, as mex3 no endogenous produces little or mRNA embryois=50 Fm in length of color.Eachfour-cell-stage theabsence viaan engineered RNAoccurs (c)Invivoproduction of double-stranded geneconstruct isa intocellsThesynthetic directly plasmid introduced of the sequences and antisense sense of both tandemarrangement RNA smallhairpin double-stranded targetgeneWhenit istranscribed, (b) by Dicerto formsiRNAlPart iscleaved TheshRNA forms(shRNA) Nature 391:806 etal. 1998, l fromA Fire

Sensetranscript

r The /oxP-Cre recombination system permits production of mice in which a geneis knocked out in a specifictissue.

siRNA 2+

Inactivating the Function of SpecificGenes in Eukaryotes r Once a gene has beencloned, important clues about its normal function in vivo can be deducedfrom the observed phenotypic effectsof mutating the gene. r Genescan be disrupted in yeast by inserting a selectable marker geneinto one allele of a wild-rype genevia homologous recombination, producing a heterozygousmutant. \7hen such a heterozygoteis sporulated, disruption of an essential genewill producetwo nonviablehaploid spores(Figure5-39). r A yeastgenecan be inactivatedin a controlledmannerby using the GALI promoter to shut off transcription of a genewhen cellsare transferredto glucosemedium.

r In the production of transgeniccells or organisms,exogenous DNA is integrated into the host genome by nonhomologous recombination (seeFigure 5-43). Introduction of a dominant-negativeallele in this way can functionally inactivate a genewithout altering its sequence. r In many organisms,including the roundworm C. elegans, double-stranded RNA triggers destruction of the all the mRNA moleculeswith the samesequence(seeFigure 5-45). This phenomenon, known as RNAI (RNA interference), provides a specificand potent means of functionally inactivating geneswithout altering their structure.

As the examplesin this chapter and throughout the book illustrate, genetic analysis is the foundation of our understanding of many fundamental processesin cell biology. By examining the phenotypic consequencesof mutations that inactivate a particular gene' geneticistsare able to connect knowledge about the sequence'structure' and biochemical

r In mice, modified genescan be incorporated into the germ line at their original genomic location by homologous recombination,producingknockouts (seeFigures5-40 and 5-41).Mouse knockoutscan providemodelsfor human genetic diseases such as cysticfibrosis. P E R S P E C T I VF EO S RT H E F U T U R E

211

Although scientistscontinue to use this classicalgenetic approachto dissectfundamentalcellular processes and biochemical pathways, the availability of iomplete genomic sequenceinformation for most of the common experimental organisms has fundamentally changed the way genetic experiments are conducted. Using various computational methods, scientistshave identified the protein-coding gene sequences in most experimentalorganismsincludingE. coli, yeast, C. elegans,Drosophila, Arabidopsis, mouse, and humans.The genesequences, in turn, revealthe primary amino acid sequenceof the encodedprotein products, providing us with a nearly complete list of the proteins found in each of the major experimentalorganisms. The approach taken by most researchershas thus shifted from discoveringnew genesand proteins to discoveringthe functions of genesand proteins whose sequencesare already known. Once an interestinggenehas beenidentified,genomic sequenceinformation greatly speedssubsequentgeneticmanipulations of the gene,including its designedinactivation, to learn more about its function. Already sets of vectors for RNAi inactivation of most defined senesin the nematode C. elegansnow allow efficientgeneric,ir..n, to be performed in this multicellular organism.Thesemethodsare now being applied to largecollectionsof genesin cultured mammalian cells and in the near future either RNAi or knockout methodswill have beenusedto inactivateeverygenein the mouse. In the past, a scientistmight spend many years studying only a single gene, but nowadays scientistscommonly study whole setsof genesar once. For example,with DNA microarrays the level of expressionof all genesin an organismcan be measuredalmost as easily as the expressionof a singlegene. One of the great challengesfacing geneticistsin the twentyfirst century will be to exploit the vast amount of available data on the function and regulation of individual genesto understandhow groups of genesare organizedto form complex biochemicalpathways and regulatory nerworks.

KeyTerms alleles 166

linkage175

ctone I /')

tlntation 765 Northern blotting 192 phenotype155

|

4

- ^

complementary DNAs (cDNAs) 181 complementation183 DNA cloning 126 DNA library 179 DNA microarray 192 dominant L66 geneknockoul'207 genomics129 genotype166 heterozygous166 homozygous165 hybridization 181 in situ hybridizatton 192 212

.

c H A p r EsR

plasmids178 polymerasechain r e a c t i o n( P C R )1 8 8 p r o b e s1 8 1 recessive165 recombinantDNA 126 recombination175 restriction enzymes176 RNA interference (RNAi)210 segregation167 Southern blotting 191 MOLECULAG RENETIC TECHNIQUES

temperature-sensrtrve mutations 170

transgenes209 vector 1-76

transfection 196 transformationL78

Review the Concepts 1, Genetic mutations can provide insights into the mechanisms of complex cellular or developmentalprocesses.How might your analysisof a geneticmutation be different depending on whether a particular mutation is recessiveor dominant? 2. Give an example of how and why temperature-sensitive mutations might be usedto study the function of essentialgenes. 3. Describehow complementation analysiscan be used to reveal whether two mutations are in the sameor in different genes.Explain why complementation analysiswill not work with dominant mutations. 4, Compare the different usesof suppressorand synthetic lethal mutations in geneticanalysis. 5. Restriction enzymesand DNA ligaseplay essentialroles in DNA cloning. How is it that a bacterium that produces a restriction enzyme does not cut its own DNA? Describe some general features of restriction enzymesites.ril/hat are the three types of DNA ends that can be generatedafter cutting DNA with restriction enzymes?\{hat reaction is catalyzedby DNA ligase? 6. Bacterial plasmids often serve as cloning vectors. Describethe essentialfeaturesof a plasmid vector. Sfhat are the advantagesand applicationsof plasmidsas cloning vectors? 7. A DNA library is a collectionof clones,eachcontaining a different fragment of DNA, inserted into a cloning vector. What is the differencebetweena cDNA and a genomic DNA library? How can you use hybridization or expressionto screen a library for a specific gene? How many different oligonucleotideprimers would need to be synthesizedas probes to screena library for the gene encoding the peptide Met-Pro-Glu-Phe-Tyr? 8. In 1993, Kerry Mullis won rhe Nobel prize in chemistry for his invention of the PCR process.Describethe three steps in each cycle of a PCR reaction. $fhy was the discoveryof a thermostableDNA polymerase(e.g.,Taq polymerase)so important for the developmentof PCR? 9. Southernand Northern blotting are powerful tools in molecular biology basedon hybridization of nucleic acids. How are these techniques the same? How do they differ? Give some specific applications for each blotting technique. 10. A number of foreign proteins have been expressedin bacterial and mammalian cells. Describe the essential featuresof a recombinantplasmid that are required for expressionof a foreign gene.How can you modify the foreign protein to facilitate its purification? What is the advantage of expressinga protein in mammalian cellsversusbacteria? 11. What is a DNA microarray? How are DNA microarrays usedfor studying geneexpression?How do experimentswith microarrays differ from Northern blotting experiments?

12. In determining the identity of the protein that corresponds to a newly discoveredgene, it often helps to know the pattern of tissueexpressionfor that gene.For example, researchers have found that a genecalledSERPINA6 is expressedin the liver, kidney, and pancreasbut not in other tissues.!7hat techniques might researchersuse to find out which tissuesexpressa particular gene?

b. A wild-type yeastcDNA library, preparedin a plasmid that contains the wild-type URA3* gene' is used to transformX cells,which are then culturedas indicated.Each black spot below representsa singleclone growing on a petri '$7hat plate. are the molecular differencesbetweenthe clones growing on the two plates?How can theseresults be used to identify the geneencodingX?

13. DNA polymorphisms can be used as DNA markers. Describethe differencesbetweenRFLR SNR and SSRpolymorphisms.How can thesemarkersbe usedfor DNA mapping studies? 14. How can linkage disequilibrium mapping sometimcs provide a much higher resolutionof genelocation than classicallinkagemapping? 15. Genetic linkage studies can usually only roughly locate the chromosomalposition of a "disease"gene.How can expression analysis and DNA sequenceanalysis help locate a diseasegenewithin the region identified by linkage mapping? 16. Gene targetingby siRNA techniquesexploits a normal micro RNA pathway that is found in all metazoansbut not in simpler eukaryotessuch as yeast.\7hat are the roles of Dicer and RISC in this pathway? 77. The ability to selectivelymodify the genome in the mousehas revolutionizedmousegenetics.Outline the procedure for generatinga knockout mouse at a specificgenetic locus.How can the loxP-Cresystembe usedto conditionally knock out a gene?\Vhat is an important medicalapplication of knockout mice? 18. Two methods for functionally inactivating a gene withmuout alteringthe genesequenceare by dominant-negative tations and RNA interference(RNAi). Describehow each method can inhibit expressionof a gene.

A culture of yeast that requiresuracil for growth (ura3-) was mutagenized,and two mutant colonies,X and Y, have been isolated.Mating type a cells of mutant X are mated with mating type o cellsof mutant Y to form diploid cells. The parental (ura3 ), X, Y, and diploid cells are streaked onto agar platescontaininguracil and incubatedat 23 "C or 32'C. Cell growth was monitored by the formation of colonieson the culture platesas shown in the figure below. Denotesgrowth of cells

ura3 '/'

ura

w

Parental X

Growthat 23'C

Parental X PCR

d. A construct of the wild-type geneX is engineeredto encode a fusion protein in which the green fluorescentprotein (GFP) is presentat the N-terminus (GFP-X) or the Cterminus (X-GFP) of protein X. Both constructs'presenton a URA3* plasmid, are used to transform X cells grown in the absenceof uracil. The transformants are then monitored for growth at 32oC, shown below at the left. At the right are typical fluorescentimagesof X-GFP and GFP-X cells grown at 23 "C in which green denotesthe presenceof green fluorescentprotein. What is a reasonableexplanation for growth o f G F P - X b u t n o t X - G F P c e l l sa t 3 2 ' C ?

\X \

\

/,

G r o w t ha t 3 2 ' C o n m e d i al a c k i n gu r a c i l

c. DNA is extracted from the parental cells, from X cells, and from Y cells and digested with a restriction enzyme.The digests are analyzedby Southern blot analysis, shown at the left below, using a probe obtained from the gene encoding X. In addition, PCR primers are used to amplify the gene encoding X in both the parental and the X cells.The primersare complementaryto regionsof DNA just external to the geneencoding X. The PCR results are shown in the gel at the right. What can be deducedabout the mutation in the X genefrom thesedata?

Southern

Analyzethe Data

Diploid

G r o w t ha t 2 3 ' C o n m e d i al a c k i n gu r a c i l

Diploid

G F P - Xc e l l

/, G r o w t ha t 3 2 ' C X-GFP

What can be deducedabout mutants X and Y from

a. the data provided?

Growth at 32 "C

G F Pl o c a l i z a t i oinn c e l l s grown at 23 'C THE DATA ANALYZE

O

213

e. Haploid offspring of the diploid cells from part (a) above are generated.XY double mutants constitute 114 of theseoffspring.Haploid X cells,Y cells,and XY cellsin liquid culture are synchronizedat a stagejust prior to budding and then are shifted from 23'C to 32 oC. Examination of the cells 24 hours later revealsthat X cellsare arresredwith small buds, Y cellsare arrestedwith large buds, and XY cellsare arrested with small buds.\Whatis the relationshipbetweenX and Y?

References Genetic Analysis of Mutations to ldentify and Study Genes Adams,A. E. M., D. Botstein,and D. B. Drubin. 1989.A yeast actin-bindingprotein is encodedby sac6,a genefound by suppression of an actin muration. Science243:231. Griffiths, A. G. F., et al. 2000. An Introduction to Genetic Analysis,Tth ed. W. H. Freemanand Company. Guarente,L.1993. Synthericenhancement in geneinteraction:a genetictoof comesof age.TrendsCenet.g:362-3o6. Hartwell, L. H. 1967.Macromolecularsynthesis of temperature-sensitive mutanrs of yeast.J. Bacteriol. 93:1662. Hartwell, L.H. 1974. Geneticcontrol of the cell divisioncycle in yeast.Science183:46. Niisslein-Volhard, C., and E.'Wieschaus. 1980.Mutations affectins segmentnumber and polariryin Drosophila.Nature 287:795-801. Simon,M. A., er al. 1991. Rasl and a putativeguaninenucleotide exchangefactor perform crucialstepsin signalingby the sevenless protein tyrosinekinase.Cell 67:701-776. Tong,A. H., et al. 2001. Systematicgeneticanalysiswith ordered arraysof yeastdeletionmurants.Science294:2364)368. DNA Cloning and Characterization Ausubel,F. M., et al.2002. Current Protocolsin Molecular Biology.Vlley. Gubler,U., and B. J. Hoffman. 1983.A simpleand very efficient method for generatingcDNA libraries. Gene 25:263-289. Han, J. H., C. Stratowa,and W. I. Rutter.1987.Isolationof fulllengthputativerat lysophospholipase cDNA usingimprovedmethods for mRNA isolationand cDNA cloning.Biochem.26:.161.7-1632. Itakura,K., J.J. Rossi,and R. B. Wallace.1984.Synthesis and use of syntheticoligonucleotides.Ann. Reu.Biochem. 53:323-356. Maniatis, T., et al. 1978.The isolation of structural senesfrom libraries of eucaryoticDNA. Cel/ 15:687-701. Nasmyth,K. A., and S. I. Reed.1980.Isolationof senesby complementationin yeast:molecularcloning of a cell-Jyclegene. Proc. Nat'l Acad. Sci.USA 77:21.19-2123. Nathans,D., and H. O. Smith. 1975.Restrictionendonucleases in the analysisand restructuringof DNA molecules.Ann. Reu.Biochem. 44:273193. Roberts,R. J., and D. Macelis.1997.REBAsE-restricrion enzymesand methylases.Nacl. Acids Res.25:248-262. Information on accessinga continuouslyupdateddatabaseon restrictionand modification enzymesat http://www.neb.com/rebase.

214

c H A P T E R5

|

M O L E C U L AG R E N E T TTCE C H N T Q U E S

Using Cloned DNA Fragments to Study Gene Expression Andrews, A.T. 1986. Electrophoresis,2d ed. Oxford University Press. Erlich, H., ed. 1.992.PCR Technology:Principlesand Applicationsfor DNA Amplification. W. H. Freemanand Company. Pellicer,A., M. Wigler, R. Axel, and S. Silverstein.1978. The transfer and stableintegration of rhe HSV thymidine kinasegene into mousecells.Cel/ 4t:1,33-1{1,. Saiki,R. K., et al. 1988. Primer-directed enzymaticamplification of DNA with a thermostableDNA polymerase.Science239:487491. Sanger,F. 1981. Determinationof nucleotidesequences in DNA. Science214:720 5-'1210. Souza,L. M., et al. 1986.Recombinanthuman granulocyte-colony stimulating factor: effectson normal and leukemic myeloid cells. Science232:61-65. 'Wahl, G. M., J. L. Meinkoth, and A. R. Kimmel. 1987. Northern and Southern6lots. Metb. Enzymol. 152:572-581. Wallace,R. B., et al. 1,987.The useof syntheticoligonucleotides as hybridizationprobes.II: Hybridization of oiigonucleotides of mixed sequence to rabbit B-globinDNA. Nzcl. Acids Res.9:879-887. ldentifying and Locating Human Disease Genes Botstein,D., et al. 1980. Constructionof a geneticlinkage map in man using restriction fragmentlength polymorphisms.Az. /. Genet.32:31.4-331.. Donis-Keller,H., et al. 1,987.A geneticlinkage map of the human genome.Cell 5l:319-337. Hartwell, et al. 2000. Genetics:From Genesto Genomes. McGraw-Hill. Hastbacka,T., et al. 1994.The diastrophicdysplasiagene encodesa novel sulfatetransporter:positional cloning by finestructurelinkage disequilibriummapping. Cell 781.073. Orita, M., et al. 1989. Rapid and sensitivedetecrionof point mutations and DNA polymorphismsusing the polymerasechain reaction. Genomics5:874. Tabor,H. K., N.J. Risch,and R. M. Myers.2002. Opinion: candidate-gene approachesfor studyingcomplex generictrairs: practicalconsiderations.Nat. Reu.Genet.3:391,-397. Inactivating the Function of Specific Genes in Eukaryotes Capecchi,M. R. 1989. Altering the genomeby homologousrecombination. Science 244 :'1,288-1,292. Deshaies,R. J., et al. 1988. A subfamily of stressproteins facili, tatestranslocationof secretoryand mitochondrial precursor polypeptides.Nature 332:800-805. Fire, A., et al. 1,998.Porentand specificgeneticinterferenceby double-strandedRNA in Caenorhabditiselegans.Nature 391:806-8'1.1. Gu, H., et al. 1994. Delerion of a DNA polymerasebeta gene segmentin T cellsusing cell type-specificgenetargeting.Science 265:L03-106. Zamore, P. D., T. Tuschl,P.A. Sharp,and D. P. Bartel.2000. RNAi: double-strandedRNA directsthe ATP-dependentcleavageof mRNA at 21 to 23 nucleotideinrervals.Cell 101:25-33. Zimmer, A. 1992. Manipulating the genomeby homorogous recombinationin embryonic stem cells.Ann. Reu.Neurosci. 15:115

CHAPTER

GENES, AND GENOMICS, CHROMOSOMES ThesebrightlycoloredRxFISH-painted chromosomes areboth anomalies and in beautifuland usefulin revealing chromosome of Clinical comparingkaryotypes of differentspecies[@Department Researchers, Incl Cytogenetics, Addenbrookes Hospital/Photo

I n previous chapterswe learnedhow the structure and comI position of proteins allow them to perform a wide variety I of cellular functions. We also examined another vital component of cells, the nucleic acids, and the processby which information encoded in the sequenceof DNA is translated into protein. In this chapter,our focus again is on DNA and proteins as we consider the characteristicsof eukaryotic nuclear and organellar genomes:the features of genesand the other DNA sequencethat comprise the genome, and how this DNA is structured and organized by proteins within the cell. By the beginning of the twenty-first century, molecular biologists had completed sequencingthe entire genomesof hundredsof viruses,scoresof bacteria,and one unicellular eukaryote, the budding yeast S. cereuisiae.In addition, the vast majority of the genome sequenceis also known for the fission yeast S. pombe, and severalmulticellular eukaryotes including the roundworm C. elegans, the fruit fly D. melanogastel,mice, and humans. Detailed analysis of these sequencingdata has revealedinsights into genome organtzation and genefunction. It has allowed researchersto identify previously unknown genesand to estimatethe total number of protein-coding genesencoded in each genome. Comparisons betweengenesequencesoften provide insight into possible functions of newly identified genes. Comparisons of genomesequenceand organizationbetweenspeciesalso help us understand the evolution of organisms. Surprisingly,DNA sequencingrevealedthat a large portion of the genomesof higher eukaryotes does not encode mRNAs or any other RNAs required by the organism. Remarkably, such noncoding DNA constitutes :98.5 percent

of human chromosomal DNA! The noncoding DNA in multicellular organisms contains many regions that are similar but not identical. Variations within some stretchesof this repetitious DNA between individuals are so great that every person can be distinguishedby a DNA "fingerprint" based on these sequencevariations. Moreover, some repetitious DNA sequencesare not found in the same positions in the genomesof different individuals of the same species.At one "junk time, all noncoding DNA was collectively termed 'We now DNA" and was considered to serve no purpose. understandthe evolutionary basisof all this extra DNA, and the variation in location of certain sequencesbetween

OUTLINE 6.1

EukaryoticGene Structure

217

6.2

C h r o m o s o m aOl r g a n i z a t i o no f G e n e s a n d N o n c o d i n gD N A

223

5.3

(Mobile) DNA Elements Transposable

226

6.4

O r g a n e l l eD N A s

236

5.5

G e n o m i c sG: e n o m e - w i d eA n a l y s i so f G e n e Structureand ExPression

6.6

StructuralOrganizationof Eukaryotic Chromosomes

6.7

M o r p h o l o g ya n d F u n c t i o n aEl l e m e n t s of EukaryoticChromosomes

215

individuals. Cellular genomesharbor symbiotic transposable (mobile) DNA elements,sequencesthat can copy themselves and move throughout the genome.Although transposable DNA elementsseemro have little funcrion in the life cycleof an individual organism, over evolutionary time they have shapedour genomesand contributedto the rapid evolution of multicellularorganisms. In higher eukaryores,DNA regions encoding proteins or functional RNAs-that is, genes-lie amidst this expanseof apparently nonfunctional DNA. In addition ro the nonfunctional DNA between genes,noncoding introns are common within genesof multicellularplants and animals.Sequencing of the same protein-coding gene in a variety of eukaryotic specieshas shown that evolutionary pressureselectsfor maintenanceof relativelysimilar sequences in the coding reglons,or exons.In contrast,wide sequence variation,evenincludingtotal loss,occursamon€lintrons,suggesting that most intron sequenceshave little functional significance.However, as we shall see,although most of the DNA sequenceof introns is not functional,the existenceof introns has favored the evolution of multidomain proteins that are common ln highereukaryotes.It also allowed the rapid evolutionof proteinswith new combinationsof functionaldomains. Mitochondria and chloroplastsalso contain DNA that encodesproteins essentialto the function of thesevital organelles.!7e shall see that mitochondrial and chloroplast DNAs are evolutionaryremnantsof the origins of theseorganelles.Comparisonof DNA sequences betweendifferent classes of bacteria and mitochondrial and chloroolast genomeshas revealedthat theseorganellesevolvedfrom in-

tracellular bacteria that developed symbiotic relationships with ancient eukaryotic cells. The sheerlength of cellular DNA is a significant problem with which cells must contend. The DNA in a single human cell, which measuresabout 2 meters in total length, must be contained within cells with diametersof less than 10 pm, a compactionratio of greaterthan 105to 1.In relativeterms,if a cell were 1 centimeter in diameter, the length of DNA packed into its nucleuswould be about 2 kilometers! Specialized eukaryotic proteins associatedwith nuclear DNA exquisitely fold and organizethe DNA so thar it fits into nuclei. And yet at the same time, any given portion of this highly compacted DNA can be accessedreadily for transcription, DNA replication, and repair of DNA damage without the long DNA moleculesbecoming tangled or broken. Furthermore, the integrity of DNA must be maintained during the processof cell division when it is partitioned into daughter cells. In eukaryotes,the complex of DNA and the proteins that organize it, called chromatin, can be visualized as individual chromosomesduring mitosis (seechapter opening figure). As we will seein this and the following chapter,the organization of DNA into chromatin allows a mechanism for regulation of geneexpressionthat is not availablein bacteria. In the first five sections of this chapter, we provide an overview of the landscapeof eukaryotic genes and genomes. First we discussthe structure of eukaryotic genesand the complexities that arise in higher organismsfrom the processingof mRNA precursorsinto alternatively spliced mRNAs. Next we discussthe main classes of eukaryoticDNA includingthe special properties of transposableDNA elementsand how they shaped

> FIGURE6-1 Overview of the structure of genes and chromosomes.DNA of higher eukaryotes consistsof uniqueand repeated s e q u e n c eO s n l y: 1 5 p e r c e not f h u m a nD N A encodesproteinsand functionalRNAsand the regulatorysequences that controltheir expression; the remainderis merelyintrons within genesand intergenicDNA between g e n e s M u c ho f t h e i n t e r g e n iD c N A ,- 4 5 percentin humans,is derivedf rom transposable (mobile)DNA elements,geneticsymbiontsthat havecontributedto the evolutionof contemporary genomes Eachchromosome consrsts of a single,long moleculeof DNA up t o : 2 8 0 M b i n h u m a n so, r g a n i z e d into increasing levelsof condensation by the histone a n d n o n h i s t o n pe r o t e i n sw i t h w h i c h i t i s intricately complexedMuch smallerDNA m o l e c u l easr e l o c a l i z eidn m i t o c h o n d r iaan d chloroplasts

" B e a d so n a s t r i n g "

Nucleosome

Major Types of DNA Sequence S i n g l e - c o pg yenes S i m p l e - s e q u e n cDeN A G e n ef a m i l i e s T r a n s p o s a b lD e N Ae l e m e n t s T a n d e m l yr e p e a t e dg e n e s S p a c e rD N A Introns

216

C H A P T E6R |

GENES G,E N O M t CASN, D C H R O M O S O M E S

contemporarygenomes.We then considerorganelleDNA and how it differs from nuclear DNA. This background preparesus to discussgenomics,computer-basedmethods for analyzingand interpreting vast amounts of sequencedata. The final two sections of the chapter addresshow DNA is physically organizedin eukaryoticcells.'Sfeconsiderthe packagingof DNA and histone proteins into compact complexes(nucleosomes)that are the fundamental building blocks of chromatin, the large-scalestructure of chromosomes,and the functional elementsrequired for chromosomeduplication and segregation.Figure 6-1 provides an overview of theseinterrelatedsubjects.The understandingof genes,genomics,and chromosomesgained in this chapter will prepareus to explore current knowledge about how the synthesis and concentration of each protein and functional RNA in a cell is regulatedin the following two chapters.

lil

Eukaryotic GeneStructure

In molecular terms, a genecommonly is defined as tbe entire nucleic acid seqwencethat is necessaryfor the synthesisof a fwnctionalgeneproduct (polypeptideor RNA). According to this definition, a geneincludesmore than the nucleotidesencoding an amino acid sequenceor a functional RNA, referred to as the coding region. A genealso includesall the DNA sequencesrequired for synthesisof a particular RNA transcript, no matter where those sequences are locatedin relation to the coding region.For example,in eukaryoticgenes,transcriptioncontrol regions known as enhancerscan lie 50 kb or more from the coding region.As we learnedin Chapter4, other critical noncoding regions in eukaryotic genesinclude the promoter, as well as sequencesthat specify 3' cleavage and polyadenylation,known as poly(A) sites, and splicing of primary RNA transcripts,known as splicesiles(seeFigure4-15). Mutations in thesesequences, which control transcriptioninitiation and RNA processing,affect the normal expressionand function of RNAs, producing distinct phenotypesin mutant organisms.We examine these various control elementsof genesin greaterdetail in Chapters7 and 8. Although most genes are transcribed into mRNAs, which encode proteins, some DNA sequencesare transcribedinto RNAs that do not encodeproteins (e.g.,tRNAs and rRNAs described in Chapter 4 and micro RNAs that regulatemRNA stability and translationdiscussedin Chapter 8). Becausethe DNA that encodestRNAs, rRNAs and micro RNAs can cause specific phenotypeswhen mutated, theseDNA regions generallyare referred to as tRNA, rRNA and micro RNA genes, even though the final products of thesegenesare RNA moleculesand not proteins. In this section,we will examine the structure of genesin bacteria and eukaryotesand discusshow their respective gene structuresinfluence geneexpressionand evolution.

M o s t E u k a r y o t i cG e n e sC o n t a i nI n t r o n s a n d P r o d u c em R N A sE n c o d i n gS i n g l eP r o t e i n s As discussedin Chapter4, many bacterialmRNAs (e.g.,the mRNA encodedby the trp operon) include the coding region

for several proteins that function together in a biological process.SuchmRNAs are said to be polycistronic. (A cistron is a genetic unit encoding a single polypeptide.) In contrast' most eukaryotic mRNAs are monocistronic; that is, each mRNA molecule encodes a single protein. This difference betweenpolycistronic and monocistronic mRNAs correlates with a fundamental differencein their translatton. Within a bacterial polycistronic mRNA a ribosome-binding site is locatednear the start site for eachof the protein-coding regions,or cistrons,in the mRNA. Translation initiation can beginat any of thesemultiple internal sites,producing multiple proteins (seeFigure 4-13a).In most eukaryoticmRNAs' however,the 5'-cap structuredirectsribosome binding, and translation beginsat the closestAUG start codon (seeFigure4-13b). As a result, translation beginsonly at this site. In many cases, the primary transcriptsof eukaryoticprotein-codinggenesare processedinto a singletype of mRNA, which is translatedto give a singletype of polypeptide(seeFigure4-15). Unlike bacterialand yeastgenes,which generallylack introns, most genesin multicellular animals and plants contain introns, which are removedduring RNA processingin the nucleus beforethe fully processedmRNA is exported to the cytosol for translation. In many cases,the introns in a geneare considerablylonger than the exons. Although many introns are :90 bp long, the median intron length in human genesis 3.3 kb. Some,however,are much longer: the longestknown human intron is 17,1.06bp, and lies within titan, a gene encoding a structural protein in muscle cells. In comparison, most human exonscontain only 50-200 basepairs. The typiprotein is :50,000 cal human geneencodingan average-size bp long, but more than 95 percentof that sequenceis present in introns and flanking noncoding 5' and 3' regions. Many large proteins in higher organisms that have repeated domains are encoded by genesconsisting of repeats of similar exons separatedby introns of variable length. An example of this is fibronectin, a component of the extracellular matrix. The fibronectin gene contains multiple copies of five types of exons (seeFigure 4-16). Suchgenesevolved by tandem duplication of the DNA encoding the repeated exon, probably by unequal crossingover during meiosisas shown in Figure6-2a.

n nits S i m p l ea n d C o m p l e xT r a n s c r i p t i o U A r e F o u n di n E u k a r y o t i cG e n o m e s The cluster of genesthat form a bacterial operon comprises a single transcription unit that is transcribed from a specific promoter in the DNA sequenceto a termination site. producing a singleprimary transcript. In other words, genesand transcription units often are distinguishablein prokaryotes since a single transcription unit contains severalgeneswhen they are part of an operon. In contrast' most eukaryotic genes are expressedfrom separatetranscription units' and each mRNA is translated into a single protein. Eukaryotic transcription units, however, are classified into two types, dependingon the fate of the primary transcript. The primary transcript produced from a simple transcription unit is processedto yield a singletype of mRNA' encoding E U K A R Y O T IG C E N Es T R U C T U R E .

217

(a) Exon duplication L1

E x o n1

Parental chromosomes

===

E x o n2

Exon 3

L1

Exon 2

Exon 1

Exon 3

===

I

I R e c o m b i n a t i o(nu n e q u acl r o s s i n go v e r )

+ L1 Recombinant cnromosomes

Exon 1

Exon 2

Exon 3

Exon 3

Exon 1

Exon 2

(b) Gene duplication B - g l o b i ng e n e

Parentat I chromosomes I I * Recombinant cnromosomes

R e c o m b i n a t i o(nu n e q u acl r o s s i n g B - s l o b i ng e n e

t

A FIGURE 5-2 Exonand geneduplication.(a)Exonduplication results fromunequal crossing overduringmeiosrs Eachparental chromosome contains oneancestral genecontaining threeexons ( n u m b e r e1d- 3 )a n dt w o i n t r o n sH o m o l o g o n uo s n c o d i nLg1 r e p e a t esde q u e n c lei es5 ' a n d3 ' o f t h eg e n ea, n da l s oi n t h ei n t r o n between exons2 and3 As discussed in Section 6 3, L1sequences havebeenrepeatedly transposed to newsitesin thegenomeoverrne course of humanevolution, sothatallchromosomes arepeppered withthem Theparental chromosomes areshowndisplaced relative to eachother,sothatthe L1sequences arealignedHomologous recombination betweenL1sequences asshownwouldgenerate one recombinant chromosome in whichthegenehasfourexons(two

copies of exon3) andonechromosome in whichthe geneis missing exon3 (b)Unequal crossing overbetweenL'1sequences alsocan generate duplications of entiregenesInthisexample, eachparental chromosome gene,andoneof the contains oneancestral B-globin recombinant chromosomes contains two duplicated genes B-globin S u b s e q u ei n d t e p e n d em nu t t a t i o ni ns t h ed u p l i c a t egde n e cs o u l d l e a dt o s l i g hct h a n g ei sn s e q u e n ct hea tm i g h tr e s u litn s l i g h t l y properties different functional proteinsUnequal of theencoded crossing overalsocanresultfromrarerecombinations between unrelated sequences Notethatthe scalein part(b)is muchlarger (b)seeD H A Fitch thanin part(a) [Part eral, 1991,ProcNat'|. AcadSci. USA88:7396 I

a singleprotein.Mutations in exons,introns,and transcriptioncontrol regions all may influence expressionof the protein encodedby a simpletranscriptionunit (Figure6-3a). In the caseof complex rranscription units, which are quite common in multicellular organisms,the primary RNA transcript can be processedin more than one way, leading to formation of mRNAs containing different exons. Each alternate mRNA, however, is monocistronic, being translated into a single polypeptide, with translation usually initiating ar the first AUG in the mRNA. Multiple mRNAs can arise from a primary transcriptin threeways, as shown in Figure6-3b. Examplesof all three types of alternariveRNA processing occur in the genesthat regulate sexual differentiation in Drosophila (see Figure 8-16). Commonly, one mRNA is produced from a complex transcription unit in some cell types, and a different mRNA is made in other cell types. For example, alternative splicing of the primary fibronectin tran-

script in fibroblasts and hepatocytesdetermineswhether or not the secretedprotein includes domains that adhereto cell surfaces(seeFigure 4-16). The phenomenonof alternative splicing greatly expands the number of proteins encoded in the genomesof higher organisms. It is estimated that -60 percent of human genesare contained within complex transcription units that give rise to alternatively spliced mRNAs encoding proteins with distinct functions, as for the fibroblast and hepatocyteforms of fibronectin. The relationshipbetweena mutation and a gene is not always straightforward when it comes to complex transcription units. A mutation in the control region or in an exon shared by alternatively spliced mRNAs will affect all the alternative proteins encoded by a given complex transcription unit. On the other hand, mutations in an exon present in only one of the alternative mRNAs will affect only the protein encodedby that mRNA. As explained in

218

.

cHAprER 6

|

G E N E sG, E N o M t c sA,N Dc H R o M o s o M E s

Transcription Units ffi ,od."rt: Eukaryotic > FIGURE 6-3 Simpleand complexeukaryotictrans€ription Simple transcription unit units.(a)A simple transcription unitincludes a regionthatencodes , 50kb , oneprotein, extending fromthe 5' capsiteto the 3' poly(A) site, l-l CaPsite andassociated controlregionsIntrons liebetween exons(light bluerectangles) andareremoved duringprocessing of the primary (dashed transcripts redlines); thustheydo notoccurin thefunctional Gene m o n o c i s t r o nmi cR N AM u t a t i o ni sn a t r a n s c r i p t i o n - c o nr et rgoilo n (a b) mayreduce or prevent transcription, thusreducing or eliminatrng Control regions protein. synthesis of theencoded A mutation withinan exon(c)may proteinwith diminished 5' resultin an abnormal activityA mutation nRNA withinan intron(d) thatintroduces a newsplicesiteresults in an protein(b) abnormally spliced mRNAencoding a nonfunctional primary transcription unitsproduce thatcanbe (b) Complex transcription units Complex transcripts processed in alternative ways.(Iop)lf a primary transcript contains Cap site alternative splicesites,it canbe processed intomRNAs with thesame 5' and 3' exonsbut differentinternalexons(Middle)lf a primary transcript hastwo poly(A) sites,it canbe processed intomRNAs with Gene (f or g) are promoters alternative 3' exons(Botton)lf alternative Exon1 produced in a celltypein which activein different celltypes,mRNA1, f isactivated, hasa different firstexon(1A)thanmRNA2 has,which mRNAT S'f----a (andwhereexon1B isproduced in a celltypein whichg isactivated or (aandb) andthosedesignated isused)Mutations in controlregions nRNA2 5' c withinexonssharedbythealternative mRNAs affectthe proteins processed encoded by bothalternatively mRNAsIn contrast, (designated mutations d ande)withinexonsuniqueto oneof the Cap site processed from alternatively mRNAs affectonlythe proteintranslated promoters thatmRNAForgenesthataretranscribed fromdifferent in differentcelltypes(bottom),mutations in differentcontrolregions (f andg) affectexpression onlyin thecelltypein whichthatcontrol Gene Exon1 regionisactive mRNAT

S'f

Poly(A)site

Exon2

f_l-

Exon3

Exon4

-___---l3, T_

Poly(A)

E x o n3

J

or Chapter 5, genetic complementationtests commonly are nRNA2 5'[---f .T used to determine if two mutations are in the same or different genes(seeFigure5-7). However,in the complex transcription unit shown in Figure 6-3b (middle), mutations d Cap site Cap site poty(A) and e would complement each other in a genetic complementation test, even though they occur in the same gene. This is becausea chromosomewith mutation d can express Gene E x o n3 Exon2 ExonlB E x o n1 A a normal protein encoded by mRNA2 and a chromosome with mutation e can express a normal protein encoded by nRNAI 5' mRNA1. Both mRNAs produced from this gene would be presentin a diploid cell carrying both mutations,generating or both protein products and hence a wild-type phenotype. mRNA2 However, a chromosome with mutation c in an exon common to both mRNAs would not complement either mutation d or e. In other words, mutation c would be in the same complementation groups as mutations d and e, even though P r o t e i n - C o d i nG g e n e sM a y B e S o l i t a r yo r B e l o n g d and e themselveswould not be in the same complementaF a m i ly to a Gene tion group! Given these complications with the genetic defThe nucleotidesequenceswithin chromosomal DNA can be inition of a gene, the genomic definition outlined at the of classified on the basis of structure and function' as shown in beginning of this section is commonly used. In the case 'We will examinethe propertiesof eachclass'beginThble 6-1. genes, gene DNA tranprotein-coding a is the sequence genes,which comprise two groups. protein-coding with ning pre-mRNA precursor, equivalent to a transcribed into a roughly 25-50 percent of the organisms, In multicellular plus required unit, any other regulatory elements scription protein-codinggenesare representedonly once in the haploid for synthesisof the primary transcript.The various proteins encoded by the alternatively spliced mRNAs expressed genome and thus are termed solitary genes.A well-studied example of a solitary protein-coding gene is the chicken from one gene are called isoforms. G E N ES T R U C T U R E . EUKARYOTIC

219

CLASS

ITNGTH

(0/o) C0PY NUMBEB lNHUMAN GEN0ME FRACTI0N 0FHUMAN GEN()ME

Protein-coding genes

0.5-2200kb

-25,000

:55" (1.8)1

Tandemlyrepeatedgenes U2 snRNA rRNAs

5 . 1k b + 43 kb+

:20 :300

FIGURE6-18 Exon shuffling via recombination between homologous interspersedrepeats. Recombination repeatsin the intronsof betweeninterspersed separategenesproducestranscriptionunits with a new combinationof exons(greenand blue) ln the exampleshown here,a double crossoverbetween two setsof A/u elements, ( t h em o s ta b u n d a n S t I N E isn h u m a n s r) e s u l t s in an exchangeof exonsbetweenthe two

called enhancersthat can operate over distancesof tens of thousandsof basepairs. Transcription of many genesis controlled through the combined effectsof severalenhancerelements.Insertionof mobile elementsnear such transcriptioncontrol regionsprobably contributed to the evolution of new combinatr,onsof enhancersequences.These in turn control which specificgenesare expressedin particular cell types and the amount oi the encoded protein produced in modern organisms,as we discussin the next chapter' Theseconsiderationssuggestthat the early view of mobile DNA elements as completely selfish molecular parasites missesthe mark. Rather,they have contributed profoundly to the evolution of higher organismsby promoting (1) the generation of genefamilies via geneduplication, (2) the creation of new genesvia shuffling of preexistingexons' and (3) formation of -o.. complex regulatory regionsthat provide multifaceted control of gene expression.Today, researchersare attempting to harnesstranspositionmechanismsfor inserting therapeuticgenesinto patients as a form of genetherapy'

Transposable(Mobile) DNA Elements r Transposable DNA elements are moderately repeated sequencestnterspersedat multiple sites throughout the g.r,o-., of highir eukaryotes. They are present less frequently in prokarYotic genomes. r DNA transposonsmove to new sites directly as DNA; first transcribed into an RNA copy of retrotransporo.t. ".. then is reverse-transcribedinto DNA which the element, ( s e eF i g u r e6 - 8 ) ' r A common feature of all mobile elementsis the presence of short direct repeatsflanking the sequence' r Enzymesencodedby transposonsthemselvescatalyzeinsertion of thesesequencesat new sitesin genomic DNA' r Although DNA transposons'similar in structure to bacterial IS elements,occur in eukaryotes(e'g', the Drosophila P element), retrotransposonsgenerally are much more abundant, especiallyin vertebrates. r LTR retrotransposonsare flanked by long terminal repeats (LTRs), similar to those in retroviral DNA; like retroviruses,they encode reversetranscriptaseand integrase. They move in the genome by being transcribed into RNA, which then undergoesreversetranscription in

G e n e1 l l ] G e n e2 I I I

I Doublecrossover betweenA/uelements { +

9enes ) NA ELEMENTS T R A N S P O S A B (LM EO B I L E D

235

(a)

G e n e1 l l l

I

Trun.ooru."excisionfrom gene

tIsarfllo I S te

G e n e2 = : =

::: I I Trencnn,

y

s a s ei n s e r t i o ni n t o g e n e2

W e a kp o l y ( A ) srgnat

(b)

G e n e ' sp o l y ( A ) srgnat

G e n e1 l l ] 3'exon T r a n s c r i p t i oann d p o l y a d e n y l a t i o n at end of downstreamexon %AAAA l l t s c n t o r ts t e

G e n e2 : : :

::: I I ORF2reversetranscription and insertion I

:: =

:::

the cytosol, nuclear import of the resulting DNA with LTRs, and integration into a host-cell chromosome (see F i g u r e6 - 1 4 ) .

@

sequencesexhibit extensivehomology with small RNAs and are rranscribedby the sameRNA poly_ A/z elements,the most common SINEsin humans, 0-bp sequences found scatteredthroughout the hu_ man genome. r Some interspersedrepeats are derived from cellular RNAs that were reverse-transcribedand inserted rnto genomic DNA at some time in evolutionarv history. Processed pseudogenes derivedfrom mRNAs lack tntrons, a featurethat distinguishesthem from pseudogenes, which aroseby sequencedrift of duplicatedgenes. bile DNA elementsmost likely influencedevolution cantly by servingas recombinationsitesand by mo_ g adjacentDNA sequences.

CHAPTER 6

|

OrganelleDNAs

Although the vast majority of DNA in most eukaryotesis found in the nucleus,some DNA is presentwithin the mitochondria of animals, planrs, and fungi and within the chloroplastsof plants. Theseorganellesare the main cellular sitesfor ATP formation, during oxidative phosphorylation in mitochondria and photosynthesisin chloroplasts (Chapter 12). Many lines of evidenceindicate that mitochondria and chloroplastsevolvedfrom bacteriathat were endocytosedinto ancestral cells containing a eukaryotic nucleus,forming endosymbionts(Figure 6-20). Over evo_ lutionary time, most of the bacterialgeneswere lost from

( s e eF i g u r e6 - 1 7 ) .

236

< FIGURE 5-19 Exonshufflingvia transpositionof a DNAtransposonor LINE retrotransposon. (a)Transposition of an exon(blue)flankedby homologous DNA transposons intoan intronof a second gene As we sawin Figure 6-10,step[, transposase canrecognize andcleave the DNAat the ends of thetransposon inverted repeatsln gene1, if thetransposase cleaves at the leftendof the transposon on the leftandat the rightendof thetransposon on the right,it cantranspose a l lt h ei n t e r v e n i n DgN A i,n c l u d i nt g h ee x o n fromgene1,to a newsitein an intronof gene2 Thenetresultisan insertion of the exonfromgene1 intogene2 (b)lntegration of an exonintoanothergeneviatransposition of a LINE c o n t a i n i nagw e a kp o l y ( As)i g n a l .f s u c ha L I N E i s i n t h e3 ' - m o sitn t r o no f g e n e 1 , t r a n s c r i p t i o fnt h eL I N Ei n t oa n R N A i n t e r m e d i am t ea yc o n t i n ubee y o n di t so w n poly(A) siteandextendintothe 3, exon, t r a n s c r i b i tnhgec l e a v a gaen dp o l y a d e n y l a t i o n s i t eo f g e n e1 i t s e l fT h i sR N Ac a nt h e nb e reverse-transcribed andintegrated by the L I N EO R F 2 p r o t e i n( s e eF i g u r 6 e- ' 7 l ) i n t oa n i n t r o no n g e n e2 , i n t r o d u c i naqn e w3 , e x o n ( f r o mg e n e1 )i n t og e n e2

nucleus. However, mitochondria and chloroplasts in to_ day's eukaryotesretain DNAs encoding some prorernsessentialfor organellarfunction, as well as the ribosomal and transfer RNAs required for synthesis of these proteins. Thus eukaryotic cells have multiple geneticsysrems:a predominant nuclear systemand secondarysystemswith their own DNA, ribosomes, and tRNAs in mitochondria and chloroplasts.

G E N E SG , E N O M t C SA, N D C H R O M O S O M E S

Endocytosisof bacterium caoableof oxidative phosphorylation

,

Endocytosisof bacterium capableof photosYnthesis

Ancestral cell

ATPsynthase t,

Bacterial genome

BacteriaI genome

./

\/ E u k a r y o t i cp l a s m am e m b r a n e

Mitochondrial Mitochondrial genome matrix

Stroma

origin of 6-20 Modelfor endosymbiotic FIGURE by an of a bacterium Endocytosis mitochondriaand chloroplasts. with two an organelle cellwouldgenerate eukaryotic ancestral fromthe eukaryotic derived theoutermembrane membranes, plasma (gray) plasma andthe inneronefromthe bacterial membrane (red)Proteins bacterial localized to the ancestral membrane

suchthatthe portionof the retaintheirorientation, membrane spacenowfacesthe proteinoncefacingthe extracellular fromthe innerchloroplast of vesicles Budding space. intermembrane in of chloroplasts suchasoccursduringdevelopment membrane, of membranes thylakoid generate the plants, would contemporary indicated are DNAs organellar The chloroplasts

M i t o c h o n d r i aC o n t a i nM u l t i p l e m t D N AM o l e c u l e s

colonies. Genetic crossesbetween different (haploid) yeast strains showed that the petite mutation does not segregate with any known nuclear geneor chromosome' In later studies, most petite mutants were found to contain deletions of MtDNA.

Individual mitochondria arclarge enough to be seenunder the light microscope, and even the mitochondrial DNA (mtDNA) can be detectedby fluorescencemicroscopy.The mtDNA is located in the interior of the mitochondrion, the region known as the matrix (seeFigure 12-6). As judged by the number of yellow fluorescent "dots" of mtDNA' a Euglena gracilis cell contains at least 30 mtDNA molecules ( F i g u r e6 - 2 1 ) . Replication of mtDNA and division of the mitochondrial network can be followed in living cells using time-lapsemicroscopy.Such studiesshow that in most organismsmtDNA replicates throughout interphase. At mitosis each daughter cell receivesapproximately the same number of mitochondria, but since there is no mechanism for apportioning exactly equal numbers of mitochondria to the daughter cells, some cells contain more mtDNA than others. By isolating mitochondria from cells and analyztngthe DNA extracted from them, it can be seenthat each mitochondrion contains multiple mtDNA molecules. Thus the total amount of mtDNA in a cell dependson the number of mitochondria, the size of the mtDNA, and the number of mtDNA molecules per mitochondrion. Each of these parametersvaries greatly between different cell types.

m t D N A l s l n h e r i t e dC y t o p l a s m i c a l l y Studies of mutants in yeasts and other single-celledorganisms first indicated that mitochondria exhibit cytoplasmic inheritance and thus must contain their own genetic system (Figure 6-22). For instance,petite yeast mutants exhibit structurally abnormal mitochondria and are incapable of oxidative phosphorylation. As a result, petite cells grow more slowly than wild-type yeasts and form smaller

,

10tr-

t

6-21 Dualstainingrevealsthe FIGURE A EXPERIMENTAL muftipfemitochondrialDNAmoleculesin a growing Euglena of two dyes:ethidium graciliscell.Cellsweretreatedwith a mixture and red fluorescence, a emits and whichbindsto DNA bromide, emits and mitochondria into specifically is incorporated which D|OC6, and emitsa redfluorescence, Thusthe nucleus a greenfluorescence. of yellow-a combination DNAfluoresce areasrichin mitochondrial and Y Hayashi fluorescence [From redDNAandgreenmitochondrial J CellSci93:565 l K Ueda,1989, o R G A N E L L ED N A s

t

237

(a)

Haploidparentswith wild-type nucleargenes

Normal mitochondrion

< FIGURE 6-22 Cytoplasmic inheritanceof the pefite mutation in yeast.Petite-strain mitochondria aredefective in oxidative phosphorylation owingto a deletion in mtDNA(a)Haploid cellsfuse to produce a diploidcellthatundergoes meiosis, duringwhichrandom segregation of parental chromosomes andmitochondria containing mtDNAoccursNotethatalleles for genesin nuclear DNA(represented by largeandsmallnuclear chromosomes colored redandblue) segregate (seeFigure 2:2duringmeiosis 5-5) Incontrast, sinceyeast -50 mtDNAmolecules normally contain percell,allproducts of meiosis usually containbothnormalandpetitemtDNAs andarecapable of (b)Asthesehaploidcellsgrowanddividemitotically, respiration the (including cytoplasm themitochondria) israndomly distributed to the daughter cellsOccasionally, a cellisgenerated thatcontains only petitemtDNAandyieldsa petitecolony. defective Thusformation of suchpetitecellsisindependent of anynuclear geneticmarker.

"Petite" mitochondrion

Diploid zygore

In the mating by fusion of haploid yeastcells,both parents contribute equally to the cytoplasm of the resulting diploid; thus inheritance of mitochondria is biparental. In mammals and most other multicellular organisms,however,the sperm contributeslittle (if any) cytoplasmto the zygote,and virtually all the mitochondria in the embryo are derived from those in the egg, not the sperm. Studiesin mice have shown that 99.99 percentof mtDNA is maternally inherited, but a small part (0.01 percent)is inheritedfrom the male parent. In higher plants, mtDNA is inherited exclusivelyin a uniparental fashion through the femaleparent (egg),not the male (pollen).

M e i o s i s :r a n d o md i s t r i b u t i o n of mitochondriato d a u g h t e rc e l l s

The Size,Structure,and CodingCapacity of mtDNA Vary ConsiderablyBetweenOrganisms

(b)

\ \Mitosis

\

Respiratory-proficient

238

.

C H A P T E6R I

Petite

Respiratoryproficient

Surprisingly,the size of the mtDNA, the number and nature of the proteins it encodes,and eventhe mitochondrial genetic code itself vary gre^iy between different organisms. The mtDNAs of most multicellular animals are :16-kb circular moleculesthat encodeintron-lessgenescompactly arranged on both DNA strands. VertebratemtDNAs encode the two rRNAs found in mitochondrial ribosomes, the 22 tRNAs used to translatemitochondrial mRNAs, and 13 proteins involved in electron transport and AIP synthesis(Chapter 12). The smallestmitochondrial genomesknown arein plasmodium, single-celledobligate intracellular parasitesrhat cause malaria in humans. Plasmodium mtDNAs are only :6 kb, encoding five proteins and the mitochondrial rRNAs. The entire mitochondrial genomesfrom a number of different metazoan organisms (i.e., multicellular animals) have now been cloned and sequenced,and mtDNAs from all these sourcesencode essentialmitochondrial proteins (Figure 6-23). All proteins encodedby mtDNA are synthesized on mitochondrial ribosomes. Most mitochondriasynthesizedpolypeptidesidentified thus far are subunits of multimeric complexesused in electrontransport. ATp svnt h e s i s ,o r i n s e r r i o no f p r o r e i n si n t o t h e i n n e r m i t o c h o n d i i a l membrane or intermembranespace.However, most of the proteins localizedin mitochondria, such as those involved in the processeslisted at the top of Figure 6-23, are encoded by nuclear genes, synthesizedon cytosolic ribosomes, and imported into the organelle by processesdisc u s s e di n C h a p t e r1 3 .

G E N E SG , E N O M I C SA, N D C H R O M O S O M E S

L i p i dm e t a b o l i s m N u c l e o t i d em e t a b o l i s m A m i n o a c i dm e t a b o l i s m

C a r b o h y d r a tm e etabolism H e m es y n t h e s i s Fe-Ssynthesis

U b i q u i n o n es y n t h e s i s Co-factorsynthesis

Chaperones S i g n a l i n gp a t h w a y s

Proteases

DNA repair,replication,etc.

Heme ryase RNA polymerase

TIM

c Cvtochrome 6-23 Proteinsencodedin mitochondrialDNA A FIGURE Onlythe and their involvementin mitochondrialprocesses. a r e d e p i c t e dM o s tm i t o i n n e r m e m b r a n e m i t o c h o n d rm i aal t r i xa n d (blue); mitochondrial areencoded bythenucleus components chondrial components processes nucleus-encoded carriedout by exclusively a r el i s t e da t t h et o p M i t o c h o n d r ci aolm p o n e nst sh o w ni n p i n ka r e genomein but bythe nuclear by mtDNAin someeukaryotes encoded in encoded invariably few components The relatively othereukaryotes. in electron l-V areinvolved mtDNAareshownin orangeComplexes phosphorylation TlM,Sec,Tat,andOxal andoxidative transport

in proteinimportandexport,andinsertion areinvolved translocases e i sa o f p r o t e i nisn t ot h e i n n e rm e m b r a n(eC h a p t e1r3 ) R N a sP 8) lt should the 5'-endof tRNAs(Chapter that processes ribozyme havea multi-subunit be notedthat the majorityof eukaryotes encoded invariantly subunits three with here, I as depicted complex (e g , Saccharomyces' in a few organisms by mtDNA However, thiscomplexis replacedby andPlasmodium), Schizosaccharomyces, Formoredetailson enzyme. single-polypeptide a nucleus-encoded, C h a p t e r1s2 a n d1 3 s e e t r a n s p o r t , a n d m e t a b o l i s m mitochondrial 19:709 Genet Trends ] 2003, al et G Burger from , lAdapted

In contrast to metazoan mtDNAs, plant mtDNAs are many times larger,and most of the DNA doesnot encodeprotein. For instance,the mtDNA in the important model plant Arabidopsis thaliana is 366,924 base pairs, and the largest known mtDNA is =2 Mb, found in cucurbit plants (e.g., melon and cucumber).Most plant mtDNA consistsof long inmobile DNA elementsrestrictedto the mitrons, pseudogenes, and piecesof foreign (chloroplast, compartment, tochondrial nuclearand viral) DNA that were probably insertedinto plant mitochondrial genomesduring their evolution' Duplicated sequencesalso contributeto the greaterlength of plant mtDNAs. Differences in the number of genes encoded by the mtDNA from various organisms most likely reflect the movement of DNA between mitochondria and the nucleus during evolution. Direct evidencefor this movement comes from the observation that several proteins encoded by mtDNA in some speciesare encoded by nuclear DNA in other, closely related species.The most striking example of this phenomenon involves the cor 11 gene, which encodes subunit 2 of cytochrome c oxidase, which constitutes complex IV in the mitochondrial electron transport chain (see Figure 12-1.6).This gene is found in mtDNA in all multicellular plants studied except for certain related speciesof legumes,including the mung bean and soybeans,in which the cox 11geneis nuclear.The cox 1I geneis completely missing from mung beanmtDNA, but a defectivecox 1/ pseudo-

gene that has accumulatedmany mutations can still be recognized in soybeanmtDNA. Many RNA transcripts of plant mitochondrial genesare edited, mainly by the enzyme-catalyzedconversion of selected C residuesto U, and occasionallyU to C' (RNA editing is discussedin Chapter 8.) The nuclear cox lI gene of mrng bean corresponds more closely to the edited cox II RNA transcripts than to the mitochondrial cox 11 genes found in other legumes. These observations are strong evidencethat the cox ll genemoved from the mitochondrion to the nucleusduring mung bean evolution by a processthat involved an RNA intermediate. Presumablythis movement involved a reverse-transcriptionmechanism similar to that by which processedpseudogenesare generatedin the nuclear genome from nucleus-encodedmRNAs. In addition to the large differences in the sizes of mtDNAs in different eukaryotes' the structure of the mtDNA also varies greatly.As mentioned above, mtDNA in most animals is a circular molecule -16 kb' However' the mtDNA of many organismssuch as the protist Tetrahymena exists as linear head-to-tail concatomersof repeating sequence. In the most extreme examples, the mtDNA of the Amoebidium parasiticum is composed of several protist 'h,rndred distinct short linear molecules'And the mtDNA of Trypanosoma is comprised of multiple maxicircles concate,r"t.d (ittt.tlocked) to thousands of minicircles encoding o R G A N E L L ED N A s

o

239

guide RNAs involved in editing the sequenceof the mitochondrial mRNAs encodedin the maxicircles.

P r o d u c t so f M i t o c h o n d r i aG l enes Are Not Exported As far as is known, all RNA transcripts of mtDNA and their translationproducts remain in the mitochondrion in which they are produced, and all mtDNA-encoded proteins are synthesizedon mitochondrial ribosomes. Mitochondrial DNA encodesthe rRNAs that form mitochondrial ribosomes, although most of the ribosomal proteins are imported from the cytosol.In animalsand fungi, all the tRNAs used for protein synthesisin mitochondria also are encoded by mtDNAs. However,in plantsand many prorozoans,most mitochondrialtRNAs are encodedby the nuclearDNA and imported into the mitochondrion. Reflectingthe bacterialancestryof mitochondria, mitochondrial ribosomesresemblebacterial ribosomesand differ from eukaryotic cytosolic ribosomesin their RNA and protein compositions,their size,and their sensitivityto certain antibiotics (seeFigure 4-22). For insrance,chloramphenicol blocks protein synthesisby bacterialand mitochondrial ribosomesfrom most organisms,but cycloheximidedoesnot. This sensitivity of mitochondrial ribosomes ro the imoortant aminoglycosideclass of antibiotics that includ., .hlo."-phenicol is the main causeof the toxicity that theseantibiotics can cause. Conversely,cytosolic ribosomes are sensitiveto cycloheximideand resisrantto chloramohenicol.I

M i t o c h o n d r i aE v o l v e df r o m a S i n g l e E n d o s y m b i o t iE c v e n tI n v o l v i n ga -l Rickettsi a i ke Bacteriu m Analysisof the mtDNA sequences from various eukaryotes, including single-celledprotists that diverged from other eu, karyotes early in evolurion, provides rtrong support for the idea that the mitochondrion had a singleorigin. Mitochondria most likely arosefrom a bacterialsymbiontwhoseclosest contemporary relatives are in the Rickettsiacedegroup. Bacteria in this group are obligate intracellular parasitei. Thus, the ancestor of the mitochondrion probably also had an intracellular life style, putting it in a good location for evolving into an intracellularuylbion,. ihe mtDNA with the largest number of encoded genesso far found is in the protist speciesReclinomonasamericana. All other mtDNAs have a subset of the R. amertcana genes)strongly implying that they evolved from a common ancestor with R. i*rrlcqna, losing different groups of mitochondrial genesby deletion and/or transfer to the nucleusover time. In organismswhose mtDNA includesonly a limited num_ ber of genes, rhe same set of mitochondrial genes are retained, independent of the phyla that includes these organ_ rsms (seeFigure 6-23, orangeproteins). One hypothesisfor why thesegeneswere never successfullytransferredto the nuclear genome is that their encoded polypeptides are too hydrophobic to cross the outer mitochondrial membrane. and 240

.

thereforewould not be imported back into the mitochondria if they were synthesizedin the cytosol. Similarly,the large size of rRNAs may interferewith their transport from the nucleus through the cytosol into mitochondria. Alternatively, these genesmay not have been transferred to the nucleus during evolution becauseregulation of their expressionrn response to conditions within individual mitochondria may be advantageous.If thesegeneswere locatedin the nucleus,conditions within each mitochondria could not influencethe exoression of proteins found in that particular mitochondrion.

M i t o c h o n d r i aG l e n e t i cC o d e sD i f f e r f r o m t h e S t a n d a r dN u c l e a rC o d e The geneticcode used in animal and fungal mitochondria is different from the standard code used in l[ prokaryotic and eukaryotic nuclear genes;remarkably, the code even differs in mitochondria from different species(Table 6-3). '$7hyand how these differencesarose during evolution is mysterious. UGA, for example, is normally a stop codon, but is read as tryptophan by human and fungal mitochondrial translation systems;however,in plant mitochondria, UGA is still recognized as a stop codon. AGA and AGG, the standardnuclear codons for arginine, also code for arginine in fungal and plant mtDNA, but they are stop codons in mammalian mtDNA and serinecodons in Droso,bila mtDNA. As shown in Table 6-3, plant mitochondria appear ro E utilize the standard genetic code. However, comparisons of the amino acid sequencesof plant mitochondrial proteins with the nucleotidesequences of plant mtDNAs suggestedthat CGG could code for either arginine (the ,,stand a r d " a m i n o a c i d ) o r t r y p r o p h a n .T h i s a f p a r e n r n o n s p e c i ficity of the plant mitochondrial code is explained by editing of mitochondrial RNA rranscripts,which can converr cyrosine residuesto uracil residues.If a CGG sequenceis edited to UGG, the codon specifiestryptophan, the standard amino acid for UGG, whereas unedited CGG codons encode the standard arginine. Thus the translation systemin plant mitochondria doesutilize the standard qeneticcode. I

M u t a t i o n si n M i t o c h o n d r i aD l N A C a u s eS e v e r a l G e n e t i cD i s e a s e isn H u m a n s The severity of diseasecausedby a mutation in mtDNA depends on the nature of the mutation and on the proportion of mutant and wild-type mtDNAs presentin a particular cell type. Generally when mutations in mtDNA are found, cells contain mixtures of wild-type and mutant mtDNAs-a condition known as heteroplasmy.Each time a mammalian somatic or germ-line cell divides, the mutant and wild-type mtDNAs segregaterandomly into the daughter cells, as occurs in yeast cells (seeFigure 6-2Zb). Thus, the mtDNA genotype, which fluctuates from one generation and from one cell division to the next, can drift toward predominantlv w i l d - t y p eo r p r e d o m i n a n r l ym u r a n r m r D N A ; . S i n c ea l l e n zymes required for the replication and growth of mammalian mitochondria, such as the mitochondrial DNA and

c H A p r EbR I G E N EcsE, N o M t c A s ,N Dc H R o M o s o M E s

MITOCHOilDBIA CODON

SIANOARO C[)DE

MAMMATS

DBOSI]PHITA NEUR()SPORAYEASTS

UGA

Stop

Trn

Trn ^_r

Trp

Trp

Stop

AGA,AGG

A.g

Stop

Ser

Arg

Arg

Arg

AUA

Ile

Met

Met

Ile

Met

IIe

AUU

Ile

Met

Met

Met

Met

IIe

CUU,CUC,CUA,CUG

Leu

Leu

Leu

Leu

Thr

Leu

PTANTS

proteins. "For nuclear-encoded p' 239; ed', Springer-Verlag. S. Andersonet al., 1981,Nature 290:457;P.Borst,in InternationalCell Biology 1980-1981,H. G' Schweiger, souRCEs: Biol' Deu' Cell In Vitro 1'986, Levings, C' S. and K. Eckenrode V. 10:478483; Sci. Trends Biochem. Raj Bhandary, 1985, and U. L. C. Breitenberger S. Covelloand M. W. Gtay,1,989,Nature34l:662-666. 22:169-176;J.M. Gualberetal., 1989,Nature34'1.:660-662;andP.

RNA polymerases,are encodedin the nucleusand imported from the cytosol, a mutant mtDNA should not be at a "replication disadvantage"lmutants that involve large deletionsof mtDNA might even be at a selectiveadvantagein replication becausethey can replicate faster. Recent researchsuggeststhat the accumulation of mutations in mtDNA is an important component of aging in mammals. Mutations in mtDNA have been observedto accumulate with aging, perhaps due to a decreasein the proofreading ability of DNA polymerase.To study this hypothesis, researchersused gene "knock-in" techniquesto replace the nuclear gene encoding mitochondrial DNA polymerase with normal proofreading activity (seeFigure 4-34) with a mutant gene encoding a polymerasedefectivein proofreading. Mutations in mtDNA accumulatedmuch more rapidly in homozygous mutant mice than in wild-type mice, and the mutant mice aged at a highly acceleratedrate (Frgure6-24). With few exceptions,all human cells have mitochondria, yet mutations in mtDNA affect only some tisThose most commonly affectedare tissuesthat have a sues. high requirementfor ATP produced by oxidative phosphorylation and tissuesthat require most of or all the mtDNA in the cell to synthesizesufficient amounts of functional mitochondrial proteins. For instance, Leber's hereditary optic neuropathy (degeneration of the optic nerve) is causedby a missensemutation in the mtDNA geneencoding subunit 4 of the NADH-CoQ reductase(complex I)' a protein required for ATP production by mitochondria. Any of severallarge deletionsin mtDNA causesanother set of diseasesincluding chronic progressiueexternal ophthalmo' plegia, characterizedby eye defects, and Kearns-Sayresyndrome, characterized by eye defects, abnormal heartbeat and central nervous system degeneration.A third condition, causing "ragged" muscle fibers (with improperly assembled mitochondria) and associateduncontrolled jerky movements,is due to a single mutation in the TIICG loop

(a)

Wild-typemouse

Homozygousmutant

(b) 100 90 80 970 t60 250

E oano

;i d

zv

10 0 o

100 2oo 3oo 400 500 600 700 800 900 1000 Age (days)

6-24 Micewith a mitochondrial FIGURE a EXPERIMENTAL DNA polymerasedefectivefor proofreadingexhibit " i c ew e r ep r e p a r e d p r e m a t u r ea g i n g .A l i n eo f " k n o c k - i nm inthe mutation w i t h a b y m e t h o dds i s c u s s ei ndC h a p t e5r i nactivates p o l y m e r a t s h e a t D N A m i t o c h o n d r i a l g e n ee n c o d i n g e r' so o f r e a d i nf ugn c t i o n(.a ) W i l d - t y paen d t h e p o l y m e r a sp ) he t i c ea t 3 9 0d a y so l d( 1 3m o n t h s T h o m o z y g o umsu t a n m mutanm t o u s ed i s p l a yms a n yo f t h ef e a t u r eosf a n a g e dm o u s e versusage (>720 days,or 24 monthsof age) (b) Plotof survival muranls' homozygous and heterozygous and mice of wild-type T h eh o m o z y g o umsu t a n t cs l e a r lhy a v ea m u c hs h o r t elri f es p a n 309:481 et al, 2005,Sclence G C Kujoth thanwild-typemice [From Gregory and Wisconsin-Madison of of JeffMiller/Universrty Part(a)courtesy PhD l Kuioth DNAS ORGANELLE

241

of the mitochondrial lysine IRNA. As a result of this mutation, the translation of several mitochondrial proteins apparentlyis inhibited. I

ChloroplastsContain LargeDNAsOften E n c o d i n gM o r e T h a n a H u n d r e dp r o t e i n s Like mitochondria, chloroplasts are thought to have evolved from an ancestral endosymbiotic photosvnthetic bacterium(seeFigure6-20). However,the endosymbiotic event giving rise to chloroplasts occurred more recently (L2-1,.5 billion yearsago) than the eventleadingto the evolution of mitochondria (1.5-2.2 billion years ago). Consequentlg contemporary chloroplast DNAs show less structural diversity than do mtDNAs. Also similar to mitochondria, chloroplasts contain multiple copies of the organellar DNA and ribosomes, which synthesizesome chloroplast-encodedproteins using the standard genetic code. Like plant mtDNA, chloroplastDNA is inherited exclusively in a uniparental fashion through the female parent (egg). Other chloroplast proteins are encoded by nuclear genes,synthesizedon cytosolic ribosomes,and then incorporated into the organelle(Chapter 13). I In higher plants, chloroplast DNAs are 120-160 kb long, depending on the species.They initially were thought to be circular DNA molecules becausein genetically tractable organisms like the model plant protozoan Chlamydomonas reinhardtii, the geneticmap is circular. However, recentstudies have revealed that plant chloroplast DNAs are actually long head-to-tail linear concatomersplus recombination intermediates between these long linear molecules. In these studies,researchershave used techniquesthat minimize mechanical breakageof long DNA molecules during isolation and gel electrophoresis,permitting analysisof megabase-size DNA. The complere sequencesof several chloroplast DNAs from higher plants have been determined in the past several years. They contain 120-135 genes, 130 in the important model plant Arabidopsis thaliana. A. thaliana chloroplast DNA encodes 76 protein-coding genes and 54 geneswith RNA products such as rRNAs and tRNAs. Chloroplast DNAs encode the subunits of a bacterial-like RNA folymeraseand expressmany of their genesfrom polycistronic operons as in bacteria (seeFigure 4-l3a). Some chloroplast genescontain introns, but theseare similar to the soecialized introns found in some bacterial genesand in mirochondrial genesfrom fungi and protozoans, rather than the introns of nuclear genes. As in the evolution of mitochondrial genomes,many genesin the ancestralchloroplast endosym_ biont that were redundant with nuclear genes -g.n., have beenlost from chloroplast DNA. Also, many .rr..rtial for chloroplast function have been transferred to rhe nuclear genome of plants over evolutionary time. Recent estimates from sequenceanalysisof the A. thaliana and cyanobacterial genomesindicate that -4,500 geneshave been transferred from the original endosymbiont to the nuclear genome. 242

CHAPTER 5

|

G E N E sG , E N O M t C SA, N D C H R O M O S O M E S

Methods similar to those used for the transformation of yeast cells (Chapter 5) have been developedfor stably introducing foreign DNA into the chloroplasts of higher plants. The large number of chloroplast DNA moleculesper cell permits the introduction of thousandsof copiesof an engineeredgeneinto eachcell, resulting in extraordinarily high levels of foreign protein production. Chloroplast transformation has recently led to the engineeringof plants that are resistant to bacterial and fungal infections, drought, and herbicides. The level of production of foreign proteins is comparable with that achievedwith engineeredbacteria, making it likely that chloroplast transformation will be used for the production of human pharmaceuticalsand possibly for the engineeringof food crops containing high levelsof all the amino acids essentialto humans. I

Organelle DNAs r Mitochondria and chloroplastsmost likely evolved from bacteria that formed a symbiotic relationship with ancestral cells containing a eukaryotic nucleus (seeFigure 6-20). r Most of the genes originally within mitochondria and chloroplastswere either lost becausetheir funcdons were redundant with nucleargenesor moved to the nucleargenome over evolutionary time, leaving different gene sets in the organellarDNAs of different organisms(seeFigure 6-23). r Animal mtDNAs are circular molecules,reflecting their probable bacterial origin. Plant mtDNAs and chloroplast DNAs generally are longer than mtDNAs from other eukaryotes, Iargely becausethey contain more noncoding regions and repetitive sequences. r All mtDNAs and chloroplast DNAs encoderRNAs and some of the proteins involved in mitochondrial or photosynthetic electron transport and ATP synthesis.Most animal mtDNAs and chloroplast DNAs also encode the tRNAs necessaryto translate the organellar mRNAs. r Becausemost mtDNA is inherited from egg cells rather than sperm, mutations in mtDNA exhibit a maternal cytoplasmic pattern of inheritance. Similarlg chloroplast DNA is exclusivelyinherited from the maternal parent. r Mitochondrial ribosomes resemble bacterial ribosomes in their structure, sensitivity to chloramphenicol, and resistanceto cycloheximide. r The genetic code of animal and fungal mtDNAs differs slightly from that of bacteria and the nuclear genome and varies between different animals and fungi (seeTable 6-3). In contrast, plant mtDNAs and chloroplast DNAs appear to conform to the standard geneticcode. r Severalhuman neuromusculardisordersresult from mutations in mtDNA. Patients generally have a mixture of wild-type and mutant mtDNA in their cells (heteroplasmy): the higher the fraction of mutant mtDNA, the more severe the mutant phenotype.

Analysis Genome-wide ff,| Genomics: of GeneStructureand Expression Using automated DNA sequencingtechniques,methods for cloning DNA fragments on the order of 100 kb in length, and computer algorithms to piece together the stored sequence data, researchershave determined vast amounts of DNA sequenceincluding nearly the entire genomic sequence of humans and many key experimental organisms. This enormous volume of data,which is growing at a rapid pace, has been stored and organized in two primary data banks: the GenBank at the National Institutes of Health, Bethesda, Maryland, and the EMBL SequenceData Base at the European Molecular Biology Laboratory in Heidelberg, Germany. These databasescontinuously exchange newly reported sequencesand make them available to scientists throughout the world on the Internet. By now, the genome sequenceshave been completeln or nearly completely,determined for hundreds of viruses and bacteria, scores of archaea,yeasts (eukaryotes),and model multicellular eukaryotes such as the roundworm C. elegans, the fruit fly Drosophila melanogaster,mice, and humans. The cost of sequencinga megabaseof DNA has fallen so low that projects are underway to sequencethe entire genome in cancer cells and compare it to the genomein normal cells from the samepatient in order to determine all the mutations that have accumulatedin that patient's tumor cells. This approach may reveal genes that are commonly mutated in all cancers,as well as genesthat are commonly mutated in tumor cells from different patients with the same type of cancer (e.g., breast versus colon cancer). Such detailed information also may eventually lead to highly individualized cancer treatments tailored to the specific mutations in the tumor cells of a particular patient. In this section, we examine some of the ways researchersare mining this treasuretrove of data to provide insights about gene function and evolutionary relationships, to identify new geneswhose encodedproteins have never been isolated, and to determine when and where genes are expressed.This use of computers to analyze sequencedata has led to the emergenceof a new field of biology: b ioinformatics.

The most widely usedcomputer program for this purpose is known as BLAST (basic/ocal alignment searchrool). The BLAST algorithm dividesthe "new" protein sequence(known as the query sequence) into shorter segments and then searchesthe databasefor significant matches to any of the The matching program assignsa high score stored sequences. to identically matched amino acids and a lower score to matchesbetweenamino acidsthat are related (e.g.,hydrophobic, polar, positively charged, negatively charged) but not identical.When a significantmatch is found for a segment'the BLAST algorithm will searchlocally to extend the region of similarity.After searchingis completed,the program ranks the matches betweenthe query protein and various known proteins accordingto their p-ualwes.This parameteris a measure of the probability of finding such a degreeof similarity between two protein sequencesby chance. The lower the pvalue, the greater the sequencesimilarity between two sequences.A p-value lessthan about 10-' usually is considered as significantevidencethat two proteins sharea common ancestor.Many alternativecomputer programs have beendeveloped in addition to BLAST that can detect relationshipsbetween proteins that are more distantly related to each other than can be detected by BLAST. The development of such methodsis currently an active areaof bioinformaticsresearch. To illustrate the power of this approach, we consider the human geneNF1 . Mutations in NF1 are associated with the inherited diseaseneurofibromatosis f in which multiple tumors develop in the peripheral nervous system' causinglarge protuberancesin the skin. After a cDNA clone of NFl was isolated and sequenced,the deducedsequenceof the NF1 protein was checked against all other protein sequencesin GenBank. A region of NF1 protein was discovered to have considerablehomology to a portion of the yeast protein calledIra (Figure6-25). Previousstudieshad shown that Ira is a GTPase-activatingprotein (GAP) that modulates the GTPaseactivity of the monomeric G protein called Ras (seeFigure 3-32). As we examine in detail in Chapter 16, GAP and Ras proteins normally function to control cell

StoredSequencesSuggestFunctionsof Newly ldentified Genesand Proteins As discussedin Chapter 3, proteins with similar functions often contain similar amino acid sequencesthat correspond to important functional domains in the three-dimensional structure of the proteins. By comparing the amino acid sequence of the protein encoded by a newly cloned gene with the sequencesof proteins of known function, an investigator can look for sequencesimilarities that provide clues to the function of the encoded protein. Becauseof the degeneracy in the geneticcode, related proteins invariably exhibit more sequencesimilarity than the genesencoding them. For this reason, protein sequencesrather than the corresponding are usuallvcompared. DNA seouences

characteristicof the disease.I Even when a protein shows no significant similarity to other proteins with the BLAST algorithm, it may nevertheless share a short sequencethat is functionally important' Such short segmentsrecurring in many different proteins, referred to as structural motifs, generally have similar functions. Several such motifs are described in Chapter 3 and illustratedin Figure3-9.To searchfor theseand other motifs in a new protein, researcherscompare the query protein sequencewith a databaseof known motif sequences.

243

GENoM|CS:GENoME-W|DEANALYS|SoFGENESTRUCTUREANDEXPRESSIoN

NF1 841 TRATFMEVLTK I LOOGTE FDTLAETVLADRFERLVE LVTMMGDOGE LP IA 890 aaaaaoaaaaaa lra 1500 lR IAFLRVF ID lV. . TNYPVNPEKHEMDKMLA iDDFir FIGURE 8-15 Modelfor cleavageand polyadenylation of pre-mRNAs in mammaliancells.Cleavage andpolyadenylation specificity factor(CPSF) bindsto the upstream AAUAAApoly(A) signalCSIFinteracts with a downstream GU-or U-richsequence a n dw i t h b o u n dC P S F f o, r m i n ga l o o pi n t h e R N Ab; i n d i n g of CFI andCFllhelpstabilize the complexBindingof poly(A)polymerase (PAP) thenstimulates cleavage at a poly(A) site,whichusually is 10-35nucleotides 3' of the upstream poly(A) signal.Thecleavage factorsarereleased, as isthe downstream RNAcleavage product, w h i c hi s r a p i d ldy e g r a d e dB.o u n dp A pt h e na d d s: 1 2 A r e s i d u east a slowrateto the 3'-hydroxyl groupgenerated by the creavage reactionBindingof poly(A)-binding proteinil (pABpil) to the initial shortpoly(A) tailaccelerates the rateof additionby pApAfter 200-250A residues pApto stoo havebeenadded,pABpll siqnals polVmenzation

Poly(A) signal [I

s',

3' Pre-mRNA

,, I CPSF, CSIF, CFI,CFII

1

residuesoccurs slowly, followed by rapid addition of up to 200-250 more A residues.The rapid phase requires the binding of multiple copies of a poty(A)-binding protein containing the RRM motif. This protein is designatedpABpII ro distinguish it from the poly(A)-binding protein presentin the cytoplasm. PABPII binds to the short A tail initially added by PAP,stimulating the rate of polymerization of additional A residuesby PAP,resulting in rhe fast phase of polyadenylation (Figure 8-15). PABPII is also responsiblefor signaling poly(A) polymerase to terminate polymerization when the poly(A) tail reachesa length of 200-250 residues,although the mechanismfor controlling the length of the tail is not yit understood. Binding of PABP to the poly(A) tail is essential for mRNA export into the cytoplasm.

N u c l e a rE x o n u c l e a s eDse g r a d eR N AT h a t l s Processed Out of Pre-mRNAs

poty(A) p o l y ( A )s i t e s i g n a l \

J.,""'un"

ATP o-€j-

ppi

slow PolYadenYlation

+ CSIF,CFl,CFll

Becausethe human genome contains long introns, only :5 percent of the nucleotides that are polymerized by RNA polymerase II during transcription are retained in mature, processedmRNAs. Although this process appears inefficient, it probably evolved in multicellular organismsbecause the processof exon shuffling facilitated the evolution of new

As mentionedearlier,the 2,,5,-phosphodiesterbond in excised introns is hydrolyzed by a debranching enzyme,yielding a linear molecule with unprotected ends that can be attacked by exonucleases(seeFigure 8-11). The predominant nuclear decaypathway is 3' -+ 5' hydrolysis by 11 exonucleases that associatewith one another in a large protein complex called the exosome.Other proteins in the complex include RNR helicasesthat disrupt base pairing and RNA-protein interactions that would otherwiseimpedethe exonucleases. Exosomesalso function in the cytoplasm, as discussedlater. In addition, the exosome appears to degrade pre-mRNAs that have not been properly spliced or polyadenylated. It is not yet clear how the

336

C H A P T E R8

|

posT-TRANSCR|pTtoNA GLE N EC O N T R O L

ATP ppi

Rapid PolYadenYlation

-zoo ou 3'

exosomerecognizesimproperly processedpre-mRNAs. But in yeast cells with temperature-sensitivemutant poly(A) polymerase(Figure8-15), pre-mRNAs are retainedat their sitesof transcription at the nonpermissivetemperature.Theseabnormally processedpre-mRNAs are releasedin cellswith a second mutation in a subunit of the exosomefound only in nuclear and not cytoplasmicexosomes(PM-Scl; 100 kD in humans). Also, exosomesare found concentratedat sitesof transcription in Drosophila polytene chromosomes,where they are associated with RNA polymeraseII elongation factors. Theseresults suggestthat the exosomeparticipatesin an as yet poorly understood quality-control mechanismthat recognizesaberrantly processed pre-mRNAs, preventing their export to the cytoplasm and ultimately leadingto their degradation. To avoid being degraded by nuclear exonucleases,nascent transcripts, pre-mRNA processing intermediates, and mature mRNAs in the nucleus must have their ends protected.As discussedabove,the 5' end of a nascenttranscript is protectedby addition ofthe 5'cap structureas soon as the 5' end emergesfrom the polymerase.The 5' cap is protected becauseit is bound by a nuclear cap-binding complex, which protects it from 5' exonucleasesand also functions in export of mRNA to the cytoplasm. The 3' end of a nascent transcript lies within the RNA polymeraseand thus is inaccessible to exonucleases(seeFigure4-12). As discussedpreviously, the free 3' end generatedby cleavageof a pre-mRNA downstream from the poly(A) signal is rapidly polyadenylatedby the poly(A) polymeraseassociatedwith the other 3' processing factors, and the resulting poly(A) tail is bound by PABPII (Figure 8-15). This tight coupling of cleavageand polyadenylation protects the 3' end from exonucleaseattack.

pre-mRNAs of higher organisms. A network of interactions between SR proteins, snRNPs' and splicing factors forms a cross-exonrecognition complex that specifiescorrect splicesites(seeFigure 8-13)' r The snRNAs in the spliceosomeare thought to have an overall tertiary structure similar to that of group II selfsplicing introns. r For long transcription units in higher organisms,splicing of exons usually begins as the pre-mRNA is still being formed. Cleavageand polyadenylation to form the 3' end of the mRNA occur after the poly(A) site is transcribed. r In most protein-coding genes' a conserved AAUAAA poly(A) signal lies slightly upstream from a poly(A) site where cleavageand polyadenylation occur. A GU- or Urich sequence downstream from the poly(A) site contributes to the efficiency of cleavageand polyadenylation. r A multiprotein complex that includes poly(A) polymerase(PAP)carries out the cleavageand polyadenylation of a pre-mRNA. A nuclear poly(A)-binding protein, PABPII, stimulatesaddition of A residuesby PAP and stops addition once the poly(A) tail reaches 200-250 residues ( s e eF i g u r e8 - 1 5 ) . r Excised introns and RNA downstream from the cleavagel poly(A) site are degradedprimarily by exosomes,multiprotein complexesthat contain eleven3' -> 5' exonucleasesas well as RNA helicases.Exosomesalso degradeimproperly processedpre-mRNAs.

fp| Regulationof Pre-mRNA Processing Processingof Eukaryotic Pre-mRNA r In the nucleus of eukaryotic cells, pre-mRNAs are associated with hnRNP proteins and processedby 5' capping, 3' cleavageand polyadenylation, and splicing before being transported to the cytoplasm (seeFigure 8-2). r Shortly after transcriptioninitiation, a cappingenzymeassociatedwith the phosphorylatedCTD of RNA polymerase II addsthe 5' cap to the nascenttranscript. Other RNA processingfactors involved in RNA splicing, 3' cleavage,and polyadenylation also associate with the phosphorylated CTD, increasingthe rate of transcriptionelongation.Consequently, transcription does not proceed at a high rate until RNA processingfactors become associatedwith the CTD, where they are poised to interact with the nascent premRNA as it emergesfrom the surface of the polymerase. r Five different snRNPs interact via basepairing with one another and with pre-mRNA to form the spliceosome(see Figure 8-11). This very large ribonucleoproteincomplex catalyzestwo transesterificationreactions that join two exons and remove the intron as a lariat structure, which is subsequentlydegraded(seeFigure 8-8). r SR proteins that bind to exonic splicing enhancer sequencesin exons are critical in defining exons in the large

Now that we've seenhow pre-mRNAs are processedinto mature, functional mRNAs' we consider how regulation of this processcan contribute to genecontrol. Recall from Chapter 5 that higher eukaryotescontain both simple and complex tran-

transcription units 1-69o7oof all human transcription units) can be processedin alternative ways to yield different mRNAs that encodedistinct proteins (seeFigure 6-3).

A l t e r n a t i v eS p l i c i n gl s t h e P r i m a r yM e c h a n i s m for RegulatingmRNAProcessing The discovery that a large fraction of transcription units in higher organisms encode alternatively spliced mRNAs and that differently splicedmRNAs are expressedin different cell types revealedthat regulation of RNA splicing is an importun, g.n"-.ontrol mechanismin higher eukaryotes.Although many examples of cleavageat alternative poly(A) sites in pre-mRNAs are known, alternative splicing of different exons is the more common mechanismfor expressingdifferent O F P R E - m R N AP R O C E S S I N G REGULATION

337

Pre-mRNAs

(a) sxl

mRNAs

q .-

z

(b) tra

? -/Y .-\

gsxt'rotein

W+ J

4

-+

t-fl-t} 1

2

?rra protein

5

(c) dsx

A FIGURE 8-16 Cascade of regulatedsplicingthat controlssex determinationin Drosophilaembryos.Forclarity,onlythe exons (boxes) andintrons(blacklines) whereregulated splicing occursare shownSplicing isindicated by reddashed linesabove(female) and bluedashed linesbelow(male) the pre-mRNAs Vertical redlinesin exonsindicate ln-frame stopcodons, whrchprevent synthesis of functional proteinOnlyfemaleembryos produce functional Sxl protein, whichrepresses splicing betweenexons2 and3 in sxlpremRNA(a)andbetweenexons1 and2 in tra pre-mRNA (b) (c)In contrast, the cooperative bindingof Traproteinandtwo SRproteins,

Rbpl andfra2,activates splicing betweenexons3 and4 and cleavage/polyadenylation Anat the 3' endof exon4 in dsxprem R N Ai n f e m a l e m b r y o sI n m a l ee m b r y o w s ,h i c hl a c kf u n c t i o n a l Tra,the SRproteins do not bindto exon4, andconsequently exon3 isspliced to exon5 ThedistinctDsxproteins produced in female andmaleembryos asthe resultof thiscascade of regulated splicing repress transcription of genesrequired for sexual differentiation of the opposite sex.[Adapted fromM J Mooreet al, 1993,in R Gesteland press, andJ Atkrns, eds,IheRNAWorld, ColdSpring pp 303-357 Harbor l

proteins from one complex transcription unit. In Chapter 4, for example, we mentioned that fibroblasts produce one type of the extracellular protein fibronectin, whereas hepatocytes produce another type. Both fibronectin isoforms are encoded by the same transcription unit, which is spliced differendy in the two cell types to yield two different mRNAs (seeFigure4-16).ln other cases,alternativeprocessingmay occur simultaneouslyin the samecell type in responseto different developmentalor environmental signals.First we discussone of the best-undersrood examplesof regulatedRNA processingand then briefly consider the consequencesof RNA splicing in the developmentof rhe nervous sysrem.

The Sxl protein, encodedby the sex-lethalgene,isthe first protein to act in the cascade(Figure 8-16). The Sxl protein is present only in female embryos. Early in development,the gene is transcribed from a promoter that functions only in female embryos. Later in development,this female-specific promoter is shut off and another promoter for sex-letbal becomesactive in both male and female embryos. However, in the absenceof early Sxl protein, the sex-lethalpre-mRNA in male embryos is spliced to produce an mRNA that contains a stop codon early in the sequence.The net result is that male embryos produce no functional Sxl protein either early or later in development. In contrast, the Sxl protein expressedin early female embryos directs splicing of the sex-lerEal pre-mRNA so that a functional sex-lethalmRNA is produced (Figure 8-16a). Sxl accomplishesthis by binding to a sequencein the pre-mRNA near the 3' end of the intron between exon 2 and exon 3, thereby blocking the proper association of U2AF and tJ2 snRNP.As a consequence, the U1 snRNP bound to the 3, end of exon 2 assembles into a spliceosomewith U2 snRNp bound to the branch point at the 3' end of the intron betweenexons 3 and 4,leading to splicing of exon 2 to 4 and skipping of exon 3. The resulting female-specificsex-lethal mRNA is translated into functional Sxl protein, which reinforces its own expressionin female embryos by continuing to cause skipping of exon 3. The absenceof Sxl protein in male embryos allows the inclusion of exon 3 and, consequently,of the stop codon that preventstranslation of functional Sxl protein.

A Cascade o f R e g u l a t e dR N AS p l i c i n gC o n t r o l s D r o s o p h i l aS e x u a lD i f f e r e n t i a t i o n One of the earliestexamplesof regulatedalternarivesplicing of pre-mRNA came from studiesof sexual differentiaiion in

splicing in Drosophila embryos. More recent researchhas provided insight into how theseproteins regulate RNA processingand ultimately lead to the creation of two different sex-specifictranscriptionalrepressorsthat suppressthe development of characteristicsof the oppositesex. 338

.

c H A p r E8R I

posr-TRANscRtploN GA ELN E coNTRoL

U2AF and U2 snRNP to the 3' end of the intron between exons 3 and 4, just as other SR proteins do for constitutively spliced exons (see Figure 8-13). The TralTra2lRbpl complexes may also enhancebinding of the cleavage/polyadenylation complex to the 3' end of exon 4.

and ActivatorsControl SplicingRepressors Splicingat Alternative Sites

d

A FIGURE 8-17 Modelof splicingactivationby Traproteinand embryos, the 5RproteinsRbpl and Tra2.In femaleDrosophila isactivated by bindingof splicing of exons3 and4 in dsxpre-mRNA Rbpl andTra2 fra/Tra2lRbp1 complexes to sixsitesin exon4 Because in theabsence of Tra,exon4 isskipped cannotbindto the pre-mRNA A, : polyadenylation in maleembryos. Seethetextfor discussion 2002,Nature 418:236] fromT.Maniatis andB Tasic, lAdapted Sxl protein also regulatesalternative RNA splicing of the transformergenepre-mRNA (Figure8-16b).In male embryos, where no Sxl is expressed,exon 1 is spliced to exon 2, which contains a stop codon that prevents synthesisof a functional transformer protein. In female embryos, however, binding of Sxl protein to the 3' end of the intron between exons 1 and 2 blocks binding of U2AF at this site. The interaction of Sxl with transformer pre-mRNA is mediated by two RRM domains in the protein (seeFigure 8-5).Vhen Sxl is bound, U2AF binds to a lower-affiniry site farther 3' in the pre-mRNA; as a result exon 1 is spliced to this alternative 3' splice site, eliminating exon2 with its stop codon. The resulting female-specifictransformer nRNA, which contains additional constitutively spliced exons, is translated into functional Transformer (Tra) protein. Finally, Tra protein regulatesthe alternativeprocessingof pre-mRNA transcribed from the double-sex gene (Figure 8-16c). In female embryos,a complex of Tra and two constitutively expressedproteins,Rbpl and Tra2, directssplicingof exon 3 to exon 4 and also promotescleavage/polyadenylation at the alternativepoly(A) site at the 3' end of exon 4-leading to a short, female-specificversion of the Dsx protein. In male embryos,which produce no Tra protein, exon 4 is skipped,so that exon 3 is spliced to exon 5. Exon 5 is constitutively splicedto exon 6, which is polyadenylatedat its 3' end-leading to a longer,male-specificversion of the Dsx protein. As a result of the cascadeof regulatedRNA processingdepictedin Figure 8-16, different Dsx proteins are expressedin male and femaleembryos.The male Dsx protein is a transcriptionalrepressor that inhibits the expression of genes required for female development.Conversely,the female Dsx protein repressestranscription of genesrequired for male development. complex Figure 8-17 illustrates how theTrafta2iRbpl is thought to interact with double-sex(dsx)pre-mRNA. Rbpl andTra2 are SR proteins, but they do not interact with exon 4 in the absenceof the Tra protein. Tra protein interactswith Rbpl and Tra2, resulting in the cooperative binding of all three proteins to six exonic splicing enhancersin exon 4. The bound Tra2 and Rbpl proteins then promote the binding of

As is evident from Figure 8-15, the Drosophila Sxl protein and Tra protein have opposite effects:Sxl prevents splicing' causingexons to be skipped, whereasTra promotes splicing. The action of similar proteins may explain the cell-typespecificexpressionof fibronectin isoforms in humans. For instance,an Sxl-like splicingrepressorexpressedin hepatocytes might bind to splicesitesfor the EIIIA and EIIIB exons in the fibronectin pre-mRNA, causing them to be skipped during RNA splicing (seeFigure4-16). Alternatively,a Tralike splicing activator expressedin fibroblastsmight activatethe splice sites associatedwith the fibronectin EIIIA and EIIIB exons' leading to inclusion of these exons in the mature mRNA. Experimental examination in some systemshas revealedthat inclusion of an exon in some cell types versusskipping of the same exon in other cell types results from the combined influenceof severalsplicing repressorsand enhancers. Alternative splicing of exons is especially common in the nervous system,generatingmultiple isoforms of many proteins required for neuronal developmentand function in both vertebratesand invertebrates.The primary transcripts from thesegenesoften show complex splicing patterns that can generate several different mRNAs' with different spliced forms expressedin different anatomical locationswithin the central nervous system.We consider two remarkable examplesthat illustrate the critical role of this processin neural function. Expression of K*-Channel Proteins in Vertebrate Hair Cells In the inner ear of vertebrates, individual "hair cells," which are ciliated neurons' respond most strongly to a specific frequency of sound. Cells tuned to low frequency (:50 Hz) are found at one end of the tubular cochlea that makes up the inner ear; cells responding to high frequency (:5000 Hz\ arefound at the other end (Figure8-18a).Cells in between the ends respond to a gradient of frequenciesbetween theseextremes.One component in the tuning of hair cells in reptiles and birds is the opening of K*ion channelsin resDonseio increasedintracellular Ca2*concentrations.The Ca2* concentration at which the channel opens determines the frequency with which the membrane potential oscillates and hencethe frequency to which the cell is tuned' The gene encoding this Ca2+-activatedK+ channel is expressedas multiple, alternatively spliced mRNAs. The various proteins encodedby thesealternative mRNAs open at different Ca2* concentrations. Hair cells with different response frequenciesexpressdifferent isoforms of the channel protein depending on their position along the length of the cochlea (see Figure 23-30). The sequencevariation in the protein is very complex: there are at leasteight regions in the mRNA where alternative exons are utilized, permitting the expression of 576 possibleisoforms(Figure8-18b).PCR analysisof mRNAs from O F P R E - m R N AP R O C E S S I N G REGULATION

339

(a)

through post-translational modifications of splicing factors playsa significantrole in modularingneuron function.

Apical h a i rc e l l ( 5 0H z )

Auditory nerve cell body

Basal h a i rc e l l (5000Hz)

Cytosol

outside) of only about 1,70-fold.Thus by coupling the transport of two Na+ ions to the transport of one glucose, the two-Na*/one-glucose symporter permits cells to accumulate a very high concentration of glucoserelative to the external

Oveview Animation: BiologicalEnergyInterconversions Exrenol

2Na+o a

OGlucose

++ Inward-facing conformation

FIGURE 11-25 Operationalmodel for the two-Na+/one_ glucosesymporter.Simultaneous bindingof Na+andglucose to the conformation with outward-facing bindingsites(stepIl) causes a conformational changein the proteinsuchthatthe boundsubstrates aretransiently occluded, unableto dissociate intoeithermedium(stepZ) In stepEt the proteinassumes a

466

C H A P T E R1 1

I

Outward-facing conformation

thirdconformation with inward-facing sitesDissociation of the boundNa* andglucose (stepB) allowsthe protern intothecytosol to revertto itsoriginaloutward-facing (step[), ready conformation to transportadditional physiology substrate[SeeE Wrightet.at,2OO4, 19:370 for detailson the structureand function of thrs and related transportersl

T R A N S M E M B R A NTER A N S P O R O T F I O N SA N D S M A L L M O L E C U L E 5

concentration. This means that glucosepresent even at very low concentrations in the lumen of the intestine or in the forming urine can be efficiently transported into the lining cells and not lost from the body. The two-Na+/one-glucose symporter is thought to contain L4 transmembrane a helices with both its N- and Ctermini extending into the cytosol. A truncated recombinant protein consisting of only the five C-terminal transmembrane o helices can transport glucose independently of Na* across the plasma membrane, down its concentration gradient. This portion of the molecule thus functions as a glucose uniporter. The N-terminal portion of the protein, including helices 1-9, is required to couple Na+ binding and influx to the transport of glucoseagainst a concentration gradient. Figure 11-25 depicts the current model of transport by Na*/glucose symporters.This model entails conformational changesin the protein analogousto those that occur in uniport transporters, such as GLUT1, which do not require a

cotransportedion (seeFigure 11-5). Binding of all substrates to their sites on the extracellular domain is required before the protein undergoesthe conformational change that transitions the substrate-bindingsites from outward to inward facing; this ensures that inward transport of glucose and Na* ions are coupled.

BacterialSymporterStructureRevealsthe Mechanismof SubstrateBinding No three-dimensional structure of a mammalian sodium symporter has been determined' but the structuresof several homologous bacterial sodium-amino acid transporters have provided considerable information about symport function. The bacterial two-Na*/one-leucine symporter shown in Figure 1'1'-26aconsistsof 12 membrane-spanning a helices.Two of the helices (numbers 1, and 6l have nonhelical segmentsin the middle of the membrane that form part of the leucine-bindingsite.

fT

!l

Symporter eod.ust:The Two-Na+/one-Leucine

structureof the two-Na-11-26 Three-dimensional A FIGURE /one-feucinesymporterfrom the bacteriumAquifex aeolicus' (a)TheboundL-leucine, ionare two sodiumions,anda chloride The shownasCPKmodelsin yellow,purpleandgreen,respectively are cthelices thatbindthe Na*or leuctne threemembrane-spanning of thetwo sodiumions brown,blue,andorange(b)Binding colored

oxygens side-chain atomsor carboxyl oxygen main-chain to carbonyl (red)that arepartof helices1 (brown),6 (blue),or 8 (orange)lt is thatoneof thesodiumions(top)isalsoboundto the important A Yamashita leucine(yellow). [From groupof the transported carboxyl 431:811 Nature 2004, et al, Yernool D see also 437 :215: Nature 2005, et al, transporters l of thisandrelated andfunction onthestructure fordetails

C O T R A N S P O RBTY S Y M P O R T E RASN D A N T I P O R T E R S

467

Amino acid residues involved in binding the leucine and the two sodium ions are located in the middle of the membrane-spanning segment (as depicted for the twoNa*/one-glucosesymporter in Figure 11-25) and are close together in three-dimensional space. This demonstrates that the coupling of substrate and ion transport in these transporters is the consequenceof direct or nearly direct physical interactions of the substrates.Indeed, one of the sodium ions (number 1 in Figure 11-26b) is bound to the carboxyl group of the transported leucine,indicating how binding of sodium and leucine are coupled. Each of the two sodium ions is bound to six oxyge., ,to-r. Sodium 1, for example, is also bound to carbonyl oxygens of several transporter amino acids as well as to carbonyl oxygens and the hydroxyl oxygen of one threonine. Equally importantly, there are no water moleculessurrounding either of the bound sodium atoms, as is the case for K+ ions in potassium channels (see Figure 11-20). Thus as the sodium ions lose their water of hydration in binding to the transporter,they bind to six oxygen atoms with a similar geometry. This reducesthe activation energy for binding of sodium ions and preventsother ions, such as porassium, from binding in place of sodium. One striking feature of the structure depicted in Figu r e 1 1 - 2 6 i s t h a t t h e b o u n d s o d i u m i o n s a n d l e u c i n ea r e occluded-that is, they cannot diffuse out of the protein to either the surrounding extracellular or cytoplasmic media. Apparently the process of crystallization of this protein with its bound substrateshas "trapped" it into an intermediate transport step (see Figure 11-25) in which the protein appears to be changing from a conformation with an exoplasmic- to one with a cytosolic-facing binding site.

Na+-LinkedCa2+Antiporter ExportsCa2*from C a r d i a cM u s c l eC e l l s In cardiac muscle cells a tbree-Na+/one-Ca2* anti\orter. rather than the plasma membrane Ca2* Alpase dis.ussej earlier, plays the principal role in maintaining a low concentration of Ca2* in the cytosol. The transporr reacrron mediated by this cation antiporter can be written 3 Na+or, * Ca2*;, ; -

3 Na+1. * C"'*o,.

Note that the movement of three Na+ ions is required to power the export of_oneCa2' ion from the .ytoto1, with a lcal*J of :2 x 10-7 M, ro the extracellularmedium, with a [C"t*] of 2 x 10-3 M, a gradientof some 10,000-fold.In all muscle cells, a rise in the cytosolic Ca2* concentration in cardiac muscle triggers conrraction; by lowering cytosolic Ca'-, operation of the Na+/Ca2+ 2lliporter reducesthe strengthof heart musclecontracrion. The Na*/K+ ATPasein the plasma membrane of cardiac muscle cells, as in other body cells, createsthe N a * concentration gradient necessaryfor export of Caz* 468

.

c H A p r E R1 1 |

by the Na*-linked Ca'* antiporter. As mentioned earlier, inhibition of the Na*/K* AIPase by the drugs ouabain and digoxin lowers the cytosolic K* concentration and, more relevant here, simultaneouslyincreasescytosolic Na*. The resulting reduced Na* electrochemicalgradient across the membranecausesthe Na+-linked Ca2* antiDorterto function less efficiently. As a result, fewer Ca21 ions are exported and the cytosolic Ca2* concentration increases, causing the muscle to contract more strongly. Becauseof their ability to increasethe force of heart muscle contractions, drugs such as ouabain and digoxin that inhibit the Na-/K- ATPase are widely used in the treatment of congestiveheart failure. I

SeveralCotransportersRegulateCytosolicpH The anaerobic metabolism of glucoseyields lactic acid, and aerobic metabolism yields CO2, which adds water to form carbonic acid (H2CO3).Theseweak acids dissociate,yielding H- ions (protons); if these excessprotons were not removed from cells, the cytosolic pH would drop precipitously, endangering cellular functions. Two types of cotransport proteins help remove some of the "excess" protons generatedduring metabolism in animal cells. One is a Na- HCO j- /Cl antiporter, which imports one Na+ ion together with one HCO3-, in exchangefor export of one Cl ion. The cytosolic enzymecarbonic anhydrasecatalyzesdissociation of the imported HCO3- ions into CO2 and an OH- (hydroxyl)ion: HCO.-

;-

CO, + OH

The OH- ions combine with intracellular protons, forming water, and the CO2 diffuses out of the cell. Thus the overall action of this transporter is to consume cytosolic H+ ions, thereby raising the cytosolic pH. Also importanr in raising cytosolic pH is a Na* /H* antiporter, which couplesentry of one Na* ion into the cell down its concenrrationgradient to the export of one H+ ron. Under certain circumstances,the cytosolic pH can rise beyond the normal range of 7 .2-7.5. To cope with the excess OH- ions associatedwith elevated pH, many animal cells utilize an anion antiporter that catalyzesthe one-for-one exchangeof HCO3- and Cl acrossthe plasma membrane.At high pH, this C/ /HCO3- antiporter exports HCO3(which can be viewed as a "complex" of OH and CO2) in exchangefor import of Cl-, thus lowering the cytosolic pH. The import of CI- down its concentration gradient (Cl -.air- ) Cl .r,oror)powers the transport. The activity of all three of these anriport proteins depends on pH, providing cells with a finely tuned mechanismfor controlling the cytosolic pH. The two antiporters that operateto increasecltosolic pH are activated when the pH of the cytosol falls. Similarly, a rise in pH above 7.2 stimulates the CI-iHCO3- antiporter, leading to a more rapid export of HCO3- and decreasein the cytosolic pH. In this manner,the cytosolic pH of growing cells is maintained very closeto pH7.4.

T R A N S M E M B R ATNREA N s p o RoTF t o N s A N D S M A L LM O L E C U L E S

A PutativeCation ExchangeProteinPlaysa Key R o l ei n E v o l u t i o no f H u m a nS k i nP i g m e n t a t i o n Sequencingof the human, mouse, and rat genomesindicates the presenceof hundreds of putative transport proteins, but the functions of most of these are as yet unknown. A particularly interesting human transporter called SLC24A5 emerged from a study of zebrafish that had abnormal skin color; in fish homozygous for the golden mutation, the eponymous black horizontal stripes were very pale (Figure 11-27a and b). Microscopy showed that the mutant fish had a much lower amount of the black pigment called melanin, and melanin vesicles,called melanosomes,were much smaller and paler than normal (Figure 11-27 c and d). Positional cloning of the golden gene demonstrated that it encodesa putative cation exchange protein termed SLC24A5. Immunofluorescence studies showed that the protein is found in intracellular

membranes,likely in the membrane of the melanosomeor its precursor,but the ions transported by SLC24A5 are not yet known. However, the amino acid sequence of the SLAC24A5 protein is closestto that of severalsodium/calcium antiporters, so the protein is likely a sodium/calcium antlporter. Most strikingly, investigators showed that the human version of SLC24A5 is highly similar in sequenceto the zebrafish protein; when the human protein is expressedin mutant golden zebrafish,it complementsthe mutant phenotype and the fish have normal black stripes. The most evolutionarily conservedform of the gene, or allele, most similar to the wild-type zebrafish gene predominates in dark-skinned African and East Asian human populations. In contrast. a version, or variant allele, of the SLC24A5 gene with a single amino acid change that is thought to encode a less active protein is found in virtually all people of European origin. Studies of the allele frequenciesin admixed populations indicate that different forms, or polymorphisms, in iust this cation transporter play a key role in determining the darkness of human skin color' Clearly much needsto be learned about the role of this transporter in cell physiology and how a single point mutation in this gene accountsfor the large differencesin skin pigmentation characteristic of individuals of European, African, and Asian origin.

t r o t e i n sE n a b l eP l a n t N u m e r o u sT r a n s p o r P Vacuolesto AccumulateMetabolitesand lons

11-27 Zebratishmutationsin the gene encodingthe A FfGURE causethe golden skin pigment cation exchangerSLC24A5 phenotype.Lateralviewsof adultwild-type(a)andgolden(b) (arrowheads) Scale bars,5 mm Insets showmelanophores zebrafish (inset, thatareon 0 5 mm) Goldenmutantshavemelanophores paler, thannormal andmoretransparent smaller, average fromwildmicrographs of skinmelanophores electron Transmission type(c) andgolden(d)larvaeshowthat goldenskinmelanophores (arrowheads fewer arethinnerandcontarn showedges) l c a lb e a r si n ( c ) a n d( d ) ,1 0 0 0n m l F r o m m e l a n o s o m e s t hnaonr m a S Scrence 310:1 782,1 etal, 2005, R L Lamason

The lumen of plant vacuolesis much more acidic (pH 3-6) than is the cytosol (pH 7.5). The acidity of vacuolesis maintained by a V-classAlP-powered proton pump (seeFigure 1,1,-9)and by a pyrophosphate-poweredpump that is unique to plants. Both of thesepumps, located in the vacuolar membrane, import H* ions into the vacuolar lumen against a concentration gradient. The vacuolar membrane also conchannels that transport these anions tains Cl- and NO: from the cytosol into the vacuole. Entry of these anions against their concentration gradients is driven by the insidepositive potential generated by the H* pumps. The comtined operation of these proton pumps and anion channels producesan inside-positiveelectricpotential of about 20 mV across the vacuolar membrane and also a substantial pH gradient (Figure 11-28). The proton electrochemical gradient across the plant vacuole membrane is usedin much the sameway as the Na* electrochemicalgradient acrossthe animal-cell plasma membrane: to power the selectiveuptake or extrusion of ions and small molecules by various antiporters. In the leaf, for example, excesssucrosegeneratedduring photosynthesisin the day is stored in the vacuole; during the night' the stored sucrose moves into the cytoplasm and is metabolized to CO2 and H2O with concomitant generation of ATP from ADP and P;. A proton/sucrose antiporter in the vacuolar membrane operatesto accumulatesucrosein plant vacuoles.The inward movement of sucrose is powered by the outward movement of H*, which is favored by its concentration C O T R A N S P O RBTY S Y M P O R T E RASN D A N T I P O R T E R S

469

H*-pumping proteins ADP+ P;

Cotransport by Symporters and Antiporters PPi

2Hlon-channel

r Cotransporters use the energy releasedby movement of an ion (usually Ht or Na*) down its electrochemicalgradient to power the import or export of a small molecule or different ion against its concenrrationgradient.

H' + +

P l a n tv a c u o l el u m e n ( p H= 3 - 6 ) Nat

H*

Ca2*

Sucrose

H+

Cytosol ( p H= 7 . 5 )

Protonantiportproteins A FIGURE 11-28 Concentration of ionsand sucroseby the plant vacuole.Thevacuolar membrane contains two typesof protonpumps(orange): (/eft)anda a V-class H* ATpase pyrophosphate-hydrolyzing protonpump(flght)thatdiffersfromall otheriontransport proteins andprobably isuniqueto plantsThese pumpsgenerate a low luminalpHaswellasan inside-positive potential electric across thevacuolar membrane owingto the inward pumpingof H* ions Theinside-positive potential powersthe movement of Cl- andNOr- fromthecytosol throughseparate (purple)Protonantiporters proteins channel (green), powered by the H + g r a d i e nat ,c c u m u l aNt ea + ,C a 2 +a, n ds u c r o si n e s i dteh ev a c u o l e . [ A f t e r P R e aa n d D S a n d e r s1, 9 8 7 ,p h y s i o t p l a n t 7 1 . t 3 1 ; J M M a a t h u i sa n d D S a n d e r s1, 9 9 2 ,C u r r O p i n C e l lB i o l . 4 . 6 6 1 ;a n d p A R e ae t a l , 1 9 9 2 . TrendsBiochem Sci. 17:348 l

gradient (lumen > cytosol) and by the cytosolic-negative potendal acro-ssthe vacuolar membrane (seeFigure 11-28). Uptake of Ca2* and Na+ into the vacuole from the cytosol against their concentration gradients is similarly mediated by proton antiporters. Understandingof the transport proteins in plant vacuolar membraneshas the potential for increasingagriculproduction in high-salt (NaCl) soils, which are found throughout the world. Becausemost agriculturally useful crops cannot grow in such saline soils, agricultural scientists have long sought to develop salt-tolerant plants by traditional breedingmerhods.\With the availability of the cloned gene encoding the vacuolar Na*/H* antiporter, researchers can now produce transgenicplants that overexpressthis transport protein, leading to increasedsequestrationof Na* in the vacuole. For insrance,rransgenlctomato plants that overexpressthe vacuolar Na*/H* antiporter can groq flower, and produce fruit in the presenceof soil NaCl concentrations that kill wild-type plants. Interestingly although the leavesof thesetransgenictomato plants accumulatelarge amounts of salt, the fruit has a very low salt content. I

470

CHAPTER 11

I

r The cells lining the small intestine and kidney tubules express symport proteins that couple the energetically favorable entry of Na+ to the import of glucose and amino acids against their concentration gradients (see Figure 11-25). r The molecular structure of a bacterial Na+-amino acid symporter revealshow binding of Na* and leucineare coupled and provides a snapshot of an occluded transport intermediate in which the bound substratescannot diffuse out of the protein. r In cardiac musclecells,the export of Ca2* is coupled to and powered by the import of Na* by a cation antiporter, which transports 3 Na* ions inward for each Ca2* ion exported. r As judged by mutations in zebrafish and polymor, phisms in humans, the presumed sodium/calcium cotransporter SLC24A5 plays a major role in forming melanin granules and in regulating the darkness of human skin pigmentation. r Two cotransportersthat are activated at low pH help maintain the cytosolic pH in animal cells very close to 7.4 despitemetabolic production of carbonic and lactic acids.One, a Na*/H* antiporter,exports excessprotons. The other, a Na*HCO3-lClcotransporrer, imports HCO3 , which dissociatesin the cytosol to yield pHraising OH- ions. r A CI-/HCO3 antiporter that is activated at high pH functions to export HCO3 when the cytosolic pH rises above normal and causesa decreasein pH. r Uptake of sucrose,Na*, Ca2*, and other substances into plant vacuoles is carried out by proton antiporters in the vacuolar membrane.Ion channelsand proton pumps in the membrane are critical in generatinga large enough proton concentration gradient to power accumulation of ions and metabolites in vacuoles by these proton antiporters (see Figure 11-28).

Transepithel iaI Transport Previous sections illustrated how several types of transporters function together to carry out important cell functions (seeFigure 11,-2).Here,we exrend this concept by focusing on the transport of several types of molecules and ions acrossthe sheetlikelayers of epithelial cells that cover

T R A N S M E M B R A NTER A N S P O ROTF I O N SA N D S M A L LM O L E C U L E 5

most external and internal surfacesof body organs. Like all epithelial cells, an intestinal epithelial cell is said to be polarized becauseits plasma membrane is organized into at least two discreteregions.Typically, the surfacethat facesthe outside of the organism, here the lumen of the intestine,is called the apical, or top, surface, and the surface that faces the inside of the organism is called the basolateral surface (see Figure 19-9). Specializedregions of the epithelial-cellplasma membrane, called cell junctions, connect the cells and provide strength and rigidity to the cell sheet (seeFigure 1,9-9for details). One of these types of cell junctions-the tight junction-is of particular interesthere sincetight junctions prevent many water-solublesubstanceson one side of an epithelium from moving acrossto the other side through the extracellular space betweencells. For this reason, absorption of nutrients from the intestinal lumen into the blood occurs by the two-stage process called transcellwlar transport: import of moleculesthrough the plasma membrane on the apical side of intestinal epithelial cells and their export through the plasma membrane on the bloodfacing (basolateral,or serosal) side (Figure 11-29). The apical portion of the plasmamembrane,which facesthe intestinal lumen, is specialized for absorption of sugars, amino acids, and other moleculesthat are produced from food by various digestive enzymes.Numerous fingerlike projections (100 nm in diameter) called microvilli greatly increasethe area of the apical surfaceand so the number of

2 Na+/glucose symporter

GLUTz Glucose

Glucose

FI

t

2Na

Na

Glucose 2 Na'

Na+76+ ATPase

Apical membrane T i g h tj u n c t i o n Cytosol Low NaH i g hK *

11-29Transcellular transportof glucosefrom the A FIGURE in the intestinallumeninto the blood.TheNa*/K*ATPase generates membrane Nat andK* concentration surface basolateral (step1) Theoutwardmovement gradients of K+ ionsthrough (notshown)generates an inside-negative K* channels nongated gradient potentialBoththe Na* concentration and membrane from potential areusedto drivethe uptakeof glucose the membrane located symporter lumenbythetwo-Na'/one-glucose the intestinal (step2) Glucose leaves thecellvia membrane in the apicalsurface located uniporter a glucose catalyzed by GLUT2, facilitated diffusion (step3) membrane in the basolateral

transport proteins it can contain, enhancingthe cell's absorptive capacitY.

Multiple TransportProteinsAre Neededto Move Glucoseand Amino AcidsAcrossEpithelia Figure 11-29 depicts the proteins that mediate absorption of glucose from the intestinal lumen into the blood and illustrates the important concept that different types of proteins are localizedto the apical and basolateralmembranesof epithelial cells.In the first stageof this process'a two-Na*/oneglucosesymporter located in microvillar membranesimports glucose, against its concentration gradient' from the intestinal lumen acrossthe apical surfaceof the epithelial cells.As noted above, this symporter couples the energeticallyunfavorable inward movement of one glucosemoleculeto the en-

port, are pumped out across the basolateralmembrane, which facesthe underlying tissue.Thus the low intracellular Na* concentration is maintained. The Na*/K* ATPasethat accomplishesthis is found exclusivelyin the basolateral membraneof intestinalepithelialcells' The coordinatedoperation of thesetwo transport proteins allows uphill movement of glucoseand amino acids from the intestine into the cell. This first stage in transcellular transport ultimately is powered by ATP hydrolysis by the Na*/l(- ATPase. In the secondstage,glucoseand amino acidsconcentrated inside intestinal cells by symportersare exported down their concentrationgradientsinto the blood via uniport proteins in the basolateralmembrane.In the caseof glucose,this movement is mediatedby GLUT2 (seeFigure 11-29).As noted earlier, this GLUT isoform has a relatively low affinity for glucosebut increasesits rate of transport substantiallywhen the glucosegradient acrossthe membranerises(seeFigure 11-4). The net result of this two-stage processis movement of Na* ions, glucose,and amino acidsfrom the intestinallumen acrossthe intestinalepithelium into the extracellularmedium that surrounds the basolateralsurfaceof intestinal epithelial cells.Tight junctions betweenthe epithelialcellspreventthese moleculesfrom diffusing back into the intestinal lumen, and eventually they move into the blood. The increasedosmotic pressurecreated by transcellular transport of salt' glucose, and amino acids acrossthe intestinal epithelium draws water from the intestinal lumen into the extracellular medium that surrounds the basolateralsurface.In a sense,salts, glucose, and amino acids "carry" the water along with them.

S i m p l eR e h y d r a t i o nT h e r a p yD e p e n d so n t h e OsmoticGradientCreatedby Absorption of G l u c o s ea n d N a An understandingof osmosisand the intestinal absorption of salt and glucose forms the basis for a simple

TRANSEPITHELIT AR L ANSPORT

471

therapy that savesmillions of lives each year, particularly in Iess-developedcountries. In these countries, cholera and other intestinal pathogens are major causesof death of young children. A toxin released by the bacteria acrivates chloride secretion by the intestinal epithelial cells into the lumen; water follows osmotically, and the resultant massive loss of water causesdiarrhea, dehydration,and ultimately death. A cure demands not only killing the bacteria with antibiotics but also rehydration-replacement of the water that is lost from the blood and other tissues. Simply drinking water does nor help, becauseit is excretedfrom the gastrointestinal tract almost as soon as it enters. Howeve! as we have just learned. the coordinated transport of glucoseand Na* acrossthe inrestinalepithelium creates a transepirhelial osmotic gradient, forcing movement of water from the intestinal lumen acrossthe cell layer and ultimately into the blood. Thus giving affected children a solution of sugarand salt to drink (but not sugar or salt alone)causesthe osmoticflow of water into the blood from the intestinallumen and leadsto rehydration.Similar sugar-saltsolu ns are the basisof popular drinks used by athletesto get gar as well as water into the body quickly and efficiently.

Cl /HCO3 antiporter at-

HC03-

Cl- channel

CI HCO3-

K+ channel

J.",oon'" anhvdrase I

coz Basolateral memorane

2

I + OH co, v o tl

x2

"

l,-- NADH+ H+ LactateI

dehydrogenase N

cH3-cH

Acetaldchyde

| \> NAD* v oHo

MITOCHONDRION

ttl

cH3-cH-C-OH Lastlc acid

NADH + H+

coz

NAD'

x2

CoA-SH

Pyruvate d e h y dr o g e n a s e

NAD+ NADH

Overallreactionsof anaerobicmetabolism: G l u c o s+e 2 A D P+ 2 P , ----> 2 ethanol+ 2 CO2+2 ATP+ 2 H2O G l u c o s e + 2 A D P +P 2 , ---> 2 lactate + 2 ATP+ 2 H1O NADH

C i t r i ca c i d cycle

NAD+

Oxidative phosphorylation

-28 ADP + -28 Pi -28 AfP + -28 H2O

A FIGURE 12-5 Anaerobicversusaerobicmetabolismof glucose.The ultimatefateof pyruvate formedduringglycolysis depends on the presence or absence of oxygen. In theformation of pyruvate fromglucose, onemolecule (byaddition of NAD+isreduced of two electrons) to NADHfor eachmolecule of pyruvate formed(seeFigure12-3, reaction6) (a)In the absence of oxygen, two electrons aretransferred from eachNADHmolecule to an acceptor molecule to regenerate NAD+,whichis required for continued glycolysis. In yeasts(/eft),acetaldehyde isthe electronacceptorandethanolisthe product. Thisprocess iscalledalcoholic fermentationwhen oxygenis limitingin muscle cells(nght),NADHreduces pyruvate to formlacticacid,regenerating NAD+. (b) In the presence of oxygen,pyruvateistransported into mitochondriaFirstit isconverted by pyruvate dehydrogenase intoonemolecule of CO2andone of aceticacid,the latterlinkedto coenzyme A (CoA-SH) to form acetylCoA, concomitant with reduction of onemolecule of NAD+to NADH.Further metabolism of acetylcoA andNADHgenerates approximately an additional 28 molecules of ATPperglucose molecule oxidized. 484

C H A P T E R1 2

I

CELLULAR ENERGETICS

2CO2 Overallreactionof aerobicmetabolism: Glucose+ 6 02 + -30 ADP + -30 Pi ----> 6 CO2+ 36 H2O+ -30 ATP

the polymer glycogen,a storageform of glucose(Chapter 2), directly to glucose 6-phosphate (without involvement of hexokinase,step [). Under theseconditions, there is a reduction in fructose 2,6-bisphosphatelevels and decreased phosphofructokinase-1activity (Figure 12-4). As a result, glucose6-phosphatederived from glycogenis not metabolized to pyruvate; rather, it is converted to glucose by a phosphataseand releasedinto the blood to nourish the brain and red blood cells,which dependprimarily on glucose as an energy fuel. In all cases,the activity of these regulated enzymesis controlled by the level of small-molecule metabolites, generally by allosteric interactions, or by hormone-mediated phosphorylation and dephosphorylation reactions (Chapter 15 gives a more detailed discussion of hormonal control of glucose metabolism in liver and muscle).

G l u c o s el s F e r m e n t e dU n d e r A n a e r o b i cC o n d i t i o n s Many eukaryotesare obligate aerobes:they grow only in the presenceof molecular oxygen and metabolizeglucose(or related sugars)completely to CO2, with the concomitant production of a large amount of ATP. Most eukaryotes, however, can generate some ATP by anaerobic metabolism. A few eukaryotesare facubatiue anaerobes:they grow in either the presenceor the absenceof oxygen. For example, annelids,mollusks,and someyeastscan live and grow for days without oxygen. In the absenceof oxygen, yeasts convert the pyruvate produced by glycolysisto one molecule each of ethanol and CO2; in these reactions two NADH molecules are oxidized to NAD* for each two pyruvates converted to ethanol, thereby regeneratingthe supply of NAD- (Figure 12-5a, left). This anaerobic degradation of glucose, called fermentation, is the basis of beer and wine production. Oxygen deprivation can also affect glucosemetabolism in animals. During prolonged contraction of mammalian skeletal muscle cells-for example, during exercise-oxygen within the muscle tissue can becomelimited and glucosecatabolism is limited to glycolysis(stageI). As a consequence, muscle cells convert the pyruvate from glycolysis to two molecules of lactic acid by a reduction reaction that also oxidizes two NADHs to NAD*s (Figure L2-5a, right). Although the lactic acid is releasedfrom the muscle into the blood, if the contractions are sufficiently rapid and strong, the lactic acid can transiently accumulate in that tissue and contribute to muscle and joint pain during exercise.Once it is secretedinto the blood, some of the lactic acid passesinto the liver, where it is reoxidized to pyruvate and either further metabolizedto C02 aerobically or converted back to glucose.Much lactate is metabolized to CO2 by the heart, which is highly perfused by blood and can continue aerobic metabolism at times when exercising, oxygen-poor skeletal muscles secretelactate. Lactic acid bacteria (the organisms that spoil milk) and other prokaryotes also generateATP by the fermentation of glucoseto lactate.

, itochondria U n d e rA e r o b i cC o n d i t i o n sM EfficientlyOxidizePyruvateand Generate ATP(Stagesll-lv) In the presenceof oxygen, pyruvate formed by glycolysis is transported into mitochondria, where it is oxidized by 02 to CO2 and H2O via a seriesof oxidation reactions. The overall process by which cells use 02 and produce COz is collectively termed cellular respiration (Figure 12-5b). Reactions in the mitochondria (stagesII-IV) generatean estimated 28 additional ATP molecules per original glucose molecule, far outstripping the ATP yield from anaerobic glucosemetabolism. Oxygen-producing photosynthetic cyanobacteria appeared about 2.7 billion years ago' The subsequentbuildup in the earth's atmosphere of sufficient oxygen during the next approximately billion years opened the way for organisms to evolve the very efficient aerobic oxidation pathway, which in turn permitted the evolution, especially during what is called the Cambrian explosion' of large and complex body forms and associatedmetabolic activities. In effect, mitochondria are ATP-generating factories, taking full advantageof this plentiful oxygen. We first describe their structure and then the reactions they employ to degrade pyruvate.

M i t o c h o n d r i aA r e D y n a m i cO r g a n e l l e s with Two Structurallyand Functionally D i s t i n c tM e m b r a n e s Mitochondria (Figure 12-6) are among the larger organelles in the cell. A mitochondrion is about the size of an E. coli bacterium, which is not surprising, becausebacteria are thought to be the evolutionary precursorsof mitochondria (see Chapter 6 and the discussionof endosymbiont hypothesis, below). Most eukaryotic cells contain many mitochondria, collectively occupying as much as 25 percent of the volume of the cytoplasm. The numbers of mitochondria in a cell, hundreds to thousandsin mammalian cells, are regulated to match the cell's requirements for ATP (e.g., stomach cells' which use a lot of ATP for acid secretion,have many mitochondria). Analysis of fluorescentlylabeled mitochondria in living cellshas shown that mitochondria are highly dynamic. They undergo frequent fusions and fissions that generate tubular, sometimesbranched networks (Figure 1'2-7),which may account for the wide variety of mitochondrial morphologiesseenin different types ofcells. Fusionsand fissions apparently play a functional role as well becausegeneticdisruptions in GTPasesuperfamily genesrequired for thesedynamic processescan disrupt function, such as maintenance of proper inner membrane electrical potential, and cause human disease,such as the neuromuscular diseaseCharcotMarie-Tooth subtype 2A. The details of mitochondrial structure can be observed with electron microscopy (seeFigure 9-8). Mitochondria have two distinct kinds of concentricallyrelated membranes. The outer membrane definesthe smooth outer perimeter of the mitochondrion. The inner membrane has numerous

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E A N D F A T T YA C I D C A T A B O L I S MG F T R SS T T E P SO F G L U C O S E

Video: Mitochondrion Reconstructedby ElectronTomography (b) F6F1complexes Intermembrane space C r i s t a ej u n c t i o n s

FIGURE 12-6 Internalstructureof a mitochondrion. ( a )S c h e m a tdi ica g r a m s h o w i ntgh ep r i n c i p m a le m b r a n a en sd compartments Thecristae formsheets andtubesby invagination o f t h ei n n e rm e m b r a naen dc o n n e ct o t t h ei n n e m r embrane throughrelatively smalluniformtubularstructures calledcnsta junctionsTheintermembrane spaceappears continuous with the lumenof eachcristaTheFeF,complexes (smallredspheres), whichsynthesize ATP, areintramembrane particles thatprotrude fromthecristae andinnermembrane intothe matrixThematrix contains the mitochondrial (small DNA(bluestrand), ribosomes

Video: MitochondrialFusionand Fission (d

E X P E R I M E N TFA L U RtE2 - 7 M i t o c h o n d r iuan d e r g o IG rapidfusionand fissionin living cells.Mitochondria labeled with a fluorescent proteinin a livingnormalmurineemoryontc fibroblast wereobserved usingtime-lapse fluorescence microscopy Several mitochondria undergoing fusion(top)or (bottom)areartificially jn blueandwith fission highllghted arrowslModified fromD C Chan, 2006,Cell125(])j241-12521

486

c H A P T E R1 2

|

CELLULAE RN E R G E T T C S

(largeyellowspheres) (b)Computerbluespheres), andgranules generated modelof a section of a mitochondrion fromchicken brain. Thismodelisbased on a three-dimensional electron microscopic image calculated froma series of two-dimensional electron micrographs recorded at regularintervals Thistechnique isanalogous to a threedimensional x-raytomogram or CATscanusedin medical imaging (yellow-green), Notethetightlypacked cristae the innermembrane (lightblue),andtheoutermembrane (darkblue)[part(a)courtesy of T. Frey;part (b) from T. Freyand C Mannella,2000, TrendsBiochem Sci 25:319l

invaginationscalled cristae (seeFigure 12-6). These membranes topologically define two submitochondrial compartments: the intermembrane space, between the outer and inner membranes,and the matrix, or central compartment, which forms the lumen within the inner membrane.'When individual mitochondria fuse, each of their distinct comparrments intermixes (e.g.,matrix with matrix, inner membrane with inner membrane). Fractionation and purification of these membranes and compartments have made it possible to determine their protein, DNA, and phospholipid compositions and to localize each enzyme-catalyzedreaction to a specific membrane or comparrment. About 1000 polypeptides are required to make and maintain mitochondria and permit them to function. Only a small number of these-13 in humans-are encoded by mitochondrial DNA genes, while the remaining proteins are encoded by nuclear genes ( C h a p t e r6 ) . The most abundant protein in the outer membrane is mitochondrial porin, a transmembrane channel protein similar in structure to bacterial porins (seeFigure 10-1S). Ions and most small molecules(up to about 5000 Da) can

readily pass through these channel proteins when they are open. Although there may be metabolic regulation of the opening of mitochondrial porins and thus the flow of metabolites across the outer membrane, the inner membrane with its cristae are the major permeability barriers between the cytosol and the mitochondrial matrix, limiting the rate of mitochondrial oxidation. Protein constitutes 76 percent of the total weight of the inner membrane-a higher fraction than in any other cellular membrane. Many of these proteins are key participants in cellular respiration. They include ATP synthase, proteins responsiblefor electron transport, and a wide variety of transport proteins that permit the movement of metabolites between the cytosol and the mitochondrial matrix. The human genomeencodes48 membersof a family of mitochondrial transport proteins. One of these is called the ADP/ATP carrier, an antiporter that moves newly synthesizedATP out of the matrix and into the inner membrane space(and subsequentlythe cytosol) in ex'!flithout this changefor ADP originating from the cytosol. essential antiporter, the energy trapped in the chemical bonds in mitochondrial ATP would not be availableto the rest of the cell. The invaginating cristae greatly expand the surface area of the inner mitochondrial membrane (see Figure 12-6), enhancing its capacity to generateATP. In typical liver mitochondria, for example,the area of the inner membrane, including cristae, is about five times that of the outer membrane. In fact, the total area of all inner mitochondrial membranes in liver cells is about 1,7 times that of the plasma membrane. The mitochondria in heart and skeletal muscles contain three times as many cristae as are found in typical liver mitochondria-presumably reflecting the greater demand for AIP by muscle cells. Note that plants have mitochondria and perform cellular respiration as well. In plants, stored carbohydrates,mostly in the form of starch, are hydrolyzed to glucose.Glycolysis then produces pyruvate, which is transported into mitochondria, as in animal cells. Mitochondrial oxidation of pyruvate and concomitant formation of ATP occur in photosynthetic cells during dark periods when photosynthesisis not possible and in roots and other nonphotosynthetic tissuesat all times. The mitochondrial inner membrane, cristae, and matrix are the sites of most reactions involving the oxidation of pyruvate and fatty acids to CO2 and H2O and the coupled synthesisof ATP from ADP and P1, with each reaction occurring in a discretemembrane or spacein the mitochondrion (Figure12-8). The last three of the four stagesof glucoseoxidation are r StageII. Conversionof pyruvate to acetyl CoA, followed by oxidation to CO2 in the citric acid cycle.Theseoxidations are coupled to reduction of NAD* to NADH and of FAD to FADH2. (Fatty acid oxidation follows a similar route, with conversion of fatty acyl CoA to acetyl CoA.) Most of the reactionsoccur in or on the membrane facing the matrIX.

r StageIII. Electron transfer from NADH and FADH2 to 02 via an electron transport chain within the inner membrane, which generatesa proton-motive force acrossthat membrane. r StageIV. Harnessingthe energy of the proton-motive force for ATP synthesisin the mitochondrial inner membrane. StagesIII and IV are together called oxidative phosphorylation.

In Stagell, Pyruvatels Oxidizedto CO2 and High-EnergyElectronsStored in ReducedCoenzymes Pyruvate formed during glycolysis in stage I in the cytosol is transportedinto the mitochondrial matrix (Figure 12-8). StageII metabolism accomplishesthree things: (1) it converts the 3-carbon pyruvate to three moleculesof CO1' Q) it generates high-energy electron carriers (NADH and FADH2) that will be used for electrontransport (stageIII); and (3) it generatesa GTP molecule, which is then converted to ATP: GTP + ADP i-

GDP + AIP

StageII can be subdivided into two distinct parts: (1) the generationof acetyl CoA plus one molecule of COz and one NADH and (2\ the conversion of acetyl CoA to two molecules of CO2 and the high-energy intermediatesNADH (3 molecules),FADH2, and GTP. Generation of Acetyl CoA \il/ithin the mitochondrial matrix, pyruvate reactswith coenzymeA, forming CO2 and acetyl CoA and NADH (Figure 12-8). This reaction, catalyzedby pyruuate - 8.0 kcaUmol)and dehydrogena.sais highly exergonic(AG"' : essentiallyirreversible. Acetyl CoA (Figure 1'2-9) plays a central role in the oxidation of fatty acids and amino acids.In addition, it is an intermediate in numerous biosynthetic reactions' including transfer of an acetyl group to histone proteins and many mammalian proteins, and synthesisof lipids such as cholesterol. In respiring mitochondria, however' the acetyl group of acetyl CoA is almost always oxidized to CO2 via the citric acid cycle. Citric Acid Cycle Nine sequentialreactions operate in a cycle to oxidize acetyl CoA to CO2. The cycle is referred to by severalnames:the citric acid cycle, the tricarboxylic acid (or TCA) cycle, and the Krebs cycle. The net result is that for each acetyl group entering the cycle as acetyl CoA, two moleculesof CO2, three of NADH' and one each of FADH2 and GTP are produced. As shown in Figure 12-1'0,the cycle begins with condensation of the two-carbon acetyl group from acetyl CoA with the four-carbon molecule oxaloacetate to yield the six-carboncitric acid (hencethe name citric acid cycle).In both reactions 4 and 5, a CO2 molecule is releasedand NAD+ is reduced to NADH. Reduction of NAD- to NADH also occurs during reaction 9; thus three NADHs

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E F T R SS T T E p SO F G L U C o 5 EA N D F A T T YA C I D C A T A B O L I S MG

487

Outer mitochondrial membrane {permeable to metabolites)

coz

Intermembrane space

StageI Glucose

2 NAD*-J I 2 NADH.f" z efP J 2 Pyruvate -

Pyruvate

-----?2COt

Acetyl CoA

C i t r i ca c i d cycre

Mitochondrlal matrix

Succinate

2 e- + 2H* + !Or---+ Hrg Fumarate

Qz

Hzo

FoF,complex

A FIGURE 12-8 Summaryof aerobicoxidationof glucoseand fatty acids.Stagel: Inthecytosol, glucose isconverted to pyruvate (glycolysis) andfattyacidto fattyarylCoA hTruvate andfattyacylCoA thenmoveintothemitochondrion Mitochondrial porins maketheouter membrane permeable to thesemetabolites, butspecific rranspon (colored proteins ovals) in theinnermembrane arerequired to import pyruvate (yellow) andfattyacids(blue)intothematrixFattyacyl groupsaretransferred fromfattyarylCoAto an intermediate carriel transported across (blueoval),andthenreattached the innermembrane to CoAon thematrixsideStagell: Inthemitochondrial matrix, pyruvate andfattyacylCoAareconverted to acetylCoAandthen oxidized, releasing CO2Pyruvate isconverted to acetylCoAwiththe formation of NADHandCOr;two carbons fromfattyacylCoAare convefted to acetylCoAwiththeformation of FADH,andNADH Oxidation of acetylCoAin thecitricacidcycleqenerates NADHand

FADH2, GTBandCO2.Stagelll: Electron transport reduces oxygen to waterandgenerates (blue)from a proton-motive force.Electrons reduced coenzymes aretransferred viaelectron-transport complexes (blueboxes) to 02 concomitant withtransport of H* ions(red)fromthe matrixto theintermembrane generating space, theproton-motive force Electrons fromNADHflowdirectly fromcomplex I to complex lll, bypassing complex ll Electrons fromFADH, flowdirectly fromcomplex ll to complex lll,bypassing complex I StagelV:ATPsynthase, the FoFl (orange), complex harnesses the proton-motive forceto synthesize ATp (purpleandgreenovals) rnthematrix,Antiporter proteins transport ADp andP;intothematrixandexporlhydroxyl groupsandATpNADH generated in thecytosol isnottransported directly to thematnxoecause theinnermembrane rsimpermeable to NAD+andNADH;instead, a (red)transports shuttlesystem electrons fromcytosolic NADHto NAD+in the matrix02 diffuses intothematrix,andCOrdiffuses our

are generatedper turn of the cycle.In reaction 7, two electrons and two protons are transferredto FAD, yielding the reducedform of this coenzyme,FADH2. Reaction 7 is distinctive becauseit not only is an intrinsic part of the citric acid cycle (stageII), but also it is caralyzedty a membrane-

attached enzyme that is an intrinsic part of the electron transport chain (stageIII). In reaction 6, hydrolysis of the high-energythioester bond in succinyl CoA is coupled to synthesisof one GTP by subsrrate-levelphosphorylation. (BecauseGTP and ATP are interconvertible,this can be

Coenzyme A (CoA) a F I G U R E l 2 ' 9T h e s t r u c t u r e o fa c e t y lC o A . T h j s c o m p o u n d i s an importantintermediatein the aerobicoxidationof pyruvate,fatty

488

c H A P T E R1 2

|

CELLULAE RN E R G E T T C S

a c i d s , a n d m a n y a m i n o a c il d t asl.s o c o n t r i b u t e s a c eg tr yolu p s i n many biosynthetic pathways

N A D H+ H HrO

coo I nu

tc-coo

HO-C-H

tl HC I

I

V /

QH, t-

coo-

iaa

vvv

crs-Aconitate

Malate

I Hro\tl

coo-

coo.1.,, UN

I CH"

t-

coo-

HC

I coo-

Z

Fumarate

//

,r Y

FADH,

t-

CH, I'

QH,

lC H "= FA

c:o I coo-

t-' coo

Succinate

H-C-COO

I nu

cooI

GDP + Pi + H2O

GTP + HSCoA

t

I

E

HO-C-H

/l

coolsocitrate

4-Keto-

glutarate C O r + N A D H+ H '

+ N A D H+ H -

12-10The citricacidcycle.AcetylCoAis metabolized a FIGURE In NADHandFADH2. electron carriers to C02andthe high-energy fromacetylCoAcondenses acetylresidue 1, a two-carbon reaction to formthe six-carbon molecule oxaloacetate with the four-carbon (2-9)eachmolecule of citrate reactions citrate.In the remaining losingtwo C02 backto oxaloacetate, converted iseventually In eachturn of the cycle,four pairsof in the process. molecules fromcarbonatoms,forminqthreemolecules areremoved electrons

of GTP andonemolecule of FADH2, of NADH,onemolecule Thetwo carbonatomsthatenterthe cyclewith acetylCoAare y lo A I n s u c c t n aat en d h i g h l i g h t ei ndb l u et h r o u g hs u c c i n C theycanno longer molecules, whicharesymmetric fumarate, haveshownthat studies lsotope-labeling denoted. be specifically thesecarbonatomsarenot lostin theturn of the cyclein which onewill be lostasC02duringthe next theyenter;on average, turns. the otherin subsequent and turn of the cycle

consideredan ATP-generatingstep.) Reaction 9 regenerates oxaloacetate,so the cycle can begin again. Note that molecular 02 does not participatein the citric acid cycle. Most enzymesand small moleculesinvolved in the citric acid cycle are soluble in the aqueousmitochondrial matrix. Theseinclude CoA, acetylCoA, succinylCoA, NAD*, and NADH, as well as most of the eight cycle enzymes. Succinate dehydrogenase(reaction 7), however, is a component of an integral membraneprotein in the inner membrane,with its active site facing the matrix. When mitochondria are disrupted by gentle ultrasonic vibration or osmotic lysis, non-membrane-boundenzymesin the citric acid cycle are releasedas very large multiprotein complexes.Within such complexes the reaction product of one enzyme is thought to pass directly to the next enzymewithout diffusing through the solution. However,much work is neededto determine the structuresof these large enzyme complexes as they exist in the cell. Since glycolysis of one glucose molecule generatestwo acetyl CoA molecules, the reactions in the glycolytic pathway and citric acid cycle produce six CO2 molecules, 10 NADH molecules, and two FADH2 molecules per glucose molecule(Table 12-1). Although thesereactionsalso generate four high-energyphosphoanhydridebonds in the form of

two ATP and two GTP molecules' this represents only a small fraction of the available energy releasedin the complete aerobic oxidation of glucose.The remaining energy is itored as high-energy electrons in the reduced coenzymes NADH and FADH2. The goal of stagesIII and IV is to recover this energy in the form of ATP.

T r a n s p o r t e risn t h e I n n e r M i t o c h o n d r i a l MembraneHelp Maintain AppropriateCytosolic and Matrix Concentrationsof NAD* and NADH In the cytosol NAD+ is required for step 6 of glycolysis (see Figure 12-3), and in the mitochondrial matrix NAD+ is reqrri..d for conversion of pyruvate to acetyl CoA and for th... tt.pt in the citric acid cycle (4, 5, and 9 in Figure 12-

FADH2 to FAD as it reduces02 to water and converts the energy stored in the high-energy electrons in the reduced

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E A N D F A T T YA C I D C A T A B O L I S MG F I R S Ts T E P SO F G L U C O S E

489

c02M0ltcljlts PBODUCED

fltAcTt0N

NAD+ Ml)LECULES FAD M{]TECULES REOUCED TONADH REDUCED T0tADH2

(OR ATP GTP)

L glucose molecule to 2 pyruvate molecules

0

2

0

z

2 pyruvates to 2 acetyl CoA molecules

a L

z

0

0

2 acetyl CoA to 4 CO2 molecules

4

t)

z

2

Total

5

10

2

+

forms of these molecules into a proton-motive force. Even though 02 is not involved in any reaction of the citric acid cycle, in the absenceof 02, this cycle soon stops operating as the intramitochondrial suppliesof NAD* and FAD dwindle due to the inability of the electron transport chain to oxidize NADH and FADH2. These observations raise the question of how a supply of NAD+ in the cytosol is regenerated. If the NADH from the cytosol could move into the mitochondrial matrix and be oxidized by the electron transport chain and if the NAD+ product could be transported back into the cytosol, regenerationof cytosolic NAD+ would be

Cytosol

,

simple. However, the inner mitochondrial membrane is impermeableto NADH. To bypassthis problem and permit the electrons from cytosolic NADH to be transfer red indirectly to 02 via the electron transport chain, cells use severalelectron shuttles to transfer electrons from cytosolic NADH to NAD+ in the matrix. Operation of the most widespreadshuttle-the malate-aspartateshwttle-is depicted in Figure 12-11. For every complete "turn" of the cycle,there is no overall change in the numbers of NADH and NAD* moleculesor the intermediatesaspartateor malate used by the shuttle.However, in the cytosol, NADH is oxidized to NAD+, which can be used

NADHcytosot NAD*cytosol

oxaroacetate \E/

Aspartate

, t",.,"

( \ - K e t olgu t a r a t eG l u t a m a t e Gluta

Mitochondrial inner membrane Glutamate

_->-_)

r r - K e t o g l u t ar a t e G l u t a m a t e

I Aspartate

\q/

FIGURE 12-11The malateshuttle.Thiscyclical series of reactions transfers electrons fromNADHin thecytosol (intermembrane space) a c r o stsh e i n n e rm i t o c h o n d r m i ael m b r a n w e ,h i c hi s i m p e r m e a b l e to NADHitself,to NAD* in the matrixThenet resultisthe replacement of cytosolic NADHwith NAD+and matrixNAD+ with NADHStepll: Cytosolic malatedehydrogenase transfers electrons fromcytosolic NADHto oxaloacetate, formingmalate. S t e pf , l : A n a n t i p o r t e( b r l u eo v a l )i n t h e i n n e rm i t o c h o n d r i a l m e m b r a nter a n s p o r m t sa l a t ei n t ot h e m a t r i xi n e x c h a n gf e or cr-ketogIutarate. StepS: MitochondriaI malatedehydrogenase converts malatebackto oxaloacetate, reducing NAD+in the matrix to NADHin the processStepE: Oxaloacetate. whichcannot CHAPTER 12

I

CELLULAR ENERGETICS

,,"nuY,1ll';,"."',

Oxaloacetate €-----------

Matrix

490

o - K e t o gI u t ar a t e

/E\

Malare

NADHmatrix NAD*m"tri"

directly crossthe innermembrane, isconverted to aspartate by additionof an aminogroupfromglutamateInthistransaminasecatalyzed reaction in the matrix,glutamate is converted to cr-ketoglutarate (redoval)exports Step[: A secondantrporter aspartate to the cytosolin exchange for glutamateStep@: A cytosoltc transamrnase converts aspartate to oxaloacetate and ct-ketoglutarate to glutamate, completing the cycleTheblue arrowsreflectthe movement of the cr-ketoglutarate, the red arrowsthe movement of glutamate, andthe blackarrowsthat of aspartate/malate lt is noteworthy that,asaspartate and malatecycleclockwise, glutamate andcr-ketoglutarate cyclein the oppositedirection

for glycolysis,and in the matrix, NAD* is reducedto NADH, which can be usedto generateATP via stagesIII and IV * NADH-.1'1" NADH.yrorot + NAD;".. * --+NAD.*y,oro1

MitochondrialOxidation of Fatty Acids GeneratesATP

o R-C

Up to now, we have focusedmainly on the oxidation of carbohydrates,namely glucose,for ATP generation.Fatty acids are another important source of cellular energy. Cells can take up either glucose or fatty acids from the extracellular spacewith the help of specifictransporter proteins (Chapter 11). Should a cell not needto immediatelyburn thesemolecules,it can store them as a polymer of glucosecalled glycogen (especiallyin muscleor liver) or as a trimer of fatty acids covalently linked to glycerol, called a triacylglycerol or triglyceride. In some cells, excessglucose is converted into fatty acids and then triacylglycerols for storage. However, unlike microorganisms, animals are unable to convert fatty acids to glucose.lWhen the cells need to burn these energy storesto make ATP (e.g.,when a resting muscle beginsto do work), enzymesbreak down glycogen to glucose or hydrolyze triacylglycerols to fatty acids, which are then oxidized to generateATP:

o cH3-(CH2)"-C- O-CH2 ^l

YI

* 3 Hro +

cH3-(cH2),-c-o-cH

oxidation. The differenceslie in the cytosolic stageI and the first part of the mitochondrial stageII. In stageI, fatty acids are convertedto a fatty acyl CoA in the cytosol in a reaction coupled to the hydrolysis of ATP to AMP and PPl (inorganic pyrophosphate)(seeFigure 12-8):

O- + HSCoA + ATP ------->

Fatty acid

o R-C-SCOA+AMP+PPI Fatty acyl CoA

Subsequenthydrolysis of PP1to two molecules of P; drives this reaction to completion. To transfer the fatty acyl group into the mitochondrial matrix, it is covalently transferred to a molecule called carnitine, moved across the inner mitochondrial membrane by an acylcarnitine transporter protein (seeFigure 12-8' blue oval), and then on th; matrix side, the fatty acyl group is released from carnitine and reattached to another CoA molecule' The activity of the acyl carnitine transporter is regulated to prevent oxidation of fatty acids when cells have adequate energy (ATP) supPlies. In the first part of stage II' each molecule of a fatty acyl CoA in the mitochondrion is oxidized in a cyclical sequence of four reactionsin which all the carbon atoms are converted two at a time to acetyl CoA with generation of FADH2 and NADH (Figure 1,2-1'2a).For example, mitochondrial oxidation of each molecule of the 18-carbon stearic acid'

?l

cH3- (cH2)"-c-o-cH2

HO-CH2

Triacylglycerol O

HO-CH

l

3 CH3-(CH2)"-C-OH + HO-CH2 Fatty acid

GlYcerol

Fatty acidsare the major energysourcefor many tissues, particularly adult heart muscle.In humans,in fact, the oxidation of fats is quantitatively more important than the oxidation of glucose as a source of ATP. The oxidation of 1 g of triacylglyceride to CO2 generatesabout six times as much AIP as does the oxidation of 1 g of hydrated glycogen. Thus triglycerides are more efficient than carbohydrates for storage of energy,in part becausethey are stored in anhydrous form and can yield more energy when oxidized and also becausethey are intrinsically more reduced (have more hydrogens)than carbohydrates.In mammals, the primary site of storageof triacylglyceridesis fat (adipose)tissue,whereasthe primary sitesfor glycogenstorage are muscle and the liver. Just as there are four stagesin the oxidation of glucose, there are four stagesin the oxidation of fatty acids.To optimize the efficiency of ATP generation,part of stageII (citric acid cycle oxidation of acetyl CoA) and all of stagesIII and IV of fatty acid oxidation are identical to those of glucose

section, the reduced NADH and FADH2 with their highenergyelectronsfrom stageII will be used in stageIII to generct; a proton-motive force that in turn is used in stageIV to power ATP synthesis.

PeroxisomalOxidation of Fatty Acids GeneratesNo ATP Mitochondrial oxidation of fatty acids is the major sourceof ATP in mammalian liver cells, and biochemistsat one time believedthis was true in all cell types. However, rats treated with clofibrate, a drug that affectsmany featuresof lipid metabolism, were found to exhibit an increasedrate of fatty acid oxidation and a large increasein the number of peroxisomes in their liver cells.This finding suggestedthat peroxisomes'as well as mitochondri a, can oxidize fatty acids' These small or-

carbonsin the fatty acyl chain, or (Cs)' medium-(C3-Crz),

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E F I R S TS T E P SO F G L U C O s EA N D F A T T YA C I D C A T A B O L I S MG

491

> FIGURE12-12 Oxidation of fatty acids in mitochondria and peroxisomes.In both mitochondrial oxidation(a)and peroxisomal oxidation(b),fatty acidsare convertedto acetylCoA by a seriesof four enzyme-catalyzed reactions(showndown the centerof the fioure) A fatty acylCoA moleculeis convertedto u..tyl CoA and a fatty acylCoA shortenedby two carbonatoms Concomitantly, one FADmolecule is reducedto FADH2and one NAD* molecule is reducedto NADH The cycleis repeatedon the shortenedacylCoA untilfatty acidswith an evennumberof carbonatomsare completelv converted to acetylCoA In mitochondrra, electrons from FADH2and NADHenterthe electrontransport chainand ultimately are usedto generateATp;the acetylCoA generatedisoxidizedrn the citricacid cycle,resulting in release of CO2and ultimately the synthesis of additionalATP Becauseperoxisomes lackthe electrontransportcomplexescomposlng the electrontransportchainand the enzymesof the citricacidcycle,oxidationof fatty acidsin these organellesyieldsno ATPlAdapted fromD L Netson principlesof andl\,4M Cox,Lehninger Biochemistry,3d ed , 2000,WorthPublishers l

( a ) M I T O C H O N D R I AOLX t D A T | O N (b)PEROXTSOMAL OXtDATION

o R- CH2-CH2-CHr-C-SCoA Fatty acyl CoA

o

H r O+ ' t / z O ,

il

R- CH2-CH: CH-C-SCoA

HrO

NADH exportedfor reoxidation

R-CHr-C-SCoA I

o

Acyl CoA shortened by two carbon atoms +

o Citricacid +cycre

l

H3C-C-SCoA Acetyl CoA

Acetyl CoA exported

stead it is transported into the cytosol for use in the synthesis of cholesterol(Chapter10) and other metabolites.

oxidation of fatty acids, which is coupled to generation of ATP, peroxisomal oxidation of fatty acids is not linked to ATP formation, and energy is releasedas heat. The reaction pathway by which fatty acids are degraded to acetyl CoA in peroxisomesis similar to that used in mito_

dases, peroxisomes contain abundant catalase, which quickly decomposesthe H2O2, a highly cytotoxic metabo_ lite. NADH produced during oxidation of fatty acids is exported and reoxidized in the cytosol; there is no need for a malate/aspartateshuttle here. peroxisomesalso lack the cit_ ric acid cycle, so acetyl CoA generatedduring peroxisomal degradation of fatty acids cannot be oxidized further: in492

c H A P T E R1 2

|

cELLULAR ENERGETTCS

First Steps of Glucoseand Fatty Acid Catabolism: Glycolysisand the Citric Acid Cycle r In a processknown as aerobicoxidation, cellsconvert the energy releasedby the oxidation ("burning") of glucoseor fatty acidsinto the terminal phosphoanhydridebond of ATp. r The complete aerobic oxidation of each molecule of glucoseproduces six moleculesof CO2 and approximately 30 ATP molecules.The entire process,which starts in the cytosol and moves into the mitochondrion, can be divided into four stages:(I) glycolysis to pyruvate in the cytosol, (II) pyruvate oxidation to C02 in the mitochondrion. (III) electron transport to generatea proton-motive force together with conversion of molecular oxygen to wate! and (IV) ATP synthesis. r The mitochondrion hastwo distinctmembranes(outerand inner) and two distinct subcompartments(intermembrane

space between the two membranesand the matrix surrounded by the inner membrane).Aerobic oxidation occurs in the mitochondrial matrix and on the inner mitochondrial membrane. r Each turn of the citric acid cycle releasestwo molecules of CO2 and generatesthree NADH molecules,one FADH2 molecule. and one GTP. r In glycolysis (stageI), cytosolic enzymesconvert glucose to two moleculesof pyruvate and generatetwo molecules each of NADH and ATP. r The rate of glucoseoxidation via glycolysisand the citric acid cycle is regulated by the inhibition or stimulation of severalenzymes,dependingon the cell'sneed for ATP. Glucoseis stored (asglycogenor fat) when ATP is abundant. r Some of the energy releasedin the early stagesof oxidation is temporarily stored in the reducedcoenzymesNADH or FADH2, which carry high-energy electrons that subsequently drive the electron transport chain (stageIII). r In the absenceof oxygen (anaerobicconditions),cellscan metabolize pyruvate to lactate or (in the case of yeast) to ethanol and CO2, in the processconvertingNADH back to NAD*, which is necessaryfor continued glycolysis.In aerobic conditions (presenceof oxygen),pyruvate is transported into the mitochondrion, where stagesII through IV occur.

chain, also known as the respiratorychain into the protonmotive force.'We first describethe logic and componentsof the electron transport chain and the pumping of protons acrossthe mitochondrial inner membrane.'Weconcludethe sectionwith a discussion of the magnitude of the proton-motive force produced by electron transport and proton pumping. In the following section,we describestageIV, focusing on the structure of the AIP synthaseand how it usesthe proton-motive force to synthesizeATP.

StepwiseElectronTransportEfficientlyReleases t h e E n e r g yS t o r e di n N A D Ha n d F A D H 2 During electron transport' electrons are released from NADH and FADH2 and eventually transferred to 02, forming H2O according to the following overall reactions: NADH + H+ + 1/z02 --+ NAD* + H2O, LG: -52'6 kcal/mol FADH2 -r 1/z02 --+ FAD + H2O, L'G : -43.4 kcal/mol Recall that the conversion of 1 glucosemolecule to CO2 via the glycolytic pathway and citric acid cycle yields 10 NADH and-2 FADH2 molecules (seeTable 12-1). Oxidation of -613 kcal/mol thesereducedcoenzymeshas a total AGo'of

r In stage II, the three-carbon pyruvate molecule is first oxidized to generateone moleculeeach of CO2, NADH' and acetyl CoA. The acetyl CoA is then oxidized to CO2 by the citric acid cycle. r Neither glycolysis(stageI) nor the citric acid cycle (stage II) directly use molecularoxygen (02). r The malate/aspartateshuttle regeneratesthe supply of for continuedglycolysis. cytosolicNAD* necessary r Like glucoseoxidation, the oxidation of fatty acids takes place in four stages.In stageI, fatty acids are converted to fatty acyl CoA in the cytosol. In stageII, the fatty acyl CoA is first converted into multiple acetyl CoA moleculeswith generationof NADH and FADH2. Then, as in glucoseoxidation, the acetyl CoA entersthe citric acid cycle. StagesIII and IV are identical for fatty acid and glucoseoxidation. r In most eukaryotic cells, oxidation of short- to longchain fatty acids occurs in mitochondria with production of ATR whereas oxidation of very long chain fatty acids occurs primarily in peroxisomes and is not linked to ATP production; the releasedenergy is converted to heat.

Slfl The ElectronTransportchain and Generationof the Proton-MotiveForce Most of the energy releasedduring the oxidation of glucose and fatty acidsto CO2 (stagesI and II) is convertedinto highenergyelectronsin the reducedcoenzymesNADH and FADH2. \7e now turn to stageIII, in which the energytransiently stored in the coenzymesis converted by an electron transport

duction of FAD, which requires lessenergy. The energy carried in the reduced coenzymescan be released by oxidizing them. The biochemical challengefaced by the mitochondrion is to transfer,as efficiently as possible' the energy releasedby this oxidation into the energy in the terminal phosphoanhydridebond in ATP. P,t- * H* + ADP3- -+ATPa- + H2o, L,G : +7.3 kcal/mol A relatively simple one-to-onereaction in which reduction of one coenzyme molecule and synthesis of one ATP occurs would be terribly inefficient, becausethe AG'' for ATP generation from ADP and P1is substantially less than for the coenzymeoxidation and much energywould be lost as heat' To efficiently recover the energ5 the mitochondrion first converts the energy of coenzyme oxidation into a protonmotive force using a seriesof electron carriers' all but one of which are integral components of the inner membrane'

ElectronTransportin Mitochondrials Coupled t o P r o t o nP u m P i n g At severalsites during electron transport from NADH and FADH2 to C,2,protons from the mitochondrial matrix are 493

THEELECTRoNTRANSPORTcHA|NANDGENERAT|oNoFTHEPRoToN-MoT|VEFoRcE

pH electrode

O, solution

c

60

o

8= 40 +P

.=-

20

a)

(J n

0 Mitochondrion EXPERIMENTAL FIGURE 12-13Electrontransferfrom NADH to 02 is coupledto proton transportacrossthe mitochondrial membrane.lf NADHisaddedto a suspension of mitochondria depleted of 02, no NADHisoxidizedWhena smallamountof O, is (arrow), addedto thesystem thereisa sharprisein theconcentration of protonsin thesurrounding mediumoutside the mitochondria pumped acrossthe inner membrane;this generares proton concentration and electricalgradientsacrossthe inner membrane (seeFigure 12-2). This pumping causesthe pH of the mitochondrial matrix to becomehigher (i.e.,the H+ concentration is lower) than that of the intermembranespaceand cytosol.An electricpotential acrossthe inner membranealso resultsfrom the pumping of H* outward from the matrix, which becomes negative with respect to the intermembrane space.Thus free energy releasedduring the oxidation of NADH or FADH2 is storedboth as an electricpotential and a proton concentrarion gradient-collectivelS the proton-motive force-across the

major source of ATP in aerobic nonphotosyntheticcells. Much evidenceshows that in mitochondria and bacteriathis processof oxidative phosphorylation dependson generarion of a proton-motive force acrossthe inner membrane (mitochondria) or bacterial plasma membrane, with electron transport, proton pumping, and ATp formation occurring simultaneously.In the laboratory, for instance, addition o] 02 and an oxidizable substratesuch as pyruvate or succinare to isolated intact mitochondria results in a net synthesisof ATP if the inner mitochondrial membrane is intact. In the presence of minute amounts of detergents that make the membrane leaky, electron transport and the oxidation of these metabolites by 02 still occurs. However, under these conditions no ATP is made, becausethe proton leak prevents the maintenance of the transmembrane proton concentration gradientand the membraneelectricporential. The coupling between elecrron transporr from NADH (or FADH2) to 02 and proron rransporr aiross the inner mitochondrial membrane can be demonstrutedexperimentally 494

.

cHAprE1 R2 I c E L L U L AER NERGETtcs

60 120 180 E l a p s etdi m e( s )

240

300

(decrease in pH) Thustheoxidation of NADHby 02 iscoupled to the movement of protons out of the matrixOncethe 02 isdepleted, the protons excess slowlymovebackintothe mitochondria (powering the synthesis of ATP)andthe pHof the extracellular mediumreturns t o i t si n i t i avl a l u e .

with isolated, intact mitochondria (Figure 12-13). As soon as 02 is addedto a suspensionof mitochondria in an otherwise O2-free solution that contains NADH, the medium outside the mitochondria transiently becomesmore acidic (increasedproton concentration), becausethe mitochondrial outer membrane is freely permeableto protons. (Remember that malate/aspartateand other shuttles can convert the NADH in the solution into NADH in the matrix.) Once the 02 is depleted by its reduction, the excessprotons in the medium slowly leak back into the matrix. From analysisof the measuredpH changein such experiments,one can calculate that about 10 protons are transported out of the matrix for every electron pair transferred from NADH to 02. To obtain numbers for FADH2, the above experiment can be repeated,but with succinateinsteadof NADH as the substrate. (Recall that oxidation of succinateto fumarate in the citric acid cycle generatesFADH2; see Figure 12-10). The amount of succinateaddedcan be adiustedso that the amount of FADH2 generatedis equivalentto the amount of NADH in the first experiment. As in the first experiment, addition of oxygen causes the medium outside the mitochondria to becomeacidic, but lessso rhan with NADH. This is not surprising becauseelectronsin FADH2 havelesspotential energy (43.4 kcal/mol) than electrons in NADH (52.6 kcal/mole). and thus it drives the translocation of fewer protons from the matrix and a smaller changein pH.

ElectronsFlow from FADH2and NADHto 02 T h r o u g hF o u rM u l t i p r o t e i nC o m p l e x e s I7e now examine more closely the energetically favored movement of electronsfrom NADH and FADH2 to the final electron acceptor,02. For simplicity, we will focus our discussion on NADH. In respiring mitochondria, each NADH molecule releasestwo electronsto the electron transDort

chain; these electrons ultimately reduce one oxygen atom (half of an 02 molecule),forming one moleculeof water: NADH--+NAD* + H* + 2e t/rOr--HrO 2e- + 2H' + As electronsmove from NADH to 02, their potential declines by 1.14 V, which correspondsto 26.2 kcal/mol of electronstransferred,or :53 kcal/mol for a pair of electrons. As noted earlier,much of this energyis conservedin the proton-motive force generatedacross the inner mitochondrial membrane. There are four large multiprotein complexesin the electron transport chain that span the inner mitochondrial membrane: NADH-CoQ reductase(complex I, >40 subunits), swccinate-CoQreductase(complex II, 4 subunits), CoQH2-cytochromec reductdse(complexIII, 11 subunits), and cytochrome c oxidase (complex IV, 13 subunits). Electrons from NADH flow from complex I to III to IV, bypassing complex II; electronsfrom FADH2 flow from complex II to III to IV, bypassingcomplex I (seeFigure 12-8). Each complex contains several prosthetic groups that participate in moving electrons.These small nonpeptide organic moleculesor metal ions are tightly and specificallyassociatedwith the multiprotein complexes(Table 12-2).

COMP()NENT PROTEIN

GROUPSPR{]STHETIC

NADH-CoQ reductase (complex I)

FMN Fe-S

Succinate-CoQreductase (complex II)

FAD Fe-S

CoQH2-cytochrome c reductase (complex III)

Heme by Heme bp1 Fe-S Heme c1

Cytochrome c

Heme c

Cytochrome c oxidase (comPlex IV)

Crru2* Heme a LutHerne a3

*-Not included is coenzyme Q, an electron carrier that is not permanently bound to a protein complex. iou*.,.' J. \Xl.De Pierre and L. Ernster, L977, Ann. Reu. Biochem' 46:201..

Heme and the Cytochromes Severaltypesof heme,an ironcontaining prostheticgroup similar to that in hemoglobin and myoglobin (Figure 12-14a\, are tightly bound (covalently or noncovalently) to a set of mitochondrial proteins called cytochromes.Each cytochrome is designatedby a letter, such as A, b, c, or c1. Electron flow through the cytochromesoccurs by oxidation and reductionof the Fe atom in the centerof the hememolecule: Fe3* + e-

.

. --> Fe2*

Becausethe heme ring in cytochromesconsistsof alternating double- and single-bondedatoms, a large number of resonance hybrid forms exist. These allow the extra electron delivered to the cytochrome to be delocalizedthroughout the heme carbon and nitrogen atoms as well as the Fe ion. The various cytochromes have slightly different heme groups and surrounding atoms (called axial ligands), which

(b)

ta) HrC:CH II

9H.

Protein

tl

CH,

l--l o2c-cH2

H,C

H2c-co2

12-14 Hemeand iron-sulfurprostheticgroupsin A FIGURE bg the electrontransportchain.(a)Hemeportionof cytochromes c reductase of CoQHz-cytochrome andbs,whicharecomponents in allhemes (complex is present ring(yellow) lll) Thesameporphyrin porphyrin ring differin the to the attached substituents Thechemical

accept chain.All hemes transport in theelectron othercytochromes (Fe-S) (b) cluster iron-sulfur Dimeric time. at a oneelectron andrelease sulfur and inorganic are two atoms: four S to is bonded Fe atom Each proteinAll Fe-S of the associated sidechains two arein cysteine at a time. oneelectron acceptandrelease clusters

O F T H E P R O T O N - M O T I VFEO R C E T R A N S P O RC T H A I NA N D G E N E R A T I O N THE ELECTRON

495

generate different environments for the Fe ion. Therefore, each cytochrome has a different reduction potential, or tendency to accept an electron-an important property dictating the unidirectional "downhill" electron flow along the chain. Just as water spontaneously flows downhill from a higher to lower potential energy state-but not uphill-so too do electronsflow in only one direction from one heme (or other prosthetic group) to another due to their differing reduction potentials. All the cytochromes, excepr cytochrome c, are componentsof integral membranemultiprotein complexesin the inner mitochondrialmembrane. fron-Sulfur Clusters lron-sulfur clustersare nonheme,ironcontaining prosrheticgroups consistingof Fe atoms bonded both to inorganicS atomsand to S atomson cysteineresiduesin a protein (Figure I2-l4b). Some Fe aroms in the cluster bear a *2 charge;othershavea *3 charge.However,the net chargeof eachFe atom is actuallybetween+2 and *3, becauseelectrons in their outermost orbitals together with the extra electron delivered via the transport chain are dispersedamong the Fe atoms and move rapidly from one atom to another.Iron-sulfur clustersacceptand releaseelecffonsone at a time. Coenzyme Q (CoQ) Coenzyme e (Coe), also called ubiquinone, is the only small-moleculeelectron carrier in the chain that is not a protein-bound prosthetic group (Figure 12-15). It is a carrier of both protons and electrons.The oxidized quinone form of CoQ can accepr a single electron to

o (coo) ubiquinone ( o x i d i z feodr m )

H3co

cH, itt (CHr-CH:C-CH2)10-H

H3CO

o I e rl

J

os e m i q u i n o n e( c o e ; ) { f r e er a d i c a l )

H3cO

CH.

H3CO

(CHr-CH:J-CH2)10-H

9H'

form a semiquinone, a charged free radical denoted by CoQ .. Addition of a secondelectronand two protons (thus a total of two hydrogen atoms) to CoQ-. forms dihydroubiquinone (CoQH2), the fully reducedform. Both Coe and CoQH2 are solublein phospholipidsand diffuse freely in the hydrophobic centerof the inner mitochondrial membrane. This is how it participatesin the electron transport chaincarrying electronsand protons betweenthe complexes. As shown in Figure 12-16, CoQ acceptselectronsreleased from NADH-CoQ reductase(complex I) or succinate-Coe reductase(complex II) and donates them to CoeH2cytochrome c reductase(complex III). Importantly, reduction and oxidation of CoQ are coupled to pumping of protons. Ifhenever CoQ acceptselectrons,it does so at a binding site on the matrix (also called the cytosolic) face of the protein complex, always picking up protons from the medium there. \Thenever CoQH2 releasesits electrons,it doesso at a srteon the intermembranespace(also called the exoplasmic)side of the protein complex, releasingprotons into the intermembrane (or exoplasmic) fluid. Thus transport of each pair of electronsby CoQ is obligatorily coupled to movement of two protons from the matrix to the intermembranespacefluid. NADH-CoQ Reductase (Complex l) Electronsare transferred from NADH to CoQ by NADH-CoQ reducrase(Figure 12-16).In bacteriathe massof this complex is about 500 kDa (-14 subunits),whereasfor the L-shapedeukaryoticcomplex it is 1 MDa (14 centraland as many as 32 accessorysubunits). NAD- is exclusively a two-electron carrier: it accepts or releasesa pair of electronssimultaneously.In NADH-Coe reductase (complex I), electronsfirst flow from NADH to FMN (flavin mononucleotide), a cofactor related to FAD, then to an iron-sulfur clusteq and finally to CoQ. FMN, like FAD, can accept two electronsbut does so one electron at a trme. Each transported electron undergoesa drop in potential of =360 mV, equivalenrto a AG'' of -16.6kcallmol for the two electrons transported. Much of this releasedenergy is used to transport four protons across the inner membrane per molecule of NADH oxidized by the complex I. Those four protons are distinct from the two protons transferredto the CoQ in the chemical reaction shown above. The overall reaction catalyzedby this complex is

U.

zut +

"-

NADH + CoQ * 6H*1. --+

t.l

J

(Reduced) (Oxidized)

NAD- * Hti. + CoeH2 + 4H+o,t

OH

(Oxidized)

Dihydroquinone 1 999H,) (fully reduced form)

H3co

cH, ?*' (CHr-CH:C-CH2)10-H

H3CO OH

A FIGURE12-15 Oxidized and reduced forms of coenzyme e (CoQ),which can carry two protons and two electrons. Because of its long hydrocarbon "tail" of isopreneunits,Coe, alsocalled ubrquinone, is solublein the hydrophobic coreof phospholipid bilayers and is verymobile.Reductionof Coe to the fully reducedform, eH2 (dihydroquinone), occursin two stepswith a half-reduced free-radical intermediate , l l e ds e m i q u i n o n e ca 496

.

c H A p r E R1 2 |

C E L L U L AERN E R G E T t c s

(Reduced)

Succinate-CoQ Reductase (Complex ll) Succinatedehydrogenase,the enzyme that oxidizes a molecule of succinate to fumaratein the citric acid cycle(and in the processgenerares the reduced coenzymeFADH2), is one of the four subunits of complex II. In this way the citric acid cycle is physically as well as functionally linked to the electron transport chain. The two electronsreleasedin conversionof succinateto fumarate are transferredfirst to FAD in succinatedehydrogenase,then to iron-sulfur clusters-regenerating FAD-and finally to Coe,

llll+ Animation:ElectronTransport

at (b) Fromsuccinate

( a ) F r o mN A D H Intermembranespace (exoplasmic) 4 H+++

zn

Exoplasmic oO

Cytosolic

4HMatrix

(cytosolic) NADH

1120+ 22H O 2H* H* H z O

zn

NAD++ H+

NADH-CoOreductase { c o m p l e xl )

CoOH2-cytochrome c reductase (complex lll)

Complex lll

Gytochrome c oxidase (complex lV)

Succinate

Fumarate+2 H*

reductase Succinate-CoO (comPlexll)

FIGURE 12-16 Multiproteincomplexesand mobileelectron (bluearrows) carriersof the electrontransportchain.Electrons (l-lV) Electron flow throughfour majormultiprotein complexes rsmediated eitherbythe lipid-soluble movement between complexes form)or reduced form;CoQHz, molecule coenzyme Q (CoQ,oxidized proteincytochrome c (cytc),Themultipleprotein thewatersoluble frompassing electrons to pump usethe energyreleased complexes protons space(redarrows) fromthe matrixto the intramembrane (a)Pathway l, flowto complex fromNADHFromNADHelectrons perpairof aretranslocated thenlllandthenlV A totalof 10 protons intothe thatflowfromNADHto O, Theprotonsreleased electrons

I are of NADHby complex matrixspaceduringoxidation lV, by complex from 02 water of in formation the consumed fromthesereactions in no netprotontranslocation resulting (via flow fromsuccinate (b)Pathway Electrons fromsuccinate released lllandthenlV Electrons ll to complex in complex FADHz) ll areused in complex to fumarate of succinate duringoxidation additional without translocating to CoQHz CoQ reduce to fromCoQHz transport of electron protons, Theremainder proceeds bythe samepathwayasfor the NADHpathwayin (a). to 02, fromsuccinate transported Thusfor everypairof electrons lV lll and complexes by protons are translocated six

which binds to a cleft on the matrix side of the transmembrane portions of complex II (Figure 72-16). The overall reaction catalyzedby this complex is

Figure 12-12).There are severalfatty acyl-CoA dehydrogenase enzymeswith specificitiesfor fatty acyl chains of different lengths.Theseenzymesmediate the initial step in a four-step processthat removestwo carbons from the fatty acyl group by oxidizing the carbon in the B position of the fatty acyl chain (thus the entire processis often referred to as B-oxidation). Thesereactionsgenerateacetyl CoA, which in turn entersthe citric acid cycle. They also generatean FADH2 intermediate and NADH. The FADH2 generated remains bound to the enzymeduring the redox reaction,as is the casefor complex II. A water-soluble protein called electron transfer flauoprotein (ETF) transfersthe high-energy electrons from the FADH2 in the acyl-CoA dehydrogenaseto electron transfer flauoprotein:ubiquinoneoxidor eductase(ETF: QO), a membraneprotein that reducesCoQ to CoQH2 in the inner membrane.This CoQH2 intermixes in the membrane with the other CoQH2 moleculesgeneratedby complexesI and II.

Succinate+ CoQ --+fumarate + CoQH2 (Reduced)

(Oxidized) (Oxidized)

(Reduced)

Although the AGo' for this reactionis negative,the released energyis insufficientfor proton pumping in addition to reduction of CoQ to form CoQH2, which then dissociates from complex II. Thus no protons are translocateddirectly acrossthe membraneby the succinate-CoQreductasecomplex, and no proton-motive force is generatedin this part of the respiratorychain. Shortly we will seehow the protons and electronsin the CoQH2 moleculesgeneratedby complex I and complex II contribute to the generationof the proton-motive force. Complex II generatesCoQH2 from succinatevia FAD/ FADH2-mediatedredox reactions.Another setof proteinsin the matrix and inner mitochondrialmembraneperformsa comparedox reactionsto generate rable setof FAD/FADH2-mediated CoQH2 from fatty acyl CoA. Fatty acyl-CoA dehydrogenase, which is a water-solubleenzyme,catalyzesthe first stepof the oxidation of fatty acyl CoA in the mitochondrial matrix (see

CoQH2-Cytochrome c Reductase(Complex lll) A CoQHz generatedeither by complex I or complex II (or ETF:QO) donates two electronsto CoQH2-cytochrome c reductase(complex III), regeneratingoxidized CoQ. Concomitantlyit releases into the intermembranespacerwo protonspreviouslypickedup on the matrix face, generatingpart of the proton-motive force

NF T H E P R O T O N - M O T I VFEO R C E T H E E L E C T R OTNR A N S P O RCTH A I NA N D G E N E R A T I OO

497

(Figure 12-16). Vithin complex III, the releasedelectronsfirst are transferredto an iron-sulfur cluster within complex III and then to cltochrome c1 or to two &-type cytochromes (by and bs, seeQ cycle below). Finally, the two electronsare transferred sequentially to two molecules of the oxidized form of cytochrome 6, a water-soluble peripheral protein that diffuses in the intermembranespace.For eachpair of electronstransferred, the overall reaction catalyzed by the CoQH2-cytochrome c reductasecomplex is CoQH2 + 2 Cytc3* *2 H+i. -+ Coe + 4 H+o,t+ 2 Cytc}+ (Reduced)

(Oxidized)

(Oxidized)

(Reduced)

The AG'' for this reaction is sufficiently negative that two protons in addition to those from CoQH2 are translocated from the mitochondrial matrix across the inner membrane for each pair of electronstransferred; this involves the proton-motive Q cycle, discussedlater. The heme protein cytochrome c and the small lipid-soluble molecule Coe play similar roles in the electron transport chain in that they both serveas mobile electron shuttles,transferring electrons(and thus energy)betweenthe complexesof the elecrronrransporr chain. Cytochrome c Oxidase (Complex lV) Cytochrome c, after being reduced by CoQH2-cytochrome c reductase(complex III), is reoxidized as it transports electrons,one at a time, to cytochrome c oxidase (complex IV) (Figure 12-16). Mitochondrial cytochrome c oxidases contain 13 different subunits, but the catalytic core of the enzyme consists of only

three subunits.The function of the remaining subunits is less well understood. Bacterial cytochrome c oxidases contain only the three catalyric subunits. Four moleculesof reduced cytochrome c bind, one at a time, to the oxidase.An electron is transferred from the heme of each cytochrome c, first to the pair of copper ions called Crr^2*,then to the heme in cytochrome a, and next to the Cub2+ and the heme in cytochrome a3 that together make up the oxygen reduction center. The electrons are finally passedto 02, the ultimate electron acceptor,yielding 4 H2O, which together with CO2 is one of the end products of the oxidation pathway. Proposed intermediatesin oxygen reduction include the peroxide anion (Ort-) and probably the hydroxyl radical iOH.), as well as unusual complexes of iron and oxygen atoms. These intermediateswould be harmful to the cell if they escaped from the reaction center, but they do so only rarely (seethe discussionof reactiveoxygen speciesbelow). During transport of four electrons through the cytochrome c oxidase complex, four protons from the matrix space are translocatedacrossthe membrane.However, the mechanism by which theseprotons are translocatedis not known. For each four electronstransferred, the overall reaction catalyzed by cytochrome c oxidase is 4 C y t c 2 * * 8 H + 1 , + 0 2 - + 4 C y t c 3 ++ 2 H z O + (Reduced)

4H+o,t

(Oxidized)

The poison cyanide, used as a chemical warfare agent, by spies to commit suicide when captured, in gas chambers to executeprisoners, and by the Nazis (Zyklon B

(a) C o m p l e xl l l d i m e r C o m p l e xl V ,-

#-

Supercomplexl/lll2llV gupspsomplexl/lll,

-

Complex I ATP synthase

-

C o m p l e xl l l d i m e r ( l l l 2 )

-

Complex lV

-

C o m p l e xl l

**-

EXPERIMENTAL FIGURE l2-17 Electrophoresis and electron microscopicimaging identifiesan electrontransportchain supercomplex containingcomplexesl, lll, and lV.(a)Membrane proteins in isolated bovineheartmitochondria weresolubilized wlth a detergent, andthe complexes andsupercomplexes wereseparated by gelelectrophoresis usingthe bluenative(BN)-PAGE method.Each blue-stained bandwithinthe gelrepresents protein the indicated complex or supercomplex, with ll12 representing a dimerof complex lll Intensity of the bluestainisapproximately proportional to theamount of complex or supercomplex present(b)Supercomplex l/lllrllV was

498

C H A P T E R1 2

I

CELLULAR ENERGETICS

extracted fromthe gel,andthe particles werenegatively stained with 1% uranylacetate andvisualized by transmission electron microscopy. lmages of 228 particles werecombined at a resolution o'f-3 4 nm to generate an averaged imageof the complex viewed fromthesidein the planeof the membrane Approximate locations of thecomplex llldimerandcomplex lV areindicated by dashed ovals; the outlineof complex I isalsoindicated by a dashed line (white).Scalebaris 10 nm. fAdapted fromE Schafer et al, 2006, ]. Biol. Chem 2A1Q2): 1537 O-1537 5 l

gas) for the mass murder of Jews and others, is toxic becauseit binds to the heme a3 in mitochondrial cytochrome c oxidase (complex IV), inhibiting cellular respiration and therefore production of ATP. Cyanide is one of many toxic small moleculesthat interfere with energy production in mitochondria. I

ReductionPotentialsof ElectronCarriersFavor ElectronFlow from NADHto 02

Electron Transport Supercomplexes Over 50 years ago Britton Chance proposed that electron transport complexes might assembleinto large supercomplexes.Doing so would bring the complexesinto closeand highly organized proximity, which might improve the speed and efficiency of the overall process.However, an alternativeview holding that the complexesbehaved as independententities diffusing freely in the inner membrane became the dominant paradigm. During the past several years, genetic, biochemical, and biophysical studies have provided very strong evidencefor the existenceof electron transport chain supercomplexes.These studies involved relatively new gel electrophoreticmethods called blue native (BN)-PAGE and colorless native (CN)-PAGE, which permit separation of very large macromolecular protein complexes, and electron microscopic analysisof their threedimensionalstructures. One such supercomplex contains one copy of complex I, a dimer of complex III (III2), and one or more copies of complex IV (Figure 1,2-1,7).The unique phospholipid cardiolipin (diphosphatidylglycerol)

is a measureof the equilibrium constant of that partial reaction. lfith the exception of the b cytochromes in the CoQH2-cytochrome c reductasecomplex, the standard reduction potential Eo' of the electron carriers in the mitochondrial respiratory chain increasessteadily from NADH to 02. For instance,for the partial reaction

Cardiolipin o

g6

+lta-o-[-o.-X-.o

do

As we saw in Chapter 2, the reduction potential E fot a partial reduction reaction

NAD* +H*

I

a-o-i-

appears to play an important role in the assembly and function of these supercomplexes.Generally not observed in other membranes of eukaryotic cells, cardiolipin has been observed to bind to integral membrane proteins of the inner membrane (e.g., complex II). Genetic and biochemical studies in yeast mutants in which cardiolipin synthesisis blocked have establishedthat cardiolipin contributes to the formation and activity of mitochondrial supercomplexes,and thus it has been called the glue that holds together the electron transport chain, though the precise mechanism remains to be defined. In addition, there is evidencethat cardiolipin may influence the inner membrane'sbinding and permeability to protons and consequentlythe proton-motive force.

+2C_

-

'NADH

-320 mV, the value of the standard reduction potential is for transfer + kcal/mol 14.8 AGo' of which is equivalentto a proceed to tends partial reaction Thus this of two electrons. toward the left, that is, toward the oxidation of NADH to NAD+. By contrast, the standard reduction potential for the partial reactron Cytochromeco*(Fe3*) + e

-

- cytochromecred(F.t*)

is +220 mV (AG"' : -5.1 kcal/mol)for transferof one electron. Thus this partial reaction tends to proceed toward the * right, that is, toward the reduction of cytochrome c (Fe3 ) to cytochrome c (Fe'-). The final reaction in the respiratory chain, the reduction of 02 to H2O 2H-

) HO< ) o

= reduced molecule

Oxidized molecule * e- -

+ 'lror+

2e- -+H2O

has a standard reduction potential of +816 mV (AG'' : -37.8 kcal/mol for transfer of two electrons),the most positive in the whole series;thus this reaction also tends to proceedtoward the right. As illustrated in Figure 12-1'8,the steady increasein Eo' values,and the correspondingdecreasein AGo' values' ofthe carriers in the electron transport chain favors the flow of electrons from NADH and FADH2 (generatedfrom succinate) to oxygen.

ExperimentU s s i n gP u r i f i e dC o m p l e x e s the Established Stoichiometryof Proton Pumping The multiprotein complexesresponsiblefor proton pumping coupled to electron transport have been identified by selectively extracting mitochondrial membranes with detergents, isolating each of the complexes in nearly pure form, and then preparing artificial phospholipid vesicles (liposomes)containing each complex. $7hen an appropriati electrondonor and electronacceptorare added to such

O F T H E P R O T O N - M O T I VFEO R C E T R A N S P O RC T HAINAND GENERATION THE ELECTRON

Redox potential tmV)

F r e ee n e r g y (kcal/mol)

60 -400 -

NADH-CoO reductase (complex l) NADH

NAD++ H+

\.v 2 e-

F u m a r a t e+ 2 H + 5U

-200

Succinate-CoO reductase(complexll) 40

H+

")

Fe-S

30

H* Cyt c.,

CoQHr-cytochrome c r e d u c t a s e( c o m p l e x l l l ) Cyt c cuu

I

20

t*

10

(complex lV)

800

2 e-

tlz Oz + 2H*

HzO

FIGURE 12-18Changesin redoxpotentialand free energy duringstepwiseflow of electronsthroughthe respiratory chain.Bluearrowsindicate electron flow;redarrows, translocation of protons across the innermitochondrial membraneElectrons pass throughthe multiprotein complexes fromthoseat a lowerreduction

potential to thosewith a higher(morepositive) (leftscale), potential with a corresponding reduction in freeenergy(rightscale)The energyreleased aselectrons flowthroughthreeof thecomplexes is sufficient to powerthe pumpingof H* ionsacross the membrane, establishing a proton-motive force

liposomes,a changein pH of the medium will occur if rhe embedded complex transporrs protons (Figure 12-19). Studies of this type indicate that NADH-Coe reductase (complex I) translocatesfour protons per pair of electrons transported, whereas cytochrome c oxidase (complex IV) t r a n s l o c a t e st w o p r o t o n s p e r e l e c t r o n p a i r t r a n s p o r t e d (or, equivalently,for every two moleculesof cytochrome c oxidized). Current evidencesuggeststhat a total of 10 protons are transportedfrom the matrix spaceacrossthe inner mitochondrial membranefor everyelectronpair that is transferredfrom NADH to 02 (seeFigure 12-1,6).Becausesuccinate-Coe reductase(complexII) doesnot transport protons and complex I is bypassedwhen the electronscome from succinatederived FADH2, only six prorons are rransported acrossthe

membrane for every electron pair that is transferred from this FADH2 to 02.

s00

c H A P T E R1 2

|

CELLULAE RN E R G E T T C S

The Q CycleIncreasesthe Numberof Protons Translocatedas ElectronsFlow Through C o m p l e xl l l Experiments such as the one depicted in Figure 12-1,9have shown that four protons are translocated across the membrane per electron pair transported from CoQH2 through CoQH2-cytochrome c reductase(complex III). Thus this complex transports two protons per electron transferred, whereas cytochrome c oxidase (complex IV) transports only one proton per electrontransferred.An evolutionarily conservedmechanism, called the Q cycle, accounts for the

(a)

-../

Phospholipid membrane

t02+ 2 Ht Hzo

(reduced)

into the intermembranespace,but one moleculeof CoQH2 is regeneratedfrom CoQ at the Q1 site (seeFigure 12-20, bottom).Thus the net result of the Q cycleis that four protons are translocatedto the intermembranespacefor every two electrons transported through the CoQH2-cytochrome c reductasecomplex and acceptedby two moleculesof cytochromec. The translocatedprotons are all derived from CoQH2, which

2Ht

lntermembrane space CoOH2 l2e I

K* Valinomycin-bound (b) Matrix

E a

q)

E -o-

GoOH2-cytochromec reductase(complex lll)

012 Elapsed time(min) FIGURE12-19 Electrontransfer from A EXPERIMENTAL reduced cytochrome c to 02 via cytochrome c oxidase (complex lV) is coupled to proton transport. The oxidasecomplexis c incorporated into liposomes with the bindingsitefor cytochrome positioned on the outersurface(a)When 02 and reducedcytochrome to 02 to form H2Oand protons c are added,electronsare transferred aretransportedfrom the insideto the mediumoutsideof the v e s i c l e sA d r u g c a l l e dv a l i n o m y c iwna s a d d e dt o t h e m e d i u mt o of H*, dissipatethe voltagegradientgeneratedby the translocation which would otherwisereducethe numberof protonsmovedacross the membrane(b) Monitoringof the medium'spH revealsa sharp drop in pH followingadditionof 02 As the reducedcytochromec and the becomesfully oxidized,protonsleakbackinto the vesicles, p H o f t h e m e d i u mr e t u r n st o i t s i n i t i avl a l u e M e a s u r e m e nst sh o w that two protonsare transportedper O atom reducedTwo electrons are neededto reduceone O atom, but cytochromec transfersonly of Cyt c'* are oxidizedfor eachO one electron;thus two molecules 1986,J Biol Chem26128254] reduced lAdaptedfrom B Reynafarleetal,

two-for-one transport of protons and electronsby complex I I I ( F i g u r e1 2 - 2 0 ) . The substratefor complex III, CoQH2, is generatedby severalenzymes,includingNADH-CoQ reductase(complex I) and succinate-CoQreductase(complex II), electron transfer flauoprotein:ubiquinone oxidoreductase(ETF:QO, during B-oxidation), and, as we shall see,by complex III itself. In one turn of the Q cycle,two moleculesof CoQH2 are oxidizedto CoQ at the Q. siteand releasea total of four protons

At Oo site: 2 CoOH2 + 2 Cyt C+ ---') ( 4 H + , z l e) 2 CoO+ 2 Cyt C+ +2 e +4 H+{outside)

(2e I At O; site: CoO + 2 e + 2 H+lmatrix -----+CoOH2 ";6sy ( 2 H ' ' 2 e \ Net O cycle (sum of reactions at Oo and O;): + CoOH2+ 2 Cyr C'+ 2 H+1661rix side) 12H*,'l t: I CoO + 2 Cyt C+ + 4 H+loutside) (2e ) t h r o u g hc o m p l e xl l l t o c y t o c h r o m ec , 4 H " P e r2 e t r a n s f e r r e d t o t h e i n t e r m e m b r a n sep a c e released A FIGURE12-20 The Q cycle.The Q cyclebeginswhen a molecule from the combinedpool of reducedCoQH, in the membranebinds to the Qo site on the intermembranespace(outel side of the portionof complexlll (steptr) There,CoQHz transmembrane space(stepEEI) and two protonsinto the intermembrane releases (stepB) One of the dissociate two electronsand the resultingCoQ protein and cytochromec1, iron-sulfur via an is transported, electrons that eachcytochromec directlyto cytochromec (stepEE) (Recall shuttlesone electronfrom complexlll to complexlV) The other b1 and bs and partiallyreduces electronmovesthroughcytochromes second,Qi, siteon the to the bound an oxidizedCoQ molecule matrix(inner)sideof the complex,forming a CoQ semiqutnone anion,Q ' (step4). The processis repeatedwith the bindingof a secondCoQH2at the Qo site(stepEt), proton release(stepEE), reductionof anothercytochromec (stepEEI),and additionof the other electronto the Q-'bound at the Qi site(stepZ) There,the additionof two protonsfrom the matrixyieldsa fully reducedCoQHz (stepsE and 9), moleculeat the Qrsite,which then dissociates (step I0) and begin freeingthe Q to bind a new moleculeof CoQ l99O, J Biol Chem B Trumpower from again the Q cycleover lAdapted BiochemSci26:4451 et al, 2001,Trends andE Darrouzet 265:11409,

NF T H E P R O T O N - M O T I VFEO R C E T H E E L E C T R OTNR A N S P O RCTH A I NA N D G E N E R A T I OO

501

obtained its protons from the matrix, as a consequenceof the reduction of CoQ catalyzedby either NADH-CoQ re, ductase (complex I) or by CoQH2-cytochrome c reductase (complex III) (seeFigure 12-16). Although seeminglycumbersome, the Q cycle optimizes the numbers of protons pumped per pair of elecrronsmoving through complex III. The Q cycle is found in all plants and animals as well as in bacteria. Its formation at a very early stageof cellular evolution was likely essentialfor the successof all life-forms as a way of convertingthe potential energyin reducedcoenzymeQ into the maximum proton-motive force acrossa membrane. How are the two electronsreleasedfrom CoQH 2 at the Qo site directed to different acceptors, either to Fe-S, cytochrome c1 and then cytochrome c (upward in Figure 1220) or to cytochrome bL, cytochrome bs, and then CoQ at the Q1 site (downward in Figure 12-20)?The answer is simple and depends on a flexible hinge in the Fe-S-containing protein subunit of complex III. Initially the Fe-Scluster is close enough to the Qo site to pick up an electron from CoQH2 bound there. Once this happens, a segment of the protein containing this Fe-Scluster swings the cluster away from the Qo site to a position near enough to the heme on cytochrome c1 for electron transfer to occur. With the Fe-S subunit in this alternate conformation, the second electron releasedfrom CoQH2 bound to the Qo site cannot move to the Fe-Scluster-it is too far away, so it takes an alternative path open to it via a somewhat less thermodynamically favored route to cytochrome 61.

The Proton-MotiveForcein Mitochondria ls Due Largelyto a Voltage GradientAcross t h e I n n e rM e m b r a n e One result of the electron transport chain is the generation of the proton-motive force (pmf), which is the sum of a transmembraneproton concentration (pH) gradient and electricpotential, or voltage, gradient. It has beenpossibleto determine experimentally the relative contribution of the two componentsto the total pmf. The relative contributions depend on the permeability of the membrane to ions other than H+. A significant voltage gradient can develop only if the membrane is poorly permeable to other cations and to anions. Otherwise, anions would leak across from the matrix to the intermembrane spacealong with the protons and prevent a voltage gradient from forming. Similarly cations leaking acrossfrom the intermembrane spaceto the matrix (exchangeof like charge) would also short-circuit voltage gradient formation. Indeed, the inner mitochondrial membrane is poorly permeablero other ions. Thus proron pumping generatesa voltage gradient that makes it energetically difficult for additional prorons ro move across becauseof charge repulsion. As a consequence,proton pumping by the electron transport chain establishesa robust voltage gradient in the context of a rather small pH gradient. Becausemitochondria are much too small to be impaled with electrodes,the electricpotential and pH gradient across the inner mitochondrial membrane cannot be determined by direct measurement. Nevertheless,it has been possible to

502

CHAPTER 12

I

CELLULAE RN E R G E T I C S

develop methods to measureindirectly these critical values. The electricpotential can be measuredby adding radioactive 42K* ions and a trace amount of valinomycin to a suspension of respiring mitochondria. Although the inner membrane is normally impermeableto K*, valinomycin is an ionophore, a small lipid-soluble molecule that selectively binds a specific ion (in this case, K*) and carries it across otherwise impermeable membranes.In the presenceof valinomycin, a2K* equilibrates across the innir membrane of isolated mitochondria in accordancewith the electric potential: the more negativethe matrix side of the membrane, the more 42K* will be attracted to and accumulatein the matrix. At equilibrium, the measuredconcentration of radioactive K+ ions in the matrix, [K6], is about 500 times greater than that in the surrounding medium, [Ko"J. Substitution of this value into the Nernst equation (Chapter 11) shows that the electricpotential E (in mV) acrossthe inner membranein respiring mitochondria is - 160 mV, with the matrix (inside) negatlve: fK'-l E : - 5 9 l o e # i : - 5 e l o g 5 0 0: - 1 6 0 m V L N o u rl

Researcherscan measurethe matrix (inside)pH by trapping pH-sensitivefluorescent dyes inside vesiclesformed from the inner mitochondrial membrane, with the matrix side of the membrane facing inward. They also can measure the pH outside of the vesicles(equivalent to the intermembrane space)and thus determine the pH gradient (ApH), which turns out to be -1 pH unit. Since a differenceof one pH unit representsa tenfold differencein H+ concentrarion, according to the Nernst equation a pH gradient of one unit across a membrane is equivalent to an electric potential of 59 mV at 20 'C. Thus, knowing the voltage and pH gradients, we can calculatethe proton-motive force, pmf, as ^ T \ - (l R q/ - 59 ApH x Ap = -p m f : v ' /H I : \t where R is the gasconstantof 1.987 call(degree.mol),7 is the temperature (in degreesKelvin), F is the Faraday constant 123,062 call(V.mol)1, and V is the transmembraneelectric potential; V and pmf are measuredin millivolts. The electric potential V acrossthe inner membraneis - 160 mV (negative inside matrix) and ApH is equivalent to :60 mV. Thus the total pmf is -220 mV, with the transmembraneelectric potential responsiblefor about 73 percentof the total.

ToxicBy-productsof ElectronTransport C a n D a m a g eC e l l s About 1-2 percent of the oxygen metabolized by aerobic organisms, rather than being converted to water, is partially reducedto the superoxideanion radical (02 ). Superoxideis unstable in aqueous biological liquids, breaking down into especially toxic hydrogen peroxide (HzOz) and then hydroxyl radicals.Theseand other reactiue

oxygen species(ROS), which contribute to what is often called cellular oxidatiue stress, can be highly toxic, because they chemically modify proteins, DNA, and unsaturated fatty acyl groups in membrane lipids, thus interfering with normal function. Indeed, ROS are purposefully generated by body defensecells (e.g.,macrophages)to kill pathogens. In humans, excessiveor inappropriate generationof ROS has been implicated in many diverse diseases,including heart failure, neurodegenerativediseases,alcohol-induced liver disease,diabetes,and aging. Although ROS can be generatedby a number of metabolic pathways, the major source of ROS appearsto be the electron transport chain, in particular mechanismscoupled to complexes I and III. The semiquinone form of ubiquinone, CoQ-. (seeFigure 72-15), an intermediateform of CoQ generatedin the Q cycle, may play a particularly important role in superoxidegeneration. To help protect against ROS toxicity, mitochondria have evolved several defensemechanisms,including the use of enzymes that inactivate superoxide first by converting it to H2O2 (Mn-containing superoxide dismutase) a n d t h e n t o H 2 O ( g l u t a t h i o n e p e r o x i d a s e ,w h i c h a l s o detoxifies the lipid hydroperoxide products formed when ROS react with unsaturated fatty acyl groups). Cardiac mitochondria also have catalase(normally only found in p e r o x i s o m e s )t o h e l p b r e a k d o w n H z O z . T h i s i s n o t s u r p r i s i n g , b e c a u s et h e m o s t o x y g e n - c o n s u m i n go r g a n i n mammals is the heart. In addition, the small molecule antioxidants c-lipoic acid and vitamin E help protect the mitochondrion from ROS. f

ElectronTransport and Generation of the Proton-Motive Force r By the end of the citric acid cycle (stageII), much of the energy originally present in the covalent bonds of glucose and fatty acids is converted into high-energy electrons in the reduced coenzymesNADH and FADH2. The energy from theseelectronsis used to generatethe proton-motive force. r In the mitochondrion, the proton-motive force is generated by coupling electron flow (from NADH and FADH2 to 02 ) to the uphill transport of protons from the matrix across the inner membrane to the intermembrane space. This processtogether with the synthesisof ATP from ADP and P1driven by the proton-motive force is called oxidative phosphorylation. r The flow of electronsfrom FADH2 and NADH to 02 is directedthrough multiprotein complexes.The four major complexesareNADH-CoQ reductase(complexI), succinate-CoQ reductase(complex II), CoQH2-cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV). r Each complex contains one or more electron-carrying prostheticgroups: iron-sulfur clusters,flavins, hemegroups, and copper ions (seeTable 12-2). Cytochrome c, which contains heme, and coenzymeQ (CoQ), a lipid-soluble small

molecule,are mobile carriersthat shuttle electronsbetween the complexes. r ComplexesI, III, and IV pump protons from the matrix into the intermembrane space.Complexes I and II reduce CoQ to CoQH2, which carries protons and high-energy electrons to complex III. The heme protein cytochrome c carries electrons from complex III to complex IV, which usesthem to pump protons and reduce molecular oxygen to water. r The high-energyelectronsfrom NADH enter the electron transport chain through complex I, whereasthe high-energy electronsfrom FADH2 (derivedfrom succinatein the citric acid cycle) enter the electron transport chain through complex II. Additional electrons derived from FADH2 by the initial step of fatty acyl-CoA B-oxidation increasethe supply of CoQH 2 avallable for electron transport. r \(ithin the inner membrane, electron transport complexes assembleinto supercomplexesheld together by cardiolipin, a specialized phospholipid. Supercomplex formation may enhancethe speedand efficiency of generation of the proton-motive force. r Each electron carrier acceptsan electron or electron pair from a carrier with a lesspositive reduction potential and transfers the electron to a carrier with a more positive reduction potential. Thus the reduction potentials of electron carriers favor unidirectional electron flow from NADH and FADH2 to 02 (seeFigure 12-1'8). r The Q cycle allows four protons (rather than two) to be translocatedper pair of electronsmoving through complex III (seeFigure 12-20). r A total of 10 H+ ions are translocated from the matrix acrossthe inner membrane per electron pair flowing from NADH to 02 (seeFigure 12-1,6),whereas 6 H* ions are translocatedper electron pair flowing from FADH2 to 02. r The proton-motive force is due largely to a voltage gradient across the inner membrane produced by proton pumping; the pH gradient plays a quantitatively less important role. r Reactive oxygen species(ROS) are toxic by-products of the electron transport chain that can modify and damage proteins, DNA, and lipids. Specificenzymes(e.g.,glutathinone peroxidase, catalase)and small molecule antioxidants (e.g.,vitamin E) help protect againstROS-induced damaqe.

the Proton-Motive Harnessing Processes Forcefor Energy-Requiring The hypothesisthat a proton-motive force across the inner mitochondrial membrane is the immediate source of energy for ATP synthesiswas proposedin 1'961by PeterMitchell. Virtually all researchersstudying oxidative phosphorylation and photosynthesisinitially reiected his chemiosmotic hypothesis. They favored a mechanism similar to the then

EO R C EF O R E N E R G Y - R E Q U I R I N PG ROCESSES H A R N E S S I NT GH E p R O T O N - M O T I V F

'

503

well-elucidated substrate-levelphosphorylation in glycolysis, in which chemical transformation of a substrate molecule (i.e.,phosphoenolpyruvate) is directly coupled to ATP synthesis.Despite intenseefforts by a large number of investigators, however, compelling evidencefor such a direct mechanismwas neverobserved. Definitive evidencesupporting Mitchell's hypothesis depended on development of techniquesto purify and reconstitute organellemembranesand membrane proteins. The experiment with vesiclesmade from chloroplast thylakoid membranes(describedin detail below) that contain ATP synthase, outlined in Figure 12-21, was one of several demonstratingthat this protein is an ATP-generatingenzyme and that ATP generation is dependenton proton movement down an electrochemical gradient.It turns out that the protons actually move tbrougD the ATP synthaseas they rraversethe membrane!

troFr --1

Bacterium

,1

Plasma memorane

Mitochondrion Intermembranesoace

Outer membrane

Matrix

,0 Fl

,.1

noe + e,

)

lLIr '

:j pH 7.5 attt'

Chloroplast

' ai:r::llr'rr""

a"

T h y l a k o i dm e m b r a n e

S o a kf o r s e v e r a m l inutes at pH 4.0

L i gh t

pH 4.0

v

ADP + P,

ATP H* li:i:li:la:l:li:illlia::iiilrrl:

,,,:"' rlr

H+ pH 4.0

.l.,1 pH 8.0

EXPERIMENTAL FTGURE 12-21Synthesis of ATpby FeFl dependson a pH gradientacrossthe membrane.lsolated chloroplast thylakoid vesicles containing FoF,particles wereequllibrated in the darkwith a buffered solution at pH4.0 Whenthe pH in the thylakoid lumenbecame 4 0, thevesicles wererapidlymixedwith a solution at pH8 0 containing ADPandP, A burstof ATpsynthesis accompanied thetransmembrane movement of protons drivenbythe 1 0 , 0 0 0 - f oHl d* c o n c e n t r a t g i orna d i e n( ltO 4 M v e r s u1sO - 8M ) I n similar experiments using"inside-out" preparations of mitochondrial membrane vesicles, an artificially generated membrane electric potential alsoresulted in ATPsynthesis c H A P T E R1 2

|

HI

,t

/---'>

A

Af P

:;::rpHa.o

I I A d d a s o l u t i o no f p H 8 . 0 II t h a t c o n t a i n sA D P a n d P 1

ADP + P;

Intermembrane space Stroma

Fl

uI illi ,riil,

504

Outer membrane

CELLULAE RN E R G E T T C S

Inner memDrane T h v l a k o i dm e m b r a n e

A FIGURE 12-22Chemiosmosis in bacteria,mitochondria, and chloroplasts. Themembrane surface facinga shaded areaisa cytosolic face;thesurface facingan unshaded, whiteareaisan exoplasmic face,Notethatthecytosolic plasma faceof the bacterial membrane, the matrixfaceof the innermitochondrial membrane, andthestromal faceof thethylakoid membrane areallequivalent Duringelectron protons transport, arealwayspumpedfromthe cytosolic faceto theexoplasmic face,creating a protonconcentration (exoplasmic gradient face> cytosolic face)andan electric potential (negative cytosolic faceandpositive exoplasmic face)across the membraneDuringthe synthesis protons of ATP, flow in the reverse (downtheirelectrochemical direction gradient) throughATPsynthase (FoFr complex), whichprotrudes in a knobat the cytosolic facein all CASCS

As we shall see,the ATP synthaseis a multiprotein complex that can be subdivided into two subcomplexescalled Fe (containing the transmembraneportions of the complex) and F1 (containingthe globular portions of the complex that sit above the membrane and point toward the matrix spacein mitochondria). Thus the ATP synthaseis

ward what becamethe stromal spaceof the chloroplast (describedin detail below). In all cases,ATP synthaseis positioned with the globular F1 domain, which catalyzesATP synthesis,on the cytosolic face of the membrane, so ATP is always formed on the cytosolic face of the membrane (seeFigure 1'2-22).Protons always flow through ATP synthasefrom the exoplasmic to the cytosolic faceof the membrane, which in the mitochondrion is from the intermembrane to the matrix space.This flow is driven by the proton motive force. InvariablS the cytosolic face has a negativeelectric potential relative to the exoplasmic face. In addition to ATP synthesis,the proton-motive force acrossthe bacterial plasma membraneis usedto power other processes,including the uptake of nutrients such as sugars (using proton/sugar symporters) and the rotation of bacterial flagella. Chemiosmotic coupling thus illustrates an important principle introduced in our discussionof active transport in Chapter 1.1.:the membrane potential, the concentrdtion gradients of protons (and other ions) across a membrane, and the phosphoanhydride bonds in ATP are equiualent and interconuertibleforms of chemical potential energy.Indeed, AIP synthesisthrough ATP synthasecan be thought of as active transport ln reverse.

often also called the FeFl complex; we will use the terms interchangeably.

T h e M e c h a n i s mo f A T PS y n t h e s i sl s S h a r e d , itochondria, A m o n g B a c t e r i aM and Chloroplasts Although bacteria lack any internal membranes,aerobic bacteria nonethelesscarry out oxidative phosphorylation by the same processesthat occur in eukaryotic mitochondria. Enzymes that catalyze the reactions of both the glycolytic pathway and the citric acid cycle are presentin the cytosol of bacteria; enzymesthat oxidize NADH to NAD* and transfer the electrons to the ultimate acceptor 02 reside in the bacterialplasmamembrane. The movement of electronsthrough thesemembranecarriers is coupled to the pumping of protons out of the cell. The movement of protons back into the cell, down their concentration gradient, is coupled to the synthesisof ATP. This general processis similar for bacteria and eukaryotes (in both mitochondriaand chloroplasts)(Figuret2-22). The bacterial ATP synthases(F6F1complex) are essentiallyidentical in structure and function to the mitochondrial and chloroplast ATP synthasesbut are simpler to purify and study. A primitive aerobic bacterium was probably the progenitor of mitochondria in eukaryotic cells (Figure 12-23). hccording to this endosymbionthypothesis,the inner mitochondrial membrane would be derived from the bacterial plasma membrane with its cytosolic face pointing toward what became the matrix space of the mitochondrion. Similarly in plants the progenitor'splasma membranebecamethe chloroplast'sthylakoid membrane and its cytosolic face pointed to-

ATPSynthaseComprisesTwo Multiprotein ComplexesTermedFsand F1 With generalacceptanceof Mitchell's chemiosmoticmechanism, researchersturned their attention to the structure and operation of the F6F1complex. The FsFl complex, or AIP synthase,has two principal components, Fe and F1, both of which are

Eukarvotic o l a s m am e m b r a n e Endocytosisof bacterium caoableof oxidative phosphorylation

I Endocytosisof bacterium capableof photosynthesis

Bacterial p l a s m am e m b r a n e

B a c t e r i apl l a s m a m e m b r a n eb e c o m e s i n n e rm e m b r a n e of chloroplast

B a c t e r i apl l a s m a m e m b r a n eb e c o m e s i n n e rm e m b r a n e of mitochondrion

a(-- .{

,-#r..\

l n n e r m e m b r a n eb u d s off thylakoid vesicles

Thylakoid memorane

M i t o c h o n d r i am l atrix

12-23 Endosymbiont hypothesis for the evolutionary a FIGURE Endocytosis of a origin of mitochondriaand chloroplasts. eukaryotic cell(stepII) wouldgenerate bacterium by an ancestral with two membranes, derived an organelle theoutermembrane plasma membrane andthe inneronefrom fromtheeukaryotic (stepE) TheF1subunitof ATPsynthase, membrane the bactenal membrane, wouldthen to thecytosolic faceof the bacterial localized

(/eft)or chloroplast mitochondrion facethe matrixof the evolving membrane, (ngrht)Budding fromthe innerchloroplast of vesicles in contemporary chloroplasts of duringdevelopment suchasoccurs with the F1subunit membranes thethylakoid plants, wouldgenerate stroma(step face,facingthe chloroplast on the cytosolic remaining faces; areaarecytosolic facinga shaded surfaces B) Membrane faces areaareexoplasmic facinqan unshaded surfaces

H A R N E s s I N G T H E P R o T o N - M o T | V E F o R c E F o R E N E R G Y - R E Q U I R | N G P R o c E s 505 sES

Animation:ProtonTranslocating, RotaryF-ATPase{lttt 100nm ------------>l

l x i ddiizziinngg aq g e nntt( e 3 l et c t r o na c c e p t o r )

12-33 PhotoelectrontransPort,the primaryevent in < FIGURE of a photonof light,oneof the photosynthesis. Afterabsorption center in the reaction pairof chlorophyll a molecules excited special (/eft)donatesviaseveral intermediates an electronto a looselybound surface of the molecule, the quinoneQ, on the stromal acceptor charge irreversible an essentially membrane, creating thylakoid (righi fhe electron cannoteasily across the membrane separation the positively centerto neutralize returnthroughthe reaction of waterto molecular a. In plantstheoxidation chlorophyll charged ll. calledphotosystem place complex in a multiprotein oxygen takes photoelectron transport photosystem I usesa similar Thecomplex the electron water,it reduces pathway,but insteadof oxidizing c a r r i eNr A D P - .

to the primary electron acceptor,quinone Q, near the stromal surfaceof the thylakoid membrane (Figure i2-33). This light-driven electron transfer,called photoelectron transport, dependson the unique environment of both the chlorophylls and the acceptor within the reaction center. Photoelectron transport, which occurs nearly every time a photon is absorbed, leavesa positive charge on the chlorophyll a closeto the luminal surface of the thylakoid membrane (opposite side from the stroma) and generatesa reduced, negatively chargedacceptor(Q-) near the stromal surface. The Q- produced by photoelectron transport is a powerful reducing agent with a strong tendencyto transfer an electron to another molecule, ultimately to NADP+. The positively charged chlorophyll a*, a strong oxidizing agent, attracts an electron from an electron donor on the luminal surface to regeneratethe original chlorophyll a. In plants, the oxidizing power of four chlorophyll a* moleculesis used,by way of intermediates,to remove four electronsfrom 2 H2O moleculesbound to a site on the luminal surfaceto form 02: 2HzO * 4 chlorophylla* --+4 H* + C2+ 4 chlorophyll a These potent biological reductants and oxidants provide all the energy neededto drive all subsequentreactions of photosynthesis:electron transport (stage 2), ATP synthesis (stage3), and CO2 fixation (stage4). Chlorophyll a also absorbs light at discretewavelengths shorter than 680 nm (see Figure 12-32). Such absorption raisesthe molecule into one of severalexcited states,whose energiesare higher than that of the first excited state describedabove,which decayby releasingenergywithin 10-12 seconds(1 picosecond,ps) to the lower-energyfirst excited state with loss of the extra energy as heat. Becausephotoelectron transport and the resulting charge separationoccur only from the first excited state of the reactron-center chlorophyll a, the quantum yield-the amount of photosynthesisper absorbedphoton-is the samefor all wavelengths of visible light shorter (and therefore of higher energy)than 680 nm. How closely the wavelength of light matches the absorption spectraof the pigmentswill determinehow likely it is that the photon will be absorbed. Once absorbed, the

photon's exact wavelength is not critical, provided it is energetic enough to push the chlorophyll into the first excited state.

I n t e r n a lA n t e n n aa n d L i g h t - H a r v e s t i n g ComplexesIncreasethe Efficiency of Photosynthesis Although chlorophyll a moleculeswithin a reaction center that are involved directly with charge separation and electron transfer are capableof directly absorbing light and initiating photosynthesis,they most commonly are energizedindirectly by energy transferred to them from other light-absorbing and energy-transferringpigments. These other pigments, which include many other chlorophyll molecules, are involved with absorption of photons and passingthe energyto the chlorophyll a molecules in the reaction center. Some are bound to protein subunitsthat are consideredto be intrinsic componentsof the photosystemand thus are called internal antennas;others are bound to proteinscomplexesthat bind to but are distinct from the photosystemcore proteins and are called light-harvesting complexes(LHCs). Even at the maximum light intensity encountered by photosynthetic organisms (tropical noontime sunlight),eachreaction-centerchlorophyll a moleculeabsorbs only about one photon per second' which is not enough to support photosynthesis sufficient for the needs of the plant. The involvement of internal antenna and LHCs greatly increasesthe efficiency of photosynthesis,especiallyat more typical light intensities,by increasingabsorption of 680-nm light and by extending the range of wavelengths of light that can be absorbedby other antennapigments. Photonscan be absorbedby any of the pigment molecules in internal antennasor an LHC. The absorbedenergyis then rapidly transferred(in l sr ssecordSurtros lereue8 puotes V ' ( 1 - 9 1 a ; n 8 r g )s s a c o r d yeraua8srqt dq snolfnu pue 'seruosrxorad'slseldorolqc

'errpuoqcolnu'(gg) unpcrler f,Iruselclopua eql ot Pelros ere aql Jo rolrolut snoenbeaql otul euErqluou suretord'a11aue3.ro aLItssorf,Eurato.rdarrluaeql uoItef,olsuEJlol spPelSurla8rel Jo 'suralord elgnlos-Jete,t\JoJ seeJel{,\\'euerqruatueqr 1o redel -lq pldl aqt olur urelord el{t Jo uorlrosul ot sPeelSurta8rel 'suralordouerquau rog 'elelduor st utelord aqr;o srsagruls r e U pu o o sJ o u o l r e l s u e rSr u r . r n Jp J t l l r JJ n J J oU E Jp u e a l l e u e B -ro ue relnllor€rJur Jo euerqruau aq] ot utolord e 1o 3ur -ta8ret oL{I 'sassacordJo sPul>l sserord sa^lo^ul rsrlJ leraue8 '3ut1,tosutalo,tdto 3ut7.a8w1. lueJeJJIPdre,t o,r,rlsessedtuocue utalotd sE ot parraJerdylensn'suotleurlsep relnllol radord rreqt ol sutalord pezrsaqluds dl,Lrau yo dra,ttlep oq1 dlradord uollrunJ ol IIOJoqr roJ suollur -ol dq suralo.rd raqlo e pecnpord rr3Lll qreer lsnu tferror IIel OqrIIE PUEOseql 'llel Oqlruo{ uollerJasroJ eJEJJns IIer eI{] ol pellrrrp eq tsntu selnJelou SuqeuSrseprrdeddlodpue saudz -ua aql sluauodtuoo se xIrlpIU relnlleterxo yo se a,rnseSrp IIe^\ pue 'snalcnu aql or paraS:eteq lsnru saseraurdyod VNC PUE etuos 'eue;quraru eurseld VNU sp qcns seurdzuealqnlos-Jate,&\ eql ol pare^rlapoq tsnu sutalord ralrodsuert pue suralordrol -dalar auoruroqdueru 'alduexa Jod 'eJpJrns eql ot ro IIor IIOJ ere IIOI lecrddr aql uqlr.,raallaue8rorelnortred p ol Paro^ITeP e ur pernpord surelo.rdJo spuDlruore;tlp eqr to JIEqse dueu se ta.ra.,u.og'losoldc eql urqlr,4 uretuer dueur puu 'seruosoqrr are sutalo.rdeseqlJo dlr.roleur crlosotdrdq pezrsaqruds '0005 rnoqe 'y1actsead e isutalord spuDl lsp^ oql Jo ruerollp 000'0I o1 dn suteluoJIIer ueITEIUtueru lerrddl 'sraq)Jeasau lrouotrJol o]oqd/oinbeN [ ]ul pue 'ouelqu.Jeul g euol4 t orlardsrosselordl aredsauerqureLurelur 'Pupuoqto]ttiJ aq11osluaLupedLuot rauur'xuleuraq] 6urpnl:u1 ut poAloAUt sutelotdltlosoy{l ate uotlettdsat aql o1pa1e6.re1 snoupA puesutelotd sutelotdauelqLreul oqf olepnsllal aq].to]paurlsap fuolar:aso^ra)orUl qbnoreq1,Losauerquayypalabtelate qtrqrrir eduol lelnllaler}ul {1nnau o} s}uar-u1t suralordpaztsaqlu{s oql +ooM] burmoqs lla) e ]o qdet6ottruuolllolabutuuelsy

sttttNVDuo cNV slNVuElEl t OINI

sNEroud9Ntnonl

U]IdVH)

overview Animation:ProteinSortingflttt Outernuclear membrane I n n e rn u c l e a r membrane

Nucleus

"\ C6osol

Membrane Matrix Peroxisome

Outermembrane

:l\

d\tu

6 t

Matrix Inner memorane Mitochondrion

Chloroplast

tr/ i

/\

\@ \

Plasma /a membrane

Lysosome l

SECRETORY PATHWAY FIGURE 13-1 Overviewof major protein-sorting pathwaysin eukaryotes. All nuclear-encoded mRNAs are translated on cytosolic ribosomesRight(nonsecretory pathways). Synthesis of proteins lacking an ERsignalsequence iscompleted (step[) Thoseproteins on freeribosomes thatcontainno targeting sequence arereleased intothe cytosol andrematn tnere (stepE). Proteins with an organelle-specific targeting sequence (pink)firstarereleased (stepZ) butthenare intothecytosol imported intomitochondria, chloroplasts, peroxisomes, or the (stepsB-E) Mitochondrial nucleus andchloroplast proteins passthroughtheouterandinnermembranes typically to enter

the matrixor stromal space, respectively Otherproteins aresortedto othersubcompartments of theseorganelles by additional sorting proteins stepsNuclear enterandexitthroughvisible poresin the nuclearenvelopeLeft(secretory pathway):Ribosomes synthesizing proteins nascent pathwayaredirected in the secretory to the rough (ER) endoplasmic (pink;steps[, reticulum byan ERsignalsequence iscompleted on the ER,theseproteins Z) Aftertranslation can moveviatransport (stepB) Further vesicles to the Golgicomplex proteins sortingdelivers eitherto the plasma membrane or to (stepsEE, @ ) Theprocesses lysosomes underlying the secretory pathway(stepsB, El, shadedbox)arediscussed in Chapter'l4

well as in the plasma membrane. Targeting to the ER generally involves nascent proteins still in the process of being synthesized.Once translocatedacross the ER membrane, proteins are assembledinto their native conformation by protein-folding catalystspresenrin the lumen of the ER. This process is monitored carefullS and only after their folding

and assemblyis complete are proteins permitted to be transported out of the ER to other organelles.Proteins are also modified in various ways after translocation into the ER. These modifications can include addition of carbohydrate groups, stabilization of protein structure through disulfide bond formation, and specificproteolytic cleavages.Proteins

534

CHAPTER 13

|

M O V T N GP R O T E t NtSN T O M E M B R A N E A SN D O R G A N E L L E S

s€s

l N V U S l A l t A U l l H t S S O U ) VS N E I O U d , l . U O l _ : l U ) tJSO N O t J _ V ) O ' l S N V U l

uortpluarJoarupseql q71M'satuoso,otuq8not potuJol'solcrse,r posoll IIErusotur dn s>1ea.rq gg q8not eql (pezrueSouoqare sllal uaql6'(7-91 arn8rg)aue.rqurauUA reqlo ruoq tf,unsrp dlleclSoloqdrorusreaddueJErns slr teql souosoqrr qlr^ pap -pnls dlesuep os sr asnEreq gg eql sE u.,\aou>l sr deru. q8not 1r -qted .{roterrasaql Sur.ratuasurelord selref,oJrer{t Ug aqt Io uonrod oql 'UE or{t Jo areJrnsoql ot punoq ere leqt sauos -oqIJ uo pazrsaqludsere daqr se suralo.rddrolarcesolur paleJ -odroJur ere sprf,e ourrue peleqel dla,rrtreorper 'sy1orqcns uI 'sllrJ reulre rltporJued qtrlr\ sluaurradxa 3ur1age1-aspd dq parepnnla lsrrJ ara.t uralord drotarcas e yo srsoqlu.,{s erJtreryedlelerpaurur rnJJo lugt stuo^ato aruanbesaq1 'demqredsrqt Surdpnrsur pesndlapm uoaq o^Er{daqt 'acuep -unge ]eer8 ur (r31ogpue UE ''3'a) de,vrqred,{rorarcasaql ;o selleue8roeql urEtuor sylec.{rotonas qJns asnpJJfl'ourlsoturar{l ot ppel terlt selnDnp olur pelerres aft wql sarudzuaarrrtsa8rp 'aluelsur.ro;'s11arreurle IEJeAes Jo sertrluenba8rel ozrsaqluds JrlpeJf,ued'surelo.rdcr;rcadsto slunoup a8rel ;o uorloJJosJoI pazqenedsare sller yo seddruretJet '(suretordxrJlpru relnllel -erxa ''3'a) suralord dlarrerrE eterfossllel Jo ile g8noqlly rardeqr 3urmo11o1 or{t ur possorppesr trodsue.rl alJrse^1o ssecordaql 'UA eql retue ,(aqrse pJrJrporueJEpue p1o;suralo.rdaorl 't'€I uorlf,osur 'oroq roprsuof,osle olN rol -deqc srqr ur possnlsrpsallaue8rorer{to eqr ot suratord 3ur Jo -la8.retol Jppurs ,{ylecgsrueqceru eJEsessacordesor{lesnpJoq 'euerqruetu UE er{l olur peuasur aq UEJsura}o.rdJruos A\oq pue UA aql ot pareSretere suratord ,(rolarcas.{\oq ssnf,srp a.\\ relder{f,srr.{lJo suorlreso,v\l tsrrJeqt uI 'selrrse,r;o Sursn; (seuososll 'r31o5 aql ot pue Surppnqq8norqr of,EJrnsIIef ro trodsuerl uretord (t) pue'uerunl UE erp eprsuruou€f,l;Iporu pue Surploy urelord (7) 'aue.rqurau UA eqt ssorre uou -EJolsuertpue srsaqtudsuralord (1) :sdarsaarqt se,rlo,rurdezn -qled drolerJaseqf rusrupqreu lrseq eql 'suralo.td{.toptt Jo -as sE dle^IlJellof, sulelord asaqr ol raJar o.&\tlIfIIduIS roJ '(4rt 't-tt arn8rg) saurososdlpue '€1og 'UA aq] Jo euprq -ruoru eqt .ro acrds leurrunl snoanbe Jrll ur aprserler{l surel -ord delr suralord Surt.ros Surzrsaqtuds ro1 pue patorJes pue -qled drolerces eruesaql .{llerluasseesn slleJ rrrod.re4na 11y

ouerquawul aql ssor)vsurolord fto1ene5lo uorle)otsue4l$l 'sarod realonu snolJnu eql q8norqr to tno pue olur surato.rd 'satuosrxoradpue 'slseld ;o uodsue.rt agt JaAoJa.n tlleurg -orolql 'errpuoqcolnu ol suratord;o Surta8reteqursep ueql ar16 de.nqred drolanas ar{t retue deql se suralo.rd ot Jnf,f,o leqt suortplrtrpou leuorlelsuerl-1sodaql Surpnlcur'Ug eqt ol surato;dJo Surla8relreloJ e.taterdeqr egr 1o rred lsrrJ eqr uI ieueJqureu eql ssoJJEJaJsuEJl 'V Ieuortrerrprunse^rrp wql KBnua Jo eJrnos eql sr reql1 isuretuopuroto.rdpJplo; atepouurofJe lr llr,ry\lo 'alels peplotun ue ur dluo q8norqr sseduer suralord leql ,{orreu os Ieuueql eqt sr 'relnrrlred u1 i.radelq oueJqurou oqt ssorf,€suralordJo reJsuprts.^aol]E wql Puupq) uotlr2?olsup4eqr Jo ernlrnrts eq] sr rEqlN 't

'z lecuanbasleu8rsaqt rc1 rclQatat oqt sI rel{lN ;seruenbasleu8rs;o sad& raqlo IUory1I saqsrn8url -srp teq.ry\pue 'atuanbaspu8rs oqt to ornleu JI{r sI leqlN 'I :suortsanb rnoJ ro^^suEol >loesl[,ry\e^'\tardeqr lEluaurPpunt 3urla3rel-uralordagl to qlPa roc srqt ur Pessnlsrpslua^,re 'palelduoc sr oupJqlrrerueql ssoJf,?uollefolsueJl eJuo saseelordrryrcadsdg uralo.rdoJnletu oql uroJt PeAoIuoJoJ€ ueuo soJuenbesyeu8rs'dleurg 'suratord roldore.r Jeqlo PUP saruonbasleu8rs.raqlola.{ uo spuedapSurlrosqons lelleue8ro le8rel eqr ulqll^/r luaurl.reduof,qns e qf,EOr ol requnt pauos dlluanbasqnse:e sutelord aruos AIV ;o stsdlo;pdq sE qJns ssarord e]qero^E dllecrle8reuauE ol uollelolsuerl Surldnor dq pa,rerqcr dllensn sl 'useldoldf, oql olul lno >1ceqSurp{s lnoqtll\ 'alyeue8touP olul uralo.rdE Jo raJsuerl Ieuonrarrprun erll redepq euerquou aqr q8nolqr ssed ol ureto.rdeqr s.^oll€ l:-tll Puueq) uotlo)olsuotT to PUDIauos ol porreJsuertsI ulel{f, utalo.rd aq} loldof,er Surpuodsa:ror aqr qlr/v\palJEJOluIseq acuanbasleu3rs u Sututeluoc utalord p aluo 'surta8rul 1o dlnr;roeds aql suralo8 ecuonbasleu8ts .seJuenbas e ur pepof,uouorleuroJur JI{} reqt Sut.rnsuesnql leu8rs ;o spur>1cr;rcedsol dyuo PuIq rpgr sutalord roldacar Jo les e serrrer allaue8ro qcea'sap4f,ad lpuBrsto sacuanbas Su4a7nx-a4wdn pe11etoslr aru esaqr :(1-E1 arn8rg eas) 'sptce ourtue saruanbespu8rs su dlporraua8 u,vloul 05-02 ';1eslt utelord eqr ;o ocuanb ;o seruenbes urqtl.ry\dllensn -es prle ouILuE eql ulqll.ry\poPorua sI uolleullsop a11aue3 -ro relnrrued e ot utelo.rd v le?rct ol uoIlEIuroJuI agt let{l 'ef,uelsurro; 1r,roo1A{ou el( 'salleur8ro JelnllaleJlul snoIJEA aqt IIE ol Surl.rosutoto:d uralo8 susru?qJalullseq elues el{l

'suo'Er',reuosarrdsep r'gr u^ oqst^"U ttf,iJi"ilirq*U*,

's;edeltq pelelndrueru ,(1aa:;aq uEf, stuolsds orlll uI qcn5 prdrl lenr;rup otul palerod.rocut sluauodtuoo urelo.rd peg -rrnd aql Sursnpetntrlsuorer ueoq e^Pq IIOI eqi ut sassalo-rd 'uorltPPP uI 'seuerg uorlprolsuErt rolpur eql Jo duetu -uer.u rElnllerPrlur lueraJJlpeql Jo I{lPe ssorf,?uollef,olsuerl ro; pa.unber stueuodulof, relnllel eqr Jo dueru pet;ttuept a,reqstsrSolorq11ar'sdatsuorleJolsuerl relncrlred elnJexJ ol elqeun sluulnlu Surd;rruaprJoJ suooJf,srtleuaS Pu€ spoqreur 'sa1nr uorlecr;rrnd leJlrueqlolg Jo uolleulqruoo e Sursn -elour llprus pup suol ot reureq e se .radepqaql Surldnrsrp lnoqtr^r euErqtueluP ssoJrepelerolsuErl eg Plnol salnJalou pue auerglueu cr;nads euo dluo uretord a8rel dlezrrreler,vloq ot potrerrp eq plnor utalo.rdua,l,t8e .ttoq :3urg;eq attnb era.'rn 'JoIJ d11erlrurssocord Surlros-utelord srql Jo saJnlee' oA/rI -elur slr olur peloru Jo eueJqueu o1laue8roJI{l olul pelJesul dlluanbesqnspue sellaueSrorrlnllef,Erlul Jo seuerqlueu eql ol pala8rel are sutalord ^\oq euluexa a.vrleldeqc :,#l""lrr", -leferlur Jo seuprqrueruaql ol Surla8rel utalo.rdruor; .{lluec -r;ru8rs sJeJlp lr dlleousrueqlatu asnef,agrordeqr lxJu JI{l 'I ur Surlros uratord Peseq-olllsa^ssnf,slPe11.'@oqPapvqs -91 arn8rg aes)raqloue to euerqrueu aql qllltl osn; uaql pue auo Jo auergueu oqr tuo{ Pnq 13qrself,Ise^IIPtus e1laue3.ro ,(rolercaseqr 3uo1epelrodsuerl a.re Jo uorlfe aqr dq dezrrqrBd 'r31o5 eql sI uolleullsoP af,Erns lyar ro 'aruosos.,{1 IBUI}esoq,,la

s l ' l ' l l N V 9 u o c N V s 3 N V U S l A l l Ao l _ N t s N E I o U d 9 N t n o N

u a e q r o t a u o s o q r r e q l s t J e r t p u r a t o J d t u e J s E ua q l u r 'losotdr eqt ur seruos af,uonbas UA enprser-0€ot -9I e Ieu8rs -ogrJ eeJJuo sur8aquratord d.rola.rras e ;o srsaqtudsJaUV ul aql ol sulaloJd Alolelra5

luo)seN sle6tel

a ) u a n b o sl e u O l Sl e u l u r a l - N ) t q o q d o r p f HV g3 qbnoraq] +o uauJnloql ol luole^tnbo'soLlosol)tu.l eql optsul aresurelordepeu Ilnnauaq] ]eq] solp)tpur6urpurlsrql luablelepqlrm ]uaurlparl{q pa,{otlsep}slt}st auetquiau Ieuloso.l)tru oq} }o lar.lleq ifrlrqeauradeql 1rIluo eseelotdeq1{q palseOrp ele sou.]osolltuJ eq} q]lM paler)osse surelordfuoleltespalaqeloql luebtalepe +o o)uosqe ro a)uosordaq] ur oseo]oldp q1^^paleol] aje soLuoso.t)Lll perltnd eq1 asol)nspue let}uola+}tp 16raldeql) uorlebnltlua>luerpet6-,{1suep e Iq ulaq+u.tot1 peletedaseq ue) pue sa;leuebro ]o uorleurqulo) snouprqurolr roqlo ueq] {lrsuep1ue,4onq teleetOqfnul e aneq 'solxosoqu sotuosor)rLU punoq aneq{eq} esne>egsau;osol.l/upalle) sal)rsa^ euseldeq1 lleursolut g3 qOnoteq16uueaqspue oue..tqLUaLU 6uunlrer,r'pezruabouoqole ueq] sllo)oql poloqelaulo)aqsurelotd pezrsaqluris {lnneu{;uo }eLllos spt)eoutL|epalaqelotpej q}tMoLUt} e Jo] palpqn)ura.resllo) srsaqlu{s ta11e {;poqs uaLUnl +ar.rq !l aql o} pozrle)ol eresurelotdfuolettes alellsuoLuap slueutuadxa 6urlaqe1 lpq] 'Ul oql regua suralo.rdtuo1a,oe5 €-€t tunglJ lvl_NllAjlUldXJ < urolojd Alololsas 1 o u o r l s a b r po p t t

t t

u ralord Joleloas ;o uor1se616

t

:tr4 L r ' #

t

|

€t Ull_dVH)

9€S

'ouErqujeur uE aqt ssorf,Epepeerql sr uratord .{rolarcaslualseu E Jo snurrurel-N er{l ,ra,oqpue sur8 -ag srsaqludsrraqt reue .{llroqs surato.rddrotertes se pazruSo -rar ere saprtdaddlod .4orl Jo uortsanbeqt pasrersrqt se qtns stueruuodxg 'sallrsel ueunl oql un{tr^^ dn pue sauros UE ]o -oqrr aseqt ,{g parrpord seprrdad.,tlod eqt (auerquetu Ug aql Jo arpJ rrlosotdr aqr or punoq sauosoqu uo pazrsaqtu.{s are surato.rdd.rola.rcas q8noqlle tEql elertsuoruap 'asealord e qlr.^apeluen ore sller palaqey-as1nd Luo{ palelosr seruos -orrnu qrrq.{\ ur 'g-91 ernSrg ur percrdapstuaurr.radxaeq1 '(eprslnoeqt uo saruosoqu) IJaJttetur eql ur puno; teql se

(e)upd] a)uanbasleu6rsX3 I opeled9 1o,4sapno; ue 6urureluotaprided{1od e 1osrsaq}u,{s uralord6uunp uJnln)t}al >rLuseldopue aq] ol po]rnr)ar1a6seulosoqupunoq-ouelqulon le)rluoprare seulosoqutrlosolI: aarJpue punoq-auelqulou]eqt oloN Ul eq1uo srsoqlu,{s ure}ord1o uorleluasa.rder )r}puloq)S(q) surelordrtolerlesuouloqlo lo trlosol{t 6urzrsaqlu{s ole asaLl} Alqeunserd1luapr^o oJesoLL]osoqt.t (aal])porl)eueun-ouelquta[! Mo] uo pau]lo]ole pue pe]oj)os0q o1 v sourosoqupoqle]le-auelquaLu arp lla) ,roadi{tsrq}{q pezrseqlu{s surelotdoq} +o }solA llo).reut)e rrleer>uede ur g3 q6noraq] o] poq)e1]esoLuosoql1o qder6ottrut uoj])oll (e) 'Ul q6nor aql lo arnpnlts Z-€l lunglJ V

AI rt

t

uolunlH3

+

auerqursruul aulosoou P"qrntiV

luebrelep qllM leall

losol^C

s l r un qn s leurosoqru

saruosoqu poqcelle qu,v\ saruoso,tctli^l

(q)

f f i / l"^ f f i nl^

, . , " f.l8f

v{$

L

l-- *-I

$'sE'

'

,

rrrrl9'6

Qqr

w

I ,

;:.

i , ...,'., l, :r,:.., '.$,1'1

suerquror.rJ uf

uaurnl uf

losol^3

Lts

l N V U S t A l t A U l l H - L S S O U f VS N t S I O U dA U O I I U ) l S l O N O T I V ) O ' l S N V U I

rau€ saurl ruOrOtJIPlE sarnlxlu uoIlJEer egl or PepPP aqt uo rotderor dUS eql ol Surpurqlq auerqueru UE Jql oJeA\ satuosolcnu tsJuJurrradxeJSaI{l uI 'JnJJo ol uoll -pJolsuErtrot JOProut luasa.rdJq lsntu sJtuosoJf,Iulqllqr^ ol xalduor auosoqrr-uratordluacseueql sle8rel urqt dUS lxaldruor a3.re1e Sururol 'lrunqns Ipuosoqrr a8.reyaql se le srsaqtudsutatord 1o a8els astrard oql eullurataP ol sp uretord tuerseu E ur aJuanbesleu8rsUE or{rqtoq or sluorurradxaluanbasqn5'(y-E1 orn8rg) srnc peuSrsapere,ry\ IIO,,\\ -f,o seruosoJf,rru sputq dlruetsuerlteql alrrtred uralordoalonuoqrrrrlosotdc eql olur lJodsuerl urolord ou 'PolalduoJ p sl eqr 01 POPp?ere sotuosorr eqJ 'ouprqtuetu oql ur parelol 'roldere.r sr srseqtudsuralord ralJE IUOls.,(s dUS UE -rr.u 'srsoqtudsuralord Surrnp luesald erE saluosorf,rluoql str pue (495) oprlred uorlruSorar-leu8rs eql arp 8ur JI -la8ret srql ur stuauodruoc .{a4 o,uo oql 'arar{t uaqt }a8-ret dluo peloruoJ acuanbesI€UBISsll sEL{Pue seluosoJJltu ;r uortruSorerecuanbas-leu8rs e'auerqueru olur pelerodJorursr uretoJd.{rolarcaslecrd& P lPql POlEJls lsntu tusruEqceru -uouep urotsds slql qll.&\ sluaruuedxe IEIIIuI 'sof,uonb relnllor ror{lo due rltr^{ tou tnq euerqr.uau Jqr qtr,lr UE -as uorterJosse ur pazrsaqludsa;e sureto.rddrolarces acurg leu8rs UE Jo alpJ pup uollrunJ aql PaIJITEIIa^Pq sotuosoqrJPunoq u^\o Jlaql ;o paddrrrs soluosorflul Pue surslord6urz^;o.rpAH-dIg o^ I ^q 'tualsdsSutzrseqluds 'ulelord drolaroase Surpocue VNUtu p a l e r l r u ls l u o r l e ) o l s u e 4l e u o t l e l s u e J l o ) -uratord salPnts e 3utzrlr1n rerJ-lloJ IerllueqJoIfl .auerqrue{u ua eql 'uorl9Jolsu€Jl sE ol uretord eqt Sulla8r€l ro; elqrsuodsardreutqceuraql qll^{ IeuorlEIsuPJlof, ol peJlelerssaro.rd€ 'oLuosoqrJ agr or punoq ylrlssr urato-rd sefuenbas 1eu3rs;o uollf,eJolul eql Jot IefIIIJJ SI lEr{l elIS (tuarseu)pazrsaqruds dlaralduocur aqt elrr{1\^. sur8aquorunl Surpurq € ruroJ soJuenbasleu8rs gE Jo erof, aql uI senplsor rrqoqdorpdq aqt snqJ 'uaunl Ug aqr ol pererolsuerl UE aql olur sutatord drotarros lsolu Jo trodsueJl aLItsnql '(97-y an3rg aas) tllosoldr pelllpotu P qlns 'rrqoqdorpdlJ PUE auosogrr er{t ur sr uralo;d ]auupr{re urqlr^.\porrnq ';;o sI acuanbasPaPPBel{l Papl^ord 'saJuenbas dlluarcrttns aq 3uo1 pe^eell Ilrls arp sprlB ounuE os Jo 0t lxeu eqt pue 'auosoqrr ourrue Ieullurol-N PaPpB tltl./lr surolord rrlosol,{r aql prre llr./!\ralel teqt eouanbasleu8rsaqr Surpnlrur ruorj aPnrlord os .ro sprrE ourruE0t rsrrJer{l 'lurod srqt tv parnpord e^Eq srel{rruasal'sanbruqlJrVNCI lueulqulorer 'uatunl 'ueunl aueJqlueul Ug oql ssorJ ol alqeun eql otul Sursn IeruosoJJrrueqt ur pozrletol oq ol uraloJdf.rolanas (aJuanbasuofE '1osol.{ceql uI suleruarurelord patalduor egl ro; ropro ur reqraSotprlull ere sprru ourrup PorJrpou oql os ro 0/ lsrrJ eqr eroteq popp€ 0q tsntu seuosorf,rr.ulEr{l sy 'aruanbesleu8rse se uorlJunJ ol uralord e to snulluJel-N sluarurrodxeasaql 'un8aq peq srseqtu.(suratord aqr;o dlrlrqeaqt qsIIoqEupr uolletntu dq eror rtqoqdorpdq pJ,ln.oqs oql otur spne ouILuEpa8reqc to uoltrnporlul eql ro aruanb -as yeu8rse ruort spIJE oultue crqoqdotpdq el{l Iero^as Jo (etuelsul 'uollJun; rIaIJrroJ roC IEII to uortalep orlcads oqt -uassosr seouanbas gg eror crqoqdorpdq eq1 leu8rs Jo acuenoes 'sllor ur PunoJsurol ;eubrs1o le^ourorpue ecuenbes;eubrs ourosoJcr.lr olur urelo.rd;o -old at{l uI luasard tou dyyensne:r sacuanbespu ,,elnleru,, l n o q i l Mu r e q c u o o s u e r ll e u o r l e l s u e r l o c -8rs snqr lauosoqrr oqt uo Surle8uola llrls sr 1r alrqzrtutalo-ld uraloroarnleN eqt urorJ pe^po]Jst oluanbasluu8ls aql'surelord drolercas '(a.ror lsoru roJ 'uotuluoJ ur JIDII a,req .,{aqlJSLi!\Jeqlolnq aql) senprsarcrqoqdorpfq U-g Jo qllorls snonulluoJ E ot tuarelpe splfe oultue pa8reqc dle.ttlrsodeJotu Jo euo uIEl -uol suralo:d drolarcasluarotllP Jo sef,uenbesleu8rs aq1 IIE 'pazrsaqtuds ag ol uralord eqr;o tred lsrlt eql 'uralord aqt 1o snurrural-N erp lp patelol sr dllurrdll aruenbasleu8rsgg uy '({tt't-tt arn8rgeas)auerquraruUfl aqr ssorreeprrdaddlod Sur.r,rorBoql to uolleJolsuEJl saleIlIuI Pue auPJqluetu :srsorllu^suralordoor]-lloJ (q) lueserdsaurosorcrLU

ecuanbes;eubrs OU JOleAOUrAr :soLuosolcrLU olul u o r l e r o o r o c uorN

t

sauerq urour ppv eLuosorcrur luasardsourosoruluou lsrseqluAsurelo.rdoorl-lloo (e)

uralotd6uttnpluesardeleseuosolltu.t (q)stsaqlu^s ;t {1uo al)lsen aql ssol)epale)olsuel] s1tsasolpueeuelqulaLu a>uanbes leu6ts stutalold oql ul pozlsaqluAs srlnq (e)saulosol)tul ]o a)uasqe q)lqMo] e 6utpoluaVNUUr aql pappestutalotd,to1er>es fuoler>es 6uturel puei11g'dIV'sVNU]'saulosoqll >r1oso1b ser-ul{zue leuot1>un; -uot uels{soarJ-llo) (g-g;etn6t3eas) e urlno paule)stsrsaqlu,is jo oal,-auosoqll aleq)tqM'sottlosor)ll.lJ souerqurauJ Ul o] luelentnbo 'seurosoqtJ pelPl)osse uleqlsdtlls'suot.*661 6urrrno;;e uorielosr ]o q)lqM'vlcl q]rMsaLuosol)lul solelaq) +o]uoulleo{ uol}elsuej} olut sutelotd,fuolallas oi peldnorsrsauiosol)tu.t +ouollelolsuell ln))o aaj]-lloff ;snoauel;nu!s siuer-utladxe ]eq}alerlsuoruep pue uolle)olsue4 t-€l lun9lJ'lvINltNluldxl < uorlelsueJl

s r r ' E N V 9 U O C N V S t N V U S I A 3 I AO t _ N tS N E I O U d 9 N t n O N

I

tr UtIdVHf

8ES

seop auerqueu UE eqt ur roldalar eqr ot punoq ser1 ue loldaf,or eql r{lr^\ 8ur>llop dg auerqruou UA eqr dUS dUS xalduror euosoqrr/urerlf,luaJseu/d1s oqt rarle dlug .UA aql o1 xaldruor eruosoqrr-urpr{f, tuJtseu ogl s8ur.rqdUS ar{J 'uetunl otul uortef,olsueJtro1 3uo1oot SunuocaqruoJJurer{f luef,seu UE ar{t olur uortef,olsuerl uralo.rdro; perrnba.re.re eqt slue.tard euosoqrr aart or{l qtr.a pue uralord d.rota.rcas ro atuosoqu eqr qrr.,talJpratur dUS aqt ur seprrdaddlodraqrg 'auprqrueu e Jo ulel{r luef,seuaqr i{roq qrl. dus aql uorlreretur 13r{l pue suretord Jo UE ar{l ol uroql sla8rel ,,(1o,rrna1as stseSSns Surpur;slql'(t-gI arn8rgees)uralord eralduroceql f.role.rrasluerseu;o sleu8rsleurr.urot-Ncrqoqdorpdqeql qtr^ 'luasqe ere seurosorf,rr.u ueq.v\ slf,protur tpqt UOIJsnoSoleueue ureluof,or rq8noqr sl tgd to ;o srsaqludsSurrrqrqurdqareqr uralord d.rola;case ;o uorte8uole s,4^olsdUS Jo acuasardeql uor8arorqoqdo-rp,(q eqI'(eS-tI orn8rg)sureql eprstrqoqdo.rp -dq dq paurl sr of,€'rnsrouur asoq^\ 1{1snor,r,ard paqrrtsep tuetsdsuorlplsuprt aa{-llel eqt uJ Uelr E surrluoJ W{ Jo ernl 'slleJ -lnrls eql Ierrelleq Jo euprqruerucnuseldoldrer{tssorf,E oqrpu' sutalord d.role.rcas A,rrrrrrqtl,ll9:ilJfi suletoJd uorlPf,olsuerl aqt ul luef,s'u ol uorlJunJ snoSoleue tsd Jo 'WC sE u.4^ou{ urelo.rd -IJ uOaIUequorlf,EJalur rar{louB punoJ elErlrur o1 ,{pea.r ue sruro;rad qlFI^\ to lprro1ceqe ol sno8 'dCC -oloruoq sr surur1o ore qrog pue 'losotdc oqr ot selcdre.r punoq aprsorqogdorplg qrrzusJnprserprre ourtue dUS {req 'raqro rraql eseeler rlreo roldarer slr pue ruorJ dueru qleo SurureruoJtsd 1o uor8er V 'uraloJd drolarcesluotspu 495 Sutlrroossrp raUV 'ereld elet uer uorterolsuert ereq^. e ur acuanbesleu8rs oql or spurg ter{t trunqns eql sr urato.rd 'auerqruaur relncrlred sql teqr eruapr^e 'sacuenbospu8rs gg ot pa]url Ug aqt uo elrs e ol oruosogrJpue urpr{l luof,seu 'roru -ssorcdllecnueqr aq uec (794) suralord aqt JoJSUEJ] solertrur'pootstapun sr Jeuuer.u E ur tou lEql ,o dUS oql Jo euo 'pue roldacer -Px3qel{l rot ploJJslsE s3 slJe qrlr{^ 'vNu rPrroepnu-Oo€E pue eqr ,{lqruassesrp sarueduoc dus dus ;o 'ssacordsrql ur roldarer -lp dJC punoq aqr 01 punoq saprrdaddlodalorrsrpxrs Jo dn epetu sr dUS eql 1o srsdlorpdll str pue dUS eql Jo elor erJrpue srsaqludsuralord drolarces luarrnf, Jno sezrreruuns9-€I orn8rg ;o Surpuets.repun 'salnrelou dJc Punoq qroq dtc Sutzlyo;pdq yo alqeder ere leqt setrs olrlfe atalduoc o.,ia,l ru;o1 daqr taqlaSor eruoJ sureloJd o,,urraql ueqm lnq ,41g ;o srsdlorpdqer{t roJ etrs JAurE etaydtuor p suretuof,ouolp llungns reqtreN'reurporataq lecr.rlauturds-opnesd E ruroJ o1 raqlaSol aruot dJC Jo alntoloru a13urse ol punog qlee 'surato.rdasaqr ISIC pue r{J{ ar{l ler{t s,vroqsqg-91 ern8rg to uonerf,ossrppue Surpurq oqt e^rrp uer srsdlorplq pue SurpurqdIC Jo e1c^,tr e ^ oq otur lq8rsur sapr,rord,snxlonbo snwnql errotreqeer{Jreer{l ruo{ '(IsrC) trunqns D roldof, -or dUS eqr pup (WS) aUS to trunqns tgd aqr yo sSoleue;o ernrrnrts eql alc ot punoq ere roldalar dus aqr to lrunqns r0aqr pue dUS to lrungns tgd er{t qroq ueq^\ pouaqr8uerrssr rolderer dUS oqr qlr.Lrxalduoo auosoqrr/urer.lrtuelspu/dUs ( l r u n q n?s9 dd u s ) (llunqns7rroldocordus) aqt Jo uortrerelul 'trunqns fl .ro11euse pup lrunqns D ue r.tJl Asrl :sllunqnso^\l to dn apetuauerqrueudE aqt uratord (q) Jo le.r8atur 'VOOZ' (q)Ued I t1€:€0€a]ual)S' le]a er)o1f iJLlor]pa]depe '966 ' urol]peldepe (e) LgLtt6 ]a ueuaa) 1 ila) ]redl le [ U taLUtp aq]1o{;qu.ressesrp 6ursnel,o)e+la}ut eq}sozlllqe}sop dCg o1srs{1olpA;1 soln)aloLU eq1to1 d1g punoqq}oqrrosrs{lotpIq sa]rso^rpeoMl ]o uotleullo]sMollelauJlple)ulaL]lLrLris-rues aq11o {lquressyloultpaq}oztltqe}s pues}tunqns urelotdeq1uoaM}aq O)elrelur aql ul llj dfg jo saln)alouJ punoqo^^iaq] laLllo q)eao] pulqAsll pueqlj uoqMpuet1D ]o oln)alouJ auoo] putq ue) q)pe pue sll qlg pue srs{1otp,iq pallotluot 6urpurq {q 419 srsuralord asoq]uoaMlaq uotlfelalut oql Moqsalellsnllt surelotd q+lpue(lolda)ol D aq] Oolouoq ]runqns dUS lo +o lellal)eq aql) sll o] punoqdf9 ]o olnlrnrlsaql (q) sa>uenbes leu6rs q]rM surell) apts osoqm (aldlnd) ])eralur spt)e ouL!e trqoqdotplq qllMpaullUel)o6rele sureluo) q)lqM'qll urureiuop oulpurq eql sMoqslopouro)ellnssrql setuanbes Ul spulqielll tsd +o leuOrs uorlrodeqto1sno6o;ou.roq sruralotdqJJleua])pq oq1(e)'(dUS) epryed uorlru6ore.r-;euErs eq1lo elnpnrls S-€l lUngll >

(dus lo lrunqnstgd ol polelor) u r e u r o p6 u r p u r q - a c u a n b e s - 1 e u q6 ; 3r s

ul aql olul

uorlp)olsuBJl utalojd ut €l)tupd uotltu6olcH lBUStSaLll l0 uorl3unj pue ajnl)njls :lselpod

ll[]

FocusAnimation:Synthesisof Secetedand Membrane-Bound Proteins mRNA

SRP

Signal sequence

SRPreceptor

GDP+ Pi

Translocon (closed)

Translocon (open)

-tl I l , t-aLa \tt\k ti....F a/ a \ sat aa a f

Folded protein

FIGURE 13-6 Cotranslational translocation. polypeptide Steps[, [: chainelongates, it chainStepE: Asthe polypeptide Oncethe ERsignalsequence emerges fromthe ribosome, passes it isbound intothe ERlumen,where throughthetranslocon channel (SRP). by a signal-recognition particle StepS: TheSRpdelivers the thesignalsequence iscleaved by signalpeptidase andisrapidly ribosome/nascent polypeptide complex to the SRpreceptor in the ER degradedStep6: Thepeptide chaincontinues to elongate as membrane. Thisinteraction isstrengthened by bindingof GTpto the mRNAistranslated towardthe 3' end Because the ribosome boththe SRP anditsreceptor. Step4: Transfer of the ribosome/nascent isattached to thetranslocon, thegrowingchainisextruded polypeptide to thetranslocon leadsto openingof thistranslocation throughthetranslocon intothe ERlumenStepsfl, S: Once channel andinsertion of the signalsequence andadjacent segment iscomplete, is released, translation the ribosome the remainder of the growingpolypeptide intothe centralpore Boththe SRpand of the proteinisdrawnintothe ERlumen,thetranslocon closes, SRPreceptor, oncedissociated fromthetranslocon, hydrolyze their andthe oroteinassumes itsnativefoldedconformation. boundGTPandthenarereadvto initiate theinsertion of another

SRP releasethe nascent chain, allowing elongation to continue at the normal rate. Thus the SRPand SRPreceptor not only help mediate interaction of a nascentsecretoryprotein with the ER membrane but also act together to permit elongation and synthesis of complete proteins only when ER membranesare present,

Passageof Growing PolypeptidesThrough the Transloconls Driven by EnergyReleased D u r i n gT r a n s l a t i o n Once the SRP and its receptor have targeted a ribosome synthesizinga secretoryprotein to the ER membrane, the ribosome and nascentchain are rapidly transferredto the translocon, a protein-lined channel within the membrane. As translation continues, the elongating chain passesdi-

rectly from the large ribosomal subunit into the central pore of the translocon. The 50S ribosomal subunit is aligned with the pore of the translocon in such a way that the growing chain is never exposed to the cytoplasm and is prevented from folding until it reaches the ER lumen ( F i g u r e1 3 - 5 ) . The translocon was first identified by mutations in the yeast gene encoding Sec61o,which causeda block in the translocation of secretoryproteins into the lumen of the ER. SubsequentlSthree proteins called the Sec61complex were found to form the mammalian translocon: Sec51cr, an integral membrane protein with 10 membrane-spanning ct helices,and two smaller proteins, termed Sec61B and Sec51"y.Chemical cross-linking experiments demonstrated that the translocating polypeptide chain comes into contact with the Sec61a protein in both yeast and

T R A N S L O C A T I OO NF S E C R E T O RPYR O T E I N A S C R O s sT H E E R M E M B R A N E

539

ArtificialmRNA

Cytosol

Sec61o Microsomal membrane

Crosslinking agent Nascent protein

Microsomal lumen

NH:

EXPERIMENTAL FIGURE 13-7 Sec61cis a translocon component.Cross-linking experiments showthatSec6lcrisa proteinsas translocon component that contactsnascentsecretory theypassintothe ERlumen.An mRNAencoding the N-terminal wastranslated in 70 aminoacidsof the secreted oroteinorolactin (seeFigure13-4b). The a cell-free system containing microsomes mRNAlackeda chain-termination onelysine codonandcontained a codon,nearthe middleof the sequence. Thereactions contained chemically modifiedlysyl-tRNA in whicha light-activated crosslinkingreagent wasattached to the lysine sidechainAlthoughthe polypeptide entiremRNAwastranslated, couldnot the completed fromthe ribosome be released withouta chain-termination codon andthusbecame"stuck"crossing the ERmembrane. Thereaction mixtures thenwereexposed to an intense light,causing the nascent proteins chainto become werenear covalently boundto whatever it in thetranslocon. Whentheexperiment wasperformed using microsomes frommammalian cells,the nascent chainbecame linkedto Sec61oDifferent covalently versions of the prolactin mRNA werecreated sothatthe modified lysine residue wouldbe placedat different distances fromthe ribosome; to Sec61c was cross-linking observed onlywhenthe modifiedlysine waspositioned withinthe translocation channel[Adapted 1992,Science fromT.A Rapoport, 258:931, andD Gorlich andT.A Rapoport, 1993, Cell75:615]1

mammalian cells, confirming its identity as a translocon component (Figure 1 3-7). lfhen microsomes in the cell-free translocation system were replaced with reconstituted phospholipid vesiclescontaining only the SRP receptor and Sec61complex, nascent secretory protein was translocated from its SRP/ribosome complex into the vesicles.This finding indicates that the SRP receptor and the Sec61 complex are the only ERmembrane proteins absolutely required for translocation. Becauseneither of these can hydrolyze ATP or otherwise provide energyto drive the translocation, the energyderived from chain elongation at the ribosome appears to be sufficient to push the polypeptide chain acrossthe membrane in one direction.

540

CHAPTER 13

I

The translocon must be able to allow passageof a wide variety of polypeptide sequenceswhile remaining sealedto small moleculessuch as ATP and amino acids. Furthermore, to maintain the permeability barrier of the ER membrane in the absenceof a translocating polypeptide, there must be some way to regulate the translocon so that it is closed in its default state, opening only when a ribosome-nascentchain complex is bound. A high-resolution structure of the Sec61 complex from the archaebacteriumMethanococcusiannaschii was recently determined by x-ray crystallograph5 which suggestshow the translocon preservesthe integrity of the membrane(Figure13-8).The 10 transmembranehelices of Sec6lcr form a central channel through which the translocating peptide chain passes.A constriction in the middle of the central pore is lined with hydrophobic isoleucine residues that may form a gasket around the translocating peptide. In addition, the structural model of the Sec61 complex (which was isolated without a translocating peptide and therefore is presumed to be in a closed conformation) reveals a short helical peptide plugging the central channel. Biochemical studies of the Sec51complex have shown that the peptide that forms the plug undergoes a significant conformational change during active translocation, and researchersthink that once a translocating peptide enters the channel, the plug peptide swings away to allow translocation to proceed. As the growing polypeptide chain entersthe lumen of the ER, the signal sequenceis cleavedby signal peptidase,which is a transmembraneER protein associatedwith the translocon (seeFigure 13-6). Signalpeptidaserecognizesa sequence on the C-terminal side of the hydrophobic core of the signal peptide and cleavesthe chain specifically at this sequence once it has emergedinto the luminal spaceof the ER. After the signal sequencehas been cleaved,the growing polypeptide moves through the translocon into the ER lumen. The translocon remains open until translation is completed and the entire polypeptide chain has moved into the ER lumen. Electron microscopy of the Sec61complex isolated from the ER of eukaryotic cells reveals that three or four copies of Sec51acoassemblein the plane of the membrane.The functional significanceof this association between translocon channels is not understood at this time. but the oligomerization of translocon channels may facilitate the association between the translocon, signal peptidase, and other luminal protein complexes that participate in the translocation process.

ATPHydrolysisPowersPost-translational Translocationof SomeSecretoryProteins in Yeast In most eukaryotes,secretoryproteins enter the ER by cotranslationaltranslocation.In yeast,however,some secretory proteins enter the ER lumen after translation has been completed. In such post-translational translocation, the translocating protein passesthrough the sameSec61translocon that is used in cotranslational translocation. However, the SRP

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

(a) Side view

i

;

;n PIug

(b) Top view

P o r er i n g

Lateralexit to lipid bilayer

Plug removeo

EXPERIMENTAL FIGURE 13-8 Structureof a bacterialSec61 complex.Thestructure of thedetergent-solubilized Sec61 complex fromthearchaebacterium (alsoknownasthe Secy M jannaschl complex) wasdetermined (a)A sideview byx-raycrystallography showsthe hourglass-shaped channel throughthe centerof the pore A ringof isoleucine residues at theconstricted waistof the poremay forma gasketthat keepsthe channel sealed to smallmolecules even polypeptide asa translocating passes throughthechannelWhenno peptideispresent, translocating thechannel isclosedby a short plug,Thisplugisthoughtto moveout of thechannel helical during translocation Inthisviewthefronthalfof proteinhasbeenremoved to bettershowthe pore.(b)A viewlookingthroughthe centerof the channel showsa region(onthe leftside)wherehelices mayseparate allowinglateralpassage of a hydrophobic transmembrane domain intothe lipidbilayer. fromA R Osborne etal, 2005.Ann Rev. [Adapted CellDev.Biology 21:529l

and SRP receptor are not involved in post-translational translocation, and in such casesa direct interaction between the translocon and the signal sequenceof the completedprotein appears to be sufficient for targeting to the ER membrane. In addition, the driving force for unidirectional translocation acrossthe ER membraneis provided by an additional protein complex known as rhe Se;63 complex and a member of the Hsc70 family of molecular chaperonesknown as BiP.The tetrameric Sec63complex is embeddedin the ER membrane in the vicinity of the translocon. whereas BiP is

within the ER lumen. Like other members of the Hsc70 famlIy, BiP has a peptide-binding domain and an ATPasedomain. These chaperonesbind and stabilize unfolded or partially folded proteins(seeFigure3-15). The current model for post-translationaltranslocation of a protein into the ER is outlined in Figure 13-9. Once the Nterminal segmentof the protein enters the ER lumen, signal peptidase cleavesthe signal sequencejust as in cotranslational translocation (step [). Interaction of BiP.ATP with the luminal portion of the Sec53complex causeshydrolysis of the bound ATR producing a conformational change in BiP that promotes its binding to an exposed polypeptide chain (step [). Sincethe Sec63complex is located near the translocon, BiP is thus activated at sites where nascent polypeptidescan enter the ER. Certain experimentssuggest that in the absenceof binding to BiP, an unfolded polypeptide slides back and forth within the translocon channel. Such random sliding motions rarely result in the entire polypeptide'scrossingthe ER membrane. Binding of a molecule of BiP.ADP to the luminal portion of the polypeptide prevents backsliding of the polypeptide out of the ER. As further inward random sliding exposesmore of the polypeptide on the luminal side of the ER membrane. successive binding of BiP.ADP moleculesto the polypeptide chain acts as a ratchet, ultimately drawing the entire polypeptide into the ER within a few seconds(stepsB and 4). O" a slower time scale,the BiP molecules spontaneouslyexchangetheir bound ADP for ATP, leading to releaseof the polypeptide, which can then fold into its native conformation (steps E and 6). The recycled BiP.ATP then is ready for another interaction with Sec53.BiP and the Sec53complex are also required for cotranslational translocation. The details of their role in this process are not well understood, but they are thought to act at an early stageof the processsuch as threading the signal peptide into the pore of the translocon. The overall reaction carried out by BiP is an important example of how the chemical energy releasedby the hydrolysis of ATP can power the mechanical movement of a protein across a membrane. Bacterial cells also use an ATPdriven process for translocating completed proteins across the plasma membrane. In bacteria the driving force for translocation comes from a cytosolic ATPaseknown as the SecA protein. SecA binds to the cytoplasmic side of the translocon and hydrolyzes cytosolic AIP. By a mechanism that is not well understood, the SecA protein pushes segments of the polypeptide through the membrane in a mechanical cvcle coupled to the hvdrolvsis of ATP.

Translocation of Secretory Proteins Across t h e E RM e m b r a n e r Synthesisof secretedproteins, integral plasma-membrane proteins, and proteins destinedfor the ER, Golgi complex, or lysosome begins on cytosolic ribosomes,which become attachedto the membraneof the ER, forming the rough ER (seeFigure 13-1, left).

T R A N S L O C A T I OO NF S E C R E T O RPYR O T E I N A S C R O S ST H E E R M E M B R A N E

541

Translocating polypeptide chain

Translocon

ER lumen NHs* Cleaved s i gn a l sequenc

BiP (bound to ATP)

FIGURE 13-9 Post-translational Thismechanism translocation. isfairlycommonin yeastandprobably in higher occurs occasionally random eukaryotes Smallarrowsinside thetranslocon represent polypeptide slidingof thetranslocating inwardandoutwardSuccessive prevents bindingof BiP.ADP to entering segments of the polypeptide the chainfromslidingout towardthecytosol[See K E Matlack etal, 1997 277:938 . Science I

r The ER signal sequenceon a nascent secretory protein consistsof a segmentof hydrophobic amino acids, generally locatedat the N-terminus. r In cotranslational translocation, the signal-recognition particle (SRP) first recognizesand binds the ER signal sequenceon a nascent secretoryprotein and in turn is bound by an SRP receptor on the ER membrane, thereby targeting the ribosome/nascentchain complex to the ER. r The SRP and SRP receptor then mediate insertion of the nascent secretoryprotein into the translocon (Sec61complex). Hydrolysis of two moleculesof GTP by the SRP and its receptor drive this docking processand causethe dissociation of SRP(seeFigures13-5 and 13-6).As the ribosome attached to the translocon continues translation, the unfolded protein chain is extruded into the ER lumen. No additional energy is required for translocation. r The translocon contains a central channel lined with hydrophobic residues that allows transit of an unfolded protein chain while remaining sealedto ions and small hydrophilic molecules.In addition, the channel is gated so that it only is open when a polypeptide is being translocated.

542

CHAPTER 13

I

r In post-translational translocation, a completed secretory protein is targetedto the ER membrane by interaction of the signal sequencewith the translocon. The polypeptide chain is then pulled into the ER by a ratcheting mechanism that requires ATP hydrolysis by the chaperone BiP, which stabilizesthe enteringpolypeptide (seeFigure 13-9). In bacteria, the driving force for post-translational translocation comes from SecA,a cytosolic ATPasethat pushespolypeptides through the translocon channel. r In both cotranslational and post-translationaltranslocation, a signal peptidasein the ER membranecleavesthe ER signal sequencefrom a secretoryprotein soon after the Nterminus entersthe lumen.

lnsertionof Proteinsinto the ERMembrane In previous chapters we have encountered many of the vast array of integral (transmembrane)proteins that are present throughout the cell. Each such protein has a unique orientation with respectto the membrane'sphospholipid bilayer.

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

Integral membrane proteins located in the ER, Golgi, and lysosomes and also proteins in the plasma membrane, which are all synthesizedon the rough ER, remain embedded in the membrane in their unique orientation as they move to their final destinationsalong the same pathway followed by soluble secretory proteins (see Figure 13-1, left).During this transport, the orientation of a membrane protein is preserved;that is, the samesegmentsof the protein always face the cytosol, whereas other segments always face in the opposite direction. Thus the final orientation of these membrane proteins is establishedduring their biosynthesison the ER membrane.In this secrion,we first seehow integral proteins can interact with membranes and then examine how severaltypes of sequences,collectively known as topogenic sequences,direct the membrane insertion and orientation of various classesof integral proteins. Theseprocessesoccur via modifications of the basic mechanism used to translocatesoluble secretoryproteins acrossthe ER membrane.

SeveralTopologicalClassesof Integral MembraneProteinsAre Synthesized on the ER The topology of a membraneprotein refers to the number of times that its polypeptide chain spansthe membrane and the orientation of these membrane-spanning segmentswithin the membrane.The key elementsof a protein that determine

coo

Cytosol

its topology are membrane-spanningsegmentsthemselves, which usually are ct-helicescontaining 20-25 hydrophobic amino acids that contribute to energeticallyfavorable interactions within the hydrophobic interior of the phospholipid bilayer. Most integral membrane proteins fall into one of the four topological classesillustrated in Figure 13-10. Topological classesI, II, and III comprise single-passproteins, which have only one membrane-spanninga-helical segment. Type I proteins have a cleaved N-terminal ER signal sequenceand are anchored in the membrane with their hydrophilic N-terminal region on the luminal face (also known as the exoplasmic face) and their hydrophilic Cterminal region on the cytosolic face. Type II proteins do not contain a cleavableER signal sequenceand are oriented with their hydrophilic N-terminal region on the cytosolic face and their hydrophilic C-terminal region on the exoplasmic face (i.e., opposite to type I proteins). Type III proteins have the same orientation as type I proteins but do not contain a cleavablesignal sequence.These different topologies reflect distinct mechanismsused by the cell to establish the membrane orientation of transmembrane segments,as discussedin the next section. The proteins forming topological classIV contain two or more membrane-spanning segments and are sometimes called multipass proteins. For example, many of the membrane transport proteins discussedin Chapter 11 and the numerous G protein-coupled receptors covered in Chapter 15 belong to this class. A final type of membrane protein

NHst

ii,,ltla itirir Exoplasmic space (ERor Golgi lumen; cell exterior)

i Cleaved s i gn aI sequence

NHs'

Type I LDL receptor InfluenzaHA orotein Insulinreceoto'. Growth hormone receptor

Type ll Asialoglycoprotein receptor

Type lll

Type lV

Cytochromep450

Transferrinreceptor Golgi galactosyltransferase Golgi sialyltransferase

A FIGURE 13-10ERmembraneproteins.Fourtopological classes proteins of integral membrane aresynthesized on the roughERas wellasa fifthtypetethered to the membrane by a phospholipid proteins anchor. Membrane areclassified bytheirorientation in the membrane andthetypesof signals theycontainto directthemthere In classes l-lv the hydrophobic segments of the proteinchainformo helices embedded in the membrane bilayer; the regions outside the membrane arehvdrophilic andfold intovarious conformations. All

G protein-coupledreceptors Glucosetransporters Voltage-gatedCa2+channels A B C s m a l l m o l e c u l ep u m p s CFTR(Cl-) channel

N Hg*

GPI-linkedprotein Plasminogen activator receptor Fasciclinll

Sec61 proteins a helicesThetypelV havemultiple transmembrane typelV to thatof G protein-coupled herecorresponds topologydepicted on theexoplasmic sideof receptors: the N-terminus seveno helices, side.Othertype on the cytosolic the membrane, andthe C-terminus numberof helices andvarious lV proteins mayhavea different E Hartmann etal, andC-terminus. orientations of the N-terminus [See andS,D,Black, andC.A Brown 1989, ProcNat'lAcad.SciUSA86:5786, 1989,JBiol Chem254:44421 I N S E R T I OO N F P R O T E I NISN T OT H E E R M E M B R A N E

543

lacks a hydrophobic membrane-spanningsegment altogether; instead, these proteins are linked to an amphipathic phospholipid anchor that is embedded in the membrane (Figure 13-10, right).

Internal Stop-Transfer and Signal-Anchor S e q u e n c eD s e t e r m i n eT o p o l o g y o f S i n g l e - P a sPsr o t e i n s \Ve begin our discussionof how membraneprotein topology is determined with the membrane insertion of integral proteins that contain a single,hydrophobic membrane-spanning segment.Two sequencesare involved in targeting and orienting type I proteins in the ER membrane,whereastype II and type III proteins contain a single,internal topogenicsequence. As we will see,there are three main types of topogenic sequencesthat are used to direct proteins to the ER membrane and to orient them within it. We have alreadybeenintroduced to one, the N-terminal ER signalsequence.The other two, introduced here, are internal sequencesknown as stop-transfer anchor sequencesand signal-anchorsequences. Type I Proteins All type I transmembraneproteins possess an N-terminal signal sequencethat targetsthem to the ER as well as an internal hydrophobic sequencethat becomesthe membrane-spanninga helix. The N-terminal signal sequence

on a nascenttype I protein, like that of a secretoryprotein, initiates cotranslationaltranslocationof the protein through the combined action of the SRP and SRP receptor.Once the Nterminus of the growing polypeptide entersthe lumen of the ER, the signalsequenceis cleaved,and the growing chain continues to be extruded across the ER membrane. However, unlike the casewith secretoryproteins,when the sequenceof approximately 22 hydrophobic amino acidsthat will become a transmembrane domain of the nascent chain enters the translocon,it stopstransferofthe protein through the channel (Figure 13-11). The structure of the Sec61complex suggests that the channelmay be able to open like a clamshell,allowing the hydrophobic transmembranesegmentof the translocating peptide to move laterally betweenthe protein domains constituting the translocon wall (seeFigure 13-8). \Whenthe peptide exits the translocon in this manner, it becomesanchored in the phospholipid bilayer of the membrane.Because of the dual function of sucha sequenceto both stop passageof the polypeptidechain through the transloconand to becomea hydrophobic transmembranesegment in the membrane bilayer,it is called a stop-transferanchor sequence. Once translocation is interrupted, translation continues at the ribosome, which is still anchored to the now unoccupied and closedtranslocon. As the C-terminus of the protein chain is synthesized,it loops out on the cytosolic side of the membrane.When translationis completed,the ribosome is

Cytosol

Open translocon

'l,.il ji;i i lr,!t rl

'fr " Signal peptidase

Nascent polypeptide chain

Stop-transfer ancnor sequence

Cleaved s i gn aI sequence

ER lumen

NHs NH:

proteins. FIGURE 13-11Positioning type lsingle-pass Step[: Afterthe ribosome/nascent chaincomplex becomes associated with a translocon in the ERmembrane, the N-terminal signal sequence iscleaved Thisprocess occurs bythesamemechanism (seeFigure astheonefor soluble proteins secretory 13-6)Steps2, untilthe hydrophobic stop-transfer anchor B: Thechainiselongated sequence issynthesized andentersthe translocon, whereit prevents the nascent chainfromextruding fartherintothe ERlumen Step@: Thestop-transfer anchorsequence moveslaterally between 544

CHAPTER 13

I

NHs

in the phospholipid the translocon subunits andbecomes anchored probably bilayer. At thistime,thetranslocon closesStepS: As synthesis continues, theelongating chainmayloopout intothe andtranslocon cytosolthroughthe smallspacebetweenthe ribosome rscomplete, Step6: Whensynthesis the ribosomal subunits are released intothe cytosol, leaving the proteinfreeto diffusein the m e m b r a n[eS e e HD o e t a,l1 9 9 6C, e l8l 5 : 3 6 a9 n, d WM o t h e s e t,a1l9 9 7 , Cell89:523l

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

releasedfrom the translocon and the C-terminus of the newly synthesizedtype I protein remains in the cytosol. Support for this mechanism has come from studies in which cDNAs encoding various mutant receptorsfor human growth hormone (HGH) are expressedin cultured mammalian cells. The wild-type HGH receptor, a typical type I protein, is transported normally to the plasma membrane. However, a mutant receptor that has charged residues inserted into the single a-helical membrane-spanningsegment or that is missingmost of this segmentis translocatedentirely into the ER lumen and is eventually secretedfrom the cell as a soluble protein. These kinds of experimentsestablishthat the hydrophobic membrane-spanningo helix of the HGH receptor and of other type I proteins functions both as a stoptransfer sequenceand a membrane anchor that preventsthe C-terminus of the protein from crossingthe ER membrane. Type ll and Type lll Proteins Unlike type I proteins, type II and type III proteins lack a cleavableN-rerminal ER signalsequence.Instead, both possessa single internal hydrophobic signal-anchorsequencethat functions as both an ER signal sequenceand membrane-anchorsequence.Recall that type II

and type III proteins have opposite orientations in the membrane (seeFigure 13-10); this differencedependson the orientation that their respectivesignal-anchorsequencesassume within the translocon.The internal signal-anchorsequencein type II proteins directs insertion of the nascentchain into the ER membrane so that the N-terminus of the chain facesthe cytosol, using the sameSRP-dependentmechanismdescribed for signal sequences(Figure 13-l2a). However, the internal signal-anchorsequenceis not cleayedand moves laterally between the protein domains of the translocon wall into the phospholipid bilayer, where it functions as a membrane anchor.As elongationcontinues,the C-terminal region of the growing chain is extruded through the transloconinto the ER lumen by cotranslationaltranslocation. In the case of type III proteins, the signal-anchor sequence, which is located near the N-terminus, inserts the nascent chain into the ER membrane with its N-terminus facing the lumen, in the opposite orientation of the signal anchor in type II proteins. The signal-anchor sequenceof type III proteins also functions like a stop-transfer sequence and prevents further extrusion of the nascentchain into the ER lumen (Figure 13-12b). Continued elongation of the

(a)

(b)

E Nascent polypeptide chain NHs* NH:*

Cposol E

E

mRNA

coo + + +

ilttffi

i,ri:j;ii:l:,1

1ru,|i NH : *

SignalER lumen a n c n o r sequence

coo FIGURE 13-12Positioningtype ll and type ilt single-pass proteins.(a)Typell proteinsStep[: Afterthe internal signalanchorsequence issynthesized on a cytosolic ribosome, it isbound by an SRP(notshown), whichdirects the ribosome/nascent chain complex to the ERmembrane Thisissimilar to targeting of soluble proteins secretory exceptthatthe hydrophobic signalsequence is not located at the N-terminus andis notsubsequently cleavedThe nascent chainbecomes oriented in thetranslocon with itsN-terminal portiontowardthecytosolThisorientation isbelieved to be mediated by the positively charged residues shownN-terminal to thesignalanchorsequence StepE:As the chaintselongated andextruded i n t ot h el u m e nt,h ei n t e r n asli g n a l - a n c hmoorv e lsa t e r a lol yu to f t h e

bilayer. translocon andanchors the chainin the phospholipid iscompleted, theC-terminus of the StepB: Onceproteinsynthesis polypeptide subunits is released intothe lumen,andthe ribosomal Step[: Assembly arereleased intothe cytosol(b)Typelll proteins. pathway to thatof typell proteins exceptthatpositively isby a similar sequence charged residues on theC-terminal sideof thesignal-anchor withinthetranslocon segment to be oriented causethetransmembrane portionoriented to the cytosol andthe N-terminal with itsC-terminal s i d eo f t h ep r o t e i inn t h eE Rl u m e nS t e p [s, B : C h a i ne l o n g a t i o n portionof the proteiniscompleted in the cytosol, of theC-terminal arereleased. M Spiess andH F Lodish, andribosomal subunits [See 1986, Cell 44:177, and H Do et al , 1996, Ce//85:369 l

I N S E R T I OO N F P R O T E I NISN T OT H E E R M E M B R A N E

545

chain C-terminal to the signal-anchor/stop-transfer sequenceproceeds as it does for type I proteins, with the hydrophobic sequence moving laterally between the translocon subunits to anchor the polypeptide in the ER membrane(seeFigure 13-11). One of the features of signal-anchor sequencesthat appears to determine their insertion orientation is a high density of positively chargedamino acids adjacentto one end of the hydrophobic segment.For reasonsthat are not well understood,thesepositively chargedresiduestend to remain on the cytosolic side of the membrane, not traversing the membrane into the ER lumen. Thus the position of the charged residuesdictates the orientation of the signal-anchorsequencewithin the translocon as well as whether or not the rest of the polypeptide chain continues to pass into the ER lumen: type II proteins tend to have positively charged residues on the N-terminal side of their signal-anchor sequence,orienting the N-terminus in the cytosol and allowing passageof the C-terminal side into the ER (Figure 1.3-L2a), whereas type III proteins tend to have positively charged residues on the C-terminal side of their signal-anchor sequence,inserting the N-terminus into the translocon and restricting the C-terminus to the cytosol (Figure 13-1,2b). A striking experimental demonstration of the importance of the flanking charge in determining membrane ori-

entation is provided by neuraminidase,a type II protein in the surface coat of influenza virus. Three arginine residues are located just N-terminal to the internal signal-anchorsequencein neuraminidase.Mutation of thesethree positively charged residues to negatively charged glutamate residues causes neuraminidase to acquire the reverse orientation. Similar experimentshave shown that other proteins, with either type II or type III orientation, can be made to "flip" their orientation in the ER membrane by mutating charged residuesthat flank the internal signal-anchorsegment.

M u l t i p a s sP r o t e i n sH a v eM u l t i p l eI n t e r n a l TopogenicSequences Figure 13-13 summarizesthe arrangementsof topogenic sequencesin single-passand multipass transmembrane proteins. In multipass (type IV) proteins, each of the membrane-spanninga helices acts as a topogenic sequence in the ways that we have already discussed:they can act to direct the protein to the ER, to anchor the protein in the ER membrane, or to stop transfer of the protein through the membrane. Multipass proteins fall into one of two types depending on whether the N-terminus extends into the cytosol or the exoplasmicspace(e.g.,the ER lumen, cell exterior). This N-terminal topology usually is determined

STA= Internalstop-transferanchorsequence SA = Internalsignal-anchorsequence

(a) Type I

( b ) T y p el l

N H 3 +Signal sequence

NH:*

(c) Type lll

NHs*

(d)Type lV-A

NHs*

coo STA

+++

coo SA

coo

+++ SA Cvtosol

Lumen COO

STA

SA

Lumen

(e) Type lV-B

Cytosol

C H A P T E R1 3

Cytosol

Lumen

+++

I

STA

SA

Cytosol

Lumen

NHqt

STA

546

sequences determine < FIGURE 13-13Topogenic orientationof ERmembraneproteins.Topogenic hydrophilic sequences areshownin red;soluble, portions sequences form in blue Theinternal topogenic or cthelices thatanchorthe proteins transmembrane of proteinsin the membrane(a)TypeI proteins segments anda singleinternal signalsequence containa cleaved (b, c)Typell andtypelll stop-transfer anchor(STA). (SA) proteins signal-anchor containa singleinternal in theorientation of these Thedifference sequence. proteins depends largely on whetherthereisa high charged aminoacids(+ + +) on density of positively (typell)or on sideof the SAsequence the N-termrnal (typelll).(d,e) SA sequence sideof the the C-terminal proteins lacka cleavable signal Nearly all multipass in theexamples shownhere. asdepicted sequence, facesthe cytosol, TypelV-Aproteins, whoseN-terminus typell S sequences andSTA containalternating TypelV-Bproteins, whoseN-terminus faces sequences followedby the lumen,beginwith a typelllSAsequence Proteins typell SAand STAsequences of alternating (oddor eachtypewith differentnumbersof ct helices even)areknown

Lumen

COO

+++ SA

STA

MOVINGPROTEINS INTO MEMBRANES AND ORGANELLES

Cytosol

SA

STA

by the hydrophobic segment closest to the N-terminus and the charge of the sequencesflanking it. lf a type IV protein has an euen number of transmembrane cr helices, both its N-terminus and C-terminus will be oriented toward the same side of the membrane (Figure 13-13d). Conversely, if a type IV protein has an odd number of a helices, its two ends will have opposite orientations ( F i g u r e1 3 - 1 3 e ) . Type lV Proteins with N-Terminus in Cytosol Among the multipass proteins whose N-terminus extends into the cytosol are the various glucose transporters (GLUTs) and most ion-channel proteins, discussedin Chapter 11. In these proteins, the hydrophobic segmentclosestto the N-terminus initiates insertion of the nascent chain into the ER membrane with the N-terminus oriented toward the cytosol; thus this a-helical segment functions like the internal signalanchor sequenceof atype II protein (seeFigure 13-12a).As the nascent chain following the first a helix elongates, it moves through the translocon until the secondhydrophobic a helix is formed. This helix prevents further extrusion of the nascentchain through the translocon;thus its function is similar to that of the stop-transferanchor sequencein a type I p r o t e i n ( s e eF i g u r e1 3 - 1 1 ) . After synthesisof the first two transmembranecl helices, both ends of the nascentchain face the cytosol and the loop between them extends into the ER lumen. The C-terminus of the nascentchain then continues ro grow into the cytosol, as it does in synthesis of type I and type III proteins. According to this mechanism,the third a helix acts as another type II signal-anchor sequenceand the fourth as another stop-transferanchor sequence(Figure 13-13d). Apparently, once the first topogenic sequenceof a multipass polypeptide initiates associationwith the translocon, the ribosome remains attached to the translocon, and topogenic sequences that subsequentlyemerge from the ribosome are threaded into the translocon without the need for the SRP and the SRP receptor. Experiments that use recombinant DNA techniques to exchangehydrophobic cr heliceshave provided insight into the functioning of the topogenic sequencesin type IV-A multipass proteins. Theseexperimentsindicate that the order of the hydrophobic a helicesrelative to each other in the growing chain largely determineswhether a given helix functions as a signal-anchor sequenceor stop-transfer anchor sequence. Other than its hydrophobicitS the specific amino acid sequenceof a particular helix has little bearing on its function. Thus the first N-terminal a helix and the subsequent odd-numbered ones function as signal-anchor sequences,whereas the intervening even-numberedhelices function as stop-transferanchor sequences. Type lV Proteins with N-Terminus in the Exoplasmic Space The large family of G protein-coupled receptors,all of which contain seventransmembranect helices,constitute the most numerous type IV-B proteins, whose N-terminus extends into the exoplasmic space.In theseproteins, the hy-

drophobic ct helix closestto the N-terminus often is followed by a cluster of positively charged amino acids, similar to a As a retype III signal-anchorsequence(seeFigure 1,3-1,2b)'. sult, the first a helix inserts the nascent chain into the translocon with the N-terminus extending into the lumen (seeFigure 13-13e).As the chain is elongated,it is inserted into the ER membrane by alternating type II signal-anchor sequencesand stop-transfer sequences,as just describedfor type IV-A proteins.

A PhospholipidAnchor TethersSomeCellSurfaceProteinsto the Membrane Somecell-surfaceproteins are anchored to the phospholipid bilayer not by a sequenceof hydrophobic amino acids but by a covalently attached amphipathic molecule, glycosylphosphatidylinositol (GPI) (Figure 1.3-1.4aand Chapter 10). These proteins are synthesizedand initially anchored to the ER membrane exactly like type I transmembrane proteins, with a cleavedN-terminal signal sequenceand internal stoptransfer anchor sequencedirecting the process (see Figure 13-11).However,a short sequenceof amino acidsin the luminal domain, adjacent to the membrane-spanningdomain, is recognizedby a transamidaselocated within the ER membrane. This enzymesimultaneously cleavesoff the original stop-transferanchor sequenceand transfersthe luminal portion of the protein to a preformed GPI anchor in the membrane (Figure 1.3-'1.4b). \X/hy change one type of membrane anchor for another? Attachment of the GPI anchor, which results in removal of the cytosol-facing hydrophilic domain from the Proteinswith GPI protein, can have severalconsequences. anchors, for example, can diffuse relatively rapidly in the plane of the phospholipid bilayer membrane. In contrast, many proteins anchored by membrane-spanningct helices are impeded from moving laterally in the membrane becausetheir cytosol-facing segments interact with the cytoskeleton. In addition, the GPI anchor targets the attachedprotein to the apical domain of the plasma membrane in certain polarized epithelial cells, as we discussin C h a p t e r1 4 .

The Topologyof a MembraneProteinOften Can Be Deducedfrom lts Sequence As we have seen,various topogenic sequencesin integral membrane proteins synthesizedon the ER govern interac'When sciention of the nascent chain with the translocon. tists begin to study a protein of unknown function, the identification of potential topogenic sequenceswithin the corresponding gene sequencecan provide important clues about the protein's topological class and function. Suppose, for example, that the gene for a protein known to be required for a cell-to-cell signaling pathway contains nucleotide sequencesthat encodean apparent N-terminal signal sequence and an internal hydrophobic sequence.These findings suggest that the protein is a type I integral membrane protein

I N S E R T I OO NF P R O T E I NISN T OT H E E R M E M B R A N E

547

drophilic amino acids negative values. Although different scalesfor the hydropathic index exist, all assignthe most positive valuesto amino acids with side chains made up of = tO Mannose mostly hydrocarbon residues(e.g.,phenylalanineand me= NH, P1-'o.01''o"thanolamine P o o* thionine) and the most negativevalues to charged amino acids (e.g., arginine and aspartate).The second step is to identify longer segmentsof sufficient overall hydrophobic% L POo PO4-+ NH3+ Fattyacyl chains ity to be N-terminal signal sequencesor internal stopTo accomtransfer sequences and signal-anchorsequences. plish this, the total hydropathic index for each successive segmentof 20 consecutiveamino acids is calculatedalong Hvdrophobic NHg* Polar J the entire length of the protein. Plots of these calculated values againstposition in the amino acid sequenceyield a hydropathy profile. (b) Figure 13-15 shows the hydropathy profiles for three GPI different membrane proteins. The prominent peaks in such transamidase Cytosol coo plots identify probable topogenicsequences as well as their position and approximate length. For example, the hydropathy profile of the human growth hormone receptor revealsthe presenceof both a hydrophobic signal sequence NHs* . at the extreme N-terminus of the protein and an internal hydrophobic stop-transfersequence(Figure 13-15a). On the Preformed G P Ia n c h o r basis of this profile, we can deduce, correctly, that the hugrowth hormone receptor is a type I integral memman Precu rsor protein. The hydropathy profile of the asialoglycobrane NlH3* protein NHs* protein receptor, a cell-surface protein that mediates Mature GPI-linked ER lumen removal of abnormal extracellular glycoproteins, reveals a protein prominent internal hydrophobic signal-anchor sequence FIGURE 13-14GP|-anchored proteins.(a)Structure of a glygives no indication of a hydrophobic N-terminal signal but (GPl)fromyeast. cosylphosphatidylinositol portion Thehydrophobic sequence(Figure 13-15b). Thus we can predict that the of the molecule iscomposed of fattyacylchains, whereas the polar (hydrophilic) asialoglycoproteinreceptor is a type II or type III membrane portionof the molecule iscomposed of carbohydrate protein. The distribution of charged residueson either side residues groupsIn otherorganisms, andphosphate boththe length of theacylchains andthe carbohydrate moieties mayvarysomewhat of the signal-anchorsequenceoften can differentiate befromthe structure shown(b)Formation proteins of GPI-anchored in tween these possibilities since positively charged amino the ERmembrane Theproteinissynthesized andinitially inserted acids flanking a membrane-spanning segment usually are i n t ot h eE Rm e m b r a naess h o w ni n F i g u r 1e 3 - 11 .A s p e c i f ti cr a n s a m i - oriented toward the cytosolic face of the membrane. For dasesimultaneously proteinwithinthe cleaves the precursor instance,in the caseof the asialoglycoproteinreceptor,exexoplasmic-facing domain,nearthe stop-transfer anchorsequence amination of the residuesflanking the signal-anchor se(red),andtransfers groupof the newC-terminus the carboxyl to the quence reveals that the residues on the N-terminal side terminal amrnogroupof a preformed GPIanchor[See C Abeijon and carry a net positive charge, thus correctly predicting that C B Hirschberg,1992,TrendsBioch Sec m i 1 7 : 3 2 , a n d K K o d u k u l ae t a l , this is a type II protein. 1992, Proc Nat'|.Acad Sci USA89:49821 The hydropathy profile of the GLUT1 glucose transporter, a multipass membrane protein, shows the presence of many segmentsthat are sufficiently hydrophobic to be membrane-spanninghelices(Figure 13-15c).The complexand therefore may be a cell-surfacereceptor for an extracelity of this profile illustrates the difficulty both in unambigulular ligand. ously identifying all the membrane-spanningsegmentsin a Identification of topogenic sequences requiresa way to multipass protein and in predicting the topology of individscan sequencedatabasesfor segmentsthat are sufficiently ual signal-anchorand stop-transfer sequences.More sohydrophobic to be either a signal sequenceor a transmemphisticated computer algorithms have been developed that brane anchor sequence.Topogenicsequencescan often be take into account the presenceof positively charged amino identified with the aid of computer programs that generare acids adjacent to hydrophobic segmentsas well as the a hydropathy profile for the protein of inrerest. The first length of and spacing between segments.Using all this instep is to assign a value known as the bydropathic index to formation, the best algorithms can predict the complex each amino acid in the protein. By convention, hydrophotopology of multipass proteins with an accuracy of greater bic amino acids are assigned positive values and hythan 7 5 percent. (a)

548

O = Inositol I = Glucosamine

CHAPTER 13

I

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

( a ) H u m a ng r o w t h h o r m o n er e c e p t o r( t y p el ) A

3 z

1 0 -1 -2 -3 N-terminus

100

200

( b )A s i a l o g l y c o p r o t e ri ne c e p t o r( t y p el l )

4

S i g n a l - a n c h osre q u e n c e

2 1 0 -1

400

300

C-terminus

( c ) G L U T(tt y p el V ) 4 z

r'i;.:'1+i i

...,,

-z

1 0 -1 -z

100

200

100

A E X P E R I M E N TFA IG L U R1E3 - 1 5H y d r o p a t h p yrofiles. profilescanidentifylikelytopogenic Hydropathy sequences in proteinsTheyaregenerated integral membrane by plottingthetotal hydrophobicity of eachsegment of 20 contiguous aminoacidsalong the lengthof a proteinPositive valuesindicate relatively hydrophobic

Finally, sequencehomology to a known protein may permit accurateprediction of the topology ol a newly discovered multipass protein. For example, the genomesof multicellularorganismsencodea very large number of multipass proteins with seventransmembranecr helices.The similaritiesbetweenthe sequences of theseproteinsstrongly suggestthat all have the sametopology as the well-studied G protein-coupled receptors,which have the N-terminus orientedto the exoplasmicsideand the C-terminusoriented to the cytosolicside of the membrane.

Insertion of Proteins into the ER Membrane r Integralmembraneproteinssynthesized on the rough ER fall into four topological classesas well as a lipid-linked t y p e ( s e eF i g u r e1 3 - 1 0 ) . r Topogenic sequences-N-terminal signal sequences,internal stop-transferanchor sequences, and internal signalanchor sequences-direct the insertion and orientation of nascentproteinswithin the ER membrane.This orientarion is retained during transport of the completedmembrane p r o r e i nt o i r s f i n a l d e s t i n a t i o n . r Single-passmembrane protelns contarn one or two topogenic sequences. In multipassmembraneproteins, each ct-helicalsegmentcan function as an internal topogenic sequence,depending on its location in the polypeptide

200

400

polarportions portions; relatively of the protein values, negative profiles for Thecomplex aremarked. sequences Probable topogenic (typelV)proteins, in part(c),oftenmustbe suchasGLUT1 multipass of to determrne the topology with otheranalyses supplemented theseoroteins

chain and the presenceof adjacent positively charged r e s i d u e s( s e eF i g u r e1 3 - 1 3 ) . r Somecell-surfaceproteins are initially synthesizedas type I proteins on the ER and then are cleavedwith their luminal domain transferredto a GPI anchor (seeFigure t3-14). r The topology of membraneproteinscan often be correctly predicted by computer programs that identify hydrophobic topogenicsegmentswithin the amino acid sequenceand generatehydropathyprofiles(seeFigure13-15).

Folding, ProteinModifications, and QualityControlin the ER Membrane and soluble secretoryproteins synthesizedon the rough ER undergo four principal modifications before they reachtheir final destinations:(1) covalentaddition and processing of carbohydtates (glycosylation) in the ER and Golgi, (2) formation of disulfidebondsin the ER, (3) proper folding of polypeptide chains and assemblyof multisubunit proteins in the ER, and (4) specific proteolytic cleavagesin the ER, Golgi, and secretory vesicles.Generally speaking, these modifications promote folding of secretory proteins into their native structures and add structural stability to proteins exposed to the extracellular environment. Modifications such as glycosylation also allow the cell to produce a vast array of chemically distinct moleculesat the cell surface

IN THEER PROTEIN M O D I F I C A T I O NFSO . L D I N GA, N D Q U A L I T YC O N T R O L

549

that are the basis of specific molecular interactions used in cell-to-cell adhesionand communication. One or more carbohydrate chains are added to the vast majority of proteinsthat are synthesizedon the rough ER; indeed, glycosylationis the principal chemicalmodification to most of these proteins. Proteins with attached carbohydrates are known as glycoproteins. Carbohydrate chains in glycoproteins may be attached to the hydroxyl group in serineand threonine residuesor to the amide nitrogen of asparagine.Theseare referred to as Olinked oligosaccharidesand Nlinked oligosaccharides,respectively.The various types of O-linked oligosaccharides include the mucin-type O-linked chains (named after abundantglycoproteinsfound in mucus)and the carbohydrate modifications on proteoglycansdescribedin Chapter 19. OIinked chains typically contain only one to four sugar residues,which are added to proteins by enzymesknown as glycosyltransferases, located in the lumen of the Golgi complex. The more common Nlinked oligosaccharides are larger and more complex,containingseveralbranchesin mammalian cells. In this section we focus on N-linked oligosaccharides, whose initial synthesisoccurs in the ER. After the initial N-glycosylationof a protein in the ER, the oligosaccharidechain is modified in the ER and commonly in the Golgi as well. Disulfide bond formation, protein folding, and assembly of multimeric proteins, which take place exclusively in the rough ER, also are discussedin this section. Only properly folded and assembledproteins are transported from the rough ER to the Golgi complex and ultimately to the cell surface or other final destination. Unfolded, misfolded, or partly folded and assembledproteins are selectivelyretained in the rough ER. Sile consider several features of such "quality control" in the latter part of this section. As discussedpreviously,N-terminal ER signal sequences are cleaved from secretory proteins and type I membrane proteins in the ER. Someproteins also undergo other specific proteolytic cleavagesin the Golgi complex or secretoryvesicles. We cover thesecleavages,as well as carbohydrate modifications that occur primarily or exclusively in the Golgi complex, in the next chapter.

A PreformedA/-LinkedOligosaccharide ls Added t o M a n y P r o t e i n si n t h e R o u g hE R Biosynthesisof all N-linked oligosaccharidesbeginsin the rough ER with addition of a preformed oligosaccharide precursorcontaining 14 residues(Figure 13-t6). The structure of this precursor is the same in plants, animals, and single-celledeukaryotes-a branchedoligosaccharide,containing three glucose(Glc), nine mannose(Man), and two N-acetylglucosamine(GlcNAc) molecules, which can be written as GIcaMane(GlcNAc)2. Once added to a protein, this branchedcarbohydrate srructureis modified by addition or removal of monosaccharidesin the ER and Golgi compartments. The modifications to N-linked chains differ from one glycoprotein to another and differ among different organisms,but a core of 5 of the 14 residuesis conservedin the structures of all N-linked oligosaccharideson secretoryand membrane proteins. 550

CHAPTER 13

I

Glc

I I

Glc

G l c N A c= N - A c e t y l g l u c o s a m i n e Man = Mannose Glc= Glucose = Conserved = Variable

Glc

Man

I I

Man

Man

II

II

Man

Man

Man

Ma n

Man

*in GlcNAc

GlcNAc

II

N H s *. . - X - A s n - X - ( S e r / T h r ) - ' . . C O O A FIGURE 13-16Commonprecursorof N-linkedoligosacprecursor charides. This14-residue of tV-linked oligosaccharides is proteins addedto nascent in the roughERSubsequent removal and in somecases addition of specific sugarresidues occurin the ERand GolgicomplexThecoreregion, composed of fiveresidues highlightedin purple,is retained in allN-linked oligosaccharides The (Asn)residues precursor canbe linkedonlyto asparagine thatare (Ser) (Thr) separated by oneaminoacid(X)froma serine or threonine on the carboxyl side.

Prior to transfer to a nascent chain in the lumen of the ER, the precursor oligosaccharideis assembledon a membrane-attached anchor called dolichol phosphate, a Iongchain polyisoprenoid lipid (Chapter 10). After the first sugar, GlcNAc, is attached to the dolichol phosphate by u pyrophosphate bond, the other sugarsare added by glycosidic bonds in a complex set of reactions catalyzed by enzymes attached to the cytosolic or luminal faces of the rough ER membrane (Figure L3-1.7).The final dolichol pyrophosphoryl oligosaccharideis oriented so that the oligosaccharide portion facesthe ER lumen. The entire 14-residueprecursor is transferred from the dolichol carrier to an asparagine residue on a nascent polypeptide as it emergesinto the ER lumen (Figure 13-18, step E). Only asparagineresidues in the tripeptide sequencesAsn-X-Ser and Asn-X-Thr (where X is any amino acid except proline) are substratesfor oligosaccharyltransferase, the enzyme that catalyzes this reaction. Two of the three subunits of this enzyme are ER membrane proteins whose cytosol-facingdomains bind to the ribosome,localizing a third subunit of the transferase,the catalytic subunit, near the growing polypeptide chain in the ER lumen. Not all

M O V T N GP R O T E | NtSN T O M E M B R A N E A SN D O R G A N E L L E S

Gytosol 5 GDP

a 4GDP 'flin

a-

e

I\

3 UDP l 3 UDP

4 GDP O

^.

,/

t\l

I

Dolichol phospha I = N-Acetylglucosamine

Completed precursor

O = Mannose A - Glucose

ER lumen

FIGURE 13-17Biosynthesis precursor. of the oligosaccharide phosphate Dolichol isa strongly hydrophobic lipid,containing 75-95 c a r b o na t o m st,h a t i s e m b e d d eidn t h e E Rm e m b r a n e T.w o (GlcNAc) tV-acetylglucosamine andfivemannose residues areadded phosphate oneat a timeto a dolichol on the cytosolic faceof the ER (stepsIl-B) Thenucleotide-sugar membrane donorsin theseand laterreactions aresynthesized in thecytosolNotethatthefirstsugar residue isattached pyrophosphate to dolichol by a high-energy linkageTunicamycin, whichblocks thefirstenzyme in thispathway, inhibits thesynthesis of allN-linked oligosaccharides in cellsAfter

pyrophosphoryl isflipped intermedrate dolichol theseven-resrdue andall four mannose face(stepZl),the remaining to the luminal areaddedoneat a time(steps5, 6). In the residues threeglucose froma laterreactions, thesugarto be addedisfirsttransferred phosphate face on thecytosolic to a carrier dolichol nucleotide-sugar face,wherethe of the ER;thecarrieristhenflippedto the luminal afterwhichthe o|gosaccharide, to the growrng sugaristransferred "empty"carrier face.[After C Abeijon isflippedbackto the cytosolic Sci17:32]1 1992,Trends Biochem andC B Hirschberg,

Asn-X-Ser/Thr sequencesbecome glycosylated,and it is not possible to predict from the amino acid sequencealone which potential N-linked glycosylation sites will be modified; for instance, rapid folding of a segment of a protein containing an Asn-X-Ser/Thr sequencemay prevent transfer precursorto it. of the oligosaccharide

Immediately after the entire precursor, GlcaMane (GlcNAc)2, is transferred to a nascent polypeptide, three different enzymes, called glycosidases,remove all three glucoseresiduesand one particular mannoseresidue (Fig, hich u r e 1 3 - 1 8 ,s t e p sZ - 4 ) . T h e t h r e eg l u c o s er e s i d u e sw are the last residuesadded during synthesisof the precursor

Dol

E

To ctsGolgi

(Man)a(GlcNAc)z

ER lumen Dol = Dolichol

o = Mannose

I = N-Acetylglucosamine

a = Glucose

FIGURE 13-18Additionand initial processing of N-linked Inthe roughERof vertebrate oligosaccharides. cells,the precursor GlcrMann(GlcNAc)2 istransferred fromthe dolichol carrier proteinassoonas to a susceptible asparagine residue on a nascent the asparagine crosses to the luminal sideof the ER(step[) In (stepEl),thentwo reactions, threeseparate firstoneglucose residue

(step4) (stepB), andfinallyonemannose residue glucose residues (stepl[) playsa residue of oneglucose Re-addition areremoved. in the ER,asdiscussed foldingof manyproteins rolein the correct 45:631, and Rev. Biochem 1985,Ann S Kornfeld, R Kornfeldand later[See M S o u s a a n d AJ P a r o d i1, 9 9 5 , E M B O l 1 4 i 4 1 9 6 )

, N D Q U A L I T YC O N T R O LI N T H E E R P R O T E I NM O D I F I C A T I O N SF,O L D I N G A

551

The oligosaccharidesattachedto glycoproteinsservevarious functions. For example, some proteins require N-linked oligosaccharidesin order to fold properly in the ER. This function has been demonstrated in studies with the antibiotic tunicamycin, which blocks the first step in the formation of the dolichol-linked oligosaccharideprecursor and therefore inhibits synthesisof all N-linked oligosaccharidesin cells (seeFigure 13-17).In the presenceof tunicamycin,the hemagglutinin precursor polypeptide (HAe) is synthesized, but it cannot fold properly and form a normal trimer; in this case,the protein remains,misfolded, in the rough ER. Moreover, mutation of a particular asparaginein the HA sequence to a glutamine residue prevents addition of an N-linked oligosaccharideto that site and causesthe protern to accumulate in the ER in an unfolded state. In addition to promoting proper folding, N-linked oligosaccharides also confer stability on many secretedglycoproteins.Many secretoryproteins fold properly and are transported to their final destination even if the addition of all N-linked oligosaccharides is blocked,for example,by tunicamycin. However, such nonglycosylatedproteins have been shown to be lessstablethan their glycosylatedforms. For instance,glycosylatedfibronectin, a normal component of the extracellularmatrix, is degradedmuch more slowly by tissueproteasesthan is nonglycosylatedfibronectin. Oligosaccharideson certain cell-surfaceglycoproteins also play a role in cell-cell adhesion. For example, the plasma membrane of white blood cells (leukocytes)contains cell-adhesionmolecules(CAMs) that are extensively glycosylated.The oligosaccharidesin these moleculesinteract with a sugar-binding domain in certain CAMs found on endothelial cells lining blood vessels.This interaction tethers the leukocytesto the endothelium and assists in their movement into tissuesduring an inflammat o r y r e s p o n s et o i n f e c t i o n ( s e e F i g u r e 1 , 9 - 3 6 ) .O t h e r c e l l - s u r f a c eg l y c o p r o t e i n s p o s s e s so l i g o s a c c h a r i d es i d e chains that can induce an immune response.A common example is the A, B, O blood-group antigens,which are O-linked oligosaccharidesattached to glycoproteins and glycolipids on the surface of erythrocytes and other cell t y p e s ( s e eF i g u r e 1 0 - 2 0 ) .

groups (-SH), also known as thiol groups, on two cysteine residuesin the same or different polypeptide chains. This reaction can proceed spontaneouslyonly when a suitable oxidant is present.In eukaryotic cells, disulfide bonds are formed only in the lumen of the rough ER; in bacterial cells, disulfide bonds are formed in the periplasmic space between the inner and outer membranes.Thus disulfide bonds are found only in secretoryproteins and in the exoplasmic domains of membraneproteins. Cytosolic proteins and organelleproteins synthesizedon free ribosomeslack disulfide bonds and depend on other interactionsto stabilize their structures. The efficient formation of disulfide bonds in the lumen of the ER dependson the enzymeprotein disulfide isomerase (PDI), which is presentin all eukaryotic cells.This enzymeis especiallyabundant in the ER of secretorycells in such organs as the liver and pancreas,where large quantities of proteins that contain disulfide bonds are produced. As shown in Figure t3-1.9a, the disulfide bond in the active site of PDI can be readily transferred to a protein by two sequential thiol-disulfide transfer reactions.The reducedPDI generated by this reaction is returned to an oxidized form by the action of an ER-residentprotein, called Ero1, which carriesa disulfide bond that can be transferredto PDI. Erol itself becomes oxidized by reaction with molecular oxygen that has diffused into the ER. In proteins that contain more than one disulfide bond, the proper pairing of cysteineresiduesis essentialfor normal structure and activity. Disulfide bonds commonly are formed between cysteinesthat occur sequentially in the amino acid sequencewhile a polypeptide is still growing on the ribosome. Such sequential formation, however, sometimesyields disulfide bonds between the wrong cysteines.For example,proinsulin, a precursor to the peptide hormone insulin, has three disulfide bonds that link cysteines 1 and 4, 2 and 6, and 3 and 5. In this case,a disulfide bond that initially formed sequentially(e.g., between cysteinesI and 2) would have to be rearranged for the protein to achieveits proper folded conformation. In cells, the rearrangementof disulfide bonds also is acceleratedby PDI, which acts on a broad range of protein substrates, allowing them to reach their thermodynamicallymost stable conformations (Figure 13-19b). Disulfide bonds generally form in a specificorder, first stabilizingsmall domains of a polypeptide, then stabilizing the interactions of more distant segments;this phenomenon is illustrated by the folding of influenza HA protein, discussedin the next sectron.

D i s u l f i d eB o n d sA r e F o r m e da n d R e a r r a n g e d b y P r o t e i n si n t h e E RL u m e n

C h a p e r o n eas n d O t h e r E RP r o t e i n sF a c i l i t a t e F o l d i n ga n d A s s e m b l yo f P r o t e i n s

In Chapter 3 we learned that both intramolecular and intermolecular disulfide bonds (-S-S-) help stabilize the tertiary and quaternary structure of many proteins. These covalent bonds form by the oxidative linkage of sulfhydryl

Although many denatured proteins can spontaneouslyrefold into their native state in vitro, such refolding usually requires hours to reach completion. Yet new soluble and membraneproteins produced in the ER generallyfold into

on the dolichol carrier, appear to acr as a signal that the oligosaccharideis complete and ready to be transferredto a protern.

O l i g o s a c c h a r i dSei d eC h a i n sM a y P r o m o t e F o l d i n ga n d S t a b i l i t yo f G l y c o p r o t e i n s

552

CHAPTER 13

|

M O V T N Gp R O T E t N |SN T O M E M B R A N E A SN D O R G A N E L L E S

( a ) F o r m a t i o no f a d i s u l f i d eb o n d

Reduced substrate prorern

Oxidized substrate protein

( b ) R e a r r a n g e m e notf d i s u l f i d eb o n d s . -qr.l 'Reduced t "' ' i PDI ic

zSH \Q.

"" Reduced PDI -SH

-

$z t"

,n.o,.,.i.',oj"i il rHi'J o""o,

e_

.",.,."L':[,l,ilXto"o.

(PDl).PDI FIGURE 13-19Actionof proteindisulfideisomerase formsandrearranges disulf idebondsviaan activesitewithtwo closely spaced cysteine residues interconverted thatareeasily between the reduced dithiolformandtheoxidized disulfide form Numbered redarrowsindicate thesequence of electron transfers Yellowbars represent disulfide bonds(a)Intheformation of disulfide bonds,the (-S )form of a cysteine proteinreacts ionized thiolin thesubstrate (9-S) bondin oxidized withthedisulfide PDIto forma disulfideproteinintermediate bondedPDI-substrate A second ionized thiolin

forminga disulfide withthisintermediate, thenreacts thesubstrate proteinandreleasing PDIPDl,in reduced bondwithinthesubstrate bondin the luminalprotein to a disulfide electrons turn,transfers PDI formof PDl.(b)Reduced theoxidized regenerating Ero1,thereby bondsby formeddisulfide of improperly rearrangement cancatalyze PDIboth reactions. Inthiscase,reduced transfer similar thiol-disulfide Thesereactions in the reactionpathway. initiates andis regenerated of the proteinis untilthe moststableconformation arerepeated F.Gilbert, 1991 l achieved ,Biochemistry30t6l9 [SeeMM LylesandH

their proper conformation within minutes after their synthesis. The rapid folding of these newly synthesizedproteins in cells dependson the sequentialaction of several proteins present within the ER lumen. We have already seen how the molecular chaperone BiP can drive posttranslationaltranslocationin yeastby binding fully synthesized polypeptidesas they enter the ER (seeFigure 13-9). BiP can also bind transiently to nascentchains as they enter the ER during cotranslationaltranslocation.Bound BiP is thought to preventsegmentsof a nascentchain from misfolding or forming aggregates,thereby promoting folding of the entire polypeptide into the proper conformation. P r o t e i n d i s u l f i d e i s o m e r a s e( P D I ) a l s o c o n t r i b u t e s t o proper folding, becausecorrect 3-D conformation is stabil i z e db y d i s u l f i d eb o n d si n m a n y p r o t e i n s . As illustrated in Figure 1,3-20,two other ER proteins, the homologous lectins (carbohydrate-binding proteins)

cdlnex,in and calreticulin, bind selectively to certain Nlinked oligosaccharideson growing nascent chains. The ligand for thesetwo lectins, which contains a single glucose residue, is generatedby a specific glucosyltransferasein the ER lumen (seeFigure 1,3-18,step Ed). This enzyme acts only on polypeptide chains that are unfolded or misfolded, acts as one of the and in this respectthe glucosyltransferase primary surveillance mechanismsto ensure quality control of protein folding in the ER. Binding of calnexin and calreticulin to unfolded nascent chains marked with glucosylated N-linked oligosaccharidesprevents aggregation of adjacent segmentsof a protein as it is being made on the ER. Thus calnexin and calreticulin, like BiP' help prevent premature, incorrect folding of segmentsof a newly made protern. Other important protein-folding catalysts in the ER Iumen are peptidyl-prolyl isomerases,a family of enzymesthat

IN THEER PROTEIN M O D I F I C A T I O NFSO , L D I N GA, N D Q U A L I T YC O N T R O L

553

(al

Oligosaccharyl transferase Dolichol oligosaccharide

M e m b r a n e - s p anni n g s helix

Cytosol

Luminal crhelix

I

ER lumen

HAotrimer Completed H A sm o n o m e r

(b) > FIGURE 13-20Hemagglutinin folding and assembly. (a)Mechanism of (HAo) trimerassembly Transtent bindingof the chaperone BiP(stepIE) to the nascent chainandof two lectins, (steplE) calnexin andcalreticulin, to certainoligosaccharide chains promotes properfoldingof adjacent segments. A totalof seven N-linked oligosaccharide portionof the chains areaddedto theluminal nascent chainduringcotranslational translocation, andPDIcatalyzes theformation of sixdisulfide bondspermonomerCompleted HA6 monomers areanchored in the membrane by a singlemembranes p a n n i nagh e l i xw i t ht h e i rN - t e r m i n u i nst h el u m e n( s t e p[ ) Interaction of threeHAechains with oneanother, initially viatheir transmembrane crhelices, apparently triggers formation of a long stemcontaining onecthelixfromthe luminalpartof eachHAs polypeptide Finally, interactions between thethreeglobular heads occur,generating a stableHA6trimer(stepB) (b)Electron micrograph of a complete influenza virionshowing trimers of HAprotein protruding (a) asspikes fromthesurface of theviralmembrane [part SeeU Tatuetal, 1995, EMBO J 14:1340, andD Hebert elal, 1997, J Cell Biol.139:613 Part(b) ChrisBjornberg/PhotoResearchers, Inc l

acceleratethe rotation about peptidyl-prolyl bonds at proline residuesin unfolded segmentsof a polypeptide:

Rotation about peptide bond

-

2, Prolyl

o*^

t11

.U

,/I

lzu\ O' NH

\

crs

trans

Such isomerizations sometimesare the rate-limiting step in the folding of protein domains. Many peptidyl-prolyl isomerasescan catalyzethe rotation of exposedpeptidyl-prolyl bonds indiscriminately in numerous proteins, but some have very specificprotein substrates. 554

CHAPTER 13

I

Many important secretory and membrane proteins synthesizedon the ER are built of two or more polypeptide subunits. In all cases,the assemblyof subunits constituring these multisubunit (multimeric) proteins occurs in the ER. An important class of multimeric secretedproteins is the immunoglobulins, which contain two heavy (H) and two light (L) chains,all linked by intrachaindisulfidebonds. Hemagglutinin (HA) is another multimeric protein that provides a good illustration of folding and subunit assembly(Figure 1320). This trimeric protein forms the spikes that protrude from the surface of an influenza virus particle. The HA trimer is formed within the ER of an infected host cell from

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

response'lre1,a 13-21The unfolded-protein < FIGURE hasa bindingsite proteinin the ERmembrane, transmembrane c o n t a i nas d o ; ec y t o s o l i c m a i n f o r B i Po n i t sl u m i n adlo m a i nt h unfolded Accumulating Step RNAendonuclease. Il: specific themfrom releasing proteins in the ERlumenbindBiPmolecules, its of lrel thenactivates lrel Dimerization monomeric mRNA activityStepsE, B: Theunspliced endonuclease by is cleaved Hacl factor thetranscription precursor encoding joined functional form to are exons two dimericlre1,andthe thatthisprocessing indicates evidence Hacl mRNACurrent generally processing althoughpre-mRNA occurstn thecytosol, Hacl into is translated occursin the nucleusStep4: Hacl activates and nucleus into the back protein, whichthenmoves catalysts' protein-folding several of genesencoding transcription 2000' etal' Bertolotti Cell'lO7:103;A 2001, etal, Ruegsegger U [See

Hacl

Spliced Hacl mRNA

E n d o n u c l e a s e - cH ua t cl Unspliced Hacl mRNA

andP Walter,1997,Ceil CellBiol 2:326;andC Sidrauski Nature 9 0 : 1 0 3l1

lrel dimer

Unfoldedproteins

Unfoldedproteins w i t h B i Pb o u n d

three copiesof a precursor protein termed HA6, which has a single membrane-spanningct helix. In the Golgi complex, each of the three HAe proteins is cleaved to form two polypeptides, HA1 and HA2; thus each HA molecule that eventually resideson the viral surfacecontains three copies of HAi and threeof HA2 (seeFigure3-10).The trimer is stabilized by interactions between the large exoplasmic domains of the constituent polypeptides,which extend into the ER lumen; after HA is transported to the cell surface,these domains extend into the extracellular space' Interactions between the smaller cytosolic and membrane-spanning portions of the HA subunits also help stabilize the trimeric protein. Studieshave shown that it takesjust 10 minutesfor the HAs polypeptidesto fold and assembleinto their proper trimeric conformation.

l m p r o p e r l yF o l d e dP r o t e i n si n t h e E RI n d u c e Expressionof Protein-FoldingCatalysts Vild-type proteins that are synthesizedon the rough ER cannot exit this compartment until they achievetheir completely folded conformation. Likewise, almost any mutation ih"t p..u..tts proper folding of a protein in the ER also blocks movement of the polypeptide from the ER lumen or membrane to the Golgi complex. The mechanismsfor retaining unfolded or incompletely folded proteins within the ER probably increase the overall efficiency of folding by keeping intermediateforms in proximity to folding catalysts, which are most abundant in the ER. Improperly folded proteins retained within the ER generallyare seenbound to the ER chaperonesBiP and calnexin. Thus theseluminal folding catalvsts perform two related functions: assisting in the

folding of normal proteins by preventing their aggregation and binding to irreversibly misfolded proteins' Both mammalian cellsand yeastsrespond to the presence

proteins that assistin protein folding' . Mammalian cells contain an additional regulatory pathway that operatesin responseto unfolded proteins in in. En. In this pathway, accumulation of unfolded pro-

F i g u r e s1 6 - 3 6 a n d 1 6 - 3 8 ) . A hereditaryform of emphysemaillustratesthe detrimental effects that can result from misfolding of proteins in the ER. This disease is caused by a point -rrt"tion in ct1-antitrypsin,which normally is secretedby

IN THEER M O D I F I C A T I O NFSO , L D I N GA, N D Q U A L I T YC O N T R O L PROTEIN

O

555

U n a s s e m b l eodr M i s f o l d e dp r o t e i n si n t h e ERAre Often Transportedto the Cytosol for Degradation

the cell altogether by degradation in the proteasome(see Figure 3-29).

Protein Modifications, Folding, and euality Control in the ER

proteases were never found. More recent studies have shown that misfolded membrane and secretoryprorelns are recognized by specific ER membrane proteins and are tar_ geted for transport from the ER lumen into the cytosol, by a processknown as dislocation or letrotrlnslocat.ion. The dislocationof misfoldedproteins out of the ER de_ pends on a set of proteins located in the ER membrane and in the cytosol that perform three basic functions. The first

r All N-linked oligosaccharides,which are bound to as_ paragineresidues,contain a core of three mannoseand two N-acetylglucosamineresidues and usually have several branches (seeFigure 13-16). O-linked olisosaccharides. which are bound to serineor threonine resid-ues, g.rr.r".. ally short, often containing only one to four ,rlg"r..ridrl... r Formarion of all N-linked oligosaccharidesbegins with assemblyof a conserved14-residuehigh-mannose precur_ sor on dolichol, a lipid in the membrane of the rough ER (seeFigure 73-17). After this preformed oligosaccharideis transferred to specific asparagine residues of nascent polypeptide chains in the ER lumen, three glucoseresidues and one mannoseresidueare removed(seeFigure 13-1g). r Oligosaccharideside chains may assist in the proper folding of glycoproteins, help protect the mature p.ot"irrc from proteolysis,participate in cell-celladhesion, fr.rrr._ ".rd tion as antigens. r Disulfide bonds are added to many secretory proteins and the exoplasmic domain of membrane proteins in the ER. Protein disulfide isomerase(pDI), presentin the ER lu_ men, catalyzesboth the formation and the rearrangement of disulfide bonds (seeFigure 13-19). r The chaperoneBiP, the lectins calnexin and calreticulin, and peptidyl-prolyl isomeraseswork together ro ensure proper folding of newly made secreroryand membrane proteins in the ER. The subunits of multimeric proteins also assemblein the ER. r Only properly folded proteins and assembledsubunits are transported from the rough ER to the Golgi complex in vesicles. r The accumularion of abnormally folded proteins and unassembledsubunits in the ER can induce increasedex_ pression of ER protein-folding catalystsvia the unfolded_ protern response(seeFigure 13-21). r Unassembledor misfolded proteins in the ER often are transported back to the cytosol, where they are degradedin the ubiquitin/proteasomepathway.

556

CHAPTER 13

I

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

Sortingof Proteinsto and Chloroplasts Mitochondria In the remainder of this chapter, we examine how proteins synthesizedon cytosolic ribosomes are sorted to mitochondria, chloroplasts, peroxisomes,and the nucleus (seeFigure 13-1).In both mitochondriaand chloroplastsan internallumen called the matrix is surrounded by a double membrane, and internal subcompartments exist within the matrix. In contrast, peroxisomesare bounded by a single membrane and have a single luminal matrix compartment. Becauseof these and other differences,we consider peroxisomesseparately in the next section. Likewise, the mechanism of protein transport into and out of the nucleus differs considerably from sorting to mitochondria and chloroplasts; this is d i s c u s s eidn t h e l a s t s e c t i o n . In addition to being bounded by two membranes'mitochondria and chloroplastsshare similar types of electron transport proteins and use an F-classATPase to synthesize ATP (seeFigure 12-2). Remarkably,gram-negativebacteria also exhibit thesecharacteristics.Also like bacterial cells' mitochondria and chloroplastscontain their own DNA, which encodesorganellerRNAs, tRNAs, and someproteins (Chapter 6). Moreover, growth and division of mitochondria and chloroplasts are not coupled to nuclear division. Rather, these organellesgrow by the incorporation of cellular proteins and lipids, and new organellesform by division of preexisting organelles.The numerous similarities of free-living bacterialcellswith mitochondria and chloroplastshave led scientiststo hypothesize that these organelles arose by the incorporation of bacteria into ancestraleukaryotic cells,

ORGANETTE TARGTT

Proteins encoded by mitochondrial DNA or chloroplast DNA are synthesizedon ribosomes within the organelles and directed to the correct subcompartmentimmediately af' ter synthesis.The majority of proteins located in mitochondria and chloroplasts, however, are encoded by genesin the nucleus and are imported into the organellesafter their synthesis in the cytosol' Apparently, over aeons of evolution' much of the geneticinformation from the ancestralbacterial DNA in these endosymbiotic organellesmoved' by an unknown mechanism, to the nucleus' Precursor proteins synthesizedin the cytosol that are destinedfor the matrix of mitochondria or the equivalent space in chloroplasts, the stroma, usually contain specificN-terminal uptake-targeting sequencesthat specifybinding to receptor proteins on the organelle surface. Generally, this sequenceis cleaved once it i.a.hes the matrix or stroma. CIearlS theseuptake-targeting sequencesare similar in their location and general function to ihe signal sequencesthat direct nascentproteins to the ER lumen. Although the three types of signalsshare some common sequencefeatures, their specific sequencesdiffer considerably,as summarizedin Table 13-1. In both mitochondria and chloroplasts, protein import requiresenergy and occurs at points where the outer and innei organellemembranesare in closecontact' Becausemitochondiia and chloroplasts contain multiple membranesand

OFSEOUEI|CE BEM()VAL PROTTIN WITHIN OFSEOUENCE LOCATII]N

OTSEOUEIICE NATURE

Endoplasmic reticulum (lumen)

N-terminus

Yes

Core of 6-12 hydrophobicamino acids, often precededby one or more basicamino acids (Arg, Lys)

Mitochondrion (matrix)

N-terminus

Yes

Amphipathichelix, 20-50 residuesin length, with Arg and Lys residueson one sideand hydrophobicresidueson the other

Chloroplast (stroma)

N-terminus

Yes

No commonmotifs;generallyrich in Ser' Thr, and smallhydrophobicresiduesand poor in Glu and AsP

Peroxisome (matrix)

C-terminus(mostproteins) N-terminus(few proteins)

No

at extreme PTSl signal(Ser-Lys-Leu) C-terminus;PTS2signalat N-terminus

Nucleus (nucleoplasm)

Varies

No

Multipledifferentkindsla commonmotif includesa short segmentrich in Lys and Arg residues

subcompartments' "Different or additional sequencestarget proteins to organelle membranes and AND CHLOROPLASTS SO M I T O C H O N D R I A S O R T I N GO F P R O T E I N T

557

> EXPERIMENTAL FIGURE 13-22lmport of mitochondrialprecursorproteinsis assayedin a cell-freesystem.Inside mitochondria, proteins areprotected from the actionof proteases suchastrypsinWhen no mitochondria arepresent, mitochondrial proteins synthesized in thecytosol are degraded by addedproteaseproteinuptake occurs onlywith energized (respiring) mitochondria, whichhavea proton electrochem icalgradient (proton-motive force)across the innermembrane The imported proteinmustcontainan appropriate uptake-targeting sequence Uptakealso requires ATPanda cytosolic extractcontaininq chaperone proteins thatmarntain the precursor proterns in an unfolded conformation Thisassay hasbeenusedto studytargeting sequences andotherfeatures of thetranslocation process

Uptaketargeting sequence

h_\{L/ t

Mitochondrial prolern

Add energized yeasr mitochondria

Yeast mitochondrial proteinsmade by cytoplasmicribosomes in a cell-freesystem

Proteintaken up into mitochondria; uptake-ta rgeting sequenceremoved and degraded

Proteinssequestered within mitochondria are resistantto trypsin ..a

Trypsin

Uptake-targeting sequenceano mitochondrial protein degraded

membrane-limited spaces,sorting of many proteins to their correct location often requires the sequentialaction of two targetlng sequencesand two membrane-boundtranslocation

A m p h i p a t h i cN - T e r m i n aSl i g n a lS e q u e n c e s D i r e c tP r o t e i n st o t h e M i t o c h o n d r i aM l atrix

. .a. , & 't' '.e ' j .r'' r t'. a' a. 'a -..3

-

pathicity of matrix-targeting sequencesis critical to their function. The cell-free assay outlined in Figure 13-22 has been widely used in studies on the import of mitochondrial precursor proteins. In this system, respiring (energized)mito_ chondria extractedfrom cellscan incorporate mitochondrial precursor proteins carrying appropriate uptake-targetingse_ quencesthat have been synthesizedin the absenceof mitochondria. Successfulincorporation of the precursor into the

translocation of secretoryproteins into the ER, which gener_ ally occurs only when microsomal (ER-derived)-.rnbn"rr., are present during synthesis(seeFigure 13-4).

M i t o c h o n d r i aP l r o t e i nl m p o r t R e q u i r e s Outer-MembraneReceptorsand Translocons in Both Membranes Mitochondrial matrix-targeting sequencesare thought to assume an a,helical conformation in which positively charged amino acids predominate on one side ofthe helix

558

'

c H A p r E R1 3 I

Figure 13-23 presentsan overview of protein import from the cytosol into the mitochondrial matrix, the route into the mitochondrion followed by most imported proteins. \Wewill discussin detail each step of protein transport into the matrix and then consider how some proteins subsequentlyare ' targeted to other compartments of the mitochondiion. After synthesisin the cytosol, the soluble precursors of mitochondrial proteins (including hydrophobic integral membrane proteins) inreract directly with the mitochondrial membrane. In general, only unfolded proteins can be im_ ported into the mitochondrion. Chaperoneproteins such as

M o v r N Gp R o r E r NrsN T oM E M B R A N A EN s Do R G A N E L L E '

coo

ATP A D P+ P ; Cytosolic Hsc70

Matrix-targeting sequence

-'.'./

NHs*

General i m p o r tp o r e (Tom40)

lmport recepror (Tom 20122\ Cytosol

+\

Outer membrane

13-23Proteinimport intothe < FIGURE proteins mitochondrialmatrix.Precursor are ribosomes on cytosolic synthesized folded or partially in an unfolded maintained Hsc70 as such chaperones, stateby bound proteinbindsto (steptr) Aftera precursor neara siteof contactwith an importreceptor (stepZ), it istransferred the innermembrane importpore(stepB) The intothe general protein thenmovesthroughthls translocating c h a n n ealn da n a d j a c e nc th a n n ei nl t h ei n n e r (stepsZl, E). Notethat membrane at rare"contactsites" occurs translocation outermembranes and inner at whichthe of thetranslocating appearto touch.Binding Hsc70and proteinbythe matrixchaperone by Hsc70helps ATPhydrolysis subsequent driveimportintothe matrixOncethe by a is removed sequence uptake-targeting from andHsc70isreleased matrixprotease protein(step6), it folds the newlyimported within activeconformation intothe mature, proteins (step of some Folding the matrix Z) [See on matrixchaperonins depends and J BiolChem271:31763, 1996, G Schatz, el al , 1997,Ann Rev.CellDevelBiol N Pfanner 13:25l

Matrix Hsc70 ADP + P; Matrix processlng protease

Cleaved targeting seq uence

cytosolic Hsc70 keep nascentand newly made proteins in an unfolded stateso that they can be taken up by mitochondria' This processrequiresATP hydrolysis' Import of an unfolded mitochondrial precursor is initiated by the binding of a mitochondrial targeting sequenceto an import receptor in the outer mitochondrial membrane. These receptors were first identified by experimentsin which antibodiesto specificproteins of the outer mitochondrial membranewere shown to inhibit protein import into isolated mitochondria. Subsequent genetic experiments, in which the genes for specific mitochondrial outer-membraneproteins were mutated, showed that specificreceptorproteinswere responsiblefor the import of different classesof mitochondrial proteins. For example, N-terminal matrix-targeting sequencesare recognized by Tom20 and Tom22. (Proteinsin the outer mitochondrial membrane involved in targeting and import are designated Tom oroteins for /ranslocon of the outet membtane.)

The import receptorssubsequentlytransfer the precursor proteins to an import channel in the outer membrane' This ch".tn.l, composed mainly of the Tom40 protein, is known ^s the general import pore becauseall known mitochondrial compartments precursor proteins gain accessto the interior 'Sfhen purified channel' this through -itt.hondrion ft tn. transmema forms Tom40 liposomes, into incorporated and brane channel with a pore wide enough to accommodatean unfolded polypeptideihain. The general import pore forms a largely p"tti". channel through the outer mitochondrial and the driving force for unidirectional transport -.*L.".t., into mitochondria comesfrom within the mitochondrion' In the caseof precursorsdestinedfor the mitochondrial matrix, transfer through the outer membrane occurs simultaneously with transfer through an inner-membranechannel composed of the Tim23 and1iml7 proteins. \Tim standsfor translocon of the lnner membrane.)Translocation into the matrix

O SO M I T O C H O N D R I A N D C H L O R O P L A S T S S O R T I N GO F P R O T E I N T

559

thus occurs at "contact sites" where the outer and inner membranesare in close proximitv.

drial membraneby interactingwith transmembraneprotein Tim44. This interaction stimulatesATp hydrolysis by matrix Hsc70, and together these two proteins are thought to power translocationof proteinsinto the matnx. Some imported proteins can fold into their final, active conformation without further assistance.Final folding of many matrrx proteins, however, requires a chaperonin. As discussedin Chapter 3, chaperoninproteins aciively facilitate protein folding in a processthat dependson ATp. For in_ stance,yeastmutantsdefectivein Hsc60, a chaperoninin the mitochondrial matrix, can import matrix proteins and cleavetheir uptake-targerrngsequencenormally, but the imported polypeptidesfail to fold and assembleinto the native tertiary and quaternarystructures.

(a)

(b)

S t u d i e sw i t h C h i m e r i cP r o t e i n sD e m o n s t r a t e l m p o r t a n tF e a t u r e so f M i t o c h o n d r i a lm p o r t Dramatic evidencefor the ability of mitochondrial matrixtargettng sequencesto direct import was obtained with chimeric proteins produced by recombinant DNA techniques. For example, the matrix-targeting sequenceof alcohol dehydrogenasecan be fused to the N-terminus of dihydrofolate reductase(DHFR), which normally residesin the cytosol. In the presenceof chaperones,which prevent the Cterminal DHFR segmentfrom folding in the cytosol, cell-free translocation assaysshow that the chimeric protein is transported into the marrix (Figure 13-24a). The inhibitor methotrexate,which binds tightly ro the acive site of DHFR and greatly stabilizes its folded conformation, renders the chimeric protein resistant to unfolding by cytosolic chaperones. \il1'hentranslocation assaysare performed in the presence of methotrexate, the chimeric protein does not iompletely enter rhe matrix. This finding demonstratesthat a precursor must be unfolded in order to traverse the imoort poresin the mitochondrialmembranes.

Bound methotrexate inhibitor

(cl

Cytosol Outer

F o l d e dDHFR Cytosol Outer membrane Intermembrane space Intermembrane space

NHr* .d9" .s9' . ((.9. \$$o

Mitochondrial matrix

Cleaved targeting sequence

,

Spacerr"qr"n.i / EXPERIMENTAL FTGURE 13-24 Experimentswith chimeric proteins elucidate mitochondrial protein import. These experiments show that a matrix-targeting sequencealonedirects

energizem d i t o c h o n d r iaan d t h e m a t r i x _ t a r g e t isnigg n atl h e n i s removed.(b) When the C-terminusof the chimericproteinis locked in the foldedstateby bindingof methotrexate, translocalonrs blocked lf the spacersequenceis long enoughto extendacrossboth

560

CHAPTER 13

I

o'2 P'm

,

transport channels, a stabletranslocation intermediate, with the targeting sequence cleaved off, isgenerated in the presence of methotrexate, asshownhere.(c)TheC-terminus of thetranslocation intermediate in (b)canbe detected by incubating the mitochondria with antibodies thatbindto the DHFR segment, followedby gold particles coatedwith bacterial proteinA, whichbindsnonspecificallv to antibody (seeFigure9-21).An electron molecules micrograph of a sectioned sample goldparticles (redarrowhead) reveals boundto the translocation intermediate at a contactsrtebetween the innerand outermembranes Othercontactsites(blackarrows) alsoareevident (a)and(b)adapted IParts fromJ Rassow et al, 1990,FEBS Letters 2Tstigo Part(c) from M SchweigereI al , j987, I CellBiol. 105:235,courtesyof W N e u o e r tl

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

Additional studies revealed that if a sufficiently long spacer sequenceseparatesthe N-terminal matrix-targeting sequenceand DHFR portion of the chimeric protein, then in the presenceof methotrexate a translocation intermediate that spansboth membranescan be trapped if enough of the polypeptideprotrudes into the matrix to preventthe polypeptide chain from sliding back into the cytosol, possibly by stably associatingwith matrix Hsc70 (Figure 13-24b).In order for such a stable translocation intermediateto form, the spacer sequencemust be long enough to span both membranes;a spacerof 50 amino acids extendedto its maximum possiblelength is adequateto do so. If the chimeracontains a shorter spacer-say, 35 amino acids-no stable translocation intermediateis obtained becausethe spacercannot span both membranes.These observationsprovide further evidence that translocated proteins can span both inner and outer mitochondrial membranes and traverse these membranesin an unfolded state. Microscopic studiesof stabletranslocation intermediates show that they accumulateat siteswhere the inner and outer mitochondrial membranes are close together, evidencethat precursor proteins enter only at such sites (Figure 1,3-24c). The distancefrom the cytosolic face of the outer membrane to the matrix face of the inner membrane at these contdct sitesis consistentwith the length of an unfolded spacer sequence required for formation of a stable translocation intermediate.Moreover, stabletranslocation intermediatescan be chemically cross-linkedto the protein subunits that comprise the translocation channelsof both the outer and inner membranes.This finding demonstratesthat imported proteins can simultaneouslyengagechannels in both the outer and inner mitochondrial membrane, as depicted in Figure 13-23. Since roughly 1000 stuck chimeric proteins can be observedin a typical yeast mitochondrion, it is thought that mitochondria have approximately 1000 general import pores for the uptake of mitochondrial proteins.

Three EnergyInputs Are Neededto lmport P r o t e i n si n t o M i t o c h o n d r i a As noted previously and indicated in Figure 13-23, ATP hydrolysis by Hsc70 chaperoneproteins in both the cytosol and the mitochondrial matrix is required for import of mitochondrial proteins. Cytosolic Hsc70 expends energy to maintain bound precursor proteins in an unfolded statethat is competent for translocationinto the matrix. The importance of ATP to this function was demonstratedin studiesin which a mitochondrial precursor protein was purified and then denatured (unfolded) by urea. \7hen tested in the cell-free mitochondrial translocation system,the denatured protein was incorporated into the matrix in the absenceof AIP. In contrast' import of the native, undenaturedprecursorrequired ATP for the normal unfolding function of cytosolic chaperones. The sequentialbinding and ATP-drivenreleaseof multiple matrix Hsc70 moleculesto a translocatingprotein may simply trap the unfolded protein in the matrix. Alternatively, the matrix Hsc70, anchoredto the membraneby the Tim44 protein, may act as a molecular motor to pull the

protein into the matrix (seeFigure 13-23).In this case'the lunctions of matrix Hsc70 andTim44 would be analogous to those of the chaperone BiP and Sec63 complex, respectivelS in post-translational translocation into the ER lumen ( s e eF i g u r e1 3 - 9 ) . The third energy input required for mitochondrial protein import is a H+ electrochemicalgradient, or protonmotive force, acrossthe inner membrane' Recall from Chapter 12 that protons are pumped from the matrix into the intermembrane space during electron transport, creating transmembranepotential across the inner membrane' In general,only mitochondria that are actively undergoing respiration, and thereforehave generateda proton-motive force acrossthe inner membrane' are able to translocateprecursor proteins from the cytosol into the mitochondrial matrix' of mitochondria with inhibitors or uncouplers of ir.u,-.n, oxidative phosphorylation' such as cyanide or dinitrophenol, dissipatesthis proton-motive force. Although precursor proteins still can bind tightly to receptorson such poisoned mitochondria, the proteins cannot be imported, either in intact cells or in cell-freesystems,even in the presenceof ATP and chaperone proteins. Scientistsdo not fully understand how the proton-motive force is used to facilitate entry of a p...rr.roi p.otein into the matrix. Once a protein is partially inserted into the inner membrane' it is subjectedto a trans-

M u l t i p l eS i g n a l sa n d P a t h w a y sT a r g e tP r o t e i n s t o S u b m i t o c h o n d r i aCl o m p a r t m e n t s Unlike targeting to the matrix, targeting of proteins to the intermembrane space,inner membrane, and outer membrane of mitochondria generally requires more than one targetlng sequenceand occurs via one of severalpathways' Figure 1325 summarizes the organization of targeting sequencesin proteins sorted to different mitochondrial locations. lnner-Membrane Proteins Three separatepathways are known to target proteins to the inner mitochondrial membrane. One pathway makes use of the same machinery that is used for targeting of matrix proteins (Figure 13-26, path A). A cytochrome oxidase subunit called CoxVa is a protein transported by this pathway' The precursor form of CoxVa' which contains an N-terminal matrix-targeting sequence recognizedby the Tom20l22 import receptor,is transferred through the generalimport pore of the outer membrane and the inier-membrane Tim23l17 translocation complex' In addition to the matrix-targeting sequence'which is cleaved during import, CoxVa contains a hydrophobic stop-transfer s.qr'r*ce. A. the protein passesthrough theTim23ll'7 chan.r.i th. stop-transfer sequenceblocks translocation of the

AND CHLOROPLASTS O SO M I T O C H O N D R I A S O R T I N GO F P R O T E I N T

561

Location of imported protein

lmported protein

Locations of targeting sequences in preprotein

Cleavageby matrix protease Alcohol d e h y d r o g e n a slel l

Matrix

*.*T**" Matrix-targeting sequence

Inner membrane PathA

Cytochrome o x i d a s es u b u n i t CoxVa

Cleavageby Hydrophobic matrix protease stop-transfersequence

Cleavageby matrix protease Path B

Mature protein

< FIGURE 13-25Targetingsequences in imported mitochondrialproteins.Most mitochondrial proteins havean N-terminal (pink)thatissimilar matrix-targeting sequence proteins but not identical in different proteins. destined for the innermembrane, the rntermembrane space, or the outermembrane haveoneor moreadditional targeting sequences thatfunctionto directthe proteins to theselocations by several different pathways. Theletteredpathways correspond to thoseillustrated in Figures 13-26and 13-27 [See W Neupert, 1997, Ann Rev. Biochem 66:863 l

Internalsequences recognizedby Oxal

ATP synthase s u b u n i t9 Internalsequencesrecognized by Tom70 receptor andTrm22 complex

Path c

ADP/ATP anttporter

Intermembrane space

PathA

Firstcleavageby matrix

Secondcleavageby protease

Cytochromeb2 Intermembrane-space-targeting sequence

Targetingsequencefor the generalimport pore path B

Cytochromec h e m el y a s e

Outer membrane

\--...P\-..^t

Stop-transferand outer-memorane localizationsequence Porin (P70)

C-terminus across the inner membrane. The membraneanchored intermediate is then transferred laterally into the bilayer of the inner membrane much as type I integral membrane proteins are incorporated into the ER membrane (see F i g u r e1 3 - 1 1 ) .

matrix via the Tom40 andTim23l17 channels.After cleav_ age of the matrix-targeting sequence,the protein is inserted into the inner membrane by a processthai requires interac_ 562

.

c H A p r E lR3

tion with Oxal and perhaps other inner-membraneproteins (Figure 13-26, parh B). Oxal is related to a bacterialprotein involved in inserting some inner-membraneproteins in bacteria. This relatednesssuggeststhat Oxal may have descendedfrom the translocation machinery in the endosymbiotic bacterium that eventually became the mitochondrion. However, the proteins forming the inner-membranechannels in mitochondria are not related to the proteins in bacterial translocons. Oxal also participates in ihe inner-membrane insertion of certain proteins (e.g., subunit II of cytochrome oxidase) that are encoded by mitochondrial DNA and synthesizedin the matrix by mitochondrial ribosomes. The final parhway for insertion in the inner mitochondrial membrane is followed by multipass proteins that

| M o v t N Gp R o r E t Nt sN T oM E M B R A NAENsD O R G A N E L L E S

Path B

Path A

Stop-transfer Matrix-targeting sequence sequence NHs NHs*

Tom40 Tom22

Cytosol

Tom20

Outer membrane

lntermembrane space Ttm23l17

; €

A Assembled protein

Mitochondrial matrix

Cleaved matrix-targeting SeQu€nCeS-_--_-

13-26Threepathwaysto the innermitochondrial A FIGURE with differenttargeting membranefrom the cytosol.Proteins pathways. viadifferent to the innermembrane aredrrected sequences via the proteins membrane pathways, the outer cross three In all A andB by pathways delivered importpore Proteins Tom40general thatis recognized sequence matrix-targeting containan N-terminal Although in theoutermembrane importreceptor bytheTom20/22 channel, 7 inner-membrane usetheTim23/1 boththesepathways proteinentersthe matrixand theydifferin thatthe entireprecursor in pathwayB MatrixHsc70 to the innermembrane thenisredirected

contain six membrane-spanning domains, such as the ADP/ATP antiporter. Theseproteins, which lack the usual N-terminal matrix-targeting sequence'contain multiple internal mitochondrial targeting sequences.After the internal sequencesare recognizedby a secondimport receptor composed of outer-membrane proteins Tom70 and Tom22, the imported protein passesthrough the outer membranevia the generalimport pore (Figure 13-26, path C). The protein then is transferredto a secondtranslocation complex in the inner membrane composedof the Tim22 and Tim54 proteins. Transfer to the Tim22l54

matrixproteins to itsrolein the importof soluble playsa rolesimilar by pathwayC containtnternal delivered (seeFigure13-23). Proteins importreceptor; by the Tom70/fom22 that arerecognized sequences (Tim22154) isused channel translocation inner-membrane a different (Tim9 Tim10) proteins and pathway. intermembrane Two in this betweenthe outerandinnerchannelsSeethe transfer facilitate andA Kuhn,2000,AnnRevCell [SeeR E Dalbey text for discussion

DevelBiol16:5l,andNPfannerandAGeissler,2OOl,NatureRevMolC Biol 2:339l

protein into the inner membrane.

. SO M I T O C H O N D R I A N D C H L O R O P L A S T S S O R T I N GO F P R O T E I N T

563

Intermembrane-Space Proteins Two pathways deliver cytosolicproteinsto the spacebetweenthe inner and outer mitochondrial membranes.The major pathway is followed by proteins, such as cytochrome b2, whose precursorscarry rwo different N-terminal targeting sequences,both of which ultimately are cleaved.The most N-terminal of the rwo seouences is a matrix-targeting sequence,which is removed by th. -a-

proteolytic cleavage,this pathway is similar to that of innermembraneproteins suchas CoxVa (seeFigure 13-26,parh A). Cytochrome c heme lyase, the enzyme responsiblefor the covalent attachment of heme to cytochrome c, illustrates a secondpathway for targetingto the intermembrane space.In this pathway, the imported protein does not con_ tain an N-terminal matrix-targetingsequenceand is deliv_ ered directly to the intermembranespace via the general

import pore without involvement of any inner-membrane translocationfactors (Figure 13-27, path B). Sincetranslocation through the Tom40 general import pore does not seemto be coupled to any energeticallyfavorable process such as hydrolysis of ATP or GTP, the mechanism rhat drives unidirectional translocation through the outer membrane is unclear. One possibility is that cytochrome c heme lyase passively diffuses through the outlr membrane and then is trapped within the intermembrane spaceby binding to another protein that is deliveredto that location by one of the translocationmechanismsdiscussedpreviously. Outer-Membrane Proteins Many of the proteinsthat reside in the mitochondrial outer membrane, including the Tom 40 pore itself and mitochondrial porin, have a B-barrel structure in which antiparallel strandsform the hydrophobic transmembranesegmentssurrounding a central channel. Such proteins are incorporated into the outer membrane by first interacting with the general import pore, Tom40, and then they are transferred to a complex known as the SAM (sorting and assembly machinery) complex, which is composedof at leastthree outer membraneproteins.presumably it is the very stable hydrophobic nature of B-barrel proteins

Path A

Path B

Intermembrane-space-targeti ng Matrix-targeting sequence /, 'NHs*

I n t e r m e mD r a n e-space-targeting sequence Protein -*..-

NHs

Tom22

Tom20

Tim23l17 Mitochondrial matrix

Cleaved

3:'J'J;'.T'"''"n FfGURE 13-27 Two pathwaysto the mitochondrial intermembrane space.pathway A, the majoronefor delivery of proteins fromthe cytosol to the intermembrane space, issimilar to pathway A for delivery to the innermembrane (seeFigure13_26) Themajordifference isthatthe internal targeting sequence in proteins suchascytochrome b2destined for the intermembrane spaceisrecognized by an inner-membrane protease. whichcleaves

564

CHAPTER 13

|

theproteinon the intermembrane-space sideof the membrane Thereleased proteinthenfoldsandbindsto itshemecofactor within the intermembrane spacePathway B involves directdelivery to the intermembrane spacethroughtheTom40general importporein the outermembrane. andA Kuhn,2OOO, [SeeR E Dalbey Ann Rev. Cett Devel Biol 16:51; N PfannerandA Geissler,2001, Nature Rev.Mol. CettBiot. 2 : 3 3 9 ;a n d K D i e k e r te t a l , 1 9 9 9 ,p r o c N a t , l A c a d S c i U S A 9 6 : 1 1 1 5 2 ] |

M O V T N GP R O T E t NtSN T O M E M B R A N E A SN D O R G A N E L L E S

that ultimately causesthem to be stably incorporated into the outer membrane, but precisely how the SAM complex facilitatesthis processis not known.

Targetingof ChloroplastStromalProteins l s S i m i l a rt o l m p o r t o f M i t o c h o n d r i a l Matrix Proteins Among the proteins found in the chloroplast stroma are the enzymesof the Calvin cycle,which function in fixing carbon dioxide into carbohydratesduring photosynthesis(Chapter 12). The large (L) subunit of ribulose 1,5-bisphosphate carboxylase(rubisco) is encodedby chloroplast DNA and synthesizedon chloroplastribosomesin the stromal space. The small (S) subunit of rubisco and all the other Calvin cycle enzymesare encodedby nuclear genesand transported to chloroplastsafter their synthesisin the cytosol.The precursor forms of these stromal proteins contain an N-terminal stromal-impolt sequence(seeTable 13-1). Experiments with isolated chloroplasts, similar to those with mitochondria illustrated in Figure 1'3-22, have shown that they can import the S-subunitprecursor after its synthesis. After the unfolded precursor entersthe stromal space,it binds transiently to a stromal Hsc70 chaperone and the N-terminal sequenceis cleaved. In reactions facilitated by Hsc60 chaperoninsthat reside within the stromal space, eight S subunits combine with the eight L subunits to yield the active rubisco enzyme. The generalprocessof stromal import appearsto be very similar to that for importing proteins into the mitochondrial matrix (seeFigure 1,3-23).At least three chloroplast outermembrane proteins, including a receptor that binds the stromal-import sequenceand a translocationchannelprotein' and five inner-membraneproteins are known to be essential for directing proteins to the stroma. Although theseproteins are functionally analogousto the receptor and channel proteins in the mitochondrial membrane,they are not structurally homologous. The lack of sequencesimilarity between these chloroplast and mitochondrial proteins suggeststhat they may have arisen independentlyduring evolutton. The available evidencesuggeststhat chloroplast stromal proteins, like mitochondrial matrix proteins, are imported in the unfolded state. Import into the stroma dependson ATP hydrolysis catalyzedby a stromal Hsc70 chaperone whose function is similar to that of Hsc70 in the mitochondrial matrix and BiP in the ER lumen. Unlike mitochondria, chloroplasts do not generatean electrochemicalgradient (protonmotive force) across their inner membrane. Thus protein import into the chloroplast stroma appears to be powered s o l e l yb y A T P h y d r o l y s i s .

ProteinsAre Targetedto Thylakoidsby MechanismsRelatedto TranslocationAcross t h e B a c t e r i aIln n e r M e m b r a n e In addition to the double membranethat surroundsthem, chloroplasts contain a series of internal interconnected

membranous sacs, the thylakoids (seeFigure 12-29). Proteins localized to the thylakoid membrane or lumen carry out photosynthesis.Many of theseproteins are synthesized in the cytosol as precursorscontaining multiple targeting sequences.For example, plastocyanin and other proteins destined for the thylakoid lumen require the successiveaction The first is an N-terminal of two uptake-targetingsequences. the protein to the directs that sequence stromal-import the rubisco S subimports pathway that same the stroma by protein from the the targets sequence second The unit. targeting these role of The lumen' thylakoid the to stroma the measuring in experiments shown has been sequences DNA recombinant by generated proteins mutant of uptake techniques into isolated chloroplasts. For instance' mutant plastocyanin that lacks the thylakoid-targeting sequence but contains an intact stromal-import sequenceaccumulates in the stroma and is not transported into the thylakoid Iumen. Four separatepathways for transporting proteins from the stroma into the thylakoid have been identified. All four pathways have been found to be closely related to analogolls t."ntport mechanismsin bacteria' illustrating the close evolutionary relationship between the stromal membrane and the bacterial inner membrane. Transport of plastocyanin and related proteins into the thylakoid lumen from the stroma occurs by a chloroplast SRP-dependentpathway that utilizes a translocon similar to SecY,the bacterial version of the Sec61complex (Figure 1'3-28,left). A second pathway for transporting proteins into the thylakoid lumen inuolues a protein related to bacterial protein SecA, which uses the energy from ATP hydrolysis to drive protein translocation through the SecY translocon. A third pathway, which targets proteins to the thylakoid membrane, depends on a protein related to the mitochondrial Oxal proiein and the homologous bacterial protein (see Figure 13-26,path B). Someproteinsencodedby chloroplastDNA and synthesizedin the stroma or transported into the stroma from the cytosol are inserted into the thylakoid membrane via this pathwaY. Finally, thylakoid proteins that bind metal-containing cofactors follow another pathway into the thylakoid lumen (Figure 13-28, rigbt). The unfolded precursorsof theseproteiis are first targeted to the stroma' where the N-terminal stromal-import sequenceis cleavedoff and the protein then folds and bittdt itt cofactor' A set of thylakoid-membrane proteins assistsin translocating the folded protein and tound cofactor into the thylakoid lumen, a processpowered by the pH gradient normally maintained across the thylakoid membrane.The thylakoid-targeting sequencethat directs a protein to this pH-dependent pathway includes two closely spacedarginine residuesthat are crucial for recognition. Baiterial cells also have a mechanismfor translocating folded proteins with a similar arginine-containing sequence across the inner membrane. The molecular mechanism whereby theselarge folded globular proteins are transported across the thylakoid membrane is currently under intense srudy.

SO M I T O C H O N D R I A N D C H L O R O P L A S T S S O R T I N GO F P R O T E I N T

555

Thylakoid-targeting sequence

Plastocya nin Precursor

coo

COO

Stromal-import --..sequence NrLr Hat

Toc complex

Toc complex

Cytosol Outer membrane Intermembrane space I n n e rm e m b r a n e ,

,)

r,..r,

Tic comprex

Stroma

""\ "".*' g'"auudrnoon sequence Fl

E,[\

/ ,tChloroplast SRP

I SRP-dependent .' pathway

Metal-binding

:-.-.,** RR Cleavedimporl sequence

Bound metal tons

Chloroplast SRPreceptor RR.b.--

Mature plastocyani n

Mature metal-binding protein

Sorting of Proteins to Mitochondria and Chloroplasts r Most mitochondrial and chloroplast proteins are en_ coded by nuclear genes,synthesizedon cytosolic ribosomes, and imported post-translationallyinto the organelles.

r Cytosolic chaperonesmaintain the precursors of mito_ chondrial and chloroplast proteins in an unfolded state. Only unfolded proteins can be imported into the organelles. 556

CHAPTER 13

I

':,!.) 'j.:

< FIGURE 13-28Transporting proteins to chloroplastthylakoids.Twoof the four pathways for transporting proteins fromthecytosol to thethylakoid lumenare shownhere In thesepathways, unfolded precursors aredelivered to thestromavia thesameouter-membrane proteins that importstromal-localized proteins. Cleavage of the N-terminal stromal-import sequence by a stromalprotease thenreveals the thylakoid-targeting (steptr) sequence At thispointthetwo pathways diverge. ln the SRP-dependent (/efr), pathway plastocyanin proteins andsimilar arekept unfolded in the stromal spaceby a setof (notshown)and,directed chaperones by thethylakoid-targeting sequence, bindto proteins thatareclosely related to the bacterial SRP, SRPreceptor, andSecY translocon, whichmediate movement into the lumen(stepZ). Afterthe thylakoidtargeting sequence is removed in the thylakoid lumenbya separate endoprotease, theproteinfoldsintoitsmatureconformation (stepB) In the pH-dependent pathway (nght),metal-binding proteins fold in the stroma,andcomplexredoxcofactors are added(stepZ). Twoarginine (RR) residues at the N-terminus of thethylakoidtargeting sequence anda pHgradient across the innermembrane arereouired for transport of thefoldedproteinintothe thylakoid lumen(stepg). Thetranslocon in thethylakoid membrane iscomposed of at leastfourproteins related to proteins in thebacterial rnnermembrane. Thethylakoid targeting sequence containing thetwo arginine residues iscleaved in thethylakoid lumen(step4) [See R Datbey andC Robinson, 1999,TrendsBiochem Sci24:1j,R E Dalbey andA Kuhn,2000, Ann Rev. CellDevelBiol. 1 5 : 5 1 ;a n d C R o b i n s oann d A B o l h u i s2.0 0 1 . NatureRev.Mol CellBiol 2:350)

Translocation in mitochondria occurs at sites where the outer and inner membranesof the organellesare close together. r Proteins destined for the mitochondrial matrix bind to receptors on the outer mitochondrial membrane and then are transferred to the general import pore (Tom40) in the outer membrane.Translocationoccursconcurrentlythrough the outer and inner membranes,driven bv the Drotonmotive force acrossthe inner membraneand ATp hyirolysis by the Hsc70 ATPasein the matrix (seeFigure 13-23). r Proteins sorted to mitochondrial destinationsother than the matrix usually contain two or more targeting sequences, one of which may be an N-terminal matrix-targeting sequence (seeFigure 13-25).

M O V T N Gp R O T E t N |SN T O M E M B R A N E A SN D O R G A N E L L E S

r Somemitochondrial proteins destinedfor the intermembrane spaceor inner membrane are first imported into the matrix and then redirected; others never enter the matrix but go directly to their final location. r Protein import into the chloroplast stroma occurs through inner-membrane and outer-membrane translocation channelsthat are analogousin function to mitochondrial channels but composed of proteins unrelated in sequenceto the correspondingmitochondrial proteins. r Proteins destined for the thylakoid have secondarytargeting sequences.After entry of these proteins into the reveals stroma,cleavageof the stromal-targetingsequences the thylakoid-targetingsequences.

many different peroxisomal matrix proteins bear a sequence of this type, known as peroxisomal-targetingsequenceL' or simply PTS1. The pathway for import of catalaseand other PTSIbearing proteins into the peroxisomal matrix is depicted in Figure 13-29. The PTSl binds to a soluble carrier protein in the cytosol (Pex5), which in turn binds to a receptor in the peroxisomemembrane(Pex14).The solubleand membraneassociatedperoxisomal import receptors appear to have a function analogous to that of the SRP and SRP receptor in targeting proteins to the ER lumen, except that the soluble ptsf -bindlng protein functions post-translationally.The protein to be imported then moves through a multimeric

r The four known pathways for moving proteins from the chloropiast stroma to the thylakoid closely resemble translocation acrossthe bacterial inner membrane (seeFigure 13-28). One of these systemscan translocatefolded protelns.

Proteins Sortingof Peroxisomal Peroxisomesare small organellesbounded by a single membrane. Unlike mitochondria and chloroplasts, peroxisomes lack DNA and ribosomes.Thus all peroxisomalproteinsare encoded by nuclear genes,synthesizedon ribosomes free in the cytosol, and then incorporated into preexisting or newly generatedperoxisomes.As peroxisomesare enlargedby addition of protein (and lipid), they eventuallydivide, forming new ones,as is the casewith mitochondria and chloroplasts. The size and enzyme composition of peroxisomes vary considerablyin different kinds of cells. However, all peroxisomescontain enzymesthat use molecular oxygen to oxidize various substratessuch as amino acids and fatty acids, breaking them down into smaller components for use in biosyntheticpathways.The hydrogenperoxide (H2O2)generated by theseoxidation reactionsis extremely reactiveand potentially harmful to cellular componentsl however, the peroxisome also contains enzymes,such catalase,that efficiently convert H2O2 into H2O. In mammals,peroxisomes are most abundant in liver cells,where they constitute about 1 to 2 percent of the cell volume.

CytosolicReceptorTargetsProteinswith a n S K LS e q u e n c ea t t h e C - T e r m i n uisn t o l atrix t h e P e r o x i s o m aM The import of catalaseand other proteins into rat liver peroxisomes can be assayedin a cell-freesystemsimilar to that used for monitoring mitochondrial protein import (seeFigure 73-22). By testing various mutant catalaseproteins in this system, researchersdiscovered that the sequenceSerLys-Leu (SKL in one-lettercode) or a related sequenceat the C-terminus was necessaryfor peroxisomal targeting. Further, addition of the SKL sequenceto the C-terminus of a normally cytosolicprotein leadsto uptake of the alteredprotein by peroxisomes in cultured cells. All but a few of the

Pex14

E Peroxisomal matrix protein

13-29PTS1directedimportof peroxisomalmatrix A FIGURE matrix andmostotherperoxisomal proteins.Step[: Catalase (red) sequence PTSluptake-targeting proteins containa C-terminal with the Pex5 Step Pex5. receptor Z: thatbindsto the cytosolic on located receptor withthe Pex'14 boundmatrixproteininteracts StepB: Thematrixprotein-Pex5 membrane. the peroxisome (Pex'l 0, proteins to a setof membrane isthentransferred complex into the for translocation necessary thatare Pexl2, andPex2) Step4: At some matrixby an unknownmechanism. peroxisomal or in the lumen,Pex5dissociates point,eitherduringtranslocation that a process to the cytosol, fromthe matrixproteinandreturns and membrane andadditional complex IhePex2/10112 involves canbe proteins not shown.Notethatfoldedproteins cytosolic ls not sequence andthatthetargeting intoperoxisomes imported , Ann andP B Lazarow,2001 P E Purdue in the matrix[See removed et al, 2000,Ann RevBrcchem S Subramani Biot17:101, Rev. CellDevel S u b r a m a n i , ,2C0e0l1l 0 5 : 1 817 59:39a 9n ; d V D a m maanid S LROTEINS . S O R T I N GO F P E R O X I S O M AP

567

translocation channel while still bound to pex5, a feature that differs from protein import into the ER lumen. At some stage either during or after entry into the matrix, pex5 dissociatesfrom the peroxisomal matrix protein and is recycled back to the cytoplasm. In conrrasr to the N-terminal uptaketargetlng sequenceson proteins destined for the ER lumen, mitochondrial matrix, and chloroplast stroma, the pTSl sequence is not cleaved from proteins after their entry into a peroxisome. Protein import into peroxisomesrequires ATp hydrolysis, but it is not known how the energyreliased from ATP is used to power unidirectional translocation acrossthe peroxisomal membrane. The peroxisome import machinery, unlike mosr sysrems that mediate protein import into the ER, mitochondria, and chloroplasts, can translocate folded proteins across the membrane. For example, catalaseassumesa folded conformation and binds to heme in the cytoplasm before traversing the peroxisomal membrane. Cell-free studies have shown that the peroxisome import machinery can transport large macromolecular objects, including gold particles of about 9 nm in diameter,as long as they have a pTSl tag attached to them. However, peroxisomal membranes do nor appear to contain large stablepore structures,such as the nuclear Dore describedin the next section. The fundamental mechanism of peroxisomal matrix protein translocation is not well understood but is a topic under active investigation. Some of the mechanisms under consideration include the idea that peroxisomal membrane proteins pex10, pex12, and pex2 may assembleto form a relatively large transmembrane channel with a gated opening of about 10-15 nm (for refer-

peroxisomal matrix and Pex5 would be releasedback into the cytosol to complete another round of cargo import. A f e w p e r o x i s o m am l a r r i x p r o r e i n ss u c h - a st h i o l a s ea r e synthesized as precursors with an N-terminal uptaketargetingsequenceknown as PTS2.These proteins bind to a different cytosolic receptor prorein, but oiherwise import is thought to occur by the same mechanismas for piSlcontaining proteins.

P e r o x i s o m aM l e m b r a n ea n d M a t r i x p r o t e i n s Are Incorporatedby Different pathways Autosomal recessivemutations that cause defective peroxisome assemblyoccur naturally in the human population. Such defectscan lead to severedeveloomental defectsoften associatedwith craniofacialabnormaiities.In Zellweger syndrome and related disorders, for example. t h e t r a n s p o r to f m a n y o r a l l p r o t e i n si n t o t h e p e r o x i s o m a l matrix is impaired: newly synthesizedperoxisomal en_ zymes remain in the cytosol and are eventually degraded. Genetic analysesof cultured cells from different Zellweser patients and of yeast cells carrying similar mutations hive 568

CHAPTER 13

I

identified more than 20 genesthat are required for peroxisome biogenesis.I Studieswith peroxisome-assembly mutants have shown that different pathwaysare usedfor importing peroxisomalmatrix proteins versusinserting proteins into the peroxisomal membrane. For example, analysis of cells from some Zellweger patients led to identification of genesencoding the putative translocation channel proteins Pex10, Pex12, and pex2. Mutant cells defectivein any one of these proteins cannot rncorporate matrix proteins into peroxisomes;nonetheless, the cellscontain empty peroxisomesthat have a normal complement of peroxisomal membraneproteins (Figure 13-30b).

( a ) W i l d - t y p ec e l l s

Stainedfor PMPTO

Stainedfor catalase

Catalase PMP70

o, .o 9' O" , ' Peroxisome (b) Pex12mutants (deficient in matrix protein import)

(c) Pex3 mutants (deficient i n m e m b r a n eb i o g e n e s i s )

EXPERIMENTAL FTGURE 13-30Studiesrevealdifferent pathwaysfor incorporationof peroxisomalmembraneand matrixproteins.Cellswerestained with fluorescent antibodies to PMP70, protein, a peroxisomal membrane or with fluorescenr antibodies to catalase, a peroxisomal matrixprotein, thenviewed in a fluorescent (a)In wild-type microscope. cells,bothperoxisomal membrane andmatrixproteins arevisible asbrightfociin numerous peroxisomal bodies(b)In cellsfroma pex12-deficient patient, catalase isdistributed uniformly throughout the cytosol, whereas PMP70 is localized normally to peroxisomal bodies(c)In cellsfrom a Pex3-deficient patient,peroxisomal membranes cannotassemble, andasa consequence peroxisomal bodiesdo notform.Thusboth catalase andPMP70 aremis-localized to thecytosol[Courtesy of Stephen Gould, Johns Hopkins Universitvl

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

Precu rsor membrane

Peroxisomal memDrane proteins

Peroxisomal ghost

Mature peroxisome

PTSl-bearing matrix protein

PTS2-bearing matrix protein

Catalase

PMPTO and biogenesis 13-31 Modelof peroxisomal FIGURE rs of peroxisomes division.Thefirststagein the de novoformation proteins intoprecursor membrane of peroxisomal the incorporation for fromthe ER Pex19 actsasthe receptor derived membranes for the Pex3andPexl6arerequired sequences membrane-targeting membrane properinsertion intotheformingperoxisomal of proteins l n s e r t i oonf a l l o e r o x i s o mm ae l m b r a nper o t e i npsr o d u c eas Mutations in any one of threeother geneswere found to block insertion of peroxisomalmembraneproteins as well as import of matrix proteins (Figure 13-30c). These findings demonstrate that one set of proteins translocatessoluble proteins into the peroxisomalmatrix but a different set is required for insertion of proteins into the peroxisomalmembrane.This situation differs markedly from that of the ER, mitochondrion, and chloroplast, for which, as we have seen,membraneproteinsand solubleproteinssharemany of the samecomponents for their insertion into theseorganelles. Although most peroxisomes are generated by division of preexisting organelles,these organellescan arise de novo by the three-stageprocessdepicted in Figure 1'3-31'.In this case, peroxisome assembly begins in the ER' At least two peroxisomal membrane proteins, Pex3 and Pex16, are inserted into the ER membrane by the mechanismsdescribed in Section13 .2. Pex3 and Pex16 recruit additional peroxisomal proteins such as Pex19 forming a specializedregion of the ER membrane that can bud off of the ER to form a peroxisomal precursor membrane. Analysis of mutant cells revealed that Pex19 is the receptor protein responsiblefor targeting of peroxisomal membrane proteins' whereas Pex3 and Pex15 are necessaryfor their proper insertion into the membrane. These three proteins are thought to be responsible for peroxisomal membrane protein assemblyin mature peroxisomesas well as during the de novo formation of new peroxisomes.The insertion of peroxisomal membrane proteins generatesmembranesthat have all the componentsnecessaryfor import of matrix proteins, leading to the formation of mature, functional peroxisomes.Division of mature peroxisomes,which largely determinesthe number of peroxisomeswithin a cell, dependson still anotherprotein,Pex11. Overexpressionof the Pex11 protein causesalatge increase in the number of peroxisomes,suggestingthat this protein controls the extent of peroxisome division. The small peroxisomesgeneratedby division can be enlargedby incorporation of additional matrix and membrane proteins via the same pathways describedpreviously'

protetns targeted of importing ghost,whichiscapable peroxisomal PTS2-bearing and PTSlpathways for importing to the matrixThe receptor of thecytosolic differonlyin the identity matrixproteins (see sequence thatbindsthetargeting (Pex5 andPex7,respectively) yields a of matrixproteins incorporation Figure13-29)Complete division requires of peroxisomes Theproliferation matureperoxisome. on the Pexl1 protein' thatdepends a process of matureperoxisomes,

Sorting of PeroxisomalProteins r All peroxisomal proteins are synthesizedon cytosolic ribosomes and incorporated into the organelle posttranslationally. r Most peroxisomal matrix proteins contain a C-terminal PTS1 taigeting sequence;a few have an N-terminal PTS2 targeting sequence.Neither targeting sequenceis cleaved after import. r All proteins destinedfor the peroxisomal matrix bind to a cytosolic carrier protein, which differs for PTSI- and PTS2-bearingproteins' and then are directed to common import receptor and translocation machinery on the peroxisomal membrane (seeFigure 13-29). r Translocation of matrix proteins acrossthe peroxisomal membrane dependson ATP hydrolysis. Many peroxisomal matrix proteins fold in the cytosol and traverse the membrane in a folded conformation, which is different than for protein import into organellessuch as the ER' mitochondrion, and chloroplast. r Proteinsdestinedfor the peroxisomal membranecontain different targeting sequencesthan peroxisomal matrix proteins and are imported by a different pathway' r Unlike mitochondria and chloroplasts, peroxisomescan arise de novo from precursor membranesprobably derived from the ER as well as by division of preexistingorganelles ( s e eF i g u r e1 3 - 3 1 ) .

TransPortinto and out of the Nucleus The nucleus is separatedfrom the cytoplasm by two membranes,which form the nuclearenvelope(seeFigure 9-1)' The nuclear envelopeis continuous with the ER and forms a part T R A N S P O R ITN T O A N D O U T O F T H E N U C L E U S

.

569

of it. Transport of proteins from the cytoplasm into the nucleus and movement of macromolecules,including mRNps, tRNAs, and ribosomal subunits, out of the nucleus occur through nuclearpores,which span both membranesof the nuclear envelope. Import of proteins into the nucleus shares some fundamental features with protein import into other organelles. For example, imported nuclear proteins carry specific targeting sequencesknown as nuclear localization sequences, or NLSs. However, proteins are imported into the nucleusin a folded state,and thus nuclear import differs fundamentally from protein translocationacrossthe membranes of the ER, mitochondrion, and chloroplast,where proteins are unfolded during translocation.In this section we discussthe main mechanism by which proteins and some ribonuclear proteins such as ribosomesenter and exit the nucleus.\Wewill also discusshow mRNAs and other ribonuclear protein complexesare exported from the nucleusby a processthat differs mechanisticallyfrom nuclear protein import.

L a r g ea n d S m a l lM o l e c u l e sE n t e ra n d L e a v et h e Nucleusvia NuclearPoreComplexes Numerous pores perforate the nuclearenvelopein all eukaryotic cells. Each nuclear pore is formed from an elaborate

structure termed the nuclear pore complex (NPC), which is one of the largest protein assemblagesin the cell. The total massof the pore structureis 60-80 million Da in vertebrates, which is about 15 times larger than a ribosome. An NpC is made up of multiple copies of some 30 different proreins called nucleoporins. Electron micrographs of nuclear pore complexesreveal a roughly octagonal,membrane-embedded structure from which eight approximately 1O0-nm-longfilaments extend into the nucleoplasm(Figure 13-32). The distal ends of thesefilaments are joined by the terminal ring, forming a structure called the nuclear basket. The membraneembeddedportion of the NPC is also attacheddirectly to the nuclear lamina, a network of lamin intermediate filaments that form a meshwork extending over the inner surface of the nuclear envelope (seeFigure 20-1,6). Cytoplasmic filaments extend from the cytoplasmic side of the NPC into the cytosol. Ions, small metabolites, and globular proreins up to 2040 kDa can passivelydiffuse through the central aqueous region of the nuclear pore complex. However, large proteins and ribonucleoprotein complexescannot diffuse in and out of the nucleus. Ratheq these macromolecules are actively transported through the NPC with the assistanceof soluble transporter proteins that bind macromoleculesand also interact with nucleoporins.

(b)

Cytoplasm

Cytoplasmic filaments

O u t e rn u c l e a r memDrane

Nu c l e a r envelope

I n n e rn u c l e a r membrane Nucleoplasm N u c l e a rl a m i n a Nuclearbasket T e r m i n a rl i n g

A FIGURE 13-32Nuclearporecomplex.(a)Nuclear envelopes mrcrodissected fromthe largenucleiof Xenopus oocytes visualized by fieldemission in-lens scanning electron microscopy. Iop:Vrewof the cytoplasmic facereveals octagonal shapeof memorane_ embedded portionof nuclear porecomplexes Bottom: Viewof the 570

'

c H A p r E R1 3 |

nucleoplasmic faceshowsthe nuclear basket thatextends fromthe membrane portion.(b)Cutaway modelof the porecomplex. [part(a) fromV DoyeandE Hurt,1997, CurrOpinCellBiol9:401; courtesy of M W G o l d b e r ga n d T . D A l l e n P a r t( b ) a d a p t e df r o m M p R o u ta n d J D A t c h i s o n . 2001, J Biol. Chem. 276:165931

M o v r N Gp R o r E r N sr N T oM E M B R A N EAsN D o R G A N E L L E '

tLs

s n l l ) n N l H l _r o t n o c N V o t - N tl u o d s N V U l _

'v8il ' '.LOgiLLl uorople) 16 fute1.t61oi{sapnor uplv ]a c urorjlauab P I areraD 1o[sepnor:66vi6lilD I q]lLus p l g i l D t ' 0 6 6 1' l p l a L U e p5Vu r o r l lQ q 6 i ; ) u o r l e q na) qu ltu l p a p n l ) u l aq}o} slN Ot^s aq}6ulpo)ua eseurlalenn.r{d iuau6elloua6lell^ pa})a}suer} e ulor;passatdxe se^^losoil)uallM^luopa,n))o snal)nuoqi ut olellsqns eua6petaaut6ua {q pe>npord e 6ursn1 uodsuel] seMll 'sllo) srq]]o uorlelnurnr:y(a1es^1) pupoluoserd )uauJrr.l) aqf snal)nuaq]o1pozrle)ol seMuralord aql ul losoil)io a)uasqe utelotd eql Oututeluot aprlded sll le SINOyAS senn snururol-N urelotd urpasserdxa SINuaOrlue-f 6715trleqlu{sp ol paldno){11e>ruieqr eletpriqoqlel ut e q]rMpolpqnlutslla)elaHpezrlrqeau.tred-uluo]t6tp eseur>1 ruau.irqr e uaqM(q) uJslloqeloul alennrrid ]uo)soronli aq}o} paztlP)ol aluo)saronll a6re;fuanstql ulse;doyi) (q) peturs)dN pueodolo^ua uralordrrlosol{> leal)nu suorllun+ ]o sqder6oi)rru aq]soneol {poqrlue:r|>adse q}!Mpoleol}alaMsll0)poln}ln)laue sr'(nno1leI) lnq lno )ealsluanllsuottrlosolI>]eq]osouelqulaul 'aseurl 'plLtteq] q]tM (e) elenntrid euseldaql sezr;rqeaured Iq paztlenstn uruolrOrp a)ua)soronllounur-ur luablalop)tuotuou ;euto11 leu6ts oq ue) slla)pornlln);orele;ououu uoqMsnal)nuaq]ol pazlle)ol e +o]uaulleo{ sllo)elaHpezrpqeeuted uotleztle)ol reol)nue o] pasn+ 'snsl)nu -uruolr6rp pue poleollun sqder6oDru rru-rseldoy{3 (e) suralord sse)ord lserluo)-aseqd llo) aq} o1suraloldspaJlp(SlN) 1o eq] urslueuoduror rr;oso1,{t alqnlos eq}saiel}suor.uap E€-El:lUngl11VINltAtutdxl v }o }uaulo^lo^ut ;euErsuorleztle)ol-real)nN eles{1 aq}ut sllo)poln}ln)pazrlrqeeLlrad utln))o o} ;o a)uasqp yodsuerlreol)nuJoarnlre] eq1'podsuell leal)nulo1 pelrnba,r are sugelo.rd rr;osoy{1t€-€t lgngll lVINl6tuldxl V oles^l +

ales^l -

s l l a cp o z r l r q e o u r r a Adq u o d L u tl e a l c n N( q ) u ; u o g 1 6 ;+9

u r u o l r D r -6

u r u o l r b rj op l c a J j ( e ) 'surelordosaqlro; roldaf,eruodurr reelfnu ? sp auole IJE uEf,snr{l pue sacuenbasS-IN ureuac ol dllce.rrppurq osl€ uel lrunqns t' urlrodurr eql 'rl q8norqr uralord o8rer eqr aptnqs ot arod Jeelfnu aql ur surotord qrr,nrslfeJelur rrunqns $ aqr sEOJer{.t\'snelcnu oql otur pal.rodsuerleq ol uralo.rd,,oBJeJ,,e ul S'IN rrseq p or spulq lunqns n aql:toliata.t 1to(tut-.toa1tnu f,rreurrporeleqe uroJ uec surgodrul o{\r oqJ '(7g-g arn8rg aas) suorleuuoJuor punoq-d(D ro -dJC reqlro ur stsrxe leql urolord C f,ueruouorue sr upy'fl uurodrur pue 'n urlrod -rur'ueg :surelordperrnberaerql per;rrndsJeqJJeeseJ'ruelsds dessesrqt Sursn 'lcetur sJdN pue edoya,ruereelJnu aql 8ur -^Eal elq.,rt IIaJ aqt Jo rno IEel ol stueuoduoc cruseldot.{c3ur -,to11e'auerqruerueruseldeql sazrlrqeerurad reqr lue8JetepE sr uruotr8rq '(Vt-tt arn8rg)uodrur r€olrnu rot perrnbarslueu -oduror crlosoldc elqnlos Surzf,yeue ro; desse orJrl ur ue pepr^ 'ua8rrue-1 a8rel -ord uralsds IIal poalrqeaured-uruolr8rpy gtAS ul euo eql ol relrurs'5151 rrseqe Surureluocsurelorduo pesnroJuodrur rpalf,nuto usruegleu aql uo 1;o,vrdlreE

'seruenbesluoreJrlPdra.Lasaql uorlruSorar eql 1o roJ sr.usrueqraualdrtlnur oq lsnu aroqr snql 'otqoqdo;pdq (oru€ls sl IV dNUuq uralord Sutputq-y51A eqr uI S'IN uE 'ruaraJJrpelrnb dlectural{J ore ss'IN rorllo speJaqd\ -ur roc 'ua8nue-1 a8rel 6yn5 ul S'IN JISBq eql ol J€lItuIs ere osoqfJo due14 'snelcnu aql olul pelrodtul sulelord roqlo sno -rerunu ur POrtIluaPIoro^/r.{lluonbesqnsseluonbas515 '(gg -g1 arn8r4) S-IN uE se suollrunJ dlluanbasuocPUEsnalf,nu eql otur uodsuErl slf,arlPlI lBtll PelerlsuoueP utalord JIIosol -dc e ol pasnJsE^{ eJuanbosslqr qtlq^l. uI suloloJd Plrqdq -s.'Q-or4 pareeur8ueqlI.&\ sluotulJadxg' pn-s.,(1-8ry-s,rr1-s,(1 :uralord oql snuruJal-J oqr JEeusprf,EouIIuE JISEquI I{fIJ Jo or;roedsE UII{tI.t Jnf,fo IIE uorlezllsf eruonbasenplsaJ-uodes -ol relnllal poJerleslql rot alqrsuodsarsuollulntu aq1 'urseld -e1dr aqt uI etelnulnf,re uo8rluP-I e8rel;o slrlrot Pal€lnu etuos spsreq,t tsl]ocPollotul-snrl^ uI snalsnueql or POzIIEJol sr uralord slql Jo ru.ro; addl-p1y1\oql 'ueSIluE-I a8rel pe11ec 6(gtAS) urotord IErr^ eql Jo ruJoI IEIurouqEue pecnpord leql 'snrll e 0t snrr^ uerturs Jo sluelnu ;o srsdleueaqr qSnorqr 'snollnu agl olul lrodsuErl pere^olsrP lsrrt eJe^\ ss-IN e^rlreles rregl slferlP IEI{I (/s7N/ 1vu8tsuotloztlozol-waPnu E uretuof, sutelord qrng 'sexaldruoc erod reelJnu q8norql snelf,nu eql olul palrodurr pue urseldoldo aqr ur pezlseql -uds ere-seseraru,(1odVNU PuEVNCI pue 'srolce; uolldlrls -ue.rt 'seuolslq sE qrns-snelrnu eql ur Punot surerord 11y

s n e l ) n Ne q l o l u ! s l e u 6 ! su o l l e z ! l e ) o ' l - r e a l ) n N suluodtul 6urureluol sulaloJdlrodsuP,r1

The mechanismfor import of cytoplasmiccargo proteins mediated by an importin is shown in Figure 13-35 (the general mechanismis the samefor either a monomeric or dimeric importin). Freeimportin in the cytoplasm binds to its cognate NLS in a cargo protein, forming a bimolecwlar cargo complex. The cargo complex then translocatesthrough the NPC channel as the importin B subunit interacts with a class of nucleoporins called FG-nucleoporins. These nucleoporins, which line the channel of the nuclear pore complex and also are found in the nuclear basket and the cytoplasmic filaments, contain multiple repeats of short hydrophobic sequencesrich in phenylalanine (F) and glycine (G) residues (FG-repeats).The hydrophobic FG-repeatsare thought to occur in regionsof extended,otherwisehydrophilic polypeptide chains that fill the central transporter channel and in some way allow the relatively hydrophobic importin complexesto traverse the channel efficiently while excluding unchaperoned hydrophilic proteins larger than 20-40 kDa. 'Sfhen the cargo complex reachesthe nucleoplasm, the importin interacts with Ran.GTP, causing a conformational changein the importin that decreasesits affinity for the NLS, releasing the cargo protein into the nucleoplasm. The importin-Ran.GTP complex then diffuses back through the NPC. Once the importin-Ran.GTP complex reachesthe cytoplasmic side of the NPC, Ran inreracts with a specific GTPase-actiuatingprotein (Ran-GAP) that is a component of the NPC cytoplasmic filaments. This stimulates Ran to hydrolyze its bound GTP to GDP, causing it to convert to a

Ra n ' G D P R a n . G T P

conformation that has low affinity for the importin, so that the free importin is releasedinto the cytoplasm, where it can participate in another cycle of import. Ran.GDP travels back through the pore to the nucleoplasm,where it encountersa specificgwaninenucleotide-exchange factor (Ran-G EF) that causesRan to releaseits bound GDP in favor of GTP. The net result of this seriesof reactionsis the coupling of the hydrolysis of GTP to the transfer of an NlS-bearing protein from the cytoplasm to the nuclear interior, thus providing a driving force for nuclear transport. The import complex travels through the pore by diffusion, a random process.Yet transport is unidirectional.The direction of transport is a consequenceof the rapid dissociation of the import complex when it reachesthe nucleoplasm.As a result, there is a concentration gradient of the importin-cargo complex across the NPC: high in the cytoplasm, where the complex assemblesand low in the nucleoplasm,where it dissociates.This concentration gradient is responsiblefor the unidirectional nature of nuclear import. A similar concentration gradient is responsiblefor driving the importin in the nucleus back into the cytoplasm. The concentration of the importinRan.GTP complex is higher in the nucleoplasm,where it assembles,than on the cytoplasmicsideof the NPC, where it dissociates.Ultimately the direction of the transporr processesis dependenton the asymmetric distribution of the Ran-GEF and the Ran-GAP. Ran-GEF in the nucleoplasm maintains Ran in the Ran.GTP state,where it promotesdissociationof the cargo complex. Ran-GAP on the cytoplasmic side of the NPC

lmportin

ti

\ra./ D P

572

CHAPTER 13

|

T

M O V T N GP R O T E t NtSN T O M E M B R A N E A SN D O R G A N E L L E S

< FIGURE 13-35 Nuclearimport. M e c h a n i sf m o r n u c l e ai m r p o rot f " c a r g o " proteins. (boftorn), In the cytoplasm a free importinbindsto the NLSof a cargoprotein, forminga bimolecular cargocomplex. ln the caseof a basicNLS,theadapterprotein importino bridges the NLSandimportinp, (not forminga trimolecular cargocomplex shown). Thecargocomplex diffuses through the NPCby interacting with successive FGnucleoporins ln the nucleoplasm, interaction of Ran.GTP withthe imoortincauses a conformational change thatdecreases its affinityfor the NLS,releasing the cargoTo supportanothercycleof import,the importinRan'GTP complex istransported backto the cytoplasm. A GTPase-accelerating protein (GAP) associated with thecytoplasmic filaments of the NPCstimulates Ranto hydrolyze the boundGTPThisgenerates a conformational changecausing dissociation fromthe importin, whichcantheninitiate anotherroundof importRan.GDP isreturned t o t h en u c l e o p l a swmh,e r ea g u a n i n e nucleotide-exchange factor(GEF) causes release of GDPandrebindinq of GTP

converts Ran.GTP to Ran.GDP, dissociatingthe importinRan.GT? complex and releasingfree importin into the cytosol.

ExportinsTransportProteinsContaining N u c l e a r - E x p oS r ti g n a l so u t o f t h e N u c l e u s A very similar mechanism is used to export proteins, tRNAs, and ribosomal subunits from the nucleus to the cytoplasm. This mechanisminitiallv was elucidatedfrom studiesof certain ribonuclear protein complexes that "shuttle" between the nucleus and cytoplasm. Such "shuttling" proteins contain a nuclear-export signal /NES/ that stimulates their export from the nucleusto the cytoplasmthrough nuclearpores,in addition to an NLS that results in their uptake into the nucleus.Experiments with engineered hybrid genes encoding a nucleusrestricted protein fused to various segmentsof a protein that shuttles in and out of the nucleus have identified at least three different classesof NESs: a leucine-rich sequencefound in PKI (an inhibitor of protein kinaseA) and in the Rev protein of human immunodeficiency virus (HIV), as well as tvvo sequences identified in fwo different heterogeneousribonucleoprotein particles (hnRNPs). The functionally significant structural features that specifynuclearexport remain poorly understood. The mechanismwhereby shuttling proteins are exported from the nucleus is best understood for those containing a leucine-richNES. According to the current model, shown in Figure 13-36a, a specificexportin, or nuclear-exportreceptor, in the nucleus,called exportin 1, first forms a complex with Ran.GTP and then binds the NES in a cargo protein. Binding of exportin 1 to Ran.GTP causesa conformational changein exportin 1 that increasesits affinity for the NES so that a trimolecular cargo compler is formed. Like importins, exportin 1 interacts transiently with FG-repeatsin FG-nucleoporins and diffusesthrough the NPC. The cargo complex dissociates when it encountersthe Ran-GAP in the NPC cytoplasmicfilaments, which stimulates Ran to hydrolyze the bound GTI shifting it into a conformation that has low affinity for exportin 1. The releasedexportin 1 changesconformation to a structurethat has low affinity for the NES, releasingthe cargo into the cytosol. The direction of the export processis driven by this dissociationof the cargo from exportin 1 in the cytoplasm, which causesa concentration gradient of the cargo complex acrossthe NPC that is high in the nucleoplasmand low in the cytoplasm.Exportin 1 and the Ran'GDP are then transportedback into the nucleusthrough an NPC. By comparing this model for nuclear export with that in Figure 13-35 for nuclear import, we can seeone obvious difference:Ran.GTP is part of the cargo complex during export but not during import. Apart from this difference,the two transport processesare remarkably similar.In both processes, associationof a transport signal receptor with Ran'GTP in the nucleoplasmcausesa conformational changethat affects its affinity for the transport signal. During import, the interaction causesreleaseof the cargo, whereasduring export, the interaction promotes associationwith the cargo. In both export and import, stimulation of Ran'GTP hydrolysis in the cytoplasm by Ran-GAP producesa conformational changein Ran that releasesthe transport signal receptor. During nu-

clear export, the cargo is also released.Importins and exportins both are thought to diffuse through the NPC channel by successiveinteractions with FG-repeatsin FG-nucleoporins. Localization of the Ran-GAP and -GEF to the cytoplasm and nucleus,respectivelSis the basisfor the unidirectional transport of cargo proteins acrossthe NPC. In keeping with the similarity in function of importins and exportins, the two types of transport proteins are highly homologous in sequenceand structure. The entire family is called the importin B familS or karyopherins. There are'l'4 karyopherins in yeastand more than20 in mammalian cells. The NESs or NLSs to which they bind have been determined for only a fraction of them. Remarkably, some individual karyopherins function as both an importin and an exportin. A similar shuttling mechanismhas beenshown to export other cargoes from the nucleus. For example, exportin-t functions to export tRNAs. Exportin-t binds fully processed tRNAs in a complex with Ran'GTP that diffuses through NPCs and dissociateswhen it interacts with Ran-GAP in the NPC cytoplasmic filaments, releasingthe IRNA into the cytosol. A Ran-dependentprocess is also required for the nuclear export of ribosomal subunits through NPCs once the protein and RNA components have been properly assembledin the nucleolus. Likewise, certain specific mRNAs that associate with particular hnRNP proteins can be exported by a Ran-dependentmechanism.

Most mRNAsAre Exportedfrom the Nucleusby a R a n - l n d e p e n d e nMt e c h a n i s m Once the processingof an mRNA is completedin the nucleus, it remains associatedwith specifichnRNP proteins rn a messengerribonuclear protein complex, or mRNP. The principle transporter of mRNPs out of the nucleus is the nRNP exporter, a heterodimericprotein composedof a large subunit called nwclearexport factor 1 (NXFI) or TAP and a small subunit, nuclear export transporter L (Nxtl). The large subunit binds to nuclear mRNPs through cooperative interactions with the RNA and other mRNP adapter proteins that associatewith nascentpre-mRNAs during transcription elongation and pre-mRNA processing.It seemslikely that multiple TAPNxtl mRNP exportersbound along the length of an nRNP assistin its export. TAPAJxII acts like a karyopherin in the sensethat both subunits interact with the FG-domains of FG-nucleoporins, allowing them to diffuse through the central channel of the NPC. Tap also binds reversiblyto the protein Gle2, which in turn binds a nucleoporin in the nuclear basket, presumably positioning the mRNP for export through the nuclear pore. A nucleoporin in the cytoplasmic filaments of the NPC is also required for mRNP export. This nucleoporin binds an RNA helicasethat is proposed to function in the dissociationof the mRNP exporter and other hnRNP proteins from the mRNP as it reachesthe cytoplasm. The TAP/Nxt1 mRNP exporters do not appear to interact with Ran, and thus the unidirectional transport of mRNA out of the nucleusrequiresa sourceof energyother than GTP hydrolysis by Ran. As the mRNP complex is transported through an NPC, the proteins associatedwith INTOAND OUT OF THE NUCLEUS TRAN5PORT

.

573

(a)

R a n . G D P R a n . G T P Exportin1

T P

D

Cargo complex

T P

u

' *

P

a\

GEF

P

(b)

< FIGURE 13-36Ran-dependent and Ran-independent nuclearexport.(a)Randependent mechanism for nuclear exportof cargoproteins containing a leucine-rich nuclearexportsignal(NES)In the nucleoplasm, the proteinexportin1 bindscooperatively to the NESof thecargoproteinto betransported andto Ran.GTP Aftertheresulting cargo complexdiffuses throughan NPCviatransient interactions with FGrepeats in FG-nucleoporins, the Ran-GAP associated with the NPCcytoplasmic filaments stimulates conversion of Ran.GTP to Ran.GDP Theaccompanying conformational changein Ranleadsto dissociation of the complexTheNES-containing cargoproteinis released intothe cytosol, whereas exportin1 andRan.GDP are transported backintothe nucleus through N P C sR a n - G Ei nFt h en u c l e o p l a tshme n stimulates conversion of Ran.GDP to Ran.GTP. (b)Ran-independent nuclear exportof mRNAs Theheterodimeric TAP/Nxt1 complex bindsto (mRNPs) mRNA-protein complexes in the nucleusAssociation of TAP/Nxt1 with components of the NPCdirecttheassociated mRNP to the centralchannel of the pore.An (Dbp5)located RNAhelicase on thecytoplasmic sideof the NPCisthoughtto provide the drivingforceby hydrolysis for movingthe mRNP throughthe pore Thehelicase also freesthe mRNAfromTAPandNxtl proteins, whicharerecycled backintothe nucleus by the Ran-dependent importprocess depicted in F i g u r 1e 3 - 3 5

Trcnscription Re-import

it are exchanged for another set of proteins in the cytoplasm, a processcalled mRNP remodeling (Figure 13-36b). Severalnuclear mRNP proteins dissociatefrom the mRNp before it reachesthe cytoplasmic side of the NpC. These remain in the nucleus, where they bind to newly synthesized nascentpre-mRNA. Other nuclear mRNP proteins, including the TAPA{xt1 mRNP exporter, are exported through NPCs into the cytoplasm. Once they reach the cytoplasmic side of the NPC, they dissociate from the mRNp with the 574

.

c H A p r E R1 3 |

help of the RNA helicase,Dbp5, which associateswith cytoplasmic NPC filaments. Recall that RNA helicasesuse the energy derived from hydrolysis of ATP to move along RNA molecules,separatingdouble-strandedRNA chains and dissociatingRNA-protein complexes(Chapter4). This leads to the simple idea that Dpb5, which associateswith the cytoplasmic side of the nuclear pore complex, acts as an ATP-driven motor to move mRNP complexes through the nuclear pore.

M o v r N Gp R o r E r N sr N T o M E M B R A N EASN D o R G A N E L L E s

After remodelingis completed,the TAP and Nxtl proteins that have beenstrippedfrom the mRNA by Dbp5 helicaseare imported back into the nucleus by an importin, where they can function in the export of another mRNP. Consequently, thesenuclear mRNP proteins shuttle between the nucleus and cytoplasm,carrying mRNPs through NPCs (Figure 13-36b).

r Most mRNPs are exported from the nucleus by a heterodimeric mRNP exporter that interacts with FG-repeats of FG-nucleoporins.The direction of transport (nucleusto cytoplasm) may result from the action of an RNA helicase associatedwith the cytoplasmic filaments of the nuclear pore complexes.

Transport into and out of the Nucleus r The nuclear envelope contains numerous nuclear pore complexes(NPCs), large, complicated structurescomposed of multiple copies of 30 proteins called nucleoporins (see Figure 1,3-32).FG-nucleoporins,which contain multiple repeatsof a short hydrophobic sequence(FG-repeats),line the central transporter channel and play a role in transport of all macromoleculesthrough nuclear pores. r Transport of macromolecules larger than 20-40 kDa through nuclear pores requires the assistanceof proteins that interact with both the transported molecule and FGrepeatsof FG-nucleoporins. r Proteins imported to or exported from the nucleus contain a specificamino acid sequencethat functions as a nuclear-localization signal (NLS) or a nuclear-export signal (NES). Nucleus-restrictedproteins contain an NLS but not an NES, whereasproteins that shuttle betweenthe nucleus and cytoplasm contain both signals. r Severaldifferent types of NES and NLS have been identified. Each type of nuclear-transport signal is thought to interact with a specific receptor protein (exportin or importin) belonging to a family of homologous proteins termed karyopherins. r A "cargo" protein bearing an NES or NLS translocates through nuclear pores bound to its cognatereceptorprotein (karyopherin), which also interacts with FG-nucleoporins. Importins and exportins are thought to diffuse through the channel, filled with a hydrophobic matrix of FG-repeats. Both transport processesalso require participation of Ran, a monomeric G protein that exists in different conformations when bound to GTP or GDP. r After a cargo complex reachesits destination (the cytoplasm during export and the nucleusduring import), it dissociates,freeing the cargo protein and other components. The latter then are transportedthrough nuclearpores in the reversedirection to participate in transporting additional moleculesof cargo protein (seeFigures13-35 and 13-36). r The unidirectional nature of protein export and import through nuclear pores results from localization of the Ran guanine nucleotide-exchangefactor (GEF) in the nucleus and of Ran GTPase-activatingprotein (GAP) in the cytoplasm. The interaction of import cargo complexeswith the Ran-GTP in the nucleoplasmcausesdissociationof the complex, releasingthe cargo into the nucleoplasm(seeFigure 13-35). Export cargo complexesdissociatein the cytoplasm when they interact with Ran'GAP localized to the NPC cytoplasmic filaments (seeFigure 13-36).

As we have seen in this chapter, we now understand many aspectsof the basic processesresponsiblefor selectively transporting proteins into the endoplasmic reticulum (ER), mitochondrion, chloroplast, peroxisome, and nucleus. Biochemical and genetic studies, for instance, have identified cis-actingsignal sequencesresponsiblefor targeting proteins recepto the correct organellemembrane and the membrane 'We also have tors that recognize these signal sequences. learnedmuch about the underlying mechanismsthat translocate proteins across organelle membranes and have determined whether energyis usedto push or pull proteins across the membrane in one direction, the type of channel through which proteins pass, and whether proteins are translocated in a folded or an unfolded state. Nonetheless'many fundamental questions remain unanswered' including how fully folded proteins move acrossa membrane and how the topology of multipass membrane proteins is determined. The peroxisomal import machinery provides one example of the translocation of folded proteins. It not only is capable of translocating fully folded proteins with bound cofactors into the peroxisomal matrix but can even direct the import of a large gold particle decoratedwith a (PTS1)peroxisomal targetingpeptide.Someresearchershave speculated that the mechanismof peroxisomal import may be related to that of nuclear import, the best-understoodexample of posttranslational translocation of folded proteins. Both the peroxisomal and nuclear import machinery can transport folded moleculesof very divergentsizes,and both appear to involve a component that cycles between the cytosol and the organelle interior-the Pex5 PTS1 receptor in the caseof peroxisomal import and the Ran-importin complex in the case of nuclear import. However, there also appear to be crucial differencesbetweenthe two translocation processes.For example, nuclear pores representlarge, stable macromolecular assembliesreadily observedby electron microscopy,whereas analogousporelike structureshave not been observedin the peroxisomal membrane. Moreover' small molecules can readily pass through nuclear pores, whereas peroxisomal membranesmaintain a permanent barrier to the diffusion of small hydrophilic molecules.Taken together,these observations suggestthat peroxisomalimport may require an entirely new type of translocation mechanism. The evolutionarily conservedmechanismsfor translocating folded proteins acrossthe cytoplasmicmembraneof bacterial cells and across the thylakoid membrane of chloroplasts also are poorly understood.A better understandingof all of theseprocessesfor translocatingfolded proteins across P E R S P E C T I VFEO SRT H E F U T U R E

575

a membrane will likely hinge on future development of in vitro translocation systemsthat allow investigatorsto define the biochemical mechanisms driving translocation and to identify the structuresof trapped translocationintermediates. Compared with our understandingof how soluble proteins are translocatedinto the ER lumen and mitochondrial matrix, our understanding of how cis-acting sequencesspecify the topology of multipass membrane proteins is quite elementary. For instance,we do not know how the translocon channel accommodatespolypeptidesthat are oriented differently with respect to the membrane, nor do we understand how local polypeptide sequencesinteract with the translocon channel both to set the orientation of transmembranespansand to signal for lateral passageinto the membrane bilayer. A better understanding of how the amino acid sequencesof membrane proteins can specify membrane topology will be crucial for decoding the vast amount of structural information for membrane proteins contained within databasesof genomic sequences. A more detailed understanding of all translocation processesshould continue to emergefrom genetic and biochemical studies, both in yeasts and in mammals. These studies will undoubtedly reveal additional key proteins involved in the recognition of targeting sequencesand in the translocation of proteins across lipid bilayers. Finally, the structural studies of translocon channels will likely be extended in the future to reveal at resolutions on the atomic scalethe conformational statesthat are associatedwith each step of the translocation cycle.

KeyTerms biomolecular cargo complex 572 cotranslational translocation 537 dislocation 555 dolichol phosphate 550 exportin 573 FG-nucleoporins572 generalimport pore 559 hydropathy profile 548 importins 571 karyopherins 574

post-translational translocation 540 Ran protein 5Z1 signal-anchorsequence544 signal-recognitionparticle

(sRP) 537 signal (uptake-targeting) sequences535 stop-transferanchor sequence544 topogenic sequences543

molecular chaperones541 Nlinked oligosaccharides550 nuclear pore complex (NPC)570

topology of a membrane protein 543 translocon 539 trimolecular cargo complex J,/J unfolded-protein response555 1

O-linked oligosaccharides550

F - ^

Review the Concepts 7. Describethe sourceor sourcesof energyneededfor unidirectional translocation across the membrane in (a) co576

CHAPTER 13

|

translational translocation into the endoplasmic reticulum (ER); (b) post-translationaltranslocation into the ER; (c) translocation across the bacterial cytoplasmic membranel and (d) translocation into the mitochondrial marnx. 2. Translocation into most organellesusually requires the activity of one or more cytosolic proteins. Describethe basic function of three different cytosolic factors required for translocation into the ER, mitochondria, and peroxisomes, respectively. 3. Describethe typical principles usedto identify topogenic sequenceswithin proteins and how thesecan be used to develop computer algorithms. How does the identification of topogenic sequenceslead to prediction of the membrane arrangementof a multipass protein? What is the importance of the arrangement of positive chargesrelative to the mem, brane orientation of a signal-anchorsequence? 4. The endoplasmicreticulum (ER) is an important site of "quality control" for newly synthesizedproteins. \7hat is meant by "quality control" in this context? I7hat accessory proteins are typically involved in the processingof newly synthesizedproteins within the ER? Cells generallydegradeER'Sfhere exit-incompetent proteins. within the cell does such degradation occur and what is the relationship of the Sec61 protein translocon and p97 to the degradationprocess? 5. Temperature-sensitive yeast mutants have been isolated that block each of the enzymatic stepsin the synthesisof the dolichol-oligosaccharideprecursor for N-linked glycosylation (seeFigure 1,3-1,7). Proposean explanation for why mutations that block synthesis of the intermediate with the structure dolichol-PP-(GlcNAc)2Man5 completely prevent addition of N-linked oligosaccharide chains to secretory proteins, whereas mutations that block conversion of this intermediate into the completed precursor-dolichol-PP(GlcNAc)2ManeGlca-allow the addition of N-linked oligosaccharidechains to secretoryglycoproteins. 6. Name four different proteins that facilitate the modification and/or folding of secretoryproteins within the lumen of the ER. Indicate which of theseproteins covalently modifies substrateproteins and which brings about only conformational changesin substrateproteins. 7. Becauseyou are interestedin studying how a particular secretoryprotein folds within the ER, you wish to determine whether BiP binds to the newly synthesizedprotein in ER extracts. You find that you can isolate some of the newly synthesizedsecretoryprotein bound to BiP when ADP is added to the cell extract but not when ATP is added to the extract. Explain this result basedon the mechanism for BiP binding to substrateproteins. 8. Describewhat would happen to the precursor of a mitochondrial matrix protein in the following types of mitochondrial mutants: (a) a mutation in the Tom22 signal receptor, (b) a mutation in the Tom70 signal receptor, (c) a mutation in the matrix Hsc70, and (d) a mutation in the matrix signal peptidase. 9. Describe the similarities and differences between the mechanismof import into the mitochondrial matrix and the chloroplast stroma.

M O V T N GP R O T E t NtSN T O M E M B R A N E A SN D O R G A N E L L E S

10. Design a set of experimentsusing chimeric proteins, composed of a mitochondrial precursor protein fused to dihydrofolate reductase(DHFR), that could be used to determine how much of the precursor protein must protrude into the mitochondrial matrix in order for the matrix-targeting sequenceto be cleaved by the matrix-processing protease (seeFigure 1,3-24). 11. Protein targeting to both mitochondria and chloroplasts involves the sorting of proteins to multiple sites within the respectiveorganelle.Briefly list thesesites.Taking the mitochondrion as an example and the proteins ADP/ATP anti-porter and cytochrome b2 as the specific cases,compareand contrast the extent to which a common mechanism is used for the site-specifictargeting of these two protelns. 12. Peroxisomescontain enzymesthat use molecular oxygen to oxidize various substrates,but in the processhydrogen peroxide forms and must be degraded.lfhat is the name of the enzyme responsiblefor the breakdown of hydrogen peroxide to water and what mechanismand associatedproteins allow for its import into the peroxisome? 13. Supposethat you have identified a new mutant cell line that lacks functional peroxisomes.Describe how you could determine experimentally whether the mutant is primarily defective for insertion/assemblyof peroxisomal membrane protelns or matrlx protelns. 14. Evidencethroughout Chapter 13 revealsthat specific motifs within polypeptidesare necessaryto direct or target these proteins acrossmembranesand into organelles. The nuclear import of proteins having a molecular mass more than approximately 40 kDa is no different, and they must be actively imported through nuclear pore complexes.What is the name given to the amino acid sequence that allows the selective transport of macromolecular cargo proteins into the nucleus?Name three proteins that are required for this import and briefly describehow they function. 'Sfhy 15. is localization of Ran-GAP in the nucleusand RanGEF in the cytoplasm necessaryfor unidirectional transport of cargo proteins containing an NES?

Analyze the Data Imagine that you are evaluating the early stepsin translocation and processingof the secretoryprotein prolactin. By using an experimental approach similar to that shown in Figure 1.3-7,you can usetruncated prolactin mRNAs to control the length of nascentprolactin polypeptidesthat are synthesized.\fhen prolactin mRNA that lacks a chain-termination (stop) codon is translated in vitro, the newly synthesized polypeptide ending with the last codon included on the mRNA will remain attached to the ribosome, thus allowing a polypeptide of defined length to extend from the ribosome. You have generateda set of mRNAs that encodesegmentsof the N-terminus of prolactin of increasing length, and each mRNA can be translated in vitro by a cytosolic translation extract containing ribosomes,tRNAs, aminoacyl-tRNA syn-

thetases,GTP, and translation initiation and elongation fac'When radiolabeled amino acids are included in the tors. translation mixture, only the polypeptide encoded by the addedmRNA will be labeled.After completion of translation, each reaction mixture was resolved by SDS poly-acrylamide gel electrophoresis,and the labeled polypeptides were identified by autoradiography. a. The autoradiogram depicted below shows the results of an experiment in which each translation reaction was carriedout eitherin the presence(+) or the absence(-) of microsomal membranes.Basedon the gel mobility of peptides synthesizedin the presenceor absenceof microsomes, deducehow long the prolactin nascentchain must be in order for the prolactin signal peptide to enter the ER lumen and to be cleaved by signal peptidase. (Note that microsomes carry significant quantities of SRP weakly bound to the membranes.)

I

!l o

-

I -

-t

-^X ar) N;

{l!'!** 1f'lil!t

--O

-+

+-

50

130 110 70 90 Sizeof mRNA(in codons)

150

Given this length, what can you conclude about the b. conformational state(s)of the nascentprolactin polypeptide when it is cleavedby signal peptidase?The following lengths will be useful for your calculation: the prolactin signal sequenceis cleavedafter amino acid 31; the channel within the ,iboro-. occupied by a nascentpolypeptide is about 150 A long; a membrane bilayer is about 50 A thick; in polypeptides with an a-helical conformation, one residue extends 1.5 A, whereas in fully extended polypeptides, one residue extendsabout 3.5 A. c. The experimentdescribedin part (a) is carried out in an identical manner except that microsomal membranes are not present during translation but are added after translation is complete. In this case none of the samples shows a difference in mobility in the presenceor absence of microsomes. lfhat can you conclude about whether prolactin can be translocated into isolated microsomes posttranslationally? d. In another experiment, each translation reaction was carried out in the presenceof microsomes,and then the microsomal membranes and bound ribosomes were separated from free ribosomes and soluble proteins by centrifugation. For each translation reaction' both the total reaction (T) and the membrane fraction (M) were resolved in neighboring gel lanes. Basedon the amounts of labeled polypeptide in the membrane fractions in the autoradiogram depicted beloq deduce how long the prolactin nascent chain A N A L Y Z ET H E D A T A

577

must be in order for ribosomesengagedin translation to engage the SRP and thereby become bound to microsomal membranes.

Tsai, B., Y. Ye, and T. A. Rapoport.2002. Retro-translocation of proteins from the endoplasmicreticulum into the cytosol. Nature Reu.Mol. Cell Biol. 3:246-255 . Sorting of Proteins to Mitochondria and Chloroplasts

l. I I 0)

--

_o .9

--

.Uto

oa dt> N;

TMTMTMTMTMTM 50

70 90 110 130 S i z eo f m R N A( i nc o d o n s )

150

References Translocation of Secretory Proteins Across the ER Membrane Egea,P. F., R. M. Srroud,and P. Walter.2005. Targetingproteins to membranes:structureof the signalrecognitionparticle. Curr. Opin. Struct. Biol. 75:213-220. Osborne,A. R., T. A. Rapoport, and B. van den Berg.2005. Protein translocationby the Sec51/SecY channel.Annu. Reu.Cell D e u .B i o l . 2 l : 5 2 9 - 5 5 0 . 'Wickner, S7.,and R. Schekman.2005. Protein rranslocation acrossbiological membranes.Science310:L452-1456. Insertion of Proteins into the ER Membrane Englund, P.T.1993. The structureand biosynthesisof glycosylphosphatidylinositolprotein anchors.Ann. Reu.Biochem.. 62:121-1.38. Goder,V., and M. Spiess.2001. Topogenesisof membraneproteins: determinantsand dynamics.FEBSLett. 504:87-93. Mothes, I7., et al. 1997. Molecular mechanismof membrane protein integrationinto the endoplasmicreticulum. Cel/ 89:523-53J. von Heijne, G. 1999. Recentadvancesin the understandingof membraneprotein assemblyand structure.Q. Reu.Biophys. 32:285-307. Protein Modifications, Folding, and euality Control in the ER . Helenius,A., and M. Aebi. 2004. Rolesof Nlinked glycansin the endoplasmicreticulum. Annu. Reu.Biochem. 73:1019-1049. Kornfeld, R., and S. Kornfeld. 1985. Assemblyof asparagine. linked oligosaccharides. Ann. Reu.Biochem. 45:631,-6G4. 'Walter. Patil, C., and P. 2001. Intracellularsignalingfrom the endoplasmicreticulum to the nucleus:the unfolded protein response in yeastand mammals. Curr. Opin. Cell Biol.73:349-355. Meusser,B., C. Hirsch, E. Jarosch,and T. Sommer.2005. ERAD: the long road ro destruction.Natwre Celt Biol.7:766-772. Sevier,C. S., and C. A. Kaiser.2002.Formation and transfer of disulphidebonds in living cells.Nature Reu.Mol. Cetl Biot. 3:836-847. . .Silberstein,S., and R. Gilmore. 1995. Biochemistry,molecular brology,and geneticsof the oligosaccharyltransferase. FASEB/. 10:849-85 8. Trombetta,E. S., and A. J. Parod. 2003. Quality control and protein folding in the secretorypathway.Annu. Reu.Cell Deu. Biol. 19:649-676.

578

CHAPTER 13

I

Koehler,C. M. 2004. New developmentsin mitochondrial assembly.Ann. Reu.Cell Deu. Biol.20:309-335. Dolezal,P.,V. Likic, J.Tachezy,and T. Lithgow 2006. Evolution of the molecular machinesfor protein import into mitochondria. Science313:31.4-31.8. Dalbey,R. E., and A. Kuhn. 2000. Evolutionarily relatedinsertion pathways of bacterial,mitochondrial, and thylakoid membrane proteins.Ann. Reu.Cell Deuel. Biol. 16:51-87. Matouschek,A., N. Pfanner,and S7.Voos. 2000. Protein unfolding by mitochondria:the Hsp70 import motor. EMBO Rept. l:404410. Neupert,'$7.,and M. Brunner.2002.The protein import motor of mitochondria.Nature Reu.Mol. Cell Biol.3:555-565. Rapaport, D. 2005. How doesthe TOM complex mediateinsertion of precursorproteins into the mitochondrial outer membrane? I. Cell Biol. 17l:479423. Robinson,C., and A. Bolhuis.2001. Protein targeringby the twin-argininetranslocationpathway.Nature Reu.Mol. Cell Biol. 2:350-356. Soll, J., and E. Schleiff.2003. Protein import into chloroplasts. Nature Reu.Mol. Cell Biol.5:198-208. Truscott, K. N., K. Brandner,and N. Pfanner.2003.Mechanismsof protein import into mitochondria. Curr. Biol. 13:R325-R337. Sorting of Peroxisomal Proteins Dammai, V., and S. Subramani.2001. The human peroxisomal targetingsignalreceptor,Pex5p,is translocatedinto the peroxisomal matrix and recycledto the cytosol. Cell 105:1,87-1,96. Gould, S. J., and C. S. Collins. 2002. Opinion: peroxisomal-protein import: is it really that complex?Nature Reu.Mol. Cell Biol. 3:382-389. Gould, S.J., and D. Valle. 2000. Peroxisomebiogenesisdisorders:geneticsand cell biology. TrendsGenet. 16:340-345. Hoepfner,D., D. Schildknegt,I. Braakman,P. Philippsen,and H. F. Tabak. 2005. Contribution of the endoplasmicreticulum to peroxisomeformation.Cell 122:85-9 5. Purdue,P. E., and P. B. Lazarow.2001. Peroxisomebiogenesis. Ann. Reu.Cell Deuel. Biol. 17:701-752. Subramani,S., A. Koller, and W. B. Snyder.2000. Import of peroxisomal matrix and membraneproteins.Ann. Reu.Biochem. 69:3994].8. Transport into and out of the Nucleus Chook, Y. M., and G. Blobel. 2001. Karyopherinsand nuclear import. Curr. Opin. Struct. Biol. ll:703-71,5. Cole, C. N., and J. J. Scarceili.2006. Transport of messenger RNA from the nucleusto the cytoplasm.Curr. Opin. Cell Biol. 18299-306. Johnson,A. \f., E. Lund, and J. Dahlberg.2002. Nuclear export of ribosomalsubunits.TrendsBiochem.Sci.27:580-585. Ribbeck, K., and D. Gorlich. 2001. Kinetic analysisof translocation through nuclearpore complexes.EMBO J. 20:1,320-1330. Rout, M. P., and J. D. Aitchison. 2001. The nuclearpore complex as a transport machine.J. Biol. Chem. 276:76593-1,6596. Schwartz,T. U. 2005. Modularity within the architectureof the nuclearpore complex. Curr. Opin. Struct. Biol. L5:221-226. Suntharalingam,M., and S. R. Wente.2003. Peeringthrough the pore: nuclearpore complex structure,assembly,and function. Deu. Cell.4:775-789.

M O V T N GP R O T E t NtSN T O M E M B R A N E A S N DO R G A N E L L E S

CHAPTER

TRAFFIC, VESICULAR AND SECRETION, ENDOCYTOSIS showingthe formationof clathrinelectronmicrograph Scanning on the cytosolic faceof the plasmamembrane coatedvesicles Washington School of Medicine University ] fJohnHeuser,

I n the previouschapter we explored how proteins are tarI g.ted to and translocatedacrossthe membranesof several I different intracellular organelles,including the endoplasmic reticulum, mitochondria and chloroplasts,peroxisomes,and the nucleus. In this chapter we turn our attention to the secretory pathway and the mechanismsthat allow soluble and membrane proteins to be deliveredto the plasma membrane and the lysosome. Ii7e will also discuss the related processesof endocytosisand autophagy,which deliver proteins and small moleculesfrom either outsidethe cell or from the cytoplasmto the interior of the lysosomefor degradation. Soluble and membrane proteins slated to function at the cell surfaceor in the lysosomeare transported to their final destination via the secretorypathway. Proteins delivered to the plasma membrane include cell-surfacereceptors, transporters for nutrient uptake, and ion channelsthat maintain the proper ionic and electrochemicalbalance across the plasma membrane.Solublesecretedproteins follow the same pathway to the cell surface as plasma membrane proteins, but instead of remaining embedded in the membrane, secreted proteins are releasedinto the aqueous extracellular environment in soluble form. Examples of secretedproteins are digestive enzymes, peptide hormones, serum proteins, and collagen. As describedin Chapter 9, the lysosomeis an organelle with an acidic interior that is generally used for degradation of unwanted proteins and the storage of small molecules such as amino acids. AccordinglS the types of proteins delivered to the lysosomal membrane are subunits of the V-classproton pump that pumps H* from the cytosol into the acidic lumen of the lysosomeas well as transporters to releasesmall molecules stored in the lvsosome into the

cytoplasm. Soluble proteins delivered by this pathway include lysosomal digestiveenzymessuch as proteases'glycosidases,phosphatases,and lipases. In contrast to the secretorypathway' which is generally used to deliver newly synthesizedmembrane proteins to their correct address,the endocyticpathway is usedto take up substancesfrom the cell surfaceinto the interior of the cell. The endocytic pathway is used to take up certain nutrients that are too large to be transported acrossthe plasma membrane by one of the transport mechanismsdiscussedin Chapter 1 1. For example, the endocytic pathway is utilized in the uptake of cholesterolcarried in LDL particlesand iron atoms carried by the iron-binding protein transferrin. In addition, the endocytic pathway can be usedremove receptorproteins from the cell surface as a way to down-regulate their activity.

OUTLINE 14.1 Techniquesfor Studyingthe Secretory Pathway

580

14.2

MolecularMechanismsof VesicularTraffic

586

14.3

EarlyStagesof the SecretoryPathway

592

14.4

Later Stagesof the SecretoryPathway

597

14.5

Endocytosis Receptor-Mediated

506

14.6

DirectingMembraneProteinsand Cytosolic Materialsto the LYsosome

612

579

A single unifying principle governs all protein trafficking in the secretoryand endocytic pathways: transport of membrane and soluble proteins from one membrane-bounded compartmentto another is mediatedby transport vesiclesthat collect " cargo" proteins in buds arisingfrom the membraneof one compartment and then deliver thesecargo proteins to the next compartment by fusing with the membraneof thar compartment. Importantly as transport vesiclesbud from one membrane and fuse with the next, the same face of the membrane remains oriented toward the cytosol. Therefore once a protein has been insertedinto the membraneor the lumen of the ER, the protein can be carried along the secretorypathway moving from one organelle ro the next without being translocatedacrossanother membraneor altering its orientation within the membrane. Similarly, the endocytic pathway usesvesicletraffic to transport proteinsfrom the plasmamembrane to the endosomeand lysosomeand thus preservestheir orientation in the membraneof theseorganelles.Figure 14-1 outlinesthe major routes for protein trafficking in the cell. Reducedto its simplest elements,the secretorypathway for delivery of newly synthesized proteins ro the plasma membrane or the lysosomeoperatesrn two stages.The first stage takes place in the rough endoplasmic reticulum (ER), as describedin Chapter 13. Newly synthesizedsoluble and membrane proteins are translocatedinto the ER, where they fold into their proper conformation and receivecovalent modifications such as N-linked and O-linked carbohydrates and disulfide bonds. Once newly synthesizedproteins are properly folded and have received their correct modifications in the ER lumen, they progressro the second stage of the secretory pathway, rransport through the Golgi. In the ER, secretory proteins are packaged into anterograde (forward-moving) transport vesicles.Thesevesiclesfusewith each other to form a flattened membrane-boundedcompartment known as the cis-Golgi cisterna. Certain proteins, mainly ER-localizedproteins, are retrieved from the cisGolgi to the ER via a different set of retrograde (backwardmoving) transport vesicles.A new cls-Golgi cisternawith its cargo of proteins physically moves from the cis position (nearestthe ER) to the trans position (farthest from the ER), successivelybecoming first a medial-Golgi cisterna and then a trans-GoIgi cisterna.This process,known as cisternalmaturation, does nor involve the budding off and fusion of anterograde transport vesicles.During cisternal maturation, enzymesand other Golgi-residentproteins are constantly being retrieved from later to earlier Golgi cisternae by retrograde transport vesicles,thereby remaining localized to the cis-, medial-, or trans-Golgi cisternae.As secreroryprorelns move through the Golgi, they can receivefurther modifications to linked carbohydratesby specific glycosyl transferasesthat are housed in the different Golgi comparrmenrs. Proteinsin the secretorypathway that are destinedfor the plasma membrane or lysosome eventually reach a complex network of membranes and vesiclestermed the trans-Golgi network (TGN). The TGN is a major branch point in the secretory pathway, and through a process known as protein sorttng, a protein can be loaded into one of at leastthree different kinds of vesiclesthat bud from the TGN. After buddine 580

C H A P T E R1 4

I

from the trans-Golgi network, the first type of vesicleimmediately moves to and fuses with the plasma membrane in a processknown as exocytosis,thus releasingits contentsto the exterior of the cell while the membraneproteinsfrom the vesicle becomeincorporatedinto the plasmamembrane.In all cell types,at leastsome proteins are loaded into such vesiclesand secretedcontinuouslyin this manner.The secondtype of vesicle to bud from the trans-Golgi network, known as secretory vesicles,are stored inside the cell until a signal for exocytosis causesreleaseof their contents at the plasma membrane. Among the proteins releasedby such regulatedsecretionare peptide hormones (e.g.,insulin, glucagon,ACTH) from various endocrinecells,precursorsof digestiveenzymesfrom pancreatic acinar cells, milk proteins from the mammary gland, and neurotransmittersfrom neurons.The third type of vesicle that buds from the trans-Golgi network is directed to the lysosome,an organelleresponsiblefor the intracellular degradation of macromolecules, and to lysosome-like storage organellesin certain cells.Secretoryproteinsdestinedfor lysosomes are first transported by vesiclesfrom the trans-Golgi network to a compartment usually called the late endosome; proteins then are transferred to the lysosome by direct fusion of the endosomewith the lysosomalmembrane. Endocytosis is related mechanistically to the secretory pathway. In the endocytic pathway, vesiclesbud from the plasma membrane, bringing membrane proteins and their bound ligands into the cell (see Figure 14-1). After being internalized by endocytosis,some proteins are transported to lysosomesvia the late endosome,whereasothers are recycled back to the cell surface. In this chapter we first discusshow our knowledge of the secretorypathway and endocytosishas expandedthrough experimental techniques.Then we focus on the generalmechanisms of membrane budding and fusion. We will seethat although different kinds of transport vesiclesutilize distinct sets of proteins for their formation and fusion, all vesiclesuse the same generalmechanismfor budding, selectionof particular setsof cargo molecules,and fusion with the appropriatetarget membrane.The following two sectionsshow how coordination betweenparticular vesicletrafficking stepscan maintain the identity (i.e.,a stableset of residentproteins) of the different compartments along the secretory pathway and how cargo selectionby vesiclesis usedto sort proteins to different intracellular locations.Next we will turn our attention to the endocytic pathway to examine how endocytosisis used to transport macromoleculesfrom the extracellularenvironment into the cell interior. Finally we will examine the variety of ways that membrane proteins and macromoleculesfrom the cell interior are transportedto the lysosomefor degradation.

Im

Techniques for Studying

the SecretoryPathway The key to understandinghow proteins are transporred through the organelles of the secretory pathway has been to develop a basic description of the function of transporr vesicles.Many components required for the formation and

V E S T C U L ATRR A F F t CS, E C R E T T OA NN , D ENDOCYTOS|S

llll+ overviewAnimation:ProteinSecretion

z

tr

Regulated / secretion /

ffi

Secretory vesicle

E

\ transGolgi network

Sortingto lysosomes

Late endosome

Transport vesicle

f .gr

transGolgi

e

medialGolgi

tr

Retrograde transportfrom l a t e rt o e a r l i e r Golgi cisternae

crs€olgi network

\

B u d d i n ga n d f u s i o no f vesiclesto ER-to-Golgi TOrmcrs-(Jorgl

!

ffi

,"*-"\

_grj *ao-&

E

RetrogradeGolgi-to-ER transoort

^/

sl*ed

-f."

\,,,,,' ER lumen

g

Rough ER I

14-1 Overviewof < FIGURE the secretoryand endocytic pathwaysof protein sorting. pathway:Synthesis of Secretory proteins an ERsignal bearing on the iscompleted sequence roughERIl, andthe newlymade polypeptide areinserted chains or crossit intothe ERmembrane 13). intothe lumen(Chapter (e.g.,ERenzymes Someproteins proteins) remain or structural are withinthe ERTheremainder vesicles packaged intotransPort El that budfromthe ERandfuse cisternae to form newcr's-Golgi proteins ER-resident Missorted proteins membrane andvesicle that needto be reusedare to the ERbYvesicles retrieved E that budfromthe cts-Golgi andfusewith the ER.Eachctswith itsProtein Golgicisterna, movesfrom content,physically the crsto the fransfaceof the Golgicomplex @ by a process called nonvesicular Retrograde maturation. cisternal vesicles transport E moveGolgiproteins to the ProPer resident In allcells, compartment. Golgi proteins moveto certainsoluble in transPort the cellsurface vesicles El andaresecreted (constitutive continuously In certaincelltYPes, secretion). proteins arestored somesoluble vesicles in secretory Z andare onlyafterthe cell released neuralor an appropriate receives signal(regulated hormonal Lysosome-destined secretion). andsoluble membrane Proteins, in vesicles whicharetransported that budfromthe trans-Golgi E, firstmoveto the late andthento the endosome lysosomeEndocytic PathwaY: andsoluble Membrane proteins takenup in extracellular thatbudfromthe Plasma vesicles membrane I alsocanmoveto viathe endosome the lysosome

Proteinsvnthesison bound ribosomes; transportof proteins co-translational into or acrossER membrane

T E C H N I Q U EF SO R S T U D Y I N GT H E S E C R E T O RPYA T H W A Y

581

fusion of transport vesicleshave been identified in the past decadeby a remarkable convergenceof the geneticand biochemical approachesdescribedin this section.All studies of intracellular protein trafficking employ some method for assaying the transport of a given protein from one compartment to another. \7e begin by describing how intracellular protein transport can be followed in living cells and then considergeneticand in vitro systemsthat have proved useful in elucidating the secretorypathway.

Transportof a ProteinThroughthe Secretory PathwayCan Be Assayedin Living Cells The classicstudiesof G. Paladeand his colleaguesin the 1960s first established the order in which proteins move from organelleto organelle in the secretoryp"ih*"y. Theseearly studies also showed that secretoryproteins are never releasedinto the cytosol, the first indication that transported proteins are always associatedwith some type of membrane,boundedintermediate. In theseexperiments,which combined pulse-chaselabeling (seeFigure 3-39) and autoradiography,radioactively labeledamino acidswere injectedinto the pancreasof a hamster.At different times after injection, the animal was sacrificed and the pancreatic cells were chemically fixed, sectioned,and subjectedto autoradiographyto visualizethe location of the radiolabeled proteins. Becausethe radioactive amino acids were administered in a short pulse, only those proteins synthesized immediately after injection were labeled, forming a distinct group, or cohort, of labeled proteins whose transport could be followed. In addition, becausepancrearicacinar cells are dedicatedsecretorycells, almost all of the labeled amino

acids in thesecells are incorporated into secretoryproteins, facilitating the observation of transported proteins. Although autoradiographyis rarely usedtoday to localize proteins within cells, these early experiments illustrate the two basic requirementsfor any assayof intercompartmental transport. First, it is necessaryto label a cohort ofproteins in an early compartment so that their subsequent transfer to Iater compartments can be followed with time. Second,it is necessaryto have a way to identify the compartment in which a labeled protein resides.Here we describetwo modern experimental procedures for observing the intracellular trafficking of a secretoryprotein in almost any type of cell. In both procedures, a gene encoding an abundant membrane glycoprotein (G protein) from vesicular stomaritis virus (VSV) is introduced into cultured mammalian cells either by transfection or simply by infecting the cells with the virus. The treated cells, even those that are not specializedfor secretion, rapidly synthesizethe VSV G protein on the ER like normal cellular secretoryproteins. Use of a mutant encoding a temperature-sensitiveVSV G protein allows researchersto turn subsequent transport of this protein on and off. At the restrictive temperature of 40 'C, newly made VSV G protein is misfolded and therefore retained within the ER by quality-control mechanismsdiscussedin Chapter 13, whereasat the permissivetemperatureof 32"C, the protein is correctly folded and is transported through the secretorypathway to the cell surface.This clever use of a temperature-sensitivemutation in effect defines a protein cohort whose subsequenttransport can be followed. In two variations of this basic procedure, transport of VSV G protein is monirored by different techniques.Studies using both of these modern trafficking assaysand Paladet

Video:Transportof VSVG-GFP Throughthe Secretorypathway 0 min

1 8 0m i n

Bts 5 ll 10

v

I thF

Plasma membrane Time(min)

A EXPERIMENTAL FIGURE 14-2 proteintransportthroughthe secretorypathway can be visualizedby fluorescence microscopy of cellsproducinga GFP-tagged membraneprotein.Culturedcells weretransfected witha hybridgeneencoding theviralmembrane glycoprotein VSVG proteinlinkedto the genefor greenfluorescent protein(GFP). A mutantversion of theviralgenewasusedsothat newlymadehybridprotein (VSVG-GFP) isretained in theERat 40 .C but isreleased for transport at 32 "C.(a)Fluorescence micrographs of cellsjustbeforeandat two timesaftertheywereshiftedto the lower temperature. Movement of VSVG-GFP fromthe ERto the Golqiand

582

C H A P T E R1 4

|

finallyto thecellsurface occurred within180minutes. Thescale baris 5 p.m (b)Plotof the levels of VSVG-GFp in the endoplasmic (ER), reticulum (pM)at different Golgi,andplasma membrane timesaftershiftto lowertemperature. Thekinetics of transport fromoneorganelle to anothercanbe reconstructed from computer analysis of thesedata.Thedecrease in totalfluorescence thatoccurs at latertimesprobably results fromslowinactivation of GFPfluorescence Jennifer Lippincott-Schwartz IFrom androret H i r s c h b e r gM, e t a b o l i s mB r a n c h N , a t i o n aIl n s t i t u t eo f C h i l dH e a l t ha n d H u m a nD e v e l o o m e nI t

VES|CULAR T R A F F t CS, E C R E T | O N A,N D E N D O C Y T O S t S

early experimentsall came to the same conclusron:In mammalian cellsvesicle-mediatedtransport of a protein molecule from its site of synthesison the rough ER to its arrival at the plasma membrane takes from 30 to 60 minutes. Microscopy of GFP-LabeledVSV G Protein One approach for observing transport of VSV G protein employs a hybrid genein which the viral geneis fused to qhegene encodinggreen fluorescent protein (GFP), a naturally fluorescent protein (Chapter 9). The hybrid geneis transfectpdinto cultured cells by techniques described in Chapter 5. \(hen cells expressing the temperature-sensitiveform of the hybrid protein (VSVGGFP) are grown at the restrictive temperature, VSVG-GFP accumulatesin the ER, which appearsas a lacy network of membranes when cells are observedin a fluorescentmicroscope. When the cells are subsequentlyshifted to a permissive temperature, the VSVG-GFP can be seento move first to the membranes of the Golgi apparatus,which are denselyconcentrated at the edge of the nucleus, and then to the cell surface (Figure 1.4-2a).By analyzing the distribution of VSVG-GFP at different times after shifting cells to the permissivetemperature' researchershave determined how long VSVG-GFP residesin each organelle of the secretorypathway (Figure 14-2b). Cis-Golgi

(a)

Detection of Compartment-Specific Oligosaccharide A second way to follow the transport of Modifications secretoryproteins takes advantageof modifications to their carbohydrate side chains that occur at different stagesof the secretorypathway. To understand this approach, recall that many secretoryproteins leaving the ER contain one or more copies of the N-linked oligosaccharide Mans(GlcNAc)2, which are synthesizedand attached to secretoryproteins in the ER (seeFigure 13-18). As a protein moves through the Golgi complex, different enzymeslocalized to the cis-, medial-, and trans-Golgi cisternae catalyze an ordered seriesof reactionsto thesecore Mans(GlcNAc)z chains, as discussed in a later section of this chapter. For instance,glycosidases that residespecificallyin the cis-Golgi compartment sequentially trim mannose residuesoff the core oligosaccharideto yield a "trimmed" form Man5(GlcNAc)2. Scientistscan use a specializedcarbohydrate-cleavingenzymeknown as endoglycosidase D to distinguish glycosylated proteins that remain in the ER from those that have entered the cisGolgi: trimmed cls-Golgi-specific oligosaccharides are cleaved from proteins by endoglycosidaseD, whereas the core (untrimmed) oligosaccharidechains on secretory proteins within the ER are resistant to cleavageby this enzyme (Figure 1'4-3a\.Becausea deglycosylatedprotein produced by endoglycosidaseD digestion moves faster on an SDS gel

Mannose trimming

+

I

Timeat 32 'C (min) 0 5 1 0 1 5 2 0 3 0 4 5 6 0

(b)

Extract slycoprotein

(ER)Resistant._ i (cls-Golgi)Sensitive

I (c)

1.0 o (Man)a(GlcNAc)z

I

(Man)s(GlcNAc),

D rreatwithendoslycosidase

'i 3 o.e aO

:'6

I

v>

0.6

Eo0.4 No cleavage, endoglycosidase D-resistant

Cleavage, endoglycosidaseD-sensitive

t = N-Acetylglucosamine O = Mannose

14-3 Transportof a membrane FIGURE A EXPERIMENTAL glycoproteinfrom the ERto the Golgicanbe assayedbasedon a D. Cellsexpressing sensitivityto cleavageby endoglycosidase with a pulse werelabeled VSVG protein(VSVG) temperature-sensitive sothat temperature aminoacidsat the nonpermissive of radioactive proteinwasretarned timesaftera return in the ERAt periodic labeled fromcells of 32"C,VSVGwasextracted temperature to the permissive moveto thec/sD.(a)Asproteins with endoglycosidase anddigested istrimmed Mans(GlcNAc)2 Golgifromthe ER,thecoreoligosaccharide compartment in the crs-Golgi that reside byenzymes to Mans(GlcNAc)2 fromproteins chains theoligosaccharide D cleaves Endoglycosidase

ti>

EE 0.2 L O

rC o o

0 T i m e( m i n ) in the ER.(b)SDSgel but notfromproteins processed in thecrs-Golgi the resistant, resolves mixtures of thedigestion electrophoresis (faster-migrating) cleaved (slower-migrating) and sensitive, uncleaved all shows,initially VSVGAsthiselectrophoretogram formsof labeled butwith timean increasing to digestion, of theVSVGwasresistant fromthe proteintransported reflecting to digestion, fractionissensitive 40'C, only at kept cells In control processed there. and ERto theGolgi after60 minutes VSVGwasdetected digestion+esistant slow-moving, to of VSVGthat issensitive (notshown).(c)Plotof the proportion of course time the reveals data, fromelectrophoretic derived digestion, 50t523 1987, Cell --; al, et ] Beckers C J [From ER Golgitransport. T E C H N I Q U EF SO R S T U D Y I N GT H E S E C R E T O RPYA T H W A Y

O

583

ClassA

Fate of secreted proteins

Normal secretion

Defective function

GlassB

ClassC

Accumulation in the cytosol

Accumulation i n r o u g hE R

Accumulation in ER-to-Golgi transoortvesicles

Accumulation in Golgi

Accumulation In secretory vesicles

Transport i n t ot h e E R

B u d d i n go f vesiclesfrom t h e r o u g hE R

Fusionof transportvesicles with Golgi

Transportfrom Golgi to secretory vesicles

Transportfrom secretoryvesicles to cell surface

EXPERIMENTAL FTGURE l4-4 phenotypes of yeastsec mutantsidentifiedstagesin the secretorypathway.These temperature-sensitive mutants canbe grouped intofiveclasses based on thesitewherenewlymadesecreted (reddots)accumulate proteins whencellsareshiftedfromthe permissive temperature to the

ClassD

ClassE

highern , o n p e r m i s s i voen e . A n a l y s i o s f d o u b l em u t a n t sp e r m i t t e d the sequentialorder of the stepsto be determined.[Seep Novick e t a | , 1 9 8 1C , e l l 2 5 : 4 6a1n, d C A K a i s e r a nRdS c h e k m al g ng . O.Cett 61:723l

than the corresponding glycosylatedprotein, these proteins A Iarge number of yeast mutants initially were identified can be readily distinguished(Figure14-3b). basedon their ability to secreteproteins at one temperature This type of assaycan be usedto track movement of VSV and inability to do so at a higher, nonpermissivetemperaG protein in virus-infectedcellspulse-labeledwith radioacture. Sfhen these temperature-sensitivesecretion (sec) mwtive amino acids. Immediately after labeling, all the extants are transferred from the lower to the higher temperatractedlabeledVSV G protein is still in the ER and is resistture, they accumulate secretedproteins at the point in the ant to digestion by endoglycosidaseD, but with time an pathway blocked by the mutation. Analysis of such mutants increasingfraction of the glycoprotein becomessensitivero identified five classes(A-E) characterizedby protern accudigestion. This conversion of VSV G protein from an endomulation in the cytosol, rough ER, small vesiclestaking proglycosidase D-resistant form ro an endoglycosidase teins from the ER to the Golgi complex, Golgi cisternae,or D-sensitive form corresponds to vesicular transport of the constitutive secretory vesicles (Figure 14-4). Subsequent protein from the ER to the cis-Golgi. Note that transport of characterization of sed mutants in the various classeshas VSV G protein from the ER to the Golgi takes about 30 minhelped elucidate the fundamental components and molecuutes as measuredby either the assaybasedon oligosaccha- lar mechanismsof vesicletrafficking that we discussin later ride processing or fluorescencemicroscopy of VSVG-GFp sectlons. (Figure 14-3c). A variety of assaysbased on specific carboTo determine the order of the steps in the pathw ay, rehydrate modifications thar occur in later Golgi compartsearchersanalyzeddouble sec mutants. For instance,when ments have been developedto measureprogression of VSV yeast cells contain mutarions in both class B and class D G protein through each stageof the Golgi apparatus. functions, proteins accumulate in the rough ER, not in the Golgi cisternae.Since proteins accumulate at the earliest blocked step; this finding shows that classB mutatrons must YeastMutants Define Major Stagesand Many act at an earlier point in the secretoryparhway than classD Componentsin VesicularTransport mutations do. These studies confirmed that as a secreted The generalorganization of the secretorypathway and many protein is synthesizedand processed,it moves sequentially of the molecular componentsrequired for vesicletrafficking from the cytosol -+ rough ER --> ER-to-Golgi transport are similar in all eukaryotic cells. Becauseof this .o.r.ruul vesicles-+_Golgi cisternae-+ secreroryvesiclesand finally is exocytoseo. The three methods outlined in this sectionhave delineated the major steps of the secretory pathway and have contributed to the identification of many of the proteins responsible for vesiclebudding and fusion. Currently eachof the individual steps in the secretorypathway is being studied in mechanisticdetail, and increasinglSbiochemical assaysand moleculargeneticstudiesare usedto study eachof thesesteps in terms of the function of individual protein molecules. 584

.

c H A p r E R1 4 |

vEstcuLAR T R A F F t cs,E c R E T t o N A,N D E N D o c y r o s t s

(b)

(a) cis-Golgi

VSV-infected wild-type cells

VSV-infected mutant cells (no N-acetylglucosamine transferasel)

medial-Golgi

trans-Golgi

Golgi isolated from uninfected wild-tyPe cells

N-Acetylglucosamine transferaseI reaction

G protein in Golgi from infected mutant cells

lncubation

I = N - A c e t y l g l u c o s a m i n e@ = G a l a c t o s e O =Mannose a=N-Acetylneuraminicacid

Addition of N-acetylglucosamine to G protein

FIGURE 14-5A cell-freeassaydemonstrates EXPERIMENTAL protein transpoftfrom one Golgi cisternato another.(a)A in thistypeof assayIn fibroblasts isessential mutantlineof cultured N-acetylglucosamine thecellslacktheenzyme thisexample, is thisenzyme cells, | (stepE in Figure14-14)ln wild-type transferase N-linked oligosaccharides andmodifies to themedr,afGolgi localized wild-type InVSV-infected of oneN-acetylglucosamine, bytheaddition to a typical on theviralG proteinismodified cells, theoligosaccharide panel.In asshownin the trans-Golgi oligosaccharide, complex the cellsurface the G proteinreaches infectedmutantcells,however,

onlytwo containing oligosaccharide high-mannose with a simpler (b)WhenGolgi residues. andfivemannose N-acetylglucosamine with Golgi mutantcellsareincubated frominfected isolated cisternae produced protein VSV G the cells, unrnfected fromnormal, cisternae Thismodification N-acetylglucosamine theadditional in vitrocontains enzymethat is movedby transport iscarriedout by transferase to the mutantctscisternae fromthewild-typemedial-Golgi vesrcles Balch et al, 1984'Cell W E in the reactionmixture[See Golgicisternae and J E Rothman 1; and 39:51 1984, Cell et al A Braell , and525;W 39:405 275:1212 1997,Science l I S6llner,

Cell-FreeTransportAssaysAllow Dissection o f I n d i v i d u a lS t e p si n V e s i c u l aTr r a n s p o r t

purified away from the donor wild-type Golgi membranes ty centrifugation. By examining the proteins that are enriched in thise vesicles,scientistshave been able to identify

In vitro assaysfor intercompartmental transport are powerful complementary approachesto studieswith yeast sec mu' tants for identifying and analyzingthe cellular components responsible for vesicular trafficking. In one application of this approach, cultured mutant cells lacking one of the enzymes that modify N-linked oligosaccharidechains in the Golgi are infected with vesicular stomatitis virus (VSV). For example, if infected cells lack N-acetylglucosamine transferaseI, they produce abundant amounts of VSV G protein but cannot add N-acetylglucosamine residues to the oligosaccharidechains in the medial-Golgi as wild-type cells do (Figure 14-5a). When Golgi membranes isolated from such mutant cells are mixed with Golgi membranes from wild-type, uninfected cells, the addition of N-acetylglucosamineto VSV G protein is restored(Figure14-5b). This modification is the consequenceof vesiculartransport of NacetylglucosaminetransferaseI from the wild-type medialGolgi to the cls-Golgi compartment from virally infected mutant cells.Successfulintercompartmentaltransport in this cell-freesystem dependson requirementsthat are typical of a normal physiologicalprocess,including a cytosolic extract, a sourceof chemicalenergyin the form of ATP and GTP, and incubation at physiological temperatures' In addition, under appropriate conditions a uniform population of the transport vesiclesthat move N-acetylglucosaminetransferaseI from the medial- to cls-Golgi can be

targeting and fusion of vesicleswith appropriate acceptor In vitro assayssimilar in general design to the -.-br".t.t. one shown in Figure 1,4-5 have been used to study various transport stepsin the secretorypathway.

Techniquesfor Studyingthe SecretoryPathway r AII assays for following t he trafficking of proteins rn living cells require a way through the secretorypathway ln and a way to identity proteins secretory to label a cohort of subsequentlyare proteins labeled where the compartments located. r Pulse labeling with radioactive amino acids can specifically label a cohort of newly made proteins in the ER' AIternatively, a temperature-sensitivemutant protein that is retained in the ER at the nonpermissivetemperaturewill be releasedas a cohort for transport when cells are shifted to the permissivetemperature. T E C H N I Q U EF SO R S T U D Y I N GT H E S E C R E T O RPYA T H W A Y

585

r Transport of a fluorescentlylabeled protein along the secretory pathway can be observed by microscopy (seeFigure 14-2). Transport of a radiolabeledprotein commonly is tracked by following comparrment-specificcovalent m o d i f i c a t i o n sr o r h e p r o t e i n . Many of the componentsrequired for intracellularprotein afficking have been identified in yeast by analysisof temperature-sensitive sec mutants defectivefor the secretionof proteins at the nonpermissivetemperarure(seeFigure 14-4). r Cell-free assaysfor intercompartmentalprorein transport have allowed the biochemicaldissectionof individual stepsof the secretorypathway. Such in vitro reactronscan be used to produce pure transport vesiclesand to rest the biochemicalfunction of individual transportproteins.

Molecular Mechanisms

I!f,|

( a )C o a t e dv e s i c l eb u d d i n g

Soluble

Membrane cargo-receptor prorern

Donor memorane

Coat proteins Cytosol

(b) Uncoatedvesiclefusion

cwosor

of VesicularTraffic

ltfl.'T..""

Small membrane-boundedvesiclesthat transporr prorelns from one organelle to another are common elemenisin the

type of vesicle, studies employing genetic and biochemical techniqueshave revealedthat each of the different vesicular transport stepsis simply a variation on a common theme. In this sectionwe explore the basicmechanismsunderlying vesicle budding and fusion, that all vesicletypeshave in common. The budding of vesiclesfrom their parent membrane is driven by the polymerization of soluble protein complexes onto the membrane to form a proteinaceousvesiclecoat (Figure 14-6a). Interactionsbetweenthe cytosolic portions of integral membraneproteins and the vesiclecoat gather the appropriate cargo proteins into the forming vesicle.Thus the coat not only gives curvature to the membrane to form a vesiclebut also acts as the filter to determine which oroteins are admitted into the vesicle. The integral membrane proteins in a budding vesicleinclude v-SNAREs, which are crucial to eventual fusion of the

protein

complex

FIGURE 14-6 Overviewof vesiclebuddingand fusionwith a target membrane.(a)Budding isinitiated by recruitment of a small proteinto a patchof donormembraneComplexes GTP-binding of coatproteins in thecytosol thenbindto thecytosolic domainof membrane cargoproteins, someof whichalsoactasreceptors that proteins bindsoluble in the lumen,therebyrecruiting luminal cargo proteins intothe buddingvesicle(b)Afterbeingreleased and shedding itscoat,a vesicle fuseswith itstargetmemorane In a process thatinvolves proteins. interaction of coqnate SNARE

reversiblepolymerization of a distinct set of protein subunits (Table 14-1). Each rype of vesicle,named for its primary coat proterns,transports cargo proteins from particular parent organellesto particular destination organelles: r COPI vesiclestransport proteins from the rough ER to the Golgi. COPI vesiclesmainly transport proteins in the retrograde irection betweenGolgi cisternaeand from the cls-Golei ack to the rough ER.

Assemblyof a ProteinCoat DrivesVesicle F o r m a t i o na n d S e l e c t i o no f C a r g oM o l e c u l e s Three types of coated vesicleshave been characterized,each with a different type of protein coat and each formed by 586

.

c H A p r E R1 4 I

r Clathrin vesiclestransport proteins from the plasma membrane (cell surface)and the trans-GoIgi network to late endosomes. Every vesicle-mediatedtrafficking step is thought to utilize some kind of vesicle coat; however, a specific coat

vEstcuLAR T R A F F t cs,E c R E T t o N A,N D E N D o c y r o s t s

TYPI VESICIE

MEDIATEI) STEP IRANSPORT

PROTEINS C()AT

GTPase ASS0CIATED

COPII

ER to cls-Golgi

1 Sec23lSec24and Secl3/Sec3 Sec16 complexes,

Sarl

COPI

cls-Golgi to ER Later to earlier Golgi cisternae

Coatomerscontalnrngseven differentCOP subunits

ARF

Clathrinand adapterprotelns

tr ans-G olgi to endosome

Clathrin* AP1 comPlexes

ARF

tr ans -Golgi to endosome

Clathrin + GGA

ARF

Plasmamembrane to endosome

Clathrin * AP2 comPlexes

ARF

Golgi to lysosome,melanosome, or plateletvesicles

AP3 complexes

ARF

.Each

vesiclescontains clathrin t1,peof Ap complex consists of four different subunits. It is not known whether the coat of AP3

protein complex has not been identified for every type ol have not yet identifiedthe vesicle.For example,researchers that move protelns the vesicles coat proteins surrounding during either membrane plasma the from the trans-Golgito secretion. constitutive or regulated The general scheme of vesicle budding shown in Figure 14-6a appliesto all three known types of coated vesicles. Experimentswith isolated or artificial membranesand purified coat proteins have shown that polymerizationof the coat proteins onto the cytosolic face of the parent membrane is necessaryto producethe high curvatureof the membranethat is typical of a transport vesicleabout 50 nm in diameter.Electron micrographs of in vitro budding reactions often reveal structufes that exhibit discrete regions of the parent membrane bearing a dense coat accompanied by the curvature characteristicof a completedvesicle(Figure14-7). Suchstructures, usually calleduesiclebwds, appearto be intermediates that are visible after the coat has begunto polymerizebut before the completedvesiclepinchesoff from the parent membrane. The polymerized coat proteins are thought to form sometype of curved lattice that drivesthe formation of a vesicle bud by adheringto the cytosolicface of the membrane'

and clathrin vesicles,this GTP-binding protein is known as ARF protein A different but related GTP-binding protein known as Sarl protein is present in the coat of COPII vesicles. Both ARF and Sarl are monomeric proteins with an overail structuresimilar to that of Ras' a key intracellular signal-transducingprotein (seeFigure 16-24)' ARF and Sarl pr"ot.inr, like Ras, belong to the GTPase superfamily of switch proteins that cycle between inactive GDP-bound and activeGTP-boundforms (seeFigure 3-32)' The cycle of GTP binding and hydrolysis by ARF and Sarl are tho,tght to control the initiation of coat assembly, as schematicafy depicted for the assemblyof COPII vesicles

A ConservedSet of GTPaseSwitch Proteins ControlsAssemblyof Different VesicleCoats Based on in vitro vesicle-buddingreactions with isolated membranesand purified coat proteins,scientistshave determined the minimum set of coat components required to form each of the three maior types of vesicles.Although most of the coat proteinsdiffer considerablyfrom one type of vesicleto another,the coats of all three vesiclescontain a small GTP-bindingprotein that acts as a regulatorysubunit to control coat assembly(seeFigure 1,4-6a).For both COPI

14-7 Vesiclebudscan be visualized FIGURE r, EXPERIMENTAL coat WhenpurifiedCOPII during in vitro buddingreactions. or artificial ERvesicles with isolated areincubated components of the coatproteins polymerization (liposomes), vesicles phospholipid curvedbuds ln highly of emergence induces surface on thevesicle notethe reaction, budding vitro in of an micrograph th electron present on the asa darkproteinlayer, coat,visible di inctmembrane 93(2):263 Ceil ] etal, 1988, K Matsuoka buds lFrom vesicle M E C H A N I S MO S F V E S I C U L ATRR A F F I C MOLECULAR

.

587

E

Sarl membranebinding, GTP exchange GTP

Cytosol

Sart

/

GDP

Hydrophobic N-terminus

\ Jec tz -

ER lumen

Sec23/Sec24

E :3:'liT'

p

.

in Figure 14-8. First, an ER membrane protein known as Sec1.2catalyzesrelease of GDp from cyrosolic Sarl.GDp and binding of GTP. The Sec12 guanine nucleotideexchangefactor apparently receivesand integratesmultiple as yet unknown signals,probably including the presenceof cargo proteins in the ER membrane that are ready to be transported. Binding of GTp causes a conformational change in Sarl that exposes its hydrophobic N-terminus, which then becomesembeddedin the phospholipid bilayer and tethers Sarl.GTP to the ER membrane. The membrane_ attached Sarl.GTP drives polymerization of cytosolic complexes of COPII subunits on the membrane.eventually leading to formation of vesicle buds. Once COPII vesiciesare releasedfrom the donor membrane, the Sarl GTpase activ_ ity hydrolyzes Sarl.GTP in the vesicle membrane to Sarl.GDP with the assistanceof one of the coat subunits. This hydrolysis triggers disassemblyof the COpII coat. Thus Sarl couples a cycle of GTp binding and hydrolysis to the formation and then dissociationof the COpII coat. ARF protein undergoesa similar cycle of nucleotide ex_ change and hydrolysis coupled to the assembly of vesicle coats composedeither of COpI or of clathrin and other coat proteins (AP complexes),discussedlater. A covalent protein modification known as a myristate anchor on the N-termi_

cre hydrotysis

P;

I

ARF'GTP with the membrane servesas the foundation for further coat assembly. Drawing on the structural similarities of Sarl and ARF to other small GTPaseswitch proteins, researchershave con_ structed genesencoding mutant versionsof the two proteins that have predictable effectson vesiculartraffic when trans-

Coatdisassembly

Uncoated vesicle FIGURE 14-8 Modelfor the role of Sarl in the assemblyand disassembly of COPIIcoats.Step[: Interaction of soluble GDp_ boundSarlwith theexchange factorSecl2, an ERinregrar membrane protein, catalyzes exchange of GTpfor GDpon Sarl In the GTP-bound formof Sar1,itshydrophobic N-terminus extends outwardfromthe protein's surface andanchors Sarlto the ER membrane. Stepf,l: Sarlattached to the membrane serves asa bindingsitefor the Sec23/Sec24 coatproteincomplex. Membrane cargoproteins arerecruited to theformingvesicle budby bindingof specific shortsequences (sorting signals) in theircytosolic regions to siteson the Sec23/Sec24 complex, Somemembrane cargoproteins alsoactasreceptors that bindsoluble proteins in the lumen.The coatiscompleted by assembly of a second typeof coatcomplex composed of Sec13 (notshown)StepB: Afterthe andSec31 vesicle coatiscomplete, the Sec23coatsubunitpromotes GTp h y d r o l y sbiysS a r l S t e p@ : R e l e a soef S a r l . G Dfpr o mt h ev e s i c l e membrane causes disassembly of thecoat.[See S Sprrngeret at, 1999, Cell97:145I

588

.

c H A p r E R1 4 |

with target membranes.Addition of a nonhydrolyzable GTp analog to in vitro vesicle-buddingreactions causesa similar blocking of coat disassembly.The vesiclesthat form in such reactions have coats that never dissociate,allowing their composition and structure to be more readily analyzed.The purified COPI vesiclesshown in Figure 14-9 were produced in such a budding reacrion.

TargetingSequenceson CargoproteinsMake SpecificMolecularContactswith Coat proteins In order for transport vesiclesto move specificproteins from one compartment to the next, vesicle buds must be able to discriminate among potential membrane and soluble cargo proteins, acceptingonly those cargo proteins that should J_ vance to the next compartment and excluding those that

vEstcuLAR T R A F F I cs,E c R E T t o N A.N D E ND O C Y T O S I S

14-9 Coatedvesiclesaccumulate FIGURE A EXPERIMENTAL of a during in vitro buddingreactionsin the presence Golgimembranes Whenisolated analogof GTP. nonhydrolyzable COPIcoatproteins, extractcontaining with a cytosolic areincubated of a Inclusion formandbudoff fromthe membranes vesicles prevents analogof GTPin the buddingreaction nonhydrolyzable shows Thismicrograph release of the coataftervesicle disassembly from generated andseparated in sucha reaction COPIvesicles prepared in thisway Coatedvesicles by centrifugation membranes andproperties theircomponents to determine canbe analyzed of L Orci] [Courtesy

should remain as residentsin the donor compartment' In addition to sculpting the curvature of a donor membrane' the vesiclecoat functions in selectingspecificproteins as cargo' The primary mechanism by which the vesicle coat selects cargo moleculesis by directly binding to specific sequences, or sorting signals, in the cytosolic portion of membrane cargoproteins(seeFigure 14-6a).The polymerizedcoat thus acts as an affinity matrix to cluster selectedmembrane cargo proteins into forming vesicle buds. Since soluble proteins within the lumen of parent organelles cannot contact the coat directly, they require a different kind of sorting signal' Soluble luminal proteins often contain what can be thought of as luminal sorting signals,which bind to the luminal domains of certain membrane cargo proteins that act as receptors for luminal cargo proteins. The properties of several known sorting signalsin membrane and soluble proteins are summarizedin Table 14-2.We describethe role of thesesignals in more detail in later sectlons.

Control Dockingof Vesicles Rab GTPases on TargetMembranes A secondset of small GTP-binding proteins, known as Rab proteins, participate in the targeting of vesiclesto the appropriate target membrane' Like Sarl and ARF, Rab proteins

LUMENAL SORTING SIGNALS (KDEL) Lys-Asp-Glu-Leu

ER-resident soluble protelns

KDEL receptorin cls-Golgi membrane

(M6P) Mannose6-phosphate

Soluble lysosomal enzymes after processingin cls-Golgi

M6P receptor in trans-Golgr membrane

Secretedlysosomal enzymes

M6P recepror in plasma membrane

Clathrin/AP2

CYTOPLASMICSORTING SIGNALS Lys-Lys-X-X(KKXX)

ER-resident membrane protelns

COPI ct and P subunits

COPI

Di-acidic(e.g.,Asp-X-Glu)

Cargo membrane proteins in ER

COPIISec24subunit

COPII

Asn-Pro-X-Tyr(NPXY)

LDL receptor in plasma membrane

AP2 complex

ClathridAP2

Tyr-X-X-lf,Eqsulatord ouos Surlcdce.r'tr ruor; Jto pnq sellrsa^IdOJ euos'paruroJ sr luarulreduoc olgrpeturalurr31og-ol-gg rr{r arurtrr{r lV 'uorteurrsapr31o3-s/,rraqt ol sacuetsrp3uo1 olotu ol sa]f,rsa^trodsuBrl ;o sare8e.rSSu a8nl asaqr 3ur1qe '(31 -ue .(s>lfeJl PeoJIrsJ,,sE qf,nru uorlf,unJ selnqnrorrr;,aq ratdeq3) snurr.uroluoxe eqt ol uoxe 3uo1aqr umop 'pau.ro; are daql ereq,vi 'dpoq ylac aql uorJ parrodsuen are sllal olrou ur salf,rso^de,tr aql ur qJnru 'r81og-su:aql ol selngnl -orJIIu 3uo1eperrodsueJtaJe,ry\ uaql seJntrnrrsra8rel esaql 'tuaw1tpdtuo) awxpawtatur r31og-o16g agl perurer sr leq.&\ Sururrol 'uorteuuo; rreql reryEdluoqs reqlo qf,po r{lr.,ra esnJ ol uees aJe,4A saprse^ pe^rrep-Ug lpro^as 'xelduroc r31og 0r{l ruorJ srelaurorJrru lBro^as PerPJol sE.4 uE 0rl1 qrrq^\ ur 's11acraqto uI 'r31o3-slr aqr rlrl{\ dpcarrp asnJuaqr pue 'turl 'UE arlt {uorJ r.uroJ ot uoos eq plnol 1 ueql ssal e^ou 's11ec oruos dCC-CASA Surureluor salf,rse^luof,seronlJ11erus u1 't.rodsuerrr31o3-ol-gg ur solprparureturaqt orur lq8rs -ur papr,ro.rd(7-y1 arn8r4 eas) ddocso.rJruref,uelsarong dq parnllnr ul d{c-cASA Io rls Pe.{\ollo} sr sllal uErlErrrrrrEru -uprt eql r{Jrq^.\ur dlsnor,Le.rd poqrrtsap stuarurredxeeq1 I ' t z r o s o l P u r qo 1O l q E u n sl leu8rs f,rprle-rp eqr teqr os UICI ;o uorrrod rrlosoldr oqr uorleruroluoc aqr a8ueqc .(eru uortelnur srql '1eu Jo -8rs Surlros f,rprre-rp aql Jo dlrurcr^ eql ur tou sr uortelnru

qlrm aprldad uot}lera}ut : I LZiGltarnleN' l-]o,{saunor [ biaqplog ZOOZ ' aqto1lt slaqlo]}eq} Ljes]o l?]a !8 x aosluMoLls lou steuelquiaLu aqt pale)tput e.ieuralordo6tereq]1oluau.r6as luau6asleurLule]-N aueJqLuaursue.r] oql pueouerqulolu olltsonlldo) aql ;o uorlrsod {1a1r1 eqt tZ)aSq}rMureLuop >rloso1,ir s,obtetaq}ur(eldtnd) leubrs )rpr)e-rp aprldadul e +ouotl)elelur riqsaltrsan lldOl ol po]tnl)alaq ue) ouerqureul aq] ul uralordo6tetV posnsenn dgpddg 6oleue Ul eq}'sarpnls lol uot}nlos urxalduor 419 alqez{lorp{quou lprnl)nr}s fueure1 alqpise LUro] ol laproul alplspunoq-d_19 slrurlpat)1le5{q auerqL.t.tauJ aresexalduor(uaarO) Ul aLl]o] po]rnJ)ar tZ)aS/a6uero) 'leo) aqt u1,{lrelal9.lre5 pue €Z)aS lldof aq]jo uorteuJro] taes pue €Z)aSsureloldteor lld6) eq1bursrrduorxe;duor fueu,ral+oernpnJlsleuorsuautrp-eelql Zl-tl lgnglJ V

ureto.rdobtec;o lueuDes aueJq uraursuelf

'UA eqt ruor; Surppng salrlso^ IIdO:) 80SCV eql q8noqrly otur Sur8e>pedstr SurFolq dq eueJgruerueurseld er{l ol '(80SCV UICf,;o trodsuerr Ierrrrouslua,rerduorlplnru sHI se umoul) ecuenbasuralord eqt ur 80S uorlrsod lp ouru -e1e1.{uaqd E Jo uorlolop E sr uouelnru UIdl uoruuroJ lsoru eql 'UE oqt Jo rno uralord UICf, aqt yo lrodsuerl .ro1dres -safau sr pue lEoJ IIdof, eqr Io lrunqns tzras aql o1 sPurg leqt yeuSrsSurtros lrprle-rp B sureluor urorord UICC eql dlluacar e^Bq sreqJreaso1 'leuueql opuop{J p leqt u.4aor{s euseld se suortrunJ 1r oreq,^a's11oclerlaqrrda;o sauerqurau-r oqt ot pal.rodsue.rlSuraq oro;eq r31og eqr ot potJodsuerl sr pue Ug aql ur uralord eueJqruerule.r8atur uE sp pazls

"Jl:T;i:il'ffilT"il,"j :xTH H ;::i:ltT,*""il

'u.AAou{l0A lou 0J3JSeUJAUI se rlf,nssulel -ord o8rBr elqnlos Jeqlo JoJ sroldacar aqr torce; Surteu n elqnlos aqr spurq teqt urolo.rdouerqureu p urpluof, ot punol uooq o^pr1s11eo lsee.{ruort salf,rse^IIdOf, par;rrnd q8nogtly 'selJrsel ere sutalord o8reo e1q IIdOI otur pepeol dle^trtcales -nlos ^toq eurruratopot >leessorpnlsSuro8uo raqlo 'self,rsa^ IIdOC olur suralord o3.recauerquoru tJorrp dleq reqr sleu8rs Ieuonppe d;rrueprot dem JopunoJp,{llua.rrncserpnlsrrlaua8 ern8rg)Ug oqr rrlort surolord euerq pue lerrruaqrolg'ftI-il -uour ureuof,yo lrodxe e^rtf,olosor{t JoJ l€rluassasr pup l€or IIdOf, eqr Jo trunqns tzres oql ot spurq luu8rs 3urilos srql 'ft-W alqel ees)(apocrouol-euoeqr ut gXC ro'n1g-y-dsy o.reeouenbassrql ur sanprserda4 eql) pu8rs 8utL,tos)tpoo -tp e urcluo) suralord osoqr dueur Jo ;o slueu8es crlosol,(c oq1 'r31og eql ol ilodsuert roJ seprsal IIdOf, otur palrnrJeJ d11err;rceds are suretord ouerqtuotu gg urelroJ ler8erur 't'tl uorrJasur aq PessnrsrP 'erntcn.rlsalrllpoJ E otur elquesse-Jlasor drqrge srqr Ilr,visE seq oslp urrqlplr 'Iel€l JeSol relrrurs 'uortezueur,(1odleoo 'surelord leor rar.{loaql ezrue8 1o dcuercrgear{t Surs€eJJur -ro ol stf,epue sexeldrucoVZI;Z)aS pue I€ltIloS eqr pue slleJelul 'Ug aqr to eJeJrnsrrlosolb aqr or dJC.IJ€S r{lr.ry\ '9IcaS pa11ec'urelord snorqr; a8reye t11eu punoq sr qJrq,,ra -rc 'selfrse^ ro] ploJtels Ildol IErnlf,uls eqr urroJ I€res olur elqruessp pup €Ilas reqr rqSnoqr sr lI 'selnlpl a>p1o3er dlsnoeueluodsuBr suralordI[JaS pue €Irrs eJnd 'ornloruls leof, erll elelduroool spurq surelo.rdI€les pue g1ca5Sursrrd -uroc xaldruof, puores e 'aurrquraru Ufl aqt uo srurol xeyd -ruor srql rery 'ZI-VI arn8rl ur u^\ogs sr tzras pue'g7ca5 'aIC.tres uoo^ueq xeldruoc lrcutet Surtlnse.laq1 pouuot '(g-y1 arn8rg aas)suretord VZ)aSpue tzres to xaldruoc e ;o Surpurq,,{qpazrrollo;ouErqueu ug eqr 01 Irps;o Surpurq sef,npure8ueqcxo srql 'IreS crlosotdc uo roJ dCC dID 'euerqruaru punoq ;o a8ueqrxa aqt saz.{1etec UA aql ur rol -ce; e8ueqcxe-aprloelrnu auruen8 e '71n9 uaq,u peraSSrrt sr salrrsal uorteturo; Xlsnorrrardpaqrrlsop sV IIdOI Jo 'selfrse^ IIdof, to uorlPrrrroJ ro; pa.lrnbersuratord l€rolas polea^or s€q sluetntu gtns Io srsdleuy '(g-y1 ern8rg eas)gg g8nor agr ur suralord olelntu -nrre pue sluelnlu ,as g ssplf,e.resuralord IIdOI ro; saua8 oqt ur suortetnruqlr,r\s]lel tsea1 'suratordIIdOI pereuBrsap 'suralord tueroJJrpyo pesodruor tnq se]rrse^ IdOl uo teqt ol relrurs lEoJ lrurlsrP E Peq seuErqurauug eql I'uort Paurrot

Video:KDELReceptorTrafficking < FIGURE 14-13Roleof the KDEtreceptorin retrievalof ER-resident luminalproteinsfrom the Golgi.ERluminal proteins, especially thosepresent at highlevels, canbe passively incorporated intoCOPII vesicles andtransported to the Golgi (stepsE and Z). Manysuchproteins beara C-terminal KDEL (Lys-Asp-Glu-Leu) (red)thatallowsthemto be retrieved sequence TheKDELreceptor, locatedmainlyin the crs-Golgi networkandin bothCOPII andCOPIvesicles, bindsproteins bearing the KDEL sortingsignalandreturnsthemto the ER(stepsEl and 4). This retrieval prevents system depletion of ERluminalproteins suchas thoseneededfor properfoldingof newlymadesecretory proteinsThebindingaffinityof the KDELreceptor isvery sensitrve to pH Thesmalldifference in the pHof the ERand Golgifavorsbindingof KDEl-bearing proteins to the receptor in Golgi-derived vesicles andtheirrelease in the ER [Adapted fromJ Semenza etal, 1990, Cell61:13491 COPIIcoat

/"

COPIVesiclesMediate RetrogradeTransport w i t h i n t h e G o l g i a n d f r o m t h e G o l g it o t h e E R COPI vesicleswere first discoveredwhen isolated Golgi fractions were incubated in a solution containing .ytorJl and a nonhydrolyzable analog of GTp (seeFiguie 14-9). Subsequentanalysisof thesevesiclesshowed that the coat is formed from large cytosolic complexes, called coatomers,composedof sevenpolypeptide subunits.yeast cells containing temperature-sensitive murations in COpI proteins accumulateproteins in the rough ER at the non_ permissive temperature and thus are categorizedas class B secmutants (seeFigure 14-4). Although discoveryof these mutants initially suggestedthat COpI vesiclesmediateER_ to-Golgi transport, subsequentexperiments showed that their main function is retrograde transport, both between Golgi cisternaeand from the cis-Golgi to the rough ER (seeFigure 14-11, right). BecauseCOpI mutanrs cannor recyclekey membrane proteins back to the rough ER, the ER gradually becomesdepleted of ER proteins such as vSNAREs necessaryfor COpII vesiclefunction. Eventually. vesicleformation from the rough ER grinds to a halt; se_ cretory proteins continue to be synthesizedbut accumu_ late in the ER, the defining characteristicof classB secmutants. The generalability of secmurants involved in either COPI or COPII vesiclefunction to eventually block both anterograde and retrograde transport illustrates the 594

o

c H A p r E R1 4 |

fundamental interdependence of these two rransporr processes. As discussedin Chapter 13, the ER contains severalsoluble proteins dedicated to the folding and modification of newly synthesizedsecretory proteins. These include the chaperone BiP and the enzymeprotein disulfide isomerase,which are necessaryfor the ER to carry out its functions. Although such ER-residentluminal proteins are not specificallyselected by COPII vesicles,their sheer abundancecausesthem to be continuouslyloadedpassivelyinto vesiclesdestinedfor the clsGolgi. The transport of thesesolubleproteins back to the ER, mediated by COPI vesicles,preventstheir eventualdepletion. Most soluble ER-residentproteins carry a Lys-Asp-GIuLeu (KDEL in the one-lettercode) sequenceat their C-terminus (seeTable 14-2). Severalexperimentsdemonstratedthat this KDEL sorting signal is both necessaryand sufficient to causea protein bearingthis sequenceto be located in the ER. For instance,when a mutant protein disulfide isomerase lacking thesefour residuesis synthesizedin cultured fibroblasts, the protein is secreted.Moreover, if a protein that normally is secretedis alteredso that it containsthe KDEL signal at its C-terminus,rhe protein is locatedin the ER. The KDEL sorting signalis recognizedand bound by the KDEL receptor, a transmembraneprotein found primarily on small transport vesiclesshuttling between the ER and the cls-Golgi and on the cis-Golgi reticulum. In addition, soluble ER-residentproteins that carry the KDEL signal have oligosaccharidechains

vEstcuLAR T R A F F t cs,E c R E T t o N A,N D E N D o c y r o s t s

s6s

V M H r _ V d A U O I _ 3 U ) l S3 H r l O S I 9 V J _ SA ' l U V 3

snqJ 'de.{\gfpd ,{rolorf,osaql ur lueulr€durof, rorlJpo ue ol tuoql SurtJodsuerlpue tuarutredruoc ralel E r.uoJtsaudz 'trodsue.rl ape.rSorlaraterpeu -ua r31og Jo UE Sur,tar.rtar .,{1a4r1 rsoru solrrse^asoqt lele^\oH '(SI-tI arnSrg)reqlo -ue ot luerutJgdtuocr31o5 euo ruorJ suralord e^otu ]eql xelduroc r31og eql qtr.&\petertosse self,rse^11erusdueu slEa^er ddocsorcrru uortf elo 'paapul 'r?1o'j-suou aql

ol -lorpaw aql ruorJ pue r.dlou-lotpaw aqt ot -sD aqr I'uort 'p.remro; suralo.rd .{rolarcas Surlrrer solJrsal lJodsueJl IIErusqrr,r\ sluaurlrBdruol Io 1rs lrlprs lllprtuossa ue sE^\ xaldruoc r31og eql reqr lq8noql sE^{tr sreoddueur roJ '(sqelsuorlEJ -lJlpour eJuanbasalrletuasa.rdore ro1 g1-71 a;n8lg aas) Jo lueruuedruof, lxau oql yo seudzue 3ur.{yporu oqt roJ saterls -qns oqr se Surrr.ras auo ur surer{f ereJpdr{ogJel luatul.redruof (>pels r3log eqr porlrpou oql qBno.rqref,uonbesur Sur,tour suratord qtr^ 'eurl .,(lgurasse ue e>lrlqrnur satBredoxelduoc r31og eql 'a1oqmoqr uO '>1cers r3log oql lrsuel daql se sural

'pla]uro) -ord d.rolo:rasol paqrplte salerpdqog:el palur1-O ro pa>lurl I t E 9 : S t w a e o t g ^ a A u u V ' S 8 61 Sp u e -51 Surd;rpou ur po^lo^ur ere leql saseroJsuenldsordppue plaluro>g ea5l r6;o9 aq1 ur sdeis 6ursse>otd ut sa)ualalltpLuol+ p a ) u r l - N+ o a t n ] ) n l l sa q ] u t s u o t i p u p n saseprsordp a.re seurdzuaaqt ,o duey41'ureluoc deqr reqr ] l n s a lu e f s a p u e q l e s o O r l o 'urelordorI16uerlpLUuJeLu e to+ slua^o0urssatotdr61o9 sou.{zua oql ol Surproooe roqloup ouo ruog .roylp r31og ;etrdril 'PuralsrJro 'sfEs eql sluaserder{ennq}edsrql oqi LuoJ}peuodu.rrstostn>eld eql losol{r Jo sluaulreduof,gns aql PaU3DEIJ to o p r l o a l ) n ur e b n su . r o t ; ' a L u et l ] p e u o ' a p u e q ) ) e s o 6 r 1eoq 1o 1 s t e 6 n s po>lJEtse ur peSue.rrEueuo orE r{rrq,{\ 'sluerulredurol tos ppe sau,izueasero+suel] trltad5 (ll dels) sanptsalasol:e1e6 -qns rnoJ ro eerql olur pezrueSrosr xeydruocr3log eq1 aql ]o tl)ea ol anprsolpoe )ruruJelneull{late-ry ue 1o a6e1ur1 {q uorlernlelNleurotsr) [q srnrrg {11eup r ,uLe ( g d a } s )s o n p t s aa. ts o l t e ; e 6o a r q t u o t l l p p el i q r 6 1 o 9 } o -sue4 aq] ur pelalduot sr 6urssatotd(E dols) pappe st aso)n+ !6loC aql qOnorql uodsuerl operboroluv al6urse pue '(E dals) penouLatoJesanptsa.i osouupLuolorJ.t oMl '(E pue E sdels)poppeere sanprsol()VN)lg) euru.reso>n16;,i1e>e-X7

'de,tr.qredd.rolarcesaql oorql 'oJaH't61ocl1erpaw aql ol uorle.rnleur leuralsr),iq senoLrL uralordoqt '(ll dals) r61o9-sr:aql ut sanptsalasouueuloorql 1o se8elsrelEl ur rnrf,o oslEfeqt 8ur>llrlJerturolord rElnf,rse^ Jo sarnleet letuaurepunJeJEsluJrulJedruorolrl oql uae.r\laq ,to lenourorJaUV sluoulueduo> pale:rpuloql 01 paztle)ola.te sureto.rdleurr.unlolqnlos 1o Surycdcaroql pue 'surelord pue dals qree 6urz,ile1et saul{zuaaqf 'slla) a}eJqayon u! oeuJa}s!) sprdqoqdsoqdauerqurau ;o Surledcaraql (selJrso^uodsuerl t6;o9-suerl pue '-Fpa.u'-sl) ulqllrvl suralo.rdo>I;6 uo suleq) a p u e q > r e s o b t l op e l u r l - N ; o 6 u r s s e > o l 6 pepunoq-euerqruou otur surolord 1o drrua e^uJeles oql V t l t l l u n g l l 'salrrse^ (aperSo;rer) IdOl pu€ (aperSo.rarue)UaOf, qloq osocnl = V plce clutuelnou;A1ecy-1y = I olur SurpeoyoSrer Jo lrlcglcads aql dq pellonuot dlareurrr O s o u u e l A =o aso]oele!=a -1nssacordparrln8er pue o^uJelosdlq8H e sr xalduroc r31og euruesocnlb;Ilacv-N = I pue UE or{l ueo,trloqsuralord;o Suruortrued aqr 'd1.reo13 't.rodsue.rr uf uloll yodsuerl e;crsen

I

_^

r6roe

z1cy11c191s1uey11 z(cyqc19)s(uey11

T lz(

z(cVNctg)s(uen) z 1 c y y c 1 9 l s 1 u e t A ) (go)V N c t

v

v-dno

gg-or-r31o5 aperSone.ralerpou solJrsolIdOl ler{l SunBc -lpul 'UE or{t or >lleq leu8rs srqr Surreaqsurolord elerrlor or elgpun are osle rnq leu8rsXX)X eqt purq ot alqeun ore dluo rou dlaOC ro nIdOJ 3ur>1ce1 sluetntu lsea,(alrlrsues -arnleradural 'UE ar{t ot tJodsueJtaperSo.uarJoJ sel3rse^ IdO) orur suratord JuEJqruoru elurod.rocur ol luarf, -rttns dresseceu pu€ qroq sr '(.raruoleorIdOl aql ur slrun -gns aprrdad,(1od ua,rasaql yo ozvrr)slrunqns fl pue o y4g3 er{t Jo xaldtuor E ol spurg rlJlrl^a 'pu8rs Sutltos XXXX slql '(Z-tI a1qe1 aas) yosoldr eqt sere; r{11{a\'luaur8es Ieurrural-) rraql to pue dre,r oql tE of,uanbasy-y-sd1-sd1 e urpluof, r3log aqr ruory UE aqt ot lleq pat.rodsue.rta.re leql surelord euerqrueu Jer{lopup roldarer lgq) or{J 'ug eql Jo reqt uerlt re^{ol dllqSas sr r31og aqr;o gd aqt esneroqUg eql ur saprrdadasaql eseeloror rnq r31og-sll:eqt ur saprrdad ,11d lC:D pulq ot elqe sr roldof,or eqr teqr rq8noql sr lr pue -IAC) or{I ,ry\ol dlrqSri slr o1 arotu spurg rotdarer pue8q te 'G.t-Vt e.rn8rg) eqr ot ueql urntor pue >lro.ryueu r31og-srr UA aqr or o^pq padecsa Surl.ros aql Sururetuor leqr IEC) leu8rs suralo.rdalqnlos elarrler o1 dlureur slce rotdacarlge) eql r81og-sr ar{l sp rEJ sE leql alerrpur s8urpurl osJrJJ'>lro,ry\tau ls?ol lE pelrodsuert uaeq pue UE ar{rr}ol e^eq tsnru suratord eseqleurl eruostE snqt !>lro,rtlaur81og-slr.ror31og-srraqr ur .,{1uo punoy seudzua Aqpazllvw ore teqt suonerrJrpou qlr,v\

^

Apotransferrin dissociatesfrom receptorat neutralpH

FIGURE canbe Thispathway cytosolic Ca2*. of elevation thatactivate by ligandbindingto GPCRs triggered to eitherthe Gooor G*oalphasubunitleading of C (stepIl). Cleavage of phospholipase activation C yieldslP3andDAG(stepZ). PlP2 by phospholipase with lP3interacts throughthecytosol, Afterdiffusing a n do p e n sC a 2 *c h a n n eilnst h em e m b r a noef t h e (stepB), causing release of reticulum endoplasmic (stepZl) Oneof storedCa2*ionsintothecytosol by a risein cytosolic responses induced several cellular C (PKC) to the of proteinkinase Ca2*is recruitment (stepE), whereit isactivated by plasma membrane membrane-associated DAG(stepEl) Theactivated k i n a scea np h o s p h o r y l vaat er i o ucse l l u l aern z y m easn d (stepZ) As theiractivity therebyaltering receptors, a aredepleted, Ca2-stores reticulum endoplasmic Ca2*channels proteinassociated with the lP3-gated in Ca'* channels bindsto andopensstore-operated membrane, allowinginfluxof extracellular the plasma 1999,Proc fromJ W Putney, Ca2*(stepEl) [Adapted Nat'lAcadSciUSA96z14669 l

(;)

,/

r"'Vil '^

kinaseC

"o oo

lP3-gated Ca2* channel

ooo OO

oO

o

A - oo o o-

i^)+

reticulum Endoplasmic

carbon-S of IP3 (seeFigure 15-29\ is hydrolyzed,yielding This compoundcannot bind to the inositol 1,4-bisphosphate. protein and thus does not stimulate IP3-gatedCa2* channel ER. Ca2* releasefrom the \ilithout some means for replenishingdepletedstores of intracellular Ca2*, a cell would soon be unableto increasethe cytosolicCa2* levelin responseto hormone-inducedIP3.Patchclampingstudies(seeFigure 11-21)haverevealedthat a plasma membrane Ca2* channel, called the store-operatedchannel, opensin responseto depletionof ER Ca2* stores.In a way that is not fully understood,depletionof Ca2* in the ER lumen leads with the IP3to a conformationalchangein a protein associated gatedCa2* channelthat allows it to bind to the store-operated Ca2* channel in the plasma membrane,causingthe latter to open (seeFigure15-30,step E). Continuous activation of certain G protein-coupled re-

tion of the cell-surface G protein-coupled receptor' How-

cytosolicCa2*, is not understood.

i no m p l e xM e d i a t e sM a n y T h e C a 2 + / C a l m o d u lC l ignals C e l l u l a rR e s p o n s etso E x t e r n a S potentiatesopening of thesechannelsby IP3, thus facilitating ihe rapid rise in cytosolicCa2* following hormone stimula-

The small ubiquitous cytosolic protein calmodulin functions as a multipurptse switch protein that mediatesmany cellular

RD E C E P T O RTSH A T A C T I V A T EP H O S P H O L I P A SCE G PROTEIN_COUPLE

65s

effects of Ca2* ions. Binding of Ca2* to four sites on calmodulin yields a complex that interacts with and modulates the activity of many enzymesand other protelns (see Figure 3-31). Becausefour Ca2* bind to calmoJulin in a cooperative fashion, a small change in the level of cytosolic Ca2* leadsto a large changein the level of activecalmodulin. One well-studied enzyme activated by the Ca2+/calmodulin complex is myosin light-chain kinase,which regulatesthe activity of myosin in muscle cells (Chapter 17). Another is cAMP phosphodiesrerase,the enzyme that degradescAMp to 5'-AMP and terminates its effects.This reaction thus links Ca2+ and cAMP, one of many examples in which two second messenger-mediated pathways interact to fine-tune certain aspectsof cell regulation. In many cells, the rise in cytosolic Ca2* following recep_ tor signaling via phospholipaseC-generated Ip3 leads io

phosphategroups from a transcription factor. An important example of this mechanism involves T cells of the immune systemin which Ca2* ions enhancethe activity of an essential transcription factor called NFAT (nuclear factor of acti-

sequencethat allows NFAT to move into the nucleus and stimulate expressionof genesessentialfor the function of T cells.

Diacylglycerol(DAG)Activatesprotein KinaseC, W h i c h R e g u l a t e sM a n y O t h e r p r o t e i n s After rts formation by phospholipaseC-catalyzed hydrolysis of PIP2, the secondary messengerDAG remains associated

phosphorylatesvarious transcription factors; depending on the cell type, these induce synthesisof mRNAs that trigger cell division.

S i g n a l - l n d u c eR d e l a x a t i o no f V a s c u l a S r mooth Musclels Mediated by cGMp-Activatedprotein K i n a s eG ffi Nitroglycerinhasbeenusedfor overa centuryas a

treatment lor the intense chest pain of angina. It was Ill known to slowly decompose in the body to nitric oxide (NO/, which causesrelaxation of the smooth muscle cells surrounding the blood vesselsthat "feed" the heart muscle itself, thereby increasingthe diameter of the blood vessels and increasing the flow of oxygen-bearingblood to the heart muscle. One of the mosr intriguing discoveriesin modern medicine is that NO, a toxic gas found in car exhaust, is in fact a natural signalingmolecule.I Definitive evidencefor the role of NO in inducins relaxation of smooth muscle came from a set of experirnentsin which acetylcholinewas added to experimentalpreparations of the smoorh muscle cells that surround blood vessels. Direct application of acetylcholineto thesecellscausedthem to contract, the expected effect of acetylcholine on these muscle cells. But addition of acetylcholine to the lumen of small isolated blood vesselscaused the underlying smooth muscles to relax, not contract. Subsequentstudies showed that in responseto acetylcholinethe endothelialcellsthat line the lumen of blood vesselswere releasingsome substancethat in turn triggeredmusclecell relaxation.That substanceturned out to be NO. \7e now know that endothelial cells contain a Go protein-coupled receptor that binds acetylcholine and activates phospholipaseC, leading to an increasein the level of cytosolic Ca2*. After Ca2* binds to calmodulin, the resulting complex stimulates the activity of NO synthase,an enzyme that catalyzes formation of NO from 02 and the amino acid arginine. BecauseNO has a short half-life (2-30 seconds),it can diffuse only locally in tissuesfrom its site of synthesis.In particular NO diffusesfrom the endothelial cell into neighboring smooth muscle cells,where ir tnggers muscle relaxation (Figure15-31). The effect of NO on smooth muscle is mediated by the secondmessengercGMP, which is formed by an intracellular NO receptor expressedby smooth muscle cells. Binding of NO to the heme group in this receptor leads to a conformational change that increasesits intrinsic guanylyl cyclase activity, leading to a rise in the cytosolic cGMp level. Most

IP3/DAG pathway. The activation of protein kinase C in different cells re_ blood vessel.In this case,cGMP acts indirectly via protein kinase G, whereasin rod cellscGMp acts directiy by bindlng to and thus opening cation channels in the plasma membrane (seeFigure 15-18). 556

'

CHAPTER 1s

I

c E L L s t c N A L r N Gr : s T G N A L TRANSDUCToN AND sHoRT-TERM CELLULARESpoNsES

Lumen of blood vessel

Acetylcholine

Endothelial cells

{

9F,.il;l

15-31The nitricoxide < FIGURE (NO)/cGMP pathwayand the relaxationof arterialsmoothmuscle.Nitricoxideis to cellsin response in endothelial synthesized in elevation andthesubsequent acetylcholine locally ca'* (n-E). No diffuses cytosolic an intracellular andactivates throughtissues in activity cyclase with guanylyl NOreceptor cells(E) Theresulting smoothmuscle nearby proteinkinase G (6 and risein cGMPactivates and muscle the of to relaxation Z), leading (ts) Thecell-surface receptor thusvasodilation alsohas factor(ANF) for atrialnatriuretic (notshown); guanylyl activity cyclase intrinsic on smooth of thisreceptor stimulation cGMP increased to leads also muscle cells PP;: relaxation muscle andsubsequent etal, C S Lowenstein pyrophosphate [See 1994,Ann lnternMed 120:227 )

Smooth musclecells

Relaxation of vascular smooth muscle also is triggered by binding of atrial natriureticfactor (ANF) and someother peptidehormonesto their receptorson smooth musclecells. The cytosolicdomain of thesecell-surfacereceptors,like the intrinsic guanylyl cyintracellular NO receptor,possesses '$fhen an increasedblood volume stretches clase activity. cardiac muscle cells in the heart atrium, they releaseANF. Circulating ANF binds to ANF receptorson the surfaceof smooth musclecellssurroundingblood vessels,inducing activation of their guanylyl cyclaseactivity and formation of cGMP. Subsequentactivationof protein kinaseG causesdilation of the vesselby the mechanismdescribedabove.This vasodilationreducesblood pressureand countersthe stimulus that provoked the initial releaseof ANF.

G Protein-Coupled ReceptorsThat Activate PhospholipaseC r Simulation of someG protein- 200 a G o o

t3u

t loo

ffi ffi

€bu 0

GTP GTP+iso GTP",6 cTP Wild type

ffi

GTP+iso GTP"yS Mutant K E YT E R M S

661

from normal individuals and patients with the pituitary tumors revealeda missensemutation in the Go, genesequence. a. To investigatethe effect of the mutation on Go, activiry wild-type and mutant G*, cDNAs were transfectedinto cells that lack the Go, gene. These cells expressa B2-adrenergicreceptor,which can be activatedby isoproterenol,a B2-adrenergic receptor agonist. Membranes were isolated from transfected cells and assayedfor adenylyl cyclaseactivity in the presenceof GTP or the hydrolysis-resistantGTP analog, GTP-1S. From the figure above, what do you conclude about the effect of the mutation on Go, activity in the presence of GTP alone comparedwith GTP-1Salone or GTP plus isoproterenol(iso)? b. In the transfected cells described in part a, what would you predict would be the cAMP levels in cells transfected with the wild-type Go, and rhe mutant G.,? What effect might this have on the cells? c. To further characterizethe molecular defectcausedby this mutation, the intrinsic GTPaseactivity present in both wild-type and mutant Go, was assayed.Assaysfor GTpaseactivity showed that the mutation reduced the k..r_6.1p(catalysis rate constantfor GTP hydrolysis)from a wild-rypevalue of 4.1 min-1 to the mutant value of 0.1 min-1. Whai do you conclude about the effect of the mutation on the GTpase activiry present in the mutant Go. subunit? How do these GTpase resultsexplain the adenylylcyclaseresultsshown in part a?

References 1 5.2 Studying Cell-Surface Receptors receptorsin hemostasis, _ Coughlin, S. R. 2005. Protease-activated thrombosisand vascularbiology.J Thromb Haemost.3:1800-1814. . Gross,A., and H. F. Lodish.2006. Cellulartrafficking and degradation of erythropoietin and NESP./. Biol. Chem.28t2624-2U1. Simonsen,H., and H. F. Lodish. 1994. Cloning by function: expressioncloning in mammalian cells.Trendsphirmacol. Sci. l5:437441.

Filipek, S., et al. 2003. G protein-coupledreceptorrhodopsin: a prospectus.Ann. Reu.Physiol. 65:851,-879. Filipek, S., et al. 2003. The crystallographicmodel of rhodopsin and its use in studies of other G protein--coupled receptors.Ann. Reu.Biophys Biomol. Struc. 32:375-397. Hurley, J. H., and J. A. Grobler. 1997. Protein kinase C and phospholipaseC: bilayer interacrionsand regulation. Cwrr.Opin. Jtruc. btol. /:JJ /-)6). Nathans,J. 1,999. The evolution and physiologyof human color vision: insightsfrom moleculargeneticstudiesof visual pigments. Neuron 24:299-31.2. Palczewski,K. 2006. G protein-coupledreceptorrhodopsin. Ann. Rev. Biochem.75:743-767. Ramsey,I. S., M. Delling, and D. Clapham.2006. An introduction to TRP channels.Ann. Reu.Physiol.6S:619-647. Singer,W. D., H. A. Brown, and P. C. Sternweis.1997.Regulation of eukaryotic phosphatidylinositol-specific phospholipaseC and phospholipase D. Ann. Reu.Biocbem.66:475-509. 15.6 G Protein-Coupled Receptors That Activate or lnhibit Adenylyl Cyclase Browner,M., and R. Fletterick.1992. Phosphorylase:a biological transducer.TrendsBiochem.Sci. 17:66-71.. Hurley, J.H. 1999. Structure,mechanism,and regulation of mammalian adenylylcyclase.J. Biol. Chem.274:7 599-7602. Johnson,L. N. 1992. Glycogenphosphorylase:conrrol by phosphorylation and allostericeffectors.FASEBJ. 6: 227 4-2282. Taylor, S. S., et al. 2005. Dynamics of signalingby PKA. Bio chim. Biophys. Acta 1754:25-37 . Lefkowitz, R. J., and S. K. Shenoy.2005. Transductionof receptor signalsby B- arrestins.Science308:512-517. ShenoSS., and R. J. Lefkowitz. 2003. Multifaceted roles of B-arrestinsin the regulationof seven-membrane spannrngreceptor trafficking and signaling.Biochem./. 375:503-515. Smith, F. D., L. K. Langeberg,and J. D. Scott.2006. The where's and when's of kinase anchoring. Trends Biochem. Sci. 3l:31,6-323. 'Witters L. A., B. E. Kemp, and A. R. Means. 2005. Churesand ladders: the searchfor protein kinases that act on AMPK. Trends Biochem. Sci.3I:1,3-16. I7ong, W., and J. D. Scott.2004. AKAP signallingcomplexes:focal points in spaceand time. Nature Reu.Mol. Cell Biol. 5:959-970.

15.3 Highly Conserved Components of Intracellular Signal-Transduction Pathways

15.7 G Protein-Coupled Receptors That Activate Phospholipase C

Cabrera-Vera,T. M. et al.2003.Insights into G protein structure, function, and regulation.Endocr. Reu. 24:765-781. I. R., and A. \X/ittinghofer.2001,. The guaninenucleotide_ _Vetter, binding switch in three dimensions.ScienceZ9+I299-I304.

Carlton, J. G., and P.J. Cullen. 2005. Coincidencederectionin phosphoinositidesignaling.Trends Cell Biol. l5'540-547. Kahl, C. R., and A. R. Means. 2003. Regulationof cell cycle progressionby calcium/calmodulin-dependent pathways.Endocr. Reu.24:.719-736. Patterson,R., D. Boehning,and S. Snyder.2004. Inositol 1, 4, 5, trisphosphatereceptorsas signalintegrators.Ann. Reu.Biochem. 73:437465.

15.4 General Elements of G protein-Coupled Receptor

Systems Bourne, H. R. 1997. How receptorstalk to trimeric G proteins. Curr. Opin. Cell Biol. 921.34-1,42. Bourne,H. R. 2001. Receptoractivation:what doesthe rhodopsin structuretell us? Trezds Pharmacol. Sci.22:587-593. Farfe\,7 ., H. Bourne, and T. Iiri. 1999. The expanding spectrumof G protein diseases. New Eng. J. Med.340. 1.012_1020. Pierce,K. L., R. T. Premont, and R. J. Lefkowitz. 2002. Seventransmembranereceptors.Nature Reu.Mol. Cell Biol. 3:639-652. Oldham, \7. M., and H. Hamm. 2006. Structural basisof function in heterotrimericG proteins.Quart Reu.Biophys.40: (ln press). 15.5 G Protein-Coupled Receptors That Regutate lon Channels _ Chin, D., and A. R. Means. 2000. Calmodulin: a prototypical calcium sensor.Trends Cell Biol. l}:322-32g.

662

CHAPTER 15

I

15.8 lntegrating Responses of Cells to Environmental Influences Papin,J. A., et al. 2005. Reconstructionof cellular signalling networks and analysisof their properties.Nature Reu.Mol. Cel[ Biol. 6:99-11'1,. Rothman, D., M. Shults,and B. Imperiali. 2005. Chemical approachesfor investigatingphosphorylationin signaltransduction networks. TrendsCell Biol. 15:502-51.0. Taniguchi,C., B. Emanuelli,and C. R. Kahn. 2006. Critical nodes in signalling pathways: insights into insulin action. Nature Reu.Mol. Cell Biol.7:85-96. 'Watson, R. T., and J. Pessin.2006. Bridging the GAp between insulin signalingand GLUT4 translocarion.TrendsBiochem. Sci. 3l:21.5-222.

C E L LS I G N A L I N G I : S I G N A LT R A N S D U C T I OANN D S H O R T - T E RcME L L U L A R REsPoNsEs

cLASSTC

EXPERIMENT

15

THEINFANCY OF SIGNALTRANSDUCTION-GTP OF cAMPSYNTHESIS STIMULATION M. Rodbellet al., 1971,J. Biol. Chem.246:1877

In the late 1960s the study of hormone action blossomedfollowing the discovery that cyclic adenosine monophosphate (cAMP) functioned as a second messenger, coupling the hormonemediated activation of a receptor to a cellular response.In setting up an experimental system to investigate the hormone-induced synthesis of cAMP, Martin Rodbell discoveredan important new player in intracellular signalingguanosine triphosphate (GTP).

Background The discoveryof GTP's role in regulating signal transduction began with studies on how glucagon and other hormones send a signal across the plasma membrane that eventually evokes a cellular response.At the outset of Rodbell's studies.it was known that binding of glucagon to specificreceptor proteins embeddedin the membrane stimulates production of cAMP. The formation of cAMP from ATP is catalyzed by a membrane-bound enzyme called adenyl cyclase.It had been proposed that the action of glucagon, and other cAMP-stimulating hormones, relied on additional molecular components that couple receptor activation to the production of cAMP. However, in studies with isolated fatcell membranes known as "ghosts," Rodbell and his coworkers were unable to provide any further insight into how glucagon binding leads to an increase in production of cAMP. Rodbell then began a seriesof studies with a newly developedcell-freesystem,purified rat liver membranes,which retained both membrane-bound and membraneassociatedproteins. Theseexperiments eventually led to the finding that GTP is required for the glucagon-induced stimulation of adenyl cyclase.

TheExperiment One of Rodbell's first goals was to characterizethe binding of glucagon to the glucagon receptor in the cell-free rat liver membrane system.First, purified rat liver membraneswere incubated with glucagon labeled with the radioactive isotope of iodine (12sI). Membranes were then separatedfrom the unbound [125I]glucagon by centrifugation. Once it was established that labeled glucagon would indeed bind to the purified rat liver cell membranes,the study went on to determine if this binding led directly to activation of adenyl cyclase and production of cAMP in the purified rat liver cell membranes, The production of cAMP in the cell-free system required the addition of ATP; the substrate for adenyl cyclase,Mg2*; and an AlP-regenerating system consisting of creatine kinase and phosphocreatine. Surprisingly, when the glucagon-binding experiment was repeated in the presenceof these additional factors, Rodbell observed a 50 percent decrease in glucagon binding. Full binding could be restored only when ATP was omitted from the reaction. This observation inspired an investigation of the effect of nucleoside triphosphates on the binding of glucagon to its receptor. It was shown that relatively high (i.e., millimolar) concentrationsof not only ATP but also uridine triphosphate (UTP) and cytidine triphosphate (CTP) reduced the binding of labeled glucagon. In contrast, the reduction of glucagon binding in the presenceof GTP occurred at far lower (micromolar) concentrations. Moreover, low concentrations of GTP were found to stimulate the dissociation of bound glucagon from the receptor.Taken together, these studies suggested that

GTP alters the glucagon receptor in a manner that lowers its affinity for glucagon. This decreasedaffinity both affects the ability of glucagon to bind to the receptor and encouragesthe dissociation of bound glucagon. The observation that GTP was involved in the action of glucagon led to a secondkey question: Can GTP also exert an affect on adenyl cyclase?Addressing this question experimentally required the addition of both ATP, as a substratefor adenyl cyclase,and GTP, as the factor being examined, to the purified rat liver membranes. However, the previous study had shown that the concentration of ATP required as a substrate for adenyl cyclase could affect glucagon binding. Might it also stimuIate adenyl cyclase?The concentration of ATP used in the experiment could not be reduced becauseATP was readily hydrolyzed by ATPases present in the rat liver membrane. To get around this dilemma, Rodbell replacedATP with an AMP analog, 5'-adenyl-imidodiphosphate (AMP-PNP), which can be converted to cAMP by adenyl cyclase,yet is resistant to hydrolysis by membrane ATPases.The critical experiment now could be performed. Purified rat liver membranes were treated with glucagon both in the presence and absence of GTP, and the production of cAMP from AMP-PNP was measured.The addition of GTP clearly stimulated the production of cAMP when comPared to glucagon alone (Figure 1) indicating that GTP affects not only the binding of glucagon to its receptor but also stimulates the activation of adenylyl cyclase.

Discussion Two key factors led Rodbell and his colleaguesto detect the role of GTP in signal transduction, whereas previous

T H E I N F A N C YO F S I G N A LT R A N S D U C T I O N _ G TSPT I M U L A T I O NO F C A M P S Y N T H E S I S

663

G l u c a g o n+ G T P

< FIGURE 1 Effectof GTPon glucagon-stimulated cAMP productionfrom AMP-PNP by purifiedrat liver membranes. In the absence of GTI glucagon stimulates cAMPformation about twofoldoverthe basallevelin theabsence of addedhormoneWhen GTPalsoisadded,cAMPproduction increases anotherfivefold [Adaptedfrom M Rodbellet al , 1971,J Biol Chem 246:1877 ]

o_ 600 @

E o 4oo

0

5

10 Minutes

studies had failed to do so. First. bv switching from fat-cellghosrsto the rat liver membrane system, the Rodbell researchersavoided contamination of their cell-freesystemwith GTP, a problem associatedwith the procedure for isolating ghosts.Suchcontamination would mask the effects of GTP on glucagon binding and activationofadenyl cyclase.Second, when AIP was first shown to influence glucagon binding, Rodbell did not simply acceptthe plausible explanation that ATP, the substrate for adenyl cyclase, also affectsbinding ofglucagon.Insread, he choseto test rhe effectson bindine of the other common nucleosidetriphosphates.Rodbell later noted that he knew commercial preparations of ATP often

664

CHAPTER 15

|

15

20

are contaminated with low concentrations of other nucleosidetriphosphates. The possibilityof contaminationsuggestedto him that small concentrations of GTP might exert large effects on glucagonbinding and the stimulationof adenylcyclase. This critical series of experiments stimulated a large number of studies on the role of GTP in hormone action. eventually leading to the discovery of G proteins, the GTP-binding proteins that couple certain receptors to the adenyl cyclase.Subsequentlyan enormous family of receptors that require G proteins to transduce their signals were identifiedin eukaryotesfrom yeast to humans. These G protein-coupled

receptorsare involved in the action of many hormones as well as in a number of other biological activities, including neurotransmissionand the immune response.It is now known that binding of ligands to their cognate G proteincoupled receptors stimulates the associated G proteins to bind GTP. This binding causestransduction of a signal that stimulates adenyl cyclaseto produce cAMP and also desensitizationof the receptor, which then releasesits ligand. Both of these affects were obs e r v e d i n R o d b e l l ' s e x p e r i m e n t so n glucagon action. For theseseminal observations, Rodbell was awarded the Nobel Prize for Physiology and Medicine in 1994.

C E L LS I G N A L I N G l : S I G N A LT R A N S D U C T I OANN D S H O R T - T E RCME L L U L A R E S p O N S E S

CHAPTER

M A Pk i n a s se i g n a l i nign e m b r y o n iDca y1 3 . 5m o u s el u n g ActivatedERKis detectedby a primaryantibodythat detects phosphorylated antibodyconjugated ERKfollowedby a secondary Bellusci, Saverio FITC@ StijnDelanghe, to greenfluorescing Denise Tefftand DavidWarburton.

cell's ability to respond to its environment is essential to its survival. Short-term responsesto environmental stimuli, which can occur rapidly and are usually reversible,most often result from modification of existing proteins, as detailed in Chapter 15. Longer term responses, which are discussedin this chapter, are usually the result of changesin transcription of genes.Transcription is influenced by chromatin structure and the cell's complement of transcription factors and other proteins (Chapter 7). Thesedetermine which genesthe cell can potentially transcribe at any given time. \7e think of theseproperties as the cell's "memory" determined by its history and responseto previous signals. But many key regulatory transcription factors are held in an inactive state in the cytosol or nucleus and become activated in responseto external signals.In this chapter we focus on how ligandsthat bind to cell-surfacereceptorstrigger activation of specifictranscription factors that, in turn, determine the precisepattern of cellular geneexpresslon. Extracellular signalsthat induce long-term responsesaffect many aspectsof cell function: division, differentiation, and even communication with other cells. Alterations in these signaling pathways cause many human diseases,including cancer,diabetes,and immune defects.In addition to the crucial roles external signalsplay in development,signals are essentialin enabling differentiated cells to respond to their environment by changing their shape, metabolism, or movement. For example, one type of transcription factor (NF-rB) ultimately impacts expressionof more than 150 genesinvolved in the immune responseto infection; NF-rcB is activated by many protein hormones that act on immune systemcells. Another family of extracellular signaling mole-

II: CELLSIGNALING SIGNALING THAT PATHWAYS GENE CONTROL ACTIVITY

cules, the cytokines, is involved in maintaining approprlate levels of blood cells such as erythrocytes (red blood cells), leukocytes(white blood cells),and platelets. In order to illustrate the variety of mechanismsused to

OUTLINE 1 6 . 1 TGFBReceptorsand the DirectActivation of Smads 16.2

15.3

CytokineReceptorsand the JAI(STAT PathwaY

672

ReceptorTYrosineKinases

679

'16.4 Activationof Rasand MAP KinasePathways

684

as SignalTransducers "16.5 Phosphoinositides

694

16.6

Activationof GeneTranscriptionby Receptors Cell-Surface Seven-5panning

16.7

PathwaysThat InvolveSignal-lnduced ProteinCleavage

66s

(a)TGFB receptors

Receptor class

( c )R e c e p t o r t y r o s i n ek i n a s e s (RTKs)

(d)G protein-coupled receptors(GPCRs) o

+ + +

-'1' ':

^^.

UP€

i'

v O

It,

.t::t:t

I I

Y P€

i

E€

l

PQ^

--&*"^ L_*

L*

I

+

Transcription factor (TF)

Smads

btAts

Various

CREB

Mechanism of TF activation

Phosphorylation in cytosol by actrvated receplor

Phosphorylation in cytosol by JAK kinase

Phosphorylation by cytosolic k i n a s ei n n u c l e u s or cytosol

Phosphorylation i n n u c l e u sb y p r o t e i nk i n a s eA

( e )W n t r e c e p t o r s ( f ) H e d g e h o g receptors

Receptor class

(g)TNFa receptors

( h ) N o t c h( D e l t a receptors)

8-,J a

t'

i

I I I I I

t' I I I I I I

I I + Transcription factor {TF}

B-Catenin

Ci (activator) NF-rB C i f r a g m e n t( r e p r e s s o r )

Notch cytosolic domain

M e c h a n i s mo f TF activation

D i s a s s e m b l yo f multiprotein c o m p l e xi n

D i s a s s e m b l yo f multiprotein c o m p l e xi n

Proteolyticrelease o f N o t c hc y t o s o l i c d o m a i n ,w h i c h a c t s w i t h n u c l e a rT F s

iEff;;::E#'h

cvtosol

FIGURE 16-1 Overviewof eight majorclasses of cell-surface receptors.In manysignaling pathways, Iigandbindingto a receptor leadsto activation of transcription factors(TFs) in thecytosol, permitting t h e mt o t r a n s l o c aitnet ot h e n u c l e uasn ds t i m u l a t(eo ro c c a s i o n a l l y repress) transcription of theirtargetgenes(a,b).Alternatively, receptor stimulation mayleadto activation proteinkinases of cytosolic that

oot)

Ir+

C H A P T E R1 6

|

Phosphorylation and degradation o f i n h i b i t o ro f NF-KBin cytosol

thentranslocate intothe nucleus andregulate the activity of nuclear TFs(c,d) In otherpathways, activeTFsarereleased fromcytosolic (e,f) or by proteolysis multiprotein complexes (g,h).Somereceptor classes cantriggermorethanoneintracellular pathway, asshownin Figure16-2 lAfterA H Brivanlou andJ,Darnell, 2OOZ, Science 295:813 I

p A T H W A y s r H A T c o N T R o L G E N EA c l v t r y C E L LS I G N A L | N Gi l : s T G N A L | N G

The kinasemay be an intrinsic part of the receptorprotein or be tightly bound to the receptor.In either case,kinase activity is activated by ligand binding, resulting-directly or indirectly-in activation of specific transcription factors located in the cytosol (Figure 1,6-1a,b).Inother pathways, receptor stimulation leads to activation of cytosolic protein kinasesthat translocate into the nucleus and phosphorylate specificnucleartranscriptionfactors (Figure16-1c,d).Binding of ligand to receptors for other types of signaling proteins causesdisassemblyof multiprotein complexesin the cytosol, releasingtranscription factors that then translocateinto the nucleus (Figure 16-1e,f).In still other signalingparhways, proteolytic cleavageof an inhibitor or the receptor itself r e l e a s e sa n a c t i v e t r a n s c r i p t i o n f a c t o r ( F i g u r e 1 6 - 1 g , h ) . Importantly, all of thesepathways are highly regulated,often by negativefeedback,in order to control the leveland duration of the signal'seffectson cellular geneexpression. :100 differenttypes A typical mammaliancell expresses of cell-surfacereceptors,many of which activate rhe sameor similar signal-transduction pathways.As shown in Figure 16-2, several classesof receptors can transduce signals by more than one pathway, and some pathways are activated to a greaterextent in certain cells than others.Moreover, many genesare regulatedby multiple transcription factors, each of which can be activated by one or more extracellular signals. Especiallyduring early development,such "cross talk" be-

Ligand (signalingmolecule)

Sig n al-transduction pathway

Receptor class

(a) TGFF,BMP

-+

TGFp receptors

Cvtokines ( b ) ( E p o ,p r o l a c t i n )

-

Cytokine receprors

+

Receptortyrosine kinases(RTKs)

Epinephrine, ( d ) g l u c a g o nm , any otners

G protein-coupled receptors(GPCRs)

(f)

Frizzled,LRP

Hedgehog

Patched, Smoothened

Transcription factor Smad proteins

Growth factors ( c ) ( E G EP D G EF G F , i n s u l i n ,I G F - 1 )

Wnt,Wg

tween signaling pathways and the resultant sequentialalterations in the pattern of gene expressioneventually can become so extensivethat the cell assumesa different developmental fate. The receivingcell's prior history and regulatory state can alter the effect of a signal; the same signal applied to different cells will elicit distinct responses. The pathways we discussin this chapter have been conservedthroughout evolution and operate in much the same manner in flies, worms, and man. The substantialhomology exhibited among many proteins in these signaling pathways has enabledresearchersto employ a variety of experimental approachesand systemsto identify and study the function of extracellularsignalingmolecules,receptors,and intracellular signal-transductionproteins.For instance,the secretedsignaling protein Hedgehog(Hh) and its receptorwere first identified in Drosophila mutants with developmentaldefects.Subsequently,the human and mouse homologs of theseproteins were cloned and shown to participate in a number of important signalingeventsduring differentiation.Abnormal activation of the Hh pathway occursin severalhuman tumors. The examplesexplored in this chapter illustrate the importance of studying signalingpathways both genetically-in flies, mice, worms, yeasts,and other organisms-and biochemically. Most of the discussion in this chapter is organized in terms of individual signalingpathways. That is, we consider the signaling molecules,their receptors,the intracellular

STAT proteins

JAK kinase Ras/MAPkinase

Various

Pl-3'kinase/PKB

FoxO,others

PLC,rP3/DAG/PKC

Various CREB

+

Disheveled +

Multiproteincomplex

p-catenin/TCF

Multiproteincomplex

Ci (activator) C; fragment (repressor)

Tumor necrosis factor 0, (TNF-s), -+ (gl l L - 1b , a c t e r i aa n d other pathogens

TNF-o,receptors, lL-1receptors, Toll receptors

+

l-rB kinase

NF-rcB

(h) Delta,Serrate

Notch

+

ADAM10/ y-secretase

Notch cytosolic domain

+

FIGURE16-2 Components and modularity of major signaling p a t h w a y s . S i g n a l i n gp a t h w a y sa r e i n i t i a t e db y t h e b i n d i n go f a signalingmolecule(the ligand),which activatesa receptorActivated pathways receptors then triggervariousintracellular signal-transduction that resultin generationof activetranscriptionfactorseitherin the cytosolor nucleusTranscription factorsthat are activatedin the C H A P T E R1 6

|

(seeFigure16-1).Manyreceptor to the nucleus translocate cytosol (RTKs), kinases receptor tyrosine receptors, cytokine including classes, signals by morethan cantransduce receptors, andG protein-coupled B;PKC: A; p63: proteinkinase onepathwayp14 : proteinkinase protein k i n a sC e ; P L C: p h o s p h o l i p aCs.e

C E L LS I G N A L I N Gl l : S l c N A L l N GP A T H W A Y ST H A T C O N T R O LG E N EA C T I V I T Y

667

signal-transductionpathway(s), the regulated transcription factors, and regulation of the pathway itself for each of the receptorclassesshown in Figure 1,6-1,.\nChapters2l and22,we examine how extracellular signalsaffect generegulation during severalcrucial developmentalstages,and in particular how cells integrate the responsesto multiple signals.In Chapter 25 we illustrate how abnormalities in severalsignal-transduction pathways describedin this chapter can lead to cancer.

TGFBReceptors and the Direct Activationof Smads 'We

begin our survey of signaling systemsthat control gene activity with one of the simplest: one family of signaling molecules(the TGFB superfamily) binds to its receptors(the TGFB receptors)and activatesone classof transcription factors (the Smads),which are located in the cytosol; activated Smads then move into the nucleus to regulate transcription (seeFigure 16-1a). Unlike many of the other signaling systems presentedin this chapter, the TGFB recepror activates only one type of transcription factor) and the transcription factor is activated by only one type of receptor.However, in spite of its simplicitg the TGFB pathway can have widely diverseeffectsin different types of cells becausedifferent members of the TGFB superfamily activate different members of the TGFB receptor family that activate different membersof the Smad class of transcriprion factors. Additionally, the sameactivatedSmadprotein will partner with different transcription factors in different cell types and thus activate different setsof genesin thesecells. The transforming growth factor p (TGFp) superfamily includesa number of relatedextracellularsignalingmolecules that play widespreadroles in regulating developmentin both invertebratesand vertebrates.One member of this superfamlIy, bone morphogeneticprotein (BMP), initially was identified by its ability to induce bone formation in cultured cells. Now called BMP7, it is usedclinically to strengthenbone after severefractures. Of the numerous BMP proteins subsequently recognized,many help induce key steps in development, including formation of mesoderm and the earliest blood-forming cells.Most have nothing to do with bones. Another member of the TGFB superfamily, now called TGFB-1, was identified on the basisof its ability to induce a malignant phenotype in certain cultured mammalian cells ("transforming growth factor"). However, the three human TGFB isoforms that are known all potently preuent proliferation of most mammalian cells by inducing synthesisof proteins that inhibit the cell cycle. TGFB is produced by many cells in the body and inhibits growth both of the secreting cell (autocrine signaling) and neighboring (paracrine signaling) cells. Loss of TGFB receptorsor any of severalintracellular signal-transductionproteins in the TGFB pathway, thereby releasingcells from this growth inhibition, occurs frequently in human tumors. TGFB proteins also promore expression of cell-adhesionmoleculesand extracellular-matrixmolecules, which play important roles in tissue organization 668

C H A P T E R1 5

|

(Chapter 19). A Drosophila homolog of TGFB, called Dpp protein, participatesin dorsal-ventralpatterning in fly embryos. Other mammalian members of the TGFB superfamily, the activins and inhibins, affectearly developmentof the genital tract. rWe consider such developmentally important TGFb proteins in Chapter 22. Despitethe complexity of cellular effectsinduced by various members of the TGFB superfamily the signaling pathway is basicallya simple one (seeFigure 16-2a). Once activated, receptorsfor theseligands directly phosphorylate and activate a particular type of transcription factor. The responseof a given cell to this activated transcription factor dependson the constellation of other transcription factors it already contains. In this section, we will progress sequentially through the TGFB pathway, considering first the family of signal molecules,then the TGFB receptors and their discovery.Next we presentinformation about how thesereceptors activate Smad transcription factors and the feedback loops that regulate signaling by this pathway. The role that TGFB plays in cancer completesour examination of TGFPSmad signaling.

A T G F FS i g n a l i n gM o l e c u l el s F o r m e d by Cleavageof an lnactivePrecursor Most animal cell types produce and secretemembers of the TGFB superfamily in an inactive form that is stored nearby in the extracellular matrix. Releaseof the active form from the matrix by protease digestion or inactivation of an inhibitor leads to quick mobilization of the signal already in place-an important feature of many signalingpathways. In humans TGFB consistsof three isoforms, TGFB-1, TGFB-2, and TGFB-3, each encoded by a unique gene and expressedin both a tissue-specificand developmentallyregulated fashion. Each TGFB isoform is synthesizedas part of a larger dimeric precursor, linked by a disulfide bond, that contains a pro-domain (often called LAP). After the precursor is secreted,LAP is cleavedoff but remains noncovalently bound to the mature TGFB via interactions betweenspecific four amino acid sequencesin each polypeptide. Most secreted TGFB is stored in the extracellular matrix as a latent, inactive complex containing the cleaved TGFB precursor and a disulfidelinked protein called latent TGFB-binding protein (LTBP). Binding of LAP by the matrix protein thrombospondin triggers releaseof mature, active dimeric TGFP. Alternatively digestion of the binding proteins by serum proteasesor by metalloproteasespresentin the matrix 'J,6-3a). can result in activation of TGFB (Figure The monomeric form of TGFB growth factors contalns three conservedintramolecular disulfide linkages. An additional cysteine in the center of each monomer links TGFB monomers into functional homodimers and heterodimers (Figure L6-3b). Much of the sequencevariation among different TGFB proteins is observedin the N-terminal regions, the loops joining the B strands, and the o. helices.Different heterodimeric combinations may increasethe functional diversity of these proteins beyond that generated by differencesin the primary sequenceof the monomer.

C E L LS I G N A L I N Gl l : S I G N A L | N GP A T H W A Y Sr H A T c o N T R o L G E N EA c T | v r r y

( a ) F o r m a t i o no f m a t u r e ,d i m e r i c T G F B SecretedTGFBprecursor

50_375 aa____________>!_rrO_r+O aa>l

ll aql dq po.re.Lrod 11e-3un1 aql ot >ltrEgsenssnruort oprxorp uoqrpr pue senssrl ot sSunl aqr uor] uo8dxo t.rodsuB.rl

uer{16oL1s{p uraloldeuerquaLU oq}sputq}eq}uteLllop leutula} - ) e p u ep u al e u r L u r o s] -lN l u o o l t s6 u r p u r q - u ru1et es e qq ) t q M 'urqdorlsrip st umoqsoslV +zel aar++oslo^alq6rqle {ilnrDerleqt s ] l q l q u] e l q l( a l d r n de)l r s6 u r p u r q - * r eeJe ^ e qa u l o s u t l ) e - lp u t q 'suralotd oM]a^eqqltqM+o11e 6ur1ur1-ssot> ]eql (anlq)sureLuop ur]re-lrnoj selduex3 (e)'sarnl)nrlsosra^!polur sluaueltl 1o url)e-Jp;or.usurelo.rd 6u11ur;-ssolr ulpv gl-l,l lUngll V

ouerqueur eurseld

urqdorlsAO

erpodolu 'srequ ssajls ' a 6 p e6 u r p e a l

uruelrl

uulcads xeuoclloS

slu Il-ssorc uuqultl

a u r zl e l c s n u r 'erpodolr; 'srequssoJls ututlce-D

suorsaLlpe ' e r p olecoj dolr,t 'rlltnolctlA u rJq[rJrl

:uollPco'l

(e)

'l'll) srNrtAVlriou)rrA :l lN3lAlAOlA CNV NOITVZINVDUO

|

1L Ull-dvH)

OEL

'11er ()) t t ue aq16urnnoqs lerlaqlrde 1olradse ;ettdeaq] uo tllt^or)tlA 1989 E lolg ila) loN ^aA ilnleN'2002' le ]0 raq)slarg , , ,,,^,, ^-.1-^^ ,^\ .,uleuloM} aq} Outnnoqs (U) :luaurll)e]1e pueq(E) pue uuI>1ue pdloepe (p) ued ss6:Sszruat)s z66l ]]lH I v puppunl I I pue 1osed,{1 v LUor] 'uo1e1a1soyb 'gzv.lgzdnleN'6L6l'xnl eqi e1I>orq1&e paldppe (q)ued uolupjg I s L!or+ 1o u.rer6er6(q) sutalotdouerqulotl.l c loIsolrnof 'soln)alou.r 'uoluer8 eq] uuIlue ate se>1ods E 9 L 9 : 2 8 V SpnS p D V l , t e N) u d ' 5 8 6 1 C p u e s r a ^ r8f u r o r + letbelurol uu]>ads)url-ssor)q)tqnn lo 'sqnqeL{}}e (e)updl ola/) OSdEIse q)ns urolord6urplo;e>se qbnorq]^lparlpul 6uoleslodsra)iep aql so]ts]uouJq)e1]e-ouerqu.taul oJeso)ods )o (lq6!t)surelordauerquroursuer] eq1o] ])osraluro] uaosoq ue) pue utrltadslo {luteulpesodL-ltol 1o uorOattru.rseldoyb eql butyoddns ueunq ur auelquou.reLuseld 6uo1aqy se]{:orq}rfue ^lparlps)ull '(d)uortelAroqdsoqd Aq pale^rDe'uuzl poirpurlo polrp qnq-pue-e1ods eq} 6utnnoqs aq ue) luourq)elle:rllr^or)ruJ se q)ns solnpnJlsa)ellns ur ouelqLUaLU uolele>1so1{r lp)ruo)aq} ,touotlezlue6;o uoJ]lall (e) 'soueJqulau auplquroruaybotqlrfuaaq1;o qdet6ot)lr..ll euseldeqi o1{11ere1e1 s}uourelr} ur}le s)url'(^lrure+ nUl) ursoour -urxrpe.r-uuza ol sluauleltlor)ltu lo luauq)e}le lelale'l 6t-l.l lun9ll v oq] ,Loraqu.ror..u e 'uuzl (p) sluor!elr+ uri)e aqi 1o{1ue1od sluaureluurlcv

s p u a( - )

s p u a( + )

u rJzS

ouerquror.!euseld (c)

uuoqdocA;g

(q) ro; troddns aqr eprrrord stuou€]rJoJJttu'xallor IIel oql u r t r o d d n s ; o a d d r p e s e q - u r r l r a d sa q r o t u o r t r p p e u I 'ospesrpsrql esnef,uec urrd>1ue pue '1'y pueq I 'ulrlcads ur suortetntu'stuarledueunq u1 'ueds olr] ratror{s e eruoq pue (s11ar poolq por;o e8elroqsp sr ereqt asn€)aq sew soxluauptapunor are sllar eqt esneraq c4["co,Laqds) -aue crldcoraqdsd.rerrparaq s€ u.lAou>l sos€esrp ol eslr SurnrB '.(1rseaa.rnldnr tpr{t sller ur tlnsor UEJ uolala>lsoldo IIal poolq par oqr ;o suratord ur sllJtep rlteuaC 's]ler ]er]eqlrqeJo euerqruaru]erelEl -oseq erJtuo uote]e>lsotdrurlce eqt or es€dlv +)/+EN eql 'e1due s{url tuaruqrelte utrtreds-urr.,t1ue ;o addl perEIOre -xe ro{ 'saddr11erdueru ur rnrco se8e>1ur1 yo saddrrelIuIS

'ardoorqtdreaqr ur padola,tepdlq8rq sr a3e1ur1srql q8noqrly 'Geyn arn8r4) 3 ut,Loqdot{'13payet uralo.rd eue;q -uorusuerl raqtoue ol L', Puoq Pe;1r':utalo.rd Surpurq -urtf,E-Cpue urrlcads e q8norqt PUE (t puvq se u, ^ou>l osle urato.rd aueJqlueursu€JlE) JJlrodsue.rl aleuogJeJlq eql ot uttt4uo Pellel ulelord e q8no.lql-stusluel{Jalu ol PeqJElle sI uIJlfJdS o.r,rfqSnorql sutalord auEJgIUeI'U 'drrlrqrxag slI qloq ar.{rorqr^{raaqt se.tr8 qt8uerrs pue ern8rg)ernlrnrls 1o eddr leu-8ut I r o , A l t e us \ q l ' F 6 I - L 1 -qsl; e SunerauaS'salncalourur.rlcedse]qIXaUxrs lnoqe 3ut - p u r q r o J s q n q s E o l r a s s l u a I u E I IrJr o r { ss s e q f ' P u r ( - ) e q l uo urlnpouodorl uratord Surddeceqr dq Pue (9'LI uollres ur lr€lap eroru uI passnrsrp)utsodruodo.rr.{q saptsrrel{l uo pezrlrqers'ql8ue1 uI sllunqns tI lnoqe Jo sluaruell; ullre srql 'daurnoIrraql ro; dresseceu lrorls uo pes€qsI >lro,^Aleu

cell-surfacestructures such as microvilli and membrane ruffles. If we think of a microvillus, it is clear that it must have an end-on attachment at the tip and lateral attach'What ments down its length. is the orientation of actin filaments in microvilli? Decoration of microvillar filaments by the 51 fragment of myosin show that it is the (* ) end at the tip (Figure 17-19c). Moreover, when fluorescentactin is added to a cell, it is incorporatedat the tip of a microvillus, showing that not only is the (+) end there but that actin filament assemblyoccurs there. At presenrit is not known how actin filaments are attachedat the microvillus tip, but a likely candidateis a formin protein. This (+ ) end orientation of actin filaments with respectto the plasma membrane is almost universally found-not just in microvilli but also, for example,in the leadingedgeof motile cells.The lateral attachmentsto the plasma membraneare believedto be provided, at leastin part, by the ERM (ezrinradixin-moesin) family of proteins. These are regulated 'When proteins that exist in a folded, inactive form. activated, often by the membrane-boundregulatory lipid PIP2 (phosphatidylinositol(4,5)P2)and subsequentphosphorylation, F-actin and membrane-protein-bindingsites of the ERM protein are exposed to provide a lateral linkage to actin filaments (Figure 17-19d). At the plasma membrane, ERM proteins can link the actin filaments directly or indirectly through scaffolding proteins to the cytoplasmic d o m a i n o f m e m b r a n ep r o t e i n s . The two types of actin membrane linkages we have discusseddo not involve areas of the plasma membrane directly attachedto other cells or the extracellularmatrix. In contrast, highly specializedregions of the plasma membrane of epithelial cells, called adherensjunctions, make contactsbetweencells(seeFigure 1,7-1,b). Other specialized regions of association,called focal adhesions,mediate attachmentof cellsto the extracellularmatrix. Thesespecialized types of attachmentsin turn connect to the cytoskeleton and are describedin more detail when we discusscell migration (Section17.7) and cells in the context of tissues ( C h a p t e r1 9 ) . Muscular dystrophies are genetic diseases often characterizedby the progressiveweakening of skeletal muscle. One of these geneticdiseases,Duchenne muscular dystrophy, affects the protein dystrophin, whose gene is located on the X-chromosome, and so the disease is much more prevalent in males. Dystrophin is a modular protein whose function is to link the cortical actin network of muscle cells to a complex of membrane proteins that link to the extracellular matrix. Thus dystrophin has an N-terminal actin-binding domain, followed by a series of spectrinlike repeats and terminating in a domain that binds the transmembrane dystroglycan complex to the extracellular matrix protein laminin (seeFigure 17-18a). In the absenceof dystrophin, the plasma membrane of muscle cells becomesweakened by cycles of muscle contraction and eventually ruptures, resulting in death of the muscle myofibril. I

Organization of Actin-BasedCellular Structures r Actin filaments are organized by cross-linking proteins that have two F-actin-binding sites. Actin cross-linking proteins can be long or short, rigid or flexible, depending on the type of structure involved (seeFigure 1,7-1'8). r Actin filaments are attachedlaterally to the plasma membrane by specificclassesof proteins, such as are seenin the red blood cell or in cell-surfacestructuressuch as microvilli (seeFigure 17-19). r The (* ) end of actin filaments can also be attached to membranes,with assemblymediated betweenthe filament end and the membrane. r Severaldiseaseshave beentraced to defectsin the microfilament-basedcortical cytoskeleton that underlies the olasmamembrane.

Myosins:Actin-Based Motor Proteins We have discussedhow actin polymerization nucleatedby the Lrp2l3 complex can be harnessedto do work. In addition to motility, cellshave a large family actin-polymerization-based of motor proteins, called myosins,that can move along actin filaments powered by ATP hydrolysis. The first myosin discovered,myosin II, was isolated from skeletalmuscle.For a long time, biologists thought that this was the only type of myosin found in nature. However, they then discoveredother types of myosins and beganto ask how many different functional classesmight exist. Today we know that there are several different classesof myosins,in addition to the myosin II of skeletalmuscle,that provide a motor function. The other classesof myosin provide a myriad of functions, such as moving organellesand other structures around cells as well as contributing to cell migration. Indeed,with the discoveryand analysis of all these actin-basedmotors and the corresponding microtubule-basedmotors describedin the next chapter, the former relatively static view of a cell has been replaced with the realization that it is incredibly dynamic-more like an organizedbut busy freeway systemwith motors busily ferrying componentsaround. To begin to understand myosins, we first discusstheir general domain organization. Armed with this information, we explore the diversity of myosins in different organisms and describein more detail some of those that are common in eukaryotes.Myosins have the amaztngability to convert the energyreleasedby ATP hydrolysis into mechanicalwork. All myosins convert ATP hydrolysis into work, yet different myosins can perform very different types of functions. For example, many moleculesof myosin II pull together on actin filaments to bring about muscle contraction, whereas myosin V binds to vesicularcargo to transport it along actin filaments. To understand how such diversefunctions can be

MOTORPROTEINS M Y O S I N SA : CTIN-BASED

731

accommodatedby one type of motor mechanism,we will investigatethe basic mechanismof how the energyreleasedby ATP hydrolysis is convertedinto work and then seehow this mechanism is modified to tailor the properties of specific myosin classesfor their specificfunctions.

M y o s i n sH a v eH e a d ,N e c k ,a n d T a i lD o m a i n s w i t h D i s t i n c tF u n c t i o n s Much of what we know about myosins comes from studies of myosin II from skeletalmuscle.In skeletalmuscle,myosin II is assembledinto so-calledbipolar thick filaments containing hundreds of individual myosin II proteins (Figure 1.7-20a)with oppositeorientationsin eachhalf of the bipolar filament. Thesemyosin filaments interdigitate with actin thin filaments to bring about muscle contraction. We will

discusshow this systemworks in a later section,but here we investigatethe properties of the myosin itself. It is possible to dissolve the myosin thick filament in a solution of ATP and high salt. The resulting solublemyosin II protein consists of six polypeptides-two associatedheavy chains and four light chains.Two light chains associatewith the "neck" region of eachheavychain (Figure1.7-20b).The soluble myosin has ATPase activity, reflecting its ability to power movementsby hydrolysis of ATP. But which domain of myosin is responsiblefor this activity? To identify functional domains in a protein, a standard approach is to cleave it into fragments with specific proteases and ask which fragmentshave the activity. Solublemyosin II can be cleaved into two pieces by gentle treatment with the protease chymotrypsin to yield two fragments, one called heavy mero-myosin (HMM: mero means "Dart of") and the other

(a)

( c ) H e a da n d n e c kd o m a i n

( b ) M y o s i nl l Head Neck

Tail

Regulatory l i g h tc h a i n

Essential

Nucleotideb i n d i n gs i t e

H e a v yc h a i n s

Regulatory l i g h tc h a i n

FIGURE 17-20Structureof myosinll. (a)Organization of myosin ll rnfilaments isolated fromskeletal muscleMyosin ll assembles intobipolar filaments in whichthetailsformtheshaftof thefilament withheads exposed. Extraction of bipolar filaments wrthhighsaltand ATPdisassembles thefilament intoindividual myosin ll molecules (b)Myosin ll molecules (lrghtblue) consist of two identical heavy chains (green andfourlightchains andblue)Thetailof theheavy chains formsa coiled-coil to dimerize; theneckregion of eachheavy chainhas two lightchainsassociated with it. Limitedproteolytic cleavage of 732

CHAPTER 17

|

myosinll generates tailfragments-LMMand S2-and the S'1motor domain (c)Three-dimensional modelof a singleS'1headdomainshows that lt hasa curved,elongatedshapeand is bisectedby a cleft.The pocketlieson one sideof thiscleft,and the actinnucleotide-binding bindingsitelieson the othersidenearthe tip of the head Wrapped aroundthe shaftof the ct-helical neckaretwo lightchainsThesechains stiffenthe neckso that it can act as a leverarm for the head Shown hereisthe ADP-bound conformation

C E L LO R G A N I Z A T I OANN D M O V E M E N Tt : M T C R O F T L A M E N T S

llll+ Animation:In Vitro Motility MyosinAssay 51 head of myosrn

light meromyosin (LMM) (Figure 17-20b). The heavy meromyosin can be further cleavedwith the proreasepapain to yield subfragment 1 (S1) and subfragment2 (52). By analyzing the properties of the various fragments-S1, 52, and LMM-it was found that the intrinsic ATPase activity of myosin residesin the 51 fragment, as doesan F-actin-binding site.Moreover, it was found that the MPase activity of the 51 fragment was greatly enhanced by the presenceof filamentous actin, so it is said to have an actin-actiuatedATPase actiuity, which is a hallmark of all myosins.The 51 fragment of myosin II consistsof the head and neck domains, whereas the 52 and LMM regionsmake up the tail domain (Figure 1720b). X-ray crystallographic analysis of the head and neck domains revealedits shape,the positions of the light chains, and the locations of the ATP-binding and actin-binding sites. The elongatedmyosin head is attached at one end to the ahelical neck (Figure 17-20c1.Two light-chain moleculeslie at the baseof the head,wrapped around the neck like C-clamps. In this position, the light chains stiffen the neck region. How much of myosin II is necessaryand sufficient for "motor" activity? To answer this question, one needsa simple in vitro motility assay.In one such assay,the slidingfilament assay,myosin moleculesare tethered to a coverslip to which is added stabilized fluorescence-labeledactin filaments. Becausethe myosin moleculesare tethered,they cannot slide; thus any force generatedby interaction of myosin headswith actin filaments forces the filaments to move relative to the myosin (Figure17-21a).If ATP is present,added actin filaments can be seento glide along the surface of the coverslip; if ATP is absent, no filament movement is observed.Using this assay,one can show that the 51 head of myosin II is sufficient to bring about movement of actin filaments. This movement is caused by the tethered myosin 51 fragments (bound to the coverslip) trying to "move" toward the (+ ) end of a filament; thus the filaments move with

17-21 Sliding-filament assayis FIGURE < EXPERIMENTAL usedto detect myosin-poweredmovement.(a)After myosin excess of a glasscoverslip, molecules areadsorbedontothe surface the coverslip thenisplacedmyosin-side unboundmyosinisremoved; throughwhichsolutions downon a glassslideto forma chamber madevisible andstableby of actinfilaments, canflow.A solution phalloidin, isallowedto flow into with rhodamine-labeled staining (Thecoverslip fromits isshowninverted in the diagram thechamber. to seethe to makeit easier on theflow chamber orientation positions of ATBthe myosinheads of the molecules ) Inthe presence in illustrated bythe mechanism walktowardthe (+) endof filaments walkingof the myosin tailsareimmobilized, Figure17-24Because of individual Movement of thefilaments. headscauses sliding lightmicroscope. in a fluorescence filaments canbe observed (b)Thesephotographs of threeactinfilaments showthe positions (numbered recorded by video intervals 1,2, 3) at 3O-second from canbe determined Therateof filamentmovement microscopy. andS Kron] of M Footer suchrecordingslPart(b)courtesy

the (-) end leading. The rate at which myosin moves an actin filament can be determined from video camera recordings of sliding-filament assays(Figure 1'7-21'b). All myosins have a domain related to the 51 domain of myosin II, which is responsiblefor their motor activity. The tail domain doesnot contribute to motility but rather defines what is moved by the S1-relateddomain. Thus, as might be expected,the tail domains can be very different and are tailored to bind specificcargoes.

M y o s i n sM a k e U p a L a r g eF a m i l yo f Motor Proteins Mechanochemical Since all myosins have related S1-motor domains with considerable similarity in primary amino acid sequence,it is possible to determine how many myosin genes, and how many different classes of myosins, exist in a sequenced genome. There are about 40 myosin genes in the human genome (Figure 1,7-22),nine in Drosophila, and five in the budding yeast. Computer analysis of the sequencerelationships between the myosin head domains suggeststhat about 20 distinct classesof myosins have evolved in eukaryotes' with greater sequencesimilarity within a classthan between' As indicated in Figure 17-22, the geneticbasis for some diseaseshas beentraced to genesencoding myosins. All myosin head domains convert ATP hydrolysis into mechanicalwork using the samegeneralmechanism.However, as we will see, subtle differences in this mechanism can have profound effects on the functional properties of different classesof myosin. How do thesedifferent classesrelateto tail domains? Amazingly if one takes just the protein sequencesof the tail domains of the myosins and uses this information to place them in classes,they fall into the same groupings as the motor domains. This implies that motor domains with specific properties have co-evolved with specific classesof tail MOTORPROTEINS M Y O S I N SA : CTIN-BASED

733

< FIGURE 17-22The myosinsuperfamilyin humans.Computer analysis of the relatedness of S1headdomains of allof the approximately 40 myosins encoded bythe humangenomeEach myosinisindicated by a line,with the lengthof the lineindicating phylogenetic relationships distance Thusmyosins connected by short linesareclosely related, whereas thoseseparated by longerlinesare moredistantly relatedAmongthesemyosins arethreeclassesmyosins l, ll,andV-widely represented amongeukaryotes, with othershavingmorespecialized functionsIndicated areexamples in whichlossof a specific myosin causes a disease andmodified [Redrawn f r o m R E C h e n e y , 2 0 0 1M , o l B i o l C e l l1 2 . l 8 O l

domains, which makes a lot of sense,suggestingthat each classof myosin has evolved to carry out a specificfunction. In every case that has been tested so far, myosins move toward the (+ ) end of an acrin filament-with one exception, myosin VI. This remarkable myosin has an insert in its head

domain to make it work in the opposite direction, and so motility is toward the (-) end of an actin filament. Myosin VI in animal cellsis believedto contribute to endocytosisby moving the endocyticvesiclesalong actin filamentsaway from the plasma membrane.Recallthat membrane-associated actin filamentshave their (+ ) endstoward the membrane,so a motor directedtoward the (- ) end would take them away from the membranetoward the centerof the cell. Among all these different classesof myosins are three especiallywell-studied ones, which are commonly found in animals and fungi: the so-called myosin I, myosin 1l and myosin V families (Figure 17-23). In humans, eight genes '14 encode heavy chains for the myosin I family, for the myosin II family, and three for the myosin V family. The myosin II class assemblesinto bipolar filaments, which is important for its involvement in contractile functions; indeed, this is the only class of myosins involved in contractile functions. The laree number of members in this

Step size

Function

1 0 - 1 4n m

Membrane association, endocytosis

5-10nm

Contraction

Vesicle

i:)

FIGURE17-23 Three common classesof myosin. MyosinI consistof a headdomainwith a variablenumberof light chains associated with the neckdomain Membersof the myosinI classare the only myosinsto havea singleheaddomain Someof these myosinsare believedto associate directlywith membranesthrough lipid interactions. Myosinlls havetwo headdomainsand two liqht

734

CHAPTER 17

|

Organelle transport

chainsperneckandarethe onlyclass thatcanassemble intobipolar filaments. MyosinVshavetwo headdomains andsixlightchainsper (brownbox)on organelles, neckTheybindspecific receptors which theytransport, All myosins in thesethreeclasses movetowardthe (+) endof actinfilaments

C E L LO R G A N T Z A T T O A N D M O V E M E N Tt : M | C R O F | L A M E N T 5

rt !f eod."rt: MyosinMovementAgainstActin Filaments Animation:Myosin-Actin Cross'Bridge Cycle ( a ) T h i c kF i l a m e n t

Coiled-coilrod /

(b)

Myosin

Actin thin filament

Il einasntB

I

ATp

H y d r o l y s i so f ATP to ADP + P,, myosin head rotates into "cocked"state i,l:rrir:

,.llrrrll

p M y o s i nh e a db i n d s actin filament

[

"Powerstroke": Releaseof I and p 'ic elasticenergy straightensmyosin; movesactinfilamentleft

ADP-l' E ADP released, ArPr\r ATPbound;

< FfGURE17-24 The myosinhead usesATPto pull on an actin of ATBthe myosinheadisfirmlyattached filament.(a)Inthe absence in living Although thisstateisveryshort-lived to theactinfilament. in death(rigor stiffness for muscle muscle, it isthe stateresponsible fromthe mortis)Step(tr): On bindingATBthe myosinheadreleases (Z): ADP andP, the ATP to hydrolyzes The head Step actinfilament. to the neckThis a rotationin the headwith respect whichinduces aselastic "cockedstate"storesthe energyreleased by ATPhydrolysis springStep(B): Myosinin the "cocked"state likea stretched energy, headcouples bindsactinStep(4): Whenboundto actinthemyosin energyto movethe actin of the elastic of P;with release release moving the filamentThisisknownasthe "powerstroke"asit involves Step neckdomain. to theendof the myosin with respect actinfilament asADPisreleased (E): Theheadremains tightlyboundto thefilament (b) of models Molecular head. by the is bound fresh ATP andbefore in "cocking" headinvolved in the myosin changes theconformational (lowerpanel)The andduringthepowerstroke thehead(upperpanel) therestof the areshownin darkblueandgreen; lightchains myosin is redlpart(a) and actin light blue, in myosinheadandneckarecolored a d a p t e d f r o mR D V a l ea n d R A M i l l i g a n2, O O 2 , S c i e n c e 2 8 8 : 8P8a r t ( b )b a s e d o n G e e v ea s n d H o l m e s2 0 0 5 ( u n p u b l i s h e d ) l

M O T O RP R O T E I N S M Y O S I N SA : CTIN-BASED

735

classreflects the need for myosin IIs with the slightly different contractile properties seen in different muscles (e.g., skeletal, cardiac, and various types of smooth muscle) as well as in nonmusclecells. The myosin II class is the only one that assemblesinto bipolar filaments. All myosin II members have a relatively short neck domain, with two light chainsper heavychain.The myosin I class is quite large, has a variable number of light chains associatedwith the neck region, and is the only one in which heavy chains are not associatedthrough their tail domains and so are single-headed. The large sizeand diversity of the myosin I class suggeststhat these myosins perform many functions, most of which remain to be determined,but some membersof this family connectactin filamentsto membranes, and others are implicated in endocytosis.Members of the myosin V classhave two heavy chains, giving a motor with two heads,long neck regionswith six light chains each, and tail regionsthat dimerize and terminate in domains that bind to specificorganellesto be transported.

ConformationalChangesin the Myosin Head CoupleATPHydrolysisto Movement The results of studies of muscle contraction provided the first evidencethat myosin headsslideor walk along actin filaments. Unraveling the mechanism of muscle contraction was greatly aided by the development of in vitro motility assaysand singlemolecule force measurements.On the basis of information obtained with these techniques and the three-dimensional structure of the myosin head, researchersdevelopeda general model for how myosin harnessesthe energy releasedby ATP hydrolysis to move along an actin filament (Figure 1,7-24 on page735).Becauseall myosinsare thought to usethe samebasic mechanism to generatemovement, we will ignore whether the myosin tail is bound to a vesicleor is part of a thick filament, as it is in muscle.One important aspectof this model is that the hydrolysisof a singleATP moleculeis coupledto each step taken by a myosin molecule along an actin filament. A question that has intrigued biologists is how myosin can convert the chemical energy releasedby ATP hydrolysis into mechanicalwork. It has beenknown for a long time that the 51 head of myosin is an AIPase, having the ability to hydrolyze ATP into ADP and P;. Biochemical analysisreveals how this works (Figure17-24a).In the absenceof ATp, the head of myosin binds very tightly to F-actin. When ATp binds, the affinity of the head for F-actin is greatly reduced and releasesfrom actin. The myosin head then hydrolyzesthe ATP, and the hydrolysis products, ADP and P1, remain bound. The energyprovided by the hydrolysisof ATP induces a conformation change in the head that results in the head domain rotating with respectto the neck. This is known as the "cocked" positionof the head (Figure17-24b).In the absenceof F-actin, releaseof P; is exceptionallyslow, the slowest part of the ATPase cycle. However, in the presenceof actin, the head binds F-actin tightly, inducing both releaseof P; and rotation of the head back to its original position, thus moving the actin filament relative to the neck domain (Figure 17-24b).In this way, binding to F-actin inducesthe move736

.

c H A p r E R1 7 |

EXPERIMENTAL FIGURE 17-25 The lengthof the myosintl neckdomaindeterminesthe rate of movement.Totestthe leverarmmodelof myosin movement, investigators usedrecombinant DNA techniques to makemyosin headsattached to different-length neck domainsTherateat whichtheymoveon actinfilaments was determined. Thelongertheleverarm,thefasterthe myosin moves, supporting theproposed mechanism. fromK A Ruppel andJ [Redrawn A Spudich, 1996, Annu ReuCellMol.Biol.12:543-5731

ment of the head and releaseof Pi, thereby coupling the two processes.This step is known as the power stroke. The head remains bound until the ADP leavesand a fresh ATP binds the head, releasingit from the filament. The cycle then repeats,and the myosin can move again againstthe filament. How is hydrolysis of ATP in the nucleotide-binding pocket converted into force? The results of structural studies of myosin in the presenceof nucleotides, and nucleotide analogs that mimic the various steps in the cycle, indicate that the binding and hydrolysis of a nucleoride cause a small conformational change in the head domain. This small movement is amplified by a "converter" region at the base of the head acting like a fulcrum and causing the leverlike neck to rotate. This rotation is amplified by the rodlike lever arm, which constitutesthe neck domain, so the actin filament moves by a few nanometers(Figure 17-24b). This model makes a strong prediction: the distance a myosin head moves actin during hydrolysis of one ATP-the myosin step size-should be proportional to the length of the neck domain. To test this, mutant myosin moleculeswere constructedwith different-lengthneck domains and the rate at which they moved down an actin filament was determined. Remarkably there is an excellentcorrespondence betweenthe length of the neck domain and the rate of movement (Figure 17-25).

Myosin HeadsTakeDiscreteStepsAlong Actin Filaments The most critical feature of myosin is its ability ro generarea force that powers movements.Researchershave usedoptical traps to measure the forces generated by single myosin molecules (Figure 17-26). In this approach, myosin is

C E L Lo R G A N r z A T r oAN N D M o v E M E N rT: M T c R o F T L A M E N T S

immobilized on beadsat a low density.An actin filament, held between two optical traps, is lowered toward the bead until it contacts the myosin molecule. When ATP is added, the myosin pulls on the actin filament. Using a mechanicalfeedback mechanismcontrolled by a computer, one can measure the distancepulled and the forces and duration of the movement (Figure 17-26). The resultsof optical trap studiesshow that myosin II does not interact with the actin filament continuously but rather binds, moves, and releasesit. In fact, myosin II spendson averageonly about 10 percent of each ATPase cycle in contact with F-actin-it is said to have a duty ratio of 10 percent. This will be important later when we consider that in muscle, hundreds of myosin heads pull on actin filaments, so that at any one time, 10 percent of the headsare engagedto provide a smooth contraction. 'When myosin II does contact F-actin, it takes discrete steps,approximately5-15 nm long (Figure1.7-27),and generates 3-5 piconewtons (pN) of force, approximately the same force as that exerted by gravity on a single bacterium. If we now look at a similar optical trap experiment with myosin V, the curves look completely different (Figure 1,7-27).Now we can easilydiscernclear stepsof about 35 nm in length. This larger step sizereflectsthe longer neck domain-the lever arm-of myosin V. Moreover, we seethat the motor takes many sequentialsteps without releasing from the actin-it is said to move processiuely.This is because its ATPase cycle is modified to have a much higher duty ratio (>70 percent)by slowing the rate of ADP release; thus the head remains in contact with the actin filament for a much larger percentageof the cycle. Sincea single myosin V moleculehas two heads,a duty ratio of )50 percentensures that one head is in contact at all times as it moves down an actin filament so that it does not fall off.

^20 5 q)

q

oO

0.5 T i m e( s ) 252 216

E r eo E .oo c o

.9 108 o 72 JO

o

0.2

0.6 0.4 T i m e( s )

0.8

17-27Measuringthe stepsizeand FIGURE EXPERIMENTAL to the Using an opticaltrapsetupsimilar processivity of myosins. the analyzed have investigators 1 7-26, in Figure described one As V (bottomtrace). of myosinll (toptrace)andmyosin behavior shownbythe peaksin thetrace,myosinll takeserraticsmallsteps andthen moves, (5-15 nm),whichmeansit bindstheactinfilament, V myosin By contrast, motor. nonprocessive a letsgo lt istherefore takesclear36-nmstepsoneaftertheother,so it hasa stepsizeof is,it doesnot let 90 of the 36 nm andis highlyprocessive-that (b)from Part (a)fromFiner 368:113 etal,1994,Nature actinfilamentIPart Acad Sci.USA97:94821 ProcNat'l M Riefetal,20OO,

M y o s i nV W a l k sH a n d O v e r H a n d D o w n an Actin Filament

17-25Opticaltrap determinesthe stepsizeand A FIGURE forcegeneratedby a singlemyosinmolecule.In an opticaltrap, on a laserisfocused by a lightmicroscope the beamof an infrared light), Iatexbead(oranyotherobjectthatdoesnot absorbinfrared andholdsthe beadin thecenterof the beamThe whichcaptures or by increasing of theforceholdingthe beadisadjusted strength an the rntensity of the laserbeam ln thisexperiment, decreasing two ootrcal trapsTheactinfilamentis actinfilamentisheldbetween of thenlowered ontoanotherbeadwith a diluteconcentration a myosinmolecules attached to it lf the actinfilamentencounters will pullon the in the presence of ATBthe myostn myosinmolecule to measure boththe whichallowsthe investigators actinfilament, takes forcegenerated andthestepsizethe myosin

The next question is, how do the two heads of myosin V work together to move down a filament? One model proposes that the two heads walk down a filament hand over hand with alternately leading heads (Figure'J-7-28a). An alternative possibility is an inchworm model, in which the leading head takes a step' the second head is pulled up behind it, and then the leading head takes another step (Figure 1,7-28b).How can one distinguish between these models? In the inchworm model, each individual head takes 36-nm steps,whereasin the walking model' eachtakes 72-nm steps.Scientistshave managed to attach a fluorescentprobe to just one neck region of myosin V and watch it walk down an actin filament: it takes 72-nm steps (Figure \7-28c), and so it walks hand over hand down a filament (72 nm is the MOTORPROTEINS M Y O S I N SA : CTIN-BASED

737

(a) Hand over hand

the properties one would expect for a motor designed to transport cargo along an actin filament.

on neck

Myosins: Actin-Based Motor Proteins r Myosins are actin-based motors powered by ATP hydrolysis. r Myosins have a motor head domain, a lever-arm neck domain, and a cargo-binding tail domain (seeFigure 17-20).

(b) Inchworm

r There are many classesof myosin, with three classes present in many eukaryotes: myosin I has a single head domain, myosin II has two headsand assemblesinto bipolar filaments, and myosin V has two headsand doesnot assemblein filaments (seeFigure 17-23).

Labelon n

r Myosins convert ATP hydrolysis to mechanicalwork by amplifying a small conformational change in their head through their neck domain when the head is bound to F-actin (seeFigure 1,7-24). (c)

r Myosin heads take discrete steps along an actin filament, which can be small (5-15 nm) and nonprocessivein the case of myosin II or large (36 nm) and processivefor myosin V.

Myosin-PoweredMovements

E 500 o

o

L

0102030405060 T i m e( s ) A EXPERIMENTAT FTGURE 17-28MyosinV hasa stepsizeof 36 nm, yet eachhead movesin 72-nmsteps,so it moveshand over hand.Twomodelsfor myosinV movement downa filament havebeensuggested. (a)Inthe hand-over-hand model,onehead bindsan actinfilament, andtheotherthenswingsaroundandbinds a site72 nm ahead.(b)In the inchworm model,the leading head moves36 nm,thenthe laggingheadmovesup behindit, allowing the leading headto takeanother36-nmstep.(c)MyosinV labeled with a fluorescent tag on justone headcanbe seento havea step sizeof 72 nm.Thusmyosrn V walkshandoverhand.[Adapted fromA Yildiz et al, 2003,Science 300:2061 l step size for each head; the step size for the double-headed motor is 35 nm). IThy is the step size of myosin V so large? If we compare its step size of 35 nm to the structure of ihe actin filament, we see that it is the same as the length between helical repeats in the filament (seeFigures 17-5 and 17-28a), so myosin V stepsbetweenequivalent binding sites as it walks down one side of an acin filament. Myosin V has presumably evolved to take large steps the size of the helical repeat of actin and to do this very processivelyso it rarely dissociatesfrom an actin filament. These are exactly 738

C H A P T E Rl 7

|

We have akeady discussedhow myosins have head and neck 'We domains responsible for their motor properties. now come to the tail regions, which define the cargoes that myosins move. The function of many of the newly discovered classes of myosins found in metazoans is not yet known. Below, we give just two examples where we have a good idea of what myosins do. Our first example is skeletal muscle,which is where myosin II was discovered.In muscle, many myosin II heads joined together in bipolar filaments, each with a short duty cycle, work together to bring about contraction. Similarly organized contractile machineries function in the contraction of smooth muscle and in stress fibers and the contractile ring during cytokinesis. We then turn to the myosin V class, which has a long duty cycle to allow thesemyosins to transport cargoesover relatively long distanceswithout dissociatingfrom actin filaments.

Myosin Thick Filamentsand Actin Thin Filamentsin SkeletalMuscleSlidePastOne Another During Contraction Muscle cells have evolved to carry out one highly specialized function-contraction. Muscle contractions must occur quickly and repetitivelg and they must occur through long distancesand with enough force to move large loads. A typical skeletal muscle cell is cylindrical, large (140 mm in length and 10-50 trr,min width), and multinucleated (containing as many as 100 nuclei) (Figure 17-29a). Within the muscle cell are many myofibrils consisting of a regular repeating array of a specializedstructure called a sarcomere (Figure 77-Z9b). A

C E L LO R G A N T Z A T T OANN D M O V E M E N Tl : M T C R O F T L A M E N T S

17-29 Structureof the skeletalmusclesarcomere. < FIGURE of fibersmadeof bundles (a)Skeletal of muscle muscles consist which a bundleof myofibrils, cells.Eachcellcontains multinucleated called structures contractile of repeating of thousands consist in muscle (b)Electron of mousestriated micrograph sarcomeres. of theZ either side On sarcomere. one showing longitudinal section, of actinthin entirely I bands,composed disksarethe lightlystained extendfrombothsidesof theZ disk thinfilaments filamentsThese in theA thickfilaments myosin with thedark-stained to interdigitate filaments actin myosin and (c) of of the arrangement band. Diagram (b)courtesy of S P Dadoune ] in a sarcomere [Part

Muscles

B u n d l eo f m u s c l ef i b e r s

Multinucleated m u s c l ec e l l

Myofibril

Nuclei

Sarcomere

The thick filaments are composedof myosin II bipolar filaments, in which the heads on each half of the filament have opposite orientations(seeFigure 1'7-20a).The thin actin filaments are assembledwith their ( + ) endsembeddedin a densely staining structure known as the Z disk (Figute 1'7-29b),so that the rwo setsof actin filaments in a sarcomerehave opposite orientations (Figure 17-30). To understand how a muscle contracts, consider the interactions between one myosin head (among the hundreds in a thick filament) and a thin (actin) filament, as diagrammed in Figure 1'7-24'Duting these cyclical interactions, also called the cross-bridgecycle,the hydrolysis of ATP is coupled to the movement of a myosin head toward the Z disk, which correspondsto the (+ ) end of the thin filament. Becausethe thick filament is bipolar, the action of the myosin heads at opposite ends of the thick filament draws the thin filaments toward the center of the thick filament and therefore toward the center of the sarcomere(Figure 17-30). This movement shortens the sarcomereuntil the ends of the thick filaments abut the Z disk. Contraction of an intact muscle results from the activity of hundreds of myosin headson a single Relaxed Actin

i _ (c)

Z disk | band ----)(-

A band --------------->letu r{Jlr{r1\ ,suorsr,rrp IIal 000'00I d.rale satuosoruol.lJ9I s]l to auo sate8er8as -sttu dyuo lsead 1eq1 petErurlsa sl rI 'suorlEcrldtuoc

(e)Uedl I uupur]]rM 1lo {sapnotslldplbollur solnqnlor)tur oloq)olaur)oq] ol sluoulqleueso)eLUq)tqM ,sullo] 0ror.])ol0ur) oq] alaqMe]rseq] oslest alaulollua)aqf olauolluo) 0ql polle)uot6atpeltulsuo) e ]e sursaqo)Iq laqlo6o] ploq '(xaldnpypq paletrldet el6ulse q]lM q)eo)sptleLuolqlrelsrsonn] seq auJosoulotqtpeletr;dnpeql stso]tulut aulosouJolqlpesuapuo) e +o s].led(q) uotldu)sop+o osearol seOelsolut papt^tpuaoq Ildtrrs seq pue'sse>o.rd snonutluo) 1r e st slso]ttyulaq] ul rn))o ]eq] sluonaoLl]pue se6e1s aq] Moqs suer6erp ]uole++tp ramol utlnqn] ro1ueetbpue VNC lo] anlq poutelssllo)Z;11d parn]lnl ur se6e1s nnoqsslauedroddn (p) .stsoltru;o se6e1sorll t€-gl lunglJ V

e 1 e ; da s e q d e l a u t aql le pau6r;e saulosoutolqO

sprleuorqcrolsts

uorlesuapuocoulosoulolqc 'uorleJedaslalse cllot!l l 'srolsecrloilr.!Aq luoLuoceldel s1rpue Ie;dsrp olnqnlorctt! aseqdlolutlo uA op)eoJg

uorlectlonpeurosoJluas ' u o t s o q o cp u e uorlecrldnp olrlosotuolLIC

aseqdord

lllll>

aseqdlalul

s!solrll :uolleturuv

APC/Cactivatedand c o h e s i n sd e g r a d e d AnaphaseA: Chromosome movementto poles A n a o h a s eB : S p i n d l ep o l e s e p a r a t i o n

Nuclearenvelopereassembly, Assemblyof contractilering

attachedby cohesins(Figure 18-34b). As discussedin more detail in Chapter 20, all theseeventsare coordinated by a rapid increasein the activity of the mitotic cyclin-CDK complex, which is a kinase that phosphorylatesmultiple protelns. The next stageof mitosis,prometaphase,is initiated by the breakdown of the nuclear envelope and the nuclear pores and disassemblyof the lamin-basednuclear lamina. Microtubules assembledfrom the spindle poles searchand "capture" chromosome pairs at specialized structures called kinetochores.Each chromatid has a kinetochore,so sister chromatids have two kinetochores, each of which becomes attached to opposite spindle poles during prometaphase. lfhen they are attached to both spindle poles, sister chromatids align equidistant from the two Prometaphase spindlepolesin a processknown as congression. continuesuntil all chromosomeshave congressed,at which point the cell entersthe next stage,metaphase,defined as the stage when all the chromosomes are aligned at the metaphaseplate. The next stage, anaphase,is induced by activation of t h e a n a p h a s e - p r o m o t i n gc o m p l e x / c y c l o s o m e( A P C / C ) . The activated APC/C leads to the destruction of the cohesins,proteins holding the sister chromatids together,so that now each separatedchromosome can be pulled to its respectivepole by the microtubules attachedto its kinetochore. This movement is known as anaphaseA. A separate

Reformationof interphasemicrotubulearray, Contractilering forms cleavagefurrow

and distinct movement also occurs: the movement of the spindle poles farther apart in a process known as inapbase B. Now that the chromosomeshave separated' the cell enters telophase,when the nuclear envelope reforms, the chromosomes decondense, and the cell is pinched into two daughter cells by the contractile ring during cytokinesis.

C e n t r o s o m eD s u p l i c a t eE a r l yi n t h e C e l lC y c l ei n Preparationfor Mitosis In order to separatethe chromosomesat mitosis, cells duplicate their MTOCs-their centrosomes-coordinately with the duplication of their chromosomesin S phase(Figure 18-35). The duplicated centrosomesseparateand become the two MTOCs-the spindle poles-of the mitotic spindle.The number of centrosomesin animal cells has to bi .'..y carefully controlled. In fact, many tumor cells have more than two centrosomes,which contribute to genetic instability resulting in missegregationof chromosomes a.td hence aneuploidy (unequal numbers of chromosomes). As cells enter mitosis, the activity of the two MTOCstheir ability to nucleate microtubules-increases greatly as they accumulate more pericentriolar material. Becausethe microtubules radiating from thesetwo MTOCs now resemble stars, they are often called mitotic asters.

783

FIGURE 18-35Relationof centrosome duplicationto the cell cycle.Afterthe pairof parentcentrioles (green) separates slightly, a d a u g h t ecre n t r i o (l eb l u eb) u d sf r o me a c ha n de l o n g a t eBsyG r , growthof the daughter centrioles iscomplete, but thetwo pairs r e m a iw n i t h i na s i n g l cee n t r o s o mcaolm p l e xE a r l iyn m i t o s i st h, e centrosome splits, andeachcentriole pairmigrates to opposite sides of the nucleusTheamountof pericentriolar material andthe activity to nucleate microtubule assembly increases greatlyin mitosisIn m i t o s i st h, e s eM T O Casr ec a l l e sd p i n d loeo l e s

astral microtubules, which extend from the spindle poles to the cell cortex (Figure 18-36). By interactingwith the cortex, the astral microtubulesperform the critical function of orienting the spindle with the axis of cell division. The secondset link the spindlepolesto the kinetochoreson the chromosomesand are therefore called kinetochore microtubules. This set of microtubules first finds the chromosomes, then attaches them through the two kinetochoresto both spindle poles and at anaphaseA transportsthem to the poles.The third set of microtubulesextendfrom eachspindlepole body toward the oppositeone and interacttogetherin an antiparallelmanner;these are calledpolar miuotubules.Thesemicrotubulesare responsible initially for pushing the duplicatedcentrosomesapart during prophase,then for maintaining the srructure of the spindle, and then for pushingthe spindlepolesapart in anaphaseB. Note that all the microtubules in each half of the symmetrical spindle have the same orientation except for some polar microtubules, which extend beyond the midpoint and interdigitate with polar microtubules from the opposite pole.

M i c r o t u b u l eD y n a m i c sI n c r e a s eD s r a m a t i c a l l yi n Mitosis

Beforewe discussthe mechanismsinvolved in this remarkable process,it is important to understandthe three distinct classes of microtubulesthat emanarefrom the spindlepoles,which is where all their (-) ends are embedded.The first classis the

Although we have drawn static imagesof the stagesof mitosis, microtubules in all stagesof mitosis are highly dynamic. As we saw above,as cellsentermitosis,the ability of their centrosomesto nucleate assembly of microtubules i n c r e a s e ss i g n i f i c a n t l y ( s e e F i g u r e 1 8 - 3 5 ) . I n a d d i t i o n , microtubulesbecomemuch more dynamic. How was this determined? In principle, you could watch microtubules and

(a)

(b)

T h e M i t o t i c S p i n d l eC o n t a i n sT h r e eC l a s s e os f Microtubules

Zone of interdigitation 'l rKinetochoreMT Kinetochore

Pole (centrosome) Polar MTs

A FIGURE18-36 Mitotic spindles have three distinct classesof microtubules.(a) In this high-voltage electronmicrograph, m i c r o t u b u l ewse r es t a i n e dw i t h b i o t i n - t a g g eadn t i - t u b u l iann t i b o d i e s to increase their size The largecylindrical objectsare chromosomes, (b) Schematic diagramcorresponding to the metaphasecellin (a) Threesetsof microtubules (MTs)make up the mitoticapparatusAll

784

CHAPTER 18

|

Chromosome

the microtubules havetheir(-) endsat the poles. Astralmicrotubules projecttowardthe cortexandarelinkedto it Kinetochore polarmicrotubules microtubules areconnected to chromosomes prolect towardthe cellcenterwith theirdistal(+) endsoverlapping Thespindle poleandassociated microtubules isalsoknownasa (a)courtesy mrtotrc aster[Part ofJ R Mclntosh I

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D T N T E R M E D T AFTt E LAMENTs

follow their individual behaviors.However, in general,there are too many microtubules in a mitotic spindle to do this. To get an averagevalue for how dynamic microtubules are, researchershave introduced fluorescent labeled tubulin into cells, which becomesincorporated randomly into all microtubules. They then bleach the fluorescentlabel in a small region of the mitotic spindle and measurethe rate at which fluorescencecomes back in a technique known as fluorescence recouery after photobleaching (FRAP) (see Figure 1,0-1,2). Sincethe recovery of fluorescenceis due to assemblyof new microtubules from soluble fluorescent tubulin dimers. this representsthe averagerate at which microtubules turn over. In a mitotic spindle,their half-life is about 15 seconds,whereas in an interphasecell, it is about 5 minutes.It should be noted that theseare bulk measurementsand individual populations of microtubulescan be more stable.as we will see. What makes microtubulesmore dynamic in mitosis?Dynamic instability is a measure of relative contributions of growth rates, shrinkage rates, catastrophes, and rescues. Analysis of microtubule dynamicsin vivo shows that the enhanced dynamics of individual microtubules in mitosis is mostly generatedby increasedcatastrophesand fewer rescues, with little change in rates of growth (i.e., lengthening) or shrinkage (i.e., shortening).Studieswith extracts from frog oocytes have suggestedthat the main factor enhancing catastrophes in both interphaseand mitotic extractsis depolymerizing by kinesin-13 proteins. This can be seenin an in vitro

T u b u l i n+ k in e s i n - 31

T u b u l i na l o n e

assaywhere microtubule assemblyfrom pure tubulin is nucleated from purified centrosomes(Figure 1'8-37a).If kinesin-13 is added into the assay,many fewer microtubules are formed. However, if the stabilizing microtubule-associatedprotein called XMAP215 is added with the kinesin-13,many microtubules are formed due to a dramatic reduction in catastrophe frequency.It turns out that the activity of kinesin-13 does not change significantly during the cell cycle, whereas the activity of XMAP215 is inhibited by its phosphorylationduring mitosis (Figure 1,8-37b).This results in much more unstable microtubules as the cell enters mitosis (Figure 1'8-37c).

l u r i n gM i t o s i s M i c r o t u b u l e sT r e a d m i lD In addition to being highly dynamic in terms of assemblyand disassembly microtubules in the mitotic spindle are treadmilling-that is, constantly adding dimers at the microtubule (+ ) end and losingthem at the (- ) end.Treadmillingcan be revealed by expressingsmall amounts of GFP-tubulin in mitotic cells, which is incorporated randomly into microtubules, giving rise to fluorescent speckleswhere the concentration of incorporated GFP-tubulin happens to be higher. These speckles orovide a marker on the microtubules, so by following them in liuing cell, one can determine if the microtubules are station" with respectto spindlepolesor moving (Figure18-38).Exary periments such as theseshow that microtubules are constantly treadmilling in prometaphase,metaphase'and anaphase.

T u b u l i n+ k i n e sni - 13 + XMAP215

(b)

.2 o^

O^

:t

ro

Tl

N (L

' c6l 0)

X

)Z

18-37 FIGURE < EXPERIMENTAT in mitosis Microtubuledynamicsincreases due to lossof a stabilizingMAP.(a)These theabilityof centrosomes reveal threepanels frompuretubulin microtubules to assemble protein (/eft);tubulinandthe destabilizing ( m i d d l e ) : k i n o r t u b u l i n , e s i n - 1a3n, d kinesin-13 protein 15 (Xenopus XMAP2 the stabilizing shows analysis Further MAPof 215kD)(ngrht). 5 isto that the majoreffectof XMAP21 3 induced by kinesin-1 catastrophes suppress in (b)Theincreased of microtubules dynamics 5 of XMAP21 isdueto the inactivation mitosis (c)Diagram the relating by phosphorylation in of microtubules stabilities dfferent Notethatin addition andmitosis. interphase and betweeninterphase stability to differential lo nucleate the abilityof MTOCs mitosis, in dramatically alsoincreases microtubules (a)fromKinoshita (seeFigure18-35). mitosis [Part Part(b)from et al, 2001,Science2g4:]340-1343 CellBiol12t267-2731 et al. 2002,Trends Kinoshita

Time (c) 0)

:>

+ .=o

Stable

Unstable

Stable MtTOSIS

785

J

o20

o i o

tn

q)

= Zca

oo' 1 0p m

0.2 0.4 0.6 0.8 1.0 Fluxvelocity(pm/min)

1.2

E X P E R I M E N TFA TLG U R1E 8 - 3 8M i c r o t u b u l ei sn m i t o s i s treadmilltoward the spindlepoles.(a)A smallamountof GFpptKl cellsto visualize tubulinwasexpressed in cultured microtubules "speckles" andgenerate alongthemdueto randomuneven incorporation (b)Byfollowingspeckles of GFP-tubulin in time,the

direction andrateof movement of microtubules canbe determined, color-coded according to the rateshownin (c) Thisanalysis shows t h a tm i c r o t u b u lter es a d m i(lol r" f l u x " )i n t h e s ec e l l sa t a b o u 0 t 7 pm/mintowardthe poles[From L A Cameron, 2006, J Cett Biol 173: 173-179 l

T h e K i n e t o c h o r eC a p t u r e sa n d H e l p sT r a n s p o r t Chromosomes

kinetochore layers,with the (+ ) ends of the kinetochore microtubules terminating in the outer layer (Figure 18-39). Yeast kinetochores are attached by a single microtubule to their pole, human kinetochores are attached by about 30, and plant chromosomesby hundreds. How doesa kinetochorebecomeattachedto microtubules in prometaphase?Microtubules nucleated from the spindle poles are very dynamic, and when they contact the kinetochore, either laterally or at their end, this can lead to chromosomal attachment (Figure 1.8-40a, steps IE and IIil). Microtubules "captured" by kinetochores are selectively

To attach to microtubules, each chromosome has a specialized structure called a kinetochore. It is located at the centromere,a constricted region of the condensedchromosome defined by centromeric DNA. Cenrromeric DNA can vary enormouslyin size;in budding yeastit is about 125 bp, whereasin humans it is on the order of 1 Mbp. Kinetochoris contarn many protein complexesto link the centromeric DNA eventually to microtubules. In animal cells,the kinetochore consists of an inner centromere and inner and outer

J o q)

-a) u

Microtubules

FIGURE 18-39Thestructureof a mammaliankinetochore. Diagram andelectron micrograph of a mammalian kinetochore 786

CHAPTER 18

|

[Modified from B McEwenet al , i 998, Chromosoma107i366:courtesvof B M c E w e nl

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D T N T E R M E D T AFTt E LAMENTs

stabilizedby reducingthe levelof catastrophes, therebypromoting the chancethat the attachment will persist. Recent studies have uncovered a mechanism involving the small Ran GTPasethat enhancesthe chancethat microtubules will encounter kinetochores.Recall that the Ran GTPasecycle is involved in transport of proteins in and out of the nucleusthrough nuclearpores (Chapter13; seeFigure 1.3-36).During mitosis, when the nuclear membrane and pores have disassembled,an exchangefactor for the Ran GTPase is bound to chromosomes,thereby generating a higher local concentration of Ran-GTP. Becausethe enzyme that stimulates GTP hydrolysis on Ran-Ran GAP-is evenly distributed in the cytosol, this generatesa gradient of Ran-GTP centeredon the chromosomes.Ran-GTP induces

the release of factors that promote the growth of microtubules, in this way biasing growth of microtubules nucleated from spindle poles toward chromosomes. Once attached to microtubules, the motor protein dynein/dynactin located at the kinetochore moves the chromosome pair down the microtubule toward the spindle pole (Figure 18-40a, step Z). In this orientation, the unoccupied kinetochore on the opposite side is pointing toward the distal spindle pole, and eventually a microtubule from the distal pole will capture the free kinetochore. The chromosome pair is now said to be bi-oriented (Figwe 78-40a, step B).\7ith the two kinetochores attached to opposite poles, the duplicated chromosome is now under tension, being pulled in both directions.

Kinesin-4

5& Tethereddynactin-dynein complex Attachmentby kinesin-7; m i c r o t u b u l ea s s e m b l y

Kinesin-13

+

-> Chromosome movement

FIGURE18-40 Chromosome Gaptureand congression in prometaphase. (a) In the first stageof prometaphase, chromosomes becomeattached,eitherto the end of a microtubule(IE) or to the sideof a microtubule(IE) The duplicatedchromosomeisthen drawn as dynein/dynactin toward the spindlepole by kinetochore-associated (E). Eventually, this motor movestowardthe (-) end of a microtubule a microtubulefrom the opoositeoolefindsand becomesattached is now saidto be biand the chromosome to the free kinetochore. then moveto a central oriented(!) The bi-orientedchromosomes point betweenthe spindlepolesin a processknown as chromosome congressionNote that duringthesesteps,chromosomearmspoint

Forcefrom dynein and m i c r o t u b u l e ds e p o l y m e r i z a t i o n b y k i n e s i n - 1 3a,n d b Y k i n e s i n - 4 o n c n r o m o s o m ea r m s

pole:thisisdueto chromokinesin/kinesinspindle awayfromtheclosest towardthe(+) endsof the armsmoving on thechromosome 4 motors of (b)Congressron oscillations bidirectional involves polarmicrotubules on shortening microtubules with onesetof kinetochore chromosomes, on theother.Ontheshortening onesideandtheothersetlengthening anda protein disassembly microtubule stimulates 3 side.a kinesin-'1 towardthe pole On movesthechromosome complex dynein/dynactin protein a CENP-E/kinesin-7 microtubules, thesidewithlengthening alsocontains Thekinetochore holdson to thegrowingmicrotubule from notshownherelModified proteincomplexes manyadditional Clevelandet al , Ce//112:407-421 l MtTOSTS

787

This tensionis a key part of the mechanismwhereby a cell properly segregates its chromosomes.'Whenthe chromosome is correctly bi-oriented, the cell sensesthe tension produced, and the attachments are stabilized. What happens if both kinetochoresaccidentallybecomeattachedto the samepole? In this situation, there is very little tensionon the kinetochore microtubules, and turnover of kinetochore microtubules is enhanced.Exactly how the cell sensestension is unclear.

D u p l i c a t e dC h r o m o s o m eAs r e A l i g n e db y M o t o r s a n d T r e a d m i l l i n gM i c r o t u b u l e s During prometaphase,the chromosomescome to lie at the midpoint betweenthe two spindlepoles,in a processknown as chromosome congression.During this process,chromosome pairs often oscillatebackward and forward before arriving at the midpoint. Chromosomecongressioninvolves the coordinated activity of severalmicrotubule-basedmotors together with microtubule treadmilling (Figure18-40b).The oscillating

behavior involves lengthening of microtubules attached to one kinetochoreand shorteningmicrotubulesattachedto the other kinetochore, all without losing their attachments.In metazoans, several microtubule-basedmotors contribute to this process.First, dynein/dynactin provides the strongest force pulling the chromosome pair toward the more distant pole. This movementrequiressimultaneousshorteningof the microtubule, which is enhancedby kinetochore-localized kinesin-13. The microtubules associatedwith the other kinetochore have to grow as the chromosomemoves.Anchored at this kinetochore is a kinesin-relatedmotor. kinesin-7.that holds on to the growing (+ ) end of the lengtheningmicrotubule.Contributing to congressionis also another kinesin, kinesin-4, associated with the chromosomearms.Kinesin-4,a (+l end-directedmotor, interactswith the polar microtubulesto pull the chromosomes toward the center of the spindle. rWhen the chromosomes have congressed to the metaphase plate, dynein/ dynactin is releasedfrom the kinetochores and streams down the kinetochore microtubules to the poles. These different

';Hn"nnun"

(-)

+-q, Sliding

--+

A FIGURE 18-41Chromosome movementand spindlepole separationin anaphase. Anaphase A movement ispowered by microtubule-shortening kinesin-13 proteins (E[) at the kinetochore a n da t t h es p i n d l p e o l e( E E ) N o t et h a tt h ec h r o m o s o maer m ss t i l l pointawayfromthespindle polesdueto associated chromokinesin/ kinesin-4 members, sothe depolymerization forcehasto be able 788

CHAPTER 18

|

to overcome theforcepullingthe armstowardthecenterof the s p i n d l eA n a p h a sBea l s oh a st w o c o m p o n e n tssl i:d i n o gf a n t i p a r a l l e l polarmicrotubules poweredby a kinesin-5 (+) end-directed motor (E[) andby dynein/dynactin located at thecellcortexpullingon (EE). [Modif astralmicrotubules iedfromCleveland erat, Cett112:40j421 I

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D T N T E R M E D T AFTt E LAMENTs

activities and opposing forces work together to bring all the chromosomes to the metaphaseplate, and when successfully there,the cell is ready for anaphase.As we discussin Chapter 20, the cell has a mechanism-the spindle checkpoint-to ensurethat anaphasedoesnot proceeduntil all the chromosomes have arrived at the metaphaseplate.

AnaphaseA Moves Chromosomes to Polesby M i c r o t u b ul e S h o r t e n i n g The onset of anaphaseA is one of the most dramatic movements that can be observedin the light microscope.SuddenlS the two sister chromatids separatefrom each other and are drawn to their respectivepoles.This appearsto work so fast becausethe kinetochore microtubules are under tension, and as soon as the cohesin attachments between chromosomes are released,the chromosomesare free to move. Experimentswith isolatedmetaphasechromosomeshave shown that anaphaseA movement can be powered by microtubule shortening,utilizing the stored structural strain releasedby removing the GTP cap. This can be nicely demonstrated in vitro. lfhen metaphasechromosomesare added to purified microtubules,they bind preferentiallyto the (+) ends of the microtubules. Dilution of the mixture to reduce the concentration of free tubulin dimers results in the movement of the chromosomestoward the (-) ends by microtubule depolymerizationat the chromosome-bound(+ ) end. Recent experiments have shown that in Drosophila two kinesin-13proteins, a classof microtubule depolymerizing proteins (see Figure 1,8-1,6),contribute to chromosome movementin anaphaseA. One of the kinesin-13proteinsis localized at the kinetochore and enhancesdisassemblythere (Figure 1.8-41,E[), and the other is localizedat the spindle pole, enhancingdepolymerizationthere (Figure18-41, Eg). Thus, at least in the fly, anaphaseA is powered in part by kinesin-13proteins specificallylocalizedat the kinetochore and spindle pole to shorten the kinetochore microtubules at both their (+ ) and (- ) endsto draw the chromosomesto the poles.

AnaphaseB SeparatesPolesby the Combined A c t i o n o f K i n e s i n sa n d D y n e i n The second part of anaphaseinvolves separationof the spindle poles in a processknown as anaphaseB' A maior contributor to this movement is the involvement of the bipolar kinesin-5proteins(Figure1.8-41,EE). Thesemotors associate with the overlapping polar microtubules, and since they are (+) end-directed motors, they push the poles apart. While this is happening, the polar microtubules have to grow to accommodate the increased distance between the spindle poles-at the same time as the kinetochore microtubules are shortening for anaphaseA! Another motor-the microtubule (- ) end-directed motor cytoplasmic dynein, localized and anchored on the cell cortex-pulls on the astral microtubules and thus helps separatethe spindle poles (Figure 18-41.,8f|).

s o n t r i b u t et o S p i n d l e A d d i t i o n a lM e c h a n i s m C Formation There are a number of casesin vivo where spindles form in the absenceof centrosomes.This implies that nucleationof microtubulesfrom centrosomesis not the only way a spindle can form. Studies exploiting mitotic extracts from frog eggs-extracts that do not contain centrosomes-show that the addition of beadscovered with DNA is sufficient to assemblea relativelynormal mitotic spindle (Figure 1842\. In this system, the beads recruit preformed microtubules, and factors in the extract cooperate to make a spindle. One of the factors necessaryfor this reaction is cytoplasmic dynein, which is proposedto bind to two microtubules and migrate to their (-) ends and thereby draw them together.

CytokinesisSplitsthe DuplicatedCell in Two During late anaphaseand telophasein animal cells, the cell assemblesa microfilament-basedcontractile ring attachedto

Add fluorescent t u b u l i na n d DNA-covered beads

Xenopus egg extracts

FIGURE 18-42Mitoticspindlescanform in EXPERIMENTAL canbe extracts the absenceof centrosomes.Centrosome-free eggsto in mitosis by centrifuging isolated fromfrogoocytes arrested material fromtheorganelles andyolk When separate a soluble together labeled tubulin(green) isaddedto extracts fluorescently

spontaneously with DNA(red),mitoticspindles with beadscovered microtubules nucleated formaroundthe beadsfromrandomly lModified from Kinoshitaet al , 2002, TrendsCellBiol 12:267-273, and Antonio et al , 2000, Cell 1O2:425l

MtTOSlS

789

the plasma membrirne that will eventually contract and pinch the cell into two, a processknown as cytokinesis(see Figure 18-34).The contractilering is a thin band of actin filamentsof mixed polarity interspersedwith myosin-II bipolar filaments (seeFigure 17-34). On receivinga signal, the ring contracts first to generatea cleauagefurrow and then to pinch the cell into two. Two aspectsof the contractilering are essentialto its function. First, it has to be appropriately placed in the cell. It is known that this placementis determined by signalsprovided by the spindle,so that the ring forms equidistant between rhe two spindie poles. Recent work with Drosophila mutants has suggestedthat componentsthat accumulateat the centralpoint of the spindleduring anaphasedirect the formation of the contractile ring. However, the molecular signals involved in coordinating spindleposition and the position of the contractilering are still being unraveled. The secondimportant aspectof the contractilering is the timing of its contraction-if it contractedbeforeall chromosomes have moved to their respectivepclles, disastrous genetlcconsequences would ensue.Again, the mechanism wherebythis timing is determinedis being unraveied. In t e r p h a s e

Prophase

P l a n tC e l l sR e o r g a n i z T e h e i r M i c r o t u b u l e sa n d B u i l da N e w C e l lW a l l i n M i t o s i s lnterphaseplant cells lack a central MTOC that organizes microtubulesinto the radiating interphasearray typical of animal cells.Instead,numerous MTOCs line the cortex of plant cellsand nucleatethe assemblyof transversebands of microtubulesbelow the cell wall (Figure18-43,left).The mtcrotubules,which are of mixed polarity, are releasedfrom the cortical MTOCs by the action of katanin, a microtubule, severingprotein; loss of katanin givesrise to very long microtubulesand misshapencells.The reasonfor this is that t h e s e c o r t i c a l m i c r o t u b u l e s ,w h i c h a r e c r o s s - l i n k e db y plant-specificMAPs, aid in laying down extracellularceliulose microfibrils, the main component of the rigid cell wall (seeFigure 19-37). Although mitotic eventsin plant cellsare generallysimilar to those in animal cells, formation of the spindle and c y t o k i n e s i sh a v e u n i q u e f e a t u r e si n p l a n t s ( F i g u r e1 8 - 4 3 ) . Plant cells bundle their cortical microtubulesinto the preprophase band and reorganize them into a spindle at prophase without the aid of centrosomes.At metaphase.

Metaphase

Telophase

Preprophase band

\" /

P hr a gm o p l a s t

C e l lo l a t e F I G U R E1 8 - 4 3 M i t o s i s i n a h i g h e r p l a n t c e l l . l m m u n olfu o r e s c e n cmei c r o g r a p h( st o p )a n d c o r r e s p o n d i ndgi a g r a m s (botton) showingarrangementof microtubules in interphase and mitoticplantcells A cortjcalarrayof mtcrotubules girdlesa cell d u r i n gi n t e r p h a s eW e b so f m i c r o t u b u l ecsa pt h e g r o w i n ge n d so f p l a n tc e l l sa n d r e m a i ni n t a c td u r i n gc e l ld i v i s i o nA s a c e l le n t e r s p r o p h a s et h , e m i c r o t u b u l easr e b u n d l e da r o u n dt h e n u c l e u sa n d r e o r g a n i z ei dn t o a s p i n d l et h a t a p p e a r s i m i l atro t h a t i n m e t a p h a s e 790

t

CHAPTER t8

|

a n i m a cl e l l s B y l a t et e l o p h a s et h, e n u c l e a m r e m b r a n eh a sr e - f o r m e d a r o u n dt h e d a u g h t e n r u c l e ai n d t h e G o l g i - d e r i v epdh r a g m o p l a shta s a s s e m b l eadt t h e e q u a t o r i apl l a t e A d d i t i o n asl m a l vl e s i c l edse r i v e d f r o m t h e G o l g ic o m p l e xa c c u m u l a taet t h e e q u a t o r i apl l a t ea n d f u s e with the phragmoplast to form the new cellplate lAdapted from R H Goddard eIal, 1994,PlantPhysiol. 104:1;mcrographs courtesy of S u s aM n W i c kl

c E L Lo R G A N r z A T r o N A N D M o v E M E N Tr : M T c R o T U B U L EASN D T N T E R M E D T AFTTEL A M E N T S

the mitotic apparatusappearsmuch the same in plant and animal cells. However, the division of the cell into two is quite different from animal cells. Golgi-derived vesicles, which appear at metaphase,are transported into the mitotic apparatusalong microtubules that radiate from each end of the spindle.At telophase,thesevesiclesline up near the center of the dividing cell and then fuse to form the phragmoplast,a membranestructurethat replacesthe animal-cell contractile ring. The membranes of the vesicles forming the phragmoplastbecomethe plasma membranes of the daughtercells.The contentsof thesevesicles,such as polysaccharideprecursorsof celluloseand pectin, form the early cell plate, which developsinto the new cell wall between the dauehter cells.

Mitosis r Mitosis-the accurate separation of duplicated chromosomes-involves a molecular machine comprising dynamic treadmilling microtubules and molecular motors. r The mitotic spindlehas three classesof microtubules,all emanating from the spindle poles-kinetochore microtubules,which attach to chromosomes;polar microtubules from each spindle pole, which overlap in the middle of the spindle; and astral microtubules, which extend to the cell cortex (seeFigure 18-36).

Wl

Intermediate Filaments

The third maior filament system of eukaryotesis collectively called intermediate filaments. This name reflects their diameter of about 10 nm, which is intermediate betweenthe 6-8 nm of microfilaments and myosin thick filaments of skeletal muscle. Intermediate filaments extend throughout the cytoplasm as well as line the inner nuclear envelope of interphaseanimal cells (Figure L8-44). Intermediate filaments have severalunique properties that distinguish them from microfilaments and microtubules. First, they are biochemically much more heterogeneous-that is' many differenr, but evolutionarily related, intermediate filament subunits exist that are often expressedin a tissue-dependent manner. Second,they have great tensile strength' which is most clearly demonstrated by hair and nail that consist primarily of the intermediate filaments of dead cells' Third, they do not have an intrinsic polarity like microfilaments and microtubules, and their constituent subunits do not bind a nucleotide. Fourth, becausethey have no intrinsic polarity, it is not surprising that there are no known motors that use them as tracks. Fifth, although they are dynamic in terms of subunit exchange,they are much more stable than microfilaments and microtubules becausethe exchangerate

r In the first stage of mitosis, prophase, the nuclear chromosomes condense and the spindle poles move to either side of the nucleus (seeFigure 18-34). r At prometaphase,the nuclear envelope breaks down and microtubules emanating from the spindle poles capture chromosomepairs at their kinetochores.Each of the two kinetochores on the duplicated chromosomes becomesattachedto the two spindle poles,which allows the chromosomes to congress to the middle of the spindle. r At metaphase, chromosomes are aligned on the metaphaseplate. The spindlecheckpointsystemmonitors unattached kinetochores and delays anaphaseuntil all chromosomesare attached. r At anaphase,duplicated chromosomes separate and move toward the spindle poles by shortening of the kinetochore microtubules at both the kinetochore and spindle pole (anaphaseA). The spindle poles also move apart, pushedby bipolar kinesin-Smoving toward the (+ ) ends of the polar microtubules(anaphaseB). Spindleseparationis also facilitated by cortically located dynein pulling on ast r a l m i c r o t u b u l e s( s e eF i g u r e1 , 8 - 4 0 , 1 8 - 4 1 ) . r Redundant mechanismscontribute to the fidelity of mitosis since the mitotic spindle has the ability to selfassemblein the absenceof MTOCs. r The position of the actin-myosin basedcleavagefurrow is determined by the position of the spindle and at cytokinesiscontracts to pinch the cell in two.

of two typesof 18-44Localization FIGURE a EXPERIMENTAL lmmunofluorescence cell' in an epithelial filaments intermediate with keratin(red)andlamin of a PtK2celldoublystained micrograph can filaments (blue)antibodies of laminintermediate A meshwork the keratin whereas membrane, the nuclear be seenunderlying membrane to the plasma extendfromthe nucleus filaments [Courteso y f R D G o l d m a n] I N T E R M E D I A TFEI L A M E N T S

791

is much slower. Indeed, a standard way to purify intermediate filaments is to subject cells to harsh extraction conditions in a detergent so that all membranes,microfilaments, and microtubules are solubilized,leaving a residuethat is almost exclusively intermediate filaments. Finally, intermediate filaments are not found in all eukaryotes. Fungi and plants do not have intermediate filaments, and insectsonly have one class,representedby two genesthat expresslamin A/C and B. These properties make intermediate filaments unique and important structuresof metazoans.The importance of intermediatefilaments is underscoredby the identification of more than 40 clinical disorders,some of which are discussedbelow, associatedwith defectsin genesencodingintermediatefilament proteins.To understandtheir contributions to cell and tissue structure, we first examine the structure of intermediatefilament proteins and how they assemble into a filament. We then describe the different classesof intermediate filaments and the functions they perform.

IntermediateFilamentsAre Assembledfrom S u b u n i tD i m e r s Intermediate filaments (IFs) are encoded in the human genome by 70 different genesin at least five subfamilies. The defining feature of IF proteins is the presenceof a c o n s e r v e do - h e l i c a l r o d d o m a i n o f a b o u t 3 1 0 r e s i d u e s (a) Head

- - - - - -R- o- -d- |

N-terminus

that has the sequencefeatures of a coiled-coil motif (see Figure 3-9a). Flanking the rod domain are nonhelical Nand C-terminal domains of different sizes,characteristic o f e a c hI F c l a s s . The primary building block of intermediatefilaments is a dimer (Figure 18-45a) held together through the rod domains that associateas a coiled-coil. Thesedimers then associate in an offset fashion to make tetramers, where the two dimers are in opposite orientations (Figure 18-45b). Tetramers are assembledend to end and interlocked into long protofilaments. Four protofilaments associateinto a protofibril, and four protofibrils associateside to side to generatethe 10-nm filament. Thus an intermediate filament has 15 protofilaments in it. Becausethe tetramer is symmetric, intermediatefilaments have no polarity. This description of the filament is basedon its structure rather than its mechanism of assembly:at presentit is not yet clear how intermediate filaments are assembledin vivo. Unlike microfilaments and microtubules, there are no known intermediate filament nucleating, sequestering,capping, or filament-severing protelns.

IntermediateFilamentsProteinsAre Expressed i n a T i s s u e - S p e c i fM i ca n n e r Sequenceanalysisof IF proteins revealsthat they fall into at least five distinct homology classes,with four classesshowing a strong correspondencebetweenthe sequenceclassand

(b) , Tail C-terminus

T^+-^-^-

(c)

Protofilament Protofibril

A FIGURE 18-45 Structureand assemblyof intermediate filaments.Electron micrographs anddrawings of lFproteindimers, tetramers, andmatureintermediate filaments fromAscan'an parasitic intestinal worm.(a)lFproteins formparallel dimersthrough a highlyconserved coiled-coil coredomain. Theglobular headsand tailsarequitevariable in lengthandsequence betweenintermediate

792

C H A P T E R1 8

I

(b)A tetramer filamentclasses. isformedby antiparallel, staggered side-by-side aggregation of two identical dimers(c)Tetramers aggregate endto endandlaterally intoa protofibril. In a mature filament, consisting of fourprotofibrils, theglobular domains form beaded clusters on thesurface[Adapted fromN.Geisler etal, 1998,,/, Mol Biol282:601; courtesy of UeliAebil

C E L LO R G A N I Z A T I O N A N D M O V E M E N Tl l : M I C R O T U B U L EASN D T N T E R M E D T A F TT EL A M E N T S

FUNCTION DISTRIBUTION PROPOSED Acidic keratins

Epithelial cells

Basickeratins

Epithelial cells

Tissuestrength and integrity

E p i t h e l i a cl e l l

Muscle, glial cells, mesenchymal cells

Sarcomere organlzatlon, lntegrlty

Dense bodies

S m o o t hm u s c l e

ry

Neurofilaments (NFL, NFM, and NFH)

Neurons

S k e l e t a lm u s c l e

Axon organization Axon

Nuclear structure and organization Nucleus

the developmentalorigin of the cell type in which the IF protein is expressed(Table18-1). Keratins that make up classesI and II are found in epithelia, classIII intermediatefilaments are generallyfound in cells of mesodermalorigin, and classIV make up the neurofilaments found in neurons. The lamins make up class V, 'We which are found lining the nucleus of all animal tissues. briefly summarize the five different homology classesand discusstheir roles in specifictissues. Keratins Keratins provide strength to epithelial cells. The first two homology classesare the so-calledacidic and basic keratins. There are about 50 genesin the human genomeencoding keratins, about evenly split between the acidic and basic classes.These keratin subunits assembleinto an obligate dimer, so that each dimer consists of one basic chain and one acidic chain; these are then assembledinto a filament as describedabove. The keratins are by far the most diverseof the IF protein families, with keratin pairs showing different expression patterns between distinct epithelia and also showing differentiation-specific regulation. Among these are the so-calledhard keratins that make up hair and nails. These keratins are rich in cysteineresiduesthat becomeoxidized to form disulfide bridges, thereby strengthening them. This property is exploited by hair stylists: if you do not

like the shapeof your hair, the disulfide bonds in your hair keratin can be reduced, the hair reshaped' and the as a disulfide bonds reformed by oxidation-known "permanent," The so-calledsoft, or cyto-keratins, are found in epithelial cells. The epidermal-cell layers that make up the skin provide a good example of the function of keratins. The lowest layer of cells, the basal layer which is in contact with the basal lamina, proliferates constantly' giving rise to cells called keratinocytes. After they leave the basal layer, the keratinocytes differentiate and expressabundant cytokeratins. The cytokeratins associatewith desmosomes,specializedattachment sites between cells, thereby providing sheets of cells that can withstand abrasion' The cells eventually die, leaving dead cells from which all cell organelleshave disappeared. This dead cell layer provides an essentialbarrier to water evaporation, without which we could not survive.The life of a skin cell, from birth to its loss from the animal as a skin flake, is about one month. In all epithelia, keratin filaments associatewith desmosomes,which link adjacent cells together, and hemidesmosomes.which link cells to the extracellular matrix, thereby giving cells and tissuestheir strength. These structures are describedin more detail in Chapter 19. In addition to simply providing structural support, there is increasing evidencethat keratin filaments provide some

I N T E R M E D I A TFEI L A M E N T S

793

organization to organelles and participate in signaltransductionpathways. For example,in responseto tissue injury, rapid cell growth is induced. In epithelial cells it has been shown that the growth signal requires an interaction between a cell-growth-signalingmolecule and a specific keratin. Desmin The classIII intermediatefilamentproteinsinclude vimentin, found in mesenchymalcells; GFAP (glial fibrillary acidic protein), found in glial cells; and desmin, found in musclecells.Desmin providesstrengthand organizationto musclecells. In smooth muscle, desmin filaments link cytoplasmic dense bodies, into which rhe conrractile myofibrils are also attached,to the plasma membraneto ensurethat cells r e s i s t o v e r s t r e t c h i n g .I n s k e l e t a l m u s c l e , a l a t t i c e c o m posed of a band of desmin filaments surrounds the sarcomere. The desmin filaments encirclethe Z disk and are cross-linked to the plasma membrane. Longitudinal desmin filaments cross to neighboring Z disks within the myofibril, and connections between desmin filaments around Z disks in adjacent myofibrils serve to cross-link myofibrils into bundles within a muscle cell. The lattice is also attached to the sarcomerethrough interactions with myosin thick filaments. Becausethe desmin filaments lie outside the sarcomere,they do not actively parricipate in generatingcontractile forces. Rather, desmin plays an essential structural role in maintaining muscle integrity. In transgenicmice lacking desmin,for example,this supporting architecture is disrupted and Z disks are misaligned. The location and morphology of mitochondria in these mice are also abnormal, suggestingthat theseintermediate

(a) 20 minutes after injection

(b) 4 hours after iniection

CHAPTER 18

|

filaments may also contribute to organization of organelles. Neurofilaments Type IV intermediate filaments consist of the three related subunits-NF-L, NF-M, NF-H (for NF light, medium, and heavy)-that make up the neurofilaments found in the axons of nerve cells (seeFigure 18-2). The three subunitsdiffer mainly in the sizeof their C-terminal domains, and all form obligate heterodimers.Experiments with transgenic mice reveal that neurofilaments are necessary to establishthe correct diameter of axons, which deter, mines the rate at which nerve impulsesare propagateddown axons. Lamins The most widespreadintermediatefilaments are the classV lamins, which provide strength and support to the inner surfaceof the nuclearmembrane(seeFigure L8-44). Lamins are the progenitor of all IF proteins, with the cytoplasmic IFs arising by geneduplication and mutation. The lamins provide a two-dimensional network lying between the nuclear envelopeand the chromatin in the nucleus.In humans, three genes encode lamins: one encodesA-type Iamin and two encodeB type. The B-type lamin appearsto be the primordial gene and is expressed in all cells, whereas A-type lamins are developmentallyregulated. Blamins are post-translationallyisoprenylated,which presumably helps them associatewith the inner nuclear envelope membrane. In addition, they bind inner nuclear membrane proteins such as emerin and lamin-associated polypeptides (LAP2). Lamins bind multiple proteins and have been proposed to play roles in large-scalechromatin organization and the spacingof nuclear pores. As cells en-

< EXPERIMENTAL FIGURE 18-46Keratin intermediatefilamentsare dynamicas solublekeratinis incorporatedinto filaments.Monomeric typeI keratinwas p u r i f i e dc ,h e m i c a l a l yb e l ewd i t hb i o t i na, n d microinjected intolivingepithelial cellsThecells werethenfixedat different timesafterinjection andstained with an antibody to biotinandwith antibodies to keratin(a)At 20 minutes after injection, the injected biotin-labeled keratinis concentrated in smallfociscattered throughthe (/eft)andhasnot beenintegrated cytoplasm intotheendogenous keratincytoskeleton (right).(b)By4 hours,the biotin-labeled (/eff) (right)display andthe keratinfilamenls identical patterns, indicating thatthe mrcroinjected proteinhasbecomeincorporated intothe existing cytoskeleton. R K MillelK Vistrom, [From andR D Goldman, 1991 ,J CellBiol113:843; courtesy of R D Goldman l

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D T N T E R M E D T AFTt E LAMENTs

ter mitosis, lamins become hyperphosphorylatedand disassemble;in telophasethey reassemblewith the reassembling nuclear membrane.

I n t e r m e d i a t eF i l a m e n t sA r e D y n a m i c Although intermediate filaments are much more stable than microtubules and microfilaments, IF protein subunits have been shown to be in dynamic equilibrium with the existing IF cytoskeleton. In one experiment, a biotin-labeled type I keratin was injected into fibroblasts; within 2 hours, the labeled protein had been incorporated into the already existing keratin cytoskeleton (Figure 18-46). The results of this experiment and others demonstratethat IF subunits in a soluble pool are able to add themselvesto preexistingfilaments and that subunits are able to dissociate from intacr filaments. The relative stability of intermediatefilamentspresents special challengesin mitotic cells, which must reorganize all three cytoskeletalnetworks in the course of the cell cycle. In particular, breakdown of the nuclear envelopeearly in mitosis depends on the disassemblyof the lamin filaments that form a meshwork supporting the membrane.As discussedin Chapter 20, the phosphorylation of nuclear lamins by a cyclin-dependentkinase that becomesacrive early in mitosis (prophase)inducesthe disassemblyof intact filaments and preventstheir reassembly.Later in mitos i s ( t e l o p h a s e ) r, e m o v a l o f t h e s e p h o s p h a t e sb y s p e c i f i c phosphatasespromotes lamin reassembly,which is critical to re-formation of a nuclear envelopearound the daughter chromosomes.The opposing actions of kinasesand phosphatasesthus provide a rapid mechanismfor controlling the assemblystate of lamin intermediatefilaments. Other intermediatefilamentsundergo similar disassemblyand reassemblyin the cell cycle.

K 1 4 k e r a t i n i s o f o r m s f o r m h e t e r o d i m e r st h a t a s s e m b l e into protofilaments. A mutant K14 with deletions in either the N- or the C-terminal domain can form heterodimers in vitro but does not assembleinto protofilaments. The expressionof such mutant keratin proteins in cells causesIF networks to break down into aggregates. Transgenicmice that expressa mutant K14 protein in the basal stem cells of the epidermis display gross skin abnormalities, primarily blistering of the epidermis,that resemble the human skin diseaseepidermolysisbullosa simplex (EBS).Histologicalexaminationof the blisteredareareveals a high incidenceof dead basalcells.Death of thesecellsappears to be caused by mechanical trauma from rubbing of the skin during movement of the limbs. Sfithout their normal bundlesof keratin filaments,the mutant basal cells become fragile and easilydamaged,causingthe overlyingepidermal layers to delaminate and blister (Figure 18-47lr.

Epidermis

Dermis

D e f e c t si n L a m i n sa n d K e r a t i n sC a u s eM a n y Diseases T h e r e a r e a b o u t 5 0 k n o w n m u t a t i o n si n t h e h u m a n gene for type-A lamin that are known to cause disease,many of which cause forms of Emery-Dreifuss muscular dystrophy (EDMD). Other mutations in the lamin-A gene cause dilated cardiomyopathy. It is not yet c l e a r w h y t h e s e t y p e - A l a m i n m u t a t i o n s c a u s eE D M D , but perhaps in muscle tissues the fragile nuclei cannot stand the stressand strains of the tissue, so they are the f i r s t t o s h o w s y m p t o m s . I n t e r e s t i n g l y ,o t h e r f o r m s o f EDMD have been traced to mutations in emerin, the l a m i n - b i n d i n gm e m b r a n ep r o t e i n o f t h e i n n e r n u c l e a re n velope. Yet other mutations in type-A lamin causeprogeria-accelerated aging. Thus the Hutchison-Gilford proge r i a ( " p r e m a t u r e l yo l d " ) s y n d r o m ei s c a u s e db y a s p l i c i n g error that results in a lamin A with a defectiveC-terminal oomaln. The structural integrity of the skin is essentialin order t o w i t h s t a n d a b r a s i o n .I n h u m a n s a n d m i c e , t h e K 4 a n d

Epidermis

Mutated

micecarryinga FIGURE 18-47Transgenic a EXPERIMENTAL mutant keratingeneexhibit blisteringsimilarto that in the sections bullosasimplex.Histological humandiseaseepidermolysis a mouse carrying anda transgenic through theskinof a normalmouse geneareshownInthenormalmouse, theskin mutantK14keratin layercovering in contactwiththe soft of a hardouterepidermal consists mouse, thetwo layers Intheskinfromthetransgenic innerdermallayer. (arrow)dueto weakening of thecellsat the baseof the areseparated of E Fuchs PCoulombe etal, 1991,Cell66:1301 epidermis. ] ; courtesy [From I N T E R M E D I A TFEI L A M E N T S

'

795

Like the role of desmin filaments in supporting muscle tissue, the general role of keratin filaments appearsto be to maintain the structural integrity of epithelial tissues by mechanically reinforcing the connectionsbetween cells. I

Intermediate Filaments r Intermediate filaments are the only nonpolarized fibrous component of the cytoskeletonand do not have associated motor proteins. Intermediate filaments are built from coiled-coil dimers that associatein an antiparallel fashion into tetramers and then into protofilaments, 16 of which make up the filament (seeFigure 18-45). r There are five major classesof intermediateproteins, with the nuclear lamins (classV) being the most ancient and ubiquitous in animal cells.The other four classesshow tissue-specific expression(seeTable 18-1). r Keratins (classesI and II) are found in animal hair and nails, as well as in cytokeratin filaments that associatewith desmosomesin epithelial cells to provide the cells and tissue with strength. r The class III filaments include vimentin, GFAP, and desmin, which provide structure and order to muscle Z disks and restrain smooth muscle from overextension. r The neurofilaments make up classIV and are important for the structure of axons. r Many diseasesare associatedwith defectsin intermediate filaments, especiallylaminopathies,which include a variety of conditions, and mutations in keratin genes,which can causeseveredefectsin skin (seeFigure 18-47).

mediate filaments to other structures.Some of these associate with keratin filaments to link them to desmosomes, which are junctions between epithelial cells that provides stability to a tissue,and hemidesmosomes,which are located at regions of the plasma membrane where intermediate filaments are linked to the extra-cellularmatrix (thesetopics are covered in detail in Chapter 19). Other plakins are found along intermediate filaments and have binding sites for microfilaments and microtubules. One of theseproteins, called plectin, can be seenby immunoelectron microscopy to provide connectionsbetweenmicrotubules and intermediatefilaments(Figure18-48).

Microfilamentsand MicrotubulesCooperateto TransportMelanosomes Studiesof mutant mice with light-coloredcoatshas uncovered a pathway in which microtubules and microfilaments cooperate to transport pigment granules. The color pigment in the hair is produced in cellscalled melanocytes,cellsvery similar to the fish and frog melanophoresdiscussedearlier (seeFigure 1,8-28).Melanocytes are found in the hair follicle at the base of the hair shaft and contain pigment-ladengranules called melanosomes.Melanosomesare transported to the dendritic extensions of melanocytes for subsequent exocytosis to the surrounding epithelial cells. Transport to the cell periphery is mediated, iust as in frog melanophores,by a kinesin family member. At the periphery, they are then handed off to myosin V and deliveredfor exocytosis.If the myosin V systemis defective,the melanosomesare not capturedand stay in the cell body. Thus microtubules are responsiblefor the long-range transport of melanosomes, whereas microfilament-based myosin V is responsible for capture and delivery at the cell

fftli| Coordinationand Cooperation betweenCytoskeletalElements So far, we have generally discussedthe three cytoskeletal filaments classes-microfilaments, microtubules, and intermediate filaments-as though they function independently of one another. However, the example that the microtubule-basedmitotic spindle determinesthe site of formation of the microfilament-basedcontractile ring is just one example of how these two cytoskeletal systems are coordinated.Here we mention some other examplesof linkages, physical and regulatory, between cytoskeletal elementsand their integration into other aspectsof celluIar organization.

IntermediateFilament-Associated Proteins C o n t r i b u t et o C e l l u l a rO r g a n i z a t i o n A group of proteins collectively called intermediate filament-associated proteins (IFAPs) have been identified that co-purify with intermediate filaments. Among these are the family of plakins, which are involved in attaching inter796

CHAPTER 18

I

FIGURE 18-48Gold-labeled A EXPERIMENTAL antibody identifiesplectincross-linksbetween intermediatefilaments and microtubules. Inthisimmunoelectron micrograph of a fibroblast cell,microtubules arehighlighted in red;intermediate filaments, in fibersbetween blue;andtheshortconnecting them,in green with gold-labeled antibodies to plectin(yellow) reveals Staining that thesefiberscontainplectin.[From T.M Svitkina, A B Verkhovsky, andG 1996, G Borisy, L CellBiol.135:991; courtesy of T.M Svitkina l

C E L LO R G A N I Z A T I OANN D M O V E M E N TI I : M I C R O T U B U L EASN D I N T E R M E D I A TFEI L A M E N T S

M i c r o t u b u l e( + ) e n d c a p t u r e\

.t, Actin assembly

Direction of oolarization cortex. This type of division of labor-long-range transport by microtubules and short range by microfilaments-has been found in many different systems,from transport in filamentous fungi to transport along axons.

< FfGURE 18-49 Cdc42regulates microfilamentsand microtubules independentlyto polarizea migrating cell.ActiveCdc42-GTP at the frontof the cell whichresults in the leadsto Racactivation. assembly of a microfilament-based leading edge(step[). Independently, Cdc42-GTP also (+) ends leadsto thecapture of microtubule andthe activation of dynein(step2; Together theseoullon microtubules to orientthe (stepB) towardthe frontof the centrosome polarizes the secretory cell.Thisreorientation pathway for the delivery alongmicrotubules molecules carriedin secretory of adhesion (stepZl). [Based inS Etiennevesicles onstudies Manneville et al, 2005,J.CellBiol170:895-901 l

r In animal cells,microtubules are generallyutilized for the Iong-range delivery of organelles,whereas microfilaments handle their local delivery. r The signalingmolecule Cdc42 coordinately regulatesmicrofilaments and microtubules during cell migration.

Cdc42CoordinatesMicrotubulesand M i c r o f i l a m e n t sD u r i n gC e l lM i g r a t i o n In Chapter 17, we discussedhow the polarity of a migrating cell is regulated by Cdc42, which resultsin the formation of an actin-basedleading edge at the front of the cell and contraction at the back (seeFigures17-44 and18-49, step [). It turns out that Cdc42 activation at the cell front also leadsto polarization of the microtubule cytoskeleton.This was originally studied in wound-healing assays(seeFigure 17-43) where it was noticed that when the cells at the edge are induced to polarize and move to fill up the scratch, the Golgi complex is moved to the front of the nucleustoward the cell front. SinceGolgi localization is dependenton the location of the MTOC (seeFigures 18-1c, 18-27), this was becausethe centrosomecame to lie in front of the nucleus.Recentstudies have suggestedhow this happens. Cdc42 activation at the front of the cell binds the polarity factor Par6, which results in the recruitment of the dynein/dynactin complex (Figure 1.8-49,step Z). Cortically localizeddynein/dynactinthen interacts with microtubules pulling on them to orient the centrosome and hence the whole radial array of microtubules (Figure 18-49, step B). This reorientation of the microtubule systemleads to the reorganization of the secretorypathway to deliver secretoryproducts, especiallyintegrins to bind the extracellular matrix, to the front of the cell for attachment to the substratum for cell migration (Figure 1,8-49,step 4).

Coordination and Cooperation between Cytoskeletal Elements r Intermediate filaments are linked both to specific attachment sites, called desmosomesand hemidesmosomes,on the plasma membrane, as well to microfilaments and microtubules(seeFigure 18-48).

In Chapters 17 and 18 we have seenhow microfilaments, microtubules, and intermediate filaments provide structure and organization to cells.'$fithout this elaboratesystem,cellswould lack all order and hence all possibility of function or division. The name "cytoskeleton" suggestsa relatively static structure on which the cell organization is hung. However, the cytoskeleton is actually a dynamic framework responding to signaltransduction pathways and operating both locally and globally to provide cellswith order to undertake their functions. In outline, we have elucidatedmany of the distinct and common functions of the three filament systems. We know many of the componentsand probably all the motors. However,in many ways this is just an exciting beginning. \fith the availablesequencedgenomesand at least in principle a complete inventory of the cytoskeletalcomponents, we have a parts list. However, a parts list is just that; what we needis to understandhow the parts come togetherin specificprocesses. A very active areaof researchtoday is to use the parts list to systematically identify the localization (through GFP fusions), functions (through RNAi knockdown), and associated partners (through isolation of protein complexes) of all cytoskeletal components. Consider that there are about 45 genes in animals that encode members of the kinesin family, yet we only know what a small subsetof them do or what cargo they carry and for what purpose. In each case it is reasonableto assumethe motors are regulated,about which very little is currently known. As we begin to put all the piecesin place, it will be increasingly possible to reconstitute specific processesin vitro. Someaspectsof the mitotic spindle have already beenreconstituted, which is an encouraging beginning, but it will be sometime before it is possibleto reconstitutethe whole process. P E R S P E C T I VFEO SRT H E F U T U R E

797

Structural biology is going play a major role becauseit will allow us to see in detail how different components work. Consider the large number of proteins that associ'We ate with the microtubule (+ ) end, the so-called*TIPs. do not know structurally how they maintain their association at the tip, and recent work has suggestedthat associations can changein different parts of the cell-again, we are only just beginning to see how these processesare regulated. Perhapsthe biggest-and most exciting-challenge is to uncover how signal-transductionpathways coordinate func'We tions between all the different cytoskeletalelements. are beginning to seeglimpsesof what is in store from the signaltransduction pathways that regulate cell polarity and allow cell migration. Although all thesestudiesare likely to be aimed at basic cell biology, as we can seefrom the studies of intraflagellar transport, such studiesoften open a window into the underlying basis of disease,from which strategiesfor treatments can be developed.The interplay between basic cell biology and medicine contributes immensely to the excitement and social value of working in this area.

KeyTerms anaphase783

kinesins 259

asters 782

kinetochores 783

axonal transportT69

lamins 794

axonerne 777

cytokinesis 783

metaphase783 microtubule-associated proteins (MAPs) 758 microtubule- o r ganizing center (MTOC) 760

desmin 794

mitosis 781

dynamic instability 764

mitotic spindle 782 plakins 796 prophase 782

basalbody 761 centromere 786 centrosome750

dyneins 759 intermediatefilamentassociatedproteins (IFAPs)795

telophase783

keratins 793

tubulin 758

protofilament 758

Review the Concepts l. Microtubules are polar filaments; that is, one end is different from the other.\(hat is the basisfor this polarity, how is polarity related to microtubule organization within the cell, and how is polarity related to the intracellular movements powered by microtubule-dependentmotors? 2. Microtubules both in vitro and in vivo undergo dynamic instability, and this type of assemblyis thought to be intrinsic to the microtubule. What is the current model that accounts for dynamic instability? 798

C H A P T E R1 8

I

3. In cells, microtubule assembly depends on other proteins as well as tubulin concentration and temperature. What types of proteins influence microtubule assembly in vivo, and how does each type affect assembly? 4. Microtubules within a cell appear to be arranged in 'S7hat specificarrays. cellular structure is responsiblefor determining the arrangement of microtubules within a cell? How many of these structures are found in a typical cell? Describe how such structures serveto nucleate microtubule assembly. 5. Many drugs that inhibit mitosis bind specifically to tubulin, microtubules, or both. What diseasesare such drugs used to treat? Functionally speaking,these drugs can be divided into two groups basedon their effect on microtubule assembly. What are the two mechanisms by which such drugs alter microtubule structure? 6. Kinesin-1 was the first member of the kinesin motor family to be identified and therefore is perhaps the bestcharacterizedfamily member. What fundamental property of kinesin was used to purify it? 7. Certain cellular components appeat to move bidirectionally on microtubules. Describehow this is possiblegiven that microtubule orientation is fixed by the MTOC. 8. The motile properties of kinesin motor proteins involve both the motor domain and the linker domain. Describethe role of eachdomain in kinesin movement,direction of movement, or both. 9. Cell swimming depends on appendages containing microtubules. What is the underlying structure of these appendages,and how do these structuresgeneratethe force required to produce swimming? 10. The mitotic spindle is often describedas a microtubulebasedcellular machine. The microtubules that constitute the mitotic spindle can be classified into three distinct types. What are the three types of spindle microtubules, and what is the function of each? 11. Mitotic spindle function relies heavily on microtubule motors. For each of the following motor proteins, predict the effect on spindle formation, function, or both of adding a drug that specifically inhibits only that motor: kinesin-5, kinesin-13,and kinesin-4. 12. The poleward movement of kinetochores, and hence chromatids, during anaphaseA requires that kinetochores maintain a hold on the shortening microtubules. How does a kinetochore hold on to shortening microtubules? 13. Anaphase B involves the separationof spindle poles. \7hat forces have been proposed to drive this separation? $7hat underlying molecular mechanismsare thought to provide theseforces? 14. Cytokinesis,the processof cytoplasmic division, occurs shortly after the separatedsisterchromatids have neared the opposite spindle poles. How is the plane of cytokinesis determined?I(hat are the respectiveroles of microtubules and actin filaments in cytokinesis? 15. Explain why there are no known motors that use intermediate filaments as tracks.

C E L LO R G A N I Z A T I O N A N D M O V E M E N TI I M I C R O T U B U L E AS N D I N T E R M E D I A TFEI L A M E N T S

Analyze the Data a. Kinesin-l contains two identical heavy chains and therefore has two identical motor domains. In contrast, kinesin-Scontains four identical heavy chains. Electron microscopic analysisof metal-shadowedkinesinsresultsin the imagesshown in the top panel.Pretreatmentof thesekinesinwith an antibody that binds specifically to the kinesin motor domain resultsin the imagesshown in the lower panel. All four images are at the same approximate magnification. What can you deduceabout the structureof kinesin-Sfrom thesedata? Kinesin-1

c. Kinesin-S can cross-bridge adjacent microtubules. Polarity marked microtubules are assembledin which tubulin attached to a red fluorescent dye is assembledto form short red microtubules, which are then elongatedwith tubulin attachedto a greenfluorescentdye. The microtubules are mixed with kinesin-S and observed by fluorescencemicroscopyas ATP is added.The following imagesshow a time sequenceof two overlapping and cross-bridgedmicrotubules as ATP is added. The arrowhead is in a fixed position. Can you explain what happens when ATP is added to microtubulescross-bridgedby kinesin-5?

Kinesin-5

No antibody

Decoratedwith k i n e s i nm o t o r d o m a i na n t i b o d y

b. To determineif kinesin-Sis a (+) or (-) end microtubule motor, polarity-marked microtubules are generatedby assemblingshort microtubulesfrom brightly fluorescenttubulin and then elongating those short, bright microtubules using less fluorescent tubulin. As a result, the microtubules are very fluorescent at one end and lessfluorescent along most of their length. A perfusion chamber is then coated with purified kinesin-s,which becomesimmobilizedon the glasssurface.The chamber is then perfused with the polarity labeled microrubules and ATI and microtubule gliding with respectto the immobilized kinesin-S is observed.The following sequenceof 'SThich imagesis obtained. end of thesemicrotubules, the bright or the lessbright end, is the (+) end? Do thesemicrotubules glide on kinesin-Swith their (+) or (-) end leading?Basedon thesedata,is kinesin-Sa (+)or (-)end microtubulemotor?

d. Eg5 is a kinesin-S family member in Xenopus. To understand Eg5 function in vivo, cells are transfectedwith RNAi directed against this motor. The following imagesare obtained of mitotic cells. ril/hat function might Eg5 play in cells?

nK2

z t o

o

o ^o |f)

o) UJ

References Structure and Organization Microtubule Badano,J. L., T. M. Teslovich,and N. Katsanis.2005. The centrosome in human geneticdisease.Natwre Reu.Mol. Cell Biol. 6:L94-205. DoxseS S. 2001. Re-evaluatingcentrosomefunction. Natwre Reu.Mol. Cell Biol.2:688-698. Dutcher,S. K. 2001. The tubulin fraternity: alpha to eta. Curr. Opin. Cell Biol. 13:49-54. Nogales,E., and H-\7 Wang. 2006. Structuralintermediatesin microtubule assemblyand disassembly:how and why? Curr. Opin. Cell Biol. 18:179-1.84.

Dynamics Microtubule Cassimeris,L.2002. The oncoprotein 18/Stathminfamily of microtubule destabilizers.Curr. ODin. Cell Biol. 14:18-24. REFEREN CES

799

Desai,A., and T. J. Mitchison. 1997. Microtubule polymerization Dyanamics.Annu. Reu.CellDeu. Biol. 13:83-7'1.7. Howard, J., and A. A. Hyman. 2003. Dynamics and mechanics of the microtubule plus end.Natwre 422:753-7 5 8. Regulation of Microtubule Structure and Dynamics Akhmanova, A., and C. C. Hoogenraad.2005. Microtubule plus-end-trackingproteins:mechanismsand functions. Curr. Opin. Cell Biol. 17:47-54. Galjart, N. 2005. CLIPs and CLASPsand cellular dynamics. Nature Reu.Mol. Cell Biol.6:487498. Kinesins and Dyneins-Microtubule-Based

Motor Proteins

Web site:kinesin home page,http://wwwproweb.org/kinesinL/ Burgess,S. A., et a|.2003. Dynein strucrureand power stroke. Nature 421:775-71,8. Dell, K. R. 2003. Dynactin policesrwo-way organelletraffic. J. Cell B iol. 160:291,-293. Dujardin, D. L., and R. B. Vallee.2002. Dynein at the cortex. Curr. Opin. Cell Biol. 14:4449. Goldstein,L. S. 2001. Kinesin molecularmotors: transport pathways,receptors,and human disease.Proc. Nat'l. Acad. Sci.

usA986999-7003. Hirokawa, N. 1998. Kinesin and dynein superfamiiyproteins and the mechanismof organelletransport. Science279:51,9-526. Hirokawa, N., and R. Takemure.2003. Biochemicaland molecular characterizationof diseaseslinked to motor oroteins.Trends Cell Biol.28:558-565. Lawrence,C. J., et aL.2004.A standardizedkinesin nomenclature.J. Cell Biol. 167:1,9-22. Schroer,T. A. 2004. Dynactin. Ann. Reu.Cell Deu. Biol. 20:759-779. Vale. R. D. 2003. The molecular motor roolbox for intracellular transport. Cell 112:467480. Vale, R. D., and R. A. Milligan. 2000. The way things move: looking under the hood of molecularmotor proteins.Science 288:88-95. 'Wordeman, L. 2005. Microtubule-depolymerizingkinesins. Curr. Opin. Cell Biol. 17:82-88. Yildiz, A., M. Tomishige,R. D. Vale, and P. R. Selvin.2004. Kinesinwalks hand-over-han d. Science303:676-67 8. Cilia and Flagella: Microtubule-Based Surface Structures 'Witman. Rosenbaum,J. L., and G. B. 2002.lntraflagellar rranspofi. Nature Reu.Mol. Cell Biol.3:813-825. Singla,V., and J. F. Reiter.2006. The primary cilium as the cells' antenna:signalingat a sensoryorganelle.Science313:629-633.

800

C H A P T E Rl 8

|

Mitosis Web site: http://www.proweb.org/kinesin/FxnSpindleMotility.html Cleveland,D. r07.,Y. Mao, Y., and K. F. Sullivan.2003. Centromeresand kinetochores:from epigeneticsto mitotic checkpoint signaling.Cell Ll2:407 42'1.. Gadde,S., and R. Heald.2004. Mechanismsand moleculesof the mitotic spindle.Curu Biol. 14:R797-R805. Heald, R., et aL.1997. Spindleassemblyin Xenopus egg roles of centrosomesand microtubule selfextracts:resDective organization.J . Cell Biol. 138:675-628. Kinoshita, K., B. Habermann,and A. A. Hyman. 2002. XMAP215: a key componentof the dynamic microtubule cytoskeleton. Trends Cell Biol. 12:267-273. Mitchison, T. J., and E. D. Salmon.2001. Mitosis: a history of division. Nature Cell Biol. 3 :El7 -821. Rogers,G. C., et aL.2004.Two mitotic kinesinscooperateto drive sisterchromatid separationduring anaphase.Nature 427:364-370. 'Wittmann, T., A. Hyman, and A. Desai.2001. The spindle:a dynamic assemblyof microtubulesand motors. Nature Cell Biol. 3:828-E34. lntermediate Filaments Intermediate Filaments Database: http://www.interfil.org/ index.php Goldman, R. D., et aI.2002. Nuclear lamins: building blocks of nuclear architecture.G enesD eu. 16:,533-547 . Herrmann, H., and U. Aebi. 2000. Intermediatefilamentsand their associates: multi-talentedstructural elementsspecifyingcytoarchitectureand cytodynamics.Curr. Opin. Cell Biol. L2:79-90. Mattout, A., et al. 2006. Nuclear lamins, diseaseand aging. Curr. Opin. Cell Biol. 18:335-341. Coordination and Cooperation between Cytoskeletal Elements ri7ebsite: Melanophores, http://www.proweb.orglkinesin"/ Melanophore.html Chang, L., and R. D. Goldman.2004.Intermediatefilaments mediatecytoskeletalcrosstalk.Nature Reu.Mol. Cell Biol. 5:

60r-613. Etienne-Manneville,S., et al. 2005. Cdc42 and Par6-PKC( regulate the spatially localized association of Dlgl and APC to control cell polarization.l. Cell Biol. l7O:895-901,. Kodama, A., T. Lechler,and E. Fuchs.2004. Coordinating cytoskeletaltracks to polarizecellular movements.l. Cell Biol. 167: 203-207. 'Wu, X., X. Xiang, andJ. A. Hammer 11I.2006.Motor proteins at the microtubule plus-end.Trends Cell Biol. 16:135-143.

C E L LO R G A N I Z A T I O N A N D M O V E M E N Tl l : M I C R O T U B U L EASN D I N T E R M E D I A TFEI L A M E N T S

CHAPTER

I tr

CELLS INTEGRATING INTOTISSUES False-color imageof a sectionthrougha desmosome from neonatalmouseepidermisElectron microscope tomography was junction,called usedto generate an imageof a specialized cellular a desmosome, that helpsholdcellsin the skintogetherCelladhesion molecules calledcadherins areblue,membranes from adjacent cellsareeachoutlrnedin red,and relatedintracellular plaqueand intermediate molecules, cytoplasmic filaments, are orangeand lightgreen,respectively Scalebaris 30 nm [FromW He,P Cowin,andD L Stokes, 2003,Science 3O2(5642): I O9-113 l

I n the development of complex multicellular organisms I such as plants and animals, progenitor cells differentiate I i n t o d i s t i n c t" t y p e s " t h a t h a v ec h a r a c t e r i s t ci co m p o s i t i o n s . structures,and functions. Cells of a given typ. oft.., gate into a tissueto cooperatively perform a common"ggr.function: muscle contracts; nervous tissuesconduct electrical impulses; xylem tissue in plants transports water. Different tissuescan be organizedinto an organ, again to perform one or more specificfunctions. For instance,the muscles,valves, and blood vesselsof a heart work together to pump blood. The coordinated functioning of many types of cells and tissuespermits the organism to move, metabolize, reproduce, and carry out other essentialactivities. Even simple animals exhibit complex tissue organization. The adult form of the roundworm Caenorhabditis eleganscontains a mere 959 cells, yet thesecells fall into 1,2 different general cell types and many distinct subtypes.Vertebrates have hundreds of different cell types, including leukocytes (white blood cells) and erythrocytes (red blood cells); photoreceptors in the retina; adipocytes,which store fat; fibroblasts in connectivetissue; and hundreds of different subtypes of neurons in the human brain. Despite their diverseforms and functions, all animal cells can be classified as being componentsof just five main classesof tissue: epithelial tissue, connectiuetissue,muscular tissue,neruous tissue, and blood. Various cell types are arranged in precise

patterns of staggering complexity to generate tissues and organs. The costs of such complexity include increased requirements for information, material, energS and time during the developmentof an individual organism. Although the physiological costs of complex tissues and organs are high, they confer the ability to thrive in varied and variable environments,a maior evolutionary advantage.

OUTLINE x dhesion: 1 9 . 1 C e l l - C ea l ln d C e l l - M a t r i A An Overview ' 1 9 . 2 C e l l - C ea J u n c t i o n sa n d T h e i r l ln d C e l I - E C M A d h e s i o nM o l e c u l e s

808

l amina 1 9 . 3 T h e E x t r a c e l l u l aMr a t r i x l : T h e B a s a L

820

'19.4 The ExtracellularMatrix ll: Connective and Other Tissues 1 9 . 5 A d h e s i v eI n t e r a c t i o nisn M o t i l e a n d N o n m o t i l eC e l l s 19.6

P l a n tT i s s u e s

833 839

801

The complex and diverse morphologies of plants and animals are examples of the whole being greater than the sum of the individual parts, more technically describedas the emergentproperties of a complex system.For example, the distinct mechanicalproperties of rigid bones, flexible joints, and contracting musclespermit vertebratesto move efficiently and achievesubstantial size. One of the defining characteristicsof animals with complex tissuesand organs (metazoans)such as ourselvesis that the external and internal surfacesof most of their tissuesand organs, and indeed the exterior of the entire organism, are built from tightly packed sheetlikelayers of cells known as epithelia. The formation of an epithelium and its subsequentremodeling into more complex collections of epithelial and nonepithelial tis-

suesis a hallmark of the development of metazoans.Sheets of tightly attachedepithelial cells act as regulatable,selective permeability barriers, which permit the generation of chemically and functionally distinct compartments in an organism (e.g.,stomach and bloodstream).As a result, distinct and sometimesopposite functions (e.g.,digestionand synthesis)can efficientlyproceedsimultaneouslywithin an organism. Such compartmentalizationalso permits more sophisticated regulation of diverse biological functions. In many ways, the roles of complex tissuesand organs in an organism are analogousto those of organellesand membranesin individual cells. The assembly of distinct tissues and their organization into organs are determined by molecular interactions at the

C e l la d h e s i o n molecules(CAMs)

T i g h tj u n c t i o n Apical surface

ADHESIONS CELL-CELL lntermediate Adapter

G a pj u n c t i o n

Desmosome

Hemidesmosome

Basal amina Connexon

surface

E

Extracellu lar m a t r i x( E C M )

FIGURE 19-1 Overviewof majorcell-celland cell-matrix adhesiveinteractions. Schematic cutaway drawingof a typical epithelial tissue, suchasin the intestines Theapical(upper) surface of thesecellsis packed withfingerlike microvilli n thatprojectinto the intestinal lumen,andthe basal(bottom) surface Z restson extracellular matrix(ECM)TheECMassociated with epithelial cellsis usually (eg, the basal organized intovarious interconnected layers lamina, connecting fibers,connective tissue), in whichlarge, interdigitating ECMmacromolecules bindto oneanotherandto the (CAMs) cellsE Cell-adhesion molecules bindto CAMson other cells,mediating cell-cell adhesions 4, andadhesion receptors bind to various components of the ECI\4, mediating cell-matrix adhesions E gothtypesof cell-surface adhesion molecules areusually integral proteins membrane whosecytosolic domains oftenbindto multiple intracellular adapterproteinsThese adapters, directly or indirectly, (actlnor intermediate linkthe CAMto thecytoskeleton filaments) 802

CHAPTER 19

I

I N T E G R A T I NCGE L L SI N T OT I S S U E S

pathwaysAs a consequence, andto intracellular signaling information canbetransferred by CAMsandthe macromolecules to whichtheybindfromthecellexterior intothe intracellular environment andviceversaln somecases, a complex aggregate of proteins CAMs,adapters, andassociated isassembled Specific localized aggregates of CAMsor adhesion receptors formvarious typesof celljunctions that playimportant rolesin holdrng tissues togetherandfacilitating communication between cellsandtheir e n v i r o n m eT n ti g . hjtu n c t i o n6s, l y i n gj u s tu n d etrh em i c r o v i l l i , prevent thediffusionof manysubstances throughtheextracellular spaces between thecellsGapjunctions Z allowthe movement throughconnexon channels of smallmolecules andionsbetween the cytosols of adjacent cellsTheremaining threetypesof junctions, junctions adherens EI, spotdesmosomes 9, andhemidesmosomes IE, tint thecytoskeleton of a cellto othercellsor the ECM [see V Vasioukhin andE Fuchs, 2001 OpinCellBiol13:761 , Curr.

cellular level and would not be possiblewithout the temporally and spatially regulated expression of a wide array of adhesivemolecules. Cells in tissuescan adhere directly to one another (cell-cell adhesion) through specializedmembrane proteins called cell-adhesionmolecules (CAMs) that often cluster into specializedcell junctions (Figure 19-1). In the fruit fly Drosophila melanogaster,at least 500 genes F4% of the total) are estimatedto be involved in cell adhesion. Cells in animal tissues also adhere indftecdy (cellmatrix adhesion) through the binding of adhesionreceptors in the plasma membrane to components of the surrounding extracellular matrix (ECM), a complex interdigitating meshwork of proteins and polysaccharidessecretedby cells into the spacesbetween them. These two basic types of interactions not only allow cells to aggregateinto distinct tissues but also provide a meansfor the bidirectional transfer of information betweenthe exterior and the interior of cells.Such information transfer is important to many biological processes,including cell survival, proliferation, differentiation, and migration. Therefore it is not surprising that defects that interfere with the adhesiveinteractions and the associated flow of information can cause or contribute to diseases,including a wide variety of neuromuscular and skeletaldisordersand cancer. In this chapter, we examine various types of adhesive moleculesand how they interact. Becauseof the particularly well-understood nature of the adhesivemoleculesin tissuesthat form tight epithelia, as well as their very early evolutionary development,we will initially focus on epithelial tissues,such as the walls of the intestinal tract and those that form skin. Epithelial cells are normally nonmotile (sessile);however, during development, wound healing, and in certain pathologic states(e.g.,cancer),epithelial cellscan transform into more motile cells.Changes in expression and function of adhesive molecules play a key role in this transformation, as they do in normal biological processesinvolving cell movement, such as the crawling of white blood cells into sites of infection. \7e therefore follow the discussionof epithelial tissueswith a discussionof adhesion in nonepithelial, developing, and motile tissues. The evolution of plants and animals diverged before multicellular organismsarose.Thus multicellularity and the molecular means for assemblingtissuesand organs must have arisen independentlyin animal and plant lineages.Not surprisingl5 then, animals and plants exhibit many differences in the organization and development of tissues.For this reason, we first consider the organization of tissuesin animals and then deal separatelywith plants.

Cell-Celland Cell-Matrix

Adhesion:An Overview We begin with a brief orientation to the various types of adhesive molecules, their major functions in organisms, and their evolutionary origin. In subsequentsections,we exam-

ine in detail the unique structuresand properties of the various participants in cell-celland cell-matrix interactions.

C e l l - A d h e s i oM n o l e c u l e sB i n dt o O n e A n o t h e r and to lntracellularProteans A large number of CAMs fall into four major families: the cadherins, immunoglobulin (Ig) superfamilS integrins' and selectins.As the schematicstructures in Figure 19-2 illustrate, many CAMs and other adhesion moleculesare mosaicsof multiple distinct domains, many of which can be found in more than one kind of protein. Some of these domains confer the binding specificity that characterizesa particular protein. Other membrane proteins, whose structures do not belong to any of the major classesof CAMs, also participate in cell-cell adhesion in various tlssues. CAMs mediate, through their extracellular domains' adhesiveinteractions between cells of the same type (homo' typic adhesion) or between cells of different types (heterotypic adhesion).A CAM on one cell can directly bind to the samekind of CAM on an adiacent cell (homophilicbinding) or to a different class of CAM (heteropbilic binding). CAMs can be broadly distributed along the regions of plasma membranes that contact other cells or clustered in discretepatchesor spots called cell iunctions. Cell-cell adhesions can be tight and long lasting or relatively weak and transient. The associationsbetween nerve cells in the spinal cord or the metabolic cells in the liver exhibit tight adhesion. In contrast, immune-systemcells in the blood often exhibit only weak, short-lasting interactions' allowing them to roll along and pass through a blood vesselwall on their way to fight an infectionwithin a tissue. The cytosol-facingdomains of CAMs recruit setsof multifunctional adapter proteins (see Figure 19-1). These adapters act as linkers that directly or indirectly connect CAMs to elementsof the cytoskeleton(Chapters1'7and 18); they can also recruit intracellular moleculesthat function in signaling pathways to control protein activity-both intracellular proteins and the CAMs themselves-and gene expression(Chapters15 and 16). In many cases'a complex aggregateof CAMs, adapter proteins, and other associated proteins is assembledat the inner surfaceof the plasma membrane. Becausecell-celladhesionsare intrinsically associated with the cytoskeleton and signaling pathways' a cell's surroundings influence its shape and functional properties ("outside-in" effects);likewise, cellular shape and function influence a cell's surroundings ("inside-out" effects). Thus connectiuity and commwnication are intimately related properties of cells in tissues. The formation of many cell-cell adhesions entails two types of molecular interactions(Figure 19-3). First' CAMs on one cell associate laterally through their extracellular domains, cytosolic domains, or both into homodimers or higher-order oligomers in the plane of the cell's plasma membrane; these interactions are called intracellular, lateral, or cls interactions. Second, CAM oligomers on one cell bind to the sameor different CAMs on an adjacentcell; : N OVERVIEW X D H E S I O NA C E L L - C E LALN D C E L L - M A T R I A

803

Homophilic interactions Cadherins (E-cadherin)

Heterophilic interactions

lg-superfamily CAMs (NCAM)

Selectins (P-selectin)

Integrins (cwF3)

-/ Fibronectin

A cadherin

U il;]::il"

Glycoprotein

T v o el l l

(-) rgoo-ain Q tii"o*ain O ::fl:'"

FIGURE 19-2 Major familiesof cell-adhesion molecules(CAUs) and adhesionreceptors.Dimeric E-cadherins mostcommonly form (self)cross-bridges homophilic with E-cadherins on adjacent cells. Members (lg)superfamily of theimmunoglobulin of CAMscanform (shownhere)andheterophilic bothhomophilic Iinkages (nonself) (forexample, linkages. Heterodimeric integrins ctvandp3 chains) functionasCAMsor asadhesion (shownhere)thatbindto receptors verylarge,multiadhesive matrixproteins suchasfibronectin, onlya smallpartof whichisshownhereSelectins, shownasdimers. contain

a carbohydrate-binding lectindomainthat recognizes specialized sugar (shownhere)andglycolipids structures on glycoproteins on adjacent cellsNotethatCAMsoftenformhigher-order oligomers withinthe planeof the plasma membrane. Manyadhesive molecules contain multiple distinct domains, someof whicharefoundin morethanone kindof CAM.Therytoplasmic domains of theseproteins areoften associated with adapterproteins that linkthemto thecytoskeleton or pathways[See to signaling R.O Hynes, 1999, Trends Celt Biol.9(12):M33, andR O Hynes, 2002,Cell110:673-687 l

these interactions are called intercellular or trans interactions. Trans interactions sometimes induce additional cis interactions and, as a consequence,yet even more trans interactions. Adhesive interactions between cells vary considerably, depending on the parricular CAMs participating and the tissue.Just like Velcro, very tight adhesion can be gener-

ated when many weak interactionsare combined, and this is especially the case when CAMs are concentrated in small, well-defined areas,such as cellular junctions. Some CAMs require calcium ions to form effective adhesions; others do not. Furthermore, the associationof intracellular molecules with the cytosolic domains of CAMs can dramatically influence the intermolecular interactions of

> FIGURE 19-3 Modelforthe generation of cell-celladhesions.Lateralinteractions between (CAMt cell-adhesion molecules withinthe plasma membrane of a cellform dimersandlargeroligomersThepartsof the molecules that participate in thesecis Interactions varyamongthe different CAMs Subsequent transinteractions betweendistal domains of CAMson adjacent cellsgenerate a Velcro-like strongadhesion between the cells[Adapted fromM S Steinberg andp M McNutt, 1999,CurrOpinCellBiol11:5541

Cell 1 Cis + trans Cis (lateral) ------->

Trans

\

-----' Cis (lateral)

.a" Trans Cis + trans

Cell2

804

C H A P T E R1 9

I

INTEGRATING C E L L SI N T O T I S S U E S

s08

MltAUlno

'l'ltl-'l']tl N V : N O | S I H C VX t U I V I A - ' l ' l l ) C N V

urlceluaTuaSopr51 urlseuoJqrc

ururue'I

surelord xlrleur a^rsegPertlnw

''3'o; (n pue'1 '1sod,fu suo3e11oc rEInqr{ (ny ed& ''3'e) Sururo; raeg5

ffi

uElalJed

'sue8lo 'senssrtr{JIq^\ur tuaurdola,rapcruol.:gure1o e8ets eql-srsaueSoqdropdlquasse enssrl Jo sa8els dlrea aql ut dlrelnrrlred 'a,rou uef, sllof, rlllq.^ uo ro q8no.rqt elrl -lEI E sepr^o.rdxrrletu aqt 'uortrppe uI .uorterluarettrppue ql.uor8 IIar lorluot leqr selntoloru Surleu8rsrelnllerertxo dueu .ro; JroAJesaJ E sp selJes osle xrrteru aql .luauuor -r^ua slr qtr-4l. e IIal Jo suorlJeratureql olplnpotu ueo dl1ec -ol pazrseqtu.(sar pue 'pape.r8ap'pelapoura.rSuraqdlruels .op plnoqs -uor ore qrlq^\ 'stuauoduroc WIT ul saSueg3 tI leq,l.r sr tI rlJr.l/r\,,!\oul llar B Surltel 'sanc letuau Pue -uoJr^ueaprrro.rduel 'enssrl E Jo snlpls lecrSolorsdqdpue atrs lpJnuotpup eql uo Surpuadapdre,r uef, r{f,rr{,r\,*lrt"er{r to uorlrsodurosaql 'uortrppe uI .sonssrtlsolu ur uors -aqpEpue !a8elrlrer ur Suruorqsnclauog ro ,qtool ,uopual e ur qt8uarls :sasod.rndcryrredsJoJ xuletu Jelnllef,EJlxo aql rolrpr slueuodurocWlg to suorleurquol tuareJlrq '(p'cV-6I a.rnSrg) s11er e8uods lretur ol rElrlurs &lculcads E r.{tr,4d Jor.{loqlpa qlr.ry\olES -e.r33e'pexnu uoqm'qcrq,tr 'speeq pa.rolor leof, ol pasn pue pagt.rnd oq upf, suralord e^rser{peasagl .srordef,ererptrns eI^ sller egr 01 purq reqr WCg eql ur suralord e^rsaqpp luersttrp ol 'lred ut 'onp sl drpurtads srql 'sarcadsraqlo eql uro{ asoqt ol lou tnq rar{to qf,Eeol erer{pe11rltsanads euo sllar aql 'pexnu arB a8uods 1o sarcadsonu ruort 'o sllal pue polprlossrp dllerrueqcaru are sa8uods Ienpr^rpur '4ro.r, orsselt s(uoslrl( setEoJf-eJqllq^\ uor.l^. l?ql s.4d.or{s 'q'eV-6I o.rn8rg :aqlaSol anssn Surploq dlererry sl Wf,E Jo uollrunt dreurrd euo teqt lya,rrsnlcuocpo.la,oqs s11ao a8uods eurrPru ur uorseqpEJo sorpn]s JrssEIf s.uoslrlfi A 'H 'Altuet? trrctr osle uer lesrr WIE eql urqtr^\ salnf,elou aqr ;o Surped ;o drrsuapeql 'xrrleru a1rrr1 dla,trrelerqlr.,r s11arpafed dlasuap dral 1o pasodruocore sue8ro dueu searequn,xuleru dllsoru sr 'aluelsur .ro3:'anssrl elrlJauuoJ oruos .senssrl Ierurug

sueBe11o3 suecllSoalo.r4

ruerettrp SuouB dyrear8d.re,txrrleur snsro^ sllet Jo seunlo^ o^nelor aq1 'adeqs IIaJ puE uorlprgrtu IIaJ snr{rpuE-uorsegpe xrler.u-ller a1e1n3arosle deql 'xrneru relnllalerlxo aqr to sluauodurocJorllo oql Surzrur8ro Jot luelJodrur are surelord osarll 'salnralour Surleu8rsJelnlloJertxapue 's.roldafaruors -eqpe oreJrns-11ar'saprreqccesdlod'suratord xrrtetu reqlo 'ua8e11oc seddr snorrpl Surpurg ro; olqrsuodsarsureruop ;o aldrrynruurptuor reql selnJelou olqrxelJ '8uoy ere 'urururel 'suralord xrrleur Pue urlf,euoJqlJ suleloJd eqr se qcns a^rsaqperllnry '(f-gt alqet) sraqro pue lsuratord xrrlBu e^rsoqperllnu elqnlos lsraqr; urroy uoqo teql suralord 'sua8 -e11ocluratordoolp ed& anbrun e 'suecdlSoaroJdopnll 1o -ur stuouoduol WJA 'uorser{pe IIaJ tJarrpur sorprpeu WJg ar{l (sllar luacrlpe uo sJotderar qlr.ry\suortJereful WJE }o acuanbasuocE sV 'reqtoue euo ol pua (Z-61 a.rn8rgaas)ace; 'sur.r8alursEqtns 's.roldacaruorseqpe -rns IIaJ aqt uo pareJol ulplrar or qtog purq xrJleru rplnllererlxe aql yo sluauoduo3

s u o l l ) u n l l a q l o p u e ' 6 u ! l p u 6 ! s' u o r s o r { p vu ! s a l e d r ) t u e dx r J l e n J e l n l l e ) e r t x la q l ' (sarrradord lecrueqearu)urelsds ,(.rotelnrrrooqr ur sprnlJ Surpuno.rrnspue sllol Jo ./r\ol;tuol -nqrnl pue reurtupl orll ro alrsnru ur Suryyndpue Surqclans s e q f , n s s a f , r o JI p u r e l x o p u e ' ( s a r l r a d o r d l e o r r u e q l o l q ) uorsaqpe ol lredsoJ gtr-^,\sWVJ Jo sotels elrlf,pur snsrel e^rtf,Eoql '(sarl.radordeyqruasua)selnf,alou uorsoqpe Jo ((soruodo.rd dlrsuep ro uorlnqrusrp eqt cneurl) alnc lerleds -a1ourSurlrerolur r{f,EeJoJ uorlprfossrp pu€ uorlerf,osse }o soleJ((uo,, pue 6(uo,,IIBJa o eqt'(serilado;d rnueudpor.uJeql) selnralou Surlceratur aql ;o ftrury;e Surpurq eqt are sller o^u uae.4leq uorsaqpp Io arnteu 0q1 eurrurelep lPrlr salqe -rrerr.dueru aql Suouy 'uorlpruroJuof, Jreql Surralle dq ro (Surrorsnlr) uorterross€ srt rrer1t Surtoruord dq rWV:)

s l n s s r l 0 1 N l s ' l ' r : l )9 N r r v u 9 l l N l

|

6t ulldvH)

908

' 1arqso{e1e]l ' ]o uoss]e]snD pS pnv AN uuv'EOOZ arnlpN'EOOZ (uMoqs}ou)uorleu.uo} LlluPJq [ 0St-0tL:S66 |e le I tB8-gLB€Zt '3 qulpauojql]+ouol]lqlqul uror3l petn6t+stp pue poueiloqs qlolq qilM (uu6e1ul ue)lolde>t uolsaqpe sluaurala ^ueul osle lelala)s 'ursueMpol speolsluauoduJo) (Nl-lluv){poqque y\fl ^o) osoq]io eluasqv'(pal) (speaqMore) uorleuro]q)uerqpo))olq]uaulteaJl puepelelost aln)elour auoqpue(an;q) e6elrper aql ozrlensr^ urlrauorqr+ o] paulels alom url)auorqr,t-rluv tA)l oql;o fttntpeoqi q)olq ro (e)aluasqeaqi u! auunu (;q6r:)luat)Uap-ue)apad pueol spurq1eq1 {poqrlueue}o (q)aluasetd to '(-la;ua:) ]eq1so^rqura ]uollllop-ll ol paMolle 6ulqluelqooJopun ua6e;;or'(;;ay)leLutou sqdetOoloqd esaql'a)tu ul slnoqol ro] oJ]r^ursrsauaooLldJor.u lo suolala)snrroqs olnleulul puesorirqr-ua elennspue;6fuentles aurJnt!uorjl polPlost luaudo;anep lelelels a^rpolap ut sllnsotsulelord l4)l auos 'tVINlt 'sanssrlasnou 6urdo;anepu! slsauaooqdlotu6ulqruelq l)olq ro; seue6aql 6u!le^!peul v 9-6! lungH ilUldxl ulpeuorqll ol serpoqlluv s-6t lun9H lvINllNluldxS v

;;nu-uece;red

1 ; n u - ;u1a b e l l o c

edfiplrnn

'(9-61 an8r4) pere,Lrrceurere 'uecal.radro 11 ue3e11or 'selncelour WIE de{ o^4.1 Jo rarpre Jo, saua8eql ueq^^ of,rur cruodrqura;o uralsdsIprolJIS ogl ur soSueql lrlpuerp aql ur uaossr sE (sonssrlJo lueudolo^ep oql Jot sof,uanbasuof, Surtetsezrapoleq uEf, suorlf,Erelur llaJ-llal pup xrrlpru -llal ur suondn.lsrq '(g-61 arn8rg)suorloeralurxrneru-llef,

sarrnbar soJnlruls Jel{lo PuE 'spuep ,fuerrllespue dreu -tueur 'slassa^poolq '3un1 aql uI sf,ESJI€ aql urro; ol (sa.rn1 -rnJls Surqf,uerqto uorleuro;) slseuoSoqdJouSurqouerq 'uoISeqPE 'aldruexa roJ IIal-ll0l sE Ila.^ase uoIsaqPE sI osle-sluerueSuerre xrrl€ru-llol uo luoPuadap .t11ecr1rrc -er pue sluerualotu 11aodq PewJoJ are sl.rBd dpoq pue

uel^looatoldoqi q]lMpaleo)aloMspeaqpalaqel^llua)soronlJ lvtv-sjvig unwwo) 'ra6rng 'Z pue slla)eaf/uedetJpuoq4eH uaarbro pou(p)pue(t) '(mo11e{) saLlpv ,\ tApueMoqr.ref I pue Lt-88:09/)S ilD'9661. pe uolsaq y\ y\pueslanbsng-zapueural aM loryilD't0021a6rn6 Xulol]paldepvl (a6uero)slat etay1otd euoDoDIW ;o sdunp-otrtr-u6utLulo,t' q}tnnole6al66es;1eraql 6urtttlsa;1ua6 :rd{loLuoqrr,Loads-seoads plaqsdunp (x0t 'uorlerrlruOeu) uorsaqpe rrd{1ouoq{q raq1a6o1 aql aloMslla)lenpl^lpul ol paMolle q]rMsolnuruJ paxrr-u raLller selebar66e turol{;rpeer 0€- lo] alel)osseal l)urlsrpolurelquasse ]nq 'sa6uods pueuolldnlslp qloq;o 6urxtuL le)lueq)aul ])elurIo sad,i1 lou op speaqpaleor(ueer6)IVH pue(por)lVlA teqt sMoLls C loued (Molla^)eatued etrpuotpt9H raUV(q) pllMaql ut 6utnnotb pueper'o uorleurquo:) (uaerO salebar66e nno;;al{ 6urr-uro; laqtaOol oMI (e)'uolseqPe pue (aOuero) etayptdeuoDoDtrysa6uods paleber66e {aq1!yy1 q}lMIluo pa}eo)aroMspeoqparolo) 11e '(gyg)eenuedeupuoLpleH q6no.rqlalqtuessealsa6uods qloq sMoqs uaqM ro (lvy\) ]eql 11arrldfioruoq rr;lrads-sagrads ) loued {;;ertueqrayl t-6t lungll 'lvlN:ll llgldxl V etaylotdeuoDoDtry roqlle+on)l aq] uorJ (lV) rolre; uorlebar66e aulrerupele.redes

Disruptions in adhesion also are characteristicof various diseases,such as metastatic cancer, in which cancerous cells leave their normal locations and spread throughout the body. Although many CAMs and adhesionreceptorswere initially identified and characterizedbecauseof their adhesive properties, they also play a major role in signaling, using many of the pathways discussedin Chapters 15 and 16. Figure 1,9-7 illustrates how one adhesion receptor,integrin, physically and functionally interactsvia adaptersand signaling kinaseswith a broad array of intracellular signaling pathways, including those initiated by receptor tyrosine kinases, to influence cell survival, gene transcription, cytoskeletal organization, cell motility, and cell proliferation. Converseln changesin the activities of signaling pathways inside of cells can influence the structuresof CAMs and adhesion receptors and thus modulate their ability to interact with other cells and ECM.

The Evolutionof MultifacetedAdhesion M o l e c u l e sE n a b l e dt h e E v o l u t i o n of DiverseAnimal Tissues Cell-cell and cell-matrix adhesionsare responsiblefor the formation, composition, architecture,and function of animal tissues.Not surprisinglS some adhesion molecules are evolutionarily ancient and are among the most highly conserved proteins in multicellular organisms. Sponges, the most primitive multicellular organisms' express certain CAMs and multiadhesive ECM molecules whose structuresare strikingly similar to those of the corresponding human proteins. The evolution of organisms with complex tissuesand organs (metazoans)has dependedon the evolution of diverse adhesion molecules with novel propertiesand functions, whose levelsof expressiondiffer in different types of cells. Some CAMs (e.g.' cadherins), adhesion receptors (e.g., integrins and immunoglobulin

ECM Ligand Bound ligand

Integrin ( a dh e s i o n receptor) Exterior Plasmamembrane

Variousadaptorsand s i g n a l i n gk i n a s e s

Cytosol

Classic s i gn al i n g pathways

Cellular responses to adhesion recepror s i gn a l i n g

Cell proliferation(cycle) Cell survival Cytoskeletalorganization C e l lm i g r a t i o n G e n et r a n s c r i p t i o n

19-7 Integrinadhesionreceptor-mediated A FIGURE signalingpathwaysthat controldiversecellfunctions.Binding o f i n t e g r i ntso t h e i rl i g a n disn d u c ecso n f o r m a t i o ncahla n g eisn n ist h t h e i rc y t o p l a s mdiocm a i n sa,l t e r n a t i nt hge i ri n t e r a c t i ow ( r c-family p r o t e i n s k i n a s e s i n c l u d s e i g n a l i n g T h e s e cytoplasmic kinase[lLK])and kinaselFAKl,integrin-linked focaladhesion kinases, v i,n c u l i nt h) a tt r a n s m si ti g n a l s , axillin a d a p t opr r o t e i n(seg , t a l i n p

cellproliferation, pathways, therebyinfluencing signaling viadiverse andgene migration, cell organization, cytoskeletal cellsurvival, pathways shownhere of the components of the Many transcription. pathways, signaling with othercell-surface-activated areshared i ndC h a p t e r1s5a n d1 6 .[ M o d i f i ferdo mW G u oa n dF G discusse Giancotti, 2OO4,Nat Rev.Mol CellBiol 5(10):8'16-826,and R O Hynes, 2002, Cell 110:,673-687l

: N OVERVIEW X D H E S I O NA C E L L - C E LALN D C E L L - M A T R I A

807

superfamily CAMs), and ECM components(type IV collagen, laminin, nidogen/entactin,and perlecan-likeproteoglycans)are highly conserved,whereasothers are not. For example,fruit flies do not have certain types of collagenor the ECM protein fibronectin that play crucial roles in mammals. A common feature of adhesiveproteins is repeating domains forming very large proteins. The overall length of these molecules,combined with their ability to bind numerous ligands via distinct functional domains, likely played a role their evolution. The diversity of adhesivemoleculesarisesin large part from two phenomena that can generatenumerous closely related proteins, called isoforms, that constitute a protein family. In some cases,the different members of a protein family are encoded by multiple genes that ,.or. ]ro" common ancestor by gene duplication and divergent evolution (Chapter 6). In other cases,a single gene produces an RNA transcript that can undergo alternative splicing to yield multiple mRNAs, each encoding a distinct protein isoform (Chapter 8). Both phenomena contribute to the diversity of some protein families such as the cadherins.Particular isoforms of an adhesiveprotein are often expressedin some cell types and tissuesbut not others.

Cell-Celland Cell-Matrix Adhesion: An Overview r Cell-cell and cell-extracellularmatrix (ECM) interactions are critical for assembling cells into tissues, controlling cell shape and function, and determining the developmentalfate of cells and tissues.Diseasesresult from abnormalities in the srrucruresor expressronof adhesion molecules. r Cell-adhesionmolecules (CAMs) mediate direct cell-cell adhesions(homotypic and heterotypic),and cell-surface adhesion receptors mediate cell-matrix adhesions (see Figure 19-1). These interactions bind cells into tissues and facilitate communication between cells and their envlronments. The cytosolic domains of CAMs and adhesionreceprors nd adapter proteins that mediate interaction with cyskeletalfibers and intracellular signaling proteins. r The major families of cell-surface adhesion molecules are the cadherins,selectins,Ig-superfamily CAMs, and integrins(seeFigure 19-2). r Tight cell-cell adhesionsentail both cis (lateral or inrracellular) oligomerization of CAMs and trans (intercellular) interaction of like (homophilic) or different (heterophilic) CAMs (seeFigure 19-3).The combinationof cis and trans interactions produces a Velcro-like adhesionbetweencells. r The extracellular matrix (ECM) is a complex meshwork of proteins and polysaccharidesthat contributes to the structure and function of a tissue. The maior classesof ECM moleculesare proteoglycans,collagens,and multiadhesivematrix proteins (fibronectin, laminin). C H A P T E R1 9

|

TNTEGRATTN CG E L L St N T O T t S s U E S

r The evolution of adhesion molecules with specialized structures and functions permits cells to assembleinto diverseclassesof tissueswith varying functions.

Cell-Cell and CelI-ECM Junctions andTheirAdhesionMolecules Cells in epithelial and nonepithelial tissuesusemany, but not all, of the samecell-cell and cell-matrix adhesionmolecules. Becauseof the relatively simple organization of epithelia, as well as their fundamental role in evolution and development, we begin our detailed discussion of adhesion with the epithelium.

E p i t h e l i aC l e l l sH a v eD i s t i n c tA p i c a l ,L a t e r a l , a n d B a s a lS u r f a c e s Cells that form epithelial tissuesare said to be polarized becausetheir plasma membranes are organized into at least two discreteregions. TypicallS the distinct surfaces of a polarized epithelial cell are called the apical (top), lateral (side),and basal (baseor bottom) surfaces(Figure 19-8). The area of the apical surface is often greatly expanded by the formation of microvilli. Adhesion molecules play essentialroles in generating and maintaining thesestructures. Epithelia in different body locations have characteristic morphologiesand functions (Figure 19-8). Stratified (multilayered)epitheliacommonly serveas barriers and protective surfaces(e.g.,the skin), whereas simple (single-layer) epithelia often selectivelymove ions and small molecules from one side of the layer to the other. For instance, the simple columnar epitheliumlining the stomachsecreteshydrochloric acid into the lumen; a similar epithelium lining the small intestine transports products of digestion from the lumen of the intestine across the basolateral surface into the blood (seeFigure 1,1,-29). In simple columnar epithelia, adhesive interactions between the lateral surfaceshold the cells together into a two-dimensional sheet,whereasthose at the basal surface connect the cells to a specializedunderlying extracellular matrix called the basal lamina. Often the basal andlateral surfaces are similar in composition and together are called the basolateral surface.The basolateralsurfacesof most simple epithelia are usually on the side of the cell closestto the blood vessels,whereas the apical surface is not in direct contact with other cells or the ECM. In animals with closed circulatory systems, blood flows through vesselswhose inner lining is composed of flattened epithelial cells called endothelial cells. The apical side of endothelial cells, which facesthe blood. is usuallv called the lwminal surfaceand the opposite basal side, thl abluminal surface. In general, epithelial cells are sessile,immobile cells, in that adhesionmoleculesfirmly and stably attach them to one another and their associatedECM. One especiallyimportant

Although hundreds of individual adhesion-moleculemediated interactions are sufficient to causecells to adhere, junctions play specialroles in imparting strength and rigidity to a tissue,transmitting information betweenthe extracellular and the intracellular space, controlling the passageof Basal ions and moleculesacrosscell layers' and serving as conduits sudace for the movement of ions and moleculesfrom the cytoplasm Basal of one cell to that of its immediate neighbor.Particularly imlamina portant to epithelial sheetsis the formation of junctions that help form tight seals between the cells and thus allow the ( b )S i m p l es q u a m o u s sheetto serveas a barrier to the flow of moleculesfrom one side of the sheetto the other. Three maior classesof animal cell junctions are prominent featuresof simple columnar epithelia (Figure 1'9-9 and Table 19-2). Anchoring iunctions and tight iunctions perform the key task of holding the tissue together. These ( c )T r a n s i t i o n a l junctions are organized into three parts: (1) adhesiveproteins in the plasma membrane that connect one cell to another cell on the lateral surfaces(CAMs) or to the extracellular matrix on the basal surfaces (adhesion receptors); (2) adapterproteins, which connect the CAMs or adhesion receptors to cytoskeletalfilaments and signaling molecules; and (3 ) the cytoskeletalfilaments themselves.Tight junctions also control the flow of solutes through the extracellular spacesbetween the cells, forming an epithelial sheet.Tight junctions are found primarily in epithelial cells' whereas (d) Stratified squamous (nonkeratinized) anchoring junctions can be seen in both epithelial and nonepithelial cells. The third class of junctions, gap iunctions, permit the rapid diffusion of small, water-solublemolecules between the cytoplasm of adjacent cells. They share with anchoring and tight junctions the role of helping a cell communicatewith its environmentsbut are structurally very different from anchoring junctions and tight junctions and do not play a key role in strengthening cell-cell and cellECM adhesions.Gap junctions, found in both epithelial and '19-8Principal typesof epithelia.Theapicaland A FIGURE cells, resemble cell-cell iunctions in plants characteristics nonepithelial cellsexhibitdistinctive surfaces of epithelial basolateral which we discussin Section19.6' calledplasmodesmata, (a)Simple cells,including of elongated epithelia consist columnar Thiee types of anchoring junctions are present in cells. tract) andcervical cells(inthe liningof thestomach mucus-secreting Two participate in cell-cell adhesion,whereasthe third par(b)Simple cells(inthe liningof thesmallintestine) andabsorptive ticipatesin cell-matrix adhesion.Adherensiwnctionsconnect composed of thincells,linethe bloodvessels epithelia, squamous (c)Transitional the lateral membranes of adiacent epithelial cells and are (endothelial andmanybodycavities cells/endothelium) shapes, layers of cellswith different of several usually located near the apical surface,just below the tight composed epithelia, (e g the (Figure 1'9-9).A circumferential belt of actin and and contraction junctions subject to expansion cavities linecertain , (nonkeratinized) line epithelia squamous bladder)(d)Stratified urinary myosin filaments in a complex with the adherensiunction resist abrasion theselinings suchasthe mouthandvagina; surfaces functions as a tension cable that can internally brace the cell of or secretion in theabsorptron do not participate andgenerally and thereby control its shape. Epithelial and some other a thinfibrous Thebasallamina, intoor out of thecavity, materials types of cells,such as smooth muscle and heart cells,are also all supports andotherECMcomponents, networkof collagen bound tightly together by desmosomes,snaplike points of tissue connective themto the underlying andconnects epithelia contact sometimescalled spot desmosomes.Hemidesmosomes,found mainly on the basal surfaceof epithelial cells, mechanismused to generatestrong, stable adhesionsis to anchor an epithelium to components of the underlying exconcentratesubsetsof these moleculesinto clusters called tracellular matrix, much like nails holding down a carpet' runctlons. Bundlesof intermediatefilaments running parallel to the cell surface or through the cell interconnect spot desmosomes T h r e eT y p e so f J u n c t i o n sM e d i a t eM a n y C e l l - C e l l and hemidesmosomes'imparting shape and rigidity to the Interactions and Cell-ECM cell. Adherensiunctions and desmosomesare found in many different types of cells; hemidesmosomesappear to be reto one another All epithelialcells in a sheetare connected strictedto epithelialcells. and the extracellular matrix bv specialized iunctions. ( a ) S i m p l ec o l u m n a r

Apical surface

'-tfl?i::

J U N C T I O NASN D T H E I RA D H E S I O NM O L E C U L E S C E L L - C E LALN D C E L L - E C M

809

(a)

(b)

Apical surface Microvillus

Tight junction A d h e r e n sj u n c t i o n Lateral surface

Actin and myosin filaments G a pj u n c t i o n Intermediate filaments

BasaI surface

Desmosome Hemidesmosome $3ssl166in6-

.(

Lonnecilvetrssue t

FIGURE 19-9 Principaltypesof celljunctionsconnectingthe columnarepithelialcellsliningthe smallintestine.(a)Schematic cutawaydrawingof intestinal epithelial cellsThebasalsurface of the cellsrests on a basallamina, andtheapical sudace isoacked with fingerlike microvilli thatproject intotheintestinal lumenTightjunctions, lyingjustunderthemicrovilli, prevent thediffusron of manysubstances between theintestinal lumenandthebloodthrouqh theextracellular

s p a c eb e t w e e cne l l sG a pj u n c t i o nasl l o wt h e m o v e m e notf s m a l l molecules andionsbetweenthe cytosols of adjacent cells.The nn s c t i o n s ,p o t r e m a i n i ntgh r e et y p e so f j u n c t i o n s - a d h e r ej u desmosomes, andhemidesmosomes-are critical to cell-cell andcell(b)Electron matrixadhesion andsignaling micrograph of a thinsection of intestinal epithelial cells,showingrelative locations of thedifferent junctionsIPart (b)C Jacobson etal,2001,]ournat CeilBiot.152:435-450 l

Desmosomesand hemidesmosomes help transmit shear forces from one region of a cell layer to the epithelium as a whole, providing strength and rigidity to the enrire epithelial cell layer. They are especiallyimportant in maintaining the integrity of skin epithelia.For instance,mutations that interfere with hemidesmosomal anchoring in the skin can lead to blistering in which the epithelium becomesdetached from its matrix foundation and e*tracellular fluid accumulates at the basolateral surface, forcing the skin to balloon outward.

many different types of cadherinsin vertebrates,because many differenr types of cells in widely diverse rissuesuse these CAMs to mediate adhesion and communication. The brain expressesthe largest number of different cadherins, presumably owing to the necessityof forming many very specificcell-cellcontactsto help establishits complex wiring diagram. Invertebrates,however, are able to function with fewer than 20 cadherins.

C a d h e r i n sM e d i a t eC e l l - C e lAl d h e s i o n s i n A d h e r e n sJ u n c t i o n sa n d D e s m o s o m e s The primary CAMs in adherensjunctions and desmosomes belong to the cadherin family. In vertebrates, rhis protein family of more than 100 members can be qrouped into at least six subfamilies, including classicalcadherins and desmosomal cadherins, which we will describe below, as well asprotocadherinsand others. The diversity of cadherins arisesfrom the presenceof multiple cadherin genesand alternative RNA splicing. It is not surprising that there are 810

.

c H A p r E R1 9 |

T N T E G R A TC I NEGL L tSN T ol s s u E s

Classical Cadherins The "classical" cadherinsinclude E-, N-, and P-cadherins.E- and N-cadherinsare the most widely expressed,particularly during early differentiation. Sheetsof polarized epithelial cells,such as those that line the small intestineor kidney tubules,contain abundant E-cadherinalong their lateral surfaces.Although E-cadherinis concentratedin adherensjunctions, it is present throughout the lateral surfaces,where it is thought to link adjacentcell membranes.The results of experimentswith L cells, a line of cultured mouse fibroblasts, demonstratedthat E-cadherinspreferentiallymediate homophilic interactions. L cells expressno cadherins and adhere poorly to themselvesor to other cells.\(hen the

ADHESI0N TYPI

0R PRINCIPAI CAMS RECEPTOBS ADtlESION

1. Adherens junctions

Cell-cell

Cadherins

Actin filaments

Shape,tension, signaling

2. Desmosomes

Cell-cell

Desmosomal cadherins

Intermediate filaments

Strength, durability, slgnallng

3. Hemidesmosomes

Cell-matrix

Integrin(ct694)

Intermediatefilaments

Shape,rigiditv'signaling

Cell-cell

Occludin,claudin,JAMs

Actin filaments

Controllingsoluteflow, signaling

Connexins, innexins, pannexlns

Possibleindirect connectlons to cytoskeleton through adapters to other junctions

Communication; small-molecule transport between cells

Anchoring junctions

Tight junctions

Communication; molecule transport between cells

E-cadheringene was introduced into L cells, the engineered L cellswere found to adherepreferentially cadherin-expressing to other cells expressingE-cadherin (Figure 19-10). These L cells expressingE-cadherin formed epithelial-like aggregates with one another and with epithelialcellsisolatedfrom lungs. Although most E-cadherins exhibit primarily homophilic binding, somemediateheterophilicinteractions. The adhesivenessof cadherins dependson the presence of extracellular Caz*, the property that gave rise to their name (calcium adhering).For example,the adhesionof L cells expressingE-cadherin is prevented when the cells are No c a d h e r i nt r a n s g e n e

bathedin a solutionthat is low in Ca2* (Figure19-10).Some adhesion moleculesrequire some minimal amount of Ca"in the extracellular fluid to function properly, whereas others (e.g.,IgCAMs) are Ca2*-independent. The role of E-cadherin in adhesion can also be demonstrated in experiments with cultured epithelial cells called Madin-Darby canine kidney (MDCK) cells (Figure 9-34)' A green fluorescent-protein-labeledform of E-cadherin has been used in these cells to show that clusters of E-cadherin mediate the initial attachment and subsequent zippering

C a d h e r i nt r a n s g e n e

adherensiunctions. Each classicalcadherin contains a single transmembrane domain, a relatively short C-terminal cytosolic domain, and five extracellular "cadherin" domains (seeFigure 1'9-2)'The extracellular domains are necessaryfor Ca2* binding and cadherin-mediatedcell-cell adhesion. Cadherin-mediated adhesion entails both lateral (intracellular) and trans (intercellular)molecularinteractions(seeFigure 1,9-3)'TheCa"C a 2 * m e d i a t e s E c a d h e r i n F I G U R 1 E 9 1 0 E X P E R I M E N T A L a binding sites,located betweenthe cadherin repeats'serveto d e p e n d e na t d h e s i o no f L c e l l s .U n d e sr t a n d a rcde l lc u l t u r e rigidify the cadherin oligomers.The cadherin oligomers subfluid,L cells in theextracellular of calcium in the presence conditions form intercellular complexes to generatecell-cell of a genethat causes seq,retttly intosheets(/eff)Introduction do not aggregate then additional lateral contacts' resulting in a and in theiragqregation adhesion in thesecellsresults of E-cadherin the expression of cadherinsinto clusters.In this way' multiup" "zippering (center) but not of calcium clumpsin the presence intoepithelial-like interactions sum to produce a very tight pt.io*-alfittity (right)Bar,60pm [From L Adams etal, 1998, Cynthia rn itsabsence adhesion. intercellular J C e l lB i o l 1 4 2 ( 4 ) : 1 0 5 - 111 9 l 811

cELL-cELLANDcELLEcMJUNcT|oNsANDTHEIRADHESIoNMoLEcULEs

Podcast:E-cadherinZipper

not just the N-terminal domains, participate by interdigitation in trans associations. The C-terminal cytosolic domain of classicalcadherins Timeafter m i x i n gc e l l s is linked to the acin cytoskeleton by adapter proteins ( hr s) : (Figure 19-12). These linkages are essential for strong 0 adhesion, apparently owing primarily to their contributing to increasedlateral associations.For example, disruption of the interactions between classicalcadherins and a- or B-catenin-two common adapter proteins that link these cadherins to actin filaments-dramatically reduces A EXPERIMENTAL FtcURE19-11 E-cadherin mediates cadherin-mediatedcell-cell adhesion. This disruption ocadhesiveconnections in culturedMDCKepithelialcells.An curs spontaneously in tumor cells, which sometimesfail to E-cadherin genefusedto greenf luorescent protein(GFp) was express cr-catenin, and can be induced experimentally by introduced intocultured MDCKcells. Thecellswerethenmixed depleting the cytosolic pool of accessibleB-catenin.The together in a calcium-containing mediumandthedistribution of cytosolic domains of cadherinsalso interact with intracellufluorescent E-cadherin wasvisualized overtime(shownin hours). lar signalingmoleculessuch as B-cateninand p12O-catenin. Clusters of E-cadherin mediate theinitialattachment andsuosequent InterestinglS B-catenin not only mediates cytoskeletal zippering up of theepithelial cells.[From Cynthia L Adams erat, 1998. attachment but can also translocate to the nucleus and J CellBiol 142(4).1105-1j191 alter gene transcription in the ITnt signaling pathway (see Figure 16-32). Cadherinsplay a critical role during tissuedifferentiaThe resultsof domain swap experiments,in which an extion. Each classicalcadherin has a characteristictissuedistracellular domain of one kind of cadherin is replaced with tribution. In the course of differentiation, the amount or the correspondingdomain of a different cadherin, have indinature of the cell-surface cadherins changes, affecting cated that the specificity of binding resides,at least in part, many aspectsof cell-celladhesionand cell migration. For in the most distal (farthestfrom the membrane)extracellular instance,the reorganizationof tissuesduring morphogendomain, the N-terminal domain. Cadherin-mediatedadheesis is often accompaniedby the conversion of nonmotile sion was commonly thought to require only head-to-head epithelial cells into motile precursor cells for other tissues interactions between the N-terminal domains of cadherin (mesenchymal cells). Such epith elial-mesenchymal tr ansioligomers on adjacent cells, as depicted in Figure 19-12. tions are associatedwith a reduction in the expressionof However, some experimentssuggestthat under some condiE-cadherin (Figure 1,9-13a,b). The conversion of epithetions, at least three cadherin domains from each molecule. lial cells into malignant carcinoma cells, such as in certain

B-Catenin

Cell 1

Plasma membrane

A FIGURE 19-12Proteinconstituents of typicaladherens junctions.Theexoplasmic domains of E-cadherin dimersclustered junctions at adherens on adjacent cellsformCa+2-dependent homophilic interactions. Thecytosolic domains of the E_cadherins binddirectly or indirectly to multiple (eg , B_ proteins adapter catenrn) thatconnect thejunctions (F-actin) to actinfilaments of the

812

c H A P T E R1 9

|

T N T E G R A T TC NE GL L St N T OT T S S U E S

Plasma memorane

Cell 2

cytoskeleton andparticipate in intracellular pathways. signaling Somewhat different setsof adapter proteins areillustrated in the two cellsto emphasize thata variety of adapters caninteract with junctionsSomeof theseadapters, adherens suchasZOj , can interact with several different CAMs[Adapted fromV Vasioukhin and E Fuchs, 2001, Curr.OpinCellBiol 13:761

( a )A d h e r e n te p i t h e l i acl e l l s ( b ) M o t i l em e s e n c h y m a l cells

The cadherin desmogleinwas identified through studies of an unusual but revealing skin diseasecalled pemphigus uulgaris, an autoimmune disease.Patients with a u t o i m m u n e d i s o r d e r s s y n t h e s i z ea n t i b o d i e s t h a t b i n d to a normal body protein. In pemphigus vulgaris the autoantibodies disrupt adhesion between epithelial cells, causing blisters of the skin and mucous membranes.The predominant autoantibody was shown to be specific for

{al Plasma

( c )C a n c e r o u cs e l l s ,n o c a d h e r i n

membrane In t e r c eIlu l ar space

N o r m a lc e l l s i n e p i t h e l i alli n i n g o f g a s t r i cg l a n d s e x p r e s sc a d h e r i n FIGURE19-13 E-cadherinactivity is lost A EXPERIMENTAL during the epithelial-mesenchymaltransition and cancer of the expression progression.A proteincalledSnailthat suppresses transitions(a) is associated with epithelial-mesenchymal E-cadherin of the NormalepithelialMDCK cellsgrown in culture (b) Expression snallgene in MDCKcellscausesthem to undergoan epithelialdetectedby of E-cadherin transition(c) Distribution mesenchymal staining(darkbrown)in thin sectionsof tissue immunohistochemical is from a patientwith hereditarydiffusegastriccancerE-cadherin boardersof normalstomachgastricgland seenat the intercellular is seenat the bordersof epithelialcells(upper right);no E-cadherin (a)and(b)fromAlfonso underlyinginvasivecarcinomacells [Panels of M A Nieto) lmagescourtesy Arias,2001,Cell105:425-431; Martinez (c)fromF.Carneiro et al , 2004,J Pathol203(2):681-687 Panel l ductal breast tumors or hereditary diffuse gastric cancer (Figure 19-13c), is also marked by a loss of E-cadherin actrvrty.

D e s m o s o m a l C a d h e r i n s D e s m o s o m e s( F i g u r e 1 9 - 1 , 4 ) contain two specializedcadherin proteins, desmoglein and desmocollin, whose cytosolic domains are distinct from those in the classical cadherins. The cytosolic domains of desmosomalcadherins interact with adapters proteins such as plakoglobin (similar in structure to B-catenin), plakophilins, and a member of the plakin family of adapters called desmoplakin. These adapters, which form the thick cytoplasmic plaques characteristic of desmosomes,in turn interact with intermediatefilaments.

D e s m o g l e i na n d desmocollin (cadherins)

lntermediatefilaments

C y t o p l a s m i cp l a q u e (Plakoglobin, desmoPlakins, plakophilins) Cytoplasmicplaques

P l a s m am e m b r a n e s

,0'2pm ,

(a)Modelof a desmosome 19-14Desmosomes. A FIGURE of intermediate to thesrdes with attachments cells epithelial between anddesmocollin CAMsdesmoglein Thetransmembrane filaments, boundto the familyAdapterproteins belongto thecadherin plakoglobin, of the CAMsinclude domains cytoplasmic (b)Electron of a thin micrograph andplakophilins desmoplakins, differentiated two cultured connecting a desmosome of section from radiate filaments of intermediate Bundles humankeratinocytes plaques that linethe inner cytoplasmic thetwo darklystaining (a),seeB M plasma membranes lPart of theadjacent surface Curr'OpinCell 1993, R Garrod, D 11:551, and 1993,Neuron Gumbiner, of R vanBuskirk l Biol5:3OPart(b)courtesy

J U N C T I O NASN D T H E I RA D H E S I O NM O L E C U L E S C E L L - C E LALN D C E L L - E C M

813

desmoglein;indeed,the addition of such antibodiesto normal skin inducesthe formation of blistersand disruption of cell adhesion.I 6

The firm epithelialcell-celladhesionsmediatedby cadherinsin adherensjunctions permits the formation of a secondclassof intercellularjunctionsin epithelia-tight junctions.

c

T i g h t J u n c t i o n sS e a lO f f B o d y C a v i t i e sa n d R e s t r i cD t i f f u s i o no f M e m b r a n eC o m p o n e n t s For polarizedepithelialcellsto function as barriersand mediators of selectivetransport, extracellularfluids surrounding their apical and basolateralmembranesmust be kept separate.Tight lunctions berween adjacent epirhelial ceils are usuallylocatedin a band surroundingthe cell just below the apicalsurfaceand help establishand maintain cell polarity (Figure79-I5). Thesespecialized junctionsform a barrier that sealsoff body cavitiessuch as the intestinallumen and the blood (e.g.,the blood-brainbarrier). Tight junctions prevent the diffusion of macromolecules and, to varying degrees,small water-solublemoleculesand ions acrossan epithelialsheetvia the spacesbetweencells. They also maintain the polarity of epiihelial cells by preventing the diffusion of membrane proteins and glycolipids betweenthe apicaland the basolateralregionsof the plasma membrane, ensuring that these regions contain different membrane components. As a consequence,movement of many nutrients acrossthe intestinal epithelium is in large part through the trdnscellular pdthway via specific membrane-boundrransportproteins (seeFigure lI-29). Tight junctions are composedof thin bands of plasmamembrane proteins that completely encircle the cell and are in contact with similar thin bands on adiacent cells. When t h i n s e c t i o n so f c e l l sa r e v i e w e di n a n e l e c t r o nm i c r o s c o D e . the lateral surfacesof adjacentcells appear to touch each other at intervals and even to fuse in the zone just below the apical surface(seeFigure 19 -9b). In freeze-fracrurepreparations, tight junctions appear as an interlocking network of ridgesand groovesin the plasmamembrane(Figure 19-15a).

Intercellular space L i n k a g eo f p r o t e i p p a , r t i c l e si n a d j a c e n t ceils

i.

n:i'

> FIGURE 19-15Tightjunctions.(a)Freeze-fracture preparation of tightjunctionzonebetween two intestinal epithelial cellsThe fracture planepasses throughthe plasma membrane of oneof the two adjacent cellsA honeycomb-like networkof rldgesandgrooves belowthe microvilli constitutes thetightjunctlonzone (b)Schematic drawingshowshow a tightjunctionmightbe formedby the linkage of rowsof proteinparticles in adjacent cells.In the insermrcrograpn ( c ) of an ultrathin sectional viewof a tightjunction, the adlacent cells canbe seenin closecontactwherethe rowsof proterns interact(c) Asshownin theseschematic drawings of the majorproteins in tight j u n c t i o n bs o , t ho c c l u d iann dc l a u d i n -c1o n t a ifno u rt r a n s m e m b r a n e helices, whereas thejunctionadhesron (JAM)hasa single molecule transmembrane domarn anda largeextracellular (a)courtesy region[part of L A Staehelin Drawing in part(b)adapted fromL A Staehelin andB E Hull, 1978,9ci An 238(5):140,andD proc Nat,l.Acad Goodenough,1999, Sci USA96:319Photograph in part(b)courtesy of S Tsukita et al, 2001,NatureRev. Mol.CellBiol.2:285 Drawing in part(c)adapted fromS Tsukita et al, 2001. rVature ReizMol. CellBiol 2:2851

814

c H A p T E R1 9

|

I N T E G R A T T NCG E L L St N T OT t S S U E S

Rows of prorern particles

N

Occludin

Claudin-1

^ \/-: -/ N>

Very high magnification revealsthat rows of protein particles 3-4 nm in diameter form the ridges seen in freezefracturemicrographsof tight junctions.In the model shown in Figure 19-15b, the tight junction is formed by a double row of theseparticles,one row donatedby eachcell. Treatment of an epithelium with the proteasetrypsin destroysthe tight junctions,supportingthe proposalthat proteinsare essential structural components of these junctions. The two principal integral-membraneproteins found in tight iunctions are occludin and clawdin When investigators engineered mice with mutations inactivating the occludin gene, which was thought to be essentialfor tight junction formation, the mice still had morphologicallydistinct tight junctions. Further analysisled to the discoveryof claudin. Each of theseproteins has four membrane-spanningct helices(Figure 19-15c).The claudin multigenefamily encodesnumerous homologousproteinsthat exhibit distinct tissue-specific patternsof expression.A group of iunction adhesionmolecules (JAMs) have been found to contribute to homophilic adhesionand other functionsof tight junctions.Thesemolecules,which contain a singletransmembranect helix, belong to the Ig superfamily of CAMs. The extracellular domains of rows of occludin,claudin, and JAM proteins in the plasma membraneof one cell apparentlyform extremelytight links with similar rows of the same proteins in an adiacentcell, creating a tight seal. Ca2*-dependentcadherin-mediated adhesionalso plays an important role in tight junction formation, stability,and functton. The long C-terminalcytosolicsegmentof occludin binds to PDZ domains in certain large cytosolicadapterproteins' Thesedomains are found in various cytosolicproteins and mediate binding to the C-termini of particular plasmamembrane proteins or to each other. PDZ-containing adapter proteins associatedwith occludin are bound, in turn, to other cytoskeletaland signaling proteins and to actin fibers.Theseinteractionsappear to stabilizethe linkage betweenoccludin and claudin moleculesthat is essential for maintaining the integrity of tight iunctions. The Ctermini of claudins also bind to the intracellular,multiplePDZ-domain-containing adaptor protein ZO-1, which is also found in adherensjunctions(seeFigure 1'9-72).Thus, as cytosolic is the casefor adherensjunctionsand desmosomes, adaptor proteins and their connectionsto the cytoskeleton are critical componentsof tight junctions. Plasma-membraneproteins cannot diffuse in the plane of the membranepast tight lunctions. Theseiunctions also restrict the lateral movement of lipids in the exoplasmic leaflet of the plasma membrane in the apical and basolate r a l r e g i o n so f e p i t h e l i a lc e l l s .I n d e e d ,t h e l i p i d c o m p o s i tions of the exoplasmic leaflet in these two regions are distinct. Essentiallyall glycolipids are present in the exop l a s m i c f a c e o f t h e a p i c a l m e m b r a n e ,a s a r e a l l p r o t e i n s linked to the membrane by a glycosylphosphatidylinositol ( G P I ) a n c h o r ( s e eF i g u r e 1 0 - 1 9 ) . I n c o n t r a s t ,l i p i d s i n t h e cytosolic leaflet in the apical and basolateral regions of epithelial cells have the same composition and can apparently diffuse laterally from one region of the membraneto the other.

A simple experiment demonstratesthe impermeability of certain tight junctionsto many water-solublesubstances. In this experiment, lanthanum hydroxide (an electron-dense colloid of high molecularweight) is injectedinto the pancreatic blood vesselof an experimental animal; a few minutes later' the pancreatic epithelial acinar cells are fixed and prepared for microscopy. As shown in Figure 19-76, the lanthanum hydroxide diffuses from the blood into the spacethat separatesthe lateral surfacesof adjacentacinar cellsbut cannot p e n e t r a t ep a s tt h e t i g h t i u n c t i o n . The barrier to diffusion provided by tight junctions is not absolute.Owing at leastin part to the varying propertiesof the different types of claudin molecules located in different tight junctions, their permeability to ions, small molecules, and water varies enormously among different epithelial tissues.In epitheliawith "leaky" tight junctions, small molecules can move from one side of the cell layer to the other through the paracellularpathway in addition to the transcellular pathway (Figure19-17). The leakinessof tight junctions can be altered by intracellular signaling pathways, especiallyG protein and cyclic AMP-coupled pathways (Chapter 15). The regulation of tight junction permeability is often studied by measuringion flux (electricalresistance)or the movement of radioactive or fluorescentmoleculesacrossmonolayersof MDCK cells. p1fll The importance of paracellular transport is illustrated in severalhuman diseases.In hereditary hypoIl magnesemia,defectsin the cldudinlS geneprevent the normaL paracellular flow of magnesium in the kidney. This results in an abnormally low blood level of magnesium, which can lead to convulsions.Furthermore' a mutation in

Apical surface of left cell

Apical surface o f r i g h tc e l l

T i g h tj u n c t i o n

LateraI surface

Lateral surface o f r i g h tc e l l

of left cell

L a n t h a n u mh y d r o x i d e (betweencells)

19-16Tightjunctionsprevent FIGURE a EXPERIMENTAL space throughextracellular passageof largemolecules are in the pancreas betweenepithelialcells.Tightjunctions hydroxide lanthanum colloid to the largewater-soluble impermeable sideof the epithelium (darkstain)administered fromthe basolateral of D Friend lCourtesv l

J U N C T I O NASN D T H E I RA D H E S I O NM O L E C U L E S C E L L - C E LALN D C E L L - E C M

T

815

P a r a c e l l u l aTrr a n s c e l l u l a r pathway pathway

memorane

A FIGURE 19-17Transcellular and paracellular pathwaysof transepithelial transport.Transcellu lartransport requires the cellular uptakeof molecules on onesideandsubsequent release o n t h eo p p o s i tsei d eb y m e c h a n i s m d is c u s s iendC h a p t e1r 1 I n paracellular transport, molecules moveextracellularly throughparts of tightjunctions, whosepermeability to smallmolecules andions depends on thecomposition of thejunctional components andthe physiologic stateof the epithelial cells[Adapted fromS Tsukita etal, 2001, NatureReuMol CellBiol2:2851

the claudinl4 genecauseshereditary deafness,apparently by altering transport around hair-cell epithelia in the cochlea of the inner ear. Toxins produced by Vibrio cholerae, which causes cholera, and several other enteric (gastrointestinaltract) bacteria alter the permeability barrier of the intestinal epithelium by altering the composirion or activiry of tight junctions. Other bacterial toxins can affectthe ion-pumping activity of membrane transport proteins in intestinal epithelial cells. Toxin-induced changes in tight junction permeability (increasedparacellulartransport) and in protein-mediatedion pumping (increasedtranscellulartransport) can result in massiveloss of internal body ions and water into the gastrointestinaltract, which in turn leadsto diarrhea and potentially lethal dehydration. I

I n t e g r i n sM e d i a t eC e l l - E C M Adhesions i n E p i t h e l i aC l ells

of anchoring junctions called hemidesmosomes(seeFigure 19-9a). Hemidesmosomescomprise several integral membrane proteins linked via cytoplasmic adaptor proteins (e.g.,plakins) to keratin-basedintermediatefilaments.The principal ECM adhesionreceptorin hemidesmosomes is integrin a684,. a member of the integrin family of proteins ( s e eF i g u r e1 9 - 2 ) .

816

.

c H A p r E R1 9 |

T N T E G R A T cTENLGL S t N T ol s s u E s

combinations, are known. A single B chain can interact with any one of multiple o chains,forming integrins that bind different ligands. This phenomenon of combinatorial diuersity allows a relatively small number of components to serve a large number of distinct functions. Although most cells expressseveraldistinct integrins that bind the sameor different ligands, many integrins are expressedpredominantly in certain types of cells. Not only do many integrins bind more than one ligand but severalof their ligands bind to multiple integrins. All integrins appear to have evolved from two ancient general subgroups: those that bind proteins containing the tripeptide sequenceArg-Gly-Asp, usually called the RGD sequence(e.g., fibronectin) and those that bind laminin. Severalintegrin o subunits contain a distinctive inserted domain, the l-domain, which can mediate binding of certain integrins (e.g.,cr1B1and e2B1) to various collagensin the ECM. Some integrins with I-domains are expressedexclusively on leukocytesand red and white blood cell precursor (hematopoietic)cells.Thesedomains recognizecell-adhesion moleculeson other cells, including members of the Ig superfamily (e.g., ICAMs, VCAMs), and thus participate in cellcell adhesion. Integrins typically exhibit low affinities for their ligands, with dissociation constants Kp between 10 6 and 10 7 mollL. However, the multiple weak interacrions generated by the binding of hundredsor thousandsof integrin molecules to their ligands on cells or in the extracellular matrix allow a cell to remain firmly anchored to its ligandexpresslngtarget. Parts of both the c and the B subunits of an integrin molecule contribute to the primary extracellular ligand-binding site (seeFigure 19-2). Ligand binding to integrins also requires the simultaneous binding of divalent cations. Like other cell-surfaceadhesivemolecules,the cytosolic region of integrins interacts with adapter proteins that in turn bind to the cytoskeletonand intracellular signalingmolecules.Most integrins are linked to the actin cytoskeleton, such as the ct6B1 and cr3B1 integrins that connect the basal surface of epithelial cells to the basal lamina via laminin. However, the cytosolic domain of the B4 chain in the a6B4 integrin in hemidesmosomes,which is much longer than those of other B integrins, binds to specializedadapter proteins that in turn interact with keratin-basedintermediatefilamenrs. As we will see,the diversity of integrins and their ECM ligands enablesintegrins to participate in a wide array of key biological processes,including the migration of cells to their correct locations in the formation of the body plan of an embryo (morphogenesis)and in the inflammatory response. The importance of integrins in diverse processesis highIighted by the defectsexhibited by knockout mice engineered to have mutations in each of almost all of the integrin subunit genes.These defectsinclude maior abnormalities in development,blood vesselformation, Ieukocytefunction. inflammation,bone remodeling,and hemosrasis. Despitetireir differences,all theseprocessesdepend on integrin-mediated regulated interactions between the cytoskeleton and either the ECM or CAMs on other cells.

ct1B1

Many types

Mainly

a291.

Many types

Mainly collagens; also laminins

(l381

Many types

Laminins

o"4pl

Hematopoietic cells

Fibronectin; VCAM-1

Ct581

Fibroblasts

Fibronectin

CI691

Many types

Laminins

ctl92

T lymphocytes

ctMp2

Monocytes

Platelets

a6g4

Epithelialcells

ICAM-2 Serumproteins(e.g.,C3b, fibrinogen,factor X); ICAM-1 S e r u mp r o t e i n s( e . g .f,i b r i n o gen,von'Willebrandfactor, vitronectin);fibronectin Laminin

'The

proteins' Some integrins are grouped into subfamilies having a common B subunit. Ligands shown in red are CAMs; all others are ECM or serum subunits can have multiple spliced isoforms w i r h d i f f e r e n t c y t o s o l i cd o m a i n s . souRCE:R. O. Hynes, L992, Cell 69:L1.

In addition to their adhesionfunction, integrins can mediate outside-inand inside-outsignaling(seeFigure 19-7). The engagementof integrins by their extracellular ligands can, through adapter proteins bound to the integrin cytosolic region, influence the cytoskeleton and intracellular signaling pathways (outside-in signaling). Conversely,intracellular signaling pathways can alter, from the cytoplasm, the structure of integrins and consequentlytheir abilities to adhereto their extracellular ligands and mediatecell-celland cell-matrix interactions (inside-out signaling). Integrinmediated signaling pathways influence processesas diverse as cell survival, cell proliferation, and programmed cell death (Chapter 27).

G a p J u n c t i o n sC o m p o s e do f C o n n e x i n sA l l o w S m a l lM o l e c u l e st o P a s sD i r e c t l yB e t w e e n A d j a c e n tC e l l s Early electron micrographs of virtually all animal cells that were in contact revealedsitesof cell-cellcontact with a characteristic intercellular gap (Figure 19-1,8a1.This feature prompted early morphologiststo call theseregionsgap junctions. In retrospect,the most important feature of thesejunctions is not the gap itself but a well-defined set of cylindrical particlesthat cross the gap and composepores connecting the cytoplasmsof adjacentcells.

In many tissues,large numbers of gap junctional particles cluster together in patches(e.g', along the lateral surfacesof epithelialcells;seeFigure 19-9). Vhen the plasma membrane is purified and then shearedinto small fragments' some piecesmainly containing patches of gap junctions are

The effectivepore size of gap junctions can be measured by inyectinga cell with a fluorescentdye covalently linked to moleculesof various sizesand observingwith a fluorescence microscope whether the dye passesinto neighboring cells' Gap junctions between mammalian cells permit the passage of moleculesas large as 1'2 nm in diameter.In insects,these junctions are permeableto moleculesas large as 2 nm in diameter.Generally speaking,moleculessmaller than 1200 Da passfreely and those larger than 2000 Da do not pass;the purr"g. of intermediate-sizedmoleculesis variable and limit.d. thus ions, many low-molecular-weight precursors of cellular macromolecules'products of intermediary metabolism, and small intracellular signaling moleculescan pass from cell to cell through gap junctions.

J U N C T I O NASN D T H E I RA D H E S I O NM O L E C U L E S C E L L - C E LALN D C E L L - E C M

'

817

(a)

Gap j un c t i o n 50 nm ,,"13 ,i1.;,,tl-,, 50 nm (c)

< F I G U R E1 9 - 1 8 G a p j u n c t i o n s . ( a )I n t h i s t h i n s e c t i o nt h r o u g ha g a p l u n c t i o nc o n n e c t i n g t w o m o u s el i v e rc e l l s t, h e t w o p l a s m a m e m b r a n e as r e c l o s e l ya s s o c i a t efdo r a d i s t a n c eo f s e v e r ahl u n d r e d n a n o m e t e r ss,e p a r a t e d b y a " g a p " o f 2 - 3 n m ( b )N u m e r o u s r o u g h l yh e x a g o n apl a r t i c l eas r ev i s i b l ei n t h i s p e r p e n d i c u l aorr, e n face,view of the cytosolicface of a regionof plasmamembrane e n r i c h e di n g a p j u n c t i o n sE a c hp a r t i c l ea l i g n sw i t h a s i m i l a rp a r t i c l e o n a n a d j a c e nct e l l ,f o r m i n ga c h a n n e cl o n n e c t i n g the two cells ( c ) S c h e m a t im c o d e lo f a g a p l u n c t i o nc o n n e c t i n g two plasma m e m b r a n e sB o t h m e m b r a n e cs o n t a i nc o n n e x o nh e m i c h a n n e l s , c y l i n d e ros f s i xd u m b b e l l - s h a p ecdo n n e x i nm o l e c u l e sT w o c o n n e x o njso i n i n t h e g a p b e t w e e nt h e c e l l st o f o r m a g a p - l u n c t i o n channel,1 5-2 O nm in diameter,that connectsthe cytosolsof the t w o c e l l s ( d ) E l e c t r o nd e n s i t yo f a r e c o m b i n a ngt a p - j u n c t i o n channeldeterminedby electroncrystallography (Left)Sideview of the completestructureorientedas in part (c) N4: membrane bilayer;E : extracellular gap; C : cytosol (Rrgrht) View looking d o w n o n t h e c o n n e x o nf r o m t h e c y t o s o pl e r p e n d i c u l at or t h e m e m b r a n eb i l a y e r sS, u p e r i m p o s eodn t h e e l e c t r o nd e n s i t ym a p a r e m o d e l so f t h e t r a n s m e m b r a ncer h e l i c e s( g o l d ) f, o u r p e r s u b u n i t ,2 4 p e r c o n n e x o nh e m i c h a n n e[lP a r(ta )c o u r t e soyf D G o o d e n o u gpha r t( b ) courtesy of N GilulaPart(d)adapted fromS j Fleishman et al ,2004,Mol Ceil 15(6):879-888 l

Connexon hemichannel

+=*4

Gapjunction channel

Cytosol

I n t e r c e l l u l agr a p

In nervoustissue,someneuronsare connectedby gap junctions through which ions pass rapidly, thereby allowing very rapid transmission of electric signals. Impulse transmission through theseconnections,calledelectricalsynapses,is almost a thousandfoldas rapid as ar chemicalsynapses(Chapter23). Gap junctions are also presentln many non-neuronaltissues, where they help to integratethe electricaland metabolicactivities of many cells.In the heart, for instance,gap junctionsrapidly pass ionic signalsamong muscle cells, which are tightly interconnectedvia desmosomes,and thus contribute to the electricallystimulatedcoordinatecontraction of cardiac muscle cellsduring a beat.As discussedin Chapter 15, someextracellular hormonal signalsinduce the production or releaseof small intracellular signalingmoleculescalled secondmessengers (e.g., cyclic AMP, IP3, and Ca2*) that regulate cellular 818

.

c H A p r E R1 9 |

T N T E G R A T cTENLGL st N r o l s s u E s

metabolism. Becausesecond messengerscan be transferred betweencellsthrough gap junctions,hormonal stimulation of one cell can trigger a coordinatedresponseby that samecell and many of its neighbors.Suchgap junction-mediatedsignaling plays an important role, for example,in the secretionof digestiveenzymesby the pancreasand in the coordinatedmuscular contractile waves (peristalsis)in the intestine.Another vivid example of gap junction-mediatedtransport is the phenomenon of metabolic coupling,or metabolic cooperation,in which a cell transfersnutrients or intermediarymetabolitesto a neighboringcell that is itself unable to synrhesizethem. Gap junctions play critical roles in the developmentof egg cellsin the ovary by mediatingthe movementof both metabolitesand signaling moleculesbetween an oocyte and its surrounding granulosacellsas well as betweenneighboringgranulosacells.

A current model of the structure of the gap junction is shown in Figure 19-1,8c,d. Vertebrategap junctions are composed of connexins, a family of structurally related transmembrane proteins with molecular weights between 26,000 and 60,000.A completelydifferentfamily of proteins,the innexins, forms the gap junctions in invertebrates.A third family of innexinJike proteins, called pannexins,was recently discovered in both vertebratesand invertebrates.Each vertebrate hexagonal particle consists of 12 noncovalently associatedconnexin molecules: 6 form a cylindrical connexin hemichannel in one plasma membrane that is joined to a connexin hemichannelin the adjacent cell membrane, forming the continuous aqueous channel betrveenthe cells. Each individual connexin molecule spansthe plasma membrane four times with a topology similar Pannexinsare capableof to that of occludin (seeFigure1,9-1,5). forming intercellular channels as well; however, pannexin hemichannelsmay also function to permit direct exchange betweenthe intracellular and extracellular spaces. There are 21 differentconnexingenesin humans,with different setsof connexinsexpressedin different cell types.This diversity togetherwith the generationof mutant mice with inactivating mutations in connexin genes,has highlighted the importanceof connexinsin a wide variety of cellular systems. Some cells expressa single connexin that forms homotypic channels.Most cells,however,expressat leasttwo connexinsl thesedifferent proteins assembleinto heteromericconnexins, which in turn form heterotypicgap-junctionchannels.Diversity in channel composition leads to differencesin channel permeability. For example, channels made from a 43-kDa connexin isoform, Cx43-the most ubiquitously expressed connexin-are more than 1OO-foldas permeableto ADP and ATP as those made from Cx32 (32 kDa). The permeability of gap junctions^can be regulated by changesin the intracellular pH and Ca'* concentration and phosphorylation of connexin. One example of the physioIogical regulation of gap junctions is mammalian childbirth. The muscle cells in the mammalian uterus must contract strongly and synchronously during labor to expel the fetus. To facilitate this coordinate activity, immediately prior to and during labor there is an approximately five- to tenfold increasein the amount of the major myometrial connexin, Cx43, and an increasein the number and size of gap junctions, which decreaserapidly postpartum. Assemblyof connexins,their trafficking within cells,and formation of functional gap junctions apparently depend on N-cadherin and its associatedjunctional proteins (e.g., a- and B-cateninsZO-1,, ZO-2) as well as desmosomalproteins (plakoglobin, desmoplakin, and plakophrlin-2). PDZ domainsinZO-l andZO-2 bind to the C-terminusof Cx43 and apparently mediate its interaction with catenins and N-cadherin. The relevanceof these relationships is particularly evident in the heart, which depends on adjacent gap junctions (for rapid coordinated electrical coupling) and adherensjunctions and desmosomes(for mechanicalcoupling between cardiomyocytes)for the intercellular integration of electricalactivity and movement required for normal cardiac function. It is noteworthy that ZO-1 servesas an adaptor for adherens (see Figure 1,9-12),tight, and gap junctions,

suggestingthis and other adapters can help integrate the formation and functions of thesediverseiunctions. Mutations in connexin genescauseat least eight huincluding neurosensorydeafness(Cx26 man diseases, and Cx31). cataractor heart malformations (Cx43, Cx45, and CxS0). and the X-linked form of Charcot-Marie-Tooth disease(Cx32), which is marked by progressive degeneration of peripheral nerves.I

Cell-Celland CelI-ECMJunctions and Their Adhesion Molecules r Polarized epithelial cells have distinct apical, basal' and lateral surfaces.Microvilli projecting from the apical surfacesof many epithelial cellsconsiderablyexpand the cells' surfaceareas. r Three maior classesof cell junctions-anchoring junctions, tight junctions, and gap junctions-assemble epithelial cells into sheetsand mediate communication between them (seeFigures19-'l' and 19-9).Anchoring junctionscan be further subdivided into adherens iunctions' desmosomes,and hemidesmosomes. r Adherens junctions and desmosomes are cadherincontaining anchoring junctions that bind the membranesof adjacentcells,giving strengthand rigidity to the entire tissue. r Cadherinsare cell-adhesionmolecules(CAMs) responsible for Ca2*-dependent interactions between cells in epithelial and other tissues.They promote strong cell-cell adhesion by mediating both lateral intracellular and intercellular interactions. r Adapter proteins that bind to the cytosolic domain of cadherinsand other CAMs and adhesionreceptorsmediate the associationof cytoskeletaland signalingmoleculeswith the plasma membrane (seeFigure 1,9-12).Strong cell-cell adhesion dependson the linkage of the interacting CAMs to the cytoskeleton. r Tight junctions block the diffusion of proteins and some lipids in the plane of the plasma membrane'contributing to the polarity of epithelialcells.They also limit and regulatethe extracellular (paracellular)flow of water and solutesfrom one sideof the epitheliumto the other (seeFigure 19-1'7). r Hemidesmosomesare integrin-containing anchoring junctions that attach cellsto elementsof the underlying extracellular matrix. r Integrins are alarge family of crBheterodimeric cell-surface proteins that mediate both cell-cell and cell-matrix adhesions and inside-outand outside-insignalingin numeroustissues. ap junctions are constructed of multiple copiesof conn proteins, assembledinto a transmembrane channel interconnectsthe cytoplasmsof two adjacentcells (see Figure 19-18). Small moleculesand ions can passthrough gap junctions, permitting metabolic and electricalcoupling of adiacentcells.

J U N C T I O NASN D T H E I RA D H E S I O NM O L E C U L E S C E L L - C E LALN D C E L L . E C M

819

The Extracellular Matrixl: TheBasalLamina In animals, the extracellular matrix helps organize cells into tissuesand coordinatestheir cellular functions by activating intracellular signaling pathways that control cell growth, proliferation, and gene expression. Many functions of the matrix require transmembraneadhesionreceptorsthat bind directly to ECM components and that also interact, through adapter proteins, with the cytoskeleton.A principal class of adhesion receptors that mediate cell-matrix adhesion is integrins (Section 19.2). However, other types of molecules also function as important adhesionreceptors. Adhesion receprors bind to three types of molecules abundantin the extracellularmatrix of all tissues: r Proteoglycans,a group of glycoproteins that cushion cells and bind a wide variety of extracellular molecules r Collagen fibers, which provide structural integrity and mechanicalstrength and resilience r Solublemultiadhesivematrix proteins, such as laminin and fibronectin, which bind to and cross-link cell-surface adhesionreceptorsand other ECM components. We begin our description of the structuresand functions of thesemajor ECM components in the context of the basal lamina-the specializedextracellular matrix sheetthat plays (a)

a particularly important role in determining the overall architecture and function of epithelial tissue. In the next section, we discuss the ECM molecules commonly found in nonepithelial tissues,including connectivetissue.

T h e B a s a lL a m i n aP r o v i d e sa F o u n d a t i o n f o r A s s e m b l yo f C e l l si n t o T i s s u e s In animals, most organized groups of cells in epithelial and nonepithelial tissuesare underlain or surrounded by the basal lamina, a sheetlikemeshwork of ECM components usually no more than 60-120 nm thick (Figure 19-191. The basal lamina is structured differently in different tissues.In columnar and other epithelia (e.g., intestinal lining, skin), it is a foundation on which only one surface of the cellsrests.In other tissues,such as muscle or fat, the basal lamina surrounds each cell. Basal laminae play important roles in regeneration after tissue damage and in embryonic development. For insrance, the basal lamina helps four- and eight-celledembryos adhere together in a ball. In the development of the nervous system, neurons migrate along ECM pathways that contain basal lamina components.In higher animals, two distinct basal laminae are employed to form a tight barrier that limits diffusion of molecules between the blood and the brain (bloodbrain barrier), and in the kidney a specializedbasallamina serves as a selectivepermeability blood filter. Thus the basal lamina is important for organizing cells into tissues and distinct compartmenrs, tissue repair, and guiding (b)

Cytosol

Basalsurface

,e:

Connective tissue

Basal amina

A FIGURE19-19 The basal lamina separating epithelial cells and some other cells from connective tissue. (a)Transmission electronmicrographof a thin sectionof cells(top)and underlying connectivetissue(bottom) The electron-dense layerof the basal laminacan be seento follow the undulationof the basalsurfaceof the cells.(b) Electronmicrographof a quick-freeze deep-etch preparationof skeletalmuscleshowingthe relationof the plasma

820

c H A P T E R1 9

|

T N T E G R A T TC NE GL L St N T OT t S S U E S

Cell-surface receptorproteins

C o l l a g e nf i b e r s

membrane,basallamina,and surroundingconnectivetissue.In this preparation, the basallaminais revealedas a meshworkof filamentousproteinsthat associates with the plasmamembraneand the thickercollagenfibersof the connectivetissue [part(a)courtesy of P FitzGerald Part(b)from D W Fawcett,1981,TheCell,2ded, SaunderV PhotoResearchers; courtesy of JohnHeuser l

migrating cells during development.It is thereforenot surprising that basal lamina components have been highly conservedthroughout evolution. Most of the ECM components in the basal lamina are synthesizedby the cells that rest on it. Four ubiquitous protein components are found in basal laminae (Figure1,9-20):

L a m i n i n ,a M u l t i a d h e s i v eM a t r i x P r o t e i n ,H e l p s C r o s s - l i nC k o m p o n e n t so f t h e B a s a lL a m i n a

t Perlecan, alarge multidomain proteoglycan that binds to and cross-linksmany ECM componentsand cell-surface molecules

Laminin, the principal multiadhesivematrix protein in basal laminae, is a heterotrimeric, cross-shapedprotein with a total molecularweight of 820,000 (Figure1'9-21).At least 15 laminin isoforms, eachcontaining slightly different polypeptide chains, have been identified. Globular LG domains at the C-terminus of the laminin a subunit mediate Ca2*dependentbinding to specificcarbohydrateson certain cellsurfacemoleculessuch as syndecanand dystroglycan,which will be described further in Section 1,9.4.LG domains are found in a wide variety of proteins and can mediate binding to steroids and proteins as well as carbohydrates. For example, LG domains in the a chain of laminin can mediate binding to certain integrins, including a6B4 integrin in hemidesmosomeson the basal surfaces of epithelial cells. Laminin is the principal basal laminal ligand of a5B4 and other integrins(Table19-3).

t Nidogen (also called entactin), a rodlike molecule that cross-linkstype IV collagen,perlecan,and laminin and helps incorporate other componentsinto the ECM

TypelV Collagenls a Major Sheet-Forming StructuralComponentof the BasalLamina

Other ECM molecules are incorporated into various basal laminae, depending on the tissue and particular functional requirementsof the basal lamina. As depictedin Figure 19-1, one side of the basallamina is linked to cells by adhesionreceptors,including cr6B4integrin in hemidesmosomes,which binds to laminin in the basal lamina. The other side of the basal lamina is anchoredto the adjacentconnectivetissueby a layer of fibers of collagenembeddedin a proteoglycan-richmatrix. In stratified squamous epithelia (e.g.,skin), this linkage is mediatedby anchoring fibrils of type VII collagen. Together,the basal lamina and collagen-anchoringfibrils form the structure calledthe basement membrane.

Type IV collagen is a principal structural component of all basallaminaeand can bind to certain integrin adhesionreceptors. CollagenIV is one of more than2} typesof collagenthat participate in the formation of distinct extracellularmatrices in various tissues(Table 1'9-4).Although they differ in certain structural features and tissue distribution, all collagensare trimeric proteinsmade from three polypeptides,usually called collagen cr chains. All three ct chains can be identical (homotrimeric) or different (heterotrimeric).A trimeric collagen moleculecontains one or more three-strandedsegments,each with a similar triple-helical structure (Figure 19-22a1.Each strand contributed by one of the a chainsis twisted into a lefthanded helix, and three such strands from the three ct chains wrap around each other to form a right-handed triple helix.

. Type IV collagen, trimeric moleculeswith both rodlike and globular domains that form a two-dimensional network t Laminins, a family of multiadhesive,cross-shapedproteins that form a fibrous two-dimensional network with type IV collagen and that also bind to integrins and other adhesionreceptors

19-20 Major protein componentsof the basal A FIGURE andlaminin eachformtwo-dimensional lamina.TypelV collagen molecules whicharecross-linked by entactin andperlecan networks,

[Adaptedf rom B Albertset al , 1994, MolecularBiologyof the Cell,3d ed , G a r l a n dp, 9 9 1 l

T H E E X T R A C E L L U L AMRA T R I X l : T H E B A S A LL A M I N A

'

821

(a)

--

^yChain (205,000MW)

BChain ( 2 1 5 , 0 0M 0 W) Bindssulfated lipids

a Chain(400,000MW)

\-Y'J

B i n d st y p e l V collagen

B i n d sc o l l a g e n , s u l f a t e dl i p i d s

s-Helical coiled coil

, 25nm ,

A FIGURE 19-22The collagentriple helix.(a)(teft)Sideviewof thecrystal structure of a polypeptide f ragment whosesequence is basedon repeating setsof threeaminoacids,Gly-X-Y characteristic q.chains(Center) of collagen Eachchainistwistedintoa left-handed helix,andthreechains wraparoundeachotherto forma right-handed triplehelix.Theschematic model(nglht) clearly illustrates thetriple (b)Viewdowntheaxisof thetriple helical natureof thestructure. (orange) helixTheprotonsidechains of theglycine residues pointinto theverynarrowspacebetweenthe polypeptide chainsin thecenterof thetriplehelix.In mutations in collagen in whichotheraminoacids glycine, replace theprotonin glycine isreplaced by largergroups that disrupt the packing of thechains anddestabilize thetriple-helical structure fromR Z Kramer etal, 2001, J MolBiol311(1):131 lAdapted ] FIGURE 19-21Laminin,a heterotrimeric multiadhesive matrixproteinfound in all basallaminae.(a)Schematic model showingthegeneral shape,location of globular domains, andcoiledcoilregionin whichlaminin's threechains arecovalently linkedby several disulfide bonds.Different regions of lamininbindto cellsurface receptors andvarious (b)Electron matrixcomponents. micrographs of intactlamininmolecule, showingitscharacteristic (/eft)andthe carbohydrate-binding crossappearance LGdomains (right).leart(a) nearthe C-terminus adapted fromG R Martin andR Timpl, 1987, Ann Rev. Cell Biol3:57,andK yamada, 199j,J BiolChem 256:12809 Part(b)fromR Timplet al , 2000,MatrixBiol 19:309; photograph at rjghtcourtesy of JUrgen Engel l

The collagen triple helix can form becauseof an unusual abundanceof three amino acids:glycine,proline, and a modified form of proline called hydroxyproline (seeFigure 2-15). They make up the characteristicrepeatingmotif Gly-X-l where X and Y can be any amino acid but are often proline and hydroxyproline and less often lysine and hydroxylysine. Glycine is essentialbecauseits small side chain, a hydrogen atom, is the only one that can fit into the crowded centerof the three-strandedhelix (Figure19-22b). Hydrogen bonds help hold the three chains together. Although the rigid peptidyl-proline and peptidyl-hydroxyproline linkages are not compatible with formation of a classic single-strandedo. helix, they stabilize the distinctive threestrandedcollagen helix. The hydroxyl group in hydroxypro-

822

CHAPTER 19

I

I N T E G R A T I NCGE L L SI N T OT I S S U E S

Iine helps hold its ring in a conformation that stabilizesthe three-strandedhelix. The unique properties of each type of collagen are due mainly to differencesin (1) the number and lengths of the collagenous, triple-helical segments;(2) the segmentsthat flank or interrupt the triple-helical segmentsand that fold into other kinds of three-dimensionalstructures;and (3) the covalent modification of the cr chains (e.g., hydroxylation, glycosylation,oxidation, cross-linking).For example, the chains in type IV collagen,which is unique to basal laminae, are designatedIVo chains.Mammals expresssix homologous IVa chains,which assembleinto a seriesof type IV collagens with distinct properties. All subtypes of type IV collagen, however, form a 400-nm-long triple helix (Figure 19-23) that is interrupted about 24 times with nonhelical segmentsand flanked by large globular domains at the C-termini of the chains and smaller globular domains at the N,termini. The nonhelical regions introduce flexibility into the molecule. Through both lateral associarionsand interactions entailing the globular N- and C-termini, type IV collagen molecules assembleinto a branching, irregular two-dimensional fibrous network that forms the lattice on which the basal lamina is built (Figure 19-23). In the kidnen a double basal lamina, the glomerular basement membrane, separatesthe epithelium that Iines the urinary spacefrom the endothelium that lines the

€28

vNil tv'l

-lvsv8

lHl_ :t xtulvt^ uv]n't'll)vulxt

lH-L

'JSeqrroLuJrl I dreuorulnd puE ornlreJ ]puor o^rssa.rSord ur Surtlnsar e8euep re]nllal sesnef,lerlt esuodserounruurruE JJo sles Surpurg srql 's8un1pue ouerqruarutuarueseq.re1n.rauo13 er{t ur punol ua8ellol AI eddr ;o surprJJ€n eqr ot purg s a r p o g r l u p , , t o l n e , ,J o ' 3 u r > l J e l l p - 1 a s( a s € o s r po u n r u t u r o l -ne erpJ d1a,rrre1a.r e 'atuotpu[,s s,atrysadpoot ul'aanotp

-u{,ss,1,tof,1y se u.4ou1 uortrpuor e 'sanrletu:ouqpr€lnlo sE ernlret leuer pup ssol Surreaq lerneurrosuos se IIO.Aa loAI urEuaf,Jo ureu e.,lrsserSord qlr.{\ PelErfosseerP surEr{f, (eJuelsur -op re1nqo13 leurturet-J eql rerle ler{t suortelnur JoC 'aJnlrpJIpueJ ot peJI ueJ 'uorteruro3:aurJn lerlrur pup poolq eqt Jo uorrertlrJerlln ro; alqrsuodsarsr qrrqm 'ernl - r n l s s r r l l u r s l l J 1 J e ' s e r . r e l p d epr r l l r J - p o o J qS u r p u n o . r . r n s

' l I g Z t S 'I g l S V l ' I 6 6 I ' e u o ] r e D ' U p u e ''spa'uosa8:ng'g pue audetr41 'U q '/g6I 'ur{n) ') :s:rcxnos lsaUrePuP^ 'W pue '7 'd 'ssa:4rrurrpurv 'sad[1 ua3o11o2 ]o uotltung puo atnpntls

xrleqeldur yo s.reruo8rlg

(tuauelduoo) poolg

suralordaueJq ureu Jue{rrrJloruoH

se8eqdoneyq

areds re1oe,t1e'poo1g

s.roldererra8uazrecs V sselJ

sureuopurrcay!xr1ar1 aldur 1o sreruo8rlg SNECV-I'IO] ESNECECJSOH

ur>lsur seuosousePrrueH urIS ur seuosousePrueH

utalord Juerqrulr

yer8arul

uralord euPJqrueuryer8elul

E[(rrnx)ro] t[(urx)r"]

IIAX IIIX

SNACV'i'IO] ANVUSI^IEWSNVUJ slJsnr.uur eurluEl IesEqreeu !peardseprrl6

uec.,{13oarord are;1ns unrorpuorlr;o uratorda.ro3

uIIs eql Jo EuItuEIIPSeq.l!\olefl rEurruElIEsEqIIV

syrrqry 3uo1 o^aI Iro.4alJu IPuorsuelUrP-

€[(,,tx)r"] t[(rrn)r'o] [(ru)zo]'[(lr)1"]

SNECVT-IO] 9NIUOH]NV

sureuoprelngop crporradf1ad.{rqrr.aruort€rf,osse IErelE-I

senssrllertrtsretur tsory

CVC punoq lureruop-re1nqo13 Ieururrel-N !11addrqtr^\uonprrosse 1errtz.1

rorunqsnoalrl'e3e1lre3

AX IIA AI

CNV SNIWUOC-JEEHS

[(Xt)go][(Xt)Zn][(XI)fn]

[(n)2"][(n)t"] SNECVI]O] CE-IVI]OSSV-'IIUSIC

elJsnu qloorus 'ur>1s 'eluaoeld'auoq'qlaat'eeu;o3

I ed.,trgrr.uueqo luorsualxeleuruJel,N .reynqol3 qtr.^aslrrqrt3uo1-ruu-g6g

[t(n)rn]'[(,t)zoz(n)tn]

€[(u)r"]

sprqgSuoy-ruu-g6g

Jorunq snoeJlr,r'eBeplre3

€[(ur)r"]

3uo1-ruu-6gE 1 ad.{rqrr,rnuar;ofslr.rqry

slJssel poolq 'alcsnru 'ui1g

senssrlIErlrlsJalur'urluep 'stueue8rl'euoq'uopuet,ur45

slt.rqry 3uo1-uru-ggg

[(r)zn]z[(r)io] SNgSV'I'IO] UY'I]IUSIC

slnsstl t/\[vIN]studlu

er{t ot osrJ eArB leqt I-X-dlD Jo saruanbas epudadr.rt Surleadar ;o ecuesa.rdaqt .{q peqsrn8urtsrp surato:d ;o dlrue; ua8elyor oqt Jo requoru e sr ua8ellor n1 eddl r 'uaqnt [655Z:50! tolgilq'r'1961 ) 9 .p!g pueoruaq)rn^ lo)uaq)ln^ aas {sapno: (q) waq) ! led 6 9 L /Ot:S LZ 1o '0002'pnelnog y paldepe uro4 (e) te;nqo16 uedl.(Motlp)lql) sureLuop f suorpelolul aql pue'(Moljeutq])s]uauJoas leur[!Jol-)uaaM]aq uaaMloq 6urpurq aprs-ol-apts oql 'oln)aloul aql+o ;err;eq-e;du1 aql Lror'sllnsoro)ueleadde {re1aqf or}t^ut poulro+ fir;rqrxal; )loM}au ua6ello)4 edfu;oqder6ottruu uoll)el] (q) )ioM]aua)tl]oeqs e 'sexaldr-uo> pue'slorlellal'sloulpulJo]'sureLuop 6urp;er,i rapro-req6rq puepeoq-o]-peaq re;nqo;6 aq] uoaMlaq suor])eralur sellaM lre]-o1-lrpl se 'sluacubas suorpelalut ;errlaqaldu1uaoMlaq lelalel oln)oloulaql ul qur) alqxal;a)nporlur {q paldnlelursrxrlaq lelll sluau6as le)rlequou a1du1 aql snurulol-Jaql]e uteuopte;nqo;6 06.rel e puesnurula] -N oql]e ureurop relnqo;6 snoua6el;oruou lleuse seqaln)olour 6uo1-ruu-ggy srql uabe;;o:41 edl{1;ouorleluasatdet (e) )tleu.raq)S 'ua6e;;or ad^f {;quressepue ornpnrls N tZ-6t lungH V 1o

@

s S n s s r io r N r s ] ' 1 3 )9 N U . v u 9 l l N l |

6l UlrdvH)

vz8

'G.Z-et ern8rdaas)purq u€f, srotdorer uorseqp€pue sluouodurol I{Jg raqlo qJlq.ry\ 01 ploJJels o>lll{seurE luroJ o1 dlprarel PUE Pue ol Puo 'e8rel aq1 r lrpralur uaSeloc n1 ed,ft to selnrolorualqrxalJ 'slueuoduoJ WJA pue srol -dara.ruorsaqpeeldrrlnur pulq reql selnf,elou uleruoPlllntu arP surolord xrrlElu a^ISOqpEIllnuJeqlo puB uIuILuE'I r 'ur.r8elur 7$9rc yo pue8rlledroulrdaql sI EuIruEIIEsEqoql ut ututruel'(1-61 a.rn8rgoos)slueuoduoJ I JJE reqlo ol palrauuof, sI uJnl uI qcrqm'eururpl l€seq oqr or sllel rol{f,ue (saurosoursopturaq ur ur.r8elurt fgn ''3'e) sJoldeJoruolseqpe oleJrns-llal r 'ueSopruTurrpluapue'(uecdlSoelo.rde) uecalrad'ue8e11oc g adlr'(ura1o.rdxulelu e^Isaqpertpure) utunuel :(97-61an -3rg aas)opurluel lesegIIe ul punoJerBsutalordINJS rnod r 'auErqr.ueulueuesEq el{r PallEl ernlf,nrls E turo} IJo^&\ -lau uo8pllotrluecefpe dlalerpeururraqr pue EuItuEIIsseg al{r taqreSol 'anssrtolrlf,euuor lueoelpe uro{ sllol to sdnor8 pazr -ue8ro ror{lo pup eqeqlrdalsotu seleredas'salncelour(WCa) 'euturel IESeqeI{I r lPlnlla)ellxl aql xrrteur relnflo]Erxe to lro^.\qsau utqt e PulruP'lleseg aql:lxlJlen

uo^i\leu Al ad^I (q)

,o,rr,"oaao I

ureuop relnqolo

rourouour tr; uebeglo3

leuruJat-3

ureuop relnqolb leuruiel-N

lecrleqelo14

lecrlsquoN

(e)

'8urrr; apsnru sloJluot IEI{l uoltJunI rrynosnruorneu eql Jo uorlf,un;s,,tpol enp dlruaredde 'sarlqeurrougealfsnul ol ro r.usr;rei^pol raqllo ppol ueo eua8 uecalred eql ut suotl -Blnru 'suerunq uI 'ululruel ol Surpurq slr etl 1{1lrerpur PUE 'sureruop Cl stl etzrXylcerrp ueralred PuIq uEJ uec,(13orls -dp roldecar uorsaqpeaqJ 'IAtrlfl oeulluel-leseq-uouPUEeeu -rurcl leseq uI punot og uer uPJOIrad'3ur1ur1-ssonpolerPoru -ueroy.redur sllnser selntelotu asaql ot Surpurqsnoauetlntuls 's.rolreJqlmor8 oprrdad,{yod pur 'sroldeooJef,eJrns-llec'(ua8 -opru'urunuel ''3'a) stueuodruocINJA roqlo Surpnlcur'sa1nc 'satlredord Surpurq -olour reqto Jo suezop or sPulq uecel.red alrlf,urlsrp o^Eq reql sulrtlf aprrrqocesdlod pue suletuoP aldrrpu slr to osnerog 'oEuItuEIIeseq to uorlf,unJ pue ornl -f,nrls aql ourlaPPuE olut ale.rodroourol dltltqe sll ol elnglrl -uor urralred srueuodtuol CVD eqt PUEutelord oqr qrog to 'utalold arc) eqt 'poqreue eJEsurpr.{f, DVD alp LIJIq^\oluo polyecdlensn sr uecdlSoalord B to luonlllsuoc ulalord ag1 'suecdlSoalo.rdpolpc ere suletl3 CVC poqJrrle dpualerroc Surureluocsuralordocdlg'(mo1eq aas)sepueqooesrpSurleed -ar yo sraudlod 3uo1ore I{rIL{,vi'(sCVC) surr,tlSourursoc.{p '(71 rerdeq3) prrv'(V'6I uonca5)palull-O Pa1utl-rV:sureqr eprreqroes.{1od tuoprroc ;o sad,b oorql suleluoo lrgl (s.re8ns e sr uecapa4 poqleup dyluapz'ocqlr.tr uralo.rde) uta1o.rdocd18 'ueceFadolu€u eql eruaq t,(doosorllul uorlf,31adq pazqensta ueq,vrsl.reed1o Surrls v lo e)uercedde eqf lr orrt8sleedor rel -nqop duetu eq1 'suleruop 3y pue suletuoPC'I o>lII-uIuIuIEI Surpnlcur 'suretuop ltullslP aAIt Jo sleadel ;o dn epeur (eA4 0h=) uralord oJof uleIuoPrllnur a8rel E to slslsuof, 'uerapa4 'aeunuel IEsEquI uetdpoatord PalaJlosroleu aql

sJolda)aue)e+Jns-lla)pue sluouodtuo) eql eulue-l +o leseg e'ue)alled slurl-ssol)'ue)^looeloJd

collagen triple-helical structure (seeFigure 1,9-22).Different collagens are distinguished by the length and chemicalmodifications of their crchainsand by the presence of segmentsthat interrupt or flank their triple-helical reglons.

their own distinct fibrils in the matrix of most connectivetissues.Although severaltypes of cells are found in connective tissues,the various ECM components are produced largely by cells called fibroblasts.

r Perlecan,a large secretedproteoglycanpresentprimarily in the basal lamina, binds many ECM componentsand adhesionreceptors.Proteoglycansconsist of membraneassociatedor secretedcore proteins covalently linked to one or more specializedpolysaccharidechains called gly(GAGs). cosaminoglycans

F i b r i l l a rC o l l a g e n sA r e t h e M a j o r F i b r o u s Proteinsin the ECMof ConnectiveTissues

TheExtracellular Matrixll: and OtherTissues Connective Connectivetissue,such as tendon and cartilage,differs from other solid tissuesin that most of its volume is made up of extracellular matrix rather than cells. This matrix is packed with insoluble protein fibers and contains proteoglycans, various multiadhesive proteins, and a specialized glycosaminoglycancalled hyaluronan.The most abundant fibrous protein in connectivetissue is collagen.Rubberlike elastin fibers, which can be stretchedand relaxed,also are presentin deformablesites (e.g.,skin, tendons,heart). The fibronectins. a familv of multiadhesivematrix proteins. form

About 80-90 percent of the collagen in the body consistsof fibrillar collagens(types I, II, and III), located primarily in connectivetissues(seeTable L9-4).Becauseof its abundance in tendon-rich tissuesuch as rat tail, type I collagenis easyto isolate and was the first collagento be charactetized.Itsfundamental structural unit is a long (300-nm)' thin (1.5-nmdiameter)triple helix (seeFigure1,9-22)consistingof two ct1(I) chains and one cr2(I)chain, each 1050 amino acids in length. The triple-strandedmoleculesassociateinto higher-orderpolymers called collagen fibrils, which in turn often aggregateinto larger bundlescalledcollagenfibers (Figure1'9-24). Quantitatively minor classesof collagen include fibrilassociatedcollagens,which link the fibrillar collagensto one another or to other ECM compoflentsi sheet-forming and anchoring collagens, which form two-dimensional networks in basal laminae (type IV) and connect the basal lamina in skin to the underlying connective tissue (type VII); transmembrane collagens, which function as adhesion reand host defense ceptors(e.g.,BP180 in hemidesmosomes);

E Rough ER ctl

-'i3tldrl'f

i'r!s:*r!:r

'-si**'rr*-r1r5!r-.

Cytosol

i'&.

tatt.Utii::r.r.r1r&:rrrei:*1',:**i*i::iiiri!ir,ii,:iar.iri:iaa{iaifriii6iii{iif.*ti{a:'

6fE Extracellular space

\..

C o l l a g e nm o l e c u l e

I

of fibrillarcollagens. < FIGURE 19-24Biosynthesis on aresynthesized ctchains Step[: Procollagen (ER) reticulum withtheendoplasmic associated ribosomes are oligosaccharides andasparagine-linked membrane, propeptide StepZ: Propeptides addedto theC-terminal linkedby to formtrimersandarecovalently associate in the Gly-X-Y residues and selected bonds, disulfide (certain prolines modified arecovalently tripletrepeats galactose arehydroxylated, andlysines lGallor galactoseto somehydroxylysines, isattached glucose lhexagonsl StepE: The prolines arecis-+ transisomerized). stabilization formatron, zipperlike facilitate modifications protein andbindingbythe chaperone of triplehelices, or the helices 13),whichmaystabilize Hsp47(Chapter of thetrimersor both premature prevent aggregation aretransported Steps4 andEl: Thefoldedprocollagens wheresomelateral to andthroughthe Golgiapparatus, a s s o c r a t ironnt os m a lbl u n d l etsa k e sp l a c eT h ec h a i n s (step6), the N- andC-terminal arethensecreted (stepZ), andthe trimers propeptrdes areremoved a s s e m bilnet of i b r i l sa n da r ec o v a l e n tcl yr o s s - l i n k e d givesthe of thetrrmers (stepts). The67-nmstaggering micrographs in electron appearance fibrilsa striated 2002, andB Brodsky, (inset)[Adapted fromA V Persikov i SA99(3):1101-11031 l c a dS c U P r o cN a t ' A

f i n r i t a s s e m b l ya n d c r o s s - l i n k i n g

EN D O T H E RT l S S U E S T H E E X T R A C E L L U L AMRA T R I X l l : C O N N E C T I VA

825

collagens, which help the body recognize and eliminate pathogens.Interestingln severalcollagens (e.g.,types XVIII and XV) are also proteoglycans with covalently attached GAGs (seeTable l9-4).

Historically, British sailors were provided with limes to prevent scurvy, Ieading to their being called "limeys." Mutations in lysyl hydroxylasegenesalso can causeconnective-tissuedefects.I

F i b r i l l a rC o l l a g e nl s S e c r e t e da n d A s s e m b l e d i n t o F i b r i l sO u t s i d eo f t h e C e l l

TypeI and ll CollagensAssociatewith N o n f i b r i l l a rC o l l a g e n st o F o r mD i v e r s e Structures

Fibrillar collagensare secretedproteins, produced primarily by fibroblasts in the ECM. Collagen biosynthesisand secretion follow the normal pathway for a secretedprotein, described in detail in Chapters 13 and 14. The collagen ct chains are synthesizedas longer precursors,called pro-c chains, by ribosomes attached to the endoplasmicreticulum (ER). The pro-a chainsundergo a seriesof covalentmodifications and fold into triple-helical procollagen moleculesbefore their releasefrom cells (seeFigure 19-24). After the secretionof procollagen from the cell, extracellular peptidasesremove the N-terminal and C-terminal propeptides. In fibrillar collagens, the resulting molecules, which consist almost entirely of a triple-strandedhelix, associatelaterallyto generatefibrils with a diameterof 50-200 nm. In fibrils, adjacent collagen moleculesare displacedfrom one another by 67 nm, about one-quarterof their length. This staggeredarray produces a striated effect that can be seenin both light and electronmicroscopicimagesof collagenfibrils (Figure 19-24, inset). The unique properties of the fibrous collagens(e.g.,types I, II, ilI) are mainly due to the formation of fibrils. Short non-triple-helical segmentsat either end of the fibrillar collagen a chains are of particular importance in the formation of collagen fibrils. Lysine and hydroxylysine side chains in thesesegmentsare covalently modified by extracellular lysyl oxidasesto form aldehydesin place of the amine group at the end of the side chain. These reactive aldehyde groups form covalent cross-linkswith lysine, hydroxylysine, and histidine residuesin adjacent molecules.These crosslinks stabilizethe side-by,side packing of collagenmolecules and generatea very strong fibril. The removal of the propeptides and covalent cross-linking take place in the extracellular spaceto prevent the potentially catastrophic assemblyof fibrils within the cell. The post-translationalmodifications of pro-o chains are crucial for the formation of mature collagenmoleculesand their assemblyinto fibrils. Defectsin thesemodifications have serious consequences, as ancient mariners frequently experienced.For example, ascorbic acid (vitamin C) is an essentialcofactor for the hydroxylases responsiblefor adding hydroxyl groups to proline and lysine residuesin pro-ct chains. In cells deprived of ascorbate,as in the diseasescuruy,the pro-o.chainsare not hydroxylated sufficientlyto form stabletriple-helicalprocollagenat normal body temperature,and the procollagenthat forms can'sfithout not assembleinto normal fibrils. the structural support of collagen, blood vessels,tendons, and skin become fragile. Fresh fruit in the diet can suDDly sufficient vitamin C to support the formation of noi-rl collagen. 826

.

cHAprER 19 |

T N T E G R A TcTEN LG L tsN T ol s s u E s

Collagensdiffer in the structuresof the fibers they form and how these fibers are organized into networks. Of the predominant types of collagen found in connectivetissues,type I collagen forms long fibers, whereasnetworks of type II collagen are more meshlike. In tendons, for instance,the long type I collagen fibers connect muscles to bones and must withstand enormous forces. Becausetype I collagen fibers have great tensile strength,tendons can be stretchedwithout being broken. Indeed, gram for gram, type I collagen is stronger than steel.Two quantitatively minor fibrillar collagens,type V and type XI, coassembleinto fibers with type I collagen, thereby regulating the structuresand properties of the fibers. Incorporation of type V collagen,for example,results in smaller-diameterfibers. Type I collagen fibrils are also used as the reinforcing rods in the construction of bone. Bones and teeth are hard and strong becausethey contain large amounts of dahllite, a crystalline calcium- and phosphate-containingmineral. Most bones are about 70 percent mineral and 30 percent protein, the vast majority of which is type I collagen. Bones form when certain cells (chondrocytesand osteoblasts)secrete collagen fibrils that are then mineralizedby deposition of small dahllite crystals. In many connectivetissues,particularly skeletal muscle, type VI collagenand proteoglycansare noncovalently bound to the sidesof type I fibrils and may bind the fibrils together to form thicker collagen fibers (Figure 19-25a).Type VI collagen is unusual in that the molecule consistsof a relatively short triple helix with globular domains at both ends. The Iateral association of two type VI monomers generatesan "antiparallel" dimer. The end-to-end associationof these dimers through their globular domains forms type VI "microfibrils." These microfibrils have a beads-on-a-string appearance,with about 50-nm-long triple-helical regions separatedby 40-nm-long globular domains. The fibrils of type II collagen, the major collagen in cartilage, are smaller in diameter than type I fibrils and are oriented randomly in a viscousproteoglycan matrix. The rigid collagen fibrils impart strength to the matrix and allow it to resist large deformations in shape. Type II fibrils are crosslinked to matrix proteoglycansby type IX collagen,another fibril-associatedcollagen.Type IX collagenand severalrelated types have two or three triple-helical segmentsconnected by flexible kinks and an N-terminal globular segment (Figure 19-25b). The globular N-terminal segment of type IX collagen extends from the fibrils at the end of one of its helical segments,as does a GAG chain that is sometimes Iinked to one of the type IX chains. These protruding nonhelical structures are thought to anchor the type II fibril to

(a)

(b)

Type I collagenfibrils

Type ll collagenfibril

abnormalities. Skin abnormalities have also been reported with type VI collagen disease.I

and Their ConstituentGAGsPlay Proteoglycans DiverseRolesin the ECM

Chondroitin su lfate

TypeVl collagen

Proteoglycan

A FIGURE 19-25 Interactions of fibrouscollagenswith (a)In tendons, nonfibrousfibril-associated collagens. typeI fibrils arealloriented in the direction of thestress aoplied to thetendon. Proteoglycans andtypeVl collagen bindnoncovalently to fibrils, coatingthesurfaceThemicrofibrils of typeVl collagen, which containglobular andtriple-helical segments, bindto typeI fibrilsand linkthemtogetherintothickerfibers(b)In cartilage, typelX collagen molecules arecovalently boundat regular intervals alongtypell fibrils A chondroitin sulfate chain,covalently linkedto thea2(lX)chainat theflexible kink,projects outwardfromthefibril,asdoestheglobular N-terminal regionlPart(a),seeR R Bruns etal, 1986, I CellBiol103:393 Part(b),seeL M Shaw andB Olson, 1991,Trends Biochem Sci18:1 91.1 proteoglycansand other components of the matrix. The interrupted triple-helical structure of type IX and related collagensprevents them from assemblinginto fibrils, although they can associatewith fibrils formed from other collagen types and form covalent cross-linksto them. Mutations affecting type I collagen and its associated proteinscausea variety of human diseases.Certain mutations in the genesencodingthe type I collagena1(I) or o2(I) chains lead to osteogenesisimperfecta, or brittle-bone disease.Becauseevery third position in a collagen ct chain must be a glycine for the triple helix to form (seeFigure 1,9-22), mutations of glycine to almost any other amino acid are deleterious, resulting in poorly formed and unstablehelices.Only one defectivea chain of the three in a collagenmoleculecan disrupt the whole molecule'striple-helicalstructureand function. A mutation in a single copy (allele) of either the cr1(I) geneor the c2(I) gene,which are located on nonsex chromosomes(autosomes),can causethis disorder.Thus it normally shows autosomal dominant inheritance. Absence or malfunctioning of collagen-fibril-associated microfibrils in muscle tissuedue to mutations in the type VI collagen genescausedominant or recessivecongenital muscular dystrophieswith generalizedmuscleweakness,respiratory insufficiency,muscle wasting, and muscle-relatedjoint

As we saw with perlecan in the basal lamina, proteoglycans play an important role in cell-ECM adhesion.Proteoglycans are a subset of secretedor cell-surface-attachedglycoproteins containing covalently linked specializedpolysaccharide chains called glycosaminoglycans(GAGs). GAGs are long linear polymers of specific repeating disaccharides.Usually one sugaris either a uronic acid (o-glucuronic acid or L-iduronic acid) or o-galactose; the other sugar is N-acetylglucosamine or N-acetylgalactosamine(Figure 1'9-26).One or both of the sugars contain at least one anionic group (carboxylate or sulfate).Thus each GAG chain bearsmany negativecharges. GAGs are classified into several major types based on the nature of the repeating disaccharideunit: heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan. A hypersulfatedform of heparan sulfate called heparin, produced mostly by mast cells, plays a key role in allergic reactions.It is also used medically as an anticlotting drug becauseof its ability to activate a natural clotting inhibitor called antithrombin III. '!(ith the exception of hyaluronan, which is discussed below, all the major GAGs occur naturally as components of proteoglycans. Like other secretedand transmembrane glycoproteins, proteoglycan core proteins are synthesized on the endoplasmic reticulum (Chapter 13). The GAG chains are assembledon these cores in the Golgi complex. To generate heparan or chondroitin sulfate chains, a three-sugar"linker" is first attached to the hydroxyl side chains of certain serine residues in a core protein; thus the linker is an Olinked oligosaccharide(Figure L9-27a). In contrast, the linkers for the addition of keratan sulfate chains are oligosaccharidechains attached to asparagine residues; such Nlinked oligosaccharides are presentin many glycoproteins (Chapter 14), although only a subsetcarry GAG chains. All GAG chains are elongated by the alternating addition of sugar monomers to form the disacchariderepeatscharacteristicof a particular GAG; the chains are often modified subsequently by the covalent linkage of small molecules such as sulfate. The mechanismsresponsible for determining which proteins are modified with GAGs, the sequenceof disaccharidesto be added, the sitesto be sulfated, and the lengths of the GAG chains are unknown. The ratio of polysaccharideto protein in all proteoglycans is much higher than that in most other glycoproteins. Function of GAG Chain Modifications As is the casewith the sequenceof amino acids in proteins, the arrangement of the sugar residuesin GAG chains and the modification of specific sugars(e.g.,addition of sulfate) in the chains can determine their function and that of the proteoglycans containing them. For example, groupings of certain modified sugars in the GAG chains of heparin sulfate proteoglycans can control the binding of growth factors to certain receptors or the activities of proteins in the blood-clotting cascade.

EN D O T H E RT I S S U E S T H E E X T R A C E L L U L AMRA T R I X l l : C O N N E C T I VA

827

( a ) H y a l u r o n a n( n < 2 5 , 0 0 0 )

coo

P(l -+ a)

9 ( 1+ 3 )

NHCOCH3 D-Glucuronicacid

lV-AcetylD-glucosamine

(b) Chondroitin(or dermatan)sulfate (n s 250\ t>u3

For years, the chemical and structural complexity of proteoglycansposed a daunting barrier to an analysis of their structures and an understanding of their many diverse functions. In recent years, investigatorsemploying classicaland state-of-the-artbiochemical techniques(e.g., capillary highpressureliquid chromatography), mass spectrometry, and genetics have begun to elucidate the detailed structures and functions of theseubiquitous ECM molecules.The resultsof ongoing studies suggest that sets of sugar-residuesequences containing some modifications in common, rather than single unique sequences,are responsiblefor specifying distinct GAG functions.A casein point is a set of five-residue(pentasaccharide) sequencesfound in a subsetof heparin GAGs that control the activity of antithrombin III (ATIII), an inhibitor of the

4l

(a)

OH D-Glucuronic acid (or L-iduronic acidl

/V-AcetylD-galactosamine

1l 3 i r - r J A - * - G a t n l i E c ) .- G l c U A - G a \-------------Y

|-Ga

l-Xyl-

Ser

L---._YJ

Linkingsugars

Chondroitin sulfate repeals

(c) Heparin/Heparansulfate(n = 200)

I ,- proteoglycan corenroiein f-

$*i

Gal= galactose G I o U A= g l u c u r o n iac c i d GalNAc= N-acetylgalactosamine Xyl = xylose

coo

(b)

I1-- Mucin-like

OH (so3-) D-Glucuronicor L-iduronic acid

I O-tinked I glycoprotein SA-Gal-GalNAc-O-Ser

NHSO3-

(cocH3)

SA

N-Acetyl- or Al-sulfoD-glucosamine (c)

I f-

o-Dystroglycan

I - Ser SA_Gal_GlcNAc_Man-O

(d) Keratansulfate(n =20-401

P(l -+ a)

M a n= m a n n o s e GlcNAc= N-acetylglucosamine SA= sialicacid '19-27Hydroxyl(O-)linked polysaccharides. (a) A FIGURE (GAG), Synthesis of a glycosaminoglycan in thiscasechondroitin is initiated sulfate, by transfer of a xylose residue to a serineresidue FIGURE 19-26The repeatingdisaccharides of glycosaminogly- in thecoreprotein, mostlikelyin the Golgicomplex, followedby cans(GAGs),the polysaccharide componentsof proteoglycans. sequential addition of two galactose residues. Glucuronic acidandNEachof thefourclasses of GAGsisformedby polymerization of acetylgalactosamine residues arethenaddedsequentially to these monomerunitsintorepeats of a particular disaccharide andsubsequent linkingsugars, formingthechondroitin sulfatechainHeparan sulfate modifications, including additionof sulfategroupsandinversion chainsareconnected to coreproteins bythesamethree-sugar linker. (epimerization) groupon carbon5 of o-glucuronic of the carboxyl (b)Mucin-type O-linked chains arecovalently boundto glycoproteins acidto yieldr-iduronic acid Heparin isgenerated (GalNAc) by hypersulfation viaan N-acetylgalactosamine monosaccharide to whichare of heparan sulfate, whereas hyaluronan isunsulfated. Thenumber covalently attached a variety of othersugars(c)Certain specialized (n)of disaccharides typically foundin eachglycosaminoglycan chain O-linked oligosaccharides, suchasthosefoundin the protein isgiven.Thesquiggly linesrepresent covalent bondsthatareoriented dystroglycan, (Man) areboundto proteins viamannose eitherabove(o-glucuronic acid)or below(r-iduronic acid)the rinq. monosaccharides. 828

C H A P T E R1 9

|

T N T E G R A T T NCG E L L St N T O T T S S U E S

A FIGURE 19-28Pentasaccharide GAGsequence that regulatesthe activity of antithrombin lll (ATlll).Setsof modified five-residue sequences in the muchlongerGAGcalledheparin with thecomposition shownherebindto ATlllandactivate it, thereby inhibiting bloodclottingThesulfategroupsin redtypeareessential for thisheparin function; the modifications in bluetypemaybe present but arenot essential Othersetsof modified GAGsequences arethoughtto regulate the activity of othertargetproteins.

key blood-clotting proteasethrombin. When thesepentasacin heparin are sulfatedat two specificposicharide sequences tions, heparin can activateATIII, thereby inhibiting clot formation (Figure 19-28). Severalother sulfatescan be presentin the active pentasaccharidein various combinations, but they are not essentialfor the anticlotting activity of heparin. The rationale for generatingsetsof similar activesequences rather than a singleunique sequenceand the mechanismsthat control GAG biosynthetic pathways, permitting the generation of such activesequences, are not well understood, Diversity of Proteoglycans The proteoglycansconstitute a remarkably diversegroup of moleculesthat are abundant in the extracellularmatrix of all animal tissuesand are also expressedon the cell surface.For example, of the five major classesof heparan sulfate proteoglycans,three are located in the extracellularmatrix (perlecan,agrin, and type XVIII collagen) and two are cell-surfaceproteins. The latter include integral membrane proteins (syndecans)and GPl-anchored proteins (glypicans);the GAG chains in both types of cellsurface proteoglycans extend into the extracellular space. The sequencesand lengthsof proteoglycancore proteins vary considerably,and the number of attachedGAG chainsranges from just a few to more than 100. Moreover, a core protein is often linked to two different types of GAG chains,generating a "hybrid" proteoglycan. The basal laminal proteoglycan perlecanis primarily a heparan sulfate proteoglycan (HSPG) with three to four GAG chains, although it sometimescan have a bound chondroitin sulfate chain. Additional diversity in proteoglycansoccurs becausethe numbers of chains,compositions, and sequencesof the GAGs attachedto otherwise identical core proteins can differ considerably.Laboratory generationand analysisof mutants with defectsin proteoglycan production in Drosopbila melanogaster lfrurt fly), C. elegans(roundworm), and mice have clearly shown that proteoglycansplay critical roles in development,most likely as modulators of various signalingpathways. Syndecansare cell-surfaceproteoglycansexpressedby epithelial and nonepithelialcellsthat bind to collagensand mul-

tiadhesivematrix proteins(e.g.,fibronectin),anchoringcellsto the extracellular matrix. Like that of many integral membrane proteins, the cytosolic domain of syndecaninteracts with the actin cytoskeleton and in some caseswith intracellular regulatory molecules.In addition, cell-surfaceproteoglycans like syndecanbind many protein growth factors and other external signalingmolecules,thereby helping to regulatecellular metabolism and function. For instance,syndecansin the hypothalamic region of the brain modulate feeding behavior in response to food deprivation. They do so by participating in the binding to cell-surfacereceptors of antisatiety peptides that help control feeding behavior. In the fed state, the syndecanextracellular domain decorated with heparan sulfate chains is released from the surfaceby proteolysis,thus suppressingthe activity of the antisatiety peptides and feeding behavior. In mice engineeredto overexpressthe syndecan-l genein the hypothalamic region of the brain and other tissues,normal control of feeding by antisatietypeptidesis disrupted and the animals overeatand becomeobese.

HyaluronanResistsCompression,Facilitates C e l lM i g r a t i o n ,a n d G i v e sC a r t i l a g e Its Gel-likeProperties Hyaluronan, also called hyaluronic acid (HA) or hyaluronate, is a nonsulfated GAG (seeFigure 1,9-26a)made by a plasmamembrane-bound enzyme (HA synthase) that is directly secretedinto the extracellular space.(A similar approach is used by plant cellsto make their ECM component cellulose.) HA is a major component of the extracellular matrix that surrounds migrating and proliferating cells, particularly in embryonic tissues.In addition, hyaluronan forms the backbone of complex proteoglycan aggregatesfound in many extracellular matrices, particularly cartilage. Becauseof its remarkable physical properties,hyaluronan imparts stiffness and resilienceas well as a lubricating quality to many types of connectivetissuesuch as jolnts. Hyaluronan molecules range in length from a few disacchariderepeatsto =25,000. The typical hyaluronan in ioints such as the elbow has 10,000 repeatsfor a total mass of 4 x 106 Da and length of 10 pm (about the diameter of a small cell). Individual segmentsof a hyaluronan molecule fold into a rodlike conformation becauseof the B glycosidic linkages betweenthe sugarsand extensiveintrachain hydrogen bonding. Mutual repulsion between negatively charged carboxylate groups that protrude outward at regular intervals also contributes to these local rigid structures.Overall' however, hyaluronan is not a long, rigid rod as is fibrillar collagen; rather, in solution it is very flexible, bending and twisting into many conformations, forming a random coil. Becauseof the large number of anionic residueson its surface, the typical hyaluronan molecule binds a large amount of water and behavesas if it were a large hydrated spherewith a diameter of =500 nm. As the concentration of hyaluronan increases,the long chains begin to entangle, forming a viscousgel. Even at low concentrations,hyaluronan forms a hydrated gel; when placed in a confining space, such as in a matrix between two cells, the long hyaluronan

E N D O T H E RT I S S U E S T H E E X T R A C E L L U L AMRA T R I X l l : C O N N E C T I V A

829

moleculeswill tend to push outward. This outward pushing createsa swelling, or turgor pressure,within the extracellular space. In addition, the binding of cations by COOgroups on the surface of hyaluronan increasesthe concentration of ions and thus the osmotic pressurein the gel. As a result, large amounts of water are taken up into the matrix, contributing to the turgor pressure. These swelling forces give connectivetissuestheir ability to resist compression forces, in contrast with collagen fibers, which are able to resist stretching forces. Hyaluronan is bound to the surface of many migrating cells by a number of adhesion receptors (e.g., one called CD44) containing HA-binding domains, each with a similar three-dimensional conformation. Becauseof its loose, hydrated,porous nature,the hyaluronan"coat" bound to cells appearsto keep cellsapart from one another,giving them the freedom to move about and proliferate. The cessationof cell movement and the initiation of cell-cell attachmentsare frequently correlated with a decreasein hyaluronan, a decrease in HA-binding cell-surfacemolecules,and an increasein the extracellular enzyme hyaluronidase, which degrades hyaluronan in the matrix. Thesefunctions of hyaluronan are particularly important during the many cell migrations that facilitate differentiation and in the releaseof a mammalian egg cell (oocyte) from its surrounding cells after ovulation. The predominant proteoglycan in cartilage, calledaggrecan, assembleswith hyaluronan into very large aggregates, illustrative of the complex structures that proteoglycans sometimesform. The backbone of the cartilageproteoglycan aggregateis a long molecule of hyaluronan to which multiple aggrecanmoleculesare bound tightly but noncovalently (Figure 19-29a). A single aggrecanaggregate,one of the largestmacromolecularcomplexesknown, can be more than 4 mm long and have a volume larger than that of abacterial cell. These aggregatesgive cartilage its unique gel-like properties and its resistanceto deformation, essentialfor distributing the load in weight-bearingJornts. The aggrecan core protein (=250,000 MW) has one N-terminal globular domain that binds with high affinity to a specific disaccharide sequencewithin hyaluronan. This specific sequenceis generated by covalent modification of some of the repeatingdisaccharidesin the hyaluronan chain. The interaction between aggrecanand hyaluronan is facilitated by a link protein that binds to both the aggrecancore protein and hyaluronan (Figure 19-29b). Aggrecan and the link protein have in common a "link" domain. =100 amino acids long, that is found in numerous matrix and cell-surface hyaluronan-binding proteins in both cartilaginous and noncartilaginoustissues.Almost certainly theseproteins arosein the course of evolution from a single ancestralgenethat encodedjust this domain.

FibronectinsInterconnectCellsand Matrix, I n f l u e n c i n gC e l lS h a p e ,D i f f e r e n t i a t i o n , and Movement Many different cell types synthesizefibronectin, an abundant multiadhesive matrix protein found in all vertebrates. 830

.

c H A p r E R1 9 |

T N T E G R A T Tc N GL st N T ol s s u E s EL

Aggrecan (b)

H v a l u r o n am n olecule

+

Linkprotein Keratan sulfate

N-terminal Hyaluronan-binding domain

Chondroitin sulfate

Linking sugars Aggrecancore protein

FIGURE 19-29 Structureof proteoglycanaggregatefrom cartilage.(a)Electron micrograph of an aggrecan aggregate from fetalbovineepiphyseal cartilageAggrecan coreproteins arebound (b)Schematic at =40-nmintervals to a molecule of hyaluronan representation of an aggrecan monomer boundto hyaluronan In aggrecan, bothkeratan sulfateandchondroitin sulfatechains are attached to the coreprotein. TheN-terminal domainof thecore proteinbindsnoncovalently to a hyaluronan molecule. Binding is facilitated by a linkprotein, whichbindsto boththe hyaluronan molecule andthe aggrecan coreprotein. Eachaggrecan coreprotein has127Ser-Gly sequences at whichGAGchains canbe addedThe molecular weightof an aggrecan monomer averages 2 x 106.The entireaggregate, whichmaycontainupwardof 100aggrecan monomers, hasa molecular weightin excess of 2 x 108andisabout aslargeasthe bacterium E coli.lPart(a)from J A Buckwalterand L Rosenberg, 1983, CollRe/Res3:489; courtesyof L Rosenberg l The discoverythat fibronectin functions as an adhesivemolecule stemmed from observations that it is oresent on the surfacesof normal fibroblastic cells, which udh.r. tightly to petri dishesin laboratory experiments,but is absentfrom the surfacesof tumorigenic (i.e., cancerous)cells, which adhere weakly. The 20 or so isoforms of fibronectin are generated by alternative splicing of the RNA transcript produced from a single gene (see Figure 4-16). Fibronecrins are essential

for the migration and differentiation of many cell types in embryogenesis.Theseproteins are also important for wound healing becausethey promote blood clotting and facilitate the migration of macrophagesand other immune cells into the affectedarea. Fibronectins help attach cells to the extracellular matrix by binding to other ECM components, particularly fibrous collagens and heparan sulfate proteoglycans, and to cellsurface adhesion receptors such as integrins (seeFigure 192). Through their interactions with adhesionreceptors(e.g., cr5B1 integrin), fibronectins influence the shape and movement of cells and the organization of the cytoskeleton.ConverselSby regulating their receptor-mediatedattachmentsto fibronectin and other ECM components,cells can sculpt the immediate ECM environment to suit their needs. Fibronectins are dimers of two similar polypeptides linked at their C-termini by two disulfide bonds; each chain is about 50-70 nm long and 2-3 nm thick. Partial digestion of fibronectin with low amounts of proteasesand analysisof the fragments showed that each chain comprises several functional regions with different ligand-binding specificities (Figure 1.9-30a).Each region, in turn, contains multiple copies of certain sequencesthat can be classifiedinto one of three types. These classificationsare designatedfibronectin type I, II, and III repeats,on the basisof similaritiesin amino acid sequence,although the sequencesof any two repeatsof a given type are not identical. These linked repeatsgive the molecule the appearanceof beadson a string. The combination of different repeats composing the regions confers on fibronectin its ability to bind multiple ligands. One of the type III repeatsin the cell-binding region of fibronectin mediatesbinding to certain integrins. The results of studies with synthetic peptides correspondingto parts of

this repeat identified the tripeptide sequenceArg-Gly-Asp, usually called the RGD sequence,as the minimal sequence within this repeat required for recognition by those integrins. In one study, heptapeptidescontaining the RGD sequence or a variation of this sequencewere tested for their ability to mediate the adhesionof rat kidney cellsto a culture dish. The results showed that heptapeptidescontaining the RGD sequencemimicked intact fibronectin's ability to stimulate integrin-mediatedadhesion,whereasvariant heptapeptides lacking this sequencewere ineffective (Figure 19-31'). A three-dimensionalmodel of fibronectin binding to integrin based on structures of parts of both fibronectin and integrin has been assembled.In a high-resolution structure of the integrin-binding fibronectin type III repeat and its neighboring type III domain, the RGD sequenceis at the apex of a loop that protrudes outward from the molecule,in a position facilitating binding to integrins (see Figure 1930b). Although the RGD sequenceis required for binding to severalintegrins, its affinity for integrins is substantiallyless than that of intact fibronectin or of the entire cell-binding region in fibronectin. Thus structural features near the RGD sequencein fibronectins (e.g.,parts of adjacentrepeats,such as the synergyregion; seeFigure 19-30b) and in other RGDcontaining proteins enhance their binding to certain integrins. Moreover, the simple soluble dimeric forms of fibronectin produced by the liver or fibroblasts are initially in a nonfunctional conformation that binds poorly to integrins becausethe RGD sequenceis not readily accessible.The adsorption of fibronectin to a collagen matrix or the basallamina or, experimentallg to a plastic tissue-culturedish results in a conformational changethat enhancesits ability to bind to cells. Possibl5 this conformational change increasesthe accessibilityof the RGD sequencefor integrin binding'

(a)

(b) Fibrin, heparan suifate coilagen binding binding

k

C e l lb i n d i n g '-------^.E l l l B ElllA

Heparan Fibrin sulfate binding binding

fypelrcpeat Type ll repeat

'ab

lllcs

Synergy regron RGD sequence

tvpe lll repeat

Integrin

A FIGURE 19-30Organizationof fibronectinand its binding isshowndockedbytwo to integrin.(a)Scale modelof f ibronectin to theextracellular domains of integrin. Onlyoneof typelll repeats whicharelinkedby disulfided bondsnear thetwo similar chains, in the dimeric theirC-termini, fibronectin molecule isshownEach of three chaincontains about2446aminoacidsandiscomposed (typel, ll,or lll repeats). The typesof repeating aminoacidsequences ElllA,ElllB-bothtypelll repeats-andlllCSdomainarevariably intothe structure at locations indicated by arrowsCirculating spliced fibronectin lacksoneor bothof ElllAandElllB. At leastfivedifferent maybe present in the lllCSregionasa resultof alternative sequences

multirepeat(seeFigure several 4-16).Eachchaincontains splicing bindingsites someof whichcontainspecific regions, containing fibrin(a sulfate, for heparan (madeup of multiple-binding repeats) integrins. andcell-surface collagen, of bloodclots), majorconstituent the cell-binding known as is also domain Theintegrin-binding from weredetermined s domains of fibronectin domainStructures (b)A high-resolution showsthat structure of the molecule. fragments (red)extends outwardin a loopfromits the RGDbindingsequence asthe typellldomainon the samesideof fibronectin compact binding (blue), high-affinity to also contributes which region synergy Cell84:161 al, 1996, D J Leahyet from ] to integrins. [Adapted

EN D O T H E RT I S S U E S T H E E X T R A C E L L U L AMRA T R I X l l : C O N N E C T I VA

831

1.4 E o

GRGDSPC

so 1 . 2

;

GRGDAPC PRGDVDC YKPGEGKRGDACEGDSG

!q 1^ @

; q)

€ o.a E 0.6

to disrupt fibronectin-integrin binding. Thus fibronectin moleculesremain bound to integrin while cell-generatedmechanical forces induce fibril formation. In effect, the integrins through adapter proteins transmit the intracellular forces generatedby the actin cytoskeletonto extracellular fibronectin. Gradually, the initially formed fibronectin fibrils mature into highly stable matrix components by covalent crossJinking. In some electron micrographic images, exterior fibronectin fibrils appear to be aligned in a seemingly continuous line with bundles of actin fibers within the cell (Figure 1,9-32b).These observations and the results from other studies provided the first example of a molecularly

o

(a)

E 0.4

GRADSPC GRGESPC GKGDSPC DREDSRC

o

* o.z 1

10 100 1000 (nmol/ml) Peptideconcentration

A EXPERIMENTAL FIGURE 19-31A specifictripeptidesequence (RGD)in the cell-bindingregion of fibronectinis requiredfor adhesionof cells.Thecell-binding regionof fibronectin contarns an integrin-binding hexapeptide sequence, GRGDSP in thesingle-letter aminoacidcode(seeFigure 2-14)Together with an additional C(C)residue terminal cysteine thisheptapeptide andseveral variants weresynthesized chemically. Differentconcentrations of each peptidewereaddedto polystyrene synthetic dishesthat hadthe (b) Fibronectin Cell Plasma Actin-containing proteinimmunoglobulin G (lgG)firmlyattached to theirsurfaces; the fibrils exterior membrane microfilaments peptides werethenchemically cross-linked to the lgG.Subsequently, cultured normalrat kidneycellswereaddedto the dishes and incubated for 30 minutes to allowadhesion. Afterthe nonbound cellswerewashedaway,the relative amountsof cellsthat had adhered firmlyweredetermined by staining the boundcellswith a dyeandmeasuring the intensity of thestaining with a spectrophotometer. Theresultsshownhereindicatethat cell adhesion increased abovethe background levelwith increasing peptideconcentration for thosepeptides containing the RGD sequence but notfor thevariants (modification lacking thissequence proc.Nat't. underlined). M D Pierschbacher andE Ruoslahti. [From 1984. A EXPERIMENTAL FIGURE 19-32Integrinsmediatelinkage Acad.Sci.USA81:5985.1 between fibronectinin the extracellularmatrix and the cytoskeleton.(a)lmmunofluorescent micrograph of a fixedcultured Microscopy and other experimental approaches (e.g., (green) fibroblast showingcolocalization of thea5p1 integrin and biochemical binding experiments) have demonstrated the actin-containing stress fibers(red).Thecellwasincubated withtwo role of integrins in crosslinking fibronectin and other ECM typesof monoclonal antibody: an integrin-specific antibody linkedto a greenfluorescing componentsto the cytoskeleton.For example,the colocalizadyeandan actin-specific antibody linkedto a red fluorescing dye.Stress fibersarelongbundles of actinmicrofilaments tion of cytoskeletalactin filaments and integrins within cells thatradiateinwardfrompointswherethecellcontacts a substratum. can be visualized by fluorescencemicroscopy (Figure I9-32a). At thedistalendof thesefibers,nearthe plasma membrane, the The binding of cell-surface integrins to fibronectin in the coincidence (green) of actin(red)andfibronectin-binding integrin matrix induces the actin cytoskeleton-dependent movement produces (b)Electron a yellowfluorescence. micrograph of the of someintegrin moleculesin the plane of the membrane.The junctionof fibronectin and actin fibers in a cultured fibroblast. ensuingmechanicaltension due to the relative movement of Individual actin-containing 7-nmmicrofilaments, components of a different integrins bound to a singlefibronectin dimer stretches stress fiber,endat the obliquely sectioned cellmembrane. The the fibronectin. This stretching promotes self-associationof microfilaments appearalignedin closeproximity to thethicker, fibronectins into multimeric fibrils. densely stained fibronectin fibrilson the outside of the cell lpart(a) The force needed to unfold and expose functional selff r o m JD u b a n d e ,t 1 a9 l 8 8J,. C eBl l i o l1. 0 7 : 1 3 8P5a r t ( b f r)o m lJ S i n g e r , associationsitesin fibronectin is much lessthan that needed 1979,Cell16:675; courtesy of l. J Singer; copyright 1979,MlT.l

832

C H A P T E R1 9

|

T N T E G R A T T NCG E L L St N T O T I S S U E S

€88

s ' l ' l : l ) l ' l [ o y \ t N o N c N V ] ' l U _ O N N t S N O U _ ) V U l t _ NI At t S I H C V

(leuorlrsoduot ,1err3o1odol pue eqr ter{t olprrp Ipf,rupr{f,or.u 'sef,pJrns -ur suorte^rosqo eseql uo parnllnJ slloJ op u?r{l reF senssrtur srsElgorqrJ esoqr a{rl ororu sar8010qd.rour pedeqs Jo -alpurds pue (uortrrelqord 11acJo soler pasearrur {llllqo* pue uorsor{pBretea:8 deldsrp suorlcun( Surroqcue ,,1e: -nlpu eroru,, eseqtqlra srselqorqrJperntlnJ 'Gt-et arn8rg) sluerurradxeornllnf,-llel ur posn ,(lerrddr unlpJtsqns telJ aqr uo Sur,tro.r8sllel ur ueessuorsaqperEIIrrqrJro IeJoJer{l uro{ dlr,rrlcBpue 'uorlnqrrlsrp 'adeqs ,uorlrsodruor ur lgqaeruos raJtIP sernlJuls Osa{I 'suorsar{pEc-t pellpt 'unlBDsqns WJA leuorsueurp-aorr{teqt ot suorseqpEurrol daqt ,sens -srl ro slloJ ruo{ pa^rrop seJrrlEru w]g Ieuorsueurp-aarql UI EJESISEIqO]qIJ UAqA. 'O IA UI SII3JrI]NSSPUNOJ PEJNIINJ -rns dlleurou ter{l luoruuorrlua WJA leuorsuourp-aaJqleql etBurxordde lyrood dluo suorlrpuoJesoql .EteJtsqnsrrlseld ro sse13reu uo ernllnr IIOOur u1\.or8srsryqorgr;ur dlluanb -eq lsotu Polpnfs uaog e^Eq sornlf,nrls o^IsaqpESurureluor -urr8etur 'sy1acyerleqrrdauouduBtu ur puno; q8noqryy 'Q-51emtu1 aas),{ttltloru IIal pue qurro;3 IIet rot sleu8rsluapuedep-uors -oqpe etelrlf,E pup sluauplrJ urlf,e ol lueruqJpl 1era1a>lsordc -tE elprpeu surelord leql patrrJossE pue roldepe JEInllaJEJlur .kt-et arn8rg aas)unceuorqr; Jo suezop ureluof, osle deql ol Surpurq 'eldurrxe ro; ,q8norqt xrJleru rplnllef,Erlxa aql ol sllal Ierleqrrdauou qletle salntJnrls elrseqpe sno -IrE^ eseql 's11ao leqaqlrdeur suortrun{ Surroqrue xulpru-llol e{l-I'(tt-5I ern8rg) salntelotu perolsnlroJ Jaqlo ro surr8elur azruSoce.r lgql serpogrluuJo esn oqr qtrl\ ddocsorcruraouac -seron[+dq pazr.rasqo dlrpeer erp sorntrnrls JSOqI .se{uosop -oq peller suorsaqperplnf,rr) ur sE se ,suorsaqperpllrrqrJ Ile,ry\ (suorsaqpp 'sexelduroc ,slceluoc ,suors pue C-t lecoy leooy -aqPE IeloJ pallBs sernlJnJls O^rsaqpssnorre^ ur salnf,elou reqlo qtr^\ perelsnlf, ore osle aupJqr.uau eurseldaqt ur surr8el -ur (s11alyeqaqlrdauouq .sllol lerlaqtrdauouur erueseqr op doqr fuorela>lsotdc pue WIE er{l uoa.{\leqa8prrq e turo; surr8 -e1ur'sr leql '(I-dI arn8rg ees) uo1a1e1so1dc eqr sluoruplrt Jo alprpourelur ot 'suralord raldepe q8norqr ,pue eururel lesrq eqt ol slloJ leryaqrrdetJauuof,surr8elur.pessnrsrpdpearle sy

luoruuolrnul leuo!suaulo-aaJqI ,!aql pue slla) uoa^,\logsleu6tsfelag surr6elul '8I pue s;a1deq3ur porelor orp 11 sadeqs rraqr d;rpou pue sllof, ladord teqt saJroJ Ief,ruer{f,ou aql ate.raue8ol pesn susrueqJeru relnllef,erlur pollerep oql 'uorsogpe Surrsel-3uo1alelpaur lerlt asoql sp lle,vr sp sllal Io tuaue^ou eqt roJ perdepe llercedsa ere t€ql suouf,e -relur e^lseqpEluersusrt eterpolu lpqt sarnlf,nrls areJrns_llaf, snorJe^ eqrJfsep azrr,uorltas sllll uI .uorlfatur Jo salrs onssrl otur sllar poolq arrq.r\ ;o 8uq,vr,e:reql sp r{Jns ,lueurolour sassocordpcrSolorg roqlo ur op laql se .uorl 11ao8ur,r1o,rur -plurotsuerl srql ur alor da1 e deld salnf,oloua^rsot{pe uors to .(uorlrsuen -sa.rdxa ur sa8ueq3 lerudqruesaurlrleqrrda eqr) sllar olrtotu erolu otur ruJoJsuel upJ sllor pqeqrrda ,(reruec ''3'e) selelscrSoloqred urelral ur pue Burleeqpuno^. ro lueur 'slleJrer{lo Jo xrrleur JEInlleJerxe -do1a,rop Surrnp'le,roelo141 ;o redel e qSnorqr ro ssorf,EI^.erJ ol elqe eq lsnu sllel lprl -aqlrdeuoueuros(senssrl lerlaqlrdauouul slsrxooslEuorsaqpE

(eproruuou)3ur1se1-3ue1 qcns q8noqrlv'uorlerluarotlrp roqr -rn; sao8rapunurnqaglrda aql Irlun ro sllar aqr yo ueds oJIIaqr lnoq8no.rql tsEI uef, pue alqprs dra,r ere uauo deql ,uorlerl -ueroJJrpSurrnp rurol erlaqrrdJur suotlrerJrur o^rseqperaryV

sllo) alrloruuoNpue elllolAu! suorpetolule^tsaqpv 'surrSetur lere,Lasdq pazruSooersr 'surelo.rdxrJleru Jeqlo otuos pue surlf,euoJq -rt ur punoJ'dsy-d15-3-ry acuenbas CCd aprrdadlrt oql r .6tel ern8rg eas)xrleur eql ol s[e] qlplle uef, snql pue (suecdpoelord'sua8elloc)sruauodruoc 'uorlerl WJg pue surr8aturroJ selrsSurpurq uretuof, daql -ueJeJJrp relnlleJ pue uoner8rur ur elor da4 e deld ler{l sural -ord xrrlBur olrsaqpertlnru ruepunqe are surlJeuorqlC r 'GZ-et e.rn8r4ees)xr.rleur al{l Jo soruedord yecrueqraruolrlourtsrP eqr ol olnqrrluof, (ueoar38e ''3'a) seyncaloruuecdlSoato;d eldulnru Jo urol -ord oroc oql or punog dllualerrocuoualnJelou ueuornpdq Ierluac e Suruleluoc sale8e.rSSeuecdlSoelord o3;e1 r 'sllaf, ol ueuornledq purq sroldecor uorseqpeoleJrnsllel urpue3 's11ao Surle.re;qordpue Sunefrtu ;o WJg ar{rJo tuau -oduoc:o[eru e sl 'CVC pererpdqdp8rq e ,ueuo.rn1ed11 r 'sroldeco.roreJrns-llarrraql ol solnf,elotuSuqeu8rs 1eu -relxo urpuer luese.rddyaqpue suorlJ€rolurxlrleruJleJ elpl -rlneJ sueJepudseql s€ qf,ns surrdlSoalo.rdore;rns-ge3 r 'uonpJlnsdq pagpour ueuo ore leqt seprrerllJrsrpIo sreur,tlodreeurl are grrg,vl'sureqc (CVC) uecdySoururesoc -.(p erour ro euo or pa>lull ,tpualeaoc suralo.rd arof, polarf, -0s ro poterf,osse-ouerqrueu lsrsuoJ suecd18oa1o.r4 r Jo '(t-6I OlqEIaes) sed.{r ue8eilor rer.{lo >lurl-ssorf,o1 ro 'slaaqs rurot ol 's[rg$ o]ur eterf,osseot suorSor Jelrlequoupue IpJrlar{rreqr ;o drrlqe aqr dq paqsrn8unsrpa;e sua8ellocsnorJel eql r 'ft2-et ern8rg aes)sraqrJpeller sarlquasse ra8rel ruro; UEJr.lcq^\ 'qrrqg pelpc sepunq otur pe>lu[-ssorcllluaye,roc orp pue 1{1e;aleyolerrossr 'pe,tea1core salnf,oloruuo8elloc -ord 'uorlarcos JaUV 'unlnf,rloJ orurseldopuaagt urqlr,vrua8 -eylocordpcqaq-eldrrr olq dlquasse rroqr pue sureqoD apptu dlll.eu;o uoTlef,rJrpouleJruar{f,eql qlr^\ ilel egr eprsursul8eq (III PUE'11'1 sadlt ''3'a) ua8ullor r€llugrJ;o srseqludsoql r 'sllol uer{t rer{rer (WJg) xrneu ;e1nl1ecenxo;o dn ePetusr atunlo^ stl Jo lsoul lErll ur senssrlprlos Jer{lo ruory sroJJIp'a8elrtrecpue uopuet sE qrns (onssrlolrlceuuo) r

senssllraqlo pue a^lpauuo) :ll xtrlen lplnlla)eJlxl aLlI

'PEerOsaPr^{ eq ol u^/\ou{,{\ou uoueruouaqde-sluauoduroc xlJleur JEInlloJ -Ertxa erll pup uoFle{so/c reynlleoerlureql uee^\laq e8prrq e Sururro; (urr8alur ue ''a'r) rotdace.ruorsaqpppeurtap [e^\

'slol3 Poolq Jo uollPluroJ eql ur urrSolursnll Jo olor arlt ol Surlseue'8urpee1golrssoJxa ol ouord are lrunqns ur.r8elur€d eql ur sDalop crlaue8qlrm a1doe4'uorleruJot lolf, ur eledrJrlJedpue surelord 3ur11oyr J€lnllererlxo or purq dyrqSrruoql uEf,urrSolursrql 'ecuenbas -uof, E sy 'ur.r8alur€dqllro ur a8ueqcleuorleruroJuor Surlel -Ef,EuE ur llnsor reqr ruseldoldc eqr ur sde,ttqledSurleu8rs ur sa8ueqc ernpur sleu8rs asagl 'sleu8rs rplnllarerlur sele -reue8 urqurorqt arudzuaSunlolJ eqr ro dev ,(q uoue,tuoe s.lalaluld er{l ro rottre} puerqollrlN uo^ pellrl urerord ry39 e8rel e ro uo8elloc or relareld e yo sroldecar Suqeu8rsraqlo erl Surpurqeql'lo1o Sunuro; E uI 'uortpurro;uoc (1uaq)elrpe -ur arll ur sr lr ssnEfeq'rop poolq E uorlEruro; eql ur eled to -rcrlrBd qrrr1l\. y1e'(urtceuo.rqrl'uaSourrqlJ''3'e) spue8rl 'o uralord s1ro1dpq8rr pulq louueJ dlpurou sralareld;o souerq -uau erusrld aql uo luasard urr8alur tdqIID oql .olets leseq slr uI 'lop poolq E Sururro; ur solnreloru Wlg qll^\ reqro8 -ol drunlc pue poolq eql ur atelnlrrl ler{l stuau8er; IIel ere srelereld'dlr,tnceSurpurqurr8etut 3ut11or1uordq parelnporu orp suourprelur xrrleru-lloJ ^{oq 1o aldurexe poo8 e sapr^t -ord ?vroleq ororu ur pessnJsrp'uorlrun; lalorEld lrElop .slla3 ror{lo ro IAtrf,a eqr grr.,ra uorlf,Erelurslr Sur.rallefqaraqr '(elerrrtce)uetq8rerls (af,uonbas .ro (ale,rrlceul) puoq rer{tre plno^. ut.r8atut aql -uof, E sV 'eterf,ossero eleredesraqtre ot slrpl oql eJJoI snql ol ro slrEl orp or Purg o1 sreldepe puE uaql ruor' olPrf,ossrP rElnllalEnur OsnEJPlnol slloJ eql to elPls lrloq€leru 0q1 ur Surpu8rsrelnlloJuJtulllqlq sa8ueqc'dlasrarruo3'sdemgred -ur Jo elEAnJEpue uotolo>lsotdceql ralle uaql ppoo srerdrpe osoqt ur sa8ueqc aqJ 'slrer oqt ruog etplf,osslpro ot purg reqlra 'lynser e se 'pue sller el{r ;o uorleredes otll ((esues,, ppoc sreldepp rplnllalerlul 'slrpt rrruseldoldc agl aleredes dlluenbasuor pue uelq8rerls ol olnf,elou eql alroJ plno,ry\ ernlrnns dllugle-nol 'ruaq aql ot slloJ rar{lo uo sWV:) ro selnrelou y{JA urelrar;o Surpurgaq1 'SurluuBIS lno-apls -ur pur ur-aprsrnoeterpou ol surr8atutyo drqtge aql roJ uoll -rueldxe olrlJuJllp ue aprzrordslapou lernlf,nJls esaql 'oleredesslrel rruseydo/c oqt pue 'Surpurgpue8tl (dltutge -q8rq) sureluop eql elrs Surpurqoqr uroJ .lelq8rl lpgl trurrad 'alels o,trlf,p ,,'lq8tells,, Jo uoneuJo1uoJ eql ur suorlrJelle

' I Z I L L - S 0 L t t t 6 Z a u a D s ' 1l0pl0a2u e u r r a r )ln ] (q)upd aas:ueLr.irar)nJ ) lo ^saunor I pupeppureA 'IOOZ' 1ara6ra9g '^aaarnleN €6LiZ'tolg 1e ilD low (e)uedllueuuor^uo,sllaloq] uo 6utpuedap L!or+ paseq-uu6alur ,fuen s11e> l{qpeurolsuorsaqpe 'adeqs puP'uorlnqulsrp aqi uotltsodulo) lo aqj (q)slueuodu-ror nll ]o xuleu leuotsuaullp -oarq]e uo lo (e)qsrpernlln)PJoa)efns atann sllaf slselqoJqt+ unnotb lel+aql uo AlDerlp 0q] uo (q)suotsaLlpe ueLUnq c-€ +osa)elrns pue(e)suorsaqpe le)o+ajearaquMoqssllo) parnlln)uo (uaer6) sornlrnrls o^rsaqpe l)elap aruo)saronllounururl o] pesnera^^spoqlaLu 'sllar lellaqlldauouut sar6oloqdlout snolJeAqll,t,\sarnl)nJlse^rseqPeolul Jalsnl) su1l6e1u1 €E-6tlUngtJ'lvtNl4tgtdxl >

s 3 n s s I o r N r s ] ] ] ) 9 N l L V U 9 3 l - N lI

Ol UrraVs:

r€8

aqr uI roqreSor punoq dlesop ere sllungns o^€ eql Jo sller 'Surpurqpue8tl drrurge-mo1 Ieurr.urol-Jcrurseldotdceqr pue drr aql lP alIS Surpurg-pue8q dluo s.tro11eelnlolotu aqt ;o 'Jlels eql Jo uorlErurotuof eql 'luag sI JOLuIPorelsI{ dn orp elrlreur eqt uI 'polr^Ilre ore sutr8alur uaqm ereld eIEr leql sa8ueqceqr to lapotu e papr,torderreqsutr8alurdq spue8q;o Surpurqeql Sune8tlsalul slueulredxa pue sorPnlsIernlJnJls arn8rg) ruro; (e,trlce)dtrulJJe-tl8tt{ to stlnser eql'Gt-6I E pup ruroJ (azrrlceur)drrulge-rrrol p :suolleluJo1uof,o1\\l ur lsrxa uer sutr8alur '11elou yr 'due1,,q Eulpulg ullEalul 'lueruo,\otu 1ac Surzrlolursuonlunt Jeqlo Pue uotle.rSlu IIOJur suoltf,eroturesaglJo alor oqr ol leflllrf, sr uorleln8er qrn5 'qtoq ro 'satltrrtlceSurpurq-pue8llrlel{l '(s1ale1uorsserdxe) sur.r8alurJo requnu eql Sunrp8ar dq suorlle.ralul xlrleru-ller peterparu-ulr8alut;o qt8uerrs aql lorluoJ dlelrsrnbxeUEJsllo3

pue tu e u a ^o w llo ) slo r tu o l6 u ; ; eu6!s uorsst{pvPe}elpoll-ulr6a}ul}o uol}eln6eu 'ro^II agl se qons 'sue8 -ro pue senssllxaldruoc :aqlo dpnls ol suollrPuof e>lII-o^IA ur eror.uaprzrordot pedola,repSuraq ere sWf,fl C-€ Jlraqr -uds pue IEJnleuqioq SurdoldruesruelsdssnoSopuy'sluo8B crtnaderaglotueqllErlualod of sllac snoroJuEf,PUEIPIurouJo '4ytu sasuodsareql Jo suoslredtuof,slturrad pur Jo sluonllls (sJJnlJnJls (IUIJ€ -uoc urelord toleur eql eloJJOS IEI{I Pellel slloJ lerlaqrrde relnrrrJ oIq-o^r^ ut atBraua8 ol 'Esrollerlaqrrde of,IAPUP dreurueru aqt strurad osle xlJl€tu (I-t eql urals.{sSurleu8rsrotdacar aseur{ autsor,(l:one; qrmor8 1eu -raprdaeql soluenlJulsutr8elut dq parerpeurSurpu8rsuI-ePIS -1no luapuadep-xlrteru(I-E eql'alduuxa rog'sgedrolunor porurolsuert dlsno.racuerIIOL{IPuE qler prleqrrde drBurureu '8utuot1cunl Sunnpord-1lltu pezIIEIJeds;o &rlrqers pue 'srsaueSoqdroureql Jo salPnls ernllnf, 11aodq palq8rlqBtq ueaq seq sllel to luetuuorllue C-t oql 1o ecueilodurr ag1 'slloJ seluedoJdrgtcads-anssneql ol atnqrrruor d1q to -eqord sJllslrelreret{f,xIrlEuI esogluI solueroulPcgtcads-ans -srl 'ilel e;o dlr.tllce pue adeqsaqr Suqlortuoc ur elor e ,(e1d (drrpqrxeg ''3'e) IIp xrJteu Jelnllererxe el{l Jo sarlredo.rd

u o r s o q pOe- e( q )

uorsoqpelecol (e)

Activationf

19-34 Model for integrin < FIGURE modelis activation.(teft)Themolecular of the basedon the x-raycrystalstructure regionof cvB3integrinin its extracellular ("bent")form,with thea low-affinity inactive, s u b u n i tn s h a d eosf b l u ea n dt h eB s u b u n i tn sites shades of red.Themajorligand-binding wherethe areat thetip of the molecule, propeller domainof thec subunit(darkblue) An RGD andBAdomatn(darkred)interact. peptideligandisshownin yellow.(Rrgrht) isthoughtto be due Activation of integrins thatinclude changes to conformational keymovements of the molecule; straightening which andBAdomains, nearthe propeller andseparation the affinityfor ligands; increases in altered resulting domains, of thecytoplasmic proteins. with adapter interactions lAdapted Opin.CellBiol et al, 2002,Curr. fromM Arnaout 2002,Cell11O:673]l 14:641, andR O Hynes,

basis of this diseasecame from the discovery that people with DMD carry mutations in the gene encoding a protein named dystrophin. This very large protein was found to be a cytosolic adapter protein, binding to actin filaments and to an adhesion receptor called dystroglycan. Dystroglycan is synthesizedas a large glycoprotein precursor that is proteolytically cleavedinto two subunits.The ct subunit is a peripheral membraneprotein' and the B subunit is a transmembraneprotein whose extracellulardomain associateswith the a subunit (Figure 1,9-35t.Multiple O-linked oligosaccharidesare attached covalently to side-chain hydroxyl groups of serine and threonine residues in the a subunit. Unlike the most abundant O-linked (also called mucin-like) oligosaccharides in which an N-acetylgalactosamine (GalNAc) is the first sugar in the chain linked directly to the hydroxy group of the side chain of serine or theonine or the linkage in proteoglycans' many of the Olinked chains in dystroglycan are directly linked to the hydroxyl group via a mannosesugar (seeFigure 1,9-271' These specializedO-linked oligosaccharidesbind to various basal lamina components' including the LG domains of multiadhesivematrix protein laminin and the proteoglycans ConnectionsBetweenthe ECM perlecanand agrin. The neurexins,a family of adhesionmoland CytoskeletonAre Defective ecules expressed by neurons' also are bound via these i n M u s c u l a rD y s t r o p h y oligosaccharides,whose detailed heterogeneousstructures The importance of the adhesion-receptor-mediated and mechanismsof synthesishave not been fully elucidated. The transmembrane segment of the dystroglycan B sublinkage between ECM components and the cytoskelewith a complex of integral membraneproteins; unit associates ton is highlighted by a set of hereditary muscle-wastingdisits cytosolic domain binds dystrophin and other adapter proeases,collectively called muscular dystrophies. Duchenne teins, as well as various intracellular signaling proteins (Figmuscular dystrophy (DMD), the most common type, is a ure 19-35). The resulting large, heteromericassemblage,the sex-linked disorder, affecting 1 in 3300 boys, that results in dystrophin gly coprotein complex (D GC), links the extracellucardiac or respiratory failure, usually in the late teens or lar matrix to the cytoskeleton and signaling pathways within early twenties. The first clue to understandingthe molecular

Integrin Expression The attachment of cells to ECM components can also be modulated by altering the number of integrin moleculesexposedon the cell surface.The cr4B1 integrin, which is found on many hematopoieticcells, offers an example of this regulatory mechanism. For these hematopoietic cells to proliferate and differentiate, they must be attached to fibronectin synthesizedby supportive ("stromal") cellsin the bone marrow. The o4B1 integrin on hematopoietic cells binds to a Glu-Ile-Leu-Asp-Val(EILDV) sequencein fibronectin, thereby anchoring the cells to the matrix. This integrin also binds to a sequencein a CAM called vascular CAM-1 (VCAM-1), which is presenton stromal cells of the bone marrow. Thus hematopoietic cells directly contact the stromal cells as well as attach to the matrix. Late in their differentiation, hematopoietic cells decreasetheir expressionof ct4B1 integrin; the resulting reduction in the number of ct4B1integrin moleculeson the cell surfaceis thought to allow mature blood cellsto detachfrom the matrix and stromal cells in the bone marrow and subsequently enter the circulation.

N M O T I L EA N D N O N M O T I L EC E L L S A D H E S I V EI N T E R A C T I O NI S

835

Laminin

Perlecan

- Basallamina

o,p-Dystroglycan -

Sarcoglycancomplex

o-linked sugar -

N - l i n k e ds u g a r

Sarcospan

Cytosol

NOS

Syntrophins

Actin FIGURE 19-35 Dystrophinglycoproteincomplex(DGC)in skeletalmusclecells.Thisschematic modelshowsthatthe DGC comprises threesubcomplexes: thect,Bdystroglycan subcomplex; the sarcoglycan/sarcospan subcomplex proteins; of integral membrane andthe cytosolic adapter subcomplex comprising dystrophin, other adapterproteins, andsignaling molecules ThroughitsO-linked sugars, bindsto components of the basallamina, B-dystroglycan suchaslamininandperlecan, proteins, andcellsurface sucnas neurexin in neuronsDystrophin-the proteindefective in Duchenne muscular dystrophy-links to the actincytoskeleton, B-dystroglycan anda-dystrobrevin linksdystrophin to the sarcoglycan/sarcospan subcomplex. (NOS) Nitricoxidesynthase produces nitricoxide,a g a s e o ussi g n a l i nmgo l e c u laen, dG R B 2 i sa c o m p o n e n o tf s i g n a l i n g pathways activated (Chapter15) by certaincell-surface receptors fromS J Winder, 2001,Trends [Adapted Biochem Sci26:118, andD E Michele andK P Campbell, 2003, J BiolChem278(18):15457-154601

muscleand other types of cells.For instance,the signalingenzyme nitric oxide synthase(NOS) is associatedthrough syntrophin with the cytosolic dystrophin subcomplex in skeletal muscle.The rise in intracellular Ca2t during musclecontraction activatesNOS to produce nitric oxide (NO), a signaling molecule that diffuses into smooth muscle cells surrounding nearby blood vessels.NO promotes smooth muscle relaxation, leading to a local rise in the flow of blood supplying nutrients and oxygen to the skeletal muscle. Mutations in dystrophin, other DGC components, laminin, or enzymesthat add the O-linked sugarsto dystroglycan can all disrupt the DGC-mediated link between the exterior and the interior of muscle cells and causemuscular dystrophies.In addition, dystroglycan mutations have been shown to greatly reduce the clustering of acetylcholine receptors on muscle cells at the neuromuscular junctions, which also is dependenton the basallamina proteins laminin and agrin. These and possibly orher effects of DGC defects apparently lead to a cumulative weakening of the mechanical stability of muscle cells as they undergo contraction and relaxation, resulting in deterioration of the cells and muscuIar dystrophy. 836

C H A P T E R1 9

I

Dystroglycan provides an elegant-and medically relevant-example of the intricate networks of connectivity in cell biology. Dystroglycan was originally discoveredin the context of studying DMD. However, it was later shown to be expressedin nonmuscle cells and, through its binding to laminin, to play a key role in the assemblyand stability of at Ieastsome basementmembranes.Thus it is essentialfor normal development (Chapter 22). Additional studies led to its identification as a cell-surface receptor for the virus that causesthe frequendy fatal human diseaseLassa fever and other related viruses,all of which bind via the same specialized O-linked sugars that mediate binding to laminin. Furthermore, dystroglycan is also the receptor on specialized cells in the nervous system-Schwann cells-to which binds the pathogenic bacterium Mycobacteriwm leprae,the causative organism of leprosy.I

INTEGRATING C E L L SI N T O T I S S U E S

l g C A M sM e d i a t eC e l l - C e lAl d h e s i o ni n N e u r o n a l and Other Tissues Numerous transmembrane proteins characterized by the presenceof multiple immunoglobulin domains (repeats)in their extracellular regions constitute the immunoglobulin (Ig) superfamily of CAMs, or IgCAMs. The Ig domain is a common protein motif, containing 70-110 residues, that was first identified in antibodies, the antigen-binding immunoglobulins, but has a much older evolutionary origin in CAMs. The human, D. melanogaster,and C. elegans genomesincludeabout765,150, and 64 genes,respectively, that encodeproteins containing Ig domains. ImmunoglobuIin domains are found in a wide variety of cell-surfaceproteins, including T-cell receptors produced by lymphocytes and many proteins that take part in adhesiveinteractions. Among the IgCAMs are neural CAMs; intercellular CAMs (ICAMs), which function in the movement of leukocytes into tissues;and junction adhesionmolecules(JAMs), which are presenrin tight junctions. As their name implies, neural CAMs are of particular importance in neural tissues.One type, the NCAMs, primarily mediate homophilic interactions. First expressed during morphogenesis,NCAMs play an important role in the differentiation of muscle, glial, and nerve cells. Their role in cell adhesion has been directly demonstrated by the inhibition of adhesion with anti-NCAM antibodies. Numerous NCAM isoforms, encoded by a single gene, are generatedby alternative mRNA splicing and by differences in glycosylation.Other neural CAMs (e.g., L1-CAM) are encoded by different genes.In humans, mutations in different parts of the L1-CAM genecausevarious neuropathologies (e.g., mental retardation, congenital hydrocephalus, and spasticity). An NCAM comprisesan extracellular region with five Ig repeats and two fibronectin type III repeats,a single membrane-spanningsegment,and a cytosolic segmentthat interacts with the cytoskeleton (seeFigure 19-2).In contrast, the extracellular region of LI-CAM has six Ig repeatsand four fibronectin type III repeats.As with cadherins,cis (intracellular) interactions and trans (intercellular) interactions

probably play key roles in IgCAM-mediated adhesion (see Figure 19-3); however, adhesion mediated by IgCAMs is Ca2*-independent. The covalent attachment of multiple chains of sialic acid, a negativelycharged sugar derivative,to NCAMs alters their adhesiveproperties. In embryonic tissues such as brain, polysialic acid constitutesas much as 25 percent of the mass of NCAMs. Possibly becauseof repulsion betweenthe many negativelychargedsugarsin theseNCAMs, cell-cellcontacts are fairly transient, being made and then broken, a property necessaryfor the developmentof the nervous system.In contrast, NCAMs from adult tissuescontain only one-third as much sialic acid, permitting more stable adhesions.

LeukocyteMovement into Tissuesls Orchestratedby a PreciselyTimed Sequence of AdhesiveInteractions In adult organisms,severaltypes of white blood cells (leukocytes) participate in the defenseagainst infection causedby foreign invaders (e.g., bacteria and viruses) and tissue damage due to trauma or inflammation. To fight infection and clear away damaged tissue, these cells must move rapidly from the blood, where they circulate as unattached, relatively quiescentcells, into the underlying tissueat sitesof infection, inflammation, or damage. We know a great deal about the movement into tissue, termed extrauasation, of four types of leukocytes:neutrophils, which releaseseveral antibacterial proteins; monocytes, the precursors of macrophages,which can engulf and destroy foreign particles;and T and B lymphocytes,the antigen-recognizingcells of the immune system (Chapter 24). Extravasation requires the successiveformation and breakage of cell-cell contacts between leukocytes in the blood and endothelial cells lining the vessels.Some of thesecontactsare mediatedby selectins,a family of CAMs that mediate leukocyte-vascularcell interactions. A key player in theseinteractionsis P-selectin,which is localized to the blood-facing surface of endothelial cells. All selectins contain a Ca2*-dependentlectin domain, which is located at the distal end of the extracellular region of the molecule and recognizesoligosaccharidesin glycoproteins or glycolipids (seeFigure 1.9-2).For example,the primary ligand for P- and E-selectinsis an oligosaccharidecalled the sialyl Lewis-x antigen, a part of longer oligosaccharides presentin abundanceon leukocyteglycoproteinsand glycolipids. Figure 19-35 illustrates the basic sequenceof cell-cell interactions leading to the extravasation of leukocytes.Various inflammatory signalsreleasedin areasof infection or inflammation first cause activation of the endothelium. P-selectin exposed on the surface of activated endothelial cells mediatesthe weak adhesion of passingleukocytes.Becauseof the force of the blood flow and the rapid "on" and "off" rates of P-selectin binding to its ligands, these "trapped" leukocytes are slowed but not stopped and literally roll along the surface of the endothelium. Among the signals that promote activation of the endothelium are

chemokines,a group of small secretedproteins (8-12 kDa) produced by a wide variety of cells, including endothelial cells and leukocytes. For tight adhesion to occur between activated endothelial cells and leukocytes, B2-containing integrins on the surfaces of leukocytes also must be activated by chemokines or other local activation signals such as platelet-actiuating factor (PAF). Platelet-activatingfactor is unusual in that it is a phospholipid, rather than a protein; it is exposed on the surface of activated endothelial cells at the same time that P-selectin is exposed. The binding of PAF or other activators to their receptors on leukocytes leads to activation of the leukocyte integrins to their highaffinity form (seeFigure 1,9-341.(Most of the receptors for chemokines and PAF are members of the G proteincoupled receptorsuperfamilydiscussedin Chapter 15') Activated integrins on leukocytes then bind to distinct IgCAMs on the surface of endothelial cells. These include ICAM-2, which is expressedconstitutivelS and ICAM-1' ICAM-1, whose synthesisalong with that of E-selectinand P-selectin is induced by activation, does not usually contribute substantially to leukocyte endothelial cell adhesion immediately after activation but rather participates at later times in casesof chronic inflammation. The resulting tight adhesion mediated by the Ca2*-independentintegrin-ICAM interactions leads to the cessation of rolling and to the spreading of leukocytes on the surface of the endothelium; soon the adhered cells move between adjacent endothelial cells and into the underlying tissue. The selective adhesion of leukocytes to the endothelium near sites of infection or inflammation thus depends on the sequential appearance and activation of several different CAMs on the surfaces of the interacting cells. Different types of leukocytes express specific integrins containing the B2 subunit: for example, aLB2 by T lymphocytes and aMB2 by monocytes, the circulating precursors of tissue macrophages.Nonetheless' all leukocytes move into tissuesby the samegeneralmechanismdepicted i n F i g u r e1 . 9 - 3 6 . Many of the CAMs usedto direct leukocyteadhesionare sharedamong different types of leukocytesand target tissues. Yet often only a particular type of leukocyte is directed to a particular tissue. How is this specificity achieved?A threeitep model has been proposed to account for the cell-type specificity of such leukocyte-endothelial-cell interactions. First, endothelial activation promotes initial relatively weak, transient, and reversiblebinding (e.g., the interaction of selectins and their carbohydrate ligands).'Without additional local activation signals,the leukocyte will quickly move on. Second,cells in the immediate vicinity of the site of infection or inflammation releaseor expresson their surfaceschemical signals(e.g.,chemokines,PAF) that activateonly specialsubsets(dependingon their complementof chemokinereceptors) of the lransiently attached leukocytes. Third, additional activation-dependent CAMs (e.g., integrins) engage their binding partners, leading to strong sustainedadhesion.Only if the proper combination of CAMs' binding partners' and activation signalsare engagedtogether with the appropriate

N M O T I L EA N D N O N M O T I L EC E L L S A D H E S I V EI N T E R A C T I O NI S

837

Animation:Cell-Cell flltt Adhesionin LeukocyteExtravasation

z

E Leukocyte (restingstate) S e l e c t i nl i g a n d (specific carbohydrate)

E Leukocyteactivation (PAFactivatesintegrin)

E n d o t h e l i aal c t i v a t i o na n d leukocyteattachmentand rolling

o"LB2 integrin

PAF receptor

lcAM-2

P-selectin

lcAM-1

-..+ Vesiclecontaining P-selectin

Extravasation

E A F I G U R1 E9 - 3 6 S e q u e n c o e f c e l l - c e liln t e r a c t i o n lse a d i n g to tight binding of leukocytesto activatedendothelialcells and subsequentextravasation.Step E: In the absence of i n f l a m m a t i oonr i n f e c t i o nl e, u k o c y t easn de n d o t h e l icael l l sl i n i n g b l o o dv e s s e a l sr ei n a r e s t i n sgt a t eS t e p Z : I n f l a m m a t osriyg n a l s r e l e a s eodn l yi n a r e a o s f i n fl a m m a t i o n i n, f e c t i o no,r b o t ha c t i v a t e restingendothelial cellsto movevesicle-sequestered selectins to t h e c e l ls u r f a c eT h ee x p o s esde l e c t i nmse d i a t e l o o s eb i n d i n go f leukocytes by interacting with carbohydrate ligandson leukocytes A c t i v a t i oonf t h e e n d o t h e l i uaml s oc a u s essy n t h e soi sf p l a t e l e t -

timing at a specificsite will a given leukocyteadherestrongly. Suchcombinatorial diversity and crosstalk allows a small set of CAMs to servediversefunctions throughout the body-a good example of biological parsimony. Leukocyte-adhesion deficiency is caused by a genetic ffi IITI . detect in the synthesisof the integrin B2 subunit. peoIil ple with this disorder are susceptibleto repeatedbacterial infections because their leukocytes cannot extravasate properly and thus fight the infection within the tissue. Some pathogenic viruses have evolved mechanisms to exploit for their own purposes cell-surfaceproteins that participate in the normal responseto inflammation. For example, many of the RNA viruses that cause the common cold (rhinoviruses)bind to and enrercellsthrough ICAM-1, and chemokine receptors can be important entry sites for human immunodeficiency virus (HIV), the cause of AIDS. Integrins appear to participate in the binding andlor inter838

c H A P T E R1 9

|

T N T E G R A T T NCG E L L St N T O T T S S U E S

F i r m a d h e s i o nv i a integrin/ICAM binding

A activating factor(PAF) and ICAM-1,bothexpressed on the cell surface. PAFandotherusually secreted activators, including c h e m o k i n et sh,e ni n d u c ec h a n g eisn t h e s h a p eosf t h e l e u k o c y t e s andactivation of leukocyte integrins suchasctlp2,whichis e x p r e s s ebdy T l y m p h o c y t eBs. T h es u b s e q u etni gt h tb i n d i n g betweenactivated integrins on leukocytes and CAMson the e 9 . , I C A M - 2a n dI C A M - 1r)e s u l tisn f i r ma d h e s i o n e n d o t h e l i u(m (extravasation) 4 andsubsequent movement intothe underlying j992,Celt68:303.] tissue E [Adapted fromR O Hynes andA Lander,

nalization of a wide variety of viruses, including reoviruses (causing fever and gastroenteritis,especiallyin infants), adenoviruses(causingconjunctivitis,acute respiratory disease), and foot-and-mouth diseasevirus (causing fever in cattle and pigs). I

Adhesive lnteractions in Diverse Motile and Nonmotile Cells r Many cells have integrin-containing aggregates(e.g.,focal adhesions,3-D adhesions,podosomes)that physically and functionally connect cells to the extracellular matrix and facilitate inside-out and outside-in signaling. r Via interaction with integrins, the three-dimensional structure of the ECM surrounding a cell can profoundly influence the behavior of the cell.

r Integrins exist in two conformations that differ in the affinity for ligands and interactions with cytosolic adapter proteins (seeFigure 19-341;switching between these two conformations allows regulation of integrin activity, which is important for control of cell adhesionand movements. r Dystroglycan, an adhesion receptor,forms a large complex with dystrophin, other adapter proteins, and signaling molecules(seeFigure 19-35). This complex links the actin cytoskeletonto the surrounding matrix, providing mechanical stability to muscle.Mutations in various componentsof this complex causedifferent types of muscular dystrophy. r Neural cell-adhesionmolecules,which belong to the immunoglobulin (Ig) family of CAMs, mediate Ca2*independentcell-cell adhesionin neural and other tissues. r The combinatorial and sequentialinteraction of several types of CAMs (e.g., selectins, integrins, and ICAMs) is critical for the specific and tight adhesionof different types of leukocytes to endothelial cells in responseto local signalsinduced by infection or inflamm a t i o n ( s e eF i g u r e 1 , 9 - 3 6 ) .

PlantTissues 'We turn now to the assemblyof plant cellsinto trssues. The overall structural organization of plants is generally simpler than that of animals. For instance,plants have

only four broad types of cells, which in mature plants form four basic classesof tissue: dermal tissue interactswith the environment; uascwlartissue transportswater and dissolved substances(e.g., sugars,ions); space-fillingground tisswe constitutesthe maior sites of metabolism; and sporogenous tisswe forms the reproductive organs. Plant tissues are organized into just four main organ systems: stems have support and transport functions' rools provide anchorage and absorb and store nutrients, leauesate the sitesof photosynthesis, and flotuers enclosethe reproductive structures' Thus at the cell, tissue,and organ levels,plants are generally lesscomplex than most animals. Moreover, unlike animals' plants do not replaceor repair old or damagedcells or tissues;they simply grow new organs. Indeed, the developmentalfate of any given plant cell is primarily based on its position in the organism rather than its lineage(Chapter 21), whereasboth are important in animals. Thus in both plants and animals a cell'sdirect communication with it neighborsis important. Most importantly for this chapter and in contrast with animals, few cells in plants directly contact one another through molecules incorporated into their plasma membranes. Instead, plant cells are typically surrounded by a rigid cell wall that contacts the cell walls of adjacentcells (Figure 1.9-37a).Also in contrast with animal cells,a plant cell rarely changesits position in the organism relative to other cells.Thesefeaturesof plants and their organization have determinedthe distinctive molecular mechanismsby

(a) Nucleus

Mitochondria

Primary wall

Pectin Cellulose microfibril Hemicellulose Plasmodesmata

, 2 0 0n m ,

P l a s m am e m b r a n e

of 19-37Structureof the plantcellwall. (a)Overview A FIGURE of a typicalplantcell,in whichthe organelle-filled theorganization by a well-defined issurrounded membrane cellwith itsplasma representation matrixcalledthe cellwall (b)Schematic extracellular . e l l u l o saen dh e m i c e l l u l oasr ea r r a n g e d l a l lo f a n o n i o nC o f t h ec e l w Thesrzeof in a matrixof pectinpolymers intoat leastthreelayers the aredrawnto scaleTosimplify andtheirseparations the polymers andothermatrix cross-links mostof the hemicellulose diagram, (e g , extensin, lignin)arenot shown(c)Fast-freeze, constituents

of the cellwallof the gardenpeatn micrograph electron deep-etch by chemical wereremoved polysaccharides pectin whichsomeof the ibrils,and microf cellulose fibers are thicker abundant The treatment (b) (arrowheads) cross-links lPart thethinnerf ibersarehemicellulose ed, Ihe 1991,in C Lloyd, andK R Roberts, fromM McCann adapted p 126 as of PtantGrowthand Form,AcademicPress, Basis Cytoskeletat T, M , Hamann G , Facette S, Brininstool C , Bauer in Somerville modified H S, Youngs Vorwerk 5, RaabT., A, Persson E, Paredez MilneJ . Osborne ,39(12)1315-1323l Part(c)fromT.FujinoandT.ltoh, 1998,PlantCellPhysiol P L A N TT I S S U E S

839

which their cellsare incorporated into tissuesand communicate with one another.

T h e P l a n tC e l lW a l l l s a L a m i n a t eo f C e l l u l o s e F i b r i l si n a M a t r i x o f G l y c o p r o t e i n s The plant extracellular matrix, or cell wall, which is mainly composedof polysaccharidesand is =O.Z pm thick, completely coats the outside of the plant cell's plasma membrane. This structure servessome of the same functions as those of the ECM produced by animal cells, even though the two structures are composed of entirely different macromolecules and have a different organization. About 1000 genesin the plant Arabidopsis are devoted to the synthesis and functioning of its cell wall, including approximately 414 glycosyltransferase and more than 316 glycosyl hydrolasegenes.Like animal cell ECM, the plant cell wall connects cells into tissues,signals a plant cell to grow and divide, and controls the shape of plant organs. It is a dynamic structure that plays important roles in controlling the differentiation of plant cells during embryogenesisand growth and provides a barrier to protect against pathogen infection. Just as the extracellular matrix helps define the shapes of animal cells, the cell wall defines the shapes of plant cells. \7hen the cell wall is digestedaway from plant cells by hydrolytic enzymes, spherical cells enclosed by a plasma membrane are left. Becausea major function of a plant cell wall is to withstand the osmotic turgor pressureof the cell (between14.5 and 435 pounds per squareinch!), the cell wall is built for lateral strength. It is arranged into layers of cellulose microfibrils-bundles of 30-36 chains of long (as much as 7 pm or greater), linear, extensively hydrogen-bonded polymers of glucose in B glycosidic linkages. The cellulose microfibrils are embedded in a matrix composed of pectin, a polymer of t-galacturonic acid and other monosaccharides, and hemicellulose,a short, highly branched polymer of severalfive- and six-carbon monosaccharides.The mechanical strength of the cell wall depends on cross-linking of the microfibrils by hemicellulosechains (Figure 19-37b, c). The layers of microfibrils prevent the cell wall from stretching laterally. Cellulose microfibrils are synthesized on the exoplasmic face of the plasma membrane from UDP-glucose and ADP-glucose formed in the cytosol. The polymerizing enzyme, called cellulose synthase, moves within the plane of the plasma membrane along tracks of intracellular microtubules as cellulose is formed, providing a distinctive mechanism for intracellular/extracellular communrcatron. Unlike cellulose,pectin and hemicelluloseare synthesized in the Golgi apparatus and transported to the cell surface, where they form an interlinked network that helps bind the walls of adjacent cells to one another and cushions them. When purified, pectin binds water and forms a gel in the presenceof Ca2* and borate ionshencethe use of pectinsin many processedfoods. As much as 15 percent of the cell wall may be composed of ex-

840

C H A P T E R1 9

|

TNTEGRATTN CG E L L St N T O T | S S U E S

tensin, a glycoprotein that contains abundant hydroxyproline and serine.Most of the hydroxyproline residues are linked to short chains of arabinose (a five-carbon monosaccharide), and the serine residues are linked to galactose.Carbohydrate accountsfor about 65 percent of extensin by weight, and its protein backbone forms an extendedrodlike helix with the hydroxyl or O-linked carbohydrates protruding outward. Lignin-a complex, insoluble polymer of phenolic residues-associateswith celIulose and is a strengthening material. Like cartilage proteoglycans, lignin resists compression forces on the matrlx. The cell wall is a selectivefilter whose permeability is controlled largely by pectins in the wall matrix. $Thereas water and ions diffuse freely acrosscell walls, the diffusion of large molecules,including proteins larger than 20 kDa, is limited. This limitation may account for why many plant hormones are small, water-solublemolecules.which can diffuse across the cell wall and interact with receptors in the plasma membrane of plant cells.

Looseningof the CellWall PermitsPlant Cell Growth Becausethe cell wall surrounding a plant cell prevents it from expanding, the wall's structure must be loosenedwhen the cell grows. The amount, type, and direction of plant-cell growth are regulated by small-moleculehormones (e.g., indoleaceticacid) called auxins. The auxin-inducedweakening of the cell wall permits the expansion of the intracellular vacuole by uptake of water, Ieadingto elongation of the cell. 'We can grasp the magnitude of this phenomenon by considering that, if all cells in a redwood tree were reduced to the size of a typical liver cell, the tree would have a maximum height of only I meter. The cell wall undergoesits greatestchangesat the meristem of a root or shoot tip. Thesesitesare where cells divide and grow. Young meristematic cells are connected by thin primary cell walls, which can be loosenedand stretchedto allow subsequent cell elongation. After cell elongation ceases,the cell wall is generally thickened, either by the secretion of additional macromoleculesinto the primary wall or, more usually, by the formation of a secondarycell wall composed of severallayers. Most of the cell eventually degenerates,leaving only the cell wall in mature tissuessuch as the xylem-the tubes that conduct salts and water from the roots through the stemsto the leaves.The unique properties of wood and of plant fibers such as cotton are due to the molecular properties of the cell walls in the tissuesof origin.

Plasmodesmata DirectlyConnectthe Cytosols o f A d j a c e n tC e l l si n H i g h e rP l a n t s The presenceof a cell wall separatingcells in plants imposes barriers to cell-cell communication-and thus cell-type differentiation-not faced by animals. One distinctive mechanism used by plant cells to communicate directly is through

specializedcell-cell junctions called plasmodesmata,which extend through the cell wall. Like gap junctions, plasmodesmata ate channelsthat connect the cytosol of a cell with that of an adiacentcell. The diameter of the channel is about 30-60 nm, and its length can vary and be greater than 1 pm. The density of plasmodesmatavaries dependingon the plant and cell type, and even the smallestmeristematic cells have more than 1000 interconnectionswith their neighbors. AIthough a variety of proteins that are physically or functionally associatedwith plasmodesmatahave been identified, key structural protein components of plasmodesmataremain to be identified. Moleculessmallerthan about 1000 Da, includinga variety of metabolic and signaling compounds (ions, sugars, amino acids),generallycan diffuse through plasmodesmata. However, the size of the channel through which molecules pass is highly regulated. In some circumstances,the channel is clamped shut; in others, it is dilated sufficiently to permit the passageof moleculeslarger than 10,000 Da. Among the factors that affect the permeabilitv of plasmodesmatais the cytosolic Ca2* concentration,*iitr an increasein cytosolic Ca2* reversibly inhibiting movement of moleculesthrough thesestructures. Although plasmodesmataand gap junctions resemble each other functionally with respectto forming channelsfor small molecule diffusion, their structuresdiffer dramatically in two significantways (Figure 19-38). The plasma membranesof the adjacentplant cellsmergeto form a continuous channel, the annulus, at each plasmodesma, whereas the membranesof cellsat a gap junction are not continuous with each other. In addition, plasmodesmataexhibit many additional complex structural and functional characteristics.For example,they contain within the channel an extensionof the endoplasmic reticulum called a desmotubule that passes through the annulus,which connectsthe cytosolsof adjacent plant cells.They also have a variety of specializedproteins at the entrance of the channel and running throughout the length of the channel, including special cytoskeletal,motor' and docking proteins that regulate the sizes and types of moleculesthat can passthrough the channel. Many types of moleculesspreadfrom cell to cell through plasmodesmata, including proteins called non-cell-autonomous proteins (NCAPs, including some transcription factors), nucleic acid/protein complexes, metabolic products, and plant viruses.It appearsthat some of theserequire specialchaperones to facilitate transport. Specializedkinasesmay also phosphorylate plasmodesmalcomponents to regulate their activities(e.g.,opening of the channels).Solublemolecules passthrough the cytosolicannulus,or sleeve(about 3-4 nm in diameter), that lies between the plasma membrane and desmotubule, whereas membrane-bound molecules or certain proteins within the ER lumen can pass from cell to cell via the desmotubule.Plasmodesmata appearto play an especially important role in regulating the development of plant cells and tissues,as is suggestedby their ability to mediate intracellular movement of transcription factors and ribonuclear protein complexes.

(a)

C e l l1 Plasma memDrane

Cell 2

Desmotubule

wall Desmotubule

28"-T

1 0 0n m

(a)Schematic modelof a 19-38Plasmodesmata. FIGURE of the an extension showingthedesmotubule, plasmodesma (ER), a plasma-membraneandtheannulus, reticulum endoplasmic of the cytosols that interconnects filledwith cytosol linedchannel of a thin sections (b) of micrographs Electron cells. adjacent (teft) plasmodesmata) individual indicate leaf(brackets sugarcane runningthrough viewshowingERanddesmotubule Longitudinal viewsof (Right) cross-sectional Perpendicular eachannulus. the connecting structures spoke in someof which plasmodesmata, (b) from K' seen. can be lPart the desmotubule to membrane olasma 18 l 184:307-3 1991 andR F Evert, , Planta Robrnson-Beers

O n l y a F e w A d h e s i v eM o l e c u l e sH a v eB e e n l d e n t i f i e di n P l a n t s Systematicanalysisof the Arabidopsls genomeand biochemical analysisof other plant speciesprovide no evidencefor the existenceof plant homologs of most animal CAMs, adhesion receptors,and ECM components.This finding is not surprising, given the dramatically different nature of cell-cell and cell-matrix/wall interactionsin animals and plants. Among the adhesive-typeproteins apparently unique to plants are five wall-associatedkinases(VAKs) and'WAK-like proteins expressedin the plasma membraneof Arabidopsis cells.The extracellularregionsin all theseproteins contain multiple epidermalgrowth factor (EGF) repeats'frequently

P L A N TT I S S U E S

841

found in animal cell-surface receptors, which may directly participate in binding to orher molecules. Some NTAKs have beenshown to bind to glycine-richproteins in the cell wall, thereby mediating membrane-wall contacts. These Arabidopsisproteins have a singletransmembranedomain and an intracellular cytosolic tyrosine kinase domain, which may participate in signaling pathways somewhat like the receptor tyrosine kinasesdiscussedin Chapter 15. The results of in vitro binding assayscombined with in vivo studiesand analysesof plant mutants have identifiedseveral macromoleculesin the ECM that are important for adhesion. For example,normal adhesionof pollen, which contains sperm cells,to the stigma or style in the female reproductive organ of the Easterlily requiresa cysteine-richprotein called stigma/stylar cysteine-richadhesin (SCA) and a specialized pectin that can bind to SCA (Figure 19-39).A small,probably ECM embedded,-10 kDa protein calledchymocyaninworks in conjunction with SCA to help direct the movement of the sperm-containingpollen tube (chemotaxis)to the ovary. Disruption of the geneencoding glucuronyltransferase1, a key enzyme in pectin biosynthesis,has provided a striking ilIustration of the imporrance of pectins in intercellular adhesion

in plant meristems.Normally, specializedpectin moleculeshelp hold the cells in meristems tightly together. Nfhen grown in culture as a cluster of relatively undifferentiated cells, called a callus, normal meristematic cells adhere tightly and can differentiate into chlorophyll-producing cells, giving the callus a green color. Eventually the callus will generateshoots. In contrast, mutant cells with an inactivated glucuronyltransferaseL gene are large, associateloosely with each other, and do not differentiate normally forming a yellow callus. The introduction of a normal glucuronyltransferase1 geneinto the mutant cells restorestheir ability to adhere and differentiate normally. The paucity of plant adhesive molecules identified to date, in contrast with the many well-defined animal adhesive molecules,may be due to the technical difficulties in working with the ECtrzUcellwall of plants. Adhesive interactions are often likely to play different roles in plant and animal biologS at least in part becauseof their differencesin development and physiology.

Plant Tissues r The integration of cells into tissuesin plants is fundamentally different from the assemblyof animal tissues,primarily becauseeach plant cell is surrounded by a relatively rigid cell wall. r The plant cell wall comprises layers of cellulose microfibrils embedded within a matrix of hemicellulose, pectin, extensin,and other lessabundant molecules. r Cellulose,alarge,linear glucosepolymer,assemblesspontaneouslyinto microfibrils stabilizedby hydrogen bonding. r The cell wall defines the shapes of plant cells and restricts their elongation. Auxin-induced looseningof the cell wall permits elongation. r Adjacent plant cells can communicate through plasmodesmata, junctions that allow molecules to pass through complex channelsconnecting the cytosols of adjacent cells (seeFigure 19-38). r Plants do not produce homologs of the common adhesive moleculesfound in animals. Only a few adhesivemoleculesunique to plants have beenwell documentedto date.

A deeper understanding of the integration of cells into tissues in complex organisms will draw on insights and techEXPERIMENTAL FTGURE 19-39An in vitro assaywas usedto niques from virtually all subdisciplines of molecular cell identify moleculesrequiredfor adherenceof pollentubesto biology-biochemistrS biophysics,microscop5 genetics,gethe stylar matrix. In thisassay, extracellular stylarmatrixcollected nomics, proteomics, and developmental biology-together (SE) fromlilystyles or an artificial matrixisdriedontonitrocellulose with bioengineering (NC).Pollen and membranes computer science.This area of cell tubescontaining spermarethenaddedand biology is undergoing explosivegrowth. theirbindingto thedriedmatrixisassessed. Inthisscanning electron micrograph, thetipsof pollentubes(arrows) An important set of questions for the future deals with canbeseenbinding to driedstylarmatrix. Thistypeof assay hasshownthatpollenadherence the mechanisms by which cells detect and respond to medepends on stigma/stylar (SCA) cysteine-rich adhesin chanical forces on them and the extracellular matrix. as well anda pectinthat bindsto SCA.[From G y Jauheral, 1997,SexplantReprod 10:1731 as the influence of their three-dimensional arrangementsand 842

.

cHAprE1 R9 |

T N T E G R A TcTENL G L tsN r o l s s u E s

interactions.A relatedquestionis how this information is used to control cell and tissuestructure and function. Theseissues involve the fields of biomechanicsand mechanotransduction. can induce distinct patternsof geneexShearor other stresses pressionand cell growth and can greatly alter cell metabolism its binding to its ECM ligands (laminin, etc'). and responsesto extracellularstimuli. MechanosensitivenonA struitural hallmark of CAMs, adhesion receptors, and selectivecation channels(NSCws)' a least some of which apECM proteins is the presenceof multiple domains that impart pear to be membersof the transient receptor potential (TRP) diversi functions to a single polypeptide chain. It is generally cation channel family, are activated by the stretch of plasma evolutionarily by membrane and are important players in mechanotransduc- agreedthat such multidomain proteins arose the distinct encoding sequences DNA of distinct the assembly tion, such as that involved in sensingsound in the ear,which is opportuprovide domains multiple encoding Genes mediated,in part, by specializedcadherins.Most of the classes domains. diversity functional and sequence enormous generate nities to of moleculesdiscussedin this chapter-ECM, adhesionreceptors, CAMs, intracellularadapters,and the cytoskeleton-are and mechanthought to play crucial roles in mechanosensing otransduction.Future researchshould give us a far more sophisticatedunderstandingof the rolesof the three-dimensional organizationof cellsand ECM componentsand the forcesacting on them under normal and pathologicalconditionsin controlling the structuresand activitiesof tissues.Applications of suchunderstandingwill provide new methodsto explorebasic celVtissuebiology and provide improved technologiesfor the searchfor novel therapiesfor disease. Although junctions help play a key role in forming stable epithelial tissues and defining the shapesand functional properties of epithelia, they are not static. Remodeling in terms of replacementof older moleculeswith more recently synthesizedmolecules is ongoing, and the dynamic properties of junctions open the door to more substantial changes when necessary(the epithelial-mesenchymaltransition durlar basis of functional cell-cell and cell-matrix attachmentsing development, wound healing, etc.). Understanding the the "wiring"-in the nervous systemand how that wiring ultimolecular mechanismsunderlying the relationship between mately peimits complex neuronal control and, indeed, the stability and dynamic changewill provide new insights into intellect required to understandmolecular cell biology' morphogenesis,maintaining tissue integrity and function, and responseto (or induction of) pathology. Numerous questions relate to intracellular signaling KeyTerms from CAMs and adhesionreceptors.Such signaling must be integratedwith other cellular signalingpathways that are acgap junction 809 adhesionreceptor 803 tivated by various external signals (e.g., growth factors) so glycosaminoglycan adherensjunction 809 that the cell responds appropriately and in a coordinated (GAG\ 822 809 anchoring lunction fashion to many different simultaneousinternal and external hyaluronan 825 basallamina 808 stimuli. It appearsthat small GTPaseproteins participate in immunoglobulin cellat least some of the integratedpathways associatedwith sigcadherin 803 adhesionmolecule naling between cellular junctions. How are the logic circuits cell-adhesionmolecule (IgCAM)835 constructed that allow cross talk between diverse signaling (cAM) 803 integrin 803 pathways? How do these circuits integrate the information cellwall839 from thesepathways?How is the combination of outside-in laminin 821 collagen 805 and inside-out signaling mediated by CAMs and adhesion multiadhesivematrix connexin819 receptorsmerged into such circuits? protein 805 'We desmosome809 can expect ever-increasingprogress in the exploration paracellular pathwaY 815 of the influenceof glycobiology (the study of biology of oligoepithelium 802 plasmodesmata841 and polysaccharides)on cell biology. The importance of epithelial-mesench Ymal proteoglycan 805 specializedGAG sequencesin controlling cellular activities' transition 833 their RGD sequence816 especiallyinteractionsbetweensomegrowth factors and extracellular matrix receptors,is now clear.With the identification of the biosynselectin803 (ECM)833 thetic mechanismsby which thesecomplex structuresare gensyndecan 829 fibrillar collagen 825 erated and the development of tools to manipulate GAG tight junction 809 fibronectin 805 structures and test their functions in cultured systemsand K E YT E R M S .

843

Reviewthe Concepts t. Using specific examples, describe the two phenomena that give rise to the diversity of adhesivemolecules. 2. Cadherins are known to mediate homophilic interactions between cells. !(/hat is a homophilic interaction. and how can it be demonstraredexperimentallyfor E-cadherins? 3. Together with their role in connecting the lateral membranes of adjacent epithelial cells, adherensjunctions play a role in controlling cell shape.What proteins and ,trrritrrr., are involved in this role? 4. IJfhat is the normal function of tight junctions? What can happen to tissueswhen tight junctions do not function properly? 'Sfhat 5. is collagen,and how is it synthesized?How do we know that collagen is required for tissueintegrity? 6. Using structural models, explain how integrins mediate outside-in and inside-out signaling. 7. Compare the functions and properties of each of three types of macromoleculesthat are abundant in the extracelluIar matrix of all tissues. 8.- Many proteoglycans have cell-signaling roles. Regulation of feeding behavior by syndecansin ttre hypothalamic region of the brain is one example.How is this reguLtion accompl-ished? You have synthesized an oligopeptide conraining an ? RGD sequencesurrounded by other amino acids. What is

the effect of this peptide when added to a fibroblast cell culture grown on a layer of fibronectin absorbedto the tissueculture dish?Vhy doesthis happen? 10. Blood clotting is a crucial function for mammalian survival. How do the multiadhesive properties of fibronectin lead to the recruitment of plateletsto blood clots? 11. How do changesin molecular connections between the extracellular matrix (ECM) and cytoskeleton give rise to Duchennemuscular dystrophy? 72, To fight infection, leukocytes move rapidly from the blood into the tissue sites of infection. lfhat is this orocess called?How are adhesionmoleculesinvolved in this process? 13. The structure of a plant cell wall needsto loosen to accommodate cell growth. What signaling molecule controls this process? L4. Compare plasmodesmatain plant cells to gap iunctions in animal cells.

Analyzethe Data Researchershave isolated two E-cadherin mutant isoforms that are hypothesizedro function differently from the isoform of the wild-type E-cadherin. An E-cadherin negative mammary carcinoma cell line was transfected with the mutant E-cadheringenesA (part a in the figure, triangles) or B (part b) (triangles)or the wild-type E-cadheringene(black circles)and

(a)

Anti-E-cadherin

80

80

60

60

E 40

540 E20

20

40 T i m e( m i n )

20

40 T i m e( m i n )

(b)

80

80

60

60

E 40

540 E20

20

40 ( m i n ) Time

844

c H A P T E R1 9

|

T N T E G R A T T NCG E L L St N T O T t S S U E S

20

40 T i m e( m i n )

compared to untransfectedcells (open circles)in an aggregation assay.In this assay,cells are first dissociatedby trypsin treatment and then allowed to aggregatein solution over a period of minutes. Aggregating cells from mutants A and B are presentedin panels a and b respectively.To demonstrate that the observedadhesionwas cadherin mediated,the cells were pretreatedwith a nonspecificantibody (left panel) or a functionblocking anti-E-cadherinmonoclonal antibody (right panel)' a. Vhy do cells transfected with the wild-type Ecadherin gene have greater aggregation than control, untransfectedcells? b. From these data, what can be said about the function of mutants A and B? Sfhy does the addition of the anti-E-cadherinmonc. oclonal antibodS but not the nonspecific antibody, block aggregation? 'What would happen to the aggregation ability of d. geneif the the cellstransfectedwith the wild-type E-c^adherin assaywere performed in media low in Ca'-?

References Cell-Cell and Cell-Matrix Adhesion: An Overview Carthew,R. \7. 2005. Adhesionproteins and the control of cell shape.Curr. Opin. Genet.Deu. 15(4\:358-363. M. Cereijido,R. G. Contreras,and L. Shoshani.2004. Cell adhesion,polarity, and epitheliain the dawn of metazoans.PhysiolReu.84:7229-1262. Gumbiner,B. M. 1,996.Cell adhesion:the molecular basisof tissue architectureand morphogenesis.Cell 84345-357. Hynes,R. O.1999. Cell adhesion:old and new questions. Trends Cell Biol. 912\:M33-M337. Millennium issue. Hynes, R. O. 2002.Integrins:bidirectional,allostericsignaling machines.Cell 170:673-687. Hynes,R. O., and Q.Zhao.2000. The evolutionof cell adhesion.J. Cell Biol. 150(21:F89-F96. putting all Ingber,D. 8.2006. Cellular mechanotransduction: . the piecestogetheragain.FASEBJ. 2O(7):811'-827 Jamora, C., and E. Fuchs.2002. Intercellularadhesion'sigNature Cell Biol.4(4):E101-E108. nallingand the cytoskeleton. transductionby cell adhesionrecep2002. Signal R. L. Juliano, tors and the cytoskeleton:functions of integrins,cadherins,selectins, and immunoglobulin-superfamilymembers.Ann. Reu.Pharmacol. Toxicol. 42:283-323. Leahy,D. J. lgg7.Implications of atomic resolution structures for cell adhesion.Ann. Reu.Cell Deuel.Biol. 13:363-393. Thiery, J. P.,and JonathanP. Sleeman.2006. Complex networks orchestraieipithelial-mesenchymaltransitions.Nature Reu.Mol. Cell Biol.7:131.-L42. Vogel,V. 2006. Mechanotransductioninvolving multimodular proteini: convertingforce into biochemicalsignals.Ann. Reu.Biophys. Biomol. Struct. 35:459488. Vogel, V., and M. Sheetz.2006.Local force and geometry senslng regulatecell functions.Nature. Reu.Mol' Cell Biol- 7 (4):265-275 . Cell-Cell and Cell-ECM Junctions and Their Adhesion Molecules The Cadherin Resource: ges/classify/ http://calcium.uhnres.utoronto.calcadherin/pub-pa index.htm

Chu. Y. S.. et al. 2004. Forcemeasurementsin E-cadherin-mediated cell doublets revealrapid adhesionstrengthenedby actin cytosk-eleton remodelingthrough Rac and Cdc42.J. CelI Biol. 167:1183-1194' Clandinin, T. R., and S. L. Zipursky. 2002. Makirrg connections in the fly visual system.Neuron 352827-841. Conacci-Sorreil,M.' J. Zhurinsky, and A. Ben-Ze'ev'2002'The cadherin-cateninadhesionsystemin signalingand cancer'J' Clin' lnuest. 109:987-99t. Fuchs,E., and S. Raghavan.2002.Getting under the skin of epidermal morphogenesis.Nature Reu-Genet' 3(3\:199J09' Gates,J., and M. Peifer.2005. Can 1000 reviews be wrong? Actin, alpiraicatenin,and adherensjunctions. Cell 123(5):769-772' Gumbiner,B. M. 2005. Regulationof cadherin-mediatedadhesion in morphogenesis.Nature Reu.Mol. Cell Biol' 6:622-634' Hatzfeld, M. 2007. Plakophilins: multifunctional proteins or Biochim' Biophys' Acta just regulatoisof desmosomaladhesion? 1773:69-77. Hobbie. L.. et al. 1987. Restorationof LDL receptoractivity in mutant celli by intercellularjunctional communication'Science 235:69-73. Hollande, F., A. Shulkes,and G' S. Baldwin' 2005' Signalingthe (277):pe73' iunctions in gut epithelium.Scl.STKE R' G' Gourdie' 2005' Zh:u, and Hunter, \(. R.' J. Barker,C. Zontrlao..l,,,d..tt-i altersconnexin43gap iunction sizeand organization by influencingchannelaccretion.Mol' Biol' Cell 16(L2):5686-5698. K' H' Liem' 2004' Jefferson,J. J., Conrad L. Leung, and Ronald Plakins,goliatirs-thatlink cell iunctions and the cytoskeleton'Nature Reu.MoL Cell Biol. 5:542-553. Laird, D. \7. 2006. Life cycle of connexinsin health and disease' Biochem.J. 394(pt.3):527-543. Lee,J. M., S. Dedhar,R. Kalluri, and E. Iil' Thompson' 2006'. transition: new insightsin signaling,deThe epiihelialimesenchymal ,relopment,and disease,l.Cell Biol. 172(7\:973-981" Li, J., V. V. Patel,and G. L. Radice.2006' Dysregulationof cell and cardiacarrhythmogenesis'Clin' Med' Res' adhesio"n'proteins 4(1,):42-52. Litjens, S. H., J. M. de Pereda,and A. Sonnenberg'2006' Current in;ish;s into i-he formation and breakdown of hemidesmosomes.TrendsCell Biol. 16(7):376-383M. D., and E. Ruoslahti' 1984' Cell attachment Pierschbacher, can be duplicated by gm^allsynthetic fragfibronectin activity of ments'of the molecule'Natur e 309(5963):30-3 3' Schtick,F., and N' Perrimon.2002. Molecular mechanismsof Ann. Reu.Cell Deuel' Biol' 18:463493' epithelialmorphogenesis. Tanoue,T., and M. Takeichi.2005. New insightsinto fat cadherins./. Cell Sci.118(pt.11\:2347-2353' Tsukita, S., M. Furuse,and M. Itoh' 2001' Nlultifunctional strandsin tighi iunctions.Nature Reu. Mol Cell Biol' 2:285-293' Vogelmann,R., M. R. Amieva, S. Falkow, and.\7' J' Nelson' 2004. ilreaking.into the epithelialapical-iunctionalcomplex-news Curr. Opin. Cell Biol' 16(11:86'93' from pathogen"hackers. The Extracellular Matrix l: The Basal Lamina Boutaud, A., et al. 2000. Type IV collagenof the glomerular basementmembranetevidencethat the chain specificityof network is encodedby the noncollagenousNC1 domains' J ' Biol' "rt.-lfy Chem. 275 :3071'6-30724. Esko, J. D., and U' Lindahl. 2001. Molecular diversityof heparan sulfate.!. CIin. Inuest. t08:169-173' Hallmann. R., et al. 2005. Expressionand function of laminins in the embryonic and mature vasculature'Pbysiol' Reu' 85:979-1000. Hohenester,E', and J. Engel.2002' Domain structureand orsanisationin'extiacellular matrix proteins' Matrix Biol'

z{Q):tts-tzg. REFERENCES

845

lozzo, R. V. 2005. Basementmembraneproteoglycans:from cel_ , lar to ceiling.Nature Reu.Mol. Cell Biol. e6l:A+A_OSe . Kanagawa,M., et al. 2005. Disruption of perlecanbinding and matrix assemblyby post-translationalor geneticdisruption of"dvs_ troglycanfunction. FEBSLett. S79lZ1l:41924796. Nakato, H., and K. Kimata. 2002.Heparansulfate fine struc_ ture and specificityof proteoglycanfunctions.Biochim. BioDhys. Acta L573:312-318. Perrimon,N., and M. Bernfield.2001. Cellular functions of proteoglycans:an overview.Sem. Cell Deuel. Biol. l2(2):65_G7. . Rosenberg,R. D., et al. 1997. Heparan sulfateproteoglycansof the cardiovascularsystem:specificstrirct.,res.-..gi but h"owis syn_ thesis regulated? J. Clin. Inuest. 99:2062-207 O. Sasaki,T., R. Fassler,and E. Hohenester.2004. Laminin: the crux of basementmembraneassembly.J Celt Biol. 164(71:959_963. The Extracellular Matrix ll: Connective and Other Tissues Canty, E. G., and K. E. Kadler. 2005. procollagentrafficking, processingand fibrillogenesis.J. Cell Sri. 119:134i_1353. . Comelli, E. M., et aL.2006.A focusedmicroarray approachto functional glycomics:transcriprionalregulation of thl giycome.G/y_ cobio logy 16(21:117-I 3 1. Couchman,J. R. 2003. Syndecans: proteoglycanregulatorsof .. cell-surfacemicrodomains?Nature Reu.Mol. "CiltS;ot."+rgZe_gn. -_ Kramer,R.Z.,J. Bella,B. Brodsky,and H. M. Berman.2001. The crystal and molecular strucrureoi.a collagenJikepeptidewith a biologically relevantsequence. . J. Mot. Biol. l\t,tlt_t+Z ,Mao, J. R., and J. Bristow.2001. The Ehlers-Danlossyndrome: on beyondcollagens.J. Clin. Inuest. 107:1-063-1,069. Sakai,T., M. Larsen,and K. yamada. 2003. Fibronectrnre_ quirementin branchingmorphogenesi s. Nature 423:g76-gg1. Shaw, L. M., and B. R. Olsen. 1991. FACIT collagens:diverse . molecular bridgesin extracellularmatrices.TrendsBichem. Sci. 16(5\:1.9'1,-194. Weiner,S.,'S7.Traub, and H. D.'Wagner. 1999.Lamellar bone: structure-function relations. J. Struc. Bi;l. 126J41,_2SS. Adhesive lnteractions in Diverse Motile and Nonmotile Cells from _ Barresi,R., and K. P. Campbell.2006. Dystroglycan: ro pathogenesir of hu-"r, disease.1. "Citt S";. lt9(pt. li.:Xlr[.5 2):1.99-207. . Bartsch,U. 2003. Neural CAMs and their role in the development and organization of myelin sheaths.Front. Biosci. g:D477_D490. Brummendorf,T., and V. Lemmon. 2001. Immunoglobulin superfamilyreceptors:cis-interactions,intracellularadaptersand alternative splicing regulate adhesron.Curr. Obin. Cett niol. 13:61.l-618. Cukierman,E., R. Pankov,and K. M. yamada. 2002. Cell rnteractionswith three-dimensionalmaffices.Curr. Opin. Cell Biol. 14:533-639. Even-Ram,S., and K. M. yamada. 2005. Cell misration in 3D matrix. Curr. Opin. Cell Biol. L7(5):524-532 Geiger,B., A. Bershadsky,R. pankov, and K. M. yamada. 2001. Transmembranecrosstalkbetweenthe extracellularmatrix and the cytoskeleton.Nature Reu.Mol. Celt Biol. 2:793-g05. Griffith, r . G., and M. A. Swartz.2005. Capturing complex 3D tissuephysiologyin vitro. Nature Reu.Mol. Cetj Uot.i(Slritt_ZZ+. Lawrence,M. B., and T. A. Springer.1991. Leukocytesroll on a , selectinat physiologicflow rates:distLction from and prerequisite for adhesionthroughinregrins.Cell 65:gS9_g73.

846

C H A P T E R1 9

|

TNTEGRATTN CG E L L St N T O T t S s U E s

Nelson, C. M., and M. J. Bissell.2005. Modeling dynamic reciprocity: engineeringthree-dimensionalculture modeli oi b.east a.chitecture,function, and neoplastictransformation.Sem. Cancer Biol. l5(51:342-352. Reizes,O., et al. 2001. Transgenicexpressionof syndecan-1un?y9rs a physiologicalcontrol of feedingbehavior by syndecan-3. Cell 106:1,05-1,1.6. _Rougon, G., and O. Hobert. 2003. New insightsinto the diversity and function of neuronal immunoglobulin supeifamily molecules. Ann. Reu.Neurosci. 26:207J3 8. Shimaoka,M., J. Takagi, and T. A. Springer.2002. Conformational regulationof integrin structureand funttion. Ann. Reu.Biophys. Biomol. Struc. 3l:48 5-51,6. Somers,,J7.S.,J. Tang, G. D. Shaw,and R. T. Camphausen. 2000. Insightsinto the molecular basisof leukocytetetlering and rolling revealedby structuresof P- and E-selectinbound to SLe(X) and PSGL-1.Cell lO3:467479. Stein,E., and M. Tessier-Iavigne.2001,.Hierarchical organiza_ tion of.guidancereceptors:silencingof netrin attraction by Slit through a Robo/DCC receprorcomplex. Science291t192b-193g. Xiong, J. P.,et al. 2001. Crystal strucrureof the extracellular segmentof integrin aYg3. Science294:339-345.

PlantTissues Bacic,A. 2005. Breakingan impassein pectin biosynthesis. Proc. Nat'l. Acad. Sci.USA 103(15):5 $9-5640. Delmer, D. P., and C. H. Haigler. 2002.The regulationof mera_ . _ bolic flux to cellulose,a major sink for carbon in plants.Metab. Eng.4:22-28. Iwai, H., N. Masaoka, T. Ishii, and S. Satoh.2000. A pectin glu_ curonyltransferasegene is essentialfor intercellular attachment iri the plant meristem.Proc. Nat'l. Acad. Sci.IISA 99:1.631,9-16324. Jorgensen,R. A., and \W.J. Lucas.2006. Teachingresources: movementof macromoleculesin plant cellsthrough plasmodesmata. Sci. STKE (3231:tr2. pollen tube guidance: Ki-, , !., J..Dong, and E. M. Lord.2004. the role of adhesionand chemotropicmolecules.Curr. Tip. Deu. Biol.6l:67-79. Lord, E. M., and J. C. Mollet. 2002.plant cell adhesion:a bioassayfacilitates discovery of the first pectin biosynthetic gene. Proc. Nat'1.Acad. Sci.USA 99:1.5843-15845. Lord, E. M., and S. D. Russell.2002.The mechanismsof oolli_ nation and fertilization in plants.Ann. Reu.Cell Deuel. Biol. 1 8 : 81 - 10 5 . L9uql,T. J., and.!7.J. Lucas. 2006.lntegrativeplant biology: , r!le of phloem long-disrancemacromolecul"i tr"ffi.-king. Ann.neu. Plant Biol. 57:203-232. Pennell,R. 1998. Cell walls: structuresand signals.Curr. Opin. Plant Biol.1:504-510. Roberts,A. G., and K. J. Oparka. 2003. plasmodesmataand rhe control of symplastictransporr.Plant Cell Enuiron.26:103-124. 'Whetten, R.If., J.J. MacKaS and R. R. Sederoff.199g. Recent advancesin understandinglignin biosynthesis.Ann. Reu.plant Physiol. Plant Mol. Biol. 49:585-609. _ Somerville,C., et al. 2004.Toward a systemsapproachto un_ derstanding plant cell w alls. Science306(57OS\ O206-2211. Zambryski, P.,and K. Crawford. 2000. plasmodesmata:sate_ keepersfor cell-to-celltransport of developmentalsignalsin plants. Ann. Reu.Cell Deuel. Biol. 16:393421.

CHAPTER

THt REGULATING EUKARYOTICCELL CYCLE against embryostainedwith anttbodies A two-cellC e/egans protein(green) a spindlecheckpoint tubulin(red)andCeBUB-1, on the is localized CeBUB-1 with DAPI(blue). DNAisstained spindlemicrotubules and kinetochore-attached chromosomes posteriorcell(nght) lt is presumed in the smaller, duringmetaphase attachmentandtenston.Thelarger, to monitorchromosome is no and CeBUB-1 anteriorcell(/eft)hasalreadyenteredanaphase, or spindlemicrotubules on the chromosomes longerdetectable embryo of thissecondcellcyclein the C e/egans Thusasynchrony presence spindle a functional of of both the observation the allows after and itsabsence checkpointproteinduringmetaphase, anaphaseIEncanadaetal initiationof ,2005,Mol BiolCe//15:1056]

control of cell division is vital to all organisms.In l\roper Pu.i..11t1ar organisms,cell division must be balanced with cell growth so that cell size is properly maintained. I If severaldivisions occur before parental cells have reached the proper size, daughter cells eventually become too small to be viable. If cells grow too large before cell division, the cells function improperly and the number of cells increases slowly. In developing multicellular organisms, the replication of each cell must be preciselycontrolled and timed to faithfully and reproducibly completethe developmentalprogram in every individual. Each type of cell in every tissue must control its replication precisely for normal development of complex organs such as the brain or the kidney. In a normal adult, cells divide only when and where they are needed.However, loss of normal controls on cell replication is the fundamental defect in cancer,an all-too-familiar diseasethat kills one in every six people in the developedworld (Chapter 25). The molecular mechanismsregulating eukaryotic cell division discussedin this chapter have gone a long way in explaining the loss of replication control in cancer cells. Appropriateln the initial experiments that elucidated the master regulators of cell division in all eukaryoteswere awardedthe Nobel prize in 2001. The term cell cycle refers to the ordered seriesof macromolecular events that lead to cell division and the produc-

tion of two daughter cells' each containing chromosomes identical with those of the parental cell' Two main molecular processestake place during the cell cycle, with resting intervals in between:during the S phaseof the cycle, the parental

OUTLINE 849

20.1

Overview of the Cell Cycleand lts Control

20.2

Control of Mitosisby Cyclinsand MPFActivity 853

20.3

KinaseRegulationDuring Cyclin-Dependent Mitosis

20.4

MolecularMechanismsfor Regulating Mitotic Events

20.5

and Ubiquitin-ProteinLigase Cyclin-CDK Control of 5 Phase

872

20.6

C e l l - C y c lCe o n t r o li n M a m m a l i a nC e l l s

879

20.7

Regulation Checkpointsin Cell-Cycle

884

20.8

Meiosis:A SpecialTypeof Cell Division

892

847

chromosomes are duplicated; in mitosis (M phase), the resulting daughter chromosomes are distributed to each daughter cell (Figure 20-1). High accuracy and fidelity are required to assurethat each daughter cell inherits the correct number of each chromosome.Further, chromosome replication and cell division must occur in the proper order in every cell division. If a cell undergoesthe eventsof mitosis before the replication of all chromosomeshas been completed, at Ieastone daughter cell will lose geneticinformation. If a second round of replication occurs in one region of a chromosome before cell division occurs, the genesencoded in that region are increasedin number out of proportion to other

OverviewAnimation:Cell-Cycle Control{tttt

Daughter cells

Chromosome decondensation, re-formationof n u c l e a re n v e l o p e , cytokinesis

Chromosome condensation, n u c l e a re n v e l o p e breakdown, chromosome segregatton

DNAsvnthesis

FIGURE 20-1 Thefate of a singleparentalchromosome throughoutthe eukaryoticcellcycle.Following (M), mitosis daughter cellscontain2n chromosomes in djploidorqanisms and 1n chromosomes in haploid organisms. In proliferating cells,G1is the periodbetween the "birth"of a cellfollowingmitosis andthe initiation of DNAsynthesis, whichmarksthe beqinnino of the S phase. At theendof theS phase, cellsenterG2containing twice the numberof chromosomes asG1cells(4nin diploidorganisms, 2n in haploid organisms). Theendof G2ismarkedbythe onsetof mitosis, duringwhichnumerous eventsleading to celldivision occur.TheG1,S,andG2phases arecollectivelv referred to as interphase, the periodbetweenone mitosis.nO tf,. next Most nonproliferating cellsin vertebrates leavethecellcvclein G,. entering the Gostate.Althoughchromosomes condense only duringmitosis, heretheyareshownin condensed formthroughout thecellcycleto emphasize the numberof chromosomes at each stageForsimplicity, the nuclear envelope is not depicted

848

C H A P T E R2 0

|

genes, a phenomenon that often leads to an imbalance of gene expressionthat is incompatible with viability. To achieve the required level of accvracy and fidelity in chromosome replication and chromosome segregation to daughter cells during mitosis, and to coordinate these with cell growth and developmental programs, cell division is controlled by checkpoint surveillancemechanismsthat prevent initiation of each step in cell division until earlier steps on which it depends have been completed. Mutations that inactivate or alter the normal operation of thesecheckpoints contribute to the generation of cancer cells becausethey result in chromosomal rearrangementsand abnormal numbers of chromosomes,which lead to mutations and changesin gene expression level that cause uncontrolled cell growth (Chapter 25). Normally, such chromosomal abnormalities are prevented by multiple layers of control mechanismsthat regulatethe eukaryotic cell cycle. In the late 1980s, it became clear that the molecular processesregulating the two key events in the cell cyclechromosome replication and segregation-are fundamentally similar in all eukaryotic cells. Initially, it was surprising to many researchersthat cellsas diverseas baker'syeastand developing human neurons use nearly identical proteins to regulate their division. However, like transcription and p.ot.in synthesis,control of cell division appearsto be a fundamental cellular processthat evolved and was largely optimized early in eukaryotic evolution. Becauseof this similariry researchwith diverse organisms,each with its own particular experimental advantages,has contributed to a growing understanding of how these events are coordinated and controlled. Biochemical,genetic, imaging, and micromanipulation techniquesall have been employed in studying various aspectsof the eukaryotic cell cycle. These studies have revealedthat cell division is controlled primarily by regulating the timing of nuclear DNA replication and mitosis. The master controllers of these events are a small number of heterodimeric protein kinasesthat contain a regulatory subunit (cyclin) and catalytic subunit (cyclin-dependent kinase, or CDK). Thesekinasesregulatethe activitiesof multiple proteins involved in DNA replication and mitosis by phosphorylating them at specificregulatory sites,activating some and inhibiting others to coordinate their activities. Regulated degradation of proteins also plays a prominent role in important cell-cycletransitions. Sinceprotein degradation is irreversible,this ensuresthat the processesmove in only one direction.

R E G U L A T T NTGH E E U K A R Y O T TC CE L LC Y C L E

Overviewof the CellCycleand Its Control \We begin our discussionby reviewing the stagesof the eukaryotic cell cycle, presenting a summary of the current model of how the cycle is regulated, and briefly describing key experimental systemsthat have provided revealing information about cell-cycleregulation. As mentioned earlier, since the fundamental molecules involved in cell-cyclecontrol are highly homologous in all eukaryotes,virtually everything learned about control of the cell cycle, whether it is from studies of yeast, sea urchins, or frogs, is relevant to control of the cell cycle in human cells.

The Cell Cyclels an OrderedSeriesof Events L e a d i n gt o C e l lR e p l i c a t i o n As illustrated in Figure 20-1', the cell cycle is divided into four major phases.Cycling (replicating)mammalian somatic cellsgrow in sizeand synthesizeRNAs and proteins required for DNA synthesisduring the G1 (first gap) phase. \7hen cells have reachedthe appropriate size and have synthesized the required proteins, they enter the S (synthesis)phase,the period in which they are actively replicating their chromosomes.After progressingthrough a secondgap phase,the G2 phase, cells begin the complicated process of mitosis, also called the M (mitotic) phase, which is divided into several stages(seeFigure20-2, top). In discussingmitosis, we commonly use the term chromosome for the replicated structuresthat condenseand become visible in the light microscopeduring the prophaseperiod of mitosis. Thus each chromosome is composed of the two daughter DNA moleculesresulting from DNA replication, plus the histones and other chromosomal proteins associated with them (see Figure 5-40). The two identical daughter DNA moleculesand associatedchromosomal proteins that form one chromosome are called sister chromatids. Sisterchromatids are attached to each other by protein cross-links along their lengths. In vertebrates'these cross-linksbecomeconfined to a singleregion of association called the centromere as chromosome condensation progresses. During interphase,the portion of the cell cycle between the end of one M phase and the beginning of the next, the outer nuclear membrane is continuous with the endoplasmic 'With the onset of mitosis in reticulum (seeFigure 9-L,9l). prophase,the nuclear enveloperetractsinto the endoplasmic reticulum in most cells from higher eukaryotes, and Golgi membranesbreak down into vesicles.As describedin Chapter 18. cellular microtubules form the mitotic apparatus' consisting of a football-shaped bundle of microtubules (the spindle) with a star-shapedcluster of microtubules radiating from eachend, or spindle pole. During the metaphaseperiod of mitosis, a multiprotein complex, the kinetochore' assembles at each centromere. The kinetochores of sister chromatids then associatewith microtubules coming from opposite spindle poles (seeFigure L8-36), and chromosomesalign

in a plane in the center of the cell. During the anaphaseperiod of mitosis, sisterchromatids separate.They initially are pulled by motor proteins along the spindle microtubules toward the oppositepoles and then are further separatedas the mitotic spindleelongates(seeFigure 18-41). Once chromosome separation is complete, the mitotic soindle disassemblesand chromosomes decondenseduring tilophase. The nuclear envelopere-forms around the segregated chromosomesas they decondense.The physical division of the cytoplasm, called cytokinesis, then yields two

the nuclear envelope,which then pinches off, forming two nuclei at the time of cytokinesis. In vertebratesand diploid yeasts' cells in G1 have a diploid number of chromosomes (2n), one inherited from .u.h p"..rrt. In haploid yeasts'cells in G1 have one of each chromosome (1n),the haploid number. Rapidly replicating human cells progressthrough the full cell cycle in about 24 hours: mitosis takes =30 minutes; G1, t hours; the S phase, 10 hours; and G2,4.5 hours. In contrast,the full cycletakes only =90 minutes in rapidly growing yeast cells' In multicellular organisms' most differentiated cells

thereby providing control of cell proliferation'

RegulatedProteinPhosphorylationand DegradationControl PassageThrough the Cell Cycle As mentioned in the chapter introduction, passagethrough the cell cycle is controlled by heterodimeric protein kinases' The concentrationsof the cyclins, the regulatory subunits of the heterodimers,increaseand decreaseas cells progress through the cell cycle' The concentrations of the catalytic s,rbr'r.titsof these kinases, called cyclin-dependent kinases (CDKs), do not fluctuate in such a characteristicmanner in yeast cells, but they have no kinase activity unless they are associatedwith a cyclin' Each CDK can associatewith a small number of different cyclins that determine the sub-

heterodimer. O V E R V I E WO F T H E C E L LC Y C L EA N D I T S C O N T R O L

O

849

l])A)

'l'll)

ltroAUV)nl lHr 9Nuvln9lu

|

0z ull-dvH)

0s8

'eseqdord pue'suotalo)sol^l o] saJoq)olaur) or.uosoLuorql srsaut)ol^) ]o ]uouiqrellepue olutsolnqnlol)tur 6uunpuorlesuopuo) aurosor.rlorqr'snleredde a;purds rrlolrru lla)ralq6nep ]o 6utlopoLuor'tel)nu llol-ralq6nep 'uMop)eerq 'uotlesuapuo)ap eq] punore sauelqulauJ o1salnqnlor)rLU adolenue Jeal)nu ^lquJesseaj oql uro,L ,Lo6ur;apourer ]o aurosourolqf ut s]lnsar'aseleqdsoqd .real)nu :srsolrLUsdels{;leeelenrpe9q63-ur;rlil rrloltLu'29 a1e1 uot])eaq] q]l^^ 6uole tL)p) 1o ]o '{1rnr1re 'e1e1duor sruorlerldarVNCa)uo sa)uauruo) u1 z9 relues11a> aql sau.rosealord {q ;63-ur;:{t )t}o}l.uurdorp6ur11nsat 'sur6uo uorleperOep rraql6ursnet'sur;>{t uollerr;dar srseqlu{s :r1o1ru.r sa1e1{urlrnblqn{1od vNc puP vNc o}e^tpeuaqi 3716y aql 'salodelpurds aql o] luauonoLu s;61-ur1:{raseqd-5 eqI saLr.rosea}ord {q uorleperbap rol uraql aulosotlolq)telly eletedes sprleurorql lalstsseoseqoeue jo lasuooq] pue'a.tautollu0) aseqd-5 sele;{urlrnbrqn{1od 6ur>1reu'ry;61-urplir 0r]] ,tosro]rqrqur lalstsaql 6u11ur; sreseqd5 saxa;duoturalord uorleperOep 1esprleLUolq) ]pql ose6!lurelord-urlrnbrqn lf S aLllIq palerlrur ;o urs]lnsoJ srqf sauloseolotd saua6+ouorldulsuerl alenr])e {q uorlepetbap uorlerr;der lo;11Ourlreu'uun)as VNCro1perrnbar Lg 'Lg-prr! a^r})eares;6]-urP^)ou 'tg {peeu1 s e 1 e 1 { u r l r n b r q n { ; o du' ar sael o6trd; - u r l r n ber'q( )n/ f d v ) x a l d u u o : ul s;63-ur1t,{r 'pauiurer6erp 'sa;od 6urloulotd-eseqdeue oql ;r1un dol aql le uMoqssrso]ruJ aletedas louuer{aqi inq ]o so6elsrofelUaql q]lM preMo]sptleuolq)telqOnep pu?'sase}eqdsoqd alpurds elrsoddo srep{: ;;a:aqf sase6rl uralord-ut}rnbrqn aql 11nd selnqnlor)tul e ; p u r d s ;6ou r u a y o qpsu es l o l o r !a l n q n l o l l t ng 1 t e l d e q Su r uralord;63-urlr{r l{qpelelnOet orpsuor}rsuerl urolord'saseur>1 'suor}rsuer} possnrsrp se 'aseqdeler-u ursnleredde ep{r-;;ar ;o uope;nEepZ-OZlungll V e;purds aql ]o salnqnloJ)rLtl ep{r-11a3 sur6r.rouorlecr;derVNO otenrlce s;63-ur;cAcoseqd-S

a s e q dg sacnpue r sebr; u r e l o l d - ur l r n b r q n

lcs

aseqd s rol s1;ecarederd s)OC-urlc^crg

slsolrurul sdels A;reealenllce s)CC-urlc^cc!lot![A

s!soullolAc pue aseqdo;a1 eseqdo16

s e c n p uers e b r ; nbrqn C/JdV urelord-u11r

olpuros

srso]rLlr ur soals alel o c n p u re s e l e q d s o q d tIcpc pue 3/cdv

llll+

srsolrlN :uolleuluvsn)oj 'saxalduroc;q3

-uqc.{r tg ;o &r,rnce pue srseqludsaql Suueln8er dq dlreur 'srusrue8roraq8rqu1 'uorl -r.rdpatrerqresruorsrlrp IIaJJo lorluoJ -urqdar y51q Surleurul ro; parrnba.rsyq3-uqc,(c asegd-5eqr pue 'suralord uonecrldar rerpo pue seseretudlodVNC (soreqd -soqdlrr aprsoelcnulxoep azrsaqludsleql sorudzueeql epnlrur osol{J 'uollEtrqda.rauosoruolltr roJ pa.rrnberseua8Suqlorluoc srolrpJ uoudtrcsuBrlSuneln8ar,(qaraqrpue Suneldroqdsoqdlq aseqd5 otur dnuo Iorluof, syq3-uqcdo tg ryO)-url)^) lg

'(67-g e;n8rg aes)sauoseerord,,(guonepe.r8ep rot uraqt Surlreur'surelord alPrlsgns efeldulfrnbrgndlod sese8rlutatord-urllnbtqn reqr IIEtraUDD1V sE PetEI^eJqqE sr rolder{J srqt uI PUe 'ataosolc{"cetTtpellPJ solulletuososlE sr xalduroc Surlouord-aseqdeue otlJ'suolllsuert e1c.{c-11ec 'xaldLuoc Sutlouto.td-asoqdouv ;o sroleln8ar da1 osle ere aqr pue g'35 'sase8rl uletord-ultrnbrqn o,r.1 '(7-97 e.rn -3rg) syq3-ur1r.{r rltollur pur 'syq3-urlcdc aseqd-5's)CJ -uqrdc IC :sesselJooJr{l olul pepl^1p alr syq3-ur1cd3

ts8

'rour_No) sr cNV l'l)A)'t'l]) 3Hl Jo MilAUrno

qrr^\ sllar tuelnru proldeq Sururo;suerr dg dyrpea;potelosr oq upr salelle luelnvr )p2 olrlrsues-aJnle.redruololrssoJoJ 'stuelntu (e1od:uorsnry-gat) cpt Jo selellp addl-p1r,t1aql pollpl ore sllor qlns '(9-g ern8rg ees) parelosrdlsea e;o; -oreql puE dllerrdocso.rcnupezruSocardlrpeer a.reelcdc 11ec aqr qSnorqr sserSordol parrnbe.rsuratordcr;rcadsur slreJOp Sursnec suorlElnru oArlrsues-aJnlerodurolqlr.,t,r susrueB -ro 'stseadeserlr qtog uI 'a1cdoaqr Jo ;o uorleln8ar parat -lp lrqrqxo leqt ro apdr 11acaql ur sdotsorlrcedsrp pollolq are ter{t sluelnurto uonelosrJoJInJesndlleoodsauooqalerl aqwod satKutotoqccosoztq)S tseaduorssr;perplardlluersrp oqt pu€ apxsrna,n)satf,wonqccrg rseed Surppnq :r]"arr", esaqrdlrruopr ot pepeeuoro^\ stuerurredxayecruraqrorqp.r" cnauaS'e1o,c lyer aqr;o saseqdhtr pue S ar{l olur .,(nua1o.rl -uof, srotf,e' elqlsnJJlpleql petensuotuopsluourradxe uorsn; -llal osoql q8noqlly 'VNC aql uo pelquressesaxalduroc uorlerqdara.rdou o.reaJaql asneJeqrolJnu zC ur Jnf,f,olou soop srsor{ludrVNC 'rolf,nu t9 dlrea ur sur8r.rouorteorldar VNCI uo pelquessp saxaldruoc uorleorldere.rdaql ele,rrloe 'saxaldurocyq3-urlcdc aseqd-5arE srotrEJaseql uPf,I{f,II{^/\^. ler{t ^{oDI ,vrou a/N 'snelf,nu zC e ul srsaqrudsVNC olnp -ur sJolf,pt oseql tnq 'srseqludsVNC orelnurrtspue touupJ rC E IIaJ to snapnu eqt rJtuo upl IIrl eseqd-5up ur srotf,EJ olqlsnJJlpsnr{J 'rallnu zC aql ur porrnrf,o aupnudqr paleq 's11acaseqd-g ot pesnJa.rerrrzC ul -EI to uorlerod.rorur ou sllar uaq^. tele,ro11 'uorsnt ror;e dp;oqs snalJnu r5 aqt ur ue8aq srseqludtVNC leqt Surtecrpur'snapnu aseqd-5aqr se sBsnolf,nurC eqr Jo VNC oqt olur peteJodrof,ursE1\{IOq IIOII^. -eyaqt'aurptudqi paleqelorperol pasodxasllar pesnJaqr pue eseqd 5 ur sllel ot pesnJaram IC ur slleJ uaqo.'dlrepurr5 'sexaldurocyq3-ur1c.{r rrlolrrrr or.{lare srol -lPJ asaql lEr{] ^\oDI .l\\ou e/N 'srsolfiu d1.reaqlr.rt pelErf,osse sassacordegt;o .{ueur o8repun ol ralf,nu aseqdralurpeJrot sllef,f,rlolru aqr;o ruseldoilr aql ur slueuodruocro luouod -tuol elqrsnJtrporuosteqr prtptrrpurSurpur;srr.lJ'(f-02 ern -3rg) pesuapuof saruosouorqf rreqt pue palJerlor sedola,rue rBOIJnurraql'srsolrru ur sllol ot posnt aram ayc.{rilal eqr to aseqdzg ro (S (IC aqt ur sller aseqd.ratur ueql6 'sllel uerleru -rupru pornllnf, glr^. sluorurrodxeuorsnJ-lleruorJ arupf,elf, -dc 11ecaql aleln8er srolrul elqrsnurpteql aruapr^arsrrJeql

suroloJd lorluo) al)^)-llo) alelosl pue fllluept ol Pesn uoog aneH suels^s leluoruuodxl asre^lo 'ase8q uralord 'sexaldruot -ultrnbrqn l/f,dv aql to uoneln8er pur )eJ -uqrdr ro s)cJ etp^rlf,pur (sry3) srorrqil{u )(l] puE purq ruql ,solrsSunerrrlrepue Jo ltqrgels pue srseqludsoql Jo uorleln8er d.rolrgrqurtE s)CJ ;o uorleldroqdsoqd 'fir,rrpp eseuDlurolo.rd )Cf, rot pa.rrnbarsuqrdc 1o uoneper8ap pur srseqludsoqr Jo uortelnSer:susrupr{Jorrr Jo ltarrB,Le q8no.rqrsyq3-uqcdr eqr 1o serlrlrlf,eosEuDIurolord aqr 8uq1o.rruor,,(q uorlcunl susrueqfau lutodlcaqc eser.{J'seuosoruorr{r pateoqder dlalerncre }o rag 'uors -tunu poJJof eql selrafer 1ec rerq8nep qJrc lpql 8ur;nsue -hlp a.re IIer 1o lrqapg q8rq dlreurproelxe aql ro; algrsuodsa.r srusrupqra{uof,uelle^rnsrurod4caq3'srseurlotdc pue asegdolal

'aseqdeuele soruosoulorqrratq8nep;o uoneredes Jo losuo pue 'srsoJrur uon€nlur 'eseqd g Jo Jo uoIlEIlIuI apnllur slurod4raqc dq pa.rotruou suorlrsuprtapdrla3 'pelalduroouaaqseq puodap .,{aqrqrrq.u uo ssecordsnorrra.rdeql 1lun palerlrw lou oJEsuou -rsurrl ap^,b-1yec roleru lerp eJnsueol alB.redoslurod4rago su u^ .ou>lsusrueqreu aJuEIIre^rnS suslueqlal4 lu!odl)aq) 'd1uo auo ur elcdr ler eqt esrolprt ot poJroJ uoDoa.rrp ue 'sutalord;o uorlepe.r8epparpl eJp slleJ 'ssalord elqrsJo^oJJr -n8ar eql dq pa.ra33r.rr are suortrsuerlosor{lesnef,og'sprlpruoJqf, relsrs uoe \teq uorseqof,f,rreuorluoJ PUEltr^rpe yq3-uqodc qroq Surleln8arlg-srsaur4or.tc pue eseqdolarrr-uoua6 e 1oOuruaetts pueuot})nl}suo) lsee{ aq]'(Jlal) sllo))tloltr.u ul oq]pueluosqesrodola^ua.ieol)nu lenpt^rput aql lo suorlerlsnllr poltelap oror.rl lo+g t-S pue1;-9 satnbrlee5 aq ]ouue)saurosotloJq) (eE€-9pueq7-;,sern6rlaas)paqsrnburlsrp aqwod 5 urseue6*tp: ed{1-plrnn a}elost ol pasnstotnpototdautes 0q] puei)elursl os 'posuopuol ]ou oJesauiosouroJq) lenpr^rpur oq1 sll0)]uelnuroql urant])o1ap sr1eq1 euaboql o] 6utpuodsol.lol pesnlunu; luauluadxe aseqdtalut reol)nuat4l'(1q60 s;1ar adolenue euaO oq]soulel{uo1otsrqlulor}palelost 36; ed,i1-plrnn VNCptr.lseld (p)'slle)aseqd.ralur ur srsolrtusa)npu!slla) uorsnj-llal 1or-uerber6 ernleraduel anrssrulladuou aql ]e ^uolo)e LlJro] pueeletrlder o11;et 'paluer-ue;du.ro) )rlolrurur Jopel elqrsn#rP lluldxl v v E-02 lunglJ lvtNll all] 6ulmolle st (oseut) e 'gZ)O) luepuedep-urltic 0ltol![A sor..uosourorqc seurosoruorqcrg ase)srq]ur)euab]uelnuloq]]o uotslaA ed{1-plrnn oq}6utute}uo) prurseld e dn sa)e] eJel oqf atnletedutel enrssrul.roduou ]eql llal oq] satuolo) r.ulo]]ou op s1ueu6etl qtnsdn o)e]]eq]slla) ]e pauilo+suel] lurelotd )pf a^tpelapaql opo)ua]ou op ]eq](/ pue q)nslsol4 lueubelly116:ruLoua6 X "6'a)saua6apn;:ursluauOet,r euo6urureluot pruseldel6urs q)el e dn sa>1e1 llo)paulJo+sueJ] ()" SE)aJnlprodutal enrssruladuou aq1]e te6eluaulnuuo pe1eld puesllo)ed{1-p1rnn uLotlpetedetd {tetqr;tru.roue6 e q}tMpouJro}sueri ereeuab16l e ul uorlelnuJ anl]tsuos-alnleteduel e q]tmsllo) ]uelnn 'sluelnu ,p) lo uotleluaure;druor leuotpunl{q fte.rq;1 rgruoueEaelst,tara)'Se utorl palelostoq ue) saue6(9g3) e;r{r uorsrnrp-;;er edfl-p;rry1t-02 lungg 1VINjt4luldxl V se6els alc^c-llocsnoueA 1 eA u o ; o cu r s l l a 3 (q)

s2303 ).

82303

I. llac crtol!hl

1;acaseqd-r9

3. gg 1euanotb sllacsrgzepr psLuloJsuelI

3" 9Zle unnorD 's vNc aelsr^aJao adAypgrnn 1o Arerqr;prr.useld Lllr/\^uIo]sueJl

sllec st9ZoPc

€s8

A ! n r l ) v l d t A o N V s N t ' l ) A ) 8 s t s o J _ t nl o ' l o u l N o )

' s J J eul E r u n qS u r p n l c u'rs 1 1 acrr l o 's8or; -d.re4na 11eor alqecrlddedprerrp e.reserceds.raqropue 'sllasut 'surqrrn 'r8un; ur sarpntsalcdc-11ar eas teql os 'pe.,lres -uoc dlq8rq ere tueql lorluor tpqt surolord ar{f puE sroteln8ar rolselu asaql 'puoras 'suralord droteln8ar apdc-11ecrelsEtu Jo raqunu llerrrs e dq pereurproor pue paleln8a.req IIE uel solnqnlorrrr.u elpurds ol saroqf,oleur>l euosoruorr{r }o tueru -r{l31lEpue 'uotleruro; alpurdsrrtotrlu (u,lirop>1eo.rq adola,rua Jreltnu 'uorlesuapuoJoruosoruoJqJse rlfns srsolnu;o sa8els dlrea aqr ur sassaco:drelnlelou xaldruor 'lsrrg 'a1odc11oc eql Jo loJluor lnoqe sarre^of,srpelqp>lr€uer o.4A1 01 pel ser -Pnls osoqJ 'srsolnu uo r{JJeosoJ ruo{ etuef uorlrsurJt JIf,dJ -lleJ lerrtrr E slortuof ler{t uratord e;o l.ra,roosrpIerrruraqJ

flrnrpy JdlNpue surl)A)Aq srsolryllo lorluo)

'Q4yl toltttJ Suttotuo.td-stsojtu4 e se ol parJeJerdJuoruruor sr )C) pue uqc"{r rnolnu e 1o pasodruor rarxrpora -teq 'uortrunl laqr qcrqm ur apdc V 11eraqr ur pol.lad eqr .{q parlsselc pue prtsrl a:e :eldeqc snll ur pessnrsrps)Cl pu? surplc asogl :s1651

g uqcd3 'y uqrd3 y uryc.,t3

suqcdcrrtotl14J uryc.{rcrlotnupue esegd-5

surlcdctC-PtW

sutTcdc addr-(

uqclc eseqd-5pue lg-are1

g uqrd3

z)cl

,I)(f, Z)Al

9)Clf,.')q] 'sluale alcdr-11ar sorpnts Jo lelrueqrorq roJ strprl -xa Jo sef,rnosaprrrords33e pazrlrrra;,{lsnouorqruds uorl sodrqruallree pue s33e oterqeue^ur pue uergrqdruy r '(y-97 ern8rgaes)eplr IIer aqr aleln8arleqr saua8yo uolterl;rtuepr eql ot pel rJJrr{{\'stuelntu(tpt) ap1'cuorsr^rp -ll0l ]sea,{ur srlpnls rlleueC r Jo uoIlEIosI eql Pe.^^'o[[e 'sllel asEqo -S ol pasnJare deqt uoq.^ sllel t5 ;o rapnu eqt ur uouef, -r1darygq etelnurls saxalduoc yq3-urlcdr eseqd-5.d1.re1 -IurS 'sllo lrtotrtu ot pesnJere daqt ueq,4asller .C p.r" tC ur adole,tuarea]Jnu agl yo dlqurassesrppup uorlpsuapuor eluosoruorr{Jasner saxelduotr )CJ-urpb rrlolrTrg 'a1ofo IIer oqr arelnSorsrotrpJ elgrsnJJrpteql petensuotuep lsrrt slle) uerleruruerupernllnJ qtr.,rrstuerurradxouorsn;-llel r

Zqll

'iqlf,

tqll'€qll 9qlf,'Sqlf, Zull'Iull €ull SZrp:)

s)(]

rrrotrl^J

yq3 aseqd-5pue r5-ase1 S)(I) I9-PIW

surpdcrrtotnu ele.I suqcdrf,rlolrrudlreapue aseqd,5erel suqcdcasegd-5dlreE surpdrt3-ele1 r3-pry,q ur1c,(c (,(1uoauo);q3

Eylsl^EUE]'S

'potertrursr dels txau erJlaroteq dlrcarroc parald -ruol sr ssacordqlea teqt ernsuo ot susrueqreru ef,uEIIreA -rns lurod>paqr dq porolruou ale suorlrsuprl apdc-11a3r

gt)pl

'saurosealord dq uorrepe;3apro; ureqt 3ur>1.reu's.rote1n8o.r elcdr-11arfa1 eleldurrrnbrqn,{yod osle sase8rluralo.rd-urtrnblg.rl,tnqnsrtlnru e3rc1 .(7-97 a.rn8rgeos)elcdc IIaJ eqt q8no.rqrIIOJE to sserSordalplnSor 'lrunqns aspur>l ruapuedap-urlodrcrldlerer B pu€ trunqns uqrdo droleyn8ere;o pasoduroo'saxeldurocyq3-ur1cd3 r

TrpJ

(d1uoeuo)uqcb cnolr141 (dpo euo)yq3

Tgwod's lt/tvN

NHI0ud/ tjstNvcuo

'(1-97 a;n8rg eas) (srsorru pue uouecrlderVNC realrnu ,(uorrecrldar zC yo uorralduot eql uae,la;eq porred eqi) pue '(uonerrlda.ry51q realJnu VNC reallnu;o por.radagr) 5 Io uolteltlur eqt pue srsotnuuao.,rtteq porrad oqt) IC '(srsolnu) oql r 1J :seseqdrnot orur popl^1psr elodr 11arcrro.,{.re1no

lorluo) sll pue al)^) lla) oql lo ^,\al^ra^o

'dSolounuretorpeds 'sroleln8er -sarceds elodr-1yec aqrrrsep ol s(ural Jo pealsur 'elrlre are daqr epdc leraua8esn IIr,4 a,vr'alqrssodre^euor.l/N IIer oqt ur uaq,r\solerrpurpue .rardeqrsrqt ur ssnrsrpa^{ reql

asoqr Jo serueu Oqr srsrl yjz ?IqeL 'reJJrpsalErqelra^ pue stsead uor; seseur>ltuapuadep-ur1c.{cpue surycdcsnorrpl (suoseer 'a1odc to setueu eql lerrrotsrq rog IIel er{r ur slurod uortrsuerl da1 aqr Jo r{Jee tnoqe srg8rsurpapr,rord aaeq saqreordde pue surelsdsletuarur.radxatuareJlrp qlr.r\ peurel -qo sl]nsal 'aas e,tr sV 'sluala droleln8er snorJe^eql to 11r,vr. slrelep rer.lunl luasard pue 'Z-02 ern8r4 ur pezrr€ruuns uorl

l'1f A)

@]r]6u{do3:(q)}rpdl sa}^)oo sndouax qdet6otrr4 (q) [a)e]o]or.ldlt\St to se6eneel))ruo^lqua {1teesnouolq>u{s71 ;o stso}LLl }slt} aq} pue uorletrldarVNC soo6rapunq)tqm 'elo6,izploldrpe e:npotd ptoldeqoql q]tM sosnlsnallnuoldaleLUa] o] snal)nuojdLuJods proldeq6ur1lnsat aq_L:E de15Apoqte;od puotas ut otuosoulorq) e q)ee jo prleuolq) auo saleutuJtla leql uotstntp ;1etletulauu.t{se puorase obrapunpue ll slsotau;o eseqdeueq6notqtpaarord {1qOrq '1sere 01 uraq]6urnno;1e aseqdelaLu ltaq] urotl s66asasealat uLteds {q uorlezr;r1re3 :fl do}S'll srsotaul;oaseqdeler-u ut pa}sale sllal 66a luaserdaro1{;lerrleuaqrsuMoqsole selnqn}olltualpurdso1

eberee;c lsr!l

sne;cnuo.rd sne;cnuo.rd aleural elen

'l'llf

)tro^uv)nl

tHr DNtiv'ln93u |

0z ulrdvH)

ts8

popouuo)saurosor.lorqr ul slsoJre onnl 66aue p;ar,io1aseqdeleuL puell slsolausa)uaururo) ,{lelerpeuuurt aybooaql lpoq rc1od1str7 uorsl^lp aql lleqslaoxa e olursaurosol.uoll) 0q] palle)lla)lleuJs lla) sno6o;ou-roq {1q6rqe puesotlosor.uotqr 1ouol1e6er6e5 1e:u1au.rLu,ise o1{;;ettleueqts ut s;;etluasetdat :I da15| srsoraur ,Loaseqdelau.r a;purds )tlollulo] pal)auuof uMoqsaJe(uooj6) salnqniorrrr-u pasdeu^s jo sred oMI I stsotoLr.i (anlq)seu,rosoLi.iorqr snoOolotitoq qlrMoleua+llnpe uP raluoo] sal^looaql sosnP)auoralsaoord {1;errblns saiboo sndouaypa}satle-zD }o +ofue^oarll Luollpo^or.uar :E dals (e)'say{roo sndouaxIo uo!}eln}eu )t}ototl.l luaLr.rlea{ seletnulllseuola1se6o.r6 s-02 lUnglJ'IVINluululdx:l v

ll slsolaul ;o eseqdelaru u1 papale 663

lsrsorow

zg u1papeue e$co6

E

.e

1

euola1se6o.t3

uorlezrlrual

Apoqrelodpuoces

(e)

Apoq re;od 1sr3

'selurl -saSord SuIPI^IP EInlsEIq eql uI suoltrsod aqI lE rueJaJJIP to ef,uosqeaqr ur s33e otur ornleru ol saldcoo aqt 'snouorqludsuou erE SUoISIAIP selElnrurlsse],{roo slleJ orur srsoraru;o qlr^ luanb polsoJJB-zD aseqdeleru tuereJJrP tE II (oJeqds 'd1a,rrl -asqns puP (s,ry\ols uaql uorsl^IP IIeJ 'EInlsEIq aqr ur s33a uor; useldoldc polsorrE uorlfeluroDrur to -Eurallv '(5-97 a;n8tg ees)11srsoreru;oaseqdeleuruI ^\olloq B Surlereue8'selcdr IIOI snouoJl{cuds'prder eroul II Polsor -rc 33e oql ol eldf,oo E ruory uorternletu atlroo q8norqr paecord uaqr sllol f,IuodrqlrIoSurllnsa.req1 'su€aq palserre-zC 'uotlp,tnloLu)Uotaut o8rapun degl 'orlrl ur srsauoSo.{Jquad1;eeJo UoISI^rPfllolllu lsJI} aqr PUEs^{ol ;o ssocord aqt -1o; uonecrlder VNC 'snelrnu etot[z Ploldp e Sunnpord euoralseSordr{tr.,ra pelpe.rrpue 3or; opure} llnpe uE;o dre,no 'snolonuord urreds proldeq eqr qll^\ sesnt uegl snolf,nuord eqr uorJ paloturr JJE sJt,{f,oosnQouay palsa.r.re-zgueql6 'sISorJu sateldruoc PUP tsor sl le))r leuos 33a proldeq 3uulnse.r eql -.rey1eseqdeleu sll ulor1 peseelarsI snolJnu 33e eqt'uttads ur srsolrl puPsal^)oo u! uorleJnlen)rlororu l dq pezqrrre;ueq1tr 's33apaller ere sllor eql a8ersstgr ry'(5 selelnurls(ran) Jol)eJ6u11ouro,r6-uotletnlen -67 arn8rg) aseqdeleurJIloIOrupuof,oseql ol ssa.rSordpue I srsorerurelua ol sal,{coo PelseJJe-zceql sef,nPulqllq,trt 'auoralsaSordauouroq ploJals oql elaJJOSslloJ uEIJeAo '(stsotaur deqr.,noqpue 'srueuod*o,u',olfi:ilffi; olEInBoJ s(alpruoJrlnpe uE 'a1etu e dq porepurtrs uoqlN '1tl37t '2-g a;n8rg aas) elodpef Surpee; to ^/\er^ro^oue roI 'Surrurur,l.sE eluJeua8 ol parrnber suoIsIAIP 11aceldtrlnut or{rro} pepaauslelrelelu eqr 1e Surlrdlcols 'uur I Jo relaue -rp e ol ozrsuI anor8,(aqr etull gllq^\ Surrnp 'sqluour 3 .ro; zC ur petserJeeluotoq pue VNC rteql alecrldar,(aql tre,ro 3or; aqr ur dole,tep saldcoo sV 'orll^ ur pererrldnp eq uef qcrq.r,rtuorleJnlEru aldf,oo to slue^e eql lno de1 tsrtl tsnur ozrr'sluorurredxaasaqt puelsJapun oL'stnaol sndouay 3ot1 eql ur uorlPJnlerualdroo to salPnlsIuory eIuEf,slsolllu soJnp -ur teql rolJEJ aql Jo uoIlPrIJIluoPI uI qSnorqr4eerq y str to eluos to uorlEzrJolf,eJeqf, PUPuorlEfrJrruapraql ssnfsrp oslp II6o/N'srsotrru ruo{ lrxe pue olur .{.rtuesyorluor leql rorreJ aqt Jo uorlpzrrelrerer{f,pue dro,rocsrpaqr pa.nro11e sad -ouax qlrl\ slueurrredxol\oq aroldxa om'uortoas srqr uI 'sluerurJedxo Ielrr.uoqlorqroJ tf,EJlxe IIer to slunorup luarf,rJtnserederd ot alqrssodlr soleur srql 'uorlezrlrlreJpue lusruleaJl euoJelsoSordno11o;lerll sluo^o aqr q8norqr l.lsnouorqcu,(sSurpaecord1e ere reqr perederd eq uer s33a pue saldcoo to sroqrunu a8rey reqr sr srsolrru ur pa^lo^ur srotreJ dpnls ol ruolsdssn{l Sursnto e8etue^pp

ss8

A|4il_)V ldhr cNV sNt'l)A) A8 StSOItA lO

'lOUrNO)

ul 'aqrrcsapuor{t pue per}rluoprse,&\ urlcdr d.roteln lrunqns -8ar egl .,'toqereg ssnJsrpe.&\tsrrg 'saqceorddu lelueurr.redxa tuoroJJlp q8norqr pezruSocer era.ry\CdW Io sluouodurof, 'petelduof, se.r\ tpql erotog 'pozrJotf,Ererlf, Ienpr^rpur or{l dlrcarrp pue dqderSolpruon{r urunloJ dq paryrrndse,r\CdW oJoloqpassedsreadlera,ras'aruosraqu;nlsr .{trlrlJe {dW oJns -PaIu ol posn ,(11erlrur desseuorloaful eldroo eql esnefafl rrotJEJsrqr to dll,rlrre IErJueBerolu aql seloueptpqt erueu E (rolf,EJSurtouord-srsolru roJ pupls uEJ osle g4yq u,{uone aqt alluerua^uoJ 'srsolru olur sllar Jo lrlua selouord fiqt '(t1Z ernSrg aas) sluaurrradxauorsnl-llal ur palpa^ -31ISJIJTOICC;EIqISNJJIP Eq] SICdW SNqI'PESUEPUOf, SOIUOS -oruorqf rrar{t puE uE aqr olul palterlor seuErqrueruJ€elJ -nu rroql'sr leql lsrsotnuparelues11ac aseqdreluroql (sller eseqdralurolur petfelur se,,\.s]leJfrletuos uerlerutuetupelsal -.redllecrlotru ruort useydor.{cueqlN 'srsoreruolur satdroo Sory;o drluo aqr sE IIe.r\se srsolrruotur sller rrleuos uErTEru -rurru Surpurl srql to drrua aqt slorluof, {dW reqt persaSSns .{lt,tuceg414 3o.g parrqrqxerpql JopeJ crloso}dce paurptuof, 'sr sllef trrlolru frlPruos uerlsr.ur.uEr.u !s33e agt olur parnt legl -eur saldroo aqt 'satdroo snQouay petsorrE-zC olur palra[ur sE1\\sllef uerletutuetu dllecnotnu petseJJe uror; urseld qrns -ordr uaql6 '(g-97 arn8rg) pezrlrrre;a.redaql lfrun 11srsorau ;o aseqdeleruur s33asnf,ouay 1o tsarrp Iernlpu eql ol relrurs sl sHI 'salnqnlorfrlu 10 dlqurassellqlqul lErll (eurrrr{llor

l 6 Z l i L L t 1 o o 7d x g t ' l L 6 l ' t ' l a i l e n

I f pupInse/\

aasl

susruebro eq}1ose6els luola#tpruol+pueelr{r 11at luala}}rp }e (EE dals),{}tntlte seyiroopalsere-29;o ldn ro] Iesse1srrlaql peprnord uorlraf urolry1 (g64) ropel 6urlor-uotd-uorlet nieu.ray{tooup suIe}uo) 'auolelsabo.rd useldoyir66e1eq1 6urnnoqs roquni tnoqltM +ouotllppe paleedeteq uet sseto.rd sautl a;dr11nr-u srql auotalsaOotd ,Loeruasqe Il srsolau ;o eseqdelau ur polseJre 661

''3'a) spunodruoc vtlm lueulee:r dg esegdeteru crlol -rtu ur pelsaJreeq uef slleJ uer1erutuetupeJnllnr 'elduexa rog 'podessesanods IIe ruog slloJ rrlolnu ur puno; uooq seq .&r,rnceg4y,q's8ory ur poro^olsrp d11er1rur q8noqrly 'srsolrruJ0lu3 sllar er{rse sesrruor{l puE sosolrruuae^4ceq sporrodaseqdra}ur aql ur srsotru to selJ eqt ur .{\ol sr dtr,rrlre g47rq'o.Lrgurad1.rea -dc snouorqcuds11 3urru.o11o; aql lnoq8norql 'ruarudolazrap rruod.rqua srsolru tsrrt aql sJelua (33e pazqrrra;) eto8trz Jo dltrrrlcECdW (uoll€zrlrlre1Sur,trolloC'II oql ure8e s11e1 Irlun aseqdelaurur petserres11eo 33a aqr ur q8q sureurorpue II srs -ororuralue sllol oql s3 ureSesasrruoql rnq'II puE I srsoreu uoo,\uaqaseqdreluroql roluo sllot oqr sE sllpJ dtr,rrloecdw '(;97 anfug) srsorau relua sllar aql se dlr^rlrp solnp 1 {dW -ur auoralsaSordqlrm tueuleerl lng Xlr^rlre CdW Jo slalel ,ry\ole^pr{saldroo pelserrp-zCpeleertun reqr puno} srar{rrees -o.r 'ollr,r ur uorleJnleu aldroo Sur.rnp sorurl lueJe;trp tE Itrlrtre 94ry .(esseot ualsds uorlcelurororruaql Sursn 'sllal f,rtodre>lne ur srsotrru IIe Jo uortertruraqt seleln8a.rreql rotJpJ fa4 aqr 'rarurporalaqyq3 -ur1c.{cf,nolru p eq ol lno pournl g4yq 'dproqs aas IIr^r e^\ sy 'G.IN ,to1crJ Surlou.totd-uouptnlpw palgr 'roloel srql .ro; desseue papr,r,o.rd os]Etnq oJlrl ur seldcooJo uorlernl?ur solelnurls reql urseydordc33a ur .rolre; E 'o uorterrJrtuepr '(g-02 arnSrg)ouorel Isrlrur eql ol pe1dpo lou Iuolsds sF{I

aql ur66eo.rnleur e 6urlereueO'(E dals)ll srsoraLU;o eseqdeler-u o1spaaroldpue(E dels)1srsoreuu sraluaeyitooeq1'(l[ dels) ey{roopelsare-29e olurpelrafuror>ruL srll srsoralx ;o eseqdeleul ul pelsorie66asndouaypazrlrpolun ue uror]useldoybaq11o ]ue)radS=uaqM'uollelnleut)tlotau salourords66asndouay Palsaileur ropel alqrsn#rpv 9-02lun9H ]vlNltNlutdxt v

z9 ugpelse.r.re eilcoO

I slsorehl

33g41ceful

ll slsolau ;o eseqdeleu u1 Pelsarre 663

raJsuell

r'1) )

'33a panylnlun agl ur tuasard oJe suorsrlrp 1ac dlrue ar{r roJ drrssacausVNUru oqr Ip (sodrgwa sniouay d1.reaur lalcdr 1ac oql ur slurod cr;nadsle pacnpord eq tsnu te,rezrLoq sVNUtu cgrcads 'suorlf,osralel ul poraprsuoJslseedur pue luaudoya,rapur rolel peteraua8sllol f,rletuosu1 '33a pezqrl -roJunoqt ur parols ere sodrqruesnflouay dlree ur sapdc 1ac paleJunrl eqr q8norql sse.rSo.rd rot porrnbar sluouodruoJrEI 'areradolou op -nlleJ el{t IIE rerlt eleJrpursuorlplJasqoasaql retpl ssnlsrpIIr^ e^^.srurod4ceqcapdo lec oql to dueur arer{,{\ sodrgrue suorsrlrp dlrea aqr ur dluo srnf,Jo ((>lJolJ lerurue Jo '(9-67 arn8rg aas) desse p elcdc-1yec,, Jo ef,uopuedepursrql uorlernleu aldcoo eql dq dtr,rrpe CdW Surdessepue sporrod 8urdre.r,raryeuseldotdr Sur,rouro.rueql pup 33e sndouay pazr -lruet e snolJnueqt 8ur^oruer,(q po.rerroxrpsE,{\uouoruou to -aqd sq1 'Jnf,f,oupf, uorldrrosuertou pup polotueJ sr snalJnu

'1unp g tatuel {q1ou.rr1 :699:95 I punlq)lposau leuadul ;o,(seunor ' ilD't861 le]asue^l1 urorlleOeneall a.rolaq 1sn[{;snolrdrxtd llo) ;1e; urP^);o ]unoureoqi ]eq]aloN (aulluerit)|erue1ur alnurul-o I snornetd aql 6ultnpa6eneelt ppqleql slla)]o uot])el]oql pue auobtepun (aurlper)pueq urplil eq] {]tsuolu! oLl] ]old(q) ulu 9Z[ ]e ure6e Jo +o pasear)ap pueuru 991 1eure6epa>1eed puequrp{taq1 sa}nutu9g ue6equeqlpueuotlpztltila+ lal+esalnutut 1eure6e6urseanur 9/ '1setluot urpaseeJ)ap u1 ur 1e{1rsua1ur firsuelur {ueppnsur;t,i: ') pasPaJlur ,{lsnonurluor pue g q)ns'surelotd se 1so171 lurodeLurl q)ea]e pa^oLuar sa;dr-ues 6urnnoqs 106SCSeql 1ouet6orpelolnv(e) ,{do>sor>rr.r.r {q abeneal> lla)}o uotpa}aplo} puelebapruelfu:e,{;od 'uotleztltual -scs ue uo srs,{;eue urelotd to,Lua)e}elaMse;dues Jeue 'pappe autuotrllaul solnuruJ sleruolut elnutul-O sem 97 6uruur6eq I ]V -sn,pue'urreds urq)n eos uot]tppe eql ^q poztltue,L ^;snouotqtu{s ]o 'soAJquaulq)ln easu! seMs66aurq)rneos,Lo uorsuedsns V urp{r rrlolrur lo uorpnr}seppue slseqluAs;er;p{r }o uolpalop aq} sllurrod{qder6orpe.ro}nv 8-02tungH tvlNtwtuldxl v (uorlezrluollsod)urtA lzl

09

-Sa s^=. JO

9Z \a f

.2

e

-

=-


l are .{aqt dla,rncago3 'y urlcdc pue 'sg uqrdc pelelar dlasolc o,\u :uonernteru etdcoo

Metaphase H i g h m i t o t i cc y c l i n High MPF activity

Late anaphase Prophase

cdhl

Polyubiquitination

4g!uq APC/C

Synthesisof mitotic cyclin

Interphase

f"[?iiT:T[:lf Telophase FIGURE 20-10 Regulationof mitoticcyclinlevelsin cycling Xenopusearlyembryoniccells.In lateanaphase, the anaphase(APC/C) promoting polyubiquitinylates complex mitoticcyclinsAsthe cyclins aredegraded by proteasomes, MPFkinase activity declines precipitously, triggering the onsetof telophase. APC/Cactivity is directed towardmitoticcyclins by a specificity factor,calledCdh7,

Control of Mitosis by Cyclinsand MPF Activity r MPF is a protein kinase that requiresa mitotic cyclin for activity. The protein kinase activity of MPF stimulates the onset of mitosis by phosphorylating multiple specific protein substrates,most of which remain to be identified. r In the synchronously dividing cells of early Xenopus and seaurchin embryos,the concentrationof mitotic cyclins(e.g., cyclin B) and MPF activiry increaseas cells enter mitosis and then fall as cells exit mitosis (seeFigures 20-7 and20-8). r The rise and fall in MPF activity during the cell cycle result from concomitant synthesisand degradationof mitotic cyclin protein (seeFigure20-9). r The multisubunit anaphase-promoting complex (APC/C) is a ubiquitin ligase that recognizesa conserved destruction box sequencein mitotic cyclins and promotes their polyubiquitination, marking the proteins for rapid degradation by proteasomes.The resulting decreasein MPF activity leads to completion of mitosis. r The ubiquitin ligase activity of APC/C is controlled so that mitotic cyclins are polyubiquitinylated only during

by G1cyclin-CDK andthereby inactivated whichis phosphorylated the calledCdcl4 removes A specificphosphatase complexes phosphate factorlatein anaphase. fromthespecificity regulatory of in G1,theconcentration factoris inhibited Oncethespecificity reaching a highenoughlevelto eventually mitoticcyclinincreases, mitosis. entryintothesubsequent stimulate

late anaphase(seeFigure 20-1'0).Deactivation of APC/C in G1 permits accumulation of mitotic cyclins during the next cell cycle. This results in the cyclical increases and decreasesin MPF activity that cause the entry into and exit from mitosis.

Kinase Cyclin-Dependent DuringMitosis Regulation The studies with Xenopws egg extracts describedin the previous section showed that continuous synthesisof a mitotic cyclin followed by its periodic degradation at late anaphase is required for the rapid cycles of mitosis observedin early Xenopus embryos. Identification of the catalytic protein kinase subunit of MPF and insight into its regulation initially came from genetic analysis of the cell cycle in the fission yeastS. pombe. An advantageof geneticstudiesis that genes involved in a processcan be identified (and readily cloned from yeasts)without any prior knowledge of the biochemical activities of the proteins they encode. S.pombe grows as a rod-shapedcell that increasesin length as it grows and then divides in the middle during mitosis to NU R I N GM I T O S I S C Y C L I N - D E P E N D EKNI N T A S ER E G U L A T I OD

859

Video: Mitosis and Cell Divisionin S. pombe Cytokinesis

,)

, (,

N u c l e a rd i v i s i o n Chromosome segregation

DNA replication Spindle formation

A FIGURE 20-11ThefissionyeastS. pombe.(a)Scanning electron micrograph of S.pombecellsat variousstagesof the cellcycle.Longcellsareaboutto enter mitosis; shortcellshavejustpassed through (b)Maineventsin the 5. pombe cytokinesis cellcycle.Notethatthe nuclear envelope doesnot disassemble duringmitosis in 5 pombeandotheryeasts[Part(a)courtesy of N Hajibagheri l

Chromosome condensation

/---l---\ (a)) \______

S p i n d l ep o l e body duplication

producetwo daughtercellsof equalsize(Figure20-1,1,1. Unlike most mammalian cells that grow primarily during G1, this yeastdoesmost of its growing during the G2 phaseof the cell cycle. Entry into mitosis is carefully regulatedin responsero cell sizein order to properly coordinate cell division with cell growth. Consequently,this organism is ideal for isolating mutants in genesthat regulateentry into mitosis sincemutations that alter the timing of mitosis yield cells of abnormal size,a readily observedphenotype. Temperature-sensitive mutants of S. pombe with conditional defectsin the ability to progressthrough the cell cycle are easily recognized becausethey causecharacteristicchangesin cell length at the nonpermissivetemperature. Many such mutants have been isolated, and fall into two groups. In the first group are cdc mvtantq which fail to progressthrough one of the phasesof the cell cycle at the nonpermissivetemperature; they form extremely long cellsbecausethey continue to grow in length, but fail to divide. In contrast, uee mutantsform smallerthan-normal cells becausethey are defectivein the proteins that normally prevent cells from dividing when they are too small. In S. pombe wild-type genesare indicated in italics with a superscriptplus sign (e.g.,cdc2* ); geneswith a recessive mutation, in italics with a superscriptminus sign (e.g.,cdc2 l.The protein encoded by a particular gene is designatedby the gene symbol in roman type with an initial capital letter (e.g., Cdc2). In this section we seehow geneticanalysesas well as structural studiesof the proteins involved allowed elucidation of the basic mechanismscontrolling entry into mitosis. First we discusshow mitotic regulatory geneswere identified in S. pombe, 850

C H A P T E R2 0

I

Cell growth

and how they were shown to be analogovs to Xenopu.sMPF. Next we explore the mechanismsused by S. pombe to regulate mitotic cyclin-CDK activity. Mammalian cells regulate mitotic cyclin-CDK in a similar manner,and we end the sectionwith an analysisof the structure of a human CDK and how its activity dependson phosphorylation-inducedconformational changes.

MPFComponentsAre ConservedBetween Lower and Higher Eukaryotes Mutations in cdc2, one of several different cdc genesin S. pombe, produce opposite phenotypesdependingon whether the mutation is recessiveor dominant (Figure 20-1,2).Recessive mutations (cdc2-) give rise to abnormally long cells, whereas dominant mutations (cdc2D) give rise to abnormally small cells, the wee phenotype. As discussedin Chapter 5, recessivemutations generally causea /oss of the wildtype protein function; in diploid cells, both allelesmust be mutant in order for the mutant phenotypeto be observed.In contrast, dominant mutations generally result in a gain in protein function, either becauseof overproduction or lack of regulation; in this case,the presenceof only one mutant allele confers the mutant phenotype in diploid cells. The finding that a loss of Cdc2 activity (cdc2- mutants) preventsentry into mitosis and a gain of Cdc2 activity (cdc2Dmutants) brings on mitosis earlier than normal identified Cdc2 as a key regulator of entry into mitosis in S. pombe. The wild-type cdc2+ gene contained in a S. pombe plasmid library was identified and isolated by its ability to

REGULATING T H E E U K A R Y O T I C E L LC Y C L E

cdc2* (wild type)

mutant

cdc2(recessive)

wild type

cdc2D (dominant)

A EXPERIMENTAL FIGURE 20-12 Recessive and dominant5. pombecdc2mutants have oppositephenotypes.Thewild-type cell(cdc2*)is depictedjust beforecytokinesis with two normal-size daughter cells.A recessive cdc2 mutantcannotentermitosis at the nonpermissive temperature andappears asan elongated cellwith a singlenucleus, whichcontains duplicated chromosomes. A dominant prematurelv cdc2D mutantentersmitosis beforereachino normalsize

fromcytokinesis aresmaller cellsresulting in Gz;thus,thetwo daughter on thannormal-theyhavetheweephenotypeUppermicrographs a cdc2-ts mutantandwt cells5 h aftershiftto the the rightcompare a cdc2D Lowermicrographs compare temperature non-permissive photos: cellfixationmethod.[Top mutantto wt usingan alternative MolecGenGenet.146t167; 1976, P Nurse, PThuriaux, andK Nasmyth, photos. P Nurse, 2002,ChemBio Chem.3:596]1 Bottom

complement cdc2- mutants (see Figure 20-4). Sequencing showed that cdc2* encodesa 34-kDa protein with homology to eukaryotic protein kinases.In subsequentstudies,researchersidentified cDNA clones from other organismsthat could complement S. pombe cdc2- mutants. Remarkably, they isolated a human cDNA encoding a protein identical with S. pombe Cdc2 in 63 percent of its residues.At the time of this experiment it was a tremendoussurprise to scientists that a human protein could perform the essentialfunctions of a protein from so distantly related an organism as S. pombe. The complementation of S. pombe cdc2- mutants by human Cdc2 was one of the first demonstrations that proteins performing fundamental cellular processes are highly conservedbetween all eukaryotic organisms. Isolation and sequencingof another S. pombe cdc gene (cdc13*), which also is required for entry into mitosis, revealedthat it encodesa protein with homology to seaurchin and Xenopws cyclin B. Further studies showed that a heterodimer of Cdc13 and CdcL forms the S. pombe MPF. Like Xenopus MPF, this heterodimer has protein kinase activity that phosphorylateshistone H1. Moreover, the H1 protein kinase activity rises as S. pombe cells enter mitosis and falls as they exit mitosis in parallel with the rise and fall in the Cdc13 protein level. These findings, which are completely analogousto the resultsobtained with Xenopus egg extracts (seeFigure 20-9a), identified Cdc13 as the mitotic cyclin in S. pombe. Further studies showed that the isolated Cdc2 protein and its homologs in other eukaryoteshave little protein kinase activity until they are bound by a cyclin. Hence, this family of protein kinases became known as cyclindependentkinases,or CDKs.

Researcherssoon found that antibodies raised against a highly conservedregion of Cdc2 recognizea polypeptide that co-purifies with MPF purified from Xenopzs eggs. Thus Xenopus MPF is also composed of a CDK (called CDKI\ plus a mitotic cyclin, cyclin B. This convergenceof findings from biochemicalstudiesin an invertebrate(seaurchin) and a vertebrate(Xenopus)and from geneticstudiesin a yeastindicatedthat entry into mitosis is controlled by analogousmitotic cyclin-CDK complexesin all eukaryotes(Figure20-2).

P h o s p h o r y l a t i oonf t h e C D KS u b u n i tR e g u l a t e s the KinaseActivity of MPF As we saw from the studiesin Xenopws egg extracts and comparable biochemicalstudiesin S. pombe, one way of regulating MPF activity is to control the stability of mitotic cyclins.Mitotic cyclins are suddenlydegradedin late anaphasebecausethey are polyubiquitinylated by the activated APC/C. A similar APC/C complex operatesin S. pombe and all eukaryotes.Sincea cyclin must be bound to a CDK for it to have significant kinase activity the degradation of mitotic cyclin causesa drop in MPF activity. However, in S. pombe and all other eukaryotes,additional layers of regulation are used by the cell to ensure that cyclin-CDK complexesare activeonly at the appropriate time in the cell cycle. These additional layers of control were first revealed by studying mutations in S. pombe genesother than cdc2* (encodingthe S. pombe CDK) or cdc13* (encodingthe S. pombe mitotic cyclin) that also affect cell size at the nonpercdc25missivetemperature.For example,temperature-sensitive nonpermissive temperature, mutants cannot enter mitosis at the producing elongatedcells.On the other hand, overexpressionof NU R I N GM I T O S I S C Y C L I N - D E P E N D EKNI N T A S ER E G U L A T I OD

861

(a)

encoded proteins with suitable expressionvectors. The deduced sequencesof Cdc25 and Weel and biochemicalstudies Deficitof Cdc25 of the proteins demonstratedthat they regulatethe kinase acor tivity of S. pombe MPF by phosphorylating and dephosphoExcessof Weel E l o n g a t e dc e l l s rylating specificregulatory sitesin the CDK subunit of MPF. ( i n c r e a s eG d 2) Figure 20-14 illustrates the functions of four proteins that regulate the protein kinase activity of the S. pombe Deficitof Weel CDK. First is the mitotic cyclin of S. pombe, which associor ateswith the CDK to form MPF with extremely low activity. Excess of Cdc25 S m a l lc e l l s Secondis the Weel protein-tyrosine kinase,which phospho(decreasedG2) rylatesan inhibitory tyrosineresidue(Y15) in the CDK subunit. Third is another kinase, designatedCDK-actiuating kinase (CAK), which phosphorylates an activating threonine (b) residue (T161). \fhen both residuesare phosphorylated, S. pombe MPF MPF is inactive. FinallS the Cdc25 phosphataseremovesthe phosphate from Y15, yielding highly active MPF. Sitespecificmutagenesisthat changedthe Y15 in S. pombe CDK to a phenylalanine, which cannot be phosphorylated, proWeel duced mutants with the wee phenotype, similar to that of E X P E R I M E N TFA L U R2E0 - 1 3C d c 2 5a n d W e e l h a v e IG wee-1, mutants. Both mutations prevent the inhibitory phosopposingeffectson S. pombe MPFactivity.(a)Cellsthat lack phorylation at Y15, resulting in the inability to properly regCdc25or Weel activity, asa resultof recessive temperature-sensitive ulate MPF activity, leading to premature entry into mitosis. genes,havetheopposite mutations in the corresponding phenotype As discussedfurther in Section20.7, the checkpoint surLikewise, cellswith multiple copies of plasmids wild-type containing * veillance systemsthat ensurethat chromosome replication is cdc25*or weel , andwhichthusproduce an excess of the encoded complete and that there is no unrepaired damage to chroproteins, (b) haveopposite phenotypes Thesephenotypes implythat mosomesor DNA before initiating mitosis function by regu(-+)by Cdc25and the mitoticcyclin-CDK complex isactivated (-l) bVWeel Seetextfor furtherdiscussion lating the activities of the inhibitory Weel kinase and the acinhibited tivating Cdc25 phosphatase.Weel and Cdc25 homologs exist in higher eukaryotes,and very similar checkpoint conCdc25 from a plasmid presentin multiple copiesper cell detrol systemsoperate in human cells. creases the lengthof G2, causingprematureentry into mitosis and small (wee) cells (Figure 20-13a). Conversely,loss-offunction mutations in the weel* genecausesprematureentry C o n f o r m a t i o n aCl h a n g e sl n d u c e db y C y c l i n into mitosis resultingin small cells,whereasoverproductionof B i n d i n ga n d P h o s p h o r y l a t i oInn c r e a s eM P F Weel protein increasesthe length of G2 and resuhs in elonActivity gated cells. A logical interpretation of these findings is that Cdc25 protein stimulatesthe kinaseactiviry of S. pombe MPF, Unlike both fission and budding yeasts,each of which prowhereas\7ee1 protein inhibits MPF activity (Figure 20-13b). duce just one CDK, vertebratesproduce several CDKs (see In subsequentstudies,the wild-type cdc25* and weel+ Table 20-1). The three-dimensional structureof one human geneswere isolated, sequenced,and used to produce the cyclin-dependentkinase (CDKZ) has been determinedand

a)

Inactive MPF

lnactive MPF

Inactive MPF

Weel

CAK

+ Y15

T161

Y 1 5 T161

Y15

@

@ @

FIGURE 20-14 Regulationof the kinaseactivityol S. pombe mitosis-promoting factor(MPF).Interaction of mitoticcyclin ( C d c 1 3w)i t hc y c l i n - d e p e n dkei nnat s(eC d c 2f )o r m sM P FT. h eC D K subunitcanbe phosphorylated at two regulatory sites:byWee'1 at tyrosine (CAK)at threonine 15 (Y15)andby CDK-activating kinase 1 6 1( T 1 6 1 )R e m o v oa fl t h ep h o s p h a ot en Y 15 b y C d c 2 5 CHAPTER 20

I

Cdc25

+ T161

Substrate-bindino surface

Y15 T161

862

Active MPF

phosphatase yieldsactiveMPFin whichthe CDKsubunitis p h o s p h o r y l aat et Td16 1 ,a n di su n p h o s p h o r y l aatteYd15 . T h e mitoticcyclinsubunitcontributes to the specificity of substrate bindingby MPF,probably byformingpartof thesubstrate-binding (crosshatch), surface whichalsoincludes the inhibitorv Y15residue

R E G U L A T I NTGH E E U K A R Y O T IC CE L LC Y C L E

(a) FreeCDK2

(b) Low-activitycyclinA-CDK2

A FIGURE 20-15Structuralmodelsof humanCDK2,which is homofogousto the 5. pombecyclin-dependent kinase(COf1.16; Free,inactive CDK2unbound to cyclinA. InfreeCDK2,theT-loop blocksaccess of proteinsubstrates to the 1-phosphate of the bound ATP,shownasa ball-and-stick model.Theconformations of the regions highlighted in yellowarealtered whenCDKisboundto cyclin A. (b)Unphosphorylated, low-activity cyclinA-CDK2complex. Conformational changes induced by binding of a domainof cyclin A (green) causethe T-loopto pullawayfromthe activesiteof CDK2,so proteins thatsubstrate canbrnd.Thect1helixin CDK2,which

intothe with cyclinA, movesseveral angstroms interacts extensively for the keycatalytic sidechainsrequired catalytic cleft,repositioning phosphotransfer equivalent reactionTheredballmarksthe position high-activity 161in 5.pombeCdc2.(c)Phosphorylated, to threonine changes induced by Theconformational cyclin A-CDK2complex. (redball)altertheshape phosphorylation of theactivating threonine greatly increasing theaffinityfor surface, of thesubstrate-binding proteinsubstrates et al.,1996, of P D Jeffrey. SeeA A Russo [Courtesy Biol3:696,1 Nature Struct.

provides insight into how cyclin binding and phosphorylation of CDKs regulatetheir protein kinase activity. Although the three-dimensionalstructures of the S. pombe CDK and most other CDKs have not been determined,their extensive sequencehomology with human CDK2 suggeststhat all these CDKs have a similar structure and are regulated by a similar mechanism. Unphosphorylated, inactive CDK2 contains a flexible region, called the T-loop, that blocks accessof protein substratesto the active site where ATP is bound (Figure 20-15a). Steric blocking by the T-loop largely explains why free CDK2, unbound to cyclin, has little protein kinase activity. Unphosphorylated CDK2 bound to one of its cyclin partners has minimal but detectableprotein kinase activity in vitro, although it may be essentiallyinactive in vivo. Extensiveinteractions betweenthe cyclin and the T-loop causea dramatic shift in the position of the Tloop, thereby exposing the CDK2 active site (Figure 2015b). Binding of the cyclin also shifts the position of the a1 helix in CDK2, modifying its substrate-binding surface. High activity of the cyclin-CDK complex requires phosphorylation of the activating threonine, located in the T-loop, causing additional conformational changesin the cyclin-CDK2 complex that gready increaseits affinity for protein substrates (Figure 20-15c). As a result, the kinase activity of the phosphorylated complex is a hundredfold greater than that of the unphosphorylated complex. The inhibitory tyrosine residue (Y15) in the S. pombe CDK is in the region of the protein that binds the ATP phosphates.Vertebrate CDK2 proteins contain an additional inhibitory residue,threonine-14(T14), that is located in the

same region of the protein. Phosphorylationof Y15 and T14 in these proteins prevents binding of ATP becauseof electrostaticrepulsion between the phosphateslinked to the protein and the phosphatesof ATP. Thus these phosphorylations inhibit protein kinase activity even when the CDK protein is bound by a cyclin and the activating residue is phosphorylated. So far we have discussedtwo mechanisms for controlling cyclin-CDK activity: (1) regulation of the concentration of mitotic cyclins as outlined in Figure 20-1.0 and (2) regulation of the kinase activity of MPF as outlined in Figure 20-L4.In Section 20.5 we shall see that the protein kinase activities of cyclin-CDK complexes can also be regulated by CDK inhibitory proteins that bind to CDKs or cyclin-CDK complexes, blocking their ability to interact with substrates.

Cyclin-DependentKinase Regulation During Mitosis r In the fission yeast S. pombe, the cdc2t gene encodesa cyclin-dependentprotein kinase (CDK) that associates with a mitotic cyclin encoded by the cdc13+ gene.The resulting mitotic cyclin-CDK heterodimer is equivalent to Xenopus MPF. Mutants that lack either the mitotic cyclin or the CDK fail to enter mitosis and, therefore' form elongated cells. r The protein kinase activity of the mitotic cyclin-CDK complex (MPF) depends on the phosphorylation state of two residues in the catalytic CDK subunit (see Figure 20-1,4). The activity is greatest when threonine 161 is D U R I N GM I T O 5 I S CYCLIN-DEPENDEK N ITN A S ER E G U L A T I O N

863

phosphorylated and is inhibited by'Wee1-catalyzedphosphorylation of tyrosine 15, which interferes with correct binding of ATP. This inhibitory phosphate is removed by the Cdc25 protein phosphatase. r The human cyclin-CDK2 complex is similar to MPF from Xenopus and S. pombe. Structural studies with the human proteins reveal that cyclin binding to CDK2 and phosphorylation of the activating threonine (equivalent to threonine 161 in the S. pombe CDK) causeconformational changes that expose the active site and modify the substrate-binding surface so that it has high activity and affinity for protein substrates(seeFigure 20-1,5}.

,

1l't

,

MolecularMechanisms for RegulatingMitotic Events In the previous sections,we have seen that a regulated increasein MPF activity inducesentry into mitosis.Presumably the entry into mitosis is a consequenceof the phosphorylation of specific proteins by the protein kinase activity of MPF. However, until recently, the vast majority of proteins phosphorylated by MPF were not determined;consequently, precisely how MPF induces enrry into mitosis is not well understood. Although many of the recently identified substratesof MPF remain to be studied,analysisof a small number of substrateshas provided examplesthat show how their phosphorylation by MPF mediatesmany of the early events of mitosis: chromosome condensation,formation of the mitotic spindle, and disassemblyof the nuclear envelope(see F i g u r e1 8 - 3 4 ) . Recall that a decreasein mitotic cyclins and the associated inactivation of MPF coincides with the later stagesof mitosis (see Figure 20-9a). Just before this, in early anaphase,sister chromatids separateand move to opposite spindle poles. During telophase, microtubule dynamics return to interphase conditions, the chromosomes decondense,the nuclear envelope re-forms, the Golgi complex is remodeled,and cytokinesisoccurs.Someof theseprocesses are triggered by dephosphorylation; others, by protein degradation. In this section, we discuss the molecular mechanisms and specificproteins associatedwith some of the eventsthat characterizeearly and late mitosis. These mechanismsillustrate how cyclin-CDK complexes together with ubiquitinprotein ligasescontrol passagethrough the mitotic phase of the cell cycle.

P h o s p h o r y l a t i oonf N u c l e a rL a m i n sa n d O t h e r ProteinsPromotesEarly Mitotic Events The nuclear envelopeis a double-membraneextensionof the rough endoplasmicreticulum containing many nuclear pore complexes (seeFigure 9-1 and Figure 13-32). The lipid bilayer of the inner nuclear membrane is supported by the

864

C H A P T E R2 0

I

Lamin tetramer

Phosphorylated l a m i nd i m e r s

A FIGURE 20-16 The nuclearlaminaand its depolymerization. (a)Electron micrograph of the nuclearlaminafrom a Xenopus oocyte Notethe regularmeshlike networkof laminintermediate f i l a m e n t sT.h i ss t r u c t u rlei e sa d j a c e nt o t t h e i n n e rn u c l e a r m e m b r a n(es e eF i g u r e1 8 - 4 4 )( b )S c h e m a tdi ci a g r a mosf t h e s t r u c t u roef t h e n u c l e alra m i n aT. w op e r p e n d i c u sl aert so f 1 0 - n m d i a m e t efri l a m e n tbsu i l to f l a m i n A s , B ,a n dC f o r mt h e n u c l e a r l a m i n a( t o p ) I. n d i v i d u a l al m i nf i l a m e n tasr ef o r m e db y e n d - t o - e n d p o l y m e r i z a t ioofnl a m i nt e t r a m e r w s ,h i c hc o n s i sot f t w o l a m i n coiled-coil dimers(middle). Theredand greencirclesrepresent r - t e r m i n a ln dC - t e r m i n ad lo m a i n sr ,e s p e c t i v e l y t h e g l o b u l aN P h o s p h o r y l a toi of n s p e c i f isce r i n er e s i d u ense a rt h e e n d so f t h e r o d l i k ec e n t r asl e c t i o n o f l a m i nd i m e r sc a u s etsh e t e t r a m e rtso (bottom)As a result, depolymerize the nuclear laminadisintegrates. Nature 323:560; courtesy of U Aebi; [Part(a)fromU Aebiet al , 1986, part(b)adapted fromA Murrayand T Hunt,1993,TheCellCycle: An lntroduction, W H Freeman andCompany l

REGULATING THE EUKARYOTIC C E L LC Y C L E

nuclear lamina, a meshwork of lamin filaments located adjacentto the inside face of the nuclear envelope(Figure 20-1,6a).The three nuclear lamins (A, B, and C) presentin vertebratecells belong to the classof cytoskeletalproteins, the intermediate filaments, that are critical in supporting c e l l u l a rm e m b r a n e s( C h a p t e r1 8 ) . Lamins A and C, which are encoded by the same transcription unit and produced by alternative splicing of a single pre-mRNA, are identical except for a 133-residueregion at the C-terminus of lamin A, which is absent in lamin C. Lamin B, encoded by a different transcription unit, is modified post-transcriptionally by the addition of a hydrophobic isoprenyl group near its carboxyl terminus. This fatty acid becomesembeddedin the inner nuclear membrane, thereby anchoring the nuclear lamina to the membrane (seeFigure 10-19). All three nuclear lamins form dimers containing a rodlike cr-helicalcoiled-coil central section and globular

head and tail domains; polymerization of these dimers through head-to-headand tail-to-tail associationsgenerates the intermediate filaments that compose the nuclear lamina ( s e eF i g u r e1 8 - 4 5 ) . Once MPF is activated at the end of G2 through eventsdescribedin the last section,MPF phosphorylatesspecificserine residuesin all three nuclearlamins. This causesdepolymerization of the lamin intermediatefilaments (Figure20-16b). The phosphorylatedlamin A and C dimers are releasedinto solution, whereas the phosphorylated lamin B dimers remain associatedwith the nuclear membrane via their isoprenyl anchor.Depolymerizationof the nuclearlamins leadsto disintegration of the nuclear lamina meshwork and contributesto disassemblyof the nuclearenvelope.The experimentsummarized in Figure 20-17 shows that disassemblyof the nuclear envelope,which normally occursearly in mitosis, dependson phosphorylation of lamin A.

(a) Interphase

(b) Prophase

(c) Metaphase

L a m i nA s t a i n

L a m i nA s t a i n

DNA stain

DNA stain

Cellswith mutanthumanlaminA

Cellswith wild-typehumanlaminA EXPERIMENTAL FIGURE 20-17 Phosphorylation of human laminA causeslamindepolymerization. Site-directed mutagenesis wasusedto prepare a mutanthuman/amrnA gene encoding a proteinin whichalanines replace the serines that normally arephosphorylated in wild-type laminA (seeFigure 2016b).As a result, the mutantlaminA cannotbe phosphorylated Expression vectors carrying thewild-type or mutanthumangene wereseparately transfected intocultured hamster cellsBecause the transfected /amlngenesareexpressed at muchhigherlevels thanthe endogenous hamster lamingene,mostof the laminA produced in transfected cellsis humanlaminA Transfected cellsat various stages in the cellcyclethenwerestained with a fluorescent-labeled

specific for humanlaminA andwith a antibody monoclonal dyethat bindsto DNA.Thebrightbandof fluorescence fluorescent for in interphase cellsstained of the nucleus aroundthe perimeter (unphosphorylated) polymerized laminA humanlaminA represents (a).In cellsexpressing humanlaminA, the diffuselamin thewild-type (b andmetaphase in prophase the cytoplasm staining throughout bandin metaphase of the brightperipheral andc)andthe absence (c)indicate littlelamin of laminA In contrast, depolymerization the mutantlaminA in cellsexpressing occurred depolymerization werefullycondensed DNAstaining showedthatthe chromosomes or mutantlaminA. eitherwild-type in cellsexpressing by metaphase lFrom R Healdand F.McKeon, 1990, Cell 61:5791

M I T O T I CE V E N T S F O RR E G U L A T I N G M O L E C U L A RM E C H A N I S M S

865

CuriouslS spontaneousdominant mutations in the lamin A/C gene ILMNA/ cause the rare syndrome Hutchinson-Guilford progeria. Patientsexpressingone of these mutant lamins A and C undergo a gready accelerated rate of aging. Other LMNA mutations causestriated muscular diseases,abnormal fat cell function, and peripheral 'While nerve cell diseases. the molecular mechanismsunderlying thesesymptoms are not understood,the observation that different mutations in the LMNA gene produce distinct syndromes suggeststhat lamin A and C perform several different functions in normal cells. If that were the case, mutations that affect one or another of these functions might produce the distinct group of symptoms that constitute the different svndromes associated with lamin A/C mutations. I MPF-catalyzed phosphorylation of specific nucleoporins (Figure20-18n) causesnuclear pore complexesto dissociate into subcomplexesduring prophase. Phosphorylation of integral membrane proteins of the inner nuclear membrane (Figure 20-1,8f|lr is thought to decreasetheir affinity for chromatin and further contribute to disassemblyof the nuclear envelope.The weakening of the associationsbetween

I

N u c l e apr o r e proteins

Cytoplasm

50nm

FIGURE 20-18 Nuclearenvelopeproteinsphosphorylated by MPF.(n) Components (NPC) porecomplex of the nuclear are phosphorylated by MPFin prophase, causing NPCs to dissociate into soluble andmembrane-associated (Z) MpF NPCsubcomplexes. phosphorylation (lNM)proteins of innernuclear membrane inhibits theirinteractions with the nuclear lamrna andchromatin(E) MPF phosphorylation of nuclear laminscauses theirdepolymerization and dissolution of the nuclear lamina(4) MPFphosphorylation of proteins chromatin induces chromatin condensation andinhibits interactions between chromatin andthe nuclear envelope[Adapted fromB Burke andJ Ellenberg, 2002,NatRev. Mol.CellBiol3:487l

866

C H A P T E R2 0

I

the inner nuclear membrane proteins and the nuclear lamina (Figure 20-18E|) and chromatin (Figure 20-1.881)allows sheetsof inner nuclear membrane to retract into the endoplasmic reticulum, which is continuous with the outer nuclear membrane. Several lines of evidence indicate that MPF-catalyzed phosphorylation also plays a role in chromosome condensation and formation of the mitotic spindle apparatus.For instance,geneticexperimentsin the budding yeastS. cereuisiae identified a family of structural maintenance of chromosomesproteins, or SMC proteins, that are required for normal chromosome segregation.These large proteins (=1200 amino acids) contain characteristicATPasedomains at their N- and C-termini and long regionsthat participate in coiledcoil structures(seeFigure6-38a). Immunoprecipitation studieswith antibodies specificfor Xenopus SMC proteins revealedthat in cycling egg extracts some SMC proteins are part of a multiprotein complex calledcondensin,which becomesphosphorylated as cells enter mitosis. $fhen the anti-SMC antibodies were used to deplete condensinfrom an egg extract, the extract lost its ability to condenseadded sperm chromatin following the initial decondensationphase. Other in vitro experiments showed that phosphorylated purified condensin binds to DNA and winds it into supercoils (seeFigure 4-8), whereas unphosphorylated condensindoes not. Theseresults and the observation that a condensin subunit is phosphorylated by MPF in vitro have led to the model that condensincomplexesare activated by phosphorylation catalyzedby MPF. Once activated, condensincomplexesare proposed to bind to DNA at intervals along the chromosome, forming successively smaller loops that result in chromosome condensation (see Figure 6-38c). Phosphorylationof microtubule-associated proteins by MPF probably is required for the dramatic changes in microtubule dynamics that result in the formation of the mitotic spindle and asters (Chapter 18). In addition, phosphorylation of proteins associatedwith the endoplasmic reticulum (ER) and Golgi complex, by MPF or other protein kinasesactivated by MPF-catalyzedphosphorylation, is thought to alter the trafficking of vesiclesbetween the ER and Golgi to favor trafficking in the direction of the ER during prophase. As a result, the Golgi complex membranes are transferred to the ER, and vesicular traffic from the ER through the Golgi to the cell surface(Chapter 14), seen in interphase cells, does not occur during mrtosrs. Many of the direct substratesof MPF have beenidentified in S. cereuisiaeby engineering a CDK mutant that can utilize an analog of ATP that is not bound by other kinases (Figure 20-1,9).This ATP analog has a bulky benzyl group attached to N5 of the adenine.This makes the analog too large to fit into the ATP-binding pocket of wild-type protein kinases. However, the ATP-binding pocket of the mutant CDK was modified to accommodate this large ATP analog. Consequentl5 only the mutant CDK can utilize this

REGULATING THE EUKARYOTIC C E L LC Y C L E

A FIGURE 20-19 ATPanalog-dependent CDKmutant. (a)Representation of the ATP-binding andcatalytic sitesof wild-type S.cerevisiae CDK(Cdc28). BoundATPanda phenylalanine sidechain (pink)ln thevicinity of thebindingpocketareshownin stickformat. (b)BulkyATPanalogs groupbound suchasthosecontaining a benzyl to the N6amrnonitrogen aretoo largeto fit intotheATP-binding pocketof wild-typeproteinkinases andthuscannotbe utilizedby

at position CDKmutant,the phenylalanine them.In the 5. cerevrslae whichlacksa largesidechain.Themutant to glycine, 88 ischanged These exhibitshighproteinkinaseactivityusingN6-(benzyl)ATP of the PKA CDKarebasedon crystalstructures modelsof 5. cerevisiae extensivehomologywith the kinase kinasedomain,whichshares et al.,2003,Nature CDK [SeeJ A. Ubersax domainof S. cerevisiae K Shahet al , 1997,Proc.Nat'lAcad.Sci.USA94:3565.1 425:859:

ATP analog as a substratefor transferring its ^y-phosphate to a protein side chain. !(hen the N5-benzyl ATP analog with a labeled "y-phosphatewas incubated with yeast cell extracts and recombinant yeast MPF containing the mutant CDK, multiple proteins were labeled. True yeast MPF in vivo substratescould be verified among these potential substratesby treatment of cells expressingthe mutant CDK in place of the wild-type protein with a similar derivative of another ATP analog that inhibits protein kinases. This derivative of the kinase inhibitor also contains a bulky substitution at the adenine N5 position so that it can bind to and inhibit only the mutant CDK. It is sterically blocked from binding to all other kinasesand consequentlyinhibits only the mutant CDK engineeredin these cells. Treatment of cells with this specific mutant CDK inhibitor resulted in the dephosphorylation of most of the putative MPF targets identified initiallS indicating that theseproteins are indeed phosphorylated by the CDK in vivo as well as in vitro. This procedure identified most of the known CDK substrates plus more than 150 additional yeast proteins. These are currently being analyzed for their functions in cell cycle processes.

(APC/C) leads to the proteasomal destruction of thesecyclins (see Figure 20-1'0). Additional experiments with Xenopus egg extracts provided evidence that degradation of cyclin B, the Xenopzs mitotic cyclin, and the resulting decreasein MPF activity are required for chromosome decondensation but not for chromosome segregation (Figure

Unlinkingof SisterChromatidsInitiates Anaphase We saw earlier that in late anaphase,polyubiquitination of mitotic cyclins by the anaphase-promoting complex

20-20a.b\. To determine if ubiquitin-dependent degradation of another protein is required for chromosome segregation, researchers prepared a peptide containing the cyclin destruction-box sequenceand the site of polyubiquitination. When this peptide was added to a reaction mixture containing untreated egg extract and sperm nuclei, decondensation of the chromosomes and, more interestingly, movement of chromosomes toward the spindle poles were greatly delayed at peptide concentrations of 20-40 pg/ml and blocked altogether at higher concentrations (Figure 20-20c). The added excess destruction-box peptide is thought to act as a substrate for the APC/C-directed polyubiquitination system, competing with the normal endogenous target proteins and thereby delaying or preventing their degradation by proteasomes. Competition with cyclin B delays cyclin B degradation, accounting for the observed inhibition of chromosome decondensation. The observationthat chromosome segregationalso was inhibited in this experiment but not in the experiment with mutant nondegradable cyclin B (see Figure 20-20b) lndicated that segregationdepends on polyubiquitination of a

M I T O T I CE V E N T S FORREGULATING M O L E C U L A RM E C H A N I S M S

867

(a) RNasetreatedextract+ mRNA encodingwild-typecyclin B

DNA

Tubulin

0101540

T i m e( m i n )

(b) RNasetreatedextract+ mutantmRNAencodingnondegradable cyclinB

DNA

Tubulin

0

10

80

15

Time(min)

(cI Untreatedextract+ cyclin B destruction-boxpeptide 1 5 - m i nr e a c t i o nt i m e

35-min reactiontime

DNA

20

868

C H A P T E R2 0

|

R E G U L A T T NTGH E E U K A R Y O T TC CE L LC Y C L E

60

80

Peptideconc. added (p.g/ml)

< E X P E R I M E N TFAI L G U R2E0 - 2 0 O n s e to f a n a p h a s e dependson polyubiquitinationof proteinsother than n i x t u r ecso n t a i n eadn u n t r e a t eodr R N a s e c y c l i nB . T h er e a c t i o m treatedXenopusegg extractand isolated Xenopusspermnuclei, p l u so t h e rc o m p o n e n itns d i c a t ebde l o wC . h r o m o s o mw e se r e v i s u a l i z ewdi t h a f l u o r e s c e D n tN A - b i n d i ndgy e .F l u o r e s c e n t r h o d a m i n e - l a b et luebdu l i ni n t h e r e a c t i o nwsa si n c o r p o r a t e d i n t om i c r o t u b u l epse, r m i t t i nogb s e r v a t i oonf t h e m i t o t i cs p i n d l e (a, b) Afterthe eggextractwastreatedwith RNase apparatus. t o d e s t r oey n d o g e n o umsR N A sa,n R N a sien h i b i t owr a sa d d e d . T h e nm R N Ae n c o d i negi t h e w r i l d - t y p cey c l i nB o r a m u t a n t n o n d e g r a d a bclyec l i nB w a sa d d e dT h et i m ea t w h i c ht h e c o n d e n s ecdh r o m o s o m ae ns da s s e m b l esdp i n d l e apparatus b e c a m vei s i b l a e f t e ra d d i t i o n o f s p e r mn u c l eits d e s i g n a t e d 0 m i n u t e sI .n t h e p r e s e n coef w i l d - t y p cey c l i nB ( a ) ,c o n d e n s e d c h r o m o s o m ae tst a c h etdo t h e s o i n d l e m i c r o t u b u la en sd s e g r e g a t et odw a r dt h e p o l e so f t h e s p i n d l eB. y4 0 m i n u t e s , h u si s n o tv i s i b l ea) ,n dt h e t h es p i n d l e h a dd e p o l y m e r i z(et d e N As t a i n i n ga)sc y c l i n c h r o m o s o mh ea s dd e c o n d e n s (eddi f f u s D B w a sd e g r a d e dI n t h e p r e s e n coef n o n d e g r a d a bclyec l i nB ( b ) , p o l e sb y 15 m i n u t e sa,s c h r o m o s o m sees g r e g a t et od t h e s p i n d l e i n ( a ) ,b u t t h e s p i n d l e microtubule d si d n o t d e p o l y m e r iaz ne dt h e c h r o m o s o m de isd n o t d e c o n d e n seev e na f t e r8 0 m i n u t e sT. h e s e o b s e r v a t i oinnsd i c a tteh a td e g r a d a t i oonf c y c l i nB i s n o t r e q u i r e d f o r c h r o m o s o msee g r e g a t i odnu r i n ga n a p h a s a e l,t h o u g h it is r e q u i r efdo r d e p o l y m e r i z a toi of ns p i n d l e m i c r o t u b u la en sd ( c )V a r i o u s c h r o m o s o mdee c o n d e n s a t idounr i n gt e l o p h a s e c o n c e n t r a t i oonfsa s h o r tp e p t i d ec o n t a i n i ntgh e c y c l i nB destruction boxwereaddedto extracts that had not beentreated w i t h R N a s et h; e s a m p l ews e r es t a i n e fdo r D N Aa t 1 5 o r 3 5 m i n u t ea s f t e rf o r m a t i o n o f t h es p i n d l e a p p a r a t uT s h et w o l o w e s t p e p t i d ec o n c e n t r a t i odnesl a y e cdh r o m o s o msee g r e g a t i oann, d t h e h i g h e cr o n c e n t r a t i ocnosm p l e t e il n y h i b i t ecdh r o m o s o m e s e g r e g a t i oInn.t h i se x p e r i m e nt th,e a d d e dd e s t r u c t i obno x p e p t i d ei st h o u g h t o c o m p e t i t i v ei n t PC/C-mediated l yh i b iA p o l y u b i q u i t i n a t oi of nc y c l i nB a sw e l la sa n o t h etra r g e tp r o t e i n is required whosedegradation for chromosome segregation lFrom et al , 1993,Cell73:1393; S L Holloway courtesy of A, W Murray J

different target protein by the same ubiquitin-protein ligasethat binds the cyclin B destructionbox and the isolated destruction-boxpeptide. As mentioned earlier, each sister chromatid of a metaphasechromosome is attached to microtubules via its kinetochore, a complex of proteins assembledat the centromere. The opposite ends of these kinetochore microtubules associatewith one of the spindle poles (seeFigure 1,8-36).At metaphase,the spindle is in a state of tension, with forces pulling the two kinetochores toward the opposite spindle poles balanced by forces pushing the spindle poles apart. Sisterchromatids do not separate,becausethey are held together at their centromeresby multiprotein complexes called coheslas.Among the proteins composing the cohesin complexes are members of the SMC protein family discussedin the previous section (seeFigure 6-38). Vhen Xenopus egg extracts were depletedof cohesin by treatment with antibodies specific for the cohesin SMC proteins, the depleted extracts were able to replicate the DNA in added

sperm nuclei, but the resulting sister chromatids did not associateproperly with each other. Furthermore, in S. cereuisiae with temperature-sensitivemutations in cohesin subunits, incubation at the nonpermissive temperature causes errors in chromosome segregationduring mitosis. Since attachment of sister chromatids to spindle fibers from opposite spindle poles requires linkage between them, this is the expectedresult if sister chromatids of thesemutant cells are not associatedduring mitosis. These findings demonstrate that cohesin is necessaryfor the cohesion between sister chromatids. Cohesin moleculesassociatewith chromosomesin late G1. Figure 20-21 presentsone model for how the circular cohesin complexes link daughter chromosomes as they replicate in S phase. According to this model, either the DNA replication fork passesthrough the cohesin circles, or cohesincircles open to let the replication fork passand then close around both daughter chromatids. This leaves cohesin links along the full length of the daughter chromatids. In some organisms, such as C. elegans,protein links persist in the chromosome arms until they are broken in anaphase. However, in S. cereuisiae and vertebrates, phosphorylation of cohesins by protein kinases that are activated by MPF causescohesin complexes to dissociatefrom the chromatid arms in late prophase. As opposed to cohesin complexes in the chromosome arms' the same type of cohesin moleculesin the vicinity of the centromeredo not dissociate,and continue to hold sister chromatids in the region of the centromere. Analysis of S. cereuisiaemutants defective in mitotic chromosomal segregation revealed that a specific isoform of a protein phosphatase called PP2A normally associateswith centromeres(observedin human chromosomesin Figure 20-22). This phosphataserapidly dephosphorylatescohesin complexes phosphorylated in late prophase by the kinase mentioned above. This occurs only in the vicinity of the centromere where the phosphatase is bound, so that centromere-associatedcohesin complexes do not dissociate during late prophase like cohesin complexesin the chromosome arms, but rather continue to link chromatids at the centromere. Further studies of yeast mutants have led to the model depicted in Figure 20-23 for how the APC/C regulates sister chromatid separation to initiate anaphase. Cohesin SMC proteins link sister chromatids at the centromere. The cross-linking activity of cohesin dependson secwrin,which is found in all eukaryotes. Prior to anaphase,securin binds to and inhibits separase,a ubiquitous protease. Once all chromosome kinetochores have attached to spindle microtubules, the APC/C is directed by a specificity factor called Cdc20 to polyubiquitinylate securin (note that this specificity factor is distinct from Cdh1, which directs the APC/C to polyubiquitinylate B-type cyclins). Polyubiquitinylated securin is rapidly degraded by proteasomes, thereby releasingseparase.Free from its inhibitor, separase cleaves a small subunit of cohesin called kleisin, breaking the protein circles linking sister chromatids. Once this link is broken, anaphasebegins,as the poleward

M I T O T I CE V E N T S FORREGULATING M O L E C U L A RM E C H A N I S M S

859

Chromatin

Replication fork

Load cohesin complexesonto

-

#

Centromere

Gl phase

S phase e

A FIGURE 20-21 Modelfor cohesinlinkageof daughter chromosomes. Thereisstrongevidence thatthe cohesin complex is circular, likeotherSMCproteincomplexes (seeFigure 6-38),but it is not knownwhethera singlecohesin ringlinksdaughter chromatids, or whethertwo rings,oneeacharoundtheseparate sister chromatids, arelinkedto eachotherlikelinksin a chain,possibly

force exerted on kinetochoresmoves sisterchromatids toward the opposite spindle poles. Because Cdc20-the specificity factor that directs APC/C to securin-is activated before Cdhl-the specificity factor that directs APC/C to mitotic cyclins-MPF activity does not decreaseuntil after the chromosomes have segregated.As a result of this temporal order in the activation of the two APC/C specificity factors, the chromosomesremain in the condensedstate and reassemblyof the nuclear envelope does not occur until chromosomes

phase G2 phase G2

Metaphase involving several linkedcohesin circles between sisterchromatids. Passage of a replication forkthrougha cohesin ringresults in linking of sisterchromatids Invertebrate cells,cohesins arereleased from chromosome armsduringprophase andearlymetaphase, andby the endof metaphase areretained onlyin the regionof the centromere. K Nasmyth andC H Haering, 2005, Ann Rev. Biochem 74:595] [From

are moved to the proper position. As we shall seein Section 20.7, Cdc20 and Cdhl are regulated by checkpoint surveillancemechanisms.Cdc20 is inhibited until every kinetochore has attached to a spindle fiber and tension is applied to the kinetochores of all sister chromatids, pulling them toward opposite spindle poles. Cdh1, on the other hand, is inhibited until daughter chromosomeshave been separatedby a sufficient distancein anaphaseto ensure that the separatedchromosomesare included in separate nuclei as the nuclear envelopesre-form and the cell divides.

Chromosome D e c o n d e n s a t i oann d R e a s s e m b l y o f t h e N u c l e a rE n v e l o p eD e p e n do n Dephosphorylationof MPFSubstrates

A F|GURE 2O-22Localizationof PP2Asubtypeat the centromereof a humanmetaphasechromosome. DNAisstained blue.A markerproteinfor centromeres wasdetected with a specific (red),aswasPP2Asubtype antibody B56a(green)Bar: 1 pm [From S Tomoyaet al , 2006, NatureM1i46l

870

C H A P T E R2 0

I

Earlier we discussedhow MPF-mediatedphosphorylation of nuclear lamins, nucleoporins,and proteins in the inner nuclear membrane contributes to the dissociation of nuclear pore complexesand retraction of the nuclear membrane into the ER. When chromosomes have separated sufficiently during anaphase, the chromosome segregation checkpoint surveillance mechanism activates the

REGULATING T H E E U K A R Y O T I C E L LC Y C L E

Securin

a A FIGURE 20-23 Regulationof cohesincleavage.Separase, protease the smallkleisin subunitof cohesin thatcancleave isinhibited complexes, beforeanaphase bythe bindingof securin microtubules and haveattached to spindle Whenallthe kinetochores isproperly andoriented, the Cdc20 the spindle apparatus assembled with theAPC/Canddirects it to specificity factorassociates

by degradation securin securinFollowing polyubiquitinylate subunit, the kleisin cleaves separase proteasomes, the released to be pulled allowingsisterchromatids circles, thecohesin breaking that ispullingthemtowardopposite apparatus apartby thespindle 2005,Ann Rev. andC H Haering, fromK,Nasmyth spindlepoles.lAdapted 74:595.1 Biochem

protein phosphatase Cdc14. Cdc14 removes phosphate groups that were added to proteins by MPF and, consequently, is an active antagonist of MPF function. Importantly, Cdc1,4 also dephosphorylates and consequently activatesthe Cdhl specificity factor. This allows Cdhl to bind to the APCiC complex, directing it to polyubiquitinylate mitotic cyclins, inducing their degradation (see F i g u r e2 0 - 1 , 0 ) . Reversal of MPF phosphorylation changesthe activities of many proteins back to their usual state in interphasecells. Dephosphorylation of condensins,histone H1 and other chromatin-associatedproteins leads to the decondensation of mitotic chromosomes in telophase. Dephosphorylated inner nuclear membrane proteins are thought to bind to chromatin once again. As a result, multiple projections of regions of the ER membrane containing these proteins are thought to associatewith the surface of the decondensingchromosomes and then fuse with one another to form a continuous double membrane around each chromosome (Figure 20-24). Dephosphorylation of nuclear pore subcomplexesallows them to reassembleinto complete NPCs traversing the inner and outer membranessoon after fusion of the ER proiections. Ran'GTR required for driving most nuclear import and e x p o r t ( C h a p t e r 1 3 ) , s t i m u l a t e sb o t h f u s i o n o f t h e E R projections to form daughter nuclear envelopesand assembly of NPCs from the nuclear pore subcomplexesthat were generatedby MPF phosphorylation of nucleoporins in prophase (Figure 20-24). The Ran'GTP concentration is highest in the microvicinity of the decondensingchromosomes becausethe Ran-guanine nucleotide-exchange factor (Ran-GEF) is bound to chromatin. Consequentln membrane fusion is stimulated at the surfacesof decondensing chromosomes, forming sheets of nuclear membrane with inserted NPCs. The reassemblyof nuclear envelopescontaining NPCs around each chromosome forms individual mini-nuclei

called karyomeres. Subsequent fusion of the karyomeres associated with each spindle pole generates the two daughter-cellnuclei, each containing a full set of chromosomes. Dephosphorylated lamins A and C appear to be imported through the reassembledNPCs during this period and reassembleinto a new nuclear lamina. Reassembly of the nuclear lamina in the daughter nuclei probably is initiated on lamin B molecules,which remain associated with the ER membrane via their isoprenyl anchors throughout mitosis and become localized to the inner membrane of the reassembled nuclear envelopes of karyomeres.

Molecular Mechanismsfor Regulating Mitotic Events r Early in mitosis, MPF-catalyzed phosphorylation of lamins A, B, and C, and of nucleoporins and inner nuclear envelope proteins causes depolymerization of lamin filaments (see Figure 20-1,6\ and dissociation of nuclear pores into pore subcomplexes,leading to disassembly of the nuclear envelope and its retraction into the ER. r Phosphorylation of condensin complexes by MPF or a kinase regulatedby MPF promotes chromosomecondensation early in mitosis. r Sister chromatids formed by DNA replication in S phase are linked at the centromere by cohesin complexes that contain DNA-binding SMC proteins and other protelns. r At the onset of anaphase, the APC/C is directed by Cdc20 to polyubiquitinylate securin,which subsequentlyis degraded by proteasomes.This activates separase,which cleaveskleisin, a subunit of cohesin,thereby unlinking sister chromatids (seeFigure 20-23).

M I T O T I CE V E N T S FORREGULATING M O L E C U L A RM E C H A N I S M S

871

Video: NuclearEnvelopeDynamics DuringMitosis

ZN

Chromatin Membrane recruitment

FIGURE20-24 Model for reassemblyof the nuclear envelope during telophase. Extensions of the endoplasmic reticulum(ER)associate with eachdecondensing chromosomeand t h e n f u s ew i t h o n e a n o t h e rf,o r m i n ga d o u b l em e m b r a n ea r o u n d the chromosomeDephosphorylated nuclearpore subcomplexes r e a s s e m b il ne t o n u c l e apr o r e s f, o r m i n gi n d i v i d u aml i n i - n u c l ec ia l l e d karyomeresThe enclosedchromosomefurtherdecondenses, and subsequent fusionof the nuclearenvelopes of all the karyomeres at e a c hs p i n d l ep o l ef o r m sa s i n g l en u c l e u sc o n t a i n i n a g f u l l s e to f chromosomesNPC: nuclearpore complex lAdapted fromB Burke andJ Ellenberg,2002, NatureRev. Mol.CellBiol 3:487I

After sisterchromatidshave moved to the spindlepoles, e APC/C is directed by Cdhl to polyubiquitinylate mitotic cyclins, leading to rheir destruction and causing the decreasein MPF activity that marks the onset of telophase. r The fall in MPF activity in telophase allows phosphatases such as Cdc14 to remove the regulatory phosphatesfrom condensin, lamins, nucleoporins, and other nuclear membrane proteins, permitting the decondensation of chromosomesand the reassemblyof the nuclear membrane, nuclear lamina, and nuclear pore complexes.

872

c H A P T E R2 0

|

r The associationof Ran-GEF with chromatin results in a high local concentrationof Ran.GTP near the decondensing chromosomes,promoting the fusion of nuclear envelope extensions from the ER around each chromosome. This forms karyomeresthat then fuse to form daughter cell nuclei (seeFigure 20-24).

Cyclin-CDK and Ubiquitin-Protein LigaseControlof S phase In most vertebrate cells, the key decision determining whether or not a cell will divide is the decisionto enter the S phase. In most cases,once a vertebrate cell has become committed to entering the S phase, it does so a few hours later and progressesthrough the remainder of the cell cycle until it ccrmpletesmitosis. The budding yeastSaccharomyces cereuisideregulatesits proliferation similarly, and much of our current understanding of the molecular mechanisms controlling entry into the S phase and the control of DNA replication originated with geneticstudiesof S. cereuisiae. S. cereuisiaecells replicate by budding (Figure 20-ZS). Both mother and daughtercells remain in the G1 period of the cell cycle while growing, although it takes the initially larger mother cells a shorter time to reacha size compatible with cell division. \Vhen S. cereuisiaecells in G1 have grown sufficiently, they begin a program of gene expression that leadsto entry into the S phase.If G1 cells are shifted from a rich medium to a medium low in nutrients before they reach a critical size,they remain in G1 and grow slowly until they are large enough to enter the S phase.However, once G1 cells reach the critical size,they becomecommitted to completing the cell cycle, enrering the S phase and proceeding through G2 and mitosis, even if they are shifted to a medium low in nutrients. The point in late G1 of growing S. cereuisiaecells when they become irrevocably committed to entering the S phaseand traversingthe entire cell cycle is called S7l4RT. As we shall seein Section20.6, a comparablephenomenonoccurs in replicatingmammaliancells. In this section, our focus is on the G1 -+ S transition as we explore the molecular eventsthat constitute START. Just as in mitosis, entry into S phase is controlled by the activity of cyclin-CDKs. However, the regulatory mechanismsgoverning the activity of these cyclin-CDKs differ from those used by mitotic cyclin-CDK complexes.We discussthe roles of G1 cyclin-CDKs and S-phasecyclin-CDKs in initiating DNA synthesisand ensuring that DNA replication occurs only onceper cell cycle,as well as how the cell cycle"resets" after mitosis, in preparation for the next cell division.

A Cyclin-Dependen Kti n a s e( C D K )l s C r i t i c a fl o r S-PhaseEntry in S. cerevisiae All S. cereuisiae cells carrying a mutation in a particular cdc gene arrest with the same size bud at the nonpermissive

R E G U L A T T NTG H E E U K A R Y O T TCCE L LC y C L E

Z^s\ \\-Z (a)

Video: Mitosis and Budding in S. cerevisiae (b) Mother cell

Daughter cell Chromosome segregation; nuclear division

Growth

START | /\ \ \ \"_, /

Spindle formation; nuclear migration

S p i n d l ep o l e bodY duplication

Q r - ) / a r oemergence DNA replication

(a)Scanning 20-25 The buddingyeast5. cerevisiae. A FfGURE stagesof the of 5. cerevisiae cellsat various electronmicrograph c e l lc y c l eT. h el a r g etrh eb u d ,w h i c he m e r g east t h ee n do f t h e G t phase,the furtheralongin the cyclethe cellis (b)Maineventsin than cellcycle.Daughter cellsarebornsmaller the 5. cerevrslae mothercellsand mustgrowto a greaterextentin G1beforethey

arelargeenoughto enterthe S phase.As in 5. pombe,tne Unlike5. doesnot breakdownduringmitosis. envelope nuclear do chromosomes the small5 cerevrslae pombechromosomes, (a) [Part to be visibleby lightmicroscopy. sufficiently not condense andL Herskowitz l of E Schachtbach courtesy

temperature(seeFigure 5-6b). Each type of mutant has a terminal phenotype with a particular bud size: no bud, intermediate-sizedbuds, or large buds. Note that in S. cereuisiae wild-type genes are indicated in italic capital letters (e.g., CDC28) and recessivemutant genes in italic lowercase letters (e.g., cdc28); the corresponding wild-type protein is written in roman letters with an initial capital (e.g.,Cdc28), similar to S. pombe proteins. Temperature-sensitivemutants in the cdc28 gene, now known to encodethe S. cereuisiaeCDK, do not form buds at the nonpermissive temperature. This phenotype indicates that Cdc28 function is required for entry into the S phase. 'When these mutants are shifted to the nonpermissivetemperature, they behavelike wild-type cells suddenly deprived of nutrients; that is, cdc28 mttant cells that have grown large enough to pass START at the time of the temperature shift continue through the cell cycle normally and undergo mitosis, whereas those that are too small to have passed

START when shifted to the nonpermissivetemperature do not enter the S phase even though nutrients are plentiful. Even though cdc28 cellsblocked in G1 continue to grow in size at the nonpermissive temperature' they cannot pass START and enter the S phase.Thus they appear as large cells with no bud. The wild-type CDC28 gene was isolated by its ability to complement mutant cdc28 cells at the nonpermissive temperature (see Figure 20-4\' Sequencing of CDC28 showed that the encoded protein is homologous to known protein kinases,and when Cdc28 protein was expressedin E. coli, it exhibited low protein kinase activity. Like S. pombe, S. cereuisiaecontains only a single cyclin-dependent protein kinase (CDK) that functions directly in cell-cycle control. Sequencecomparisons have shown that the CDKs in the two speciesare highly homologous. The difference in the phenotypes of S. pombe and S' cereuisiaecells with temperature-sensitivemutations in

I G A S EC O N T R O LO F 5 P H A S E A N D U B I Q U I T I N - P R O T ELI N CYCLIN-CDK

873

their CDK genes can be explained by the physiology of the two yeasts.In S. pombe cells growing in rich media, cell-cyclecontrol is exerted primarily at the G2 -+ M trans i t i o n ( i . e . ,e n r r y t o m i t o s i s ) .I n m a n y S . p o m b e C D K r e cessivemutants, including those initially isolated which gave the phenotype depicted in Figure 20-12, enough CDK activity is maintained at the nonpermissivetemperature to permit cells to enter the S phase, but not enough to permit entry into mitosis. Such mutant cells are observedto be elongatedcells arrestedin G2. At the nonpermissive temperature, cultures of completely defective CDK mutants include some cells arrestedin G1 and some arrestedin G2, depending on their location in the cell cycle at the time of the temperature shift. Conversely,cellcycle regulation in S. cereuisiaeis exerted primarily at the G 1 - + S t r a n s i t i o n ( i . e . ,e n t r y t o r h e S p h a s e ) .T h e r e f o r e , partially defectivemutants of CDK are arrestedin G1, but completely defective CDK mutanrs are arrested in either G1 or G2, depending on their location in the cell cycle at the time of the temperature shift. These observations demonstratethat both the S. pombe and the S. cereuisiae CDKs are required for entry into both the S phase and mrtosrs.

(a)

36"C

Wild-typecells

Coloniesform

Coloniesform

Colonies form

Arrestedin G1

(b) cdc2Sts cells

(c)

Excess wild-type G 1c y c l i n

cdc2Sts cells transformed with high-copy G 1c y c l i n plasmid

ThreeG1CyclinsAssociatewith S. cerevisiae CDKto Form S-Phase-Promoting Factors By the late 1980s,it was clearthat mitosis-promotingfactor (MPF) is composedof two subunits:a CDK and a mitotic Btype cyclin required to activate the catalytic subunit. By analogy, it seemed likely that S. cereuisiaecontains an Sphase-promoting factor (SPF)that phosphorylates and regulates proteins required for DNA synthesis.Similar to MpE, SPFwas proposedto be a heterodimercomposedof the S. cereuisiaeCDK and a cyclin, in this caseone that acts in G1 (seeFigure20-2). To identify this putative G 1 cyclin, researcherslooked for genesthat, when expressedat high concentration,could suppress certain temperature-sensitivemutations jn the S. cereuisiae CDK. The rationale of this approach is illustrated in Figure 20-26. Researchersisolated two such genes, designated CLN1 and CLN2. Using a different approach, researchersidentified a dominant mutation in a third sene c a l l e dC L N J Sequencingof the three CLN genesshowed that they encodedrelated proteins, each of which includesan =100residue region exhibiting significant homology with Btype cyclins from sea urchin, Xenopus, human, and S. pombe. This region encodesthe cyclin domain that interacts with CDKs and is included in the domain of the human cyclin shown in Figure 20-15b, c. The finding that the three CIn proteins contain this region of homology with mitotic cyclins suggestedthat they were the sought-after S. cereuisiaeG1 cyclins. (Note that the homologous CDKbinding domain found in various cyclins differs from the destruction box mentioned earlier,which is found only in B-type cyclins.)

874

C H A P T E R2 0

|

I Equilibrium v shifted

C o l o n i e sf o r m

Coloniesform

A E X P E R I M E N TFAI L G U R2E0 - 2 6 G e n e se n c o d i n gt w o S. cerevisiaeG.,cyclinswere identified by their ability to suppressa temperature-sensitive mutant CDK.Thisgenetic screen i s b a s e do n d i f f e r e n c ei nst h e i n t e r a c t r o bn es t w e e n G1 cyclinsandwild-typeandtemperature-sensitive (ts)S. cerevrsiae C D K s( a )W i l d - t y pcee l l sp r o d u c a e n o r m aC l D Kt h a ta s s o c i a t e s w i t h G r c y c l i n sf o, r m i n gt h e a c t i v eS - p h a s e - p r o m o ft a i ncgt o r ( S P Fr)e, s u l t i nign c o l o n yf o r m a t i o a n t b o t ht h e p e r m i s s i va en d t h e n o n p e r m i s stievm e p e r a t u(rie , 2 5 ' a n d 3 6 ' C ) ( b )S o m e cdc29"mutantsexpress a mutantCDKwith low affinityfor G., c y c l i na t 3 6 " C .T h e s em u t a n t sp r o d u c e n o u g hG 1c y c l i n - C D K ( S P Ft o ) s u p p o rgt r o w t ha n dc o l o n yd e v e l o p m eantt 2 5 " C ,b u t n o t a t 3 6 ' C ( c )W h e nc d c 2 8 t ' c e lw l se r et r a n s f o r m ewdi t h a S c e r e v i s i age n o m i lci b r a r cy l o n e di n a h i g h - c o ppyl a s m i dt h , ree t y p e so f c o l o n i efso r m e da t 3 6 ' C : o n ec o n t a i n ead p l a s m i d carryingthe wild-typeCDC2B gene;the othertwo contained p l a s m i dcsa r r y i negi t h e tr h e C L N Io r C L N 2g e n e I n t r a n s f o r m e d c e l l sc a r r y i ntgh e C l N To r C L N 2g e n e t, h e c o n c e n t r a t i o n f the encoded G 1c y c l i ni s h i g he n o u g ht o o f f s e t h e l o w a f f i n i t yo f t h e m u t a nC t D Kf o r a G , c y c l i a n t 3 6 ' C , s ot h a te n o u g h S P Ff o r m st o s u p p o ret n t r yi n t ot h e S p h a s ea n ds u b s e q u e n t m i t o s i sU. n t r a n s f o r mceddc 2 ? tc' e l l sa n dc e l l st r a n s f o r m e d w i t h p l a s m i dcsa r r y i nogt h e rg e n e sa r ea r r e s t eidn G 1a n dd o n o t f o r m c o l o n i e s [ S e eJ A H a d w i g e re t a l , 1 9 8 9 , p r o c N a t ' l A c a d S c i U S A8 6 : 6 2 5 5 l

R E G U L A T T NTGH E E U K A R Y O T TCCE L LC Y C L E

the mitotic cyclin caused a shortened Gz and premature entry into mitosis, whereas inhibition of the mitotic cyclin by mutation resulted in a lengthenedG2 (seeFigure 20-1'2)' Thus these results confirmed that the S. cereuisiaeCIn proteins are G1 cyclins that regulate passagethrough the G1

Gene-knockout experiments showed that S. cereuisiae cells can grow in rich medium if they carry any one of the three G1 cyclin genes.As the data presentedinFigure20-27 indicate, overproduction of one G1 cyclin decreasesthe fraction of cells in G1, demonstrating that high levels of the G1 cyclin-CDK complex drive cells through START prematurely. Moreover, in the absenceof all three of the G1 cyclins, cells becomearrestedin G1, indicating that a G1 cyclin-CDK heterodimer, or SPF, is required for S. cereuisiae cells to enter the S phase. These findings are reminiscent of the results for the S. pombe mitotic cyclin with regard to passagethrough G2 and entry into mitosis. Overproduction of

Podcast:G1-cyclinControl of Entry into S-phase High-levelexpressionof G1cyclin

Cln3 expression vector

GAL| promoter

No G,'cYclinexPression

G,'cyclin gene

(a) Wild-typecells + empty vector

t

t

o

o

E

(c) cln 1- | cln2- | cln3- cells + G1cyclinvector

(b) Wild-typecells + G1cyclinvector

+ G l c( 1 3 5m i n )

c

c)

E

+ G l c( 1 3 5m i n )

= 6)

G1

G2

Fluorescence ->

O

G1

G2

Fluorescence -->

20-27Gl cyclinis requiredfor 5. FIGURE A EXPERIMENTAL cerevisiaecellsto enter 5 phase,and overexpressionof G1cyclin prematurelydrivesthem into the 5 phase.Theyeastexpression (top)carriedoneof the three5. vectorusedin theseexperiments whichis Gr cyclingeneslinkedto the strongGAI7 promoter, cerevisiae the Todetermine ispresent in the medium. turnedoff whenglucose to a fluorescent proportion of cellsin Gr andGz,cellswereexposed througha fluorescencedyethat bindsto DNAandthenwerepassed (see DNAcontentof G2 the Figure 9-28) Since cell sorter activated cellsin the candistinguish thisprocedure cellsistwicethatof G1cells, with an empty phases(a)Wildtypecellstransformed two cell-cycle of cellsin Gr and the normaldistribution vectordisplayed expression (b)In (Glc) of glucose. glucose addition and after of G2in the absence

G2 G1 Fluorescence -->

with the G1cyclin wild-typecellstransformed of glucose, the absence percentage of cells a higher-than-normal vectordisplayed expression of the G1cyclin overexpression in the S phaseandG2because of the Gr the Gr period(topcurve)'Whenexpression decreased glucose, the cell of by addition off shut was vector cyclinfromthe to normal(bottomcurve)'(c)Cellswith returned distribution with the Gr in allthreeG1cyclingenesandtransformed mutations

Cell591127 I etal, 1989, fromH E Richardson phase. [Adapted

I G A S EC O N T R O LO F S P H A S E A N D U B I Q U I T I N . P R O T E LI N CYCLIN-CDK

875

open-readingframe that inhibits translation initiation at the Cln3 open reading frame. This inhibition is diminished when nutrients are in abundance,leading to activation of the TOR pathway and the subsequentincreasein translation initiation factor activity (seeFigure g-30). Since Cln3 is a highly unstable protein, its concenrratron fluctuates with the translation rate of its mRNA. Consequently, the amount and activity of mid-G1 cyclin-CDK complexes, which depend on the concentration of the mid-G1 cyclin protein, are largely regulated by the nutrient level. Once sufficient mid-G1 cyclin is synthesizedfrom its mRNA, the mid-G1 cyclin-CDK complex phosphorylates and activates two related transcription factors, SBF and MBF. These induce transcription of the late-G1 cyclin genes,CLNI and CLN2, whose encodedproteins accelerate entry into the S phase. Thus regulation of CLN3 mRNA translation in responseto the concentrationof nutrients in the medium is thought to be primarily responsible for controlling the length of G1 in S. cereuisiae.In addition to the late-G1 cyclins, SBF and MBF also stimulare tran-

called early S-phasecyclins.Inactivation of Cdhl allows the S-phasecyclin-CDK complexesto accumulate in late G1. The specificity factor Cdhl is phosphorylated and inactivated by both late-G1 and B-type cyclin-CDK complexes, and thus remains inhibited throughout S, G2, and M phaseuntil late anaphasewhen the Cdc14 phosphatase is activated and removes the inhibitory phosphate from

cdh1. D e g r a d a t i o no f t h e S - P h a s Ien h i b i t o rT r i g g e r s DNAReplication As the S-phasecyclin-CDK heterodimersaccumulatein late G1, they are immediately inactivated by binding of an inhibitor, called Sic1, that is expressedlate in mitosis and in early G1. BecauseSicl specificallyinhibits B-type cyclinCDK complexes, but has no effect on the G1 cyclin-CDK complexes,it functions as an S-phaseinhibitor. Entry into the S phaseis defined by the initiation of DNA replication. ln S. cereuisiaecells this occurs when the Sicl inhibitor is precipitouslydegradedfollowing its polyubiquitination by the distinct ubiquitin-protein ligasecalled SCF mentioned earlier (Figure 20-28; seealso Figure20-2). Once Sicl is degraded,the S-phasecyclin-CDK complexesinduce DNA replication by phosphorylatingseveralproteins in prereplication complexesbound to replication origins. This mechanism for activating the S-phasecyclin-CDK complexes-that is, inhibiting them as the cyclinsare synthesizedand then precipitously degradingthe inhibitor-permits the suddenacrivation of large numbers of complexes,as opposedto the gradual increasein kinaseactivity that would result if no inhibitor were presentduring synthesisof the S-phasecyclins. We can now seethat regulated proteasomal degradation directed by two ubiquitin-protein ligase complexes, SCF and APC/C, controls three major transitions in the cell cycle: onset of the S phase through degradation of Sicl by SCF, the beginning of anaphasethrough degradation of securin by the APC/C, and exit from mitosis through degradation of B-type cyclins by the APC/C. The ApC/C is

are required for initiation of DNA synthesis, they are

Polyubiquitinationof p h o s p h o r y l a t eS di c l ; proteasomal degradation

-_____+

z

s6 @@ $

Mid-lateG''

FIGURE 20-28 Controlof S phaseonset in S. cerevisiaeby regulatedproteolysisof the S-phaseinhibitor,Sic1.TheS_phase cyclin-CDK (Clb5-CDK complexes andClb6-CDK) beqinto accumutate in G1,butareinhibited bySlc1. Thisinhibition pr.u.nt,initiation of DNA replication untilthe cellisfullyprepared. G1cyclin-CDK complexes assembled in lateG1(Cln1-CDK andCln2-CDK) phosphorylate Siclat 876

.

cHAprER20

|

S phase

multiple sites(step[), marking it for polyubiquitination bytheSCF ubiquitinligase, andsubsequent proteasomal (stepZ). degradation TheactiveS-phase cyclin-CDK complexes thentriggerinitiation of DNA (stepB) by phosphorylating synthesis components of pre-initiation complexes assembled on DNAreplication originsin earlyG1 lAdapted fromR W Kingetal,1996,Science2T4:1652l

R E G U L A T T NTGH E E U K A R y o l c C E L Lc y c l E

20-29 ActivitYof 5. < FIGURE ae cycli n-CDKcomPlexes cerevisi through the courseof the cell cycle. bandsis Thewidthof the colored proportional to the approximately proteinkinase or proposed demonstrated cyclin-CDK of the indicated activity produces Mid-G1cyclin-CDK complexes. a single S cerevisiae cln3-cDK whose kinase cyclin-dependent (CDK) ,'z cyclins, by thevarious iscontrolled actrvity duringdifferent whichareexpressed portions of the cellcycle.

Mitotic cyclin-GDKs

Late S-phase/ early M-phase cyclin-CDKs ctb3,4CDK

Late-G1cyclin-CDKs Cln1.2-CDK

clb5,6-cDK EarlyS-phasecyclin-CDKs

directed to polyubiquitinylate the anaphaseinhibitor securin by the Cdc20 specificity factor (seeFigure20-23). The APC/CCdc20 complex also directs the degradation of S-phasecyclins and much of the mitotic cyclin, but sufficient mitotic cyclin remains to maintain chromosome condensation until late anaphase.Then, another specificity factor, Cdh1, targets the APC/C to the remainingB-typecyclins(seeFigure20-10). In contrast to the APC/C, the SCF ubiquitin-protein ligaseis not regulatedby phosphorylation of specificityfactors, but rather by phosphorylationof its substrate,Sic1. Sicl is phosphorylated by G1 cyclin-CDKs (see Figure 20-28). It must be phosphorylated at at least six sites,which are relatively poor substratesfor the G1 cyclin-CDKs, before it is bound sufficiently well by SCF to be polyubiquitinylated. This difference in strategy for regulating the ubiquitinprotein ligase activities of SCF and APC/C probably occurs becausethe APC/C has severalsubstrates,including securin and B-type cyclins, which must be degraded at different times in the cycle. In contrast, entry into the S phaserequires the degradation of only a single protein, the Sicl inhibitor. Also, the requirement for phosphorylating multiple weak sitesin Sicl delaysthe onset of S phaseuntil G1 cyclin-CDK activity has reachedits peak and virtually all other G1 cyclinCDK substrateshave been phosphorylated. An obvious advantage of proteolysis for controlling passagethrough these critical points in the cell cycle is that protein degradation is an irreversible process, ensuring that cells proceed irreversibly in one direction through the cycle.

Multiple CyclinsRegulatethe KinaseActivity of 5. cerevisraeCDKDuring Different Cell-CyclePhases theybethroughtheSphase, Asbuddingyeastcellsprogress gin transcribing genes encoding two additional B-type

tosis, with the help of two other mitotic cyclins. Theseadditional mitotic cyclins are expressedwhen S. cereuisiaecells complete chromosome replication and enter G2. They function as late mitotic cyclins,associatingwith the CDK to form

R e p l i c a t i o na t E a c hO r i g i n l s l n i t i a t e dO n l y O n c e D u r i n gt h e C e l lC Y c l e As discussed in Chapter 4, eukaryotic chromosomes are replicated from multiple replication origins' Initiation of replication from these origins occurs throughout S phase'

I G A 5 EC O N T R O LO F S P H A 5 E C Y C LNI - C D KA N D U B I Q U I T I N . P R O T E LI N

.

877

However, no eukaryotic origin initiates more than once per S phase. Moreover, the S phase continues until replication from multiple origins along the length of each chromosome results in complete replication of the entire chromosome. Thesetwo factors ensurethat the correct genecopy number is maintained as cells proliferate. Yeast replication origins contain an 11-base-pairconservedcore sequenceto which is bound a hexamericprotein, the origin-recognition complex (ORC), required for initiation of DNA synthesis.DNase I footprinting analysis(Figure 7-17) and immunoprecipitation of chromatin protelns crosslinked to specificDNA sequences (Figure7-31\ duringvarious phasesof the cell cycle indicate that the ORC remains associatedwith origins during all phasesof the cycle. Several additional replication initiation factors required to initiate

DNA synthesisat origins were identified in geneticstudiesin S. cereuisiae.These DNA replication initiation factors associate with the ORC at origins during G1, but not during G2 or M. During G1 the various initiation factors assemblewith the ORC into a prereplication complex ar each origin (Figure20-30). The restriction of origin "firing" to once and only once per cell cycle in S. cereuisiaeis enforcedby the alternating cycle of B-type cyclin-CDK activity levels through the cell cycle: Iow in telophasethrough G1 and high in S, G2, and M through anaphase(seeFigure 20-29). As we just discussed, S-phasecyclin-CDK complexes become active at the beginning of S phase when their specific inhibitor, Sic1, is degraded. The prereplication complexes assembledat origins early in G1 (Figure 20-30, step [) initiate DNA synthesisin

or,I 'L

Prereplication complex

E

cYcrin-cDK l3;;""

pola-primase

l-

Af

+

dNrPs, NrPs

':lifu -\ s Pors€'-

+ dNTPs,NTPs,ORCs

878

c H A p T E R2 0

|

]

R E G U L A T T NTGH E E U K A R Y O T TC CE L LC Y C L E

< FIGURE 20-30 Assemblyand regulationof p r e r e p l i c a t i ocno m p l e x e sS. t e p[ : D u r i n ge a r l yG 1 , u n p h o s p h o r y l arteepdl i c a t i oi n i t i a t i ofna c t o r sa s s e m ool en a n o r i g i n - r e c o g n i tci o n m p l e(xO R Cb) o u n dt o a r e p l i c a t i o n o r i g i nt o g e n e r a tae p r e r e p l i c a t icoonm p l e xS t e p[ : I n t h e S p h a s eS, - p h a scey c l i n - C DcKo m p l e x easn dD D K phosphorylate components of the prereplication complex S t e pS : T h i s l e a d st o b i n d i n go f C d c 4 5a, c t i v a t i oonf t h e h e x a m e rM i cC M h e l i c a s ewsh, i c hu n w i n dt h e p a r e n t a l D N As t r a n d sa,n dr e l e a soef t h e p h o s p h o r y l a tCeddc 6a n d C d t l i n i t i a t i ofna c t o r sR P Ab i n d st o t h e r e s u l t i nsgi n g r e s t r a n d eD d N A .S t e p4 : l n i t i a t i oonf s y n t h e sbi sy t h e D N A p o l y m e r a cs re- p r i m a s( see eF i g u r 4e - 3 1 ) S . t e pE : O t h e r components necessary for replication fork movement are recruited, and bidirectional synthesis awayfromthe o r i g i nc o n t i n u east e a c hf o r k O R Cb i n d st o t h e o r i g i n s e q u e n ci n e t h e d a u g h t edro u b l e - s t r a n dDeN d A ,b u t t h e p h o s p h o r y l a ti e nd i t i a t i ofna c t o r cs a n n oat s s e m b al e p r e r e p l i c a t icoonm p l eox n i t . B - t y p e c y c l i n - C DcK omplexes maintainthe initiationfactorsin a phosphorylated state t h r o u g h o ut ht e r e m a i n d eorf S ,G 2 ,a n de a r l ya n a p h a s e (fop) Thesefactorscannotassemble into new p r e r e p l i c a t icoonm p l e x eusn t i lt h e ya r ed e p h o s p h o r y l a t e d b y C d c 1 4p h o s p h a t aasnedB - t y p e c y c l i nasr ed e g r a d e d f o l l o w i n tgh e i rp o l y u b i q u i t i n a tbi oynt h e A p C / Ci n I a t e a n a p h a sS e e v e r a ld d i t i o n a f al c t o r sr e q u i r efdo r replication arenot shown

S phasewhen they are phosphorylatedby the S-phasecyclinCDKs and a secondheterodimericprotein kinase, DDK, expressedin G1 along with other proteins involved in DNA replication (step E). Although the complete set of proteins that must be phosphorylated to activate initiation of DNA synthesishas not yet been determined,there is evidencethat phosphorylation of at least one subunit of the hexameric and of another initiation factor called Cdc6 is MCM belica.se required. Following their phosphorylation, the helicaseunwinds the DNA, and the resulting single-strandedDNA is bound by the single-strandedbinding protein RPA and other replication factors (Figure 20-30 steps B, 4, and 5; see also Figure4-31). As the replication forks progressaway from each origin' the phosphorylated initiation factors are displacedfrom the chromatin. However, ORC complexes immediately bind to the origin sequencein the replicated daughter duplex DNAs and remain bound throughout the cell cycle (seeFigure 2030, step [). Origins can fire only once during the S phase becausethe phosphorylated initiation factors cannot reassembleinto a prereplication complex. Consequentlgphosphorylation of componentsof the prereplication complex by S-phasecyclin-CDK complexes and the DDK complex simultaneously activates initiation of DNA replication at an origin and inhibits re-initiation of replication at that origin. As we have noted, B-type cyclin-CDK complexesremain active throughout the S phase, G2, and early anaphase'maintaining the phosphorylated state of the replication initiation factors that prevents the assembly of new prereplication complexes(step[). When the Cdc14 phosphatase is activated in late anaphaseand the APC/C-Cdh1 complex triggers degradation of all B-type cyclins in telophase,phosphateson the initiation factors are removed by the unopposed Cdc14 phosphatase. This allows the reassembly of prereplication complexesduring early G1. As discussedpreviously, the inhibition of APC/C activity in G1 setsthe stagefor accumulation of the S-phasecyclins needed for onset of the next S (1 ) phase.This regulatory mechanismhas two consequences: prereplication complexes are assembledonly during G1, when the activity of B-type cyclin-CDK complexes is low, and (2) each origin initiates replication one time only during the S phase, when S phase cyclin-CDK complex activity is high. As a result, chromosomal DNA is replicated only one time each cell cycle.

r Once active mid-G1 cyclin-CDK complexes accumulate in mid-late G1, they phosphorylate and activate two transcription factors that stimulate expression of the late-G1 cyclins, as well as enzymesand other proteins required for DNA replication, and the early S-phaseB-type cyclins. r The late-G1 cyclin-CDK complexes phosphorylate and inhibit Cdh1, the specificity factor that directs the anaphase-promotingcomplex (APC/C) to B-type cyclins, thus permitting accumulation of S-phaseB-type cyclins in late G1. r S-phasecyclin-CDK complexesinitially are inhibited by Sic1. Polyubiquitination of Sicl by the SCF ubiquitinprotein ligasemarks Sicl for proteasomal degradation' reieasing activated S-phasecyclin-CDK complexesthat trigger onset of the S phase (seeFigure 20-28). Late S-phase/earlyM-phase B-type cyclins, expressed ter in the S phase,form heterodimerswith the CDK that also promote DNA replication and initiate spindle formation early in mitosis. t Late M-phase B-type cyclins, expressedin G2' form heterodimers with the CDK that stimulate mitotic events. r In late anaphase,the specificity factor Cdhl is activated by dephosphorylationand then directsAPC/C to polyubiquitinylate all the B-type cyclins' Their subsequentproteasomal degradation inactivates MPF activity, permitting exit from mitosis (seeFigure 20-1'0). r DNA replication is initiated from prereplication complexesassembledat origins during early G1. S-phasecyclinCDK complexessimultaneouslytrigger initiation from prereplication complexes and inhibit assembly of new prereplication complexes by phosphorylating components of the prereplication complex (seeFigure 20-30). r Initiation of DNA replication occurs at each origin, but only once, until a cell proceedsthrough anaphase,when activation of APC/C leads to the degradation of B-type cyclins. The block on re-initiation of DNA replication until replicated chromosomes have segregated assures that daughter cells contain the proper number of chromosomes per cell.

Controlin Mammalian Cell-Cycle Cells In multicellular organisms, precise control of the cell cycle

Cyclin-CDKand Ubiquitin-Protein LigaseControl of S phase t S. cereuisiaeexpressesa single cyclin-dependentprotein kinase (CDK), which interactswith severaldifferent cyclins during different phasesof the cell cycle (seeFigure 20-29). r Three G1 cyclins are active in G1. The concentration of the mid-G1 cyclin mRNA does not vary significantly through the cell cycle, but its translation is regulatedby the availability of nutrients.

ing G1, entering the G6 state (seeFigure 20-1)' Some differentiatedcells (e.g.,fibroblasts and lymphocytes)can be stimulated to reenter the cycle and replicate. Many postmitotic C E L L - C Y C LCEO N T R O LI N M A M M A L I A N C E L L S

879

(a)

DNAstain

BrdU stain

BrdU-positivecells (c)

BrdU-negativecells

Control

s 100

+ Anti-cyclinD

o

3ao

.2

oOU

o j40 dr

\s

Anti-cyclinD stain

20 U

10 14 12 to TimewhenantibodvandBrdUadded (hoursafteradditionof growthfactors)

A EXPERIMENTAL FtcURE20-31 Microinjection experiments with anti-cyclinD antibody demonstratethat cyclinD is requiredfor passagethrough the restrictionpoint. TheGsarrested mammalian cellsusedin theseexperiments passthe restriction point14-16hoursafteraddition of growthfactorsand enterthe S phase6-8 hourslater.(a)Outlineof experimental protocol. At various times1O-16hoursafteradditionof growth factors(Il), somecellsweremicroinjected with rabbitantibodies against cyclinD (Z). Bromodeoxyuridine (BrdU), a thymidine analog, wasthenaddedto the medium(E), andthe uninjected controlcells (/eft)and microinjected experimental cells(nghf)wereincubated for an additional 16 hours.Eachsample wasthenanalvzed to determine the percentage of cellsthathadincorporated BrdUintonewly synthesized DNA(4), indicating thattheyhadentered the S phase. (b)Analysis of controlcellsandexperimental cellsiniected with anticyclinD antibody 8 hoursafteradditionof growthfactors. Thethree micrographs showthesamefieldof cellsstained16 hoursafter

addition of BrdUto the medium. Cellswerestained with different fluorescent agentsto visualize DNA(top),BrdU(middle), andanti(bottom). cyclinD antibody Notethatthetwo cellsin thisfield injected (theredcellsin the bottom with anti-cyclin D antibody micrograph) did not incorporate BrdUintonuclear DNA,asindicated bytheirlackof staining in the middlemicrograph (c)percentage of controlcells(bluebars)andexpenmental cells(redbars)that incorporated BrdU.Mostcellsinjected with anti-cyclin D antibodies 10or 12 hoursafteraddition of growthfactorsfailedto enterthe S phase, indicated by the low levelof BrdUincorporation In contrast, anti-cyclin D antibodies hadlittleeffecton entryintothe S phaseand DNAsynthesis wheninjected at 14or 16 hours,thatis,aftercells hadpassed the restriction point.Theseresults indicate thatcyclinD is required to passthe restriction point,butoncecellshavepassed the restrictjon point,theydo not require cyclinD to enterthe S phase (b)and(c)adapted 6-8 hourslater.IParts fromV Baldin et al..1993,Genes & Devel. T:812.1

M a m m a l i a nR e s t r i c t i o nP o i n t l s A n a l o g o u st o STARTin Yeast Cells Most studies of mammalian cell-cycle control have been done with cultured cells that require certain polypeptide growth factors (mitogens) to stimulate cell proliferation. 880

C H A P T E R2 0

|

R E G U L A T T NTGH E E U K A R Y O T TCCE L LC Y C L E

Binding of thesegrowth factors to specificreceptor proteins that span the plasma membrane initiates a cascadeof signal transduction that ultimately influences transcription and cell-cyclecontrol (Chapters15 and 16). Mammalian cells cultured in the absenceof growth factors are arrestedwith a diploid complement of chromosomes in the Ge period of the cell cycle. If growth factors are added to the culture medium, thesequiescentcellspassthrough the restriction point 14-15 hours later, enter the S phase 6-8 hours after that, and traversethe remainder of the cell cycle (seeFigure 20-2).The restriction point is the time after addition of growth factors when cells no longer require the presenceof growth factors to enter S phase.Like START in yeast cells, the restriction point is the point in the cell cycle at which mammalian cells becomecommitted to entering the S phase and completing the cell cycle, which takes about 24 hours for most cultured mammalian cells.

Multiple CDKsand CyclinsRegulatePassageof M a m m a l i a nC e l l sT h r o u g ht h e C e l lC y c l e Unlike S. pombe and S. cereuisiae,whicheachproduce a single cyclin-dependentkinase (CDK) to regulate the cell cycle, mammalian cells use a small family of related CDKs to regulate progressionthrough the cell cycle. Four CDKs are expressed at significant levels in most mammalian cells and play a role in regulating the cell cycle. Named CDK1, 2, 4, and 6, these proteins were identified by the ability of their cDNA clonesto complement certain cdc yeastmutants or by their homology to other CDKs. Like S. cereuisiae,mammalian cells express multiple cyclins. Cyclin A and cyclin B, which function in the S phase,G2, and early mitosis, initially were detectedas proteins whose concentration oscillatesin experimentswith synchronously cycling early sea urchin and clam embryos (seeFigure 20-8). Homologous cyclin A and cyclin B proteins have been found in all multicellular animals examined.The cDNAs encoding three related human D-type cyclins and cyclin E were isolated basedon their ability to complement S. cereuisiaecells mutant in all three genesencodingG1 cyclins.The relativeamounts of the three D-ryp. cyclins expressedin various cell types differ' Here we refer to them collectivelyas cyclin D. Cyclins D and E are the mammalian mid- and late-G1cyclins, respectively. Experiments in which cultured mammalian cells were microinjected with anti-cyclin D antibody at various times after addition of growth factors demonstrated that cyclin D is essentialfor passagethrough the restriction point (Figure20-31'). Figure 20-32 presentsa current model for the periods of the cell cycle in which different cyclin-CDK complexesact in Gs-arresredmammalian cells stimulated to divide by the addition of growth factors. In the absenceof growth factors, cultured Ge cells expressneither cyclins nor CDKs; the absenceof these critical proteins explains why Ge cells do not progressthrough the cell cycle and replicate. Table 20-1, presentedearly in this chapter, summarizesthe various cyclins and CDKs that we have mentioned and the portions of the cell cycle in which they are active. The cyclins fall

into two major groups' G1 cyclins and B-type cyclins, which function in S, G2, and M. Although it is not possibleto draw a simple one-to-onecorrespondencebetweenthe functions of the severalcyclins and CDKs in S. pombe, S. cereuisiae'and vertebrates, the various cyclin-CDK complexes they form can be broadly consideredin terms of their functions in mid-G1, IateG1, S, and M phases.All B-type cyclins contain a conserveddestruction box sequencethat is recognizedby the APC/C-Cdh1 ubiquitin-protein ligase,whereas G1 cyclins lack this sequence. Thus the APC/C regulates only the activity of those cyclinCDK complexesthat include B-type cyclins.

RegulatedExpressionof Two Classesof Genes ReturnsGs MammalianCellsto the Cell Cycle Addition of growth factors to Gg-arrestedmammalian cells induces transcription of multiple genes,most of which fall into one of two classes-early-responseot delayed-response genes-depending on how soon their encoded mRNAs appear.Transcription of early-responsegenesis induced within a few minutes after addition of growth factors by signaltransduction cascadesthat activate preexisting transcription factors in the cytosol or nucleus (Chapter 16). Many of the early-responsegenesencodetranscription factors' such as cFos and c-Jun, that stimulate transcription of the delayedresponsegenes. Mutant, unregulated forms of both c-Fos anJ c-Jun are expressedby oncogenicretroviruses (Chapter 25); the discoverythat the activatedviral forms of theseproteins (v-Fos and v-Jun) can transform normal cells into cancer cells led to identification of the normal, regulated cellular forms of thesetranscription factors. After peaking at about 30 minutes following addition of growth factors, the concentrations of the early-response mRNAs fall to a lower level that is maintained as long as growth factors are present in the medium. This decreasein early-responsemRNA levels is mediated by early-response protelns. Expression of delayed-responsegenes depends on proteins encoded by early-response genes. Some delayedresponsegenesencode additional transcription factors (see below); others encode mid- and late-G1 cyclins and CDKs' The mid-G1 cyclins and their associating CDKs are ex-

centrations fall precipitously.As a consequence'the cells do not passthe restriction point and do not replicate' In addition to being controlled by transcription of the

ter l6,leading to activation of the mTOR pathway and the resulting activation of translation initiation factors (see cELLs c E L l - c y c L Ec o N T R o Ll N M A M M A L I A N

t

881

Mitotic cyclin-CDKs CyclinA-CDK1 CyclinB-CDK1

< FIGURE 20-32Activityof mammaliancyclin-CDK complexesthrough the courseof the cell cycle. Go Cultured G6cellsareinduced to divideby treatment with growthfactorsThewidthof thecolored bandsis proportional to the proteinkinase \ actrvity Mid-Gl cyclin-CDKs approximately "CyclinD" refers of the indicated complexes to allthree Cyclin D-CDK4 D-typecyclins Cyclin D-CDK6

I

I

l S-phase cyclin-CDK CyclinA-CDK2

Late-G1cyclin-CDK CyclinE-CDK2

Figure 8-30).As a resuk, translationof cyclin D mRNA and other mRNAs is stimulated. Agents that inhibit activation of translation initiation factors, such as TGF-8, inhibit translation of cyclin D mRNA and thus inhibit cell proliferation.

PassageThrough the Restrictionpoint Depends on Phosphorylationof the Tumor-Suppressor Rb Protein Somemembersof a small family of related transcription factors, referred to collectively as E2F factors, are encoded by delayed-response genes.These transcription factors activate genesencoding many of the proteins involved in DNA synthesis. They also stimulate transcription of genesencoding the late-G1cyclin, the S-phasecyclin, and the S-phaseCDK. Thus the E2Fs function in late G1 similarly to the S. cereuisiae tanscription factors SBF and MBF. In addition, E2Fs autostimulate transcription of their own genes.E2Fs function as transcriptional repressorswhen bound to Rb protein, which in turn binds histone deacetylaseand methylasecomplexes.As discussedin Chapter 7, histone deacetylationand methylation of specific histone lysines causeschromatin to assumea condensed,transcriptionally inactive form. Rb protein was initially identified as the produc of the prototype tumor-suppressorgene,RB. The products of tumor-suppressor genesfunction in various ways to inhibit progression through the cell cycle (Chapter 25). Loss-of-function mutations in RB are associatedwith the diseaseh ereditary retinoblastoma. A childwith this disease inherits one normal RB+ allele from one parent and one mutant RB- allele from the other. If rhe Rt+ allele in any of the trillions of cells that make up the human body becomesmutated to a RB- allele,then no functional Rb pro_ tein is expressedand the cell or one of its descendanisis likely to become cancerous. For reasons that are not 882

C H A P T E R2 0

|

understood,this generallyhappensin a retinal cell leading to the retinal tumors that characterizethis disease.Subsequently it was discoveredthat Rb function is inactiyated in almost all cancer cells, either by mutations in both alleles of RB, or by abnormal regulation of Rb phosphorylation.I Rb protein is one of the most significant substratesof mammalian G1 cyclin-CDK complexes. Phosphorylation of Rb protein at multiple sites prevents its associationwith E2Fs, thereby permitting E2Fs to activatetranscription of genesrequired for entry into S phase. As shown in Figure ZO-33, phosphorylation of Rb protein is initiated by the mid-G1 cyclin-CDK complexesin mid G1. Once the late-G1cyclin and CDK are induced by phosphorylation of some Rb, the resulting late-G1 cyclin-CDK complex further phosphorylates Rb in late G1. Sfhen late-G1 cyclin-CDK accumulatesto a Mid Gl

Late G1

FIGURE 20-33 Regulationof Rband E2Factivitiesin mid-lateGr. Stimulation of Gocellswith mitogens inouces expression of CDK4,CDK6,D-typecyclins, andthe E2Ftranscription factors, allencoded by delayed-response genes.Rbproteininitially inhibits E2FactivityWhensignaling frommitogens issustained, the resulting cyclinD-CDK4/6 complexes beginphosphorylating Rb, releasing someE2F, whichstimulates transcription of the genes encoding cyclinE,CDK2,andE2Fitself(autostimulation) Thecyclin E-CDK2 complexes furtherphosphorylate Rb,resulting in positive feedback loops(bluearrows) thatleadto a rapidrisernrne expression andactrvity of bothE2FandcyclinE-CDK2 asthecell approaches the G1-+ Stransition

R E G U L A T T NTGH E E U K A R Y O T TC CE L LC Y C L E

critical threshold level, further phosphorylation of Rb by the late-G1 complex continues even when mid-G1 cyclin-CDK activity is removed. This is one of the principal biochemical eventsresponsiblefor passagethrough the restriction point. At this point, further phosphorylation of Rb by the late-G1 cyclin-CDK occurs even when mitogens are withdrawn and mid-G1 cyclin-CDK levels fall. SinceE2F stimulates its own expressionand that of the late-G1 cyclin and CDK, positive cross-regulationof E2F and late-G1 cyclin-CDK produces a rapid rise of both activities in late G1. As they accumulate,S-phasecyclin-CDK and mitotic cyclin-CDK complexesmaintain Rb protein in the phosphoryIated state throughout the S, G2, and early M phases.After cells complete anaphaseand enter early G1 or Ge, the fall in cyclin-CDK levels leads to dephosphorylation of Rb. As a consequence,hypophosphorylated Rb is available to inhibit E2F activity during early G1 of the next cycle and in Gearrestedcells.

C y c l i nA l s R e q u i r e df o r D N A S y n t h e s i sa n d C D K 1f o r E n t r yi n t o M i t o s i s High levels of E2Fs activate transcription of the cyclin A gene as mammalian cells approach the G1 -+ S transition. (Despite its name, cyclin A is a B-type cyclin, seeTable 201.) Disruption of cyclin A function inhibits DNA synthesis in mammalian cells,suggestingthat cyclin A is the S-phase cyclin and that, along with CDK2, it may function like S. cereuisiaeS-phasecyclin-CDK complexes to trigger initiation of DNA synthesis.There is also evidencethat the mammalian late-G1cyclin-CDK complexesalso contribute to activation of prereplication complexes. Note that CDK2 complexes with both the late-G1 and the S-phasecyclins (seeFigure 20-32). Three related CDK lnhibitory proteins, or CKls (p27*'ot,p57*t", and p21cIP),appearto sharethe function of the S. cereuisiaeS-phaseinhibitor Sicl (seeFigure 20-28). Phosphorylation of p27*tnt by late-G1cyclin-CDK targets it for polyubiquitination by the mammalian SCF complex (see Figure 20-28). The mechanisms for degrading p2lcrP and pSZrtPzare lesswell understood. The activity of mammalian cyclin-CDK2 complexes is also regulated by phosphorylation and dephosphorylation mechanisms similar to those controlling the S. pombe mitosis-promoting factor, MPF (see Figure 20-1.4). The Cdc25A phosphatase,which removes the inhibitory phosphate from CDK2, is a mammalian equivalent of S. pombe Cdc25 except that it functions at the G1 -+ S transition rather than the G2 -+ M transition. The mammalian phosphatase normally is activated late in G1, but is degradedin the responseof mammalian cellsto DNA damageto prevent the cellsfrom enteringS phase(seeSection20.7). Once late-G1cyclin-CDK and S-phasecyclin-CDK are activated by Cdc25A and the S-phaseinhibitors have been degraded, DNA replication is initiated at prereplication complexes. The general mechanism is thought to parallel that in S. cereuisiae(seeFigure 20-30), although small differencesare found in vertebrates.Phosohorvlation of DNA

replication preinitiation complexes at replication origins by late-G1cyclin-CDK and S-phasecyclin-CDK likely promotes initiation of DNA replication. As in yeast, phosphorylation of these initiation factors likely prevents reassemblyof prereplication complexes until the cell passesthrough mitosis, thereby assuring that replication from each origin occurs only once during each cell cycle. In metazoans, a second small protein, geminin, contributes to the inhibition of reinitiation at origins until cells complete a full cell cycle. Geminin is expressedin late G1; it binds and inhibits replication initiation factors as they are releasedfrom preinitiation complexes once DNA replication is initiated during S phase (Figure 20-30, step B), contributing to the inhibition of re-initiation at an origin. Geminin contains a destruction box at its N-terminus that is recognizedby the APC/C-Cdh1, causingit to be polyubiquitinylated in late anaphaseand degraded by proteasomes.This frees the replication initiation factors, which are dephosphorylatedby Cdc14 phosphatase, to bind to ORC on replication origins forming preinitiation complexesduring the following G1 phase. The principal mammalian CDK in G2 and mitosis is CDK1 (seeFigure 20-32). This CDK, which is highly homologous with S. pombe CDK, associateswith cyclins A and B. The mRNAs encoding either of thesemammalian cyclins can promote meiotic maturation when injected into Xenopus oocytesarrestedin G2 (seeFigure 20-6), demonstrating that they function as mitotic cyclins. In somatic vertebrate cells, cyclin A-CDK1 and cyclin B-CDK1 function together as the equivalentof the S. pombe MPF (mitotic cyclin-CDK). The kinase activity of these mammalian complexes also is regulatedby proteins analogousto those that control the activity of the S. pombe MPF (see Figure 20-141. The inhibitory phosphate on CDK1 is removed by Cdc2SC phosphatase, which is analogous to S. pombe Cdc25 phosphatase. In cycling mammalian cells, cyclin B is first synthesized Iate in the S phaseand increasesin concentrationas cellsproceed through G2, peaking during metaphaseand dropping after late anaphase.This parallels the time course of cyclin B expressionin Xenopwscycling egg extracts (seeFigure 20-9). In human cells, cyclin B first accumulatesin the cytosol and then enters the nucleus iust before the nuclear enveloperetracts into the ER early in mitosis. Thus MPF activity is controlled not only by phosphorylation and dephosphorylation but also by regulation of its import into the nucleus.In fact, cyclin B shuttles between the nucleus and cytosol, and the changein its localization during the cell cycle results from a changein the relative ratesof import and export. As rn Xenopus eggsand S. cereuisiae,cyclins A and B are polyubiquitinylated by the APC/C-Cdh1 complex during late anaphase and then are degradedby proteasomes(seeFigure 20-1'0).

Two Typesof Cyclin'CDKlnhibitors Contribute to Cell-CycleControl in Mammals As noted above, three related CKIs-p21crP, p27KrPr,and p57KlP2-inhibit late-G1 cyclin-CDK and S-phasecyclinCDK activity and must be degradedbefore DNA replication C E L L - C Y C LCEO N T R O LI N M A M M A L I A N C E L L S

883

can begin. These same CDK inhibitory proteins also can bind to and inhibit the other mammalian cyclin-CDK complexes involved in cell-cycle control. As we discuss later, p21tt' plays a role in the responseof mammalian cells to DNA__damage.Experiments with knockout mice lacking p27*t" have shown that this CKI is particularly importani in controlling generalizedcell proliferation soon after birth. Although p27KrP1knockouts are larger than normal, most develop normally otherwise. In contrast, p57'tnt knockouts exhibit defectsin cell differenriation, and most die shortly after birth owing to defectivedevelopmentof various organs. A second class of cyclin-CDK inhibitors called lNK4s (lzhibitors of Ainase4) includes severalsmall, closelyrelated proteins that interact only with the mid-G1 CDKs, CDK4 and CDK5, and thus function specificallyin controlling the mid-G1 phase.Binding of INK4s to CDK4 and CDK5 blocks their interaction with cyclin D and hencetheir protein kinase activity. The resulting decreasedphosphorylation of Rb protein prevents transcriptional activation by E2Fs and entry into the S phase.One INK4 calledp16 is a tumor suppressor, like Rb protein discussedearlier.The presenceof two mutant p16 allelesin a large fraction of human cancersis evidence for the important role of p1.6 in controlling the cell cycle (Chapter25).

r The activity of S-phasecyclin-CDK, induced by high E2F activity, initially is held in check by CKIs, which function like an S-phaseinhibitor, and by the presenceof an inhibitory phosphate on CDK2, the S-phaseCDK. Proteasomal degradation of the inhibitors and activation of the Cdc25A phosphatase,as cells approach the G1 -+ S transition, generateactive S-phasecyclin-CDK. Along with the late-G1 cyclin-CDK, this complex activatesprereplication complexesto initiate DNA synthesisby a mechanismsimilar to that in S. cereuisiae(seeFigure 20-30). r Cyclin A-CDK1 and cyclin B-CDK1 induce the events of mitosis through early anaphase. Cyclins A and B are polyubiquitinylated by the anaphase-promotingcomplex (APC/C) during late anaphaseand then are degraded by proteasomes, r The activity of mammalian mitotic cyclin-CDK complexesis regulatedby phosphorylation and dephosphorylation similarly to the mechanism in S. pombe, with the Cdc25C phosphataseremoving inhibitory phosphates(see Figure20-1,4). r The activities of mammalian cyclin-CDK complexesalso are regulated by CDK inhibitors (CKIs), which bind to and inhibit each of the mammalian cyclin-CDK complexes,and INK4 proteins, which block passagethrough G1 by specifically inhibiting G1 CDKs (CDK4 and CDK5).

Cell-CycleControl in Mammalian Cells r Various polypeptide growth facrors called mitogens stimulate cultured mammalian cells to proliferate by inducing expressionof early-responsegenes.Many of these encode transcription factors that stimulare expressionof delayed-responsegenes encoding the G1 CDKs, G1 cyclins, and E2F transcription factors. r Once cells passthe restriction point, they can enter the S phase and complete S, G2, and mitosis in the absenceof growth factors. r Mammalian cells use severalCDKs and cyclins to regulate passagethrough the cell cycle. Cyclin D-CDK4 and cyclin D-CDK6 function in mid to late G1; cyclin E-CDK2, in late G1 and early S; cyclin A-CDK2, in S; and cyclin A-CDK1 and cyclin B-CDK1 in G2 and M through anaphase(seeFigure 20-32). r Unphosphorylated Rb protein binds to E2Fs, converting them into transcriptional repressors.Phosphorylation of Rb by the mid-G1 cyclin-CDK liberates E2Fs to activare transcription of genes encoding the late-G1 cyclin and CDK, as well as other proteins required for the S phase. E2Fs also autostimulate transcription of their own genes. r The late-G1cyclin-CDK further phosphorylatesRb, further activating E2Fs. Once a critical level of late-G1cyclinCDK has been expressed,a positive feedback loop with E2F results in a rapid rise of both activities that drives passagethrough the restriction point (seeFigure 20-33).

884

C H A P T E R2 0

I

Checkpoints in Cell-Cycle Regulation Before proceeding, let's review the major steps in the eukaryotic cell cycle summarized in Figure 20-34.In continuously cycling cells,cyclin-CDK complexesare absentin early G1. Hypophosphorylated DNA replication initiation factors are free to bind to ORC complexesat DNA replication origins, generating prereplication complexes that are inactive until they are phosphorylated by an S-phase cyclin-CDK (step E). In mid-G1, mid-G1 cyclin-CDKs are expressedand phosphorylate the APC/C specificity factor Cdh1, inactivating it and allowing newly synthesizedB-type cyclins (and geminin in vertebrates) to accumulate when they are expressed(stepZ).The mid-G1 cyclin-CDKsalso phosphorylate specific transcription facrors, activating expression of late-G1 and S-phasecyclins (and CDK in vertebrate somatic cells) (step S). However, as B-type cyclins are expressed, they are immediately bound by inhibitors. \fhen G1 cyclinCDK activities reach peak levels, they phosphorylate these inhibitors at multiple sites (step @), marking them for polyubiquitination by the SCF ubiquitin-protein ligase, and subsequentdegradation by proteasomes(step g). This rapid degradation of S-phasecyclin-CDK inhibitors releasesS-phasecyclin-CDK activities,which phosphorylate key regulatory sitesin prereplication complexes,stimulating initiation of DNA replication at multiple origins (step 6).

REGULATING THE EUKARYOTIC C E L LC Y C L E

CdcA phosphatase activatesCdhl and APC/C-Cdh1/proteasome degradesmitotic cyclins

r

APCIC-Cdc2Ol proteasome

,a--\ /

\

\*/

Telophaseand cytokinesis

333.1ii" s

E

Anaphase Early G1

DNA prereplication complexesassemble at origins

z

Mid-late Gl

z Metaphase

G. cyclin-CDKactivates E eipression of S-phasecyclin-

Restriction point

CDK comoonents

q

Cdc25phosphatase activatesmitotic cyclinCDKs,which activate early mitotic events

tr--:----g

G, cyclin-CDKinactivatesCdhl

'{

tr wdtl!trp S-phase cycrin-cDK w activatesprereplication comprexes

d[

G. cyclin-CDKphosphorylates 5-'phaseinhibitor

SCFrproreasome

g:3;:::..fl,e,l_fo?r'".0 inhibitor

DNA replication A FIGURE20-34 Fundamental processesin the eukaryotic cell cycle.Seethe text for discussion

Mitotic cyclin-CDKs are expressedin late S phase and G2. When DNA replication has been completed, they are activated by Cdc25 phosphatase,and either theS or other protein kinasesthat they activate,phosphorylatespecificregulatory sitesin more than a hundred proteins including histone H1, condensins and cohesins, additional chromatinassociatedproteins, microtubule-associatedproteins, nuclear lamins, inner nuclear membrane proteins, and nuclear pore complex proteins. These multiple, specific phosphorylations induce the early eventsof mitosis including chromosome condensation,remodeling of microtubules into the mitotic spindle apparatus, and, in animals and plants, retraction of the nuclear envelopeinto the ER (step Z). Once every kinetochore of each sister chromatid has attached to spindle microtubule fibers during metaphase,inhibition of the Cdc20 specificity factor is lifted. This results in

active APC/C-Cdc20 and polyubiquitination and proteasomal degradationof securin (step E ). Securindegradationreleasesthe proteolytic activity of separase,which then cleaves the cohesin rings at centromeresthat hold sister chromatids together.The forces exerted by the mitotic spindle apparatus then pull the releasedsister chromatids toward opposite spindle poles. The resulting sudden separation of all sister chromatids marks the beginning of anaphase. Once the daughter chromosomes have separated sufficiently to ensure equal segregationof all chromosomes to daughter cells during cytokinesis,the Cdc14 phosphataseis activated. Cdc1,4dephosphorylatesand activatesthe Cdhl APC/C specificity factor, resulting in the polyubiquitination and proteasomal degradation of all B-type cyclins (and geminin in vertebrates),and consequently,the loss of MPF activity (step p). Sites on the multiple proteins that were

N C E L L . C Y C LR EE G U L A T I O N C H E C K P O I N TIS

885

phosphorylated by cyclin-CDKs are dephosphorylated by Cdc1.4.This returns the proteins to their interphase functions, resulting in decondensationof chromosomes,formation of an interphasemicrotubule cytoskeletonwith a single microtubule organiztngcenter,and reassemblyof the nuclear envelopeduring telophase,followed by cytokinesis.The dephosphorylated DNA replication-initiation factors (released by geminin degradation in vertebrates)then reassemble preinitiation complexeson ORC complexesbound to repli, cation origins in daughter cells, in preparation for the next cell cycle (step[).

Successfulcompletion of the cell cycle has severalgeneral requirements.Each processsummarizedin Figure 20-34 must go to completion beforesubsequentstepsare undertaken,and the stepsmust occur in the correct order.Catastrophicgenetic damagecan occur if cellsprogressto the next phaseof the cell cycle beforethe previousphaseis properly completed.For example, when S-phasecellsare induced to enter mitosis by fusion to a cell in mitosis, the MPF presentin the mitotic cell forcesthe chromosomesof the S-phasecell to condense.This premature entry into mitosis results in fragmentation of the Sphasechromosomes,a disastrousconsequence for a cell.

KINASESAND PHOSPHMASES CAK kinase

Activatescyclin-CDKs

Veel kinase

Inhibitscyclin-CDKs

Cdc25phosphatase

Activates cyclin-CDKs

cdc14 phosphatase

Activatescdhl to inhibit mitotic cyclin-cDK

Cdc25Aphosphatase

Acrivares verrebrare S-phase cyclin-CDK

Cdc25Cphosphatase

Acrivatesverrebraremitotic cyclin-CDK

ATM/MR kinases

Checkpointcontrols,activateChkl/Chk2 kinases

Chkl/Chk2 kinases

Checkpointcontrols,inactivateCdc25Cand Cdc25Aphosphatases to inducecell-cyclearrest

INHIBITORY PROTEINS Bindsand inhibits S-phase cyclin-CDKs CKIs p27KIP1,p57*"t, and p21cIP

Bind and inhibit cyclin-CDKs

INK4

Binds and inhibits mid-G1 CDKs

Mad2

Spindle-assemblycheckpoint control, binds Cdc20 and prevents onset of anaphaseand inactivation of B-type cyclin-CDKs

Rb

Binds E2Fs, preventing transcription of multiple cell cycle genes

UBIQUITIN-PROTEINLIGASES SCF

Degradation of phosphorylated Sicl or p27KrP1to activare S-phasecyclin-CDKs

APC/C + Cdc20

Inducesdegradationof Securin,initiating anaphase.Inducespartial degradation of B-type cyclins

APC/C + Cdhl

Induces complete degradation of B-type cyclins to initiate teiophase,and geminin in metazoans to allow formation of prereplication complexes on DNA replication origins

886

C H A P T E R2 0

|

R E G U L A T T NTGH E E U K A R Y O T TC CE L LC Y C L E

'We have seenhow progression through the cell cycle is governed by precise regulation of the activities of multiple cyclin-CDK complexes. TabIe 20-2 summarizes the various types of regulators of cyclin-CDK activity. The key cellcycle events of DNA replication and chromosome segregation must be accomplishedwith extraordinary accuracy and fidelity. To ensure that these processesoccur correctly and in the proper order, cells have evolved multiple additional levels of regulation controlling these fundamental cell-cycle events. Collectively, these additional regulatory mechanismsare known as checkpoints(Figure20-35). Several examplesof cell-cyclecheckpointshave beendiscussed earlier in the chapter. In this section' we consider these and additional checkpoints in terms of the major cell-cycle processessummarizedabove.Control mechanismsthat operateat thesecheckpoints ensure that chromosomes are intact and that each stageof the cell cycle is completed before

Another example of the importance of order of events in the cell cycle concernsattachmentof kinetochoresto microtubules of the mitotic spindle during metaphase.If anaphaseis initiated before both kinetochoresof a replicated chromosome become attached to microtubules from opposite spindle poles, daughter cells are produced that have missing or extra chromosomes,an outcome called nondisjwnction. Nflhennondisjunction occurs in mitotic cells, it can lead to the misregulation of genes,and contribute to the development of cancer.When nondisjunction occurs during the meiotic division that generatesa human egg, Down syndrome can occur from trisomy of chromosome 21, resulting in developmental abnormalities and mental retardation. Other mechanismscan also generatetrisomy. (Trisomy of any of the human chromosomes can occur, but for every other chromosome except chromosome 21, trisomy results in embryonic lethality or death shortly after birth.) I

E

a

Spindle'Position checkPoint

Spindle-assembly checkpoint Mad2 I

I

I

APc/c-cdh1

C d c 1 4+

p o l y u b i q u i t i n a t i ol ulalord rrtolr{u asaqr dq uonpldrot{dsoqdpalnber eql esnpreq pelE^rlrE sr ase.redas pue pessarderopE sr 0zJpl uaq,u .ra,re -rrLoq'pe,reap e;e deql .soseur{ulalord Jrlolrur oqf dq pofel -ftoqdsoqd are daqr uoq^\ uorlprJossrpol tuelsrserare saxald -ruof urseqof,SurureluoJ-gJaUesnpf,eqsr.uJpauosoruoJr{J

I tzzliost tolg 'spl))rN ila) t'0002 I U p u p s r l n r l e dt a n a s l l l p u e l s t s o t a u j ust l l o ) + o sluauodu.rotalqnlos .lo sa;purds pue oql liq saulosoruolql q]tM aq] lou paleoosseaJeleq] srolte; {q pautLxlalap ole saulosor.Llotq> 6u11ur1 sulsaqo)]o {tlllqetsaql pue salnqn}or)il.u alpurdsqlrnnsaroqlolaut) aq] ]eql ele)tputsllnsalasaql ol paqteue atennr{aq} lo uorler)osse a;purdsqtrqan1o luepuadapur(e;odalrsoddoaql pleMol ptleurolql raqlo oqt pue e;od a;puldsauo preMolptleu.lotq)auo ,.a.r){11eu.lou peletedassaulosoruorq) aql ,^lleltulrs.Qq6l) alpurds ll stsotoLu ro Qla/)alpurdsI stsoraur aql ol paq)epelaqlaqM (laqlo aql ll srsoraur prennoltredsno6o;oLuoq laqlo aql pue alod a;purdseuo pterrnol tred

eqt ur peurelureru eJe sprleuorrlJ .rarq8nep uee^\leg sa8e>1ur1 urseqo3 'I srsoreur;ouorte8e.rSes auosotuoJrlcpozqerceds eql .ro; perrnbar are urqso8nqs pue gleg Jo saruedord asoql 'II srsorou Surrnp passardxa '(,,lrtrds uerplenS,, ro; eseuedef) lou sr urqso8nqs'ra,ta,vr.o11 utqso8nqspellef, urelord ra4uq e ,tq urtetuorqf, Jrreurortuer ol punoq sl vzdd Jo IUJoJosrrr;rcads-a.reuorluef,eql I srs -oreruSur.rnq'ase.rBdas dq pazreelo aq ol soseur>l urelord crlol -rtu asaqldq pereydroqdsoqdaq tsnu tr teql ur ursreplf,nolTru eqr ruory sreJlrposle gteg 'sespur>lurelord f,nolru dq palel -drogdsoqd sr lr ueq^,roseqdord ur alprf,osslplou saop xald -ruof,er{l 'xelduroc urseqof,oqt ur urslell f,nolrru aql seceyda.r 8JeU ueq^\ asnetoqI srsoreruroJ sJoJtrpusruEqrer.usrql '(vW-OZ e.rn8rg eos)aseqdeueur uorlp.rBdasprlpruorgf,3urmo11e pue oreruorl -uel oqt lE uorseqor Sulleunurla ,tou lo pareld.loqdsoqd

snobolouoq auo' e r),i;;eu.lou peleredes saulosotlolq) | stsoraut petredaq1 sluoualoLuaseqdeue paleldluotpeqsoulosouJoJLl) poq)eueltoqlq]tmsalpurds qloq 'salnutu01 laUV salpurds Jaqlo:lorlloue leurouJraq]ol paq)ellellol alaMsotrjosouloJLl) o1alpurds auo L.l-tol+ puesou,tosouloJLlf sotlosol.ilolqf ll stsotaul aulosonotrto1posnsemalpaouuorlelndrueurottru.t I stsoraLu e uaql llo)pasnle;0urs e urluesatdalaMsatuosoulo.tq) palel)osse raq] qlrmsa;purds;o sad,{iLl}oq}eq}ospasn}oraMll pueI stsolau .sauosoutorq)oql qllm palet)osse urseyi>oleu.uads taddoqssetg surelold[q paurrurelapeJesau,losoruoJq) )!]oteut lo uolsaqo) pue sluaura^outaseqdeuy Zt-02 lunDg IVINJU\lUldXl V

,,*oal elputds ll srsolayg

I

elpulds lslsolahl or.!osoruoJuc

saLuosouJoJ qc I stsotaul pelelndtuelA

ll srsoraur p a t e l n o r ule l

saurosouloJt{c I srsolaul

ou_rosouioJ qc ll srsotoul polelnotueulun

polelnoIueulun

'I srsororuSur.rnpsalod alpurds arues oqt ruor; Surleupue solnqnloJf,Iluqll.ry\olEIJossEol satuosoruorqcsnoSoloruol{Jo spneurolll qroq to; parrnbar sr 'xalduoc uralord rr;neds-srsorourJeI{louE'uqodouotraqr

3'l),^,)1'll) )lro

'GyV-OZ ern8rg ees)s11arurra8 otut elB8erSasol spnptu -ol{f II srsorou;o aseqdeueSurrnp grag lenpr^rpur s.4aolle 'I JrJOruoJluaJ ;o a3eaeo13 srsoreu ur uorleSerSesSurrnp polerf,osseurerueJ seuosotuoJl{r snoSoloruoq Jo sPIlEru -orqJ eqr 't1nse.re sV 'oreruorlueJ eqt to uot8e.reql ur a8e -^Ealr ruort 8JeU slralord a.roqrotaut{ aq} qtl^\ potelJosse urolo.rdcqnads-srsorerue lng 'pe.LeapsI stuJeotuosoluoJqJ oqr ur SloU '1 srsoreur;oaseqdeuedpee;o lesuo eqt tV r 'srsoraruSur.rnplrunqns uIsal{of,uISIeplf,Ilollu aqt secelda:'greg 'lrunqns ulseqof,pazqercedsV 'I sISoIOu 1o eseqdeterupue eseqdord Sur.rnp.reqleSolseuosoruoJqf, snoSolouoq eqr Surploq ro; alqrsuodsorore Jalossorf, otll ol IElsrP sPllsulorqf, ueo./v\loq s{ull-ssorJ ulsaqof, se spnpurorrlc leluered snoSolouoq Jo spnetuortlJ se IIO.&\ (re,to SurssoD)uorteurquro)eJ'aotstnan? 's ul . uee./\^.leq 'I srsororuSurrnp uonecrlder VNC Jo uon -erlrur Surlourord ur saxaldurol )CJ-ullcdc tg to uoltf,unJ eql surrolred 7oru1cr;reads-sISoIOJAJ'slsolatu lnoqSnorqr passardar sr suqcdc IC ]o uolssardxe 'austnan? 'S uI r 'slsoletuol anbtun are sutalord aruoslnq 'srs -oraur Suro8repunslle) uI uoltf,un' osle sllal Surprrrrpdler -rtolrru ur uorlJun' teql suralord ayrdc-1ecel{t Jo lsolN r 'sJnf,ro dlqer.rr,rur soluosotuoJqrsnoB lsotulE -olouoq luo^e uollEurqluof,eJeuo Jo spIlEtuoJI{Juee.ryuoq lseal lV 'srsdeuLspelleJ ssoJorde ur sqt8uelrreql 3uo1erred sauosouroJqc snoSolouroq parecqdar '1 stsoraurSurrnq r '(SE-OZorn8tg aas) ru.re8 1or cnoterue;dploldlP E tuo{ s11ar proldeq ecnpord ol uoIsI^Ip 1er 1o selodr o^\r dq Pe^\ollo1 r uorlecrldar auosoruorr{f, 1o alodr auo se^lolul sIsoIetrAJ

uorsr^lolla) lo adfi1lerrad5v:slsolaw

'POzrlrqElssr sarorltroleul>lOql 'uoIS ol luotur{f,el}EelnqnloJJnu 'peleraua8 st uotsual oluo -ue1atBrauaSlEql epelu are sluauqf,EllE lllun ureSr salnqnl 'salngnloJJllu lf,3rrof,ul -orJrru Purq or ruaqt Surlqeue er{l espalers.ragr;aypurdsSuo.rrllaqt ol t{JBllB letll seroqrol -eur1'uorsuat Jo ef,uesgeaqt ut olqelsun sI selnqnlorf,Ilrl ol ecurg '(gy-97 ern8rg aos) salod aqr or Surpurq eror.{f,olour>l pa11ndSuragruorl uaql luele.rd sauosoruorqc snoSolouloq uae^^.]aquorlEutqruof,ar dq paleraueS Blerusetql osnEleg (e1odolpurds ar.uesoqt ruor; Sunuoc selngnlorJltu ol tllelle sprtpurorqJ relsrs Jo sarorlJolourl PaluolJo-of,aqr qSnoqr uazle) uotsual Jopun oslr aJE salnqnloJf,Itu Pol€If,ossu -aJoqJolourl '1 aseqdeleur f,Iloletu Surrnq 'suollf,eJlP alIS -oddo ur palpd orp seJoqf,olaul>laql asnPf,eqpadolazrapst uorsuol 'sa1odlcerrol eql uloJt selnqnloJJllu eql ol sJnf,fo 'lerrarrroH 'uOIIJOJIPaIuESeqr ur pe1lnd tuatuqf,PllE uaqm erE seroqJoleuDl qrog esnEJeqselnqnlorJllu eql uo uors -uat ecnpord lou op Suruelroqs elngnlorf,Iur pue suralord

UV)nl 3Hr gNllv'ln9lu

|

0z ulldvH)

868

roloru 'eseqdelaur ut dlree sPIlBIuorql relsIS ol tlJel -re alod alpurds8uo.rmoqr ruory salnqnlorJllu;1 'aseqderaur Sur.rnp uolsuet Jepun ag salngnloJlllu eqr iEI{l se.rtnb -a.r'sa.tlosureqlselnqnlorJrlu aql;o drtpqels aqr Pu? 'soJol{f -olaurl ot salnqnlorcrur alpurds to lueurl{relre elgels reqr oJuapr^aalq€raPlsuol paprurorde,req lseed ut sluarutred -xa crlaua8puE slueulJedxo uortepdlueluorf,Ilu IEUoITIPPV

luoruq)euv alpulds ledord ol solnqlrluo) salnqnlol)!lNalpulds uo uolsual 'suoIlfaJIp olrsoddo ruo.r;SururocsolnqnloJJlluJo sPue + aqt dluo putq ueo degl os uoltrlualro alrsoddo eql ut sPDeruoJqf,rolsls to salrsSurputq olnqnloJJltu elpurdseqr durell ol PasodoJdare ere legl xaldruoc uqodouotu aql Jo sllunqns raqlo pessa.rdxa aql 's11arJItotIuI Pue 11 slsolauI ut uqodouoru Jo eJuasqe eqr u1 'ayod alpurds orueseql uror; Sunuoc salnqnlorf,Ilu Jo puo + oqr qtl^{ lf,EJetutdaqr reqr os uolleluolJo olues eql uI seJoqJolaull rolsls agl uI salls Surpurg olnqnloJf,Ilu sdurep xelduroc urlodouour aql rEI{r laPout eql or pel e^Eq srFS -3Jesoql 'II sISoIeuuI sllal PUEsllol f,Ilolllu uI ssJoqf,oleul{ rE punoJ osle oJB teql urlodouotu qll^ xaldwot ut1 -odouotu p lurot leql surelord o^{l POIEa^ersutalo.rdPalaqel -d{C qll^\ selPnls ddocsorctur ef,uelsoronlJ o^I^ uI PUE urlodouoru qll^ lferelul leql sulelord ;o uotlerlllluePl 'II ur sPllEruorqf,Jo sPllBtuoJqf, slsoletu 'se1odelpurds etrsoddo urorl f,rtol[u ara,v\deql q8noqf se SurleueruasolnqnloJf,Itualpurds tllll{ pelelf,ossc I srsolalu 'pole,ttlcrur sem'utloiou ;o asrqdelaur uI sPIlEtuoJI{JJOIsIS -ow pelleJ utalo.rd e Surpooua (7 srsorezzSutrnp luoul{f,El -lz alnqntoJf,ru relodouozz) LWVW'euo ueq.tt letll PolEe^eJ 'slsollru lou sauo8asoqtJo I{oEO}o rnoIJoDI rrlerualsd5 PUE 'S srsoraurur passerdxesaua8papo,rer lel{l avtsrnanl Jo sls -tr1eue'letrcoJtlru VNC qlrzrrue8eq aotstnanz'S uI uolseqof,

utaro.rdtt IoJpartnbar rersls eror{f,oleul>t " ""]l:iltjll?Xlru,

sISoIauI sI ;o uorleSorSasJIloIaIu lJOJrol JoJ IEJIIITJ II Pu? I ur selnqnlontur redord eql ol sorol{soleull JalsIS}o lueu -r{rEDEl(lsnotzrgg 'Paluauo-o) eg ol PIESere seroqf,oleull relsrs eql !e1od elpurds ataPs eql uIoU Surleueura sopqnl -orJrru elpurds ot y)elle seJoqJolaurl -ralsts 'eseqdeteur 'lse.Iluoc uI 'sllel rarqSnep lueJeJlIPol splletu I srsoreu te sI sIqI 'paluauo -orrlf, ratsls 1o uotle8at8es ro' Ierluesse -tq ot pIESare saroqf,oleul>leqr tsalod elpurds aflsoddo "q uo.ry Surteuetue solnqnlorrltu alpurds ol qJrile soroqf, -olaull ralsts '11slsolatuPue slsolltu uI 'JaIIJPopassnJSIPsV

I slsolew u! seloq)olau!) rslsls sluauo-o) xalduo) u!lodouolNotll 'se1od elrsoddoor palnd aq or elpurds 'peleap sI UISaI{oJJlJetuoJluaf, spDeuoJqJ JetsISeql 8utlro11e ur SreU 'aseqdelaur II srsolotuJo Pua oql lE PalEAIlte sI aser -edas uequrt'dpuenbasuo3 'eseqdord 11sISoIeu Surmp dlrlrt -lp )qJ-uIIJdc ctlotttu uI asIJPuoJeseqr dq paleAItJPerB leHl sosrur{ utolord JIlo}Iu aqr dq uonelrfioqdsoqd urorl parcar -o.rd lou sI ulsar{of, f,IJauoJluaf, PUE uIlEuoJqJ f,IJeuoJluof,

668

sldl)No) IHI Milntu

'surrodoalf,nu'surrusl -rapun Jerreq reqr d1o>1r1 rEalf,nu aqr eJE draa, sruooslI 'sarde.raql leuon Jo slrunqns PUE cdl{ .9 -ueluof, s,depol 01 tuetsrsord1a3;e1er€ teqt reJuef,;o sad,tr dg pareln8e; seseur>lro CdW Jo setprlsqns u,,vrou>I aerql lsureBedlercadsa 'sar8aterlscrlnede.roqlo^rlraJJaoJourrele lnoqe sn 11aleue8 Laam?qt lo uonez iloJtuor alcdc-11ao .eua8 -rralrererlf,eql u8rsep ot algrssod ag rq8rul lr (sturodlf,or{l pup slonuof, pTp aarn aql sr luqltr leql6 ;ad,fuoueqd alcdc-1eclnoqe poolsrepun era^\ erour tI 'srsoldode elnpur eqt roJ relncllred faam eql Io uorlezrralf,ereqJ pue d.rerr.ocsrp ol e8eruep crleua8 luarrrJJnsreJJnseJuonbosuocE sE pup w 'aqu.tofl'5 tsead uorssrJaql qtr^{ sorpnls srq roJ pazruSo 'lou 'pe.rreder -rar sE-&\ op sllar reJuEJ esoql sr a8eurep lng esrnN InEd rrs 'slsrtuJrJsopdr-y1ecJJrqt ol peprp^te VNC oql .S elldJ IIOoeqt lserr€ leql slurod>1eaqceycdc-11ac ale,r, se-&\ourf,rparuro dSolorsdqdur azr:d eqr ,1697 u1 Itlun IOqoN 'dderaqloruaqcro ddureql uonerppr rltr^\ -nle sllor IpurroN tsrsolru Jelu0 pelnpur a8euep VN(I e^rsuelxe ol elqerouln^ ilrelncrr sllel s3 drlrrpre {dtr trq asrr prder aql ureldxa or dlaq uorleru -rBd s.racuec sad& cryroedso{Errr ueJ slurod>lcor{oosat{l -JoJur sql sooP 1r\oH ;o iSZJPC to elEDsqnsagt sr lErllN'pelE^ .asereqdsoqd Io eluasqe oqt lele^\oH 'addroueqd JeJueJ eql ur llnseJ -nrp sr Szrpl taW dq pareldroqdsoqduaq4y 'sndouay u1 .V leql sluaura8uerreerVNC pue suorletnur eldrrlnru to uorl Szrpl agt sr CdI{ Jo selp4sqns eql to euo -elnlunf,Je oql or pol teql srurodleaqo elcdc-11ef, ur sl)eJap t srsol o^pq ueuo sllel JOJUEJ'sJOf,uEJ tuaruleeJl aql ro; dJrel -IIu lIXa ol Jo IIec e ro; padorlsapoq tsntu g urlodol?qt set?frpur -ncrlred 'saluanbesuoc tuecrlruBrs oAEq IIL& IoJtuoJ af,ueprlolpr{/N ralua ol IIOoE ro; parrnber sr g urIJ isrsolrr.u elcdc-1yeo .sodrqruapur -,{c reqr seleJrpuratuoprle 1o sloadse palrerep aseql Surpuelsrapun letueur:adxa reql6 s33e ur surelord urlcdc yo uol4ezrtelretrr{J pue drezrocsrpaql 'red pelepnnle uaaq tou e^Er1 srsorau ur slq roJ ozrrd aqt perer.{stunH rurl .t {Jo1$, I00Z 1eqo51 I 'llal glpo ur aseqd Suunp sprlpuonlJ Jo uorlp8ar8esanbrun eql arlJepun teql srusluuqf,alu aqr 1{1re1ur5 'dlprr.rleuludse oprrrrp rer{r sllel 5 yo Sururlr aql ol nedsar qlr./r\zg ul 11ece pue tg ur IIel uI pauluJalap eJE soluosoruorr.lf,rolq8nep Jo uorlpzrl€Jol e 3ursnl Jo rleue oqr tlrpord cW izC ltg-dlarnreurerd uorr -rrqdar eqr pue stsauqotdc;o aueld aqr ^\oq lnoqe ureuar suorlsonb VNq arerlrur1p,tralcdc IIoJ er{r;o seseqd3urmol1o; .7, luetr41 'stuslueS.lo relnllorrtFur Jo luarudolaureplururou aql 0r{1 r{rrq^\ ur sllar '11acaseqd-5 up pasnJ ueqlN qlrlr\ Jo ur eloJ IEJnTJJe sdeld uorsrlrp IIaf, JrJleruu,(se teldeqo gau ;dSolens srql serlJepunleql dreurqceur oql ur urpol a,&\sV 'q1ec req8rq ur tulod>loeqcuorlp8ar8es relnf,Olorueqr sr rpqlN iOlqrsrs^errrpuE lEuoDf,OJrprun sr alf, 'lurod4raqr dlqruasse-a1p -euosoruorr{f,or{t ur .l -dc tIlpf, Jo pue 11acaqr q8norqr a8essedlegl sernsuo,tSarerrsteq1tr -urds aqt ul ZpEW;o uorleln8al Jo rusrueqceu pue lonuof, 'pootsrepun aql tnoqe peureelag ol sur?ueJ rlf,nru'esrzr,ra>1r1 slda)uo) eqt /v\a!^eu dl.rood e.relurod>1caqce8uruep-yNq eql ul pue UIV tr trIV elelrtf,E tpql susruerlf,eruaqt lnq 'slurodloeqo alcdc-11ac ;o uorlerado agt rnoqe dpuaoe.rpeJa^oosrpueeq ser{ rlf,nry 'saxelduror yq3-urlclc tsoru ur lrunqns )C3 aW Jo dlrz'rl zgg OseuDl 268 srsoreu -JE osEuDIeql eleln8ar uJnl ur surelord esaqrlpegonluooeJe eursordl-urolo-rd 1eer1tr p;g surltf,i t5 esereqdsoqdSZIpO pue oseur>llaalN Jo senrlnte ar{r ^ orl 769 srsdeuds 788 stopel JTg lnoge pauJeeleq or suretuarqcny,q'elpurdsf,rlolru In1rlneaq rJ:8ulrnf,as xoq uorlf,nnsaP aqr;o dlqruessp ur sllnseJ selnqnlorf,rru uorlBzruB8ro reqt 8t8 Jo -ar Olq?>lJsureJ eql PUEuorl?suePuoJOruosoruoJr{J ol speal rls (Jds) 8t8 (s)af,) rolceJ Surtouro.rd-eseqd-5 surelo.rd eseql Jo uorlef,rJrpou oqr .^aoqpuelsrepun ol ouop sascurl luepuadap-uqcdt eg ol sureuar >lro1!ar{rnl/r{ 'ssxolduloc yq3-urlcdo crlotrur 918 rorrqrquraseqd-5 Z6g rato Sulsso.rr dq pareldroqdsoqd sale.nsqnsSurd;uuapr ur dpuaror apetu 9yg surltf,i aseqd-5 998 ursuepuol ueeq seq ssarSo.rdI€rluelsqnsterlrea passnrsrpsV 'peururel 918 tJS 598 surseqol -ap eq 01 urpruer sluauoduoc raqlo 'uorlecqdo.ry51q ererr ;gg rurod uorlJrrlser ts8 su:) -rur ol sexalduol )CJ-urlcdr aseqd-5dq parel,fuoqdsoqdaq 288 ularord qU g2g srurod>paqr lsnu lur{l xelduroc uorlecqdarard eqr;o sluauoduroc euros 3^Er{srerlf,rsaserqSnoqrle 'acuElsurrod 'pere^of, ,88 slloJ luacsarnb asereqdsoqd PaIJIluePr 799 97cp3 -slP eq ol uletuar sllefeP suaes J59 uraro:d ggd ggg asereqdsoqd IEJrluJ dueur'paqsqqptsalle,/$. y1cp3 .1\.ouuorleln8ar epdo-1ac;o rr3o1 prauo8 eqr q8noqryy 999 uqrdc Jrlolnu 8t8 repro radord eql ur rnf,fo alodcoqr yo sasegdeqt terll sJJns .rolce;dlrcr;rceds3734y rs8({drr) 'e1cdc -ue d8etens srql er{l Jo tue^a xau aql roJ IIel eqt eted rolre; Surlourord-srsolrur 0S8(l/ldv)xalduroc -erd ol pue elclc eqr;o dets e ete^n)v ol :suonf,un' tupl Surlourord-eseqdeue -rodur o.ry$seq IIaJ droleln8ar 088 suaSolur qceg 'sloJluot relnf,elotu lue,re esegt sarlrepun rr3o1 InJrln€eqV 'Vt-02 arn8rg ur peurllno suJal ^e) Iorluor apdc-1eo orlodrrlna to Iepotu aqt or pal seq sread S/ rsel eqt ro^o qJrpesaralcdc-1er;o aced elqe>lreuraroql 'ernlnJ eql ur slueulearl o^rtf,aJJo eJotu ;o u8rsep I aqr ^{olle IIrl\4.pe^lo^u sessacordrelnrelour aql;o Surpuets

t'l)A)

ol pesn eq uer Xlgruesse elnqnlorlnu slue,ra.rdreqr Snrp e 'alozepocog 'g urlc^,toaper8ap tou op pue eseqdeueral -uo lou op pale^rpe uoag spq rurodlcaqr srqt qlnl^\ ur sllof, 'snq1 'aseqdeueolur Surpeacordruo{ saroqroraul{ per{rel 'J -leun qlr.ry\sllar slue^eJd rurodloaqc olpurds aq1

rr-srr+,8ffl3**' *.s#

,,r;:,,,;;;;ili&.*'Se _

,.

*

,,:,..{if"1!!i|

l luoc !tc^5

0e0z9rzr0r8

9

r

0

0e 0Z 9t Zt 0t

uorlrppe/lux

I

I

t

0

( u r u )a u r l

uolllppe oN

_

'l'll)

)rro^uv)nl

3Hr gNlLv'ln9lu |

0z ulrdvH)

006

'urlodouour 'greg (q) (c) '7aru1(e) :8urmo11oy agl ureld oql Jo uollf,un1cr;nads-srsotaur -xfl 'slua^e uorsr^rp-llar esaql to L{rpeuI dlenbrun uollf,unJ suralord atuos 'Jalamo11'sutalo.rdorueseql;o dueur 8ur,t1o,t -ur sassalo.rdsnoSoleur IIEJa o oJEslsolltu pue sIsoIaW '/I iluIoo>lraqr E tE tsorre alcdc-11acafroJue ot s)eJ Io uollelrlleur ol peel elErlsgns slqr Jo uoDel,troqdsoqd eqr saop ,ry\oH 'ggd (WIV dq peleldroqdsoqd sI elertsqns raqro reqzt erxeto: WIV) eua8111y seprsefl'(pa1e1nw-etselcar8uela7 ar{l ur uorlplnru uollf,unJ-Jo-ssole st etselcet8uelal EIxEIE roJ srseg rrtaua8 eql 'reJuer Jo af,uaPlrul Peseerrul Pue 'dcuarcr;apounrurur'uotlereue8epornautuoJt JeJJnserselJel -3ue1a1elxale reproslp dretrpa.raqaql qrr^\ s1EnPI^IPUI'9I la8eurep VNCI qllA\ sllel ro; Sunerpetuur deld ggd saop elor 13I{lN 'SI tsar.realcdc-11ar

;(e) rred tuorJ paul€lgo leqr puodaq PPE selpnrs osaql uollPluroJul leq^. eulluroleP 'ern8g 3ur.uo11o;oql Sursll iuorteurtrnbrqn Surunuexe ro1 aleuorler eqr sr rer{l1 '(sole8nluor qn-uqr,,{c)uoueunrnbrqn g uqcdr Jot osealeJJOI;EserurlsnorJel lE pessesse ueql eJel\ 'uorlrppe pltxo oql'lprurou *rtJ f,g tserreuro{ paseoler'stf, ueqf .raq8rqsp,ry\slf,Ertxe eql ur uralord srqf ;o tunolu€ Iel -ot eqt tegl os 'eseqdelau lE polserr? 'slcB.rtxa33a snQouay 'q ot peppe se.l.r/JuX snouaSoxa'sarpnlsleuorrrppeuI

*

oeo, ; ; ;;

paFtoap_1lux

; Tffi::r^

iI,JUX roJ uonJunJ alqrssod E lnoqu aprlord elep eseqt op uou -prurolur reqr1 '1(oleqtolq uralsolN arlt uo u^\oqs sE (pJurur -ralaPg urlcdr slunoruPeqr 1o PUEuorlrPPe+zPJ rette seurl snorrpl re paldues uogt orol\^.lrerlxe aqr Jo sronbllV '*.e3 '(dpogrl Jo uorlrppe lq lsarre aseqdelarutuort peseelaruaqr -ue eldures tsrrJ eqt sE tueruteorl erues eql lnoqlr^\ rnq L1luy* or paroalqns)paraldap->1coru oro^\ to LNX;o peraldapraqrra ararr'aseqdeloruur palsaJJe'slcenxa33a sndouay 'e .\IL_I9169I 'tolg ''p to oltelese) 'g '[ ees)pessasse ueql ila)'['SO0Z are^\ srsolrruq8norqr lrsuerl uo sacuanbasuoJoql 'LJuXel; -xe Jo uonrppe q8no.rqrstrerlxe eql ur petuatu8neuaaq seq ro 'lr lsure8epasrerdpoqnlupue Sursnslcerlxo ruory polaldep ueoq sEr{reqrre/Jux r{f,rr{.ry\ ur ue>l?lrepunueeq e^eq serPnls 'uretord sqt '(3734y) auos Jo uonf,unJ orll aleprlnla o1 -olcdrTxalduol Surtourord-aseqdeueaqr ot spurq uretord srgl 's33a snf,ouay Jo slrerlxe ur perJrluepruaaq seq '11uy 'urotord ,vrau E fi.llueea1 'pezrrelrereqr ueaq azreq alcdc ilel aqr q8norqr trsupn elelnSor leql surelo.rdeqr ;o .{uey,q

elec aql azfleuv

;eruoua8aqr;o drqaptyeqr a,rrasa.rdor dleq slurod4ceqc elcdr-1ec op 1v\oH;o1odc11ac e er{l ur JnJro sturod4oaqcop ereql1 lrurod>1ceqcalcdr-11ac sr reqlN 'aptstnan2'5 lsrad Surppnq aql ur slurod4ceqcalcdc 'azr.rd -ller Jo uonezr.ret)eteqJ srg ro; pe8pelmou>1ceselt '[o^tl"H 'tI PuEIoe] IaqoN I00Z eql;o tuardrcarPrII{t eqt seseqdS ar{ror paaco.tdol IIel oql 'elerq lroll€ ot rg arel ol Pru uI Pasralaro>lerqaqi sI ^ oH p sp slf,e utelord qU eqr ^{oq eqlrJseq,,'e1cdc11ac ayrdc-11ao aqt eTprq ralsplu,, egr palleJ ueaq seg urelord qU eq1 '€I Jo 'un[-,r pue sod-,rSurporua sosnJl^orlardq uorfcalur (c) 'q urlcdo;o uotssardxere,lo(e) :s1ort 'uorlrun; qU Jo ssol (q) -uoo turod-uoltrlrlsar ;o sseddqE or PEal's11ac .racueoauros ur punoJ oJE L{f,II{^/r'suotlelnur 8ur.tro11o;ogl ^\oq ureldxg 'slorluof, tutod-uotlctrlseJ asol dgecrd,tf sllel raluE3 lrulod uorlrrDser eqr Jo uorlrulJoP lEuollrunt eqr sI rEI{lN 'zI iolJ^l IIal rod of,uo dpo pue acuo polecrlder sr eurouo8eJllua aql lEI{l ernsuool deld sexalduror)CJ-uIIldc eseqd-5op elor 1er1n''aotstnatac 'g u1 'salodrelna ut sut8rroaldrtpru tuory selelllut uotleotlda.r 'II y51q 'rauueru dlaurn e ur paraldruol oq ol aseqd 5 rod 'soxaldruocyq3-utlcdo rrlotrur pue eseqd-5to uolle^Ilf,E aql olourord syq3-uqcdc -prl'u saIlI^ItJpaql qlrg^\ ut sde,vr, IC-elel eargl etueN puE Jo 'W pue (zC (S q8norqt uotsso.rSord az,llew sexalduror;,1q3 -urpdc ad,{1-galdrrlnu PuE (IC q8norqr uorssarSordezdle -ler s)C3-ur1c.b rg-ere1 PuP -plur 'aztstnata)'5 u1 'aseqd tuenbasqnse olul uotssarSordaJnsueaseqd auo to slua^a st uotleln8ar alldc-11acJo ornteal uotuuloJ V '0I eqr r?qr 'Wd (p) 'Ie"A\ (r) ')Vl (q) 'urlcfc (r) :surotord8ur -./r\ollo;aqi dq pereFpour sr dtrrrrlce)Cf, n\oq ureydxg '6 lssaoo.rdsrqr uI dIC.uEU PUECgC-uEU Jo selor eqr are ter{lN 'relrnu 1ac-rarq8nepturot ol esnt ueql lBI{l sareuo -d.re1 u.ro1 ot aluosotuorl{t I{reo punore esnJ Ug 0gl uro{ '8 suorsuetxe adolauruaJeelfnu 'aseqdolal Jo pue eql rV 'aseqdeuele spltetuorqc relsls uorle.redesJrll seloru 1o -ord 3734y ol{r qrlq.^adq slueaeJo selrasaql egIrJSeC 'L 'SISOlIUI

qSnorqr uotssa.rSordPuE uollJun' sll speu€ sutelord esaql 'ursuaPuof, Jo qreo ;o uoueldroqdsoqd eq] .4oq eqlrf,seq

activate the spindle checkpoint. Cells in nocodazole become arrestedin early mitosis becausethey cannot form a spindle, and thus all kinetochoresremain unattached.To determineif XnfT is required for a functional spindle checkpoint, Xenopus egg extracts, arrested in metaphase,were subjectedto various protocols (see the following figure): untreated (-nocodazole), or treated with nocodazoleand either mockdepleted (preimmune) or immuno-depleted of XnfT (ctXnfT). The extracts were then treated with Ca2* to overcome arrest, and aliquots of the extracts were assessedat various times for cyclin B, as shown on the Nfesternblot beIow. \fhat can you conclude about XnfT from thesedata?

Time(min) 0

e* Preimmune

10

20

*a....it

{q{t*

q*'

**,a

30

40

W

50

w

60

ry

Roux, K. J., and B. Burke. 2006. From pore to kinetochoreand back: regulatingenvelopeassembly.Deu. Cell ll:276-278. a universaltrigger for sister \7irth, K. G., et al. 2005. Separase: chromatid disiunction but not chromosomecycle progression./. Cell Biol. 172:847-860. Yanagida,M. 2005. Basicmechanismof eukaryotic chromosome segregation. Phil. Trans.R. Soc.Lond. B Biol, Sci. 360:609-621. Cyclin-CDK and Ubiquitin-Protein Ligase Control of S-phase Bell, S. P.,and A. Dutta. 2002. DNA replication in eukaryotic cells.Ann. Reu.Biochem. 7l:333-37 4. Deshaies,R. J. 1999. SCF and Cullin/Ring H2-basedubiquitin ligases.Ann. Reu.Cell Deuel.Biol. 15:435467. Diffley, J. F.2004. Regulationof earlyeventsin chromosome replication.Curr. Biol 14:R77 8-R7 86. Nakayama, K. L, and K. Nakayama. 2005. Regulationof the cell cycle by SCF-type ubiquitin ligases.Semin. Cell Deu. Biol. 16:323-333. Reed,S. l. 2006. The ubiquitin-proteasomepathway in cell cycle control. ResubsProbl. Cell Dffer. 42:147-L81. Cell-CycleControl in Mammalian Cells

o-Xnfl

rx.*

References Overview of the Cell Cycle and lts Control Morgan, D. O. 2005. The Cell Cycle:Principlesof Control. New SciencePress. Nasmyth, K. 2001. A prize for proliferation. Cell 107:689-701,. Control of Mitosis by Cyclins and MPF Activity Doree, M., and T. Hunt. 2002.From Cdc2 to Cdkl: when did the cell cycle kinaseioin its cyclin partner?J. Cell Sci. Il5:246L-2464. Masui, Y. 2001. From oocytematuration to the in vitro cell cycle: the history of discoveriesof Maturation-PromotingFactor (MPF) and CytostaticFactor (CSF).Differentiation 69:1-t7. Cyclin-Dependent Kinase Regulation During Mitosis Nurse, P.2002. Cyclin dependentkinasesand cell cyclecontrol (Nobel lecture\.Chembiochem. 3 :596-603. Molecular Mechanisms for Regulating Mitotic Events Hirano, T. 2005. Condensins:organizingand segregatingthe genome.Cut Biol. 15:R255-R275. Kline-Smith,S. L., S. Sandall,and A. Desai.2005. Kinetochorespindlemicrotubule interactionsduring mitosis. Curr. Opin. Cell Biol.77:3546. Meyer, H. H. 2005. Golgi reassemblyafter mitosis:the AAA family meetsthe ubiquitin iamrly.Biochim. Biophys. Acta 1744:t08-11.9. Nasmyth, K., and C. H. Haering. 2005. The structureand function of SMC and kleisin complexes.Ann. Reu.Biochem. 74:595-648. Nigg, E. A. 2001. Mitotic kinasesas regulatorsof cell division and its checkpoints.Nature Reu.Mol. Cell Biol.2:21-32. Peters,J. M. 2006. The anaphasepromoting complex/cyclosome:a machinedesignedto destroy.Nature Reu.Mol. Cell Biol. 7:644-656.

Barr, F. A. 2004. Golgi inheritance:shakenbut not stirred./. Cell Biol. t64:95 5-9 58. DePamphilis,M. L., et aI.2006. Regulatingthe licensingof DNA replication origins in metazoa.Curr. Opin. Cell Biol. 18:231-239. Ekholm, S. V., and S. L Reed.2000. Regulationof G(1) cyclindependentkinasesin the mammalian cell cycle.Curr. Opin. Cell Biol. t2:675-684. Machida, Y.J., J. L. Hamlin, and A. Dutta. 2005. Right place, right time, and only once: replication initiation in metazoans.Cell 123:1.3-24. Porter,L. A., and D. J. Donoghue.2003. Cyclin 81 and CDKI: nuclearlocalizationand upstreamregulators.Prog. Cell Cycle Res. 5:335-347. Sears,R. C., and J. R. Nevins. 2002. Signalingnetworks that link cell proliferation and cell fate.J. Biol. Chem. 277:11.617-1.1.620. Sherr,C. J.2001,.The INK4a/ARF network in tumour suppression.Nature Reu.Mol. Cell Biol. 2:731.-737. Checkpoints in Cell-CycleRegulation Bartek,J., C. Lukas, and J. Lukas. 2004' Checkingon DNA damagein S phase.Nature Reu.Mol. Cell Biol. 5:792-804. Cheeseman,I. M., and A. Desai.2004' Cell division: feeling tenseenough?Nature 428:32-33. Gottifredi, V., and C. Prives.2005. The S phasecheckpoint: when the crowd meets at the fork. Semin. Cell Deu. Biol16:355-368. Hartwell, L. H. 2002. Yeast and cancer (Nobel lecture). BiosciRep.22:373-394. Kastan,M. B., and J. Bartek. 2004' Cell-cyclecheckpointsand cancer.Natur e 432:316-323. Kitagawa, R., and M. B. Kastan.2005' The AlM-dependent DNA damage signaling pathway. Cold Spring Harbor Symp' Quant. Biol. TO:99-I09. Lambert, S., and A. M. Carr. 2005. Checkpointresponsesto replication fork barriers.Biochimie 87:59I-602. Lew, D. J., and D. J. Burke. 2003. The spindleassemblyand spindleposition checkpoints.Ann. Reu' Genet.37:25l-282. McGowan, C. H., and P. Russell'2004.The DNA damage response:sensingand signaling.Curr. Opin. Cell Biol. 16:629-633. Nasmyth, K. 2005. How do so few control so many? Cell 120:739-746. REFERENCES

901

Pereira,G., and E. Schiebel.2001. The role of the yeastspindle pole body and the mammalian centrosome in regulating late mitotic events.Curr. Opin. Cell Biol. 13:762-769. Seshan,A., and A. Amon. 2004. Linked for life: temporal and spatial coordination of late mitotic events.Curr. Opin. Celt Biol. 16:4148. Stark, G. R., and !7. R. Taylor. 2006. Control of the G2lM transition. Mol. Biotechnol. 32:227-248. Stegmeier, F., and A. Amon. 2004. Closing mitosis: the functions of the Cdc14 phosphataseand its regulation.Ann. Reu.Genet. 38:203-232. Takeda,D. Y., and A. Dutta. 2005. DNA replication and progression through S phase. Oncogene 24:2827-2843.

902

C H A P T E R2 0

|

R E G U L A T T NTGH E E U K A R Y O T TCCE L I

Uhlmann, F. 2003. Separaseregulation during mitosis. Biochem, Soc. Symp. 7 02243-251.. Meiosis: A Special Type of Cell Division Marston, A. L., and A. Amon. 2004. Meiosis:cell-cyclecontrols shuffle and deal. Nature Reu. Mol. Cell Biol. 5:983-997. Petronczki,M., M. F. Siomos,and K. Nasmyth. 2003. Un m6nage a quatre: the molecular biology of chromosome segregationin meiosis.Cell 112:423440. 'Watanabe, Y.2004. Modifying sisterchromatid cohesionfor meiosis.I. Cell Sci. 117:401,74023.

cLASStC

EXPERIMENT

2O

FROMTHESEA:THEDISCOVERY CELLBIOLOGYEMERGING OF CYCLINS T. Evanset al., 1983,Ce//33:391.

From the first cell divisions after fertilization to aberrant divisions that occur in cancers, biologists have long been interested in how cells control when they divide. The processesof cell division have been separated into stages known collectively as the cell cycle. While studying early development in marine invertebrates in the early 1980s, Joan Ruderman and Tim Hunt discovered the cyclins, key regulators of the cell cycle.

tein synthesis from the maternal mRNA is required at the earlieststages of development.Ruderman and Hunt, while teaching a physiology course at the Marine Biological Lab in'Woods Hole, Massachusetts,began a set of experiments designed to uncover the genesthat were expressedat this point as well as the mechanismby which this burst of protein synthesis was controlled.

The Experiment Background The question of how an organism develops from a fertilized egg continues to drive a large body of scientific research. \(hereas such research was classically the concern of embryologists, the developing understanding of gene expressionin the 1980s brought new approaches to answer this question. One such approach was to examine the pattern of gene expression in both the oocyte and the newly fertilized egg. Ruderman and Hunt were among the biologists who took this approach to the study of early development. Biologists had well characterized the early development of a number of marine invertebrate systems. During the early stages of development, the embryonic cells grow synchronously, which allows an entire population of cells to be studied at the same stageof the cell cycle. Researchershad established that a large portion of the mRNA in the unfertilized oocyte is not translated. Upon fertilization, these maternal mRNA are rapidly translated. Previous studieshad shown that when fertilized eggs are treated with drugs that inhibit protein synthesis, cell division could not take place. This suggestedthat the initial burst of pro-

In a collaborative project, Ruderman and Hunt looked at regulation of gene expression in the fertilized egg of the surf clam Spisulasolidissima.Ifhereas it was known that overall protein synthesis rapidly increasedupon fertilization, they wanted to find out whether the proteins expressedin the earliest stageof development,the two-cell embryo, were different from those expressedin the unfertilized egg. $7hen either oocytes or two-cell clam embryos are treated with radioactively labeled amino acids,the cell takes up the amino acids, which are subsequently incorporated into newly synthesized proteins. Using this technique, Ruderman and Hunt monitored the Pattern of protein synthesisby breaking open the cells,separatingthe proteinsusing SDS-polyacrylamidegel electrophoresis (SDS-PAGE),and then visualizing the radioactively labeled proteins by autoradiography. \fhen they compared the pattern of protein synthesis in the oocyte with that in the two-cell embryo, they saw that three different proteins that were either not expressed or expressedat an extremely low level in the oocyte were highly expressedin the embryo. In a subsequentstudy,Ruderman examined the pattern of protein expression in the oocytes of the

starfish Asterias forbesi as they mature. She again observedthe increased expressionof three proteins of similar size to those that she and Hunt had seenin surf clam embryos. Soon afterward, in a third studS Hunt examined the changesin protein expressionduring the maturation and fertilization of sea urchin oocytes. This time he performed the experiment in a slightly different manner. Rather than treating the oocytes and embryo with radioactively labeled amino acids for a set time period, he labeled the cells continuously for more than 2 hours, removing samples for analysis at 10minute intervals. Now, he could monitor the changes in protein expression throughout the early stagesof development. As had been shown in other organisms,the pattern of protein synthesis was altered when the sea urchin oocyte was fertilized. Three proteinsrepresentedby three prominent bands exon an autoradiograph-were not in the but embryos, pressedin the intensity of the Interestinglr oocytes. over time; changed bands one of these the band was intense at the early time points, then barely visible after 85 minutes. It increasedin intensity again between 95 and 105 minutes. The intensity of the band, representing the amount of the protein in the cell, appearedto be oscillating over time. This suggestedthat the protein had been quickly degradedand then synthesized agarn, Becausethe time frame of the experiment coincided with early embryonic cell divisions, Hunt next asked whether the synthesis and destruction of the protein was correlated with progressionof the cell cycle. He examined a portion of cells from each time point under a microscope, counting the number of cells dividing at each time

OF CYCLINS C E L LB I O L O G YE M E R G I N GF R O M T H E S E A :T H E D I S C O V E R Y

903

< FIGURE 1 Thisfigure comparesthe changinglevelsof sea urchincyclin(drawn in blue)with a control protein (drawn in purple)as early embryoniccellsprogressthrough the cell cycle.Theoverall levelof cyclinincreases overtime,andthenit is rapidly destroyed asthe cellsapproach Thispatternappears division. to repeatthrougheachcelldivisionMeanwhile, the overall levelof thecontrolproteincontinues to increase throughout thetimeperiod of the experiment[Adapted fromI Evans et al, 1983,Ce//33:391 ]

x o .;c 5 U

0)

2 hrs point where samples had been taken for protein analysis. Hunt then correlated the amounr of the protein present in the cell with the proporrion of cells dividing at each time point. He noticed that the level of expression of one of the proteins was highest before the cell divided and lowest upon cell division (seeFigure 1), suggestinga correlation with the stage of the cell cycle. \flhen the sameexperiment was performed in the surf clam, Hunt saw that two of the proteins that he and Ruderman had describedpreviously displayedthe same pattern of synthesisand destruction. Hunt called theseproteins cyclins to reflect their changing expression through the cell cycle.

904

C H A P T E R2 0

|

Discussion The discovery of the cyclins heralded an explosion of investigation into the cell cycle. It is now known that these proteins regulate the cell cycle by associating with cyclin-dependentkinases, which in turn regulate the activities of a variety of transcription and replication factors, as well as other proteins involved in the complex alterations in cell architecture and chromosome structure that occur during mitosis. In brief, cyclin-CDK complexes direct and regulate through the cell cycle. As with so many key regulators of cellular functions, it was soon shown that the cyclins discovered in sea urchins and surf clams are conservedin eukaryotes

R E G U L A T T NTGH E E U K A R Y O T TC CE L LC Y C L E

from yeastto man. Sincethe identification of the first cyclins, scientistshave identified at least 15 other cyclins that regulate all phasesof the cell cycle. In addition to the basicresearchinterest in these proteins, the cyclins' central role in cell division has made them a focal point in cancer research. Cyclins are involved in the regulation of severalgenesthat are known to play prominent roles in tumor development. Scientists have shown that at least one cyclin, cyclin D1, is overexpressed in a number of tumors. The role of these proteins in both normal and aberrantcell division continuesto be an active and exciting area of researchtoday.

CHAPTER

CELLBIRTH, AND LINEAGE, DEATH

All nucleiare cerebellum. Cellsbeingborn in the developing labeledin red;the greencellsaredividingand migratinginto internallayersof the neuraltissue [Courtesyof TalRaveh, MatthewScott,and JaneJohnsonl

uring the evolution of multicellular organisms, new mechanismsaroseto diversify cell types,to coordinate their production, to regulate their size and number, to organize them into functioning tissues,and to eliminate extraneous or aged cells. Signaling befween cells becameeven more important than it was for single-celledorganisms.The mode of reproduction also changed,with some cells becoming specializedas germ cells (e.g., eggs, sperm), which give rise to new organisms, as distinct from all other body cells, called somatic cells. Under normal conditions somatic cells will never be part of a new individual. The formation of working tissues and organs during development of multicellular organisms dependsin part on specific patterns of mitotic cell division. A series of such cell divisions akin to a family tree is called a cell lineage. A cell lineage traces the birth order of cells, the progressive restriction of their developmental potential, and their differentiation into specialized cell types (Figure 21.-1.).CeIl lineagesare controlled by cell-intrinsic (internal) factorscells acting according to their history and internal regulatorsas well as by cell-extrinsic (external) factors such as cell-cell signals and environmental inputs. A cell lineage begins with stem cells, unspecializedcells that can potentially reproduce themselvesand generate more-specialized cells indefinitely. Their name comes from the image of a plant stem, which grows upward, continuing to form more stem, while sending off leaves and branches to the side. A cell lineage ultimately culminates in formation of terminally differentiated cells such as skin cells, neurons, or

muscle cells. Terminal differentiation generally is irreversible, and the resulting highly specializedcells often cannot divide; they survive, carry out their functions for

cells is more limited than that of the stem cells from which they arise. (Although some researchersdistinguish between precursor and progenitor cells' we will use these terms interchangeably.) Once a new precursor cell type is created, it often produces transcription factors characteristic of its fate. These transcription factors coordinately activate' or repress'

OUTLIN E The Birth of Cells:Stem Cells,Niches, and Lineage

906

21.2

Cell-TypeSpecificationin Yeast

921

21.3

Specificationand Differentiationof Muscle

924

21.4

Regulationof AsymmetricCell Division

930

21.5

Cell Death and lts Regulation

935

21.1

90s

The Birthof Cells:StemCells, Niches, and Lineage (al

Cellgtov,tth/

@

Cell death

\

@ Gell lineage

A FIGURE 21-1 Overviewof the binh, lineage,and deathof cells.Following growth,cellsare"born" asthe resultof symmetric or asymmetric celldivision(a)Thetwo daughter cellsresulting from symmetric division areessentially identical to eachotherandto the parental cell Suchdaughter cellssubsequently canhavedifferent fatesif theyareexposed to different signals. Thetwo daughter cells resulting fromasymmetric division differfrombirthandconsequently havedifferent fatesAsymmetric division commonly is preceded by the localization (greendots)in onepartof of regulatory molecules the parentcell (b)A series of symmetric and/orasymmetric cell divisions, calleda celllineage, givesbirthto eachof the specialized celltypesfoundin a multicellular organism. Thepatternof cell lineage canbe undertightgenetic controlprogrammed celldeath occurs duringnormaldevelopment (eg , in thewebbingthatinitially develops whenfingersgrow)andalsoin response to infection or poison. A series programmed of specific events, calledapoptosis, is activated in thesesituations batteriesof genesthat direct the differentiation process.For instance,a few key regulatory transcription factors createthe different mating types of budding yeastand similarly, a small number of such factors produced in sequencetrigger the steps in forming differentiated muscle cells from precursors. 'We discussboth theseexamplesin this chapter. Typically we think of cell fates in terms of the differentiated cell types that are formed. A quite different cell fate, programmed cell death, also is absolutely crucial in the formation and maintenanceof many tissues.A precisegenetic regulatory system, with checks and balances,controls cell death just as other geneticprograms control cell differentiation. In this chapter,then, we considerthe life cycle of cellstheir birth, their patterns of division (lineage),and their death. These aspectsof cell biology convergewith developmental biology and are among the most imporrant processes regulated by the signaling pathways discussedin earlier chapters.

905

C H A P T E R2 1

I

C E L LB I R T H ,L I N E A G EA , ND DEATH

Many descriptions of cell division imply that the parental cell gives rise to two daughter cells that look and behaveexactly like the parental cell: that is, cell division is symmetric, and the progeny retain the same properties as the parental cell. But if this were always the case,none of the hundreds of differentiatedcell types presentin complex organismswould ever be formed. Differencesamong cells can arise when two initially identical daughter cells diverge upon receiving distinct developmentalor environmental signals.Alternatively, the two daughter cellsmay differ from "birth," with each inheriting different parts of the parental cell (seeFigure 21-1). Daughter cells produced by such asymmetric cell division may differ in size,shape, andlor composition, or their genes may be in different statesof activity or potential activity. The differencesin these internal signalsconfer different fates on the two cells. Here we discusssome generalfeaturesof how different cell types are generated,culminating with the best-understood complex cell lineage, that of the nematode Caenorhabditis elegans.In later sections,we focus on examples of the molecular mechanisms that determine particular cell types in yeast,Drosophila, and mammals.

Stem CellsGive Riseto Both Stem Cellsand D i f f e r e n t i a t i n gC e l l s Stem cells, which give rise to the specializedcells composing the tissues of the body, exhibit several patterns of cell division (Figure 21.-2).A stem cell may divide symmetrically (a) Maintainstem-cell population @

@

-\

-\

@ @ @ g

@

@--

-t-

-l

@ O @ 9==Diff","ntiatedcerl

(b) Increasestem-cells

@@@ -\

-]-

Stemcetl

-1-

@ @ @ @@ o

(c) Increasedifferentiatingcells

@

@@@@

-\

-q

'-\

.._1-

@o @@og @@@0

FIGURE 21-2 Patternsof stem-celldivision.Divisions of stem cells(yellow) mustmaintain population, thestem-cell sometimes increase the numberof stemcells,andat the righttimeproduce differentiating cells(green)(a)Stemcellsthatundergoasymmetric produce divisions onestemcellandonedifferentiating cell Thisdoes not increase the population of stemcells(b)Somestemcellsin a population maydividesymmetrically to increase theirpopulation, whichmaybe usefulin normaldevelopment or duringrecovery from injury, whileat the sametimeothersaredividing asymmetrically asin (a) (c)In a thirdpattern, somestemcellsmaydivideasin (b),while at the sametimeothersproduce two differentiating progeny. fromS Morrison rt41:1068 andJ Kimble, [Adapted 2006,Nature I

to yield two daughter stem cells identical to itself. Alternatively, a stem cell may divide asymmetrically to generate a copy of itself and a derivative stem cell that has morerestrictedcapabilities,such as dividing for a limited period of time or giving rise to fewer types of progeny compared with the parental stem cell. A pluripotenl (or multipotent) stem cell has the capability of generating a number of different cell types, but not all. For instance, a pluripotent blood stem cell will form more of itself plus multiple types of blood cells,but never a skin cell. In contrast,a unipotent stem cell dividesto form a copy of itself plus a cell that can form only one cell type. For example stem cells in the intestinecontinuously reproducethemselves,while the other daughtercell differentiatesinto an intestinalepithelialcell, as we discussin greaterdetail below In many cases,asymmetric division of a stem cell generatesa progenitor cell, which embarks on a path of differentiation, or even a terminally differentiating cell. The two critical properties of stem cells that together distinguish them from all other cells are the ability to reproduce themselvesindefinitely, often called self-renewal, and the ability to divide asymmetrically to form one daughter stem cell identical to itself and one daughter cell of more restrictedpotential. Many stem-celldivisions are symmetric, producing two stem cells, but at some point some progeny need to differentiate. In this way, mitotic division of stem cells can either enlargea population of undifferentiated cells or maintain a stem-cell population while steadily producing a stream of differentiating cells. Although some types of precursorcellscan divide symmetrically to form more of themselves,they do so only for limited periods of time. Moreover, in contrast to stem cells,if a precursor cell divides asymmetrically,it generatestwo distinct daughter cells, neither of which is identical to the parental precursorcell. The fertilized egg, or zygote,is the ultimate totipotent cell becauseit has the capability to generateall the cell types of the body. Although not technicallya stem cell because it is not self-renewing,the zygote does give rise to cells with stem-cell properties. For example, the early mouse embryo passesthrough an eight-cellstagein which each cell can form every tissue;that is, they are totipotent. Thus the subdivision of body parts and tissuefates among the early embryonic cells has not irreversibly occurred at the eight-cellstage.At the 15-cell stage,this is no longer true; some of the cells are committed to particular differentiation paths.

Restricted Cell FatesAre Progressively D u r i n gD e v e l o p m e n t The eight cells resulting from the first three divisions of a mammalian zygote (fertilized egg) all look the same' As demonstratedexperimentally in sheep,each of the cells has the potential to give rise to a completeanimal. Additional divisions produce a mass, composed of =64 cells, that sepa-

ECTODERM Centralnervoussystem Retinaand lens Cranialand sensory Gangliaand nerves Pigmentcells HJad connectivetissue Epidermis Hair M a m m a r yg l a n d s

,ilIs

!

Skull Head,skeletalmuscle Skeleton Dermisof skin Connectivetissue Urogenitalsystem Heart B l o o d ,l y m p h c e l l s Spleen

ENDODERM Stomach Colon Liver Pancreas Urinary bladder Epithelialpartsof trachea lungs Pharynx thyroid intestine

21-3 Fatesof the germ layersin animals.Someof A FIGURE arelisted of thethreegermlayers thetissuederivatives

rates into two cell types: trophectoderm' which will form extra-embryonic tissueslike the placenta, and the inner cell mass, which gives rise to the embryo proper' The inner cell mass eventually forms three germ layers, each with distinct fates. One layer,the ectoderm,will make neural and epidermal cells: another. the mesoderm,will make muscle and connective tissue; the third layer' the endoderm, will make gut epithelia (Figure21-3). Once the three germ layers are established,they subsequently divide into cell populations with different fates' For instance,the ectoderm becomesdivided into those cells that are precursorsto the skin epithelium and those that are precursors to the nervous system. There appears to be a progressive restriction in the range of cell types that can be formed from stem cells and precursor cells as development proceeds.An early embryonic stem cell' as we've seen' can lo.- .u.ty type of cell, an ectodermal cell has a choice between neural and epidermal fates, while a keratinocyte precursor can form skin but not neurons. Another restriction that occurs early in animal development is the setting aside of cells that will form the germ line-the stem cells and precursor cells that eventually will

is widespread(though not universal) among animals' In contrast, plants do nothing of the sort; meristems,growing tips of ,ooi, and shoots,can often give rise to germ-line cells and there is no germ-line lineageset aside early' One consequenceof the early segregationof germ-line cells is that the loss or rearrangement of genesin somatic cells will not affect the inherited genome of a future zygote' Nonetheless,although segmentsof the genomeare rearranged and lost during developmentof lymphocytesfrom hematopoietic precursors, most somatic cells seem to have an intact genome, equivalent to that in the germ line (Chapter 24)' Evidence that at least some somatic cells have a complete and functional genomecomesfrom the successfulproduction

, ND LINEAGE , I C H E SA : T E M C E L L SN T H E B I R T HO F C E L L SS

907

of cloned animals by nuclear-transfercloning. In this procedure, the nucleus of an adult (somatic) cell is introduced into an egg that lacks its nucleus; the manipulated egg, which contains the diploid number of chromosomes and is equivalentro a zygote,thenis implanted into a foster mother. The only sourceof geneticinformation to guide development of the embryo is the nuclear genome of the donor somatic cell. The frequent failure of such cloning experiments, however, raises questions about how many adult somatic cells do in fact have a complete functional genome.Even the successes,like the famous cloned sheep ,,Doll%', often have medical problems. The extent to which diffeientiated cells harbor fully functional genomesis still not fully understood. A cell could, for example, have an intact genome,but be unable to properly reactivate certain genes due to inherited chromatin states. These observationsraise two important questions:How are cell fates progressively restricted during development? Are these restrictions irreversible?In addressingthese questions, it is important to remember that a cell's capabilitiesin its normal in vivo location may differ from what it is capable of doing if manipulated experimentally. Thus the observed limits to what a cell can do may result from natural regulatory mechanismsor may reflect a failure to find conditions that reveal the cell's full potential. Although our focus in this chapter is on how cells become different, their ability to r.-"in the samealso is critical to the functioning of tissuesand the whole organism. Non-dividing differentiated cells with particular characteristics often retain these features for many decades.Stem cells that divide regularly, such as a skin stem cell, must produce one daughter cell with the properties of the parental cell, retaining its characterisric composition, shape,behavior,and responsesto specificexternal signals. Meanwhile, the other daughter cell, with its own distinct inheritance as the result of asymmetric cell division, embarks on a particular differentiation parhway, which may be determined both by the signalsthe cell receivesand by

intrinsic bias in the cell's potential, such as the previous activation of certain genes.

The Complete Cell Lineageof C. e/egansls Known In the development of some organisms,cell lineagesare under tight genetic control and thus are identical in all individuals of a species.In other organisms the exact number and arrangement of cells vary substantially among different individuals. The best-documentedexample of a reproducible pattern of cell divisions comes from the nematode C. elegans.Scientistshave traced the lineage of all the somatic cells in C. elegansfrom the fertilized egg to the mature worm by following the development of live worms using Nomarski differential interference contrast (DIC) microscopy (Figure 21 -4). About L0 rounds of cell division, or fewer, create the adult worm, which is about 1 mm long and 70 pm in diameter.The adult worm has 959 somatic cell nuclei (hermaphrodite form) or 1031 (male).The number of somatic cells is somewhat fewer than the number of nuclei because some cells contain multiple nuclei (i.e., they are syncytia). Remarkably, the pattern of cell divisions starting from a C. elegans fertllized egg is nearly always the same. As we discuss later in the chapter, many cells that are generated during development undergo programmed cell death and are missing in the adult worm. The consistency of the C. elegans cell lineage does not result entirely from each newly born cell inheriting specific information about its destiny.That is, their birth cells are not necessarily..hard wired" by their own internal inherited instructions to follow a particular path of differentiation. In some cases, various signals direct initially identical cells to different fates, and the outcomes of these signals are consistent from one animal to the next. The first few cell divisions in C. elegansproduce six different founder cells, each with a separate fate as shown in

Video:C. elegansCrawli

A FfGURE 21-4 Newly hatchedlarva of C. elegans.Someof the959somatic-cell nucleiin the hermaphrodite formare visualized in thismicrograph obtained by differential interference

908

C H A P T E R2 1

|

C E L LB | R T H ,L | N E A G E A , ND DEATH

contrast(DlC)microscopy, sometimes calledNomarski microscopy. Themosteasily seenarethe intestinal nuclei, whichappearasround discs. J E Sulston andH.R Hovilz,1977, [From Devel. Biol.56:110 I

Figure 2t-5a, b. The initial division is asymmetric, giving rise to P1 and the AB founder cell. Further divisions in the P lineageform the other five founder cells. Someof the signals controlling division and fate asymmetry are known. For example, Wnt signalsfrom the P2 precursor control the asymmetric division of the EMS cell into E and MS founder cells. 'Wnt signaling (seeFigure 1.6-32)is also used in other asymmetric divisions in worms. Someof the embryonic cells function as stem cells, dividing repeatedlyto form more of themselvesor another type of precursor cell, while also generating differentiated cells that give rise to a particular tissue. The completelineageof C. elegansis shown in Figure 21-5c. This

organism has beena powerful model systemfor geneticstudies to identify the regulators that control cell lineagesin time and space.

HeterochronicMutants ProvideCluesAbout Controlof Cell Lineage Intriguing evidencefor the geneticcontrol of cell lineagehas come from isolation and analysisof heterochronic mutants. In thesemutants, a developmentalevent typical of one stage of development occurs too early (precocious development) or too late (retarded development). An example of the

of C. elegansEmbryogenesis

I Video:Time-Lapse

AB Neurons EMS Hypodermis P h a r y n g e am l uscle E MS Bodymuscle Gut Bodymuscle P h a r y n g e am l uscle Neurons Glands P4D S o m a t i cg o n a d Germcells Other

c Hypodermis Bodymuscle Neurons Bodymuscle

line

Epidermis

21-5 C. eleganslineage.(a)Patternof the firstfew A FfGURE of the to formation with P0(thezygote) andleading starting divisions (yellow isasymmetric, highlight). Thefirstdivision sixfoundercells in the P divisions producing P1andAB,a foundercell.Further generate the otherfivefoundercellsNotethatmorethan lineage or type(e 9., muscle canleadto thesametissue onelineage to it isthe precursor TheEMScellisso namedbecause neurons). with beginning The lineage and mesoderm. the endoderm mostof whicharesetaside cells, the P4cellgivesriseto allof the germ-line

Somaticgonad giveriseto All theotherlineages asin mostanimals. veryearly, of the of thefirstfew divisions cells.(b)Lightmicrographs somatic asin part thefoundercellswith cellslabeled embryothatgenerate (c)Full of organelles (a).Thetextureof the cellsshowsthe presence tissues of the some showing worm, the of body entire of the lineage few relatively cellundergoes formedNotethatanyparticular (b)fromEinhard Schierenberg fewerthan15.lPart typically divisions, K6lnl Universitet Institut, Zoologisches

M E L L SN, I C H E SA, N D L I N E A G E T H E B I R T HO F C E L L S : S T E C

909

former is premature occurrenceof a cell division that yields a cell that differentiatesand a cell that dies; as a result, the Iineagethat should have followed from the dead cell never happens.An example of the latter is the delayed occurrence of a lineage,causing juvenile structures to be produced, incorrectly, in more mature animals. In both cases,the character of a parental cell is, in essence,changed to the character of a cell at a different stageof development. The study of heterochronicgeneshas been important to understandingthe mechanismsof developmentand generegulation. One example of precocious development in C. elegans comes from loss-of-function mutations in the lin-14 gene, which cause premature formation of a certain neural precursor,the PDNB neuroblasr (Figure 2t-6a). The lin-14 geneand severalothers found to be defectivein heterochronic worm mutants encode RNA-binding or DNA-binding proteins, which presumably coordinate expression of other genes. Two other genes (lin-4 and let-7\ discovered in heterochronic C. elegansmutants were initially puzzlingbecause they appearedto encodesmall RNAs that do not encodeany

protein. To discover the products of these genes,scientists first determinedwhich piecesof genomic DNA could restore gene function, and therefore proper cell lineage,to mutants defective in each gene. They then did the same thing with genomic DNA from the corresponding genomic regions of different speciesof worm. Comparison of the "rescuing" fragments from the different species revealed that they shared common short sequenceswith little protein-coding potential. The short RNA molecules encoded by lin-4 and let-7 were subsequently shown to inhibit translation of the mRNAs encoded by lin-14 and other heterochronic genes (Figure 21-6b). These small RNAs, called micro RNAs (miRNAs), are produced by RNA polymerase II and are complementary to sequencesin the 3' untranslated parts of target mRNAs. The miRNAs direct post-transcriptional silencing of mRNAs by hybridizing to them and blocking translation or stimulating degradation (see Figure 8-25). Temporal changesin the producion of lin-4, let-7, and other miRNAs during the life cycle of C. elegansserve as a regulatory clock for cell lineage.

L3t4 lin-14, lin-28 mRNA \

,r-\

-

I

+ (a)

L t N - 1 4L, t N - 2 8 proteins

Adult lin-14, Iin-28 mRNA \/\.a..Ar *g'.d"\-^

lin-4 let-7 RNA RNA

/in41 mRNA

L1

\/-

/in41 mRNA

let-7 RNA

I v LIN-41 protein

L2

/in-29mRNA

I

c I

/in-29 mRNA

I

v

LtN_29 protein

q)

o

(r .:g

FIGURE 21-5 Timingof celldivisionsduringdevelopmentof RNAto the 3' untranslated (UTRs) regions of lin-14andlin-28 C. elegans.(a)Thepatternof celldivision for the V5 cellof C. elegans mRNAs prevents translation of thesemRNAs intoproteinThis isshownfor normal(wild-type) wormsandfor a heterochronic occurs following thefirstlarval(11)stage,permitting development to mutantcalledlin-14.InIhe lin-14mutant,the patternof celldivision proceed to the laterlarvalstagesStarting in thefourthlarvalstage (redarrows) thatnormally occurs onlyin thesecond larval (14),production stage(12) of let-7RNAbegins.tt hybridizes to lin-14,tin-28, occurs in thefirstlarvalstage(11),causing the pDNBneuroblast to be andlin-41mRNAs, preventing theirtranslation proteinisan LIN-41 generated prematurely. Inthemutant,theV5 cellbehaves duringL1 inhibitorof translation of thelin-29mRNA,so the appearance of /eflikecell"X" (purple) normally doesin 12.Theinference isthatthe LIN- 7 RNAallowsproduction protein, of LIN-29 whichisneeded for 14 proteinprevents L2-type celldivrsions, although precisely how it generation of adultcelllineages LIN-4mayalsobindto rn-4l RNA doesso is unknown.(b)Twosmallregulatory RNAs,/rn-4andlet-7, at laterstagesOnlythe 3' UTRs of the mRNAs aredepicted. lAdapted seryeascoordinating timersof geneexpression Bindingof thelin_4 from B J Reinhartet al , 2000, Nature403:901 I 910

.

c H A p r E R2 1

|

c E L LB I R T H L , I N E A G EA, N D D E A T H

miRNAs have been identified in many other animals including vertebratesand insects.More than 300 are encoded in the human genome,perhapsas many as a thousand. Since production of miRNAs is temporally and spatially regulated, they are likely to control a broad range of events,perhapsincluding qimed events as in C. elegans.How production of theseregulatory miRNAs is temporally controlled is not yet known, but they have turned out to play many roles in regulating geneexpression(Chapter 8).

CulturedEmbryonicStem CellsCan Differentiateinto VariousCellTypes Embryonic stem (ES) cells can be isolated from early mammalian embryos and grown in culture (Figure 2'J.-7a).CuItured ES cellscan differentiateinto a wide range of cell types, either in vitro or after reinsertion into a host embryo. When grown in suspensionculture, human ES cells first differentiate into multicellular aggregates,called embryoid bodies, which resembleearly embryos in the variety of tissuesthey form. When these are subsequentlytransferred to a solid medium, they grow into confluent cell sheetscontaining a variety of differentiatedcell types including neural cellsand pigmented and non-pigmented epithelial cells (Figure 21-7b).

Under other conditions, ES cells have been induced to differ'What entiate into precursors for various types of blood cells. propertiesgive ES cells their remarkable plasticity?A variety of actors play a role: signaling proteins, DNA methylation, micro RNAs, transcription factors, and chromatin regulators can all affect which genesbecomeactive (Chapters7 and 8). During the earlieststagesof embryogenesis,as the fertilized egg begins to divide, both the paternal and maternal DNA becomes demethylated (see the discussion of DNA methylation in Chapter 7). This happens becausea key maintenancemethyltransferase(Dnmtl), which normally is present in the nucleus, is transiently excluded from the nucleus.During the first few cell divisions the pattern of methylation is reset,erasingearlier epigeneticmarking of the DNA and creating a condition where cells have greater potential for diverse pathways of development. Mice engineeredto lack Dnmtl die as early embryos with drastically undermethylated DNA. ES cells prepared from such embryos are able to divide in culture, but in contrast to normal ES cells cannot undergo in vitro differentiation. The properties of mouse ES cell are critically dependent on the action of three transcription factors produced shortly aker ferttlization: Nanog, Sox2, and Oct4. The genesthat are bound by thesefactors have been identified using chromatin

Embryoid body

cells Dissociate I

@@

Replate |

Neurons

IG < E X P E R I M E N TFA L U R2E1 - 7 Embryonicstem (ES)cellscan be maintainedin cultureand canform differentiatedcell types.(a)Human aregrownfrom cleavage-stage blastocysts produced by in vitrofertilization. embryos fromthe Theinnercellmassisseparated and tissues extra-embryonic surrounding platedontoa layerof fibroblast cellsthathelp cells cells.Individual the embryonic to nourish of EScells, andformcolonies arereolated for manygenerations whichcanbe maintained andcanbe storedfrozen.(b)In suspension into humanEScellsdifferentiate culture. calledembryoid aggregates multicellular bodies(fop) Afterembryoidbodiesare solidmedium, to a gelatinized transferred cell furtherintoconfluent theydifferentiate a varietyof differentiated sheetscontaining celltypesincludingneuralcells(middle),and cells epithelial pigmented andnonpigmented (a)and(b)adapted (bottom).[Parts fromJ S 19:193.1 Stem Ce//s etal. 2001, Odorico

""0e,

Established ES cell cultures

E p i t h e l i acl e l l s C E L L SN, I C H E SA, N D L I N E A G E T H E B I R T HO F C E L L S : s T E M

911

rmmunoprecipitation experiments (see Figure 7-37). Each protein is found at more than a thousand chromosomelocations. At about 350 locations, all three proteins are found. DNA microarrays have revealedwhich genesare active in ES cells.About half of the 350 loci where all three transcription factors accumulateare at or near genesthat are transcribedin ES cells.The target genesregulatedby thesetranscription factors encode a wide variety of proteins, including the Oct4, Nanog, and Sox2 proteins themselves,signalingcomponenrs of the BMP and JAICSTAT pathways, and chromatin factors. Chromatin regulators that control gene transcription (Chapter 7) are also important in ES cells. ln Drosophila, polycomb group proteins form complexes that maintain gene repressionstatesthat have been previously established by DNA-binding transcription factors. Two mammalian protein complexes related to the fly Polycomb proteins, PRC1 and PRC2, are produced in ES cells. Early mouse embryos lacking components of PRC2 have abnormal development of the inner cell mass (the embryo proper), and ES cells cannot be made from embryos lacking PRC2 functions. The PRC2 complex of proteins acts by adding methyl groups to Iysine27 of histone H3, thus altering chromatin structure to repressgenes.Remember that this type of regulation is distinct from methylation of DNA. The possibility of using stem cells therapeutically to restore or replace damaged tissueis fueling much research on how to recognize and culture these remarkable cells from embryosand from various tissuesin postnatal(adult) animals. For example, if neurons that produce the neurotransmitter dopamine could be generatedfrom stem cellsgrown in culture, it might be possibleto treat people with Parkinson'sdisease who have lost such neurons. For such an approach to succeed, a way must be found to direct a population of embryonic or other stem cells to form the right type of dopamine-producing neurons, and rejection by the immune system must be prevented. One way to prevent immune rejection is to use adult stem cells from a patient to produce therapeutic cells for that same patient. This is exactly what is done at present in some bone marrow transplants, as we shall seebelow. However it is not yet possibleto isolate adult stem cells with similar capabilities for most other tissues.In animal experiments, embryonic stem cells have proven considerablymore adept than adult stem cells at forming a variety of tissues.One approach for exploiting the advantagesof ES cells while reducing immunological rejection may be to insert a nucleusfrom a patient into the environment of an embryonic cell, replacing the endogenous nucleus with one that will confer patient-specific properties

Recent work has been directed at exploring whether embryonic or adult stem cells can be induced to differentiate into cell types that would be useful therapeutically. For example, mouse ES cells have been treated with inhibitors of phosphatidylinositol-3 kinase, a regulator in one of the 912

.

cHAprER21

|

C E L LB I R T H ,L I N E A G EA . ND DEATH

phosphoinositide signaling pathways (Chapter 16). The treated EScells differentiate into cells that resemblepancreatic B cells in their production of insulin, their sensitivity to glucose levels,and their aggregationinto structuresreminiscent of pancreas structures. Implantation of these differentiated cells into diabetic mice restored their growth, weight, glucose levels, and survival rates to normal. Many important questions must be answered before the feasibility of using human stem cells for such purposes can be assessedadequately.I Apart from their possiblebenefit in treating disease,ES cells have already proven invaluable for producing mouse mutants useful in studying a wide range of diseases,developmental mechanisms, behavior, and physiology. By techniques describedin Chapter 5, it is possible to eliminate or modify the function of a specificgene in ES cells (seeFigure 5-40). Then the mutated ES cells can be employed ro produce mice with a gene knockout (seeFigure 5-41). Analysis of the effectscausedby deleting or modifying a gene in this way often provides clues about the normal function of the geneand its encodedprotein. \7e will now examine the properties and regulation of some postnatal (adult) stem cells, descendantsof ES cells, that build the various organs and tissuesin animals.

Adult Stem Cellsfor Different Animal Tissues O c c u p yS u s t a i n i n gN i c h e s Many differentiatedcell types are sloughedfrom the body or have life spans that are shorter than that of the organism. Diseaseand trauma also can lead to loss of differentiated cells. Since differentiated cells generally do not divide, they must be replenished from nearby stem-cell populations. Postnatal (adult) animals contain stem cells for many tissues including the blood, intestine, skin, ovaries and testes,muscle, and liver. Even some parts of the adult brain, where little cell division normally occurs, have a population of stem cells. In muscle and liver, stem cells are most important in healing, as relatively little cell division occurs in the adult tissuesat other times. Stem cells need the right microenvironment to maintain themselves.In addition to intrinsic regulatory signals-like the presenceof certain regulatory proteins-stem cells rely on extrinsic regulatory signals from surrounding cells to maintain their status as stem cells. The location where a stem-cellfate can be maintained is called a stem-cellniche by analogy to an ecologicalniche, which is a location that supports the existenceand competitive advantageof a particular organism. The right combination of intrinsic and extrinsic regulation, imparted by a niche, will create and sustain a population of stem cells. In order to investigate or use stem cells, they must be found and characterized. It is often difficult to identify stem cells precisely becausethey may lack distinctive shapesor gene expression.Much of the time, many stem cells do not divide particularly rapidly, being held in reserve, dividing slowly if at all, until stimulated by signals that convey the need for new cells. For example, an inadequate oxygen

supply can stimulate blood stem cellsto divide, and injury to the skin can stimulate regenerativecell division starting with the activation of stem cells.Somestem cells,however, including those that form the continuously shed epithelium of the intestine,are continuously dividing, usually at a slow rate. One approach for identifying stem cells in a mixed cell population depends on their relatively slow rate of division. In this approach, a type of pulse-chaseexperiment, cells are provided with a brief pulse of labeled DNA precursors,such as bromodeoxyuridine(BrdU), then later examined to seewhich cellsare labeled.After the BrdU pulse, cells that are not dividing will not be labeled at all, and rapidly dividing cellswill dilute the BrdU label with normal unlabeled nucleotides(the chase).Stem cells, in contrast, will incorporate BrdU during their slow division process. Since they divide relatively rarely, stem cells will retain the BrdU label longer than most other cells, marking them as label-retaining cells. This sort of label retention is often a useful way to identify stem cells.

In the fly ovary, the niche where oocyte precursors form and begin to differentiateis located next to the tip of the germarium (Figure 21-8a). There are two or three germline stem cellsin this location next to a few cap cells,which create the niche by secretingtwo transforming growth factor p (TGFp) proteins (Dpp and Gbb) and Hedgehog(Hh) protein (Figure21-8b). Thesesecretedprotein signalswere introduced in Chapter 16. Vhen the stem cells divide, they produce two daughters,one of which remains adjacentto the cap cells and is therefore a stem cell like the mother cell. The other daughter divides to produce two cystoblast cells that will differentiate into germ-line cells. The cystoblast cellsembark on a path of differentiationbecausethey are too far from the cap cells to receivethe cap cell-derived signals,including Hh, Dpp, and Gbb, and direct cell-cell ( a ) S t e mc e l l sa n dn i c h e si n f l y g e r m a r i u m Inner sheath

""lS

Somatic

Differentiating

stemcell cystobrast

Germ-line Stem Cells The germ line is the line of cells that produces oocytes and sperm. It is distinct from the somatic cells that make all the other tissuesbut are not passed on to progeny.The germ line, like somatic-celllineages,has stem cells. Stem-cellnicheshave been especiallywell defined in studiesof germ-linestemcellsinDrosophila and C. elegans. Germ-line stem cells are presentin adult flies and worms and the location of the stem cells is well known. The stem cells were identified by BrdU label retention.

> FfGURE21-8 Drosophilagermariumand the signalsthat of the germarium createits stem-cellniches.(a)Cross-section stem stemcells(yellow) andsomesomatic showingfemalegerm-line fromthem andthe progeny cellsderived cells(gold)in theirniches (green), which stemcellsproduce cytoblasts Thegerm-line folliclecells intooocytes; thesomatic stemcellsproduce differentiate (brown)thatwill maketheeggshellThecapcells(darkgreen)create a n dm a i n t a itnh en i c h ef o r g e r m - l i nset e mc e l l sw, h i l et h ei n n e r stemcells(b) the nichefor somatic sheathcells(blue)produce pathways of germ-line stem thatcontrolthe properties Signaling molecules-the TGFBproteins DppandGppas cellsThesignaling of theseligands to by capcellsBinding wellasHh- areproduced of the on the surfaceof a stemcellresultsin repression receptors MadandMed Repression of factors, bamgenebytwo transcription of bam activation stemcellsto renew,whereas barnallowsgerm-line promotes proteins, ArmandZpg,which differentiation Twosurface physically in linkcapcellsandstemcells,arealsoimportant nicheCellsout of reachof Arm andZpg maintaining thestem-cell ratherthanrenewMicroRNAsareincreasingly differentiate including ascritical regulators of celldifferentiation, recognized germ-line an essential cellsSomeof thembindto the Piwiprotein, pathways germ-line regulator in bothcapandstemcells(c)Signaling of somatic stemcells.TheWnt signal thatcontrolthe properties (Wg)isproduced by the innersheathcellsandis received Wingless (Fz)on a somatic receptor stemcell Hh issimilarly bythe Frizzled signal produced, andisreceived by the PtcreceptorBothreceptors stemcells resulting in self-renewal of somatic to controltranscription CellDevelBiol21:605 fromL LiandT.Xie,2005,Ann Rev. I lAdapted

(b) Signalsthat creategerm-line stem-cellniche

Signalsthat create somaticstdm:cell

Germ-line stem cell lnner Cap cell . S e c r e t e sH h s i g n a l , . R e c e i v e sH h t h r o u g h s h e a t h c e l l . Secretestwo TGF0 Ptc receptor,promoting . Secretestwo s i g n a l s ,W g s i g n a l sD , p p& G b b . self-renewal. . R e c e i v e sD p p a n d . P r o d u c e sA r m andHh. . Produces G b b t h r o u g hT G F B and Zpg surface r e c e p t o rs u b u n i t sI a n d Arm protein proteins. . H a sP l W l p r o t e i n ll, promoting on its self-renewal. surface. in the nucleus. . TGFFprotein c a u s e signals activationof Mad and Med transcription factors to repress bam gene and allow self-renewal. . P r o d u c e sA r m a n d Z p g surface proteins,which interactwith themselves on the cap cell. . H a s P l W lp r o t e i ni n t h e nucleusto promote stem cell fate.

Somatic stem cell . ReceivesWg signals through the Fz receptors, promoting self-renewal. . R e c e i v e sH h s i g n a lt h r o u g h the Ptc receptor, Promotlng self-renewal. . P r o d u c sA r m , which lnteracts with Arm on i n n e rs h e a t h cell.

T H E B I R T HO F C E L L SS: T E MC E L L SN, I C H E SA, N D L I N E A G E

913

interactions mediated by the cell-surfaceproteins Arm and Zpg, which together direct a cell to remain a stem cell. Both cap cells and germ-line stem cells produce Piwi proteins, which bind micro RNAs. Piwi proteins and their bound miRNAs regulate gene expression and control germ-line cell development in a wide variety of animals as well as stem-celldevelopmentin plants. Thus they consti, tute an ancient mechanism of developmentalregulation. Separatesomatic stem cellsin the germarium produce follicle cellsthat will make the eggshell.The somatic stem cells have a niche too, created by the inner sheath cells, which produce Wingless(Wg) protein-a fly Wnt signal-and Hh protein (Figure 2l-8c). Thus two different populations of stem cells can work in close coordination to produce different parts of an egg. Micro RNAs control the division properries of Drosophila female germ-line stem cells. The Dicer prorein, a doublestranded RNase, produces micro RNAs (seeFigure 8-25). Germ-line stem cells with dicer mutations fail to pass successfullythrough the G1 to S transition of the cell cycle; as a result, the population of stem cells and therefore oocytes is depleted.The absenceof miRNA function causes,directly or indirectly, increasedfunction of the p21127cyclin-dependent kinaseinhibitor. As discussedin Chapter 20, p21127normally restricts G1 to S transitions by regulating cyclin E-CDK complexes.Thus the net effect of the absent miRNA function, which permits increasedp21,127activity, is to restrict cell division. In worms the long tubelike arms of the gonads have tips where a cell called a distal tip cell crearesa stem-cellniche (Figure21-9).The transmembraneprotein Delta, produced by the distal tip cell, binds to the Notch receptor on the

Distal tip cell

Germ-line stem cell

Mitotic stem cell

Meiotic stem cell

FfGURE 21-9 C. elegansgerm-linestem-cellniche.A crosssection of thetip of a gonadarmshowsstemcellsin theirnicheand progeny derived fromthem Thesingledistaltip cell(green) in each gonadarmcreates andmaintains the nrcheSelf-renewing mitotic stemcellsproduced bythe germ-line stemcellsconvert to meiosis whentheymovebeyondthe rangeof the Deltasignalfromthedistal tip cell.Duringthesestages, thecellsareonlypartially separated by ("Y" shapes) membranes andaretherefore a syncytium lAdapted from L Li and T. Xie, 2005, Ann Rev.Cell Devel.Biot.21:605l

914

C H A P T E R2 1

I

C E L LB I R T H ,L I N E A G EA , ND DEATH

germ-line stem cells. The DeltaA{otch signaling pathway (see Figure 1,6-36) promotes mitotic division of worm germ-line stem cells, thus creating more stem cells. Meiosis (i.e., germ-line differentiation) is blocked by the Delta signal until the stem cells move beyond the range of the signal from the distal tip cell. Mutations that activare Notch in the germ-linestemcells,evenin the absenceof Delta signal, cause a gonadal tumor with massive numbers of extra germ-line stem cells, due to excessivemitosis and little merosls. The identification and characterization of Drosophila and C. elegansgerm-line stem cells were important because they showed convincingly the existenceof stem-cell niches and permitted experiments to identify the niche-made signals that causecells to become and remain stem cells. Thus a stem-cell niche is a set of cells and the signals they produce, not just a location. Identification of specificmolecules that maintain the stem-cell state in Drosophila and C. elegans brought an unexpected bonus: Some of these molecules are also used to form a stem-cell niche and control stem-cell fates in mammals. For example, germ-line stem cells in the mouse testis are dependenton a TGFB signaling protein (GDNF) derived from somatic cells. Each seminiferous tubule contains exactly one germ-line stem cell, which divides asymmetrically to re-create itself and to produce a spermatogonial cell. This cell proliferates and its progeny becomespermatocytes,which go through the extraordinary differentiation processthat builds a sperm. The niche is created by a specializedregion of the Sertoli cell along with a myoid cell and a basementmembrane produced by the myoid cell, though many details of the molecular signals remain to be explored. Skin/Hair Stem Cells in Mammals Epithelial stem cells that give rise to skin and hair in mammals are located in hair follicles and in the basal layer of the epithelium between follicles. In the hair follicle, the stem cells occupy a niche called the bulge (Figure 21-10a). The stem cells divide asymmetrically to produce more stem cells and to make precursor cells of at least two kinds. One type of precursor will rise toward the surface of the skin and form keratinocytes, the major cell type of skin, which is a multilayered epithelium (the epidermis). Other cells emergefrom the stem cells to become hair-matrix progenitors that move down deeper in the hair follicle and form a complex set of structures including the hair itself. The molecular regulatorsin the skin stem-cellniche are incompletely known. However, as in flies, TGFB signals, arising from mesenchymalcells that surround the bulge cells, and a Wnt signal, arising from the dermal papilla, are important in controlling stem-cell renewal and differentiation into either skin or hair (Figure 21-10b). Evidence '$fnt for the importance of signaling came from manipulations of the expression of B-catenin, a protein that helps link certain cell-cell junctions to the cytoskeleton (see Figure 1,9-12)and also functions as a signal transducerin the

(a) Crosssectionof hair follicle

cell f Basal l*l tre stemcett

Hair

B u l g es t e m c e l l lnterfollicular e p i t h e l i u m( l F E )

(b) Signalsthat controlthe stem-cellniche in skin

'

Basement

Dermal papilla

)

(Dermap l apilla & bulge)

K

I Stem Dkk cell Wif sFRP

I

(Bulge)

FIGURE 21-10Skin/hairstem-cell nichein mammalsand the signalsthat controlit. (a)A hairfollicle showing stemcells(yellow) in thebulgeandinterfollicular stemcells(lightgreen) amongthebasal epithelial cells(green) outside thehairfollicle. Theprogeny of bulge stemcellsmigrate downto contribute to hairformation nearthe dermalpapillaThedarkgreencellshaveyetto beginterminal differentiation; theyaretransient amplifying cellsthatarestilldividing but cannotgenerate stemcells.Notethatonlythe basallayerof epithelial cellsadjacent to thebasement membrane areshown. Overlaying thesebasalcells,outsideof thefollicle,areseveral layers (b)Signaling of differentiating keratinocytes. eventsin the stem-cell nicheThesource of eachsignal isindicated in parentheses. A Wnt signalpromotes formation of newhaircells, but in thebulge-home (Dkk.Wif. andsFRP) of the stemcells-at leastthreeWnt inhibitors blockdifferentiation andpreserve the stem-cell stateAwayfromthose inhibitors, the lackof Wnt signaling allowsdifferentiation of stemcells intoskincells(keratinocytes). BMBwhichbelongs to theTGFB family proteins, isproduced of signaling by mesenchymal cellsadjacent to the bulge.Duringdevelopment, whennewhairisto begrown,thedermal papilla makesNoggin, whichblocks the BMPsignal, andmoreWnt, whichovercomes the inhibitors, allowingstemcellsto differentiate into haircells. BMPs havecomplex andincompletely understood rolesin skindevelopment, andtheirfunctions change withthestageof development fromF.M Wattetal,2006,CuffOpinGenetDevel. [Adapted 16:51 8,andL LiandI Xie,2005, Ann,ReuCellDevel. Biol.21:605 l

'!fnt

pathway (seeFigure L6-32). Activation of B-catenin changesthe fate of cells from epidermis (skin) to hair. In contrast, removal of B-cateninfrom the skin of engineered mice eliminates formation of hair cells. Epithelial stem cells then form only epidermis, not hair cells. Thus B-catenin acts as a switch that controls which type of precursor arises from epithelial stem cells. \fnt signals also have stimulatory effects on cell division, which can be

restrained by'Sfnt pathway inhibitors, such as DKK and sFRP,that are present in the bulge-a Battle of the Bulge of sorts. Newly formed keratinocytes move toward the outer surface, becoming increasingly flattened and filled with keratin intermediate filaments (Chapter 18). It normally takes about 15-30 days for a newly "born" keratinocyte in the lowest Iayer of the skin to differentiate and move to the topmost layer. The "cells" forming the topmost layer are actually dead and are continually shed from the surface. In addition to keratinocytes, skin contains dendritic epidermal T cells, an immune-system cell that produces a certain form of the T-cell receptor (Chapter 24). Ifhen dendritic epidermal T cells are geneticallymodified so they do not produce T-cell receptors,wound healing is slow and lesscomplete than in normal skin. Normal healing is restored by addition of keratinocyte growth factor. The current hypothesisis that when dendritic epidermal T cells recognizeantigenson cells in damaged tissue,they respond by producing stimulating proteins, such as keratinocyte growth factor, that promote the production of more keratinocytes and wound healing. Many other signals-including Hedgehog, calcium, and transforming growth factor a (TGFct)control the production of skin cells from stem cells. Discovering how all these signalswork together to control growth and stimulate healing will advanceour understandingof diseasessuch as psoriasisand skin cancer and perhapspave the way for effectivetreatments.I Intestinal Stem Cells In contrast to epidermis,the epithelium lining the small intestine is a single cell thick (see Figure 19-8). This thin layer is enormously important for keeping toxins and pathogens from entering our bodies; it also transports nutrients essentialfor survival from the intestinal lumen into the body (seeFigure 11-29). The cells of the intestinal epithelium continuously regenerate from a stem-cell population located deep in the intestinal wall in By identifying the Iabelpits called crypts (Figure 21,-1,1'a). retaining cells in the intestinal epithelium, researchersdetermined that the stem cells are located precisely four or five cells above the bottom of a crypt. The niche is created at the level of the by mesenchymalcells that abut the crypts 'Wnt signal, a BMP stem cells. These cells produce a (TGFB) signal, and possibly a ligand for the Notch receptor on stem cells (Figure 21-1'1b). Overproduction of Bcatenin in intestinal cells leads to excess proliferation of 'Sfnt those cells, as though they were receiving too much signal (which stabilizesB-catenin).Blocking the function of B-catenin by interfering with the TCF transcription factor that it activates abolishes the stem cells in the intestine. Thus $fnt signaling, acting through p-catenin, plays a critical role in maintaining the intestinal stem-cell population. BMP has the opposite effect, promoting differentiation and restraining the effect of Wnt.

, ND LINEAGE , I C H E SA : T E M C E L L SN r H E B I R T HO F C E L L SS

915

Podcast:Stem Cellsin the lntestinal Epithelium (b) Directionof cell migration

Proliferationzone (c) Intestinalstem cell signalingpathways

I Mesenchymalcells

Intestinal stem cells produce precursor cells that proliferate and differentiate as they ascendthe sides of crypts to form the surface layer of the finger-like gut projections calleduilli, acrosswhich intestinal absorption occurs. Pulsechaselabeling experiments with BrdU have shown that the time from cell birth in the crypts to the loss of cells at the tip of the villi is only about 2 to 3 days. Thus enormous numbers of cells must be produced continually to keep the epithelium intact. The production of new cells is preciselycontrolled: too little division would eliminate villi and lead to breakdown of the intestinal surface; roo much division would create an excessivelylarge epithelium and also might be a step toward cancer.Indeed, mutations that inappropriately activate Wnt signal transduction are a major contributor to the progression of colon cancer, as we shall see in Chapter 25. Neural Stem Cells The great interestin the formation of the nervous system and in finding better ways to prevent or treat neurodegenerativediseaseshas made the characterization of neural stem cells an important goal. The earliest stagesof vertebrate neural development involve the rolling up of ectoderm to form the neural tube, which extends the length of the embryo from head to tail (Figure 2l-12a).

916

CHAPTER 21

|

C E L LB t R T H L , t N E A G EA, N D D E A T H

< FIGURE 21-11 lntestinalstem-cellnicheand projections the signalsthat control it. (a)Finger-like of the innersurface of the intestine calledvilliare dividedby deeppitscalledcryptsThesingle-cell thick protects epithelium usfrominfection andallows selective of usefulnutrients intothe blood transoort (b)An intestinal stream. cryptshowingthe intestinal stemcells(yellow), theirproliferating mitoticprogeny (lightgreen), (darkgreen)in thefinal andprecursors stagesof differentiation Mesenchymal cells,shownin blue,createthisstem-cell niche.Paneth cells(orange), locatedat the baseof the crypt,secrete antimicrobial defenseproteinscalleddefensins. Theseproteinsform poresin bacterial membranes, leading to deathof the bacteria. Signaling eventsin thestem-cell niche.Wnt promotestem-cell signals fates,offsetting BMPsignals thatpushtowarddifferentiation. Nogginrestrains the proliferation, BMPsignals andthuspromotes stem-cell whereas Dkkrestrains Wnt signaling whengrowthis not needed.TheNotchreceptorisalsoinvolved, althoughitsligandon mesenchymal cellsisunknown. fromL LiandT.Xie,2005,Ann.ReuCellDevel lAdapted Biol.21:6051

Initially the neural tube is composed of a single layer of cells, the neural stem cells (NSCs); these will give rise to the entire central nervous system (brain and spinal cord). Labeling and tracing experimentshave shown where neural cells are born and where they go after birth. The most active region of cell division is the subuentricular zone, which has the properties of a stem-cellniche and is named for its proximity to the central fluid-filled uentricle. The embryonic neural stem cells that line the ventricle divide can symmetrically, produce two daughter stem cells side-by-side (Figure 21.-1.2b),or asymmetrically, produce a cell that remains a stem cell and another that migrates radially outward. The migrating cells are often transient amplifying (TA) cells, which in turn divide to form neural precursors called neuroblasts. Once formed, TA cells and neuroblasts migrate radially outward and form successive layers of the neural tissue in an inside-out order, whereas the stem cells remain in contact with the ventricle (seeFigure 21-12b). Newly formed cells therefore traverse the layers of preexisting cells before taking up residenceon the outside. Tracing experiments with viruses have shown that a neuroblast can produce two daughters,one a neuron and one a glial cell. In these experiments, altbrary of defective

N e u r a lp l a t e

(b)

o o

.z q)

E E o A p i c a lQ

Basal

< FfGURE21-12 Formationof the neuraltube and division a portionof the of neuralstem cells.(a)Earlyin development, fromthe restof the embryonic ectodermrollsup and separates c e l l sT h i sf o r m st h e e p i d e r m (i sg r a ya) n dt h e n e u r atlu b e( b l u e ) betweenthe two, neuralcrestcellsform and Nearthe interface t o c o n t r i b u tteo s k i np i g m e n t a t i onne, r v ef o r m a t i o n , t h e nm i g r a t e c r a n i o f a c si akle l e t o nh,e a r vt a l v e sp,e r i p h e r na el u r o n sa,n do t h e r for whichwe are a rodof mesoderm Thenotochord, structures provides that affectcellfatesin the signals named(chordates), n e u r atlu b e( C h a p t e2r2 ) f h e i n t e r i oor f t h e n e u r atlu b ew i l l v e n t r i c l eN s eural become a s e r i eos f f l u i d - f i l l ecdh a m b e rcsa l l e d d d j a c e nt ot t h ev e n t r i c l ei ns t h es u b v e n t r i c u l a r s t e mc e l l sl o c a t e a r a d i a l loyu t w a r dtso z o n ew i l ld i v i d et o f o r mn e u r o ntsh a t m i g r a t e (b) Neural stemcellsin the system. layers of the nervous form the (top),alongtheir zonecandividesymmetrically subventricular a p i c a l - b a saaxli st,o g i v er i s et o s i d e - b y - s iddaeu g h t esrt e mc e l l s , stemcellscan Alternatively, both in contactwith the ventricle. e n ed a u g h t etrh a ti sa d i v i d ea, l o n gt h e i ro t h e ra x i st,o p r o d u c o s t e mc e l la b l et o s e l f - r e n eawn da d a u g h t ecre l l c, a l l e da t r a n s i e n t a n dd i f f e r e n t i a t e a m p l i f y i n(gT A c) e l l t, h a tb e g i n tso m i g r a t e is (bottom)A keydifference betweenthe two patternsof division fromL R wolpert of the mitoticspindle[Adapted the orientation Press 2ded, Oxford University of Development, l et al 2OO2Principles

c

-r-L \,/ - Mitotic

o

E E o

+"

TA cell

retroviruses,each able to infect only once and containing a unique DNA sequence,was prepared (Figure 21-13a). Any cell infected by a singlevirion would give rise to a clone of cells that all carry that particular virus' DNA sequence.In this way, all the cellsthat derivedfrom a singleneural stem cell or TA cell can be identified as a clone (Figure 21-13b). T h e r e s u l r so f t h e s et r a c i n g e x p e r i m e n t sw e r e s u r p r i s i n g . First, some neuronswere found to have migrated considerable distanceslaterallS abandoning their radial migration to the outer cortical layer. Second,in some cases,a single neuron and singleglial cell, sharedthe sameviral DNA sequence.A neural precursorhad evidentlybeeninfectedand had then divided once to give rise to two quite different cell types. Most mammalian brain cells stop dividing by adulthood but some cells in the subventricularzone and at least one other part of the brain continue to act as stem cells and generatenew neurons (Figure 21,-1,4).In the subventricular zone of adults, the neural stem cells are astrocytes, a somewhat confusing nomenclature since more

traditionally "astrocyte" meant atype of glia cell. Evidently neural stem cells are a subsetof astrocytesnot previously r e c o g n i z e df o r t h e i r s p e c i a l s t e m - c e l l q u a l i t i e s . N e u r a l stem cells have some properties of astrocytes,such as producing glial fibrillary acidic protein (GFAP), but they also can divide asymmetrically to renew themselves and to produce TA cells. The subventricular stem-cell niche is created by mostly unknown signals from the ependymal cells that form a layer just inside the neural tube (lining the ventricle) and by endothelial cells that form blood vesselsin the vicinity (Figure 21-14b). The endothelial cells, and the basal lamina they form, are in direct contact with neural stem cells and are believed to be essentialin forming the niche. Each neural stem cell extends a single cilium through the ependymal cell layer to directly contact the ventricle. Though the function of the cilium is unknown, it may function as an antenna for receiving signals that would otherwise be inaccessibleto the neural stem cell. The signalsthat create the niche are not completely characterized,but there is evidencefor a blend of factors including FGFs, BMPs, IGR VEGR TGFo, and BDNF. The BMPs appear to favor astrocyte differentiatron over neural differentiation, and over-expressionof IGF (insulinlike growth factor) causesmice to develop with abnormally large brains. Later stagesof neural developmentare d i s c u s s e di n C h a p t e r 2 3 . Hematopoietic Stem Cells Like the intestinalepithelium the blood is a continuously replenishedtissue.The stem cells

C E L L SN, I C H E SA, N D L I N E A G E T H E B I R T HO F C E L L S : s T E M

917

(a) Crosssectionof whole developingbrain

Distinctive oligonucleotide sequence

(b)

A EXPERIMENTAL FIGURE 21-13 Retrovirusinfectioncan be usedto tracecelllineage.(a)Engineered viralgenome. Thelong (LTRs) terminalrepeats arestandard retroviral repeats. Viralproteins required for infection areencoded in thegagandin A-envgenes PLAP isan introduced genefor an alkaline phosphatase Detection of thisenzyme by histochemical staining isusedto determine which cellscarrya virusTheoligonucleotide sequence, synthesized by providing randomnucleotides, isdifferent in eachvirusandcanbe amplified by PCR,usingprimers for sequences thatarein allviruses (purplearrows), andthensequenced A library of morethan107 distinct viruses wasmade Because theseviruses lackthegenes required for production of newvirionsin infected cells,each defective viruscaninfectonlyonce.(b)Tissue section showingcells infected with defective virusesTheDNAfromeachstained cloneof cellscanbe extracted andamplified by PCRto determine the sequence of the infecting virus.Cellsdescended fromthesame initially infected cellwill havethe sameoligonucleotide sequence, whereas separate infection events willgivedifferent sequences. [From J A G o l d e ne t a l , 1 9 9 5 ,P r o c N a t ' l .A c a d S c i U S A 9 2 : 5 7 0 4 1

that give rise to the different types of blood cells are located in the bone marrow in adult animals. All types of blood cells derive from a single type of pluripotent hematopoietic stem cell, which givesrise to the more-restrictedmyeloid and lympboid stem cells (Figure 21-15). Although myeloid and lymphoid stem cells are capable of self-renewal, each type is committed to one of the two major hematopoietic lineages. Thus these cells function as both stem cells and precursor c el l s . After hematopoietic stem cells form, numerous extracellular growth factors called cytokines regulateproliferation and differentiation of the precursor cells for various blood-cell lineages.Each branch of the blood-cell lineage tree has different cytokine regulators, allowing exquisite control of the production of specificcellstypes. For example, after a bleeding injury, when all blood cells are needed, multiple cytokines are produced. But when a 918

CHAPTER 21

I

C E L LB I R T H L , I N E A G EA, N D D E A T H

Ependymal cells Neuroblast Transit-amplifying c el l s Lateral ventricle

NSC

Expansion Neuroblast

A FIGURE 21-14 Neuralstem-cellniche.(a)Cross-section of the developing nervous system showing ventricle, the lateral a fluid{illed spaceinsidethe neuraltube Theareajustsurrounding theventricle, calledthe subventricular zone,isthesiteof stemcellsfromwhich neuralprecursors arise(b)Theneuralstemcells(yellow), a subset of (blue)andadjacent astrocytes, arein contactwith bloodvessels ependymal cells(pink),bothof whichprovide signals or direct populationNeuralstemcells contacts thatmaintain the stem-cell (NSC) divideto renewthemselves population andto forma dividing (TA)cells(lightgreen;seeFigure21-12b).IA of transitamplifying (darkgreen), cellsin turndivideto formneuroblasts whichgiverise to neuronslAdapted fromL LiandT.Xie,2005,Ann Rev. CellDevelBiol 2 1 : 6 0l 5

person is traveling at high altitude and needs additional erythrocytes,erythropoietin-a cytokine that acts only on erythrocyte precursors-is produced. Erythropoietin activates several different intracellular signal-transduction pathways, leading to changes in gene expression that p r o m o t e f o r m a t i o n o f e r y t h r o c y t e s( s e eF i g u r e 1 6 - 6 ) . I n contrast GM-CSR a different cytokine, stimulates production of granulocytes, macrophages,eosinophils, and megakaryocytes.

b-L5r

Granulocytes(phagocyticimmune cells) I L - 3G , M - C S ES C EI L - 6 M,CSF

Monocytes(macrophageprecursors) Myeloid stem cell

I1.3G , M-CSF

@

Pluripotent HSC

E o s i n o p h i l (si m m u n ec e l l sa c t i v ei n allergicreactions.fighting parasites)

lr

(HSc)

f /\

/--:-\

/

, M-CSF S C E T p o ,l L - 3 G

n

E#) o-t" v

Pluripotent HSC

Megakaryocytes (platelet-formi ng cells)

Lymphoid stem cell tL-2,lL-7,lL-12,sDF-l, El T2

T-CFC

@ B-CFC

ti^-hd

TNltr-d

i r-\) T and B cellsof the immune system

A FIGURE 21-15 Formationof blood cellsfrom hematopoieticstem cellsin the bone marrow.Pluripotent stem (bluecurved cells(yellow) maydividesymmetrically to self-renew arrow)or divideasymmetrically to forma myeloidor lymphoid stem t r e e na) n da d a u g h t ecre l lt h a ti sp l u r i p o t e nl i tk et h e c e l l( l i g h g parental cell Althoughmyeloidand lymphoid stemcellsare capable of self-renewal, eachtypeiscommitted to oneof the two majorhematopoietic lineagesDepending on the typesand present, amountsof cytokines the myeloidand lymphoid stemcells generate differentprecursor whichareincapable cells(darkgreen), of self-renewal. Precursor cellsarecalledcolony-forming cells (CFCs) because of theirabilityto form colonies containing the differentiated celltypesshownat right Thecolonies aredetected

that havehadtheirown hematopoietic on the spleenof animals r l l si n t r o d u c eF du r t h e r ce c e l l se l i m i n a t eadn dt h e p r e c u r s o proliferation, anddifferentiation commitment, cytokine-induced typesof bloodcells cellsgiveriseto the various of the precursor (red process areindicated this that support the cytokines Someof E: Eo : eosinophil; labels). GM : granulocyte-macrophage; B : B-cell; CFU= T : T-cell; mega: megakaryocyte; erythrocyte; : : factor;lL unit;CSF colony-stimulating colony-forming Tpo : SCF: stemcellfactor;Epo: erythropoietin; interleukin; factor;TGF: transforming TNF: tumornecrosis thrombopoietin; ligand: factor;FLT-3 growthfactor;SDF: stromalcell-derived fromM 3. [Adapted kinasereceptor tyrosine ligandfor fms-like ProcNat'lAcadSciUSA95:6573]1 1998, Socolovskyetal,

The hematopoietic lineageoriginally was worked out by injecting the various types of precursor cells into mice whose precursor cells had beenwiped out by irradiation. By observing which blood cells were restored in these transplant experiments,researcherscould infer which precursors (e.g., GM-CFC, BFU-E) or terminally differentiated cells (e.g., erythrocytes, monocytes) arise from a particular type of precursor. RemarkablS a single hematopoietic stem cell is sufficient to restore the entire blood system of an irradiated mouse. The first step in theseexperimentswas to separatethe different types of hematopoietic precursors.

This separationis possiblebecauseeach type of precursor produces unique combinations of cell-surface proteins that can serve as type-specific markers. If bone marrow extracts are treated with fluorochrome-labeledantibodies for these markers, cells with different surface markers can be separated in a fluorescence-activatedcell sorter (see F i g u r e9 - 2 8 ) . The frequency of hematopoietic stem cells is about 1 cell per 104 bone marrow cells.Activation of the Hoxb4 gene in embryonic stem cells drives the formation of hematopoietic stem cells. (As describedin Chapter 22, Hoxb4 also plays a C E L L SN, l C H E SA, N D L I N E A G E T H E B I R T HO F C E L L S : s T E M

919

role in pattern formation along the head-to-tail body axis.) The Bmi geneis also required for self-renewalof hematopoietic stem cells, and of neural stem cells as well. This geneencodes a polycomb-type chromatin regulator protein that repressescertain genesincluding some Hox genes,a group of developmentally important genes discussedin Chapter 22. Bmil is a component of the PRC1 protein complex that we discussedabove in relation to embryonic stem cells. Thus membersof the polycomb group of proteins are important in both embryonic and adult stem cells. Like other stem cells, hematopoietic stem cells are residents of a niche. The niche is formed by spindle-shapedcells on the surface of the bone in the bone marrow. N-cadherin fastensthe stem cells to theseniche cells.A Delta-like ligand produced by niche cells signals to Notch receptors on the stem cells, and several other growth factor-receptor pairs stimulate their self-renewaland differentiation into mveloid or lymohoid stemcells. Stem cells can become cancerous. For example, Ieukemia is a cancer of the white blood cells. This type of canceris marked by two types of cells:leukemictumor cells, which arise from differentiated white blood cells and have limited growth abilities,and the more dangerous leukemic tumor stem cells with unlimited growth abilities. The leukemia tumor stem cells,which are capableof initiating a new tumor on their own, are present in a human tumor about once for every million dividing leukemic cells. Thus the bulk of the leukemic cells are not able to grow a new tumor. Therefore to provide an enduring cure, treatments must ensure the death or limited mitosis of the tumor stem cells. This is particularly challenging because many cancer stem cells divide slowly or not at all for substantial periods, making them selectively resistant to chemotherapy drugs and irradiation-both treatments that target rapidly dividing cells. To date, bone marrow transplants, a treatment for leukemia and other blood disorders, represent the most successfuland widespreaduse of stem cells in medicine.In 1959 a patient with end-stage(fatal) leukemia was irradiated to destroy her cancer cells. She was transfusedwith bone marrow cells from her identical twin sister, thus avoiding an immune response,and was in remission for three months. This promising beginningled to present-day treatments that can often lead to a complete cure for Ieukemia. The stem cells in transplanted bone marrow can generate neq functional blood cells in patients with certain hereditary blood diseasesand in cancerparientswho have received irradiation andlor chemotherapy. Both chemotherapy and irradiation destroy the bone marrow cells as well as cancer cells. Bone marrow transplants go beyond eliminating cancer cells as done with irradiation. Instead.an immune attack on the leukemic cells can be mounted by the injected donor cells. More than two dozen diseasesare now commonly treated with bone marrow transplantation. These include

920

C H A P T E R2 1

I

C E L LB I R T H ,L I N E A G EA , ND DEATH

leukemias and anemiasof various types, lymphomas, severe combined immunodeficiency, and certain autoimmune disorders. The effectivenessof bone marrow transplants varies among diseasesand patients from minor improvement to full cures.Much researchis under way to employ other types of stem cells in the treatment of diseasesinvolving nonblood tissues.I You have probably noticed that all the molecular regulators of stem cells are familiar signal proteins rather than dedicated regulators that specialize in stem-cell control. Each type of signal is usedrepeatedlyto control cell fates and proliferation. Theseare ancient signaling systems,at least a half billion years old, for which new useshave emergedas cells, tissues,organs, and animals have evolved new variations. \7e will seemany more examplesof such evolutionary conservation in the next chapter on development.

MeristemsAre Nichesfor Stem Cellsin PostnatalPlants Stem cells in plants are located in meristems,populations of undifferentiatedcells found at the tips of growing shoots. Shoot apical meristems (SAMs) produce leaves and shoots as well as more stem cells that constitute the nearly immortal meristems.Meristems can persist for thousands of years in long-lived speciessuch as redwood trees and bristlecone pines. As a plant grows, the cells "left behind" the meristemsare encasedin rigid cell walls and can no longer grow. SAMs can split to form branches, each branch with its own SAM, or be converted into floral meristems (Figure 21-16). Floral meristemsgive rise ro the four floral organs-sepals, stamens, carpels, and petals-that form flowers. Unlike SAMs, floral meristems are gradually depletedas they give rise to the floral organs. A meristem is a stem-cellniche, but much remains to be learned about how the niche is created and maintained. Numerous genes have been found to regulate the formation, maintenance, and properties of meristems. Many of thesegenesencodetranscription factors that direct progeny of stem cells down different paths of differentiation. For instance,a hierarchy of regulators, particularly transcription factors, controls the separation of differentiating cells from SAMs as leaves form; similarlS three types of regulators control formation of the floral organs from floral meristems (seeFigure 22-36). In both cases,a cascadeof gene interactions occurs, with earlier transcription factors causing production of later ones. At the same time, cells are dividing and the differentiating ones are spreading away from their original birth sites. One signal rhat creates the plant niche is ZwillelPinhead, which encodesa protein related to the Piwi protein that supports stem-cell niches in animals (seeFigure 21-8). These are "argonaute" family proteins, which repress genes in response to certain small RNA molecules.I

(a) Regionsof shoot apical and floral meristems

(b) Fatesof cells in L2 layer

but may have different fates if they are exposed to different external signals (seeFigure 21-1). r Pluripotent stem cellscan produce more than one type of descendantcell, including in some casesa stem cell with a more-restricted potential to produce differentiated cell types. r Embryonic developmentof C. elegansbeginswith asymmetric division of the fertilized egg (zygote).The lineageof all the cells in adult worms is known and is highly reproducible (seeFigure21-5). r Short regulatory RNAs (micro RNAs) control the timing of developmentalcell divisions by preventing translation of mRNAs whose encoded proteins control cell lineages(see Figure2L-5). r Cultured embryonic stem cells (ES cells) are capable of giving rise to many kinds of differentiated cell types. They are useful in production of geneticallyaltered mice and offer potential for therapeuticuses.Specifictranscription factors and chromatin regulators are important in giving ES cellstheir unusualproperties. r Stem cells are formed in niches that provide signals to maintain a population of nondifferentiating stem cells.The niche must maintain stem cells without allowing their excessproliferation and must block differentiation. r Germline cells give rise to eggsor sperm. By definition' all other cells are somatic cells.

(a)In A FIGURE 21-16 Cellfates in meristemsoI Arabidopsrs. sections, with theselongitudinal cellnucleiarerevealed by staining propidium iodide,whichbindsto DNA.Iop:Theshootapical (SAM)produces meristem shoots,leaves, and moremeristem. Flower production occurswhenthe meristem from leaf/shoot swrtches production to flowerproduction, with an increase in the concomitant (FMs), numberof meristem cellsto formfloralmeristems asshown here Middle:Cellsin a SAMexhibitdifferentfatesand behaviors. zone(PZ,green)to produce Cellsdividerapidlyin the peripheral leaves andin the ribzone(Rib,blue)to produce centralshoot structures. Cellsin thecentralzone(CZ,red)dividemoreslowly, producing an ongoingsource of meristem andcontributing cellsto Thelayers coloredblue, the PZandRib.Eottorn: of the meristem, (cloned) pinkandyellowhere,areeachderived fromthe same precursor cell.Scale bars,50 pm (b)Colorcodingshowsthefatesof positions cellsin different in the L2 layer. Thecolorcodedoesnot to that in part(a).[Part(a)fromE Meyerowitz, 1997, Cell correspond micrographs courtesy of ElliotMeyerowitz Part(b)afterC Wolpert 88:299; of Development,2d University Press et al, 2002,Principles ed, Oxford l

r Populations of stem cells associatedwith the gonads, skin, intestinal epithelium and most other tissuesregenerate differentiated tissuecells that are damagedor sloughed or becomeaged (seeFigures21'-8-21-11',21,-14)' r In the blood cell lineage, different precursor types form and proliferate under the control of distinct cytokines (see Figure 21-1.5).This allows the body to specifically induce the replenishmentof some, or all, of the necessarytypes. r Stem cells are prevented from differentiating by specific controls that operatein the niche. A high level of B-catenin, a component of the tJfnt signaling pathway' has been implicated in preservingstem cells in the skin and intestine by directing cells toward division rather than differentiation states. r Plant stem cells persist for the life of the plant in the meristem. Meristem cells can give rise to a broad spectrum of cell types and structures(seeFigure 21-1'61'

Specificationin Yeast Cell-Type The Birth of Cells:Stem Cells,Niches,and Lineage r In asymmetriccell division, two different types of daughter cells are formed from one mother cell. In contrast, both daughter cells formed in symmetric divisions are identical

In the previous section, we saw that stem cells and precursor/progenitor cells produce progeny that embark upon specific differentiation paths. The elegant regulatory mechanisms of differentiation are referred to as cell-type specification Specification usually involves a combination

C E L L - T Y PS EP E C I F I C A T I OI N Y E A S T

921

of external signalswith internal signal-transductionmechanisms like those describedin Chapters 15 and 15. The transition from an undifferentiated cell to a differentiating one often involves the production of one or a small number of transcription factors. The newly produced transcription factors are powerful switches that trigger the activation (and sometimesrepression)of large batteriesof subservientgenes. Thus an initially modest change can causemassivechanges in geneexpressionthat confer a new character on the cell. Our first example of cell-type specification comes from the budding yeast, S. cereuisiae.We introduced this useful unicellular eukaryote way back in Chapter 1 and have encountered it in several other chapters. S. cereuisiaeforms three cell types: haploid a and ct cells, and diploid a/ct cells. Each type has its own distinctive set of active genes;many other genesare active in all three cell types.In a parrern common to many organisms and tissues,cell-type specification in yeast is controlled by a small number of transcription factors that coordinate the activities of many other genes.Similar regulatory featuresare found in the responsesof higher eukaryotic cells to environmental signals and in the specification and patterning of cells and tissues during development (Chapter 22). DNA microarray studies have provided a genome-wide picture of the fluctuations of geneexpressionin the different cell types and different stagesof the S. cereuisiaelife cycle (seeFigure 5-29 for an explanation of the DNA microarray technique). Among other things, these studies identified 32 genesthat are transcribedat more than twofold higher levels in o. cells than in a cells.Another 50 genesare transcribed at more than twofold higher levelsin a cells than in crcells.The products of these 82 genes,which initially are activated by cell-type specificationtranscription regulators,convey many of the critical differencesbetweenthe two cell types. The results clearly demonstratethat changesin expressionof only a small fraction of the genome,in this case(2 percent of the =5000 yeast genes,can significantly alter the behavior and properties of cells.Transcription of a much larger number of genes,about 25 percent of the total assayed,differed substantially in diploid cells compared with haploid cells. These differencesin expressionpatterns make sensesince a and ct cells are very similar (hence, expression of relatively few genes differ between them), whereas haploid and diploid cellsare quite different.

Mating-TypeTranscriptionFactorsSpecify Cell Types Each of the three S. cereuisiaecell types expressesa unique set of regulatory genesthat is responsiblefor all the differencesamong the three cell types. All haploid cells express certain haploid-specificgenes;in addition, a cells expressaspecific genes, and o. cells express cr-specificgenes.In a"/a diploid cells, diploid-specific genes are expressed,whereas haploid-specific,a-specific,and a-specific genesare not. As illustrated in Figure 21,-17,three cell type-specific transcription factors (o.L,u2, and a1) encoded atthe MAT locus act in combination with a general transcription factor called

922

CHAPTER 21

|

C E L LB t R T H L , t N E A G EA, N D D E A T H

(a) a cell

----:: \

J- MArE-T

(b) ocell

ll

MATU2

MATaI

+CC+>@] (c) a/crcell

MATU2

MATU|

FIGURE 21-17Transcriptional controlof celltype-specific genesin S. cerevisiae. Thecodingsequences carriedat theMAT locusdifferin haploida anda cellsandin diploidcells.Threetypespecific transcription factors(ct1,o2, anda1)encoded attheMAT locusactwith MCM1,a constitutive transcription factorproduced by allthreecelltypes,to produce patternof gene a distinctive expression in eachof the threecelltypes.asg : a-specific geneVmRNAs; genes/mRNAs; cr59: a-specific hsg : haploidspecific Aenes/mRNAs.

MCMI, which is expressedin all three cell types, to mediate cell type-specific gene expressionin S. cereuisiae.Thus the actions of just three transcription factors can set the yeast cell on a specific differentiation pathway culminating in a particular cell type. From the DNA microarray experiments we know one effect of these key players: the activation or repressionof many dozensof genesthat control cell charactenstrcs. MCM1 was the first member of the MADS family of transcription factors to be discovered.(MADS is an acronym for the initial four factors identified in this family.) The DNA-binding proteins composing this family dimerize and contain a similar N-terminal MADS domain. In Section21.3 we will encounter other MADS transcription factors that participate in development of skeletal muscle. MADS transcription factors also specify cell types in floral organs (see Figure 22-35). Acting alone, MCM1 activatestranscription of a-specificgenesin a cells and of haploid-specificgenesin both ct and a cells (seeFigure 21,-17a,b).In haploid o cells,

(a) a cells

cr-specificURS

MCMl dimer

No tanscriptionof a-specificgenes

(b) o cells

cr-specific URS

a-specificURS

Transcription of a-specific genes

s2 dimer

Transcription of a-specific genes

a-specificURS

however,the activity of MCM1 also is determined by its association with the o.1.or a2 transcription factor. As a result of this combinatorial action, MCM1 promotes transcription of a-specific genesand repressestranscription of a-specific genesin cr cells. Now let's take a closer look at how MCM1 and the MAT-encoded proteins exert their effects.

M C M l a n d a 1 - M C M 1C o m p l e x e A s ctivate GeneTranscription In a cells,homodimeric MCM1 binds to the so-calledP box sequencein the upstream regulatory sequences(URSs)of aspecificgenes,stimulating their transcription (Figure 21-18a). Transcription of o.-specificgenesis controlled by two adjacent sequences-the P box and the Q box-located in the URSs associatedwith these genes.Although MCM1 alone binds to the P box in a-specificURSs,it does not bind to the P box in a-specific URSs. Thus a cells do not rranscribe aspecificgenes. In ct cells, which produce the ct1 transcription factor encoded by MATa, the simultaneous binding of MCM1 and cr,1to PQ sites occurs with high affinity (Figure 21-18b1. This binding turns on transcription of cr-specific genes. Therefore, a-specific transcription is a simple matter of a single transcription factor binding to its target genes,while ct-specifictranscription requires a combination of two factors-neither of which can activate target genesalone.

a2-MCM1 and c2-a1 ComplexesRepress Transcription Highly specificbinding occurs as a consequenceof the interaction of o2 with other transcription factors at different sites in DNA. Flanking the P box in each a-specificURS are two cr2-binding sites. Both MCM1 and a2 can bind independently to an a-specificURS with relatively low affinity. In cr cells, however, highly cooperative, simultaneous binding of both ct2 and MCM1 proteins to thesesitesoccurs with high affinity. This high-affinity binding repressestranscription of

No transcription ol a-specific genes

< FIGURE 21-18 Activityof MCMl in a and a yeastcells.MCMl bindsasa dimerto anda-specif ic upstream the Psitein ct-specific (URSs), regulatory sequences whichcontrol transcription of o-specific aenesanda-specific (a)In a cells,MCMl genes,respectively. genes. stimulates transcription of a-specific MCMl doesnot bindefficiently to the Psitein o-specrfic URSs in the absence of o1 protein (b)In o cells,theactivity of MCMl ismodified withcr1or o2 Theo1-MCM1 by itsassociation complex stimulates transcription of a-specific genes, whereas theo2-MCM1complex blocks genes. transcription of a-specific Theo2MCMl complex alsoisproduced in diploid cells,whereit hasthesameblocking effect (see on transcription of a-specific Aenes i l o u r ez t - t / c ) .

a-specific genes,ensuring that they are not expressedin e cellsand diploid cells (seeFigure 21.-1.8b,right).MCM1promotes binding of a2 to an a-specificURS by orienting the two DNA-binding domains of the ct2 dimer to the a2-binding sequencesin this URS. Sincea dimeric cr2molecule binds to both sitesin an cr-specificURS, each DNA site is referred to as a half-site. The relative positions of both half-sitesand their orientation are highly conserved among different aspecificURSs. Combinations of transcription factors create additional specificity in gene regulation. The presenceof numerous c2binding sitesin the genome and the "relaxed" specificity of ct2 protein may expand the range of genesthat it can regulate. For instance,in a/ct diploid cells, cr2 forms a heterodimer with a1 that repressesboth haploid-specificgenesand the geneenThe example of a2 suggests coding a1 (seeFigure 21,-1,7c). that relaxed specificitymay be a generalstrategyfor increasing the regulatory range of a single transcription factor.

PheromonesInduceMating of a and a Cellsto Generatea Third CellType An important feature of the yeast life cycle is the ability of haploid a and c cells to mate, that is, attach and fuse giving rise to a diploid ala cell (seeFigure 1-5). Each haploid cell type secretesa different mating factor, a small polypeptide pheromone, and expressesa cell-surfaceG protein-coupled receptor that recognizesthe pheromone secretedby cells of the other type. Thus a and a cells both secreteand respond Binding of the mating factors to pheromones(Figure 21,-1,9). to their receptorsinducesexpressionof a set of genesencoding proteins that direct arrest of the cell cycle in G1 and promote attachment/fusion of haploid cells to form diploid cells. In the presenceof sufficient nutrients, the diploid cells will continue to grow. Starvation, however, induces diploid cells to progressthrough meiosis,each yielding four haploid spores.If the environmental conditions becomeconduciveto vegetative growth, the spores will germinate and undergo mitotic division. C E L L . T Y PS E P E C I F I C A T I OI N Y E A S T

923

TT

a factor\ at

a

a

a-factorreceptor

Cell-cyclearresl Morphogenesis

mate. The STE12 gene encodesa transcription factor that binds to a DNA sequencereferred to as the pheromoneresponsiveelement,which is presentin many different a- and a-specificURSs.Binding of mating factors to cell-surfacereceptors induces a cascadeof signaling events, resulting in phosphorylation of various proteins including the Ste12protein (seeFigure l6-28a). This rapid phosphorylation is correlated with an increasein the ability of Ste12 to stimulate transcription. It is not yet known, however, whether Ste12 must be phosphorylatedto stimulate transcription in responseto pheromone. Interaction of Ste12 protein with DNA has been studied most extensively at the URS controlling transcription of STE2, an a-specific gene encoding the receptor for the cr pheromone. Pheromone-inducedproduction of the o receptor encoded by STE2 increasesthe efficiency of the mating process.Adjacent to the a-specificURS in the STE2 geneis a pheromone-responsive element that binds Ste12. When a cells are treated with ct pheromone, transcription of the STE2 geneincreasesin a processthat requiresSte12protein. Ste12 protein has been found to bind most efficiently to the pheromone-responsiveelement in the STE2 URS when MCM1 is simultaneouslybound to the adjacent P site. I(e saw previously that MCM1 can act as an activator or a repressor at different URSs depending on whether it complexeswith a1 or o.2.In this case,the function of MCM1 as an activator is stimulated by the binding of yet another transcription factor, Ste12, whose activity is modified by extracellular signals.

ruucrear tusion J

Cell-Type Specification in Yeast D i p l o i dc e l l

+Nutient7/ Mitotic growth

\llrtri"ntt Meiosis

\

oo oo Sporulation

A FIGURE 21-19 Pheromone-induced matingof haploidyeast cells.Thea cellsproduce o matingfactoranda-factorreceptor; the a cellsproduce a factorando.-factor receptorBinding of the mating factorsto theircognatereceptors on cellsof theopposite typeleads to geneactivation, resulting in matingandproduction of diploid cellsInthe presence of sufficient nutrients, thesecellswillgrowas diploidsWithoutsufficient nutrients, cellswill underqomeiosis and formfour haploid spores. Studies with yeast mutants have provided insights into how the a and ct pheromones induce mating. For instance, haploid yeast cells carrying mutations in the sterile 12 (STE12) locus cannot respond to pheromones and do not 924

C H A P T E R2 1

I

C E L LB I R T H ,L I N E A G EA , ND DEATH

r Specificationof each of the three yeast cell types-the a and cr haploid cells and the diploid a"/acells-is determined by a unique set of transcription factors acting in different combinations at specific regulatory sites in the yeast genome (seeFigure 21,-1,7). r Some transcription factors can act as repressorsor activators depending on the specificregulatory sitesthey bind and the presenceor absenceof other transcription factors bound to neighboringsites. r Binding of mating-type pheromones by haploid yeast cells activates expressionof genesencoding proteins that mediate mating, thereby generatingthe third yeastcell type (seeFigure 21-19).

Specification and Differentiation of Muscle An impressivearray of molecular strategies,some analogous to those found in yeast cell-type specification,have evolved to carry out the complex developmentalpathways that characterizemulticellular organisms.Muscle cells have been the focus of many such studiesbecausetheir developmentcan be studied in cultured cells as well as in intact animals. Early

advancesin understandingthe formation of muscle cells (myogenesis)came from discovering regulatory genes that could convert cultured cells into muscle cells. Then mouse mutations affecting those geneswere created and studied to learn the functions of the proteins encoded by these genes, following which scientistshave investigatedhow the muscle regulatory genescontrol other genes. Recent microarray studies have looked for genes whose transcription differs in various subtypesof muscle in mice. These studies have identified 49 genes out of the 3000 genesexamined that are transcribed at substantially different levelsin red (endurance)muscle and white (fast response) muscle. Clues to the molecular basis of the functional differences between red and white muscle are likely to come from studying those 49 genesand their products. Here we examine the role of certain transcription factors in creating skeletal muscle in vertebrates.These muscle regulators illustrate how coordinated transcription of sets of target genescan produce differentiated cell types and how a cascadeof transcriptional eventsand signals is necessaryto coordinate cell behaviors and functions.

EmbryonicSomitesGive Riseto Myoblasts Vertebrate skeletal myogenesis proceeds through three stages:determination of the precursor muscle cells, called myoblasts,which commits them to a muscle cell fate; proliferation and in some casesmigration of myoblasts; and their terminal differentiation into mature muscle (Figure 21,-20). In the first stage,myoblasts arise from blocks of mesoderm cells, called somites,that are located next to the neural tube in the embryo. Specificsignalsfrom surrounding tissueplay an important role in determining where myoblasts will form in the developing somite. At the molecular level, the "decision" of a mesoderm cell to adopt a muscle cell fate reflectsthe activation of genesencodingparticular transcription factors. As myoblasts proliferate and migrate, say, to a developing limb bud, they becomealigned, stop dividing, and fuse to form a syncytium (a cell containing many nuclei but sharing 'We a common cytoplasm). refer to this multinucleate cell as a myotube. Concomitant with cell fusion is a dramatic rise in the expressionof genesnecessaryfor further muscle development and function. The specific extracellular signals that induce determination of each group of myoblasts are expressedonly transiently. These signalstrigger production of intracellular factors that maintain the myogenic program after the inducing signalsare gone. We discussthe identification and functions of these myogenic proteins, and their interactions, in the next severalsections.

M y o g e n i cG e n e sW e r e F i r s tl d e n t i f i e di n S t u d i e s with CulturedFibroblasts Myogenic genesare a fine example of how transcription factors control the progressivedifferentiation that occurs in a

Dermomyotome (givesrise to dermis of skin and muscle precursors)

Somite MyoD/MyfS

| [

O"ter-lnation

+ .-:\

€)(D\r9G Myoblasts

M y o g e n iann dM E F s| [ l

e r o t i t e r a t i oann d / o rm i g r a t i o n

*

Premusclemasses

I andcellfusion I p Oitterentiation v Myotube (muscle celll

21-20 Threestagesin developmentof vertebrate A FIGURE spheres of embryonic areepithelial muscle. Somites skeletal becomedetermined cells,someof which(themyotome) mesoderm (tr). Afterthe fromothertissues signals afterreceiving asmyoblasts proliferate andmigrateto the limbbudsandelsewhere myoblasts (Z), theyundergo intomultinucleate terminaldifferentiation (B). Keytranscription factors cells,calledmyotubes muscle skeletal programarehighlighted in yellowSee that helpdrivethe myogenic a l s oF i g u r2e1 - 2 3 .

cell lineage. In vitro studies with the fibroblast cell line designated C3H 1.0TV2have played a central role in dissecting the transcription control mechanisms regulating skeletal myogenesis.When incubated in the presenceof 5azacytidine (a cytidine derivative that cannot be methylated), C3IH l\Tyz cells differentiate into myotubes. Upon entry into cells, S-azacytidineis converted to 5-azadeoxycytidine triphosphate and then is incorporated into DNA in place of deoxycytidine. Becausemethylated deoxycytidine residuescommonly are present in transcriptionally inactive DNA regions, replacementof cytidine residueswith a derivative that cannot be methylated may permit activation of genespreviously repressedby methylation. The high frequency at which azacytidine-treated C3H 10Ty2 cells are converted into myotubes suggestedto early workers that reactivation of one or a small number of closely linked genesis sufficient to drive a myogenic program. To test this hypothesis, researchersisolated DNA from C3H 10T\/z cells grown in the presence of S-azacytidine and transfectedit into untreated cells. The observation that 1 in 104 cells transfected with this DNA was converted into a myotube is consistentwith the hypothesisthat one or a small set of closely linked genesis responsiblefor converting fibroblasts into myotubes. Subsequentstudies led to the isolation and characterization of four different but related genes that can convert S P E C I F I C A T I OANN D D I F F E R E N T I A T I OONF M U S C L E

925

> EXPERIMENTAL FIGURE 21-21Myogenicaenesisolatedfrom azacytidine-treated cellscan drive myogenesiswhen transfectedinto other cells.(a)WhenC3H'10T%cells(afibroblast cellline)aretreated with azacytidine, theydevelop intomyotubes at highfrequency. Toisolatethe genesresponsible for converting azacytidine-treated cellsinto myotubes, allthe mRNAs fromtreated cellsfirstwereisolated fromcellextracts on an oligo-dT column. Because of theirpoly(A) tails,mRNAs areselectively retained on this column. TheredmRNAs represent molecules in azacytidineenriched treatedcells; the pinkonesareallothermRNAsStepsI andE:The isolated mRNAs wereconverted to radiolabeled cDNAs.StepB: Whenthe cDNAsweremixedwith mRNAsfromuntreatedC3H10f1/z (lightred)produced cells,onlycDNAs derived frommRNAs by both azacytidine-treated cellsanduntreated Theresulting cellshybridized. double-stranded DNAwasseparated fromthe unhybridized cDNAs (darkblue)produced onlyby azacytidine-treated cells.StepZl: The cDNAsspecific for azacytidine-treated cellsthenwereusedasprobes to screen a cDNAlibrary fromazacytidine-treated cells(seeFigure5-16). At leastsomeof the clonesidentified with theseprobescorrespond to genesrequired (b)Eachof thecDNAclones for myogenesis identified in part(a)wasincorporated intoa plasmid carrying a strongpromoter. StepsI and E: C3H10T1/z cellswerecotransfected with each plasmid recombinant plusa second plasmid carrying a geneconferring resistance to an antibiotic calledG418;onlycellsthat haveincorporated the plasmids willgrowon a mediumcontaining G418.Oneof the selected clones, designated myoD,wasshownto driveconversion of C3H10f1/z cellsintomuscle cells, identified bytheirbinding of labeled antibodies against protein(stepE) [See myosin, a muscle-specific RL Davis etal,1987,Cell51:987 l

C3I{ 10T1/z cells into muscle. Figure 21-21 oudines rhe experimental protocol for identifying and assaying one of these genes,called the myogenic determination (myoD) gene. C3H 10Ty2 cells transfectedwith myoD cDNA and those treatedwith S-azacytidineboth formed myotubes.The myoD cDNA alsowas able to converra number of other cultured cell linesinto muscle.Basedon thesefindings, the myoD genewas proposedto play a key role in muscledevelopment.A similar approach identified three other genes-myogenin, myfS, and mrf4-that also function in muscledevelopment.

Two Classesof RegulatoryFactorsAct in Concertto Guide Productionof MuscleCells The four myogenic proteins-MyoD, Myf5, myogenin, and MRF4-are all members of the basic helix-loop-helix (bHLH) family of DNA-binding transcription factors (see Figure 7-26). Near the center of these proteins is a basic DNA-binding (B) region adjacentto the HLH domain, which mediatesdimer formation. Flanking this central DNA- binding/dimerizationregion are tvvo activation domains. We refer to the four myogenic bHLH proteins collectively as muscle regulatory factors, or MRFs (Figure21-22a). bHLH proteinsform homo- and heterodimersthat bind to a 6-bp DNA site with rhe consensussequenceCANNTG (N : any nucleotide). Referred to as the E box, this sequenceis presentin many different locations within the genome (on a purely random basis the E box will be found every 256 nucleotides).Thus some mechanism(s)must ensurethat MRFs 926

C H A P T E R2 1

I

C E L LB I R T H ,L I N E A G EA , ND DEATH

(a) Screenfor myogenicgenes .\A'iNNNAAANA .vttuwtAiAniA

Total mRNA from azacvtidine-treated cells lncubatewith reverse transcriptaseand [32P]dNTPs RemovemRNAs 1AA^fl\AAA/TA .IAAAATINA^AA

3 2 P - l a b e l e dc D N A s

Hybridizewith excessof mRNAs from untreated C3H 10Tt/zcells

Discard

()

.I A NN .\,\'\N,\'\

'\,\'V A,I,\,!

32P-labeled cDNAs specificfor azacytidine-treated cells

ScreencDNA library from treatedcells

lsolatecDNAscharacteristic of azacytidine-treatedcells

(b) Assay for myogenicactivityof myoD cDNA

H 1OTt/zcell

E

z

Transfectwith a olasmid carryingmyoD cDNA and a plasmid conferringresistance to G418 Selecton G418-containing m e d i u mf o r c e l l st h a t tookup bothplasmids

(a) Structure of muscle-regulatoryfactors {MRFs) DNAbinding/dimerization H z N Transactivationl B

I

HLH lTransactivation

cooH

MEF-interactinq domain (b) Structure of myocyte-enhancingfactors (MEFs) DNAbinding/dimerization HzN

MADSIMEF

I

Transactivation

tion with myogenin (Figure21-22b). The synergisticaction of the MEF homodimer and MRF-E2A heterodimer is thought to drive high-levelexpressionof muscle-specificgenes. Knockout mice and Drosophila mutants have been used to explore the roles of MRF and MEF proteins in conferring myogenic specificityin intact animals, extending the work in cell culture. These experimentsdemonstratedthe importance of three of the MRF proteins (MyoD, Myf5, and myogenin) and of MEF proteins for distinct steps in muscle development (see Figure 21,-20).The function of the fourth myogenic protein, Mrf4, is not entirely clear.

cooH

M R F - i n t e r a c tdi nogm a i n FIGURE 21-22 Generalstructuresof two classes of (muscle transcriptionfactorsthat participatein myogenesis. MRFs regulatory factors), suchasmyogenin andMyoD,arebHLH(basic helixproteins produced loop-helix) onlyin developing muscleMEFs (myocyte-enhancing factors), whichareproduced in several tissues in addition to developing muscle, belong to theMADSfamily. The myogenic activity of MRFs isenhanced bytheirinteraction with MEFs Thedomain structures of theproteins areshown,including (B),helix-loop-helix (HLH), transactivation, basic MADSandMEF domains,

specificallyregulatemuscle-specific genesand not other genes containing E boxesin their transcription-controlregions.One clue to how this myogenicspecificityis achievedwas the finding that the DNA-binding affinity of MyoD is tenfold greater when it binds as a heterodimercomplexedwith E2A, another bHLH protein, than when it binds as a homodimer.Moreover, in azacytidine-treatedC3H 10Ty2 cells,MyoD is found as a heterodimercomplexed with E2A, and both proteins are required for myogenesisin these cells. The DNA-binding domains of E2A and MyoD have similar but not identicalamino acid sequences, and both proteins recognizeE box sequences. The other MRFs also form heterodimerswith E2A that have properties similar to MyoD-E2A complexes. This heterodimerization restricts activity of the myogenic transcription factors to genesthat contain at leasttwo E boxeslocated closeto each other. Since E2A is expressedin many tissues,the requirement for E2A is not sufficient to confer myogenic specificity.Subsequent studies suggestedthat specific amino acids in the bHLH domain of all the MRFs confer myogenic specificity by allowing MRF-E2A complexesto bind specificallyto another family of DNA-binding proteins called myocyte enhancing factors, or MEFs. MEFs were consideredexcellent candidatesfor interaction with MRFs for two reasons.First, many muscle-specificgenescontain recognition sitesfor both MEFs and MRFs. Second, although MEFs cannot induce myogenic conversionof azacytidine-treated C3H 10T% cells by themselves,they enhancethe ability of MRFs to do so. This enhancementrequires physical interaction between a MEF and MRF-E2A heterodimer. MEFs belong to the MADS family of transcription factors and contain a MEF domain, adjacent to the MADS domain, that mediatesinterac-

Differentiationof Myoblastsls Under Positive and NegativeControl Powerful developmentalregulators like the MRFs cannot be allowed to run rampant. In fact, their actions are ctrcumscribedat severallevels.First, production of the muscleregulators is activatedonly in mesodermcellsin responseto locally acting signals,suchas Hedgehog,'Wnt,and BMP, that are produced at the right time and place in the embryo. Other proteinsmediateadditional mechanismsfor assuringtight control over myogenesis:chromatin-remodelingproteins are needed to make target genesaccessibleto MRFs; inhibitory proteins can restrict when MRFs act; and antagonistic relations between cell-cycle regulators and differentiation factors like MRFs ensurethat differentiatingcellswill not divide, and vice versa.All thesefactorscontrol when and where musclesform. Activating Chromatin-Remodeling Proteins MRF proteins control batteriesof muscle-specificgenes,but can do so only if chromatin factors allow access.Remodeling of chromatin, which usually is necessaryfor gene activation, is carried out by large protein complexes(e.g.,S'$fVSNFcomplex) that have ATPaseand perhaps helicaseactivity. These complexes recruit histone acetylasesthat modify chromatin to make genesaccessibleto transcription factors (Chapter 7). The hypothesis that remodeling complexes help myogenic factors was tested using dominant-negativeversions of the ATPaseproteins that form the cores of thesecomplexes.(Recall from Chapter 5 that a dominant-negativemutation producesa mutant phenotype even when a normal allele of the '$fhen genescarrying these dominantgene also is present.) negative mutations were transfectedinto C3H 10T72 cells, the subsequentintroduction of myogenic genes no longer converted the cells into myotubes. In addition, a musclespecific gene that is normally activated did not exhibit its usual pattern of chromatin changesin the doubly transfected C3H t0T1/z cells. These results indicate that transcription activation by myogenic proteins dependson a suitable chromatin structure in the regions of muscle-specificgenes. MEF2 recruits histone acetylasessuch as p300/CBR through another protein that serves as a mediator, thus activating transcription of target genes.Chromatin immunoprecipitation experimentswith antibodies against acetylated histone H4 show that the acetylatedhistone level associated with MEF2-regulatedgenesis higher in differentiatedmyotubes than in myoblasts (seeFigure 7-37). A N D D I F F E R E N T I A T IO OF NM U S C L E SPECIFICATIO

927

Inhibitory Proteins Screeningfor genesrelated to myoD led to identification of a related protein that retains the HLH dimerization region but lacks the basic DNA-binding region and henceis unable to bind to E box sequencesin DNA. By binding to MyoD or E2A, this protein inhibits formation of MyoD-E2A heterodimersand hencetheir high-affinity binding to DNA. Accordingly, this protein is referred to as Id, for inhibitor of DNA binding. Id prevents cells that produce MyoD andE2A from activating transcription of the musclespecific gene encoding creatine kinase. As a result, the cells remain in a proliferative growth state. When these cells are induced to differentiate into muscle (for instance, by the removal of serum, which contains the growth factors required for proliferative growth), the Id concentration falls. MyoD-E2A dimers now can form and bind to the regulatory regions of target genes, driving differentiation of C3H 10Ty2 cellsinto myoblast-likecells. The role of histone deacetylases, which inhibit transcription, in muscle developmentwas revealedin experimentsin which scientists first introduced extra myoD genesinto cultured C3H 10T1/zcells to raise the level of MyoD. This resulted in increasedactivation of target genesand more rapid differentiation of the cells into myotubes. However, when genesencodinghistonedeacetylases also were introduced into the C3H 10Ty2 cells, the muscle-inducingeffect of MyoD was blocked and the cellsdid not differentiateinto myotubes. The explanation for how histone deacetylases inhibit MyoDinduced muscledifferentiation came from the surprisingfinding that the musclegeneactivator MEF2 can bind, through its MADS domain, to a histone deacetylase.This interaction, which can preventMEF2 function and muscledifferentiation, is normally blocked during differentiation becausethe histone deacetylaseis phosphorylatedby a Ca2*lcalmodulin-dependent protein kinase;the phosphorylateddeacetylasethen is moved from the nucleus to the cytoplasm. Taken together,theseresults indicate that activation of muscle genesby MyoD and MEF2 is in competition with inactivation of musclegenesby repressivechromatin structures. Cell-CycleProteins The onsetof terminal differentiation in many cell types is associatedwith arrest of the cell cycle, most commonly in G1, suggestingthat the transition from the determined to differentiated state may be influenced by cell-cycle proteins including cyclins and cyclin-dependent kinases(Chapter20). For instance,certain inhibitors of cyclin-dependentkinasescan induce muscledifferentiationin cell culture, and the amounts of these inhibitors are markedly higher in differentiating muscle cells than in nondifferentiating ones in vivo. Converseln differentiation of cultured myoblasts can be inhibited by transfecting the cells with DNA encodingcyclin D1 under the control of a constitutively active promoter. Expression of cyclin D1, which normally occursonly during G1, is induced by mitogenic factors in many cell types and drives the cell cycle (seeFigure 20-32). The ability of cyclin DL to prevent myoblast differentiation in vitro may mimic aspectsof the in vivo signals that antagonize the differentiation pathway. The antagonism between negative and positive regulators 928

C H A P T E R2 1

I

C E L LB I R T H ,L I N E A G EA , ND DEATH

of G1 progression is likely to play an important role in controlling myogenesisin vivo.

C e l l - C e lS l i g n a l sA r e C r u c i a fl o r D e t e r m i n a t i o n a n d M i g r a t i o no f M y o b l a s t s As noted akeady,after myoblasts arisefrom somites,they must move to their proper locations and form the correct attachments as they differentiate into musclecells (Figure 21,-23).Expression of myogenic genes often occurs after elaborate events that tell certain somite cells to delaminate from the somite epithelium and guide their subsequentmovementsto muscle assemblysites. A transcription factor, Pax3, is produced in the subsetof somite cells that will form muscle.Pax3 appearsto be at the top of the regulatory hierarchy controlling muscleformation in the body wall and limbs. Myoblasts that will migrate, but not cells that remain behind, also produce a transcription factor called Lbx1. If Pax3 is not functional, Lbxl transcripts are not produced and myoblastsdo not migrate. Both Pax3 and Lbxl can affect expressionof myoD. The departure of myoblasts from somitesalso dependsupon reception of a secreted protein signal appropriately called scatter factor, Qr hepatocyte grocuth factor (9F/HGF). This signal, which is produced by embryonic connectivetissuecells (mesenchyme) in the limb buds, attracts migrating myoblasts, thus directing them to their proper destination. Production of SF/HGF is previously induced by still other secretedsignals.If the SF/HGF signal or its receptor on myoblasts is not functional, somite cells will produce Lbxl but not go on to migrate; thus no muscleswill form in the limbs. Expressionof the myogenin and mrf4 genesdoes not begin until migrating myoblasts approach their limb-bud destinations(seeFigure 21.-201.

Dermamyotome (gives rise to dermis of skin and to muscle)

N e u r a lt u b e Myoblasts (migratefrom the myotome to form skeletaland limb muscles)

Epidermis

o

Notochord

Sclerotome (givesriseto skeletal structures suchas vertebrae) FIGURE 21-23 Embryonicdeterminationand migrationof myoblastsin mammals.Afterformation of the neuraltube,each somiteformssclerotome, whichdevelops intoskeletal structures, and dermomyotome, whichgivesriseto the dermisof theskinandto the muscles. Lateral myoblasts migrate fromthe dermomyotome to the limbbud;medialmyoblasts develop intothetrunkmuscles. The givesriseto the connective remainder of a dermomyotome tissueof the skin [Adapted fromM Buckingiam, 1992,Trends Genet8:1441

Vertebrate myogenesis myogenin mRNA

E2,A(general)

^. /V\^+

6;;A I mrfal?l

\ -

Myf5, MyoD*

f

-1 MYogenin

I {others) | Muscleprecursor genes

Precursor determination

Differentiation

< FfGURE 21-24 Comparisonof genes that regulatevertebratemyogenesisand factors bHLHtranscription fly neurogenesis. functions in determination of haveanalogous precursor cellsandtheir neuralandmuscle intomature subseouent differentiation the andmuscle cellsIn bothcases, neurons genes proteinsencodedby the earliest-acting (/eft)areunderboth positiveand negative controlby otherrelatedproteins(bluetype) fromY N Janand Seetextfor details.[Adapted L Y Jan.1993,Cell75:827 |

Fly neurogenesis

genes

Emc

precursor genes

'We

have touched on just a few of the many external signalsand transcription factors that participate in development of a properly patterned muscle. The function of all these regulatory molecules must be coordinated both in spaceand in time during myogenesis.

bHLHRegulatoryProteinsFunctionin Creation of Other Tissues Four bHLH transcription factors that are remarkably similar to the myogenic bHLH proteins control neurogenesisin Drosophila. Similar proteins appearto function in neurogenesis in vertebrates and perhaps in the determination and differentiation of hematopoieticcells. The neurogenicDrosophila bHLH proteins are encoded by an -100-kb stretch of genomic DNA, termed the achaete-scutecomplex (AS-C), containing four genesdesignated achaete (ac), scute (sc), letbal of scute (l'sc), and asense1'a/.Analysis of the effects of loss-of-function mutations indicate that the Achaete (Ac) and Scute (Sc) proteins participate in determination of neuronal stem cells, called neuroblasts in flies, while the Asense (As) protein is required for differentiation of the progeny of these cells into neurons. (Note that the term neuroblastsrefers to stem cells in flies but to precursor cells in mammals.) These functions are analogousto the roles of MyoD and Myf5 in muscle determination and of myogenin in differentiation. Two other Drosophila proteins, designated Da and Emc, are analogous in structure and function to vertebrate E2A and Id, respectively. For example, heterodimeric complexes of Da with Ac or Sc bind to DNA better than the homodimeric forms of Ac and Sc. Emc, like Id, lacks any DNA-binding domain; it binds to Ac and Sc proteins, thus inhibiting their associationwith Da and binding to DNA. The similar functions of these myogenic and neurogenic proteins are depicted in Figure 21-24.

A family of bHLH proteins related to the Drosophila Achaete and Scute proteins has been identified in vertebrates. One of these,called neurogenin, controls the formation of neuroblasts. In situ hybridization experiments showed that neurogenin is produced at an early stagein the developing nervous system and induces production of NeuroD, another bHLH protein that acts later (Figure 21'-25a). Injection of large amounts of neurogeniz mRNA into Xenopas embryos further demonstratedthe ability of neurogenin to induce neurogenesis(Figure 21,-25b).The function of neurogenin is analogous to that of the Achaete and Scute in Drosophila; likewise, NeuroD and Asensemay have analogous functions in vertebratesand Drosophila.

Specification and Differentiation of Muscle r Development of skeletal muscle begins with the signalinduced determination of certain mesodermcellsin somites as myoblasts. Following their proliferation and migration, myoblasts stop dividing and differentiate into multinucleate muscle cells (myotubes) that express muscle-specific proteins (seeFigure 21'-20). r Four myogenic bHLH transcription factors-MyoD, myogenin, Myf5, and MRF4, collectively called muscleregulatory factors (MRFs)-associate with E2A and MEFs to form large transcriptional complexesthat drive myogenesisand expressionof muscle-specificgenes. r Dimerization of bHLH transcription factors with different partners modulates the specificity or affinity of their binding to specificDNA regulatory sites,and also may prevent their binding entirely. r The myogenic program driven by MRFs dependson the SITVSNF chromatin-remodeling complex, which makes targetgenesaccessible. S P E C I F I C A T I OANN D D I F F E R E N T I A T I OONF M U S C L E

929

(b) (a)

neurogenin mRNA

neuroDmRNA

B-tubulin mRNA

neurogeninmRNA

A EXPERIMENTAL FIGURE 21-25tn situ hybridizationand injectionexperimentsdemonstratethat neurogeninacts (a)Sections before NeuroDin vertebrateneurogenesis. of rat neuraltubeweretreatedwith a probespecific for neurogenrn mRNA (/eft)or neuroDmRNA(nght)Theopenspacein the centeristhe ventricle, andthe cellsliningthiscavityconstitute the subventricular layerAll the neuralcellsarebornin thesubventricular layerandthen migrate outward(seeFigure 21-12), As illustrated in these micrographs, neurogenin mRNAisproduced in proliferating neuroblasts in the subventricular layer(arrow), whereas neuroD

mRNAis present in migrating neuroblasts thathaveleftthe (b)Oneof thetwo cellsin earlyXenopus ventricular zone(arrow). embryos wasinjected with neurogenrn mRNA(inj)andthenstained with a probespecific for neuron-specific mRNAs encoding B-tubulin (/eft)or NeuroD(right).Theregionof the embryoderivedfromthe uninjected cellservedasa control(con)Theneurogenrn mRNA induced a massive increase in the numberof neuroblasts expressing neuroDmRNAandneurons expressing mRNAin the region B-tubulin of the neuraltubederived fromthe injected cell lrromQ Maetal, 1996,Cell87:43; courtesyof D J Anderson l

r The myogenic program is inhibited by bindingof Id protein to MyoD, which blocks binding of MyoD to DNA, and by histone deacetylases,which repress activation of target genesby MRFs.

may yield unequal daughter cells, for example, one that remains attachedto a stalk and one that developsflagella used for swimming. Essential to asymmetric cell division is polarization of the parental cell and then differential incorporation of parts of the parental cell into the two daughters(Figure 21-26). A variety of molecular mechanismsare employed to createand propagate the initial asymmetry that polarizes the parental cell. In addition to being different, the daughter cells must often be placed in a specific orientation with respectto sur'When rounding structures. stem cells divide asymmetrically, the cell that remains in contact with niche signalswill persist as a stem cell. Therefore, the mitotic spindlesand cell polarity must be aligned with the overall tissueso that differentiating cellsmove off in the right direction, and so that at least one daughter remains in the stem-cellniche to perpetuatethe stem-cellpopulation. This phenomenonis exemplified in the division of neural stem cells during embryonic development ( s e eF i g u r e2 1 - 1 2 b ) . We begin with an especiallywell-understood example of asymmetric cell division, the budding of yeast cells, and move on to recently discoveredprotein complexesimportant for asymmetric cell divisions in multicellular organisms.Nfe seein the yeast example an elegant systemthat links asymmetric division to the processof controlling cell type.

r Migration of myoblasts to the limb buds is induced by scatter factor/hepatocytegrowth factor (SF/HGF), a protein signal secretedby mesenchymalcells (seeFigure 21, 23). Myoblastsmust expressboth the Pax3 and Lbxl transcription factors to migrate. r Terminal differentiation of myoblasts and induction of muscle-specificproteins do not occur until myoblasts stop dividing and migrating. r Neurogenesisin Drosopbila dependsupon a set of four neurogenicbHLH proteins that are conceptuallyand structurally similar to the vertebrate myogenic proteins (see Figure21-24 ). r A related vertebrate protein, neurogenin, is required for formation of neural precursors and also determines their fate as neurons or glial cells.

Regulationof Asymmetric Cell Division During embryogenesis,the earliest stagein animal development, asymmetriccell division often createsthe initial diversity that ultimately culminares in formation of specific differentiatedcell types. Both stem cells and precursor cells can divide asymmetricallyvia similar mechanisms,although the details vary with the tissue. Even in bacteria, cell division

930

C H A P T E R2 1

|

C E L LB t R T H ,L t N E A G EA , ND DEATH

YeastMating-TypeSwitchingDependsupon AsymmetricCell Division S. cereuisiaecells use a remarkable mechanismto control the differentiation of the cells as the cell lineage progresses. 'Sfhether a haploid yeast cell exhibits the ct or a maring rype

/-\-o-Bud

t0)

\^/

Polarized parentalcell

Division

'O Localizeddeterminants ( p r o t e i no r m R N A )

@o *o"n,

\ Asymmetric localization of cell-fate determinants

HOtt Sr

M

\

\tttoHOtranscription \

)o

\"switching

'O@o

MDMD

A FIGURE 21-26 Generalfeaturesof asymmetriccelldivision. Various mechanisms canleadto asymmetric distribution of (red proteins cytoplasmic components, suchasparticular or mRNAs parental dots),thereby forminga polarized cell Division of a polarized cellwill be asymmetric if the mitoticspindle isoriented so cytoplasmic aredistributed unequally thatthe localized components if thespindle is to thetwo daughter cells,asshownhere.However, positioned relative differently to the localized cytoplasmic components, cells. division of a polarized cellmayproduce equivalent daughter

is determined by which genesare present at the MAT locus (seeFigure 2t-1,71.As describedin Chapter 7, the MAT locus in the S. cereuisiaegenome is flanked by two "silent," transcriptionally inactive loci containing the alternative o or a sequences(see Figure 7-33\. A specific DNA rearrangement brings the genesthat encodethe cr-specificor a-specific transcription factors from thesesilent loci to the active MAT locus where they can be transcribed. Interestingly,some haploid yeast cells can switch repeatedly between the cr and a types. Mating-type switching occurs when the a allele occupying the MAT locus is replacedby the a allele,or vice versa.The first step in this processis catalyzed by HO endonuclease,which is expressedin mother cells but not in daughtercells.Thus mating-typeswitching occursonly in mother cells (Figure 21-27). Transcription of the HO gene is dependenton the S\fVSNF chromatin-remodelingcomplex (seeFigure 7-431,the samecomplex that we encounteredearlier in our discussionof myogenesis.Daughter yeastcellsarising by budding from mother cells contain a protein called Ashlp (for Asymmetric synthesisof HO) that prevents recruitment of the SWUSNFcomplex to the HO gene,thereby preventingits transcription.The absenceof Ashl from mother cellsallows them to transcribethe HO gene. Recentexperimentshave revealedhow the asymmetry in the distribution of Ashl betweenmother and daughter cells is established.ASHL mRNA accumulatesin the growing bud that will form a daughter cell due to the action of a myosin motor protein (Chapter 17). This motor protein, called

21-27 Switchingof matingtype in haploidyeast A FIGURE by buddingformsa largermothercell(M)andsmaller cells.Division (D), bothof whichhavethe samematingtypeasthe cell daughter Themothercellcanswitchmating cell(crin thisexample) original typeduringG, of the nextcellcycleandthendivideagain,producing on transcription depends a type.Switching two cellsof theopposite of Ashl protein. onlyin the absence of the HOgene,whichoccurs cannot Ashl protein, cells,whichproduce daughter Thesmaller theydivideto form switch;aftergrowingin sizethroughinterphase, cellsandarrowsindicate cell.Orange a mothercellanddaughter switchevents

Myo4p, moves the ASHL mRNA, as a ribonucleoprotein complex, along actin filaments in one direction only, toward the bud (Figure 21,-281.Two connector proteins, termed She2p and She3p (for S$7l5p-dependentHO expression) tether the ASH1 mRNA to the Myo4p motor protein. By the time the bud separatesfrom the mother cell, the mother cell is largely depletedof ASHI mRNA and thus can switch mating type in the following G1 before additional ASH1 mRNA is produced and before DNA replication in the S phase. Budding yeastsuse a relatively simple mechanismto create molecular differencesbetween the two cells formed by division. In higher organisms,as in yeast,the mitotic spindle must be oriented in such a way that each daughter cell receivesits own set of cytoplasmic components. Genetic studies in C. elegansand Drosophila have revealedthe key participants, a first step in understandingat the molecular level how asymmetriccell division is regulatedin multicellular organisms.To illustrate thesecomplexities,we focus on asymmetric division of neuroblastsin Drosophila'

ProteinsThat RegulateAsymmetryAre Localizedat OppositeEndsof Dividing Neuroblastsin DrosoPhila Fly neuroblasts,which are stem cells, arise from a sheet of ectoderm cells that is one cell thick. As in vertebrates' the Drosophila ectoderm forms both epidermis and the nervous system, and many ectoderm cells have the potential to O F A S Y M M E T R I CC E L LD I V I S I O N REGULATION

931

Video: ASHl mRNA Localization > FIGURE 21-28 Model for restrictionof mating-type switchingto mother cellsin S. cerevisiae. Ashl protein prevents a cellfromtranscribing theHOgenewhoseencoded proteininitiates the DNArearrangement that results in matingtypeswitching froma to cror a to a Switching occurs onlyin the mothercell,afterit separates froma newlybuddeddaughter cell, because of the presence of Ashl proteinonlyin the daughter cell Themolecular basisfor thisdifferential localization of Ashl isthe one-way transport of ASHImRNAintothe bud.A linkingprotein, She2p, bindsto specific 3' untranslated sequences in theASHI mRNAandalsobindsto She3pproteinThisproteinin turnbinds to a myosinmotor,Myo4p,whichmovesalongactinfilaments into the bud [See S KoonandB J Schnapp, 2001,Curr. Biology 1 1 : R 1I6 6

assumeeither a neural or epidermal fate. Under the control of genes that become active only in certain cells, some of the cells increasein size and begin to loosen from the ectodermal layer. At this point, the delaminating cells use the DeltaArlotch signaling pathway to mediate lateral inhibition of their neighbors, causing them to retain the epidermal fate (seeFigures 16-35 and 22-42). The delaminating cells move inside and becomesphericalneuroblasts,while the prospective epidermal cells remain behind and close up to form a tight sheet.This processgenerates50 neuroblasts

ASHI mRNA

\,.--

Bud

VV

in each body segment, which will give rise to about 700 neurons per segment. Once formed, the neuroblastsundergo asymmetric divisions,at each division recreatingthemselvesand producing a ganglion mother cell (GMC) on the basal side of the neurobIast (Figure 21-29). A single neuroblast will produce several GMCs; eachGMC in turn forms two neurons.Dependingon where they form in the embryo and consequent regulatory events,neuroblastsmay form more or fewer GMCs. NeurobIasts and GMCs in different locations exhibit different

EctodermafCell Divisionsin the DrosophilaEmbryo > F|GURE 21-29 Asymmetriccelldivisionduring Drosophila neurogenesis. Theectodermal sheet(tr) of theearlyembryo givesriseto bothepidermal cellsandneuralcells.Neuroblasts, the stemcellsfor the fly neryous system, areformedwhenectoderm cellsenlarge, separate fromthe ectodermal epithelium, andmove intothe interiorof the embryo(Z-4). Eachneuroblast thatarises divides asymmetrically to recreate itselfandproduce a ganglion mothercell,or GMC(E) Subsequent divisions of a neuroblast produce moreGMCs,creating a stackof theseprecursor cells(6) EachGMCdivides (Z). Neuroblasts onceto giveriseto two neurons andtheirneuronal descendants canhavedifferent fatesdepending on theirlocationThemicrograph showsan asymmetrically dividing Drosophila neuroblast. Theapicalend(red)willforma newneuroblast andthe basalend(blueandred)willforma GMC.Themicrotubules arelabeled in green[photograph courtesy of Dr.C Q Doe,University of Oregon l

E

z E

E E

Z Neur

932

G H A P T E R2 t

I

C E L LB t R T H ,L t N E A G EA . ND DEATH

Ectodermalcells

Surface epidermis It tnt"rio,.nervous system -,|

patterns of gene expression, an indicator of their fates. Analysis of fly mutants led to the discovery of key proteins that (1) establishapical-basalpolarity in neuroblasts,(2) align the mitotic spindle of dividing neuroblastswith their polarity, and (3) direct formation of daughtercellswhose fate and size differ from that of neuroblasts.Genetic studies of asymmetric cell divisions in the C. elegansearly embryo independently led to the discovery of important cell division asymmetry proteins. The machinery controlling asymmetric cell division is highly conservedand readily recognizedfrom worms to flies to mammals, indicating conservationof protein functions for more than half a billion years. Basal and apical protein complexes congregate during each neuroblast division, disperse,and then localize again for the next round of division. Four protein complexes, which we denote as MPSB, BPR DSL, and IPLG, govern the entire process(Figure 21,-30\: t BPP, an apical complex also known as the PAR complex, is responsiblefor defining the end of the cell that will remain a neuroblast.It comprisesBazooka and Par6, both of which containPDZ domains. and aPKC. an atypical isoform of protein kinase C. t IPLG, a secondapical complex, is composedof lnscuteable (Insc),Partner of inscuteable(Pins),Locomotion defects (Loco), and G;, a heterotrimericG protein (Chapter 15). This complex is critical for orienting the spindleduring asymmetric divisions. r DSL, a complex that is fairly evenly distributed around the cell, is composedof Discs-large(Dlg), Scribble(Scrib),

Proteincomplexesr I

see

IPLG

MPSB

*

K/ INTERPHASE NEUROBLAST

+

Neuroblast

GMC

ANAPHASE NEUROBLAST

21-30 Localizedproteincomplexesthat control A FIGURE neuroblast, the BPP asymmetriccelldivision.(a)Inthe Drosophila in ectoderm cellsandin delaminating islocalized apically complex (steps isalso complex 21-29)TheIPLG neuroblasts Il-B in Figure (notshown) fairly isdistributed localized. TheDSLcomplex apically by BPP, theMPSB to regulation In response aroundthecells. evenly into localizes to thebasalside,whereit willbe incorporated complex (GMC). genes in that encode ganglion Mutations mother cell the andaretherefore polarized proteins celldivision disruptasymmetric filaments transport alongcytoskeletal fatal.Motorprotein-mediated 2001, C Q DoeandB Bowerman, theMPSB basalcomplex. localizes [See 7005,Curr.OpinCellBiol.17:4751 andA Wodarz, Curr.OpinCellBiol 13:68,

and Lethal giant larvae (Lgl). Lgl reversibly associateswith the cytoskeleton.The DSL complex is mostly employed in localizing basal proteins. t MPSB,a basalcomplex, confersGMC cell fate. It includes the coiled-coil scaffold protein Miranda, the homeodomainclasstranscription factor Prospero, the RNA-binding protein Staufen,and a translational repressorprotein called Brain tumor (Brat). Vith the key players in neuroblast asymmetric division introduced. let's examine their functions more closely' Apical Complexes and Spindle Orientation For localized protein complexesto be differentially incorporated into two daughter cells, the plane of cell division must be appropriately oriented. In dividing fly neuroblasts, the mitotic spindle first aligns perpendicularto the apical-basalaxis and then turns 90 degreesto align with it at the same time that the basal complexesbecome localizedto the basal side (Figure 21.-31).The apical IPLG and BPP complexes,which are already in place before spindle rotation, control the final orientation of the spindle. This is supported by the finding that mutations in any of the components of these complexes eliminate the coordination of the spindle with apical-basal polarity, causingthe spindle orientation to becomerandom. The two apical protein complexeshave different roles in spindle orientation. First, the BPP complex responds to extrinsic cues from the overlying ectoderm to form an apical crescent at late interphase. In this way' the BPP complex aligns neuroblastpolarity with the surrounding tissueso that the apical side of the neuroblast is always next to the ectoderm. Second,the BPP complex recruits the IPLG complex to the apical cortex, and the BPPcomplex anchors one spindle pole to the apical cortex, thereby aligning the spindle along the apical-basalaxis. The direct link with the spindle is mediated by the NuMA protein, which joins the Pins protein of the IPLG complex to microtubules. The IPLG complex is sufficient for anchoring the spindle and promoting asymmetric division. Basal Complex and the Determination of GMC Fate During fly neuroblast divisions,the MPSB complex becomes localized to the basal cortex prior to each division and remains there while the basal part of the neuroblast becomesa new GMC (seeFigure 21,-30)'Miranda provides a scaffold for the other three proteins in the complex (Prospero' Staufen, and Brat) and is needed to muster them near the basal plasma membrane.After each division, MPSB proteins in the basallylocated daughtercell inhibit neuroblastproperties and confer GMC properties. Prospero negatively reguIatestranscription of cell-cyclegenes'which remain active in the dividing neuroblast. Brat post-transcriptionally inhibits the transcription factor Myc, a positive regulator of cell division and negativeregulator of cell size.In this way Brat helps keep the GMC small and restrainsits division- Genetic studies lrovide support for such involvement of Prospero and Brat in determinationof GMCs. For example,brat mutations O F A S Y M M E T R I CC E L LD I V I S I O N REGULATION

'

933

Video: Mitotic Spindle Asymmetry in Drosophila Neuroblast Cell Division

l

A EXPERIMENTAL FIGURE 21-31Time-lapse fluorescence imaging revealsrotation of the mitotic spindlein asymmetricalfydividing neuroblasts.EarlyDrosophila embryos wereinjected with a hybridgenecomposed of thegeneencoding greenfluorescent protein(GFP) fusedto the geneencoding Tau,a proteinthatbindsto microtubules At thetop aretime-lapse images of a singledividingneuroblast in a ilveembryo. Thebasalsideisat the top,andtheapicalsideat the bottom At time0, equivalent to

cause GMCs to enlarge into neuroblastsand keep dividing, whereas loss of ProsperocausesGMCs to remain small but maintain neuroblast-stylegeneexpressionand proliferation. How doesthe MPSBcomplex becomelocatedbasallyprior to each neuroblast division? The answer is more complex than Ashlp localizationin yeast.Both apical BPPand uniform cortical DSL complexesare involved. The BPPcomplex controls MPSB localization. The atypical protein kinase C (apKC), a component of the BPPcomplex, phosphorylatesand thus inactivates the Lgl protein, a component of the DSL complex. Lgl is required to bring MPSB proteins ro the basal cortex. Since aPKC is located at the apical end of the cells, Lgl is active only in basalregions.The restriction of active Lgl to basalcortex explains how MPSB proteins are brought to the basal cortex where they causeone daughter cell to becomea GMC. How doesactive, basalLgl control localization of MpSB? Although the full story is nor yer known, genetic and biochemical studies show that actin, myosin II, and myosin VI are involved. For instance,drug-induced disruption of actin microfilaments blocks MPSB targeting to the neuroblastcortex. Myosin VI binds Miranda (the "M" of MpSB) directly and is also required for basaltargeting of the MpSB complex. Asymmetry of Daughter Cell Size A notable feature of neuroblast asymmetric division is the pronounced difference in the sizeof neuroblastsand GMCs. This cell-sizedifference is regulatedby the Pinsand Go1componentsof the IpLG complex. The IPLG complex is brought to the apical cortex by virtue of the association of its Insc component with the Baz componentof the BPPcomplex. \fhen a neuroblastdividesto produce a GMC and a neuroblast,the GMC is usually considerably smaller. The spindle is oriented, as we have described,in the apical-basaldirection, and at metaphaseof the neuroblast division, the two halves of the spindle are about 934

C H A P T E R2 1

|

C E L LB t R T H ,L t N E A G EA , ND DEATH

prophase, thetwo centrosomes arevisible on opposite sidesof the cell.These functionasthespindle poles;asmitosis proceeds, the microtubules formingthe mitoticspindle areassembled fromthe poles (seeFigure18-34). (at 32,64,and80 seconds), In successive images the bipolarspindle canbeseemto formandrotate90 degrees to alignwith theapical-basal axis,asschematically depicted at the bottom.[From JA Kaltschmidt etal, 2000,Nature CellBiol.2:7; courtesy ofJ Kaltschmidt andA H, Brand, Wellcome/CRC lnstitute, Cambridge University.l

equal in size.However the two centrosomes,one at each pole of the spindle, behave differently. The basal centrosome, marking the pole where the GMC will form, has few astral microtubules,while the apical centrosomeenlargesand grows a bushy mass of astral microtubules that make that pole of the dividing neuroblast considerably larger (Figure 21-32). INTERPHASE NEUROBLAST

ANAPHASE NEUROBLAST

Centrosome Astral microtubules Neuroblast

-+ Cytoskeleton: I

nctin microfilaments

I

Microtubules

GMC microtubules

Chromosome

A FIGURE 21-32 Orientationof mitoticspindleand difference in daughtercell size in asymmetricdivisionof neuroblasts. Interactions between spindle microtubules andthe lpLGapical complex (red)liejustunder orientthespindle. Actinmicrofilaments thecellsurface (blue)radiate at alltimes.Microtubules fromthe centrosome duringinterphase andthenassemble intothe mjtotic spindle, attached to theduplicated centrosomes, duringcelldivision. Notethe biased location of thecentrosome duringinterphase, at the apicalendof thecell Theasymmetry in daughter cellsizebegins with differential assembly of astralmicrotubules, whichareconsiderably shorter andlessabundant in thebasalendof thedividing cell,which formsthedaughter ganglion mothercell(GMC). fromC e, [Adapted DoeandB Bowerman, 2001,Curr.OpinCellBiol 13:681

Genetic tests have shown redundant control of the cellsize asymmetry betweenthe two daughter cells. If either the BPP apical complex or the IPLG apical complex is functional, the cells formed will be the usual small GMC and large neuroblast. In contrast, a double mutant with defects in both the BPPand IPLG complexes(e.g.,a double pins and baz mutant) produces two daughter cells that are equal in size. Double mutations that inactivate the Gg and G" subunits, but not the Go1subunit, of the Gi protein component of the IPLG complex, causenumerous astral microtubules to form on both centrosomes.Over-production of the G9 protein has the opposite effect-no astral tubules on either centrosome. From thesegeneticanalyseswe may conclude that a normal function of the G9 protein is to selectivelyprevent assembly of astral microtubules at the basal centrosome. This regulation would involve the action of Gp/G" subunits that are not part of the apical IPLG complexes.Indeed, Gp is uniformly distributed all around the neuroblast cortex. Heterotrimeric G proteins like the one in the IPLG complex often are controlled by a G protein-coupled receptor that, when activated, dissociatesthe trimer by binding G. and releasingactive GB/G, subunits (Chapter 15). No sign of a G protein--coupledreceptor has been found in the search for proteins controlling neuroblast asymmetry.Instead Pins and Loco, componentsof the IPLG complex, substitutefor a receptor in triggering dissociation of the inactive heterotrimeric G protein. Pins and Loco are partially redundant; mutating both causesdefectsequivalentto mutation of either the Gs or G, subunit. Pins and Loco bind to GDP'G.1and act like guanine nucleotidedissociationinhibitors, thereby keeping G6 associatedwith GDP and allowing G6'GDP and GB/G.'to act upon their (as yet unknown) targets.As would be expectedfor a typical G protein cycle,a GTPase-activating protein (GAP) and a GDP exchangefactor (GEF) have also been found to regulate neuroblast division asymmetry.The GAP reaction inactivates the G protein by breaking down GTP to GDP, while the GEF reaction rechargesthe G protein for activity by bringing in a new GTP. The mechanism by which the G protein component of the IPLG complex regulatesthe activity of the centrosomeremains unknown. Summary of Asymmetry-Determining Protein Complexes The initial courseof eventsin polarizing and organizing asymmetric cell division can be summarized as having three phases:(1) establishmentof cell polarity, (2) alignment of the mitotic spindle with cell polarity, and (3) specification of distinct sibling fates. For phase 1, the BPP complex is already apically located '$fhen some of those cells sink beneath in all ectoderm cells. the surfaceto becomeneuroblasts,the apical localization of BPPpersists.The IPLG complex becomesapically located after the BPP complex. Acting through the evenly distributed DSL complex, the two apical complexesdirect the basal localization of MPSB. For phase2, the orientation of the spindle relative to the ectoderm is an indirect outcome of the apically located BPP complex, which links the already oriented ectoderm to the IPLG complex. Mitotic spindle microtubules are joined to

the apical IPLG complex by the fly NuMA protein, thus orienting the spindle. For phase 3, as asymmetric cell divisions commence, each neuroblastrenewsitself while budding off a smaller daughter GMC in the interior (basal) direction. Different sibling fates are specified by proteins incorporated into the daughter cells. Neuroblasts have stem-cell properties and are determined to retain that stem-cell fate by aPKC, a patt of the BPP complex that promotes neuroblast self-renewal.Brat and Prospero, componentsof the MPSB complex, are located in the smaller interior (basal)daughter cell and promote GMC differentiation. The G protein component of the IPLG complex activates astral microtubule assembly in a dividing neuroblast, determining the size of the two poles and hence of the daughter cells. Since IPLG is apically localized, the apical daughter cell (a neuroblast)is larger than the basal daughtercell (a GMC). From this summary,we can seehow a set of protein complexes coordinates multiple critical events during asymmetric cell division: localization and activation of asymmetry regulators, differential segregationof cell fate-determining regulatory proteins, orientation of the spindle, and generation of different sizeddaughter cells.

Regulation of Asymmetric Cell Division r Asymmetric cell division requires polarization of the dividing cell, which usually entails localization of some cytoplasmic components, and then the unequal distribution of thesecomponentsto the daughter cells (seeFigure 21'-26). r In the asymmetric division of budding yeasts' a myosindependenttransport systemcarries ASH1 mRNA into the bud (seeFigure21'-28). r Ashl protein is produced in the daughter cell soon after division and prevents expression of HO endonuclease, which is necessary for mating-type switching. Thus a daughter cell cannot switch mating type, whereas the mother cell from which it arisescan (seeFigure 21'-27). r Asymmetric cell division in Drosophila neutoblastsdepends on two apical protein complexes (BPP' IPLG), a basal complex (MPSB), and an evenly distributed complex (DSL). The basal proteins are incorporated into the ganglion mother cell (GMC) and contain proteins that determine cell fate (seeFigure 21,-30). r Asymmetry factors exert their influence at least in part by controlling the orientation of the mitotic spindle, so that asymmetricallylocalizedproteins and structuresare differentially incorporated into the two daughter cells (see Figure 21,-32). r The atypical protein kinase C (aPKC) in the BPP apical complex phosphorylatesthe LGL protein, a component of the DSL complex, but can do so only in the apical region, since that is where BPP is located. Nonphosphorylated LGL, which therefore exists only in basal regions, is active in bringing MPSB to the cortex where it requires the actin cytoskeletonto be anchored. O F A S Y M M E T R I CC E L LD I V I S I O N REGULATION

93s

r The general processof asymmetric cell division and the protein complexes controlling it are highly conserved through evolutionary tlme.

majority of cells generatedduring brain developmentsubsequently die. Cellular interactionsregulatecell death in two fundamentally different ways. First, most if not all cellsin multicellular organismsrequire signalsto stay alive. In the absenceof such survival signals,frequently referredto as trophic factors,cells activatea "suicide" program. Second,in some developmental contexts, including the immune sysrem,specific signals induce a "murder" program that kills cells.lfhether cellscommit suicide for lack of survival signals or are murdered by killing signalsfrom other cells, death is mediated by a common molecular pathway. In this section,we first distinguish

CellDeathand lts Regulation Programmedcell death is a counter-intuitivebut essential cell fate. Cell death keeps our hands from being webbed, our embryonic tails from persisting,our immune sysrem from respondingto our own proteins, and our brain from being filled with uselesselectricalconnections.In fact, the

Video:CellsUndergoingApoptosis'&) (b)

Mildconvolution C h r o m a t i nc o m p a c t i o n and margination Condensationof cytoplasm

B r e a k u po f n u c l e a re n v e l o p e N u c l e a rf r a g m e n t a t i o n Blebbing C e l lf r a g m e n t a t i o n

Phagocytosis

Apoptotic body Apoptoticcell Phagocyticcell

FIGURE21-33 Ultrastructural features of cell death by apoptosis.(a)Schematic drawingsillustrating the progression of morphologicchangesobservedin apoptoticcells Earlyin apoptosis, densechromosome condensation occursalongthe nuclearperiphery. Thecellbodyalsoshrinks,althoughmostorganelles remainintact Laterboth the nucleusand cytoplasmfragment,forming apoptotic

936

c H A p T E R2 1

|

C E L LB t R T H ,L t N E A G EA , ND DEATH

bodies, whicharephagocytosed bysurrounding cells(b)photomicrographs comparing a normalcell(fop)andapoptotic cell(bottom) Clearly visiblein the latteraredensespheres of compacted chromatin asthenucleus (a)adapted begins to fragmentlPart fromJ Kuby, 1997, lmmunology,3ded,W H Freema& n C o , p 5 3 P a r t ( b ) f r o mM . J A r e n d s and A H Wyllie,1991,lnt'\. Rev.Exp Pathol.32:2231

programmed cell death from death due to tissueinjurg then considerthe role of trophic factors in neuronal development, and finally describe the evolutionarily conserved effector pathway that leadsto cell suicideor murder.

P r o g r a m m e dC e l lD e a t hO c c u r sT h r o u g h Apoptosis The demiseof cells by programmed cell death is marked by a well-definedsequenceof morphological changes,collectively referredto as apoptosis,a Greek word that means "dropping off" or "falling off," as leavesfrom a tree. Dying cells shrink and condenseand then fragment, releasingsmall membranebound apoptotic bodies, which generally are engulfed by other cells (Figure 21-33; see also Figure 1-19). The nuclei condenseand the DNA is fragmented.Importantly, the intracellular constituents are not releasedinto the extracellular milieu where they might have deleteriouseffectson neighboring cells.The stereotypicalchangesin cells during apoptosis, like condensation of the nucleus and engulfment by surrounding cells, suggestedto early workers that this type of cell death was under the control of a strict program. This program is critical during both embryonic and adult life to maintain normal cell number and composition. The genesinvolved in controlling cell death encodeproteins with three distinct functions: r "Killer" proteins are required for a cell to begin the apoptotrc process.

'$fhen neurons grow to make developing nervous system. sometimesover muscles, or to neurons connectionsto other will eventually grow than more cells considerabledistances, in the spinal located are bodies cell neurons' survive. The processes extend far their ganglia, while cord and adjacent prevail make connections Those that regions. these outside die. fail to connect that those and survive; In the early 1900s the number of neurons innervating the periphery was shown to dependupon the sizeof the tissueto which they would connect, the so-called "tatget field." For instance, removal of limb buds from the developing chick embryo leads to a reduction in the number of sensoryneurons and motoneurons innervating the bud (Figure 21,-34). ( a ) A m p u t a t i o no f d e v e l o p i n gl i m b b u d

O p t i cc u p and lens

Chickembryo

S p i n a lc o r d Limbbud

S p i n a lc o r d (crosssection)

Motor neurons

r "Destruction" proteins do things like digest DNA in a dying cell. r "Engulfment" proteins are required for phagocytosisof the dying cell by another cell. At first glance, engulfment seemsto be simply an after-death cleanupprocess,but some evidencesuggeststhat it is part of the final death decision.For example, mutations in killer genes always preventcells from initiating apoptosis,whereasmutations that block engulfmentsometimesallow cells to survive that would normally die. That is, cells with engulfment-gene mutations can initiate apoptosis but sometimesrecover.Engulfment involves the assemblyof a halo of actin around the dying cell, triggeredby apoptosisproteinsthat activateRac, a monomeric G protein that helps regulate actin polymerizatron (seeFigure 17-42).A signalon the surfaceofthe dying cell also stimulates a receptor on neighboring cells, which initiates membrane changesleading to engulfment. In contrast to apoptosis,cellsthat die in responseto tissue damageexhibit very different morphologicalchanges,referred to as necrosis.Typically,cells that undergo this processswell and burst, releasingtheir intracellular contents, which can damagesurroundingcellsand frequentlycauseinflammation'

NeurotrophinsPromoteSurvivalof Neurons The earlieststudiesdemonstratingthe importance of trophic factors in cellular development came from analysesof the

(b) Transplantation of extra limb bud

Motorneuron generation:

100o/o

100o/o

100o/o

100o/o

I Motoon"rronapoptosis Y

Motor neuron survival:

50Yo

lOYo

50Yo

75o/o

21-34 The survivalof motor FIGURE A EXPERIMENTAL neuronsdependson the sizeof the muscletarget field they of a limbbudfromonesideof a chick innervate.(a)Removal in the number in a markeddecrease embryoat about2 5 daysresults embryo, side.In an amputated on the affected of motorneurons on bothsides aregenerated of motorneurons normalnumbers remain manyfewermotorneurons (middldLaterin development, the than on limb missing the with on the sideof the spinalcord percent motor the of 50 (bottom). about only that Note side normal survive(b) normally aregenerated thatoriginally neurons of an extralimbbudintoan earlychickembryo Transplantation on thestdewith effect,moremotorneurons produces the opposite fromD side normal the than on [Adapted tissue target additional Connections, of Neural Theory A Trophic 1988,BodyandBrain: Purves, andT M Jessell, J H Schwartz, andE R Kandel, Press, University Harvard of NeuralScience,4thed , McGraw-Hill,,p 1054, Figure53-11 l 2OOO.Principles C E L LD E A T HA N D I T S R E G U L A T I O N

937

Conversely, grafting additional limb tissue to a limb bud leads to an increasein the number of neurons in corresoonding regions of the spinal cord and sensoryganglia. InJeed, incremental increasesin the target-field size are accompanied by commensurateincremental increasesin the number of neurons innervating the target field. This relation was found to result from the selectivesurvival of neurons rather than changes in their differentiation or proliferation. The observation that many sensoryand motor neurons die after reaching their peripheral target field suggestedthat these neurons compete for survival factors produced by the target tISSUC.

Subsequentto these early observations,scientistsdiscovered that transplantation of a mouse sarcoma tumor into a chick led to a marked increasein the numbersof certain types of neurons.This finding implicated the tumor as a rich source of the presumed trophic factor. To isolate and purify this factor, known simply as nerve growth factor (NGF), scientistsusedan in vitro assayin which outgrowth of neurites from sensory ganglia (nerves)was measured. Neurites are extensionsof the cell cytoplasmthat can grow to become the long wires of the nervous system, the axons and dendrites(seeFigure23-2).The later discoverythat the submaxillary gland in the mouse also produceslarge quantities of NGF enabledbiochemiststo purify and to sequenceit. A homodimer of two 118-residuepolypeptides,NGF belongs to a family of structurally and functionally related trophic factors collectively referred to as neurotrophins. Brainderived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) also are membersof this protein family. Neurotrophins bind to and activare a family of recepror tyrosine kinases called Trks (pronounced .,tracks',). (The general structure of receptor tyrosine kinasesand the intracellular signaling pathways they activate are covered in Chapter 16.) Each neurotrophin binds with high affinity to one Trk receptor: NGF binds to TrkA; BDNR to TrkB; and NT-3, to TrkC. NT-3 can also bind with lower affinity to

both TrkA and TrkB. Binding of these factors to their receptors provides a survival signal for different classesof neurons. As neurons grow from the spinal cord to the periphery, neurotrophins produced by target tissues bind to Trk receptors on the growth cones of the extending axons, promoting survival of neurons that successfullyreach targets.In addition, lerrotrophins bind to a distinct type of receptor called p75Nr* lNtR : neurotrophin recepior) but with lower affinity. However, p75*t* forms heteromultimeric complexeswith the different Trk receptors;this association increasesthe affinity of Trks for their ligands. Depending on the cell type, binding of NGF and BDNF to p75*t' in the absence of TrkA may promore cell death rather than prevent it. (The phenomenon of multiple neurotrophins interacting with multiple similar receprors is comparable to EGF-like ligands and their HER receptors,illustrated in Figure 1,6-18). To critically addressthe role of the neurotrophins in development, scientistsproduced mice with knockout mutations in each of the neurotrophins and their receptors.These studiesrevealedthat different neurotrophins and their corresponding receptorsare required for the survival of different classes of sensory neurons (Figure 21,-35). For instance, pain-sensitive (nociceptive) neurons, which express TrkA, are selectivelylost from the dorsal root ganglion of knockout mice lacking NGF or TrkA, whereas TrkB- and TrkCexpressing neurons are unaffected in such knockouts. In contrast, TrkC-expressing proprioceptive neurons, which detect the position of the limbs, are missing from the dorsal root ganglion in TrkC and NT-3 mutanrs.

A Cascadeof CaspaseProteinsFunctionsin One Apoptotic Pathway Neurotrophins and other signals that keep cells alive act upon an evolutionarily conservedcell-deathcontrol system. Key insights into the molecular mechanismsregulating cell

> EXPERIMENTAL FTGURE 21-35 Different WildType classesof sensoryneuronsare lost in knockoutmicelackingdifferenttrophic Spinal M echano receotors factorsor their receptors. In animals lacking cord nervegrowthfactor(NGF)or its receptor Dorsalroot TrkA, ganglion (pain-sensing) smallnociceptive (blue) neurons thatinnervate theskinaremissingThese Propioceptive neurons express TrkAreceptor neurons andinnervate NGF-producing targets. In animals lacking Nociceptive (NT-3) eitherneurotrophin-3 or itsreceptor neurons TrkC,largepropioceptive (red) neurons innervating muscle spindles aremissingMuscle produces NT-3andthe propioceptive neurons express TrkCMechanoreceptors (orange), anotherclass of sensory neurons in the dorsal rootganglion, areunaffected in thesemutants. [Adaptedfrom W D Snldet I 994, Cett77:627 ]

Motoneurons

938

c H A p T E R2 1

|

C E L LB t R T H L , t N E A G EA, N D D E A T H

Mutant NGF_L or TrkA-/-

Mutant NT-37_ or TrkC-L

Video: ProgrammedCell Death in C. elegansEmbryonic Development 21-36 Mutationsin the ced-3 FIGURE < EXPERIMENTAL gene block programmedcell death in C. elegans.(a)Newly mutantlarvacarrya mutationin theced-7gene.Because hatched of deadcells,highly engulfment in thisgeneprevent mutations (arrows), their facilitating deadcellsaccumulate refractile in boththe (b)Newlyhatched larvawith mutations visualization deadcellsin of refractile ced-l andced-3genes.Theabsence that no celldeathsoccurredThus thesedoublemutantsindicates HM celldeath.lFrom for programmed CED-3proteinisrequired Ellisl Hilary courtesyof 1986, Ce//91:818; H R Horvitz, Ellisand

death came from genetic studies using C. elegans. Of the 947 nongonadal cells generated during development of the adult hermaphroditeform, 131 cellsundergo programmed cell death. Specific mutations have identified four genes whose encoded proteins play an essentialrole in controlling programmed cell death during C. elegans development: ced-3, ced-4, ced-9, and egl-1.In ced-3 or ced-4 mutants, for example,the 131 "doomed" cells survive (Figure 21,-36).The mammalian proteins that correspond most closely to the worm CED-3, CED-4, CED-9, and EGL-1 proteins are indicated in Figure 21-37. In discussingthe worm proteins we will include the mammalian names in parenthesesto make it easier to keep the relationships clear. The confluenceof geneticstudiesin worms and studies on human cancer cells first suggestedthat an evolutionarily conservedpathway mediatesapoptosis.The first mammalian apoptotic gene to be cloned, bcl-2, was isolated from human follicular lymphomas. A mutant form of this gene, created in lymphoma cells by a chromosomal rearrangement,was shown to act as an oncogenethat promoted cell survival rather than cell death (Chapter 25). The chromosomerearrangementioins the coding region of the bcl-2 gene to an immunoglobulin gene enhancer. The combination results in over-production of BcI-2 protein that keepscancer cells alive when, otherwise, they would becomeprogrammed to die. The human Bcl-2 protein and

worm CED-9 protein are homologous' and a bcl-2 transgene can block the extensive cell death found in ced-9 mutant worms even though the two proteins are only 23 petcent homologous. Thus both proteins act as regulators that suppressthe apoptotic pathway (seeFigure 21'-37)'ln addition, both proteins contain a single transmembrane domain and are localized to the outer mitochondrial, nuclear, and endoplasmic reticulum membranes, where they serveas sensorsthat control the apoptotic pathway in responseto external stimuli. As we discussbelow, other regulators promote apoptosis. In the worm apoptotic pathway' CED-3 (caspase9) is required to destroy cell components during apoptosis. CED-4 (Apaf-1) is a protease-activating factor that causes autocleavageof (and by) the CED-3 precursor protein, creating an active CED-3 proteasethat initiates cell death (seeFigure 21.-37).Cell death does not occur in ced-3 and ced-4 single mutants or in ced-9/ced-3 do'lble mutants' whereas all cells die during embryonic Iife in ced-9 mutants' so the adult form never derrilops.Thesegeneticstudiesindicate that the CED3 and CED-4 are "killer" proteins required for cell death' that CED-9 (Bcl-2) suppressesapoptosis,and that the apoptotic pathway can be activatedin all cells.Moreover, the absenceof cell death in ced-9/ced-3double mutants suggests that CED-9 acts "upstream" of CED-3 to suppressthe apoptotic pathway. The mechanismby which CED-9 (Bcl-Z)controls CED-3 (caspase9) is now known. CED-9 protein, which is normally tethered to the outside of mitochondria, forms a complex with CED-4 (Apaf-l), thereby preventing activation of CED3 by CED-4. As a result, the cell survives.This mechanism fits with the genetics,which shows that the absenceof CEDt has no effeit if CED-3 is also missing (ced-3/ced-9double mutants have no cell death). The crystal structure of the trimeric CED-4/CED-9 complex revealsa huge contact surface between each of the two CED-4 molecules and the single CED-9 molecule (Figure 21'-38it.The large contact

C E L LD E A T HA N D I T S R E G U L A T I O N

939

(a) Nematodes

(b) Mammals A p o p t o t i cs t i m u l i

EGL-1 disrupts the CED-4/CED-9 complex comes from the crystal structure of EGL-1 (Bid/Bim) complexedwith CED-9 (Bcl-2).In this complex,the BH3 domain forms rhe key part of tEGLTI ( B H 3o n r y ) the contact surfacebetweenthe two proteins. CED,9 has a difA I F | | HTRA2/OMI ferent conformation when bound by EGL-1 than when bound by CED-4. This finding suggeststhat EGL-1 binding distorts EndoG CED-9, making its interactionwith CED-4 lessprobable and Iess stable. Once EGL-1 causesdissociationof the CED4ICED-9 complex, the releasedCED-4 dimer dimerizesagain to make a tetramer, which then activates CED-3. Cell death soon follows (Figure 21-39). Evidence for similar events has beenfound in human cultured cells. Evidencethat the steps describedhere are sufficient for caspaseactivation comes from experiments in which the eventswere reconstitutedin vitro (i.e., in solution) with purified components. CED-3, CED-4, a truncated CED-9 that lacked its transmembranemitochondrial membrane anchor, and EGL-1 were purified, as was a CED-4/CED-9 complex. Effectors [tAP;-] Purified CED-4 (Apaf-1) was able to acceleratethe autocatalysisof purified CED-3 (caspase9), but addition of the truncated CED-9 (Bcl-2) to the reaction mixrure inhibited C e l l u l a tra r g e t s the autocleavage.When the CED-4/CED-9 complex was mixed with CED-3, autocleavagedtd not occur, but addition Apoptosis of EGL-1 to the reaction restored CED-3 autocleavage. The effector proteins in the apoptotic pathwaS the cas,,c.\vJ-.r pases,are named becausethey contain a key cysteineresiduein the catalytic site and selectivelycleaveproteins at sitesjust Cterminal to aspartateresidues.Caspases work as homodimers. A FIGURE 21-37 Evolutionaryconservation of apoptosis pathways.Simrlar proteins, shownin identical play colors, corresponding rolesin bothnematodes andmammals(a)ln nematodes, the proteincalledEGL-1 bindsto CED-9on the surface of mitochondria; thisinteraction releases CED-4 fromthe CED9/CED-4 complex. FreeCED-4 thenactivates autoproteolysrs of the caspase CED-3, whichdestroys cellproteins to driveapoptosis These relationships areshownasa geneticpathway, with EGL-linhibiting CED-9, whichin turn inhibits CED-4ActiveCED-4 a/B-fold activates CED_3 (b)In mammals, homologs of the nematode proteins andother CED-4 proteins regulate apoptosts. TheBcl-2proteinlssimilar to CED_9 in To promoting cellsurvival by preventing activation Apaf-1,whichis mitochondrial s i m i l at o r C E D - 4T w oB H 3 - o npl yr o t e i n B memDrane s ,i da n dB i m ,i n h i b iBt c l _t2o Helical allowapoptosis. Apoptotic stimulidamagemitochondria, leading to oclp-fold domain release of several proteins thatstimulate celldeath.In parrrcurar, cytochrome c released frommitochondria activates Apaf_1, whichin turnactivates caspase-9 Thisinitiator caspase thenactivates effector Helical caspases-3 and-7, eventually leading to celldeath Seetextfor domain discussion of othermammalian (SMAC/DIABLO proteins andlAps) thathaveno nematode homologs[Adapted fromS J Riedl andy Shi,

I

I

I tril] I I

faEDd

I 6)

X

2004, Nature Rev.Mol Cell Biol 5(11\:8911

surface makes the associationhighly specific, but in such a

BH3 domain. SinceEGL-1 lacks most of the other domains of CED-9, EGL-1 is calleda BH3-only protein. The closestmammalian BH3-only proteins are Bim and Bid. Insieht into how 940

CHAPTER 21

|

C E L LB t R T H L , t N E A G EA, N D D E A T H

Winged helix

FIGURE 21-38Structureof the CED-4/CED-g proteincomplex. Thecrystal structure hastwo CED-4 molecules associated withone CED-9 molecule (darkblue)serves TheC-terminus of CED-9 to tether thecomplex to themitochondrial membrane CED-4 iscomposed of (CARD, fourdomains o/g folds,wingedhelixdomain, andanother helical domain)EachCED-4 molecule hasa boundATpanda Mgr* ionwhicharevisible asa cluster of orangeandblueatomswithineach subunit[Based onN Yanetal, 2005, Nature 437:831 I

EGL-1

cED-4 dimer

I D-g/EGL-1 Mitochondrion

A

FEm-t-y-tr"s*l

FfGURE 21-39 Activationof CED-3proteasein C. elegans. thattrigger protein, to signals in response whichisproduced EGL-1 with CED-9 on dimerfromitsassociation CED-4 celldeath,displaces (tr) ThefreeCED-4 with dimercombines of mitochondria thesurface (E), whichcatalyzes of the theconversion to forma tetramer another (anenzymatically precursor of a protease) inactive zymogen CED-3 (B). Thiseffector to protease thenbegins caspase intoactiveCED-3 leading to cell apoptosis, andthusinitiate destroy cellcomponents 437|831 fromN Yanetal, 2005,Nature death(4) [Adapted I

with one domain of each stabilizing the active site of the other. The principal effector caspasein C. elegansis CED-3, while humans have 15 different caspases.AII caspasesare initially made as procaspasesthat must be cleavedto become active. Such proteolytic processingof proproteins is used repeatedlyin blood clotting, generationof digestiveenzymes' and generationof hormones.In vertebrates,initiator caspases (e.g.,caspase-9)are activated by autoproteolysisinduced by other types of proteins (e.g., Apaf-l), which help the initiators to aggregate.Activated initiator caspasescleaveeffector caspases(e.g.,caspase-3)and thus quickly amplify the total caspaseactivity level in the dying cell. The various effector caspasesrecognizeand cleaveshort amino acid sequencesin many different target proteins. They differ in their preferred target sequences.Their specific intracellular targets include proteins of the nuclear lamina and cytoskeletonwhose cleavage leadsto the demiseof a cell. In mammals and flies but not worms, apoptosisis regulated by severalother proteins(seeFigure21-37, right).For instance,a family of inhibitor of apoptosisproteins (IAPs)' provides another way to restrain both initiator and effector IAPs haveone or more zinc-bindingdomainsthat can caspases. bind directly to caspasesand inhibit their proteaseactivity. (Baculovirus, a type of insect virus, produces a protein that thus preventingan insimilarly binds to and inhibits caspases, stop a viral infection besuicide to fected cell from committing inhibition of caspasesby The viruses be made.) can fore new needs to undergo a cell problem when creates a IAPs, however, since again, picture once the enter Mitochondria apoptosis. they are the source of a family of proteins, called SMAC/DIABLOs, that inhibit IAPs. After cell injury, SMAC/DIABLOs

releasedfrom mitochondria bind to IAPs in the cytosol' thereby blocking the IAPs from binding to caspases.By relieving IAPmediated inhibition, SMAC/DIABLOs promote caspaseactivity and cell death. Three other mitochondria-associated proteins-Ht ra2l Omi serine protease' apoptosis-inducingfactor (AIF), and endonucleaseG-also help to kill cells upon their releasefrom mitochondria following cell injury. Htra2l Omi cleavesIAPs, thus relieving their restraint of apoptosis' Since this regulation is catalytic, Htta2lOmi is a more powerful antagonist of IAPs than is SMAC/DIABLO. AIR a flavoprotein that normally acts as a NADH oxidase, is cleaved by Droteasesand moves to the nucleus where it causeschromoio-e .o.rd.tsation and DNA fragmentation. These effectsare so not all apoptosisinvolvescaspases' caspase-independent'

Pro-ApoptoticRegulatorsPermitCaspase Activation in the Absenceof TrophicFactors Having introduced the major participants in the apoptotic pathway, we now take a closer look at the workings of the miiochondrial membrane proteins that regulate apoptosis' Although the normal function of CED-9 andBcl-2 is to suppress the cell-death pathwaS other intracellular regulatory proteins promote apoptosis.The first pro-apoptotic regulator to be identified, named Bax, is related in sequenceto CED-9 and Bcl2. Overproduction of Bax induces cell death rather than protectingcellsfrom apoptosis,as CED-9 and Bcl-2 do. Thus this

the intracellular signaling pathways regulating them. Some Bcl-2 family members preserveor disrupt the integrity of the outer mitochondrial membrane, thereby controlling releaseof mitochondrial proteins such as cytochrome c' In normal healthy cells' cytochrome c is localized betweenthe inner and outer mitochondrial membrane,but in cellsundergo-

ducesapoptosis.A variety of death-inducing stimuli causeBax to move from the cytosol to the outer mitochon-ono-.ti drial membranewhere they oligomerize' Bax homodimers, but not Bcl-2 homodimers ot Bcl-2lBax heterodimers, permit influx of ions through the mitochondrial membrane' It remains unclear how this ion influx triggers the releaseof cytochrome c' The effect of Bcl-2 family members on the permeability of the mitochondrial outer membrane has been mimicked in

C E L LD E A T HA N D I T 5 R E G U L A T I O N .

941

of Bcl-2 family membersappearsto reflect their generalability to alter mitochondrial membranes.In addition to increasedpermeability, mitochondria normally undergo dramatic changesin their nerwork morphology by fusion and fission during the cell-death process. Bcl-xl, a vertebrate member of the Bcl-2 family, and CED-9 introduced into mammalian cells, can induce mitochondrial fusion. Thus theseproteins appear to have profound abilities to engineer the properties of outer mitochondrial membranes. Once cytochrome c is releasedinto the cytosol, it binds to Apaf-1 (the mammalian homolog of CED-4) and promotes activation of a caspasecascadeleading to cell death (seeFigure 21-37, right).ln the absenceof cytochrome c, monomeric Apaf-1 is bound to dATp. After binding cytochrome c, Apaf-1.cleavesits bound dATp into dADp and undergoes a dramatic assembly process into a disc-shaped heptamer,a 1,.4megadaltonwheel of death called the apoptosome (Figure 21-40). The apoptosomeservesas an acrivation machine for initiator and effector casDases.

SomeTrophicFactorsInduceInactivationof a Pro-ApoptoticRegulator We saw earlier that neurotrophins such as nerve growth factor (NGF) prorect neurons from cell death. In the absenceof trophic factors, however, the nonphosphorylated form of a pro-apoptotic protein called Bad is associatedwith Bcl-2l

Bcl-xl at the mitochondrial membrane (Figure 21-41a). Binding of Bad inhibits the anti-apoptotic function of Bcl-2/ Bcl-xl, thereby promoting cell death. Phosphorylated Bad, however,cannot bind to Bcl-2lBcl-xl and is found in the cytosol complexed to the phosphoserine-bindingprotein 14-33. Hence, signaling pathways leading to Bad phosphorylation would be particularly attractive candidates for transmitting survival signals. A number of trophic factors including NGF have been shown to trigger the PI-3 kinase signaling pathway, leading to activation of protein kinase B (seeFigure 16-30). Activated protein kinase B phosphorylatesBad at sitesknown to inhibit its pro-apoptotic activity. Moreover, a constitutively active form of protein kinase B can rescue cultured neurotrophin-deprived neurons, which otherwise would undergo apoptosis and die. These findings support the mechanism for the survival action of trophic factors depicted in Figure 21-41b. In other cell types, different trophic factors may promote cell survival through post-translational modification of other componentsof the cell-deathmachinery. Another mechanism by which neurotrophins can affect apoptosis, this time positively, involves p75tt*, the lowaffinity neurotrophin receptormentionedabove.This protein can either promote or inhibit apoptosisdependingon the cellular context. In certain neurons, neurotrophin signals such as BDNF stimulate apoptosis by acting through p75NrR. In these neurons, cleavageof p75MR by a membrane-bound

E

Cytochromec releasefrom mitochondriabinds Apaf-1 dATP hydrolysis

E

Procaspase9 recruitment to Apaf-1

___+

Caspase3 and IAP recruitmentto Apaf-1 Apoptosome

A FIGURE 21-40Assemblyof the mammalianapoptosome.In theabsence of apoptosis triggers, Apaf-1exists in the cytosot asan inactive monomer boundto dATpApaf-1contains multiple WD4O repeats, a dATP-binding CED-4 domain,anda CARDdomain.Step istriggered, [: Whenapoptosis damage to mitochondria allows release of cytochrome c, whichbindsto Apaf-1Thisinteraction leads to hydrolysis of the bounddATpto dADpanda changein the conformation of Apaf-1.Stepf,l: In itsextended conformation, 942

.

cHAprER 21

|

C E L LB I R T H ,L I N E A G EA . ND DEATH

Apaf-1assembles intoa seven-subunit complex, theapoprosome Step$: Interaction of theapoptosome withthe initiator procaspase_ 9 stimulates theautocleavage anddimerization of the procaspase, whichis necessary for itsactivity. Activecaspase-9 thenactsupon effectorcaspases suchascaspase-3 Inhibitorof apoptosis proteins (lAPs) arealsoboundbythe apoptosome, although theirexact actionstherearenot understoodlAdapted lromZf. Schafer andS Kornbluth, 2006,Devel. Cell10:5491

(b) Presenceof trophic factor: Inhibition of caspaseactivation

(a) Absence of trophic factor: Caspaseactivation '-

Trophicfactor receptor

E

Plasmamembrane

Death.@^ r-l /

Cleavageof substrates

/

6;;b /

pathwaysleadingto 21-41 Proposedintracellular A FIGURE cell to trophic factor-mediated by apoptosis or cell death of a trophicfactor, survivalin mammaliancells.(a)Inthe absence pro-apoptotic proteinBadbindsto the anti-apoptotic the soluble intothe mitochondrial proteins whichareinserted Bcl-2andBcl-xl, proteins (tr). Badbindingprevents theanti-apoptotic membrane protein. pro-apoptotic with Bax,a membrane-bound frominteracting in the channels Baxformshomo-oligomeric As a consequence, an as-yet-unknown ionflux[Z] Through membrane that mediate c intothe of cytochrome thisfluxleadsto the release mechanism,

whereit bindsto the adapterproteinApaf-1(p), promoting cytosol, thatleadsto celldeath(Z|) (b)In somecells, cascade a caspase Pl-3kinase bindingof a trophicfactor,suchasNGF(Il) stimulates protein B kinase of activation the downstream to leading activity, Badthenformsa (PKB), Bad Phosphorylated whichphosphorylates in the withthe 14-3-3protein(U ). WithBadsequestered complex proteins caninhibitthe Bcl-2lBcl-xl the anti-apoptotic cytosol, c of cytochrome the release of Bax(B), therebypreventing activity and B Pettman from cascade. the caspase of [Adapted andactivation 20:633 Neuron l C E Henderson,1998,

releasesthe receptor'sintracellular proteasecalled^y-secretase, domain, which is associatedwith a DNA-binding protein called NRIF. The cleavageof p75MR leadsto the ubiquitination of NRIF and its movement to the nucleus where it stimulates is apoptosis,perhaps by regulating transcription.1-Secretase cleavage the intramembrane protease that catalyzes the same of the receptor Notch, thus activating it, and also of amyloid precursor protein (APP) in the genesisof Alzheimer'sdisease (seeFigures1.6-36and L6-37).

tor (TNFa), which is releasedby macrophages,triggers the cell death and tissue destruction seen in certain chronic inflammatory diseases (Chapter 24). Another important death-inducingsignal,the Fasligand' is a cell-surfaceprotein produced by activated natural killer cells and cytotoxic T iymphocytes. This signal can trigger death of virus-infected cells, some tumor cells, and foreign graft cells. Both TNF and Fas ligand act through cell-surface "death" receptorsthat have a singletransmembranedomain and are activatedwhen ligand binding brings three receptor molecules into close proximity. The trimeric receptor complex attracts a protein called FADD (Fas-associateddeath domain), which servesas an adapter to recruit and in some way activatecaspase-8,an initiator caspase'in cellsreceiving a death signal. The death domain found in FADD is a sequence that is present in a number of proteins involved in apoptosis.Once activated,caspase-8activatesother caspases

Tumor NecrosisFactorand RelatedDeath SignalsPromoteCell Murder by Activating Caspases Although cell death can arise as a default in the absenceof survival factors, apoptosis can also be stimulated by positively acting death signals.For instance,tumor necrosisfac-

C E L LD E A T HA N D I T S R E G U L A T I O N

943

and the amplification cascadebegins.To test the ability of the Fas receptor to induce cell death, researchersincubated cells with antibodiesagainstthe receptor.Theseantibodies,which bind and cross-link Fas receptors,were found to stimulate cell death, indicating that activation of the Fasreceptoris sufficient to trigger apoptosis.

Cell Death and tts Regulation r All cells require trophic factors to prevent apoptosisand thus survive. In the absenceof these factors, cells commit suicide. r Genetic studies in C. elegansdefined an evolutionarily conservedapoptotic pathway with three major components: membrane-boundregulatory proteins, cytosolic regulatory proteins, and effector proteasescalled caspasesin vertebrates(seeFigure 21-37). r Once activated,apoptotic proteasescleavespecificintracellular substratesleading to the demise of a cell. proteins (e.g., CED-4, Apaf-1), which bind regulatory proteins and caspases,are required for caspaseactivation (seeFigures 21-39 and21-40\. r Pro-apoptotic regulator proteins (e.g., Bax, Bad) promote caspaseactivation, and anti-apoptotic regulators (e.g., Bcl-2) suppressactivation. Direct interactions between pro-apoptotic and anti-apoptotic proteins lead to cell death in the absenceof trophic facors. Binding of extracellular trophic factors can trigger changesin these interactions,resulting in cell survival (seeFigure 21-41). r The Bcl-2 family contains both pro-apoptotic and antiapoptotic proteins; all are single-passtransmembrane proteins and engagein protein-protein interactions. Bcl-2 molecules can restrain the release of cytochrome c from mitochondria, inhibiting cell death, while pro-apoptotic factors stimulate membrane breakdown that allows cytochrome C to escape,bind to Apaf-1, and thus activatecaspases. r Binding of extracellular death signals, such as rumor necrosisfactor and Fas ligand, to their receptors activates an associatedprotein (FADD) that in turn triggers the caspasecascadeleading to cell murder.

Cell birth, lineage, and death, which lie at the heart of the

a y,ardlong, pulsating multinucleate muscle cells,exquisitely light-sensitiveretina cells,ravenousmacrophagesthai recognize and engulf germs, and all the hundreds of other cell types. Regulators of cell lineageproduce this rich variety by controlling two critical decisions: (1) when and where to 944

c H A p T E R2 1

|

C E L LB t R T H ,L t N E A G EA , ND DEATH

activate the cell cycle (Chapter 20) and (2) whether the rwo daughter cells will be the same or different. A cell may be just like its parent, or it may embark on a new path. Cell birth is normally carefully resrricted to specific localesand times, such as the basallayer of the skin or the root meristem. Liver regenerateswhen there is injury, but liver cancer is prevented by restricting unnecessarygrowth at other times. Cell lineageis patterned by the asymmetric distribution of key regulatorsto the daughter cellsof a division. Some of theseregulators are intrinsic to the parent cell, becoming asymmetrically distributed during polarization of the cell; other regulators are external signals that differentially reach the daughter cells. Asymmetry of cells becomes asymmetry of tissues and whole organisms. Our left and right hands differ only as a result of cell asymmetry. Somecells persist for the life of the organism, but others such as blood and intestinal cells turn over rapidly. Many cells live for awhile and are then programmed to die and be replaced by others arising from a stem-cellpopulation. programmed cell death is also the basisfor the meticulous elimination of potentially harmful cells, such as autoreacriveimmune cells, which attack the body's own cells, or neurons that have failed to properly connect. Cell-death programs have also evolved as a defenseagainst infection, and virusinfected cells are selectivelymurdered in responseto death signals.Viruses,in turn, devote much of theiieffort to evading host defenses.For example, p53, a transcription factor that sensescell stressesand damage and activatestranscription of pro-apoptotic membersof the b cl-2 genefamilS is inhibited by the adenovirusE1B protein. It has been estimated that about a third of the adenovirus genome is directed at evading host defenses.Cell death is relevant to toxic chemicals as well as viral infections; malformations due to poisons often originatefrom excessapoptosis. Failures of programmed cell death can lead to uncontrolled cancerous growth (Chapter 25). The proteins that prevent the death of cancer cells therefore become possible targets for drugs. A tumor may contain a mixture of cells, some capable of seedingnew tumors or continued uncontrolled growth, and some capable only of growing in place or for a limited time. In this sensethe tumor has its own stem cells, and they must be found and studied, so they become vulnerable to medical intervention. One option is to manipulate the cell-death pathway by sending signals that will make cancer cells destroy themselves. Much attention is now being given to the regulation of stem cells in an effort to understand how dividing populations of cells are created and maintained. This has clear implications for repair of tissue: for example, to restore damagedeyes,torn cartilage,degeneratingbrain tissue,or failing organs. One interesting possibility is that some populations of stem cells with the potential to generateor regeneratetissue are normally eliminated by cell death during later development. If so, finding ways to selectivelyblock the death of these cells could make regenerationmore likely. Could the elimination of such cells during mammalian developmentbe the difference between an amphibian capable of limb regeneration and a mammal that is not?

KeyTerms apoptosis937 apoptosome 942 asymmetriccell division 905 Bcl-2 famtly 941. BPP complex 933 940 caspases 905 lineage cell death signals943 determination 925 differentiation 905 ectoderm 907 embryonicstem (ES)cells911 endoderm 907 ganglion mother cell

(GMC)e32 germline907 heterochronicmutants 909 MAT locus 922 mating factor 923

920 meristems 907 mesoderm microRNAs (miRNAs)910 MPSBcompIex933 muscleregulatoryfactors (MRFs)926 neurotrophins938 nuclear-transfer cloning908 pluripotent907 precursor(progenitor) cells905 somaticcells905 stemcells905 stem-cellniche912 transientamplifying(TA) cells905 totipotent 907 trophic factors935

mating-type switching 931

Review the Concepts 1. What two properties define a stem cell? Distinguish between a totipotent stem cell, a pluripotent stem cell, and a precursor (progenitor) cell. 2. Where are stem cells located in plants? $7hereare stem cells located in adult animals?How doesthe concept of stem cell differ between animal and plant systems? 3. ln 1997, Dolly the sheepwas cloned by a techniquecalled somatic cell nuclear transfer (or nuclear-transfercloning). A nucleus from an adult mammary cell was transferred into an egg from which the nucleus had been removed. The egg was allowed to divide several times in culture, then the embryo was transferred to a surrogate mother who gave birth to Dolly. Dolly died in 2003 after mating and giving birth herself to viable offspring. \Uhat does the creation of Dolly tell us about the potential of nuclear material derived from a fully differentiatedadult cell?Doesthe creationof Dolly tell us anything about the potential of an intact, fully differentiated adult cell? Name three types of information that function to preserve cell type. \Which of these types of information was shown to be reversibleby the Dolly experiment? 4. The roundworm C. eleganshas proven to be a valuable model organism for studiesof cell birth, cell lineage,and cell death.'What properties of C. elegansrender it so well suited for these studies?Vhy is so much information from C. elegans experimentsof use to investigatorsinterestedin mammalian development? 5. How are retroviruses used in tracing experiments that map cell lineages?

6. In the budding yeast S. cereuisiae,what is the role of the MCM1 protein in the following? a. transcription of a-specificgenesin a cells b. blocking transcription of ct-specificgenesin a cells c. transcription of a-specificgenesin ct cells d. blocking transcription of a-specificgenesin ct cells 7. ln S. cereuisiae, what ensures that a and a cells mate with one another rather than with cells of the same mating type (i.e.,a with a or ct with a)? 8. Exposure of C3H L0Tlz ceIls to 5-azacytidine, a nucleotide analog, is a model systemfor muscle differentiation. How was S-azacytidinetreatment used to isolate the genes involved in muscle differentiation? 9. Through the experiments on C3H 1'0Ty2 cells treated with 5-azacytidine,MyoD was identified as a key transcription factor in regulating the differentiation of muscle. To what generalclassof DNA-binding proteins doesMyoD belong? How do the interactions of MyoD with the following proteins affect its function? (alE2A, (b) MEFs' (c) Id. L0. The mechanismsthat regulate muscle differentiation in mammals and neural differentiation in Drosophila (and probably mammals as well) bear remarkable similarities. 'What proteins function analogousto MyoD, myogenin, Id, and E2A in neural cell differentiation in Drosophila? Based on these analogies, predict the effect of microinjection of myoD mRNA on the developmentof Xenopus embryos. 11. Predict the effect of the following mutations on the ability of mother and daughter cells of S. cereuisiaeto undetgo mating-type switching following cell division: a. loss-of-functionmutation in the HO endonuclease b. gain-of-function mutation that renders HO endonuclease gene constitutively expressed independent of SIUTI/SNF c. gain-of-function mutation in SVUSNF that renders it insensitiveto Ashl 12. Asymmetric cell division often relies on cytoskeletalelements to generareor maintain the asymmetricdistribution of cellular factors. In S. cereuisiae, what factor is localized to the bud by myosin motors? In Drosophila neuroblasts,what factors are localized apically by microtubules ? 13. How do studiesof brain developmentin knockout mice support the statementthat apoptosis is a default pathway in neuronal cells? 'S7hat morphologic features distinguish programmed 14. necrotic cell death? TNF and Fas ligand bind and cell death to trigger cell death. Although the receptors cell-surface death signal is generatedexternal to the cell, why do we consider the death induced by these moleculesto be apoptotic rather than necrotic? 15. Predict the effects of the following mutations on the ability of a cell to undergo apoptosls: a. mutation in Bad such that it cannot be phosphorylated by protein kinase B (PKB) b. c.

overexpressionof Bcl-2 mutation in Bax such that it cannot form homodimers R E V I E WT H E C O N C E P T S

'

945

One common characteristicof cancer cells is a loss of function in the apoptotic pathway. Vhich of the mutations listed above might you expect to find in some cancer cells? 16. How do IAPs (inhibitor of apoptosis proteins) interact with caspasesto prevent apoptosis?How do mitochondrial proteins interactwith IAPs to preventinhibition of apoptosis?

Analyze the Data The immortal-strand hypothesispostulatesthat when a stem cell divides asymetrically to produce one "new" stem cell and one progenitor cell, the new stem cell receivesthe sister chromatids containing the oldest strand of DNA (the immortal strand). The other daughter cell, a progenitor cell that eventually gives rise to differentiated cells. receivesthe sister chromatids containing more recent DNA strands (see diagram below). If the immortal-strand mechanismactually operates,it would prevent the accumulation of mutations in adult stem cells that otherwise would occur during each round of DNA replication.

hypothesis, researchersrecently conducted the following studies: a. Satellite cells were isolated from mouse muscle fibers and cultured in vitro in the presenceof BrdU, a nucleotide analog that is incorporated into DNA during replication. After 4 days in BrdU, all satellitecells in the culture were extensively labeled with BrdU (pulse), as expected if these cells underwent symmetric divisions. The cells were then incubated for 18 hours in the absenceof BrdU (chase), a period of time that correspondsto approximately two cell divisions in these cells. The images below show two examples of muscle satellite cells undergoing division after this 18-hr incubation in the absenceof BrdU. The blue labeling (Hoechst) shows total DNA, the red labeling shows where BrdU-containing DNA is located.

Phase

Oldest strand Stem cell

ord strand New strand

I

s-ntaru Phase

J Mitosis

Asymmetric c e l ld i v i s i o n

Stem cell

Progenitorcell

Muscle satellitecells are progenitorsfor myoblastsand are the source of cells that result in muscle growth after birth and muscle repair after in;'ury. The satellite cells can replenish themselves,suggestingthat they also have properties of stem cells. To test the immortal-strand 946

C H A P T E R2 1

|

C E L LB t R T H ,L t N E A G EA , ND DEATH

The majority of the cells appear like the dividing cell in the top panel, but about 1.5 percent of dividing cells appear like that in the lower panel. Can you explain theseobservations? Given that mice have 40 chromosomes,could the segregation of the BrdU-labeled sister chromatids, observedin the lower panel, have occurred by chance? b. Satellitecells were subjectedto a BrdU pulse-chase experiment similar to that describedin part (a) above and then were assessedfor the production of Numb, a protein whose presence or absence allows two daughter cells to adopt different developmentalfates.The micrographs below show a dividing cell stainedfor Numb (green)and for BrdUcontaining DNA (red). \fhat do you expect to be the outcome of the daughter cell that acquiresNumb? How might you determine if Numb is involved in generatingco-segregation of the older DNA strands?

c. Suppose you conducted a pulse-chaseexperiment using an establishedcultured cell line, but asymmetric divisions like that observedin the lower panel in part (a) above were not observed.Explain this result.

References The Birth of Cells: Stem Cells, Niches, and Lineage Aurelio, O., T. Boulin, and O. Hobert. 2003. Identificationof spatial and temporal cuesthat regulatepostembryonicexpressionof axon maintenancefactors in the C. elegansventral nervecord. Deuelopment13U599-6'1.0. Buszczak,M., and A. C. Spradling.2006. Searchingchromatin for stem cell identity. Cell 125:233-236. Chopra, V.S.,and R. K. Mishra. 2005. To SIR with Polycomb: linking silencingmechanisms.Bioessays27:1,19-121,. Copelan,E.A. 2006. Hematopoieticstem-celltransplantation. N. Engl. J. Med.354:1.81.3-1.826. Edenfeld,G., J. Pielage,and C. Klambt.2002. Cell lineagespecification in the nervous system.Curr. Opin. Genet.Deuel. 122473477. Feinberg,A. P.,R. Ohlsson,and S. Henikoff. 2005. The epigenetic progenitor origin of human cancer.Nature Reu.Genet. T:21,-33. Golden,J. A., S. C. Fields-Berr5and C. L. Cepko. 1995.Construction and characterizationof a highly complex retroviral library for lineageanalysis.Proc. Nat'|. Acad. Sci.U SA 9225704-5708. Hatfield, S.D., et al. 2005. Stemcell division is regulatedby the micro RNA pathway.Nature 435:974-978. Hochedlinger,K., and R. Jaenisch.2006. Nuclear reprogramming and pluripotency.Nature 441:1.061.-1067. Hori, Y., et aL.2002.Growth inhibitors promote differentiation of insulin-producingtissuefrom embryonic stem cells.Proc. Nat'|. Acad. Sci. USA 99:161.05-1.6710. Huelsken,J., et al. 2001. B-Catenincontrolshair follicle morphogenesisand stemcell differentiationin the skin. Cell 105:533-545. Li, L., and T. Xie. 2005. Stemcell niche: structureand function. Ann. Reu.Cell Deuel. Biol.2L605-631. Marshman, E., C. Booth, and C. S. Potten.2002. The intestinal epithelialstemcell. Bioessays24;9I-98. Morrison, S.J., and J. Kimble. 2005. Asymmetric and symmetric stem-celldivisions in developmentand cancer.Nature 441:1,068-1.074. Orkin, S. H. 2000. Diversificationof haematopoieticstemcells to specificlineages.Nature Reu.Genet. l:57-64. Reinhart,B. J., et al. 2000. The 21-nucleotide/er-7RNA regulates developmentaltiming in Caenorhabditis elegans.Nature 4032901,-906. SanchezAlvardo, A.2006. Planarianregeneration:its end is its beginning.Cell 124:241-245. Shafritz,D.A., et al. 2006. Liver stemcellsand prospectsfor liver reconstitutionby transplantedcells.Hepatology 43(2 Suppl 1):S89-9 8. Watt, F. M., C. Lo Selso,and V. Silva-Vargas.2006.Epidermal stem cells:an update. Curr. Opin. Genet.Deuel. 16:518-524. '!7u, H., and Y. E. Sun. 2006. Epigeneticregulation of stemcell differentiation.Pediatr.Res.59(4 Pt 2):21R-25R. Cell-Type Specification in Yeast Bagnat,M., and K. Simons.2002. Cell surfacepolarization during yeastmating. Proc. Nat'|. Acad. Sci.USA 99214183-14188. Coic, E., G-F.Richard, and J. E. Haber.2006. Cell cycle-dependentregulationof Saccharomyces cereuisiaedonor preference during mating-typeswitchingby SBF (Swi4/Swi6)and Fkh1. Mol. Cell Biol. 26:.5470-5480. Cosma,M. P.2004. Daughter-specificrepressionof Saccharomyces cereuisiaeHO: Ashl is the commander.EMBO Rep. 5:953-957.

Dittmar, G. A., et aL.2002.Role of a ubiquitin-like modification s. Science29522442-2446. in polarizedmorphogenesi Dohlman, H. G., and J. W. Thorner. 2001. Regulationof G protein-initiatedsignaltransductionin yeast:paradigmsand principles. Ann. Reu.Biocbem. 70:703-7 54. Hall. I. M.. et a\.2002. Establishmentand maintenanceof a heterochromatindomain. Science297:2232-2237. Kirchmaier,A. L., and J. Rine. 2005. Cell cyclerequirementsin Mol. Cell cereuisiae. assemblingsilent chromatin in Saccharomyces Biol. 26:852-862. Lau, A., H. Blitzblau, and S. P. 8e11.2002.Cell-cyclecontrol of the establishmentof mating-type silencing in S. cereuisiae.Genes Deuel. 16:2935-2945. Miller, M. G., and A. D. Johnson.2002' White-opaqueswitching in Candida albicans is controlled by mating-type locus homeodomain proteinsand allows efficientmating. Cell 110:293-302. Takizawa,P. A., and R. D. Vale. 2000' The myosin motor, Myo4p, binds Ashl mRNA via the adapterprotein, She3p.Proc. Nat'l. Acad. Sci.USA 97:5273-5278. Specification and Differentiation of Muscle Bailen P.,T. Holowacz, and A. B. Lassar.2001. The origin of skeletal muscle stem cells in the embryo and the aduk. Curr. Opin. Cell Biol.13:679-689. Berkes,C.A, and S.J. Tapscott.2005. MyoD and the transcriptional control of myogenesis.Semin.Cell. Deuel.Biol- 162585-595. Buckingham,M., S. Meilhac, and S. Zaffuan.2005. Building the mammalian heart from two sourcesof myocardial cells.Nature Reu. Genet.6:826-835. Dhawan, J., and T. A. Rando. 2005. Stemcells in postnatal actimyogenesis:molecularmechanismsof satellitecell quiescence, vation and replenishment.TrendsCell Biol. t5:666-673. Gustafsson,M. K., et aI.2002. Myf5 is a direct target of longrange Shh signalingand Gli regulationfor musclespecification. G enesDeuel.16z11'4-126. McKinsey,T. A., C. L. Zhang, and E. N. Olson. 2002. Signaling chromatin to make muscle.Curr. Opin. Cell Biol.74:763-772. Pipes,G. C., E. E. Creemers,and E. N. Olson. 2006. The myocardin family of transcriptional coactivators:versatileregulators of GenesDeuel.2O:1545-1'555. cell growth, migration,and myogenesis. Yan, 2., et al. 2003. Highly coordinatedgeneregulationin mouseskeletalmuscleregeneration./. Biol. Chem.278:8826-8836. Regulation of Asymmetric Cell Division Bellaiche,Y., and M. Gotta. 2005' HeterotrimericG proteins and regulationof sizeasymmetryduring cell division. Curr' Opin. Cell Biol. 17:658-663. Betschinger, J., and J. A. Knoblich. 2004.Dare to be different: asymmetric ..ll dirritiott in Drosopbila, C. elegansand vertebrates. Curr. Biol. 74:R67 4-68 5. Betschinger, J., K. Mechtler,and J. A. Knoblich. 2003. The Par complex directs asymmetric cell division by phosphorylating the cytoskeletalprotein Lgl. Nature 422:326-330. Betschinger, J., K. Mechtler,and J. A. Knoblich' 2006. Asymmetric segregationof the tumor suppressorbrat regulatesself-renewal in Drosopbila neural stem cells. Cell 124:1241-1253. and -independBhalerao,S., et al. 2005.Localization-dependent in Drosopbila. specification ent roles of numb contribute to cell-fate Curr.Biol. 15:l 583-l 590. Bowman, S. K., et al.2006.The Drosophila NuMA Homolog Mud regulatesspindleorientation in asymmetriccell division. Deuel. Cell 102731-742. Cleary,M. D., and C. Q. Doe. 2005. Regulationof neuroblast competence:multiple temporal identity factors specifydistinctneu.o.rr^l f"t", within a single early competencewindow GenesDeuel. 20:429434. REFEREN CE

947

Cowan, C.R., and A. A. Hyman. 2004. Asvmmetriccell division in C. elegans:cortical polarity and spindlepositioning.Ann. Reu. Cell D euel.Biol. 20:4274 53. Fichelson,P.,et al. 2005. Cell cycle and cell-fatedetermination rn Drosophila neural cell lineages.Trends Genet. 212413420. Heidstra,R., D.'Welch,and B. Scheres.2004. Mosaic analyses using marked activation and deletionclonesdissectA rabidopsisSCARECROW action in asymmerriccell division. GenesDeuel. iS|De+-D69. Helariutta, Y., et al. 2000. The SHORT-ROOT geneconrrols radial patterning of the Arabidops,sroot through radial signaling.Cel/ 101:555-557. Hutterer, A., et al. 2004. Sequentialroles of Cdc42. Par-6. aPKC,and Lgl in the establishmintof epithelialpolarityduring Drosophila embryogenesis . Deuel. Cell 6:845-854. Kipreos,E. T. 2005. C. eleganscell cycles:invarianceand stem cell divisions.Nature Reu.Mol. Cell Biol.6:766-776. Lee, C-Y., K. J. Robinson,and C. Q. Doe. 2006.Lg|, Pins and aPKC regulateneuroblastself-renewalversusdifferentiation.Nature 439:594-598. Lee, C-Y., et al. 2006. Brat is a Miranda cargo protein that promotes neuronal differentiationand inhibits neuroblastself-renewal. Deuel. Cell 1O2441,449. Lu, H., and D. Bilder.2005. Endocyticcontrol of epithelialpolariry and proliferation in Drosophila. Nature Cell Biol. 7:1,232-1239. Nance,J. 2005. PAR proteins and the establishmentof cell polarity during C. elegansdevelopment.Bioessays27:126-135. O'Donnell, K.A., et al. 2005. c-Myc-regulatedmicro RNAs modulate E2F1 expression.Nature 4352839-843. Petritsch,C., et al. 2003. The Drosophila myosin VI Jaguaris required for basalprotein targetingand correct spindleorientation in mitotic neuroblasrs.Deuel. Cell 42273-28I. . !!"ll P.J., et al. 2003. A polarity complex of mPar-5and atypical PKC binds, phosphorylatesand regulatesmammalian Lgl. Na-ture Cell Biol. 5:30'l-308. Shapiro,L., H. H. McAdams, and R. Losick. 2002. Generatins and exploiting polarity in bacteria.Science298:1942-1946. Siegrest,S. E., and C. Q. Doe. 2005. Microtubule-inducedpins/ Go; cortical polarity in Drosopbila neuroblasts.Cell 123:1323-1,335. Siegrest,S. 8., and C. Q. Doe. 2005. Extrinsic cuesorient the cell division axis in Drosophila embryonic neuroblasts.Deuelopment 1332529-536. Siller,K. H., C. Cabernard,and C. Q. Doe. 2006.The NuMArelatedMud protein binds Pins and regulatesspindleorientation in Drosophila neuroblasrs.Nature Cell Biol. 8:594-500. 'Wang, H., and 17. Chia. 2005. Drosophila neuralprogenitor po_ larity and asymmetricdivision. Biol. Cett 97:63-74. Wodarz, A. 2005. Molecular control of cell polarity and asvmmetric cell division tn Drosophila neuroblasts.i"rr. Opln. Celt Biol. L7:475481. . Zarnescu,D. C., et al. 2005. FragileX protein funcrionswith lgl and the par complex in flies and mice.Deuil. Cell 8:43-52. Zgurski, J. M., et al. Asymmetricauxin responseprecedes asymmetricgrowrh and differentiationof asymmetricieafI and asymmetricleaf2 Arabidopsrsleaves.Plant Cell 17:77-91. Cell Death and lts Regulation Aderem, A. 2002. How to eat somethingbiggerthan your head. Cell ll0:5-8. Alvarez-Garcia,I., and E. A. Miska. 2005. Micro RNA functions in animal developmentand human disease.Deuelo\ment 132:46534662.

948

C H A P T E R2 1

I

C E L LB I R T H ,L I N E A G EA , ND DEATH

Ambrose,V. 2003. Micro RNA pathways in flies and worms: growth, death, fat, stress,and timing. Cell 113:673-676. Baehrecke,E. H. 2002. How death shapeslife during development. Nature Reu. Molec. Cell Biol. 32779-787. Bao, Q., S. T. Riedl, and Y. Shi. 2005. Structureof Apaf-1 in the auto-inhibited form: a critical role for ADP. Cell Cycle 8: 1001-1003. Brennecke,J., et al. 2003. bantam encodesa developmentally regulated micro RNA that controls cell proliferation and regulates the proapoptotic genehid,in Drosophila. Cell 113z25-36. Cory, S., and J. M. Adams. 2002. The Bcl2 family: regularors of the cellular life-or-death switch. Nature Reu. Cancer 2: 647-656. Estaquier,J., and D. Arnoult. 2006. CED-9 and EGL-I: a duo also regulating mitochondrial network morphology. Molec. Cell 2l:730-732. Hay, B. A., and M. Guo. 2005. Caspase-dependent cell death in Drosophila. Ann. Reu.Cell Deuel. Biol.22:623-650. Green,D. R., and G. Kroemer.2004.The pathophysiologyof mitochondrial cell death. Science305:626-629. 'Weinberg. 2002. Taking the study of cancer Jacks, T., and R. A. cell survival to a new dimension.Cell 11l:923-925. Kinchen,J. M., et al. 2005. Two pathwaysconvergeat CED-10 to mediate actin rearrangementand corpse removal in C. elegans. Nature 434:93-99. Kinchen,J. M., and M. O. Hengartner.2005. Talesof cannibalism, suicide,and murder: programmedcell death in C. elegans. Curr. Top. Deuel. Biol. 65 1,45. Lakhani, S. A., et al. 2006. Caspases3 and 7:key mediatorsof mitochondrial eventsof apoptosis.Science10:847-851. Marsden, V. S., and A. Strasser. 2003. Control of apoptosisin the immune system:Bcl-2, BH3-only proteins and more. Ann. Reu. Immunol. 2l:71-'1,05. Penninger,J. M., and Kroemer,G. 2003. Mitochondria, AIF, and caspases-rivaling for cell death execution. Nature Cell Biol. 5:97-99. Riedl, S.J., and Y. Shi. 2004. Molecular mechanismsof caspase regulation during apoptosis. Nature Reu. Molec. Cell Biol. 5:897-907. Schafer,2.T., and S. Kornbluth. 2006.The apoptosome:physiological, developmental,and pathologicalmodesof regulation. Deuel. Cell 10:549-561,. Vaccari, T., and D. Bilder. 2005. The Drosophila tumor suppressor vps25 preventsnonautonomousoverproliferationby regulating notch trafficking. Deuel. Cell 9:687-698. Xu, P, M. Guo, and B. A. Hay. 2004. Micro RNAs and the regulation of cell death. TrendsGenet.20:6L7-624. Yan, N., et al.2004. Structural,biochemical,and functional analysesof CED-9 recognitionby the proapoptotic proteins EGL-1 and CED-4. Molec. Cell 24999-1006. Yan, N., et al. 2005. Strucrureof the CED-4-CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans.Nature 437 :831-837 . Yan, N., and Y. Shi. 2005. Mechanisms of apoptosis through structural biology. Ann. Reu. CelI Deuel. Biol.2L: 35-56. Zuzarte-Luis, V., and J. M. Hurle. 2002. Programmedcell death in the developing limb. Int'\. J. Deuel. Biol. 46; 871-876.

CHAPTER

Froma singlecellto a humanembryo.Twentyhoursafterthe fertilization of a humanegg,the maleand femalepronucleiare aboutto fuseand combinethe geneticinformationfrom fatherand mother.Forty-six dayslaterthe embryo,2 cm long,is beginningto developorgansand tissues, nourishedby bloodenteringthrough the umbilicalcord ICourtesy of TheLennartNilsson AwardBoard]

ust as notes and chords blend into a symphong genes, proteins, and cells act as an integrated system during embryogenesis,the development of an embryo. Signals flow within and between cells, and massivewaves of gene expressionallow thousands of types and shapesof cells to form (Chapters 7, 15, 16). For normal embryogenesis,the cell cycle must be regulated, as describedin Chapter 20, so that cell growth and division occur at the right times and places;cell lineageslike those describedin Chapter 21 must be organized in time and space; mechanisms described in Chapters 17-19 must organizecells into tissues,organs, and whole bodies; and cell death (Chapter 2L) must be programmed so that the webbing-not the fingers-is removed. In this chapter we explore the regulation of early-stage animal embryos to observe developmental mechanisms in context.'We concentrateon insectsand mammals,with some examplesfrom other animal species,as well as plants. After a brief summary of early development,we describehow eggs and sperm are made, how fertilization occurs, and the special genetic properties of early mammalian cells. Then we look at the earliestcell divisions in mammalian development and the creation of different layers of tissues.The formation of repeating segmentsin animal embryos and the genesthat eventually causethose segmentsto differ are discussednext. !7e also examine severalparticularly informative aspectsof later animal development, including formation of the leftright asymmetry of the bodS control of cell fates in the early nervous system, and the patterning of limbs. As we cover various topics, we will see how different experimental approaches-lineage tracing, genetic screens,mosaic animals, manipulations of signaling proteins, and transplantation-

THEMOLECULAR CELLBIOLOGYOF DEVELOPMENT

have been used to discover and analyze key molecular and cellular eventsthat build animals. Let's start by thinking about a simple situation in which a sheet of cells has formed through cell division, but all the cells are identical. To form a working tissue,each cell has to do its iob. Somemay divide, some may bend, some may send out a signal. Each cell must somehow learn its location and fate, and start to differentiate appropriately' Differentiation may entail activation of certain genes,production of particular proteins, an increase or decrease in cell division, changed shape,changed surfaceproperties and adhesion to other cells, the releaseof secretedsignals,the acquisition of electrical activity, polarization along one or more axes,

OUTLINE 22J

Highlightsof DeveloPment

950

22.2

and Fertilization Gametogenesis

953

22.3 Cell Diversityand Patterningin Early Vertebrate Embryos

22.4 Control of Body Segmentation:Themesand Variationsin Insectsand Vertebrates

969

22.5 Cell-TypeSpecificationin EarlyNeural Development

22.6 Growth and Patterningof Limbs

985 990

949

migration, or a combination of any of these. A mistake in any aspectof cell differentiation early in developmentcan be fatal to the organism. The fascination of developmentalcell biology lies in discovering how the integrated system of development works and why it is so successfuldespitevariations in environment, inherited genes, cell numbers, and nutrition. At the same time, this field offers a new way to explore evolution and human origins: how animal forms and speciesarose,are maintained, and change. In addition, many diseasesare most readily understood in the context of normal developmental processesgone awry. And it all beginswith a singlecell!

Highlightsof Development Single-celledorganismscan go through complex developmental lfe cycles during which their shapesand behaviors may changedramatically. An example is the life cycle of the malariaplasmodiumdiscussedin Chapter 1 (seeFigure 1-4). In this chapter,however,we concentrateon developmentof multicellularanimals.The earlieststagesof animal development accomplishseveralcrucial goals:combination of maternal and paternalgenomesin a new organism;an increase in the number of cells; formation of three main layers of cells, the first step in creation of different cell and tissue types; and the laying out of the main organization of the embryo-front to back, head to tail, and left to rieht.

DevelopmentProgresses from Egg and Sperm t o a n E a r l yE m b r y o The development of a new organism begins with the fusion of male and female gametes:an egg (oocyte), carrying a set of chromosomesfrom the mother, and a sperm, carrying a set of chromosomes from the father. The gametes,or sex cells, are haploid becausethey have gone through meiosis and thus contain only one set of chromosomes(seeFigure 5-3). They combine in a processcalledfertilizarion,creating the initial single cell, the zygote, that has two sets of chromosomes (one maternal and one paternal) and is therefore diploid. The zygote beginsto divide in a processcalled cleavage, which produces a mass of cells that often look rather similar to eachother (Figure22-1).The progenyof theseinitial cells will gradually become different, forming all the organsand tissues. Early in embryogenesisthe cells divide into two distinct sets: germ-line cells, which will give rise to gametes, and somatic cells,which will form most of the body but are not passedon to future progeny. Germ-line cells are carriers of genetic alterations and, in some cases,inherited disease states, but anything that happens to the DNA of somatic cells cannot be passedon to future generations. In order for a functioning organism to develop, the embryo must becomepolarized. That is, cells on one side (leftright or head-tail or front-back) behavedifferently than cells on another side. The information that sets the oolarities of Firstcleavage 2-cell stage 30 hours Oviduct

--J v

4 davs -Z^

ffi

2 0h o u r s

trrtorrtuYrl{ 5 days

,ffi r9 \xqs

/r-w)pl '^--q.J

:e;1 e] \1_ \re./ F1 Blastocyst

\)

{',7o pe1reqr (o8e1sepuarrnt)e^rpl petuau8ase otur sdolarr 'usrueS.ro -ap olrque aqt oloq,tt E r.uro} ol orurl uoqs dpur .odrqruodg -zerrv ue'uonezrlnral 1o ,,{epI u1{tl/A. 1err,,{ruds aql ur suoSoqdrotusBuorlf,unt uec ,salncelou palerf,ossE lle,ry\ se 'srotce; uortdr.rrsuerl snql .slloJ Ipnpr^rpur otur runrlfo -uds aql to uorsr^rpqns oro;aq opqdoso.tq ur uortpurrurel -oP efet-llar;o sa8elstsorlrEaeql ur pesn ore r.lrrq^\'sluarper8 tuJot saplelotu errJos'soueJqruorueuseld ssoJf,ol Bur,req lnoqlrl\ ruseldoldcuoturuof,eqt ur oloru uec salncalou drol -ep8er 'luaurdolarrap Jo srnor{ ^4.eJlsrr} eql rot unrllcuds e sr od.rguradg eqr asneJeg.lno prel ere ueld dpoq eqr;o sued ,eruara;urnJJrf, roleu agl 'e8els dl.reasrql uela lE ur sllel lnq ,acuelsur ,eprselq dg .lo; 09 pue 3uo1s11oc 00I rnoqe dpo sr ar{J 'sllel zrra;dla.rrreya.r seq odrqrue aql ueq,,\4. Fe sroleln8ar Sutllortuoc-ura$Ed]ueuodur aql;o dueru ,slerurue u1 11e 'sonssrl (uorl IEuJOluror{l olur doleirapdllenluo,ropup -elnrlse8 uorsre^lJasureqt ,oprsure^ou slleJpqaqrrde aqr to Jo eruosuoo5'41od qlr^{ pallrt sr rorrelur esorl^.,eplselq etlr

'l'll)

uvtn)l'tot/!

lHl_

|

zz utldvH)

016

Jo areJrnsaql uo sller Ierleqrrdeos ro 0009 eql tuo{ po^rrap are sanssnornrnJIIV '$yZ-ZZ e.rn8rg)eFlselg ro ,urrepolselq relnller aqt Surleraua8'relcnu aql punoJe r.uroJseuerqruetu 'os ro rnor{ lxau eqt Sur.rnp :wnpo$plq 11ac Twlicur(s arTt Surur.ro;'uottBztlllroJraryesrnor{ t-Z rnoqe ef,etrnsaqt qlear rellnu oql 'euerqr.ueu etuseld aqt pre,trol preluno aler8 -rru o1 ur8aqdeql'epr,rrprellnu eql sV 'PIZ-ZZ e.rn8rg)euerq -tuaru EursEIdpue urseldoldc uoururoJ E rllr,ry\.urnrfcuu(s e '11ec 33e peteallnuulnur e aleraua8 ,{aql'suorsr,trp lyocdq peru -Bdtuoccelou ete suorsr^rpJpolf,nuoseqtesnef,afl.setnurtu t dyuo slsel leqt aseqd 5 alcdr-11are ul perdooVNC Ieurosoru (alo,fuEInap -oxp apqdosot(I ro' lo qI I 09I orrluo eql qtr^\ u,{\ou>lprder lsoru eqt sr uoueJrlde; y51q srql .solnulu 0I d.ra.,la lnoqe Surrrnocouorsrlrp gcee 'prde.rpue snouorqcu,(s an alo8,\z oql Jo suorsr^rprpalrnu €I rsrrJ aqt ,ayqdosot.q ur ralcnuord opuroJpue elpruto uorsnJpuu uortpzrTruotrartv

p a a d su l os!)raxf ue sl luaudo;eneg ellLldosotQ^Fe1

lr|repolselq relnllec

sllec uaaMloq sMorrn] o6e^ealc ]o u o r s u o l x ep u e u o r l e 6 u o ; el p a l c n N

uilepolselq le!ilcuAS

'lr+r]uaos '2661'Ip ,aluarMel lo]le e I pro]xo llaM))elg 7o6w4e61aq-l V il .LUjapolselq sLuerberp :plemoqpn sqdelboloqdl d V 1o,isa;rnot ;erybu{s oql pu0touelsod aql 'sllolaurl-u.rre6 o1esuanr6qltqM,sllo) ]o ]e a;odpa1let-os oq]atoN rol)nuIenpr^tpul ]o ral)nueq11ouorleOat6os ,uorlezuelnlla) punoleluaptna alesouelqulau teuv luasetd lle) ereofuqueaJtluo oq] 6urpunolns ueq] laqlo seuelqulauJ oN ]eq] uuseldoyir uoLuu.to) e ulq]tMuteuolq)tqM,tal)nulenptntpul 6ur{1teno .auelqujaLu se6lnqate;tnss,ieldsrp ofuqu.ra aq] 'uotlpztJelnlla) aJolaB euseldeq1ol preMlnopelerbru.r puepoLuro] a^pqtal)nu0009]noqe plunuorstntp paruedu.ro))e {q st uotslntp leellnN sqdet6ot>ruL 11at }ou u o J l ) e l pa u es u l e l 6 e rupt p o l p J l s nal llte( q )u J l a p o t s eJl e ql n l l a ) 'sgseueEortrqure o1(e)unryiru{soq} u-tol}se6e15 ellqdosotq Apea 6ur.rnpurapolselq telnlls) aql lo uotleu roJ IZ-ZZlungll >

useldolAc 6 u r p u n o : l n sq l r a n

u o r l e r 6 r t up u e u o t s t ^ t pt e e l J n N ( e )

,4#. srsauaEof.rqr.ul eltqdosoJqlo 6ut6eul asdpl-aru4 :oaptn

(al Drosophi Ia developmental stages

Egg

E m b r y o n i cd e v e l o p m e n t -1 day T h r e e l a r v a ls t a g e s -4 days Third instar larva

Pupa

of Dorsal and its translocation into nuclei. As a result, Dorsal enters ventral nuclei at a high level' lateral nuclei at a modest level, and dorsal nuclei not at all (Figure 22-23)' Thus Dorsal acts in a graded fashion and has the property of a morphogen, even though this protein is not secreted'The differential entry of the Dorsal transcription factor into nuclei, which controls subsequentcell fates, is governed by a complex signalingprocessthat starts during oogenesisin the follicle cellsand is transmitted to the developingembryo' We will not pursue the details of dorsal-ventral patterning further and will instead concentrate on anterior-posterior patternlng. To decipher the molecular basis of cell-fate determination and patterning along the three body axes, investigators have cloned genesidentified in screensfor mutations that affect the body plan; determinedthe spatial and temporal patterns of mRNA production for each gene and the distributhe tion of the encodedproteins in the embryo; and assessed effectsof mutations on cell differentiation, tissuepatterning' and the expressionof other regulatory genes.The principles of cell-fatedetermination and tissuepatterning learned from Drosophila have proved to have broad applicability to animal development.

( b ) l m a g i n a l d i s c s ,p r e c u r s o r st o t h e a d u l t

TranscriptionalControl Specifiesthe Embryo's Anterior and Posterior 'We

turn now to determination of the anterior-posterior axis in the early fly embryo while it is still a syncytium' The processbeginsduring oogenesiswhen maternal mRNAs ptoiuced by nurse cells are transported into the oocyte and become localized in discrete spatial domains (see Figure

22-22 Major stagesin the developmentol Drosophila A FIGURE intoa e99develops and locationof imaginaldiscs.(a)Thefertilized in a few hoursThelarva,a cellularization andundergoes blastoderm throughthree in about1 dayandpasses form,appears segmented (instars) Pupation intoa prepupa. overa 4-dayperiod,developing stages of theadultflyfromthe takes=4-5days,endingwiththeemergence discsareset pupalcase.(b)Groups cellscalledimaginal of ectodermal thesegive Duringpupation, sitesin thelarvalbodycavity. asideat specific cellsgiveriseto bodypartsindicatedOtherprecursor riseto thevarious (a) structures. andotherinternal thenervous system, lPart adultmuscle, in E W etal,'1969, fromJ W Fristrom Part(b) adapted Kaye Suyama courtesy of UtahPress, University inBiology, onProblems Hanly. ed, ParkCitySymposium o Jdrl

to the oocyte as a dowry from the mother. The dorsal-ventral control systeminvolves differential transport of a transcription factor called Dorsal into the nuclei of the syncytial embryo. This transcription factor, related to the vertebrate NF-rB protein, is presentin its inactive form throughout the cytoplasm of the syncytial embryo. A signal proteln concentrated on the ventral side of the embryo triggers the NF-rB signalingpathway (seeFigure 1'6-35),leadingto activation

anterior cell fates. Bicoid protein is a homeodomain-type transcription factor that activates expression of certain anterior-specific genes discussed later' In the syncytial fly embryo, Bicoid away from f,rotein spreadsthrough the common cytoplasm end where it is produced from the localized ,h. "rrt.riot mRNA. As a result' a Bicoid protein gradient is established along the anterior-posterior axis of the syncytial embryo' Sincethe effectsof Bicoid are concentration-dependent'it is acting as a morphogen. Evidence that the Bicoid protein gradiint determines anterior structures was obtained ihro,tgh injection of synthetic bicoid mRNA at different locations in the embryo. This treatment led to the formation of anterior structures at the site of iniection, with progressively more posterior structures forming at increasing distancesfrom the iniection site' Another test was to make flies that produced extra anterior Bicoid protein; in these flies, the anterior structures expanded to occupy a greater proportion of the embrYo.

AND VERTEBRATES . T :H E M E SA N D V A R I A T I O N 5I N I N S E C T S C O N T R O LO F B O D Y S E G M E N T A T I O N

971

(a) Wild type

Thresholdabove which fr4listgene is activated

-= o

I-iI I I Il"i"l"T"k Ventralmesoderm (b) Mutant-no Dorsal

Thresholdabove which fwrsf gene is activated

,:) c o a

o

(c) Mutant-nuclear Dorsal everywhere n U

Thresholdabove which fr4listgene is activated

o

All mesoderm

A gradientof the transcription ral cellfates in the early omologof vertebrate NF-xB(See Figure16-35)(a)Inwild-type embryos, moreDorsal proteinenters the nucleion theventral sideOnceinside a nucleus, Dorsal activates targetgenessuchasfwist,whichencodes a transcription factorthat directsmesoderm formationThedifferential entryof Dorsalthus

creates a dorsal-ventral polarityin twrstexpression and mesoderm induction. Inactive cytoplasmic NF-nB on thedorsal sidefailsto activatetwist.(b)A mutantlackingDorsalmakesno mesoderm cells. (c)Conversely, in a mutantthathasDorsal in thenucleiof allcells, all cellsdifferentiate asmesoderm[Adapted fromL Wolpert etal, principles of Development,2ndedition, Oxford Press,Figure5-14 l

orly in parallel (Figure 22-25a-c). Analysis of the hunchback gene revealed that it contains three low-affinity and three high-affinity binding sites for Bicoid protein. Experiments with syntheric genescontaining either all high-affinity or all low-affinity Bicoid-binding sitesdemonstratedrhat the affinity of the site determines the threshold concentration of Bicoid at which gene rranscription is activated (Figure 22-25d, e). In addition, the number of Bicoid-binding sites occupied at a given concentration has been shown to determine the amplitude, or level, of the transcription response. 972

CHAPTER 22

|

THEMOLECULAC R E L LB | O L O G YO F D E V E L O P M E N T

llll;

Development overviewAnimation:GeneControlin Embryonic 1 5 0m i n

1 6 0m i n

1 8 0m i n

22-24 Maternallyderivedbicoid FIGURE < EXPERIMENTAL mRNAis localizedto the anterior region of early Drosophila to the with anterior shownarepositioned embryos.All embryos situ hybridization in experiment, In this the top leftanddorsalat for bicoidmRNA labeledRNAprobespecific with a radioactively 2 5-3.5 hoursafter sections on whole-embryo wasperformed f romthesyncytial thetransition Thistimeperiodcovers fertilization probe After excess gastrulation of to the beginning blastoderm (dark mRNA bicoid maternal to probe hybridized wasremoved, h iyc o i dp r o t e i ni sa s i l v egr r a i n sw) a sd e t e c t ebdy a u t o r a d i o g r a pB t r a n s c r i p t i foanc t o rt h a ta c t sa l o n ea n dw i t h o t h e rr e g u l a t o tros anterior of certaingenesin the embryo's controlthe expression photographs of courtesy 335:25; 1988, tVature PW Ingham, regionfFrom I n g h a m W P l

2 1 0m i n

Findings from studiesof Bicoid's ability to regulatetranscription of the hunchback geneshow that variations in the levels of transcription factors, as well as in the number or affinity of specificregulatory sequencescontrolling different targetgenes,or both, contribute to generatingdiversepatterns of gene expressionduring Drosophila development.Similar mechanismsare employedin other developingorganisms.

TranslationInhibitorsReinforceAnteriorPosteriorPatterning Cell fates at the posterior end of the fly embryo are specified by a different mechanism-one in which control is at the

Bicoid protein gradient in embryo

Copies of bicoidgene in mother

Promoter

Expressionpattern

translational level rather than the transcriptional level' As

uniformly distributed throughout the embryo, its translation is prevented in the posterior region by another maternally deiived protein called Nanos, which is localized to the posterior end of the embryo' The set of genesrequired for posterior locali zation of Nanos protein is also required for germ-line cells to form at the posterior end of the embryo' bn. of thesegenes(staufen)is necessaryfor developmentof primordial germ cells (PGCs) in zebrafish. Thus at Ieast so-e g.t--line regulators have existed since fish and flies had a common ancestor.

!

(a)

0

o

Anterior -> Posterior

hunchback (b)

(c)

(d)

1

2

2

(e) 2

S U U

Highaffinity Bicoidbinding site

Lowaffinity Bicoidbinding site

Synthetic High-affinityBicoidb i n d i n gs i t e s

Synthetic

--\rrz-

J-----------} IF

Low-affinityBicoidb i n d i n gs i t e s

22-25 MaternallyderivedBicoid FIGURE < EXPERIMENTAL hunchback(hb) gene embryonic of the controls expression (a-c) the numberof lncreasing axis' anterior-posterior the along gradient in theearly theBicoid bicoidgenesin motherflieschanged e m b r y ol ,e a d i n tgo a c o r r e s p o n d icnhga n g ei n t h e g r a d i e not f genein the proteinproduced from thehunchback Hunchback threehighpromoter contains hunchback genome. fhe embryo's flies sites.Transgenic Bicoid-binding affinityandthreelow-affinity promotercontaining genelinkedto a synthetic a reporter carrying sites(e)were sites(d)or four low-affinity eitherfour high-affinity gradrent in the protein Bicoid same the to response preparedIn by a promoter genecontrolled of the reporter embryo,expression moreposteriorly sitesextended Bicoid-binding high-affinity carrying sites.This low-affinity gene carrying of a reporter thandidtranscription that activates Bicoid of concentration thatthe threshold resultindicates the Bicoid-binding of the affinity on depends transcription hunchback fashion[Adapted othertargetgenesin a similar regulates site Bicoid Ce//68:201l from D St Johnstonand C NUssleln-Volhard,1992,

AND VERTEBRATES C O N T R O L O F B O D Y S E G M E N T A T I O N : T H E M E SA N D V A R I A T I O N SI N I N S E C T S

973

Figure 22-26 lllustrates how translational regulation by Nanos helps to establish the anterior -+ posterior Hunchback gradient neededfor normal fly development.Translational repression of hunchback mRNA by Nanos depends (a)

Fenilized egg

Early embryo

Hunchback protein derived from maternalRNA

Nanos protein

(b) Nanos protein

h b m R N A( m a t e r n a l )

InsectSegmentationls Controlledby a Cascade of TranscriptionFactors

Anterior

Posterior

I +

Nano protei

I

-@en

;;-+AAAAAAA3' mRNA

Nanos promotes deadenylation

Translationof hb mRNA

No hb translation

I

v

I

I

v

v

Anterior development

Abdominal development

Hb protein

h b m R N A( m a t e r n a l )

posterior

Anterior

A FIGURE 22-26 Roleof Nanosproteinin excludingmaternally derivedHunchback(Hb) protein from the posteriorregionof Drosophilaembryos.(a)Bothnanos(blue)andhunchback (red) mRNAs derived fromthe motheraredistributed uniformly in the fertilized eggandearlyembryo. Nanosprotein, whichisproduced onlyin the posterior region, subsequently inhibits translation of maternal hb mRNAposteriorly. (b)Diffusion of Nanosproteinfromits siteof synthesis in theposterior _+ regionestablishes a posterior anterior Nanosgradient. A complex of Nanosandtwo otherproteins inhibits translation of maternal hb mRNA. Asa consequence, maternally derived Hbproteinisexpressed in a gradedfashion that parallels andreinforces the Hbproteingradient resulting fromBrcoid_ controlled transcription of theembryos hb gene(seeFigure 22_25) el al, 1997 [SeeC Wreden 124:30151 , Development 974

.

c H A p r E R2 2

|

on a specific sequencein the 3'-untranslated region of the mRNA, the Nanos-responseelements (NRE). Along with two other RNA-binding proteins, Nanos binds to the NRE in hwnchbac&mRNA. The results of genetic and molecular studies suggest that Nanos promotes deadenylation of hunchback mRNA and thereby decreasesits translation. In the absenceof Nanos, the accumulation of maternal Hb protein in the posterior region leads to failure of the posterior structures to form normally, and the embryo dies. Conversely,if Nanos is produced in the anterior, thereby inhibiting the production of Hb from both maternal and embryonic hunchback mRNA, anterior body parts fail to form, againa lethal consequence.Translational control due to the action of an inhibitor, mRNA localization, or both, may be a widely used strategy for regulating development. For instance, specific mRNAs are localized during the development of muscle cells, and during cell division in the budding yeastSaccharomycescereuisiae(seeFigure 2I-28).

In both insectsand vertebrates,the anterior-posterior(headtail) axis is divided into a set of repeats,or more preciselyrepeats with variations: vertebrae and associatedganglia in vertebrates,body segmentsin insects.Specificgenescontrol subdivision of the embryo into repeats, while other genes control the differences between repeats. As noted already, not all vertebraehave attached ribs, and only some of an insect'sbody segmentshave legs growing from them. Ve discussthe genescontrolling segmentationof insectsin this section and the rather different type of regulation underlying vertebrate segmentationin the next section. Then we delve into the genesthat control differencesbetween segmenrs. Once the gap geneshave been properly activated in the Drosophila embryo, the next stepson the road to body segmentation are controlled by a transcription-factor (TF) cascade in which one TF controls a gene encoding another TR which in turn conrrols expressionof a third TF. At each step, more than one genemay be regulated.Sucha TF cascadecan generatea population of cellsthar may all look alike but differ at the transcriptional level. TF cascadeshave both a temporal and a spatial dimension. At each step in a cascade.for instance.RNA polymeraseand ribosomescan take more than an hour to produce a transcription factor from its correspondingmRNA. Spatial factors come into play when cells at different positions within an embryo synthesizedifferent transcription factors. The rough outline of cell fates that is laid down in the syncytialfly embryo is refined into a systemfor preciselycontrolling the fates of individual cells.Discovery of the relevant regulators came from a genetic screenfor mutants with altered embryo body segments.In addition to hunchback, four other gap genes-Krilppel, knirps, giant, and tailless-are transcribed in specificspatial domains beginning about 2 hours after fertilization (Figure 22-27a). Expression of these genes, like that of hunchbacft,is regulatedfirst by marernal factors and then by cross-interactionsamong the gap genes.

T H EM o L E c u L A R c E L LB t o l o c y o F D E V E L o p M E N T

/z^s\ \\"2

of SegmentationGenesin a DrosophilaEmbryo Video:Expression

( a ) G a p - g e n ep r o t e i n s

( b ) H u n c h b a ca knd Krrippel

(c)

Hunchback

Kriippel

n rps

{|'

22-27 Gapgenesand pair-rule FIGURE A EXPERIMENTAL genesare expressedin characteristic spatialpatternsin early permeabilized werestained embryos Drosophilaembryos.Fixed, protein for a particular antibodies specific withfluorescence-labeled to the leftanddorsal with anterior shownarepositioned All embryos for individually embryos werestained at thetop (a)Thesesyncytial of byfourof thefivegapgenesTranscription encoded the proteins by Hunchback, IheKruppel,knirps,andgiant gapgenesis regulated to embryowasdoublystained andCaudal(b)Thissyncytial Bicoid, protein(green)The protein(red)andKruppel Hunchback visualize in part proteinvisible posterior hereisonlyweaklyvisible Hunchback ( a )d u et o t h ep l a n eo f f o c u sT h ey e l l o wb a n di d e n t i f i et h s er e g i o n i n w h i c hp r o d u c t i oonf t h e s et w o g a pp r o t e i nosv e r l a p (sc )I n t h i s eg meb r y oF, u s htia r a z u( b l u ea) n dE v e n - s k i p p e d blastoderm-sta genesffz andeve, (brown)proteins, by the pair-rule encoded to the in stripesEachstripecorresponds areexpressed respectively, primordial about14segments cellsof onebodysegmentAltogether

canbe of segmentation evidence areformedNomorphological of for the RNAor proteinproducts seenat thisstage,but staining plan body a segmented of genesreveals the beginnings pair-rule (lower primordia (d)Therelationship betweenthe earlysegment gene(darkgray),and of onepair-rule stripes expression embryo), thatareformed(upperlarva)is larvalsegments the eventual fromheadto segments, the different Thecolorsindicate depicted. larval The larva' to embryo from tail,andhowtheycorrespond develops Notethateachsegment externally. headis barelyvisible that isaboutfour cellswide(inthe head-tofroma prrmordium andabout60 cellsaroundHalfof the segments taildirection) geneandhalffrom the pair-rule fromcellsthat express develop a different express interstripes "interstripes" not; the do that the segments A : abdominal segments; gene.T : thoracic pair-rule et al, 1992,Cell59:23].Part(b)courtesy fromG Struhl IPart(a)adapted of Part(d)courtesy Lawrence of Peter of M LevinePart(c)courtesy

All the gap-geneproteins are transcription factors. Becausethese proteins are distributed in broad overlapping p e a k s ( F i g u r e2 2 - 2 7 b ) , e a c h c e l l a l o n g t h e a n t e r i o r - p o s t e rior axis contains a particular combination of gap-gene proteins that activates or repressesspecific geneswithin that cell. Indeed, something like a battle ensues,because some gap proteins repressthe transcription of genes encoding other gap proteins. Although they have no known extracellular ligands, some gap proteins resemblenuclear receptors, which are intracellular proteins that bind lipophilic ligands (e.g., steroid hormones) capable of crossing the plasma membrane. Most ligand-nuclear re-

ceptor complexes function as transcription factors (see Figure 7-50). The sequencesimilarity between gap proteins and nuclear receptors suggeststhat gap genesmay have evolved from genes whose transcription was controlled by signalsthat could cross membranes,such as the steroid hormones.The use of such signal-controlledgenes' rather than TF cascades,could explain how early cell-fate specificationoperatesin animals that do not have a syncytial stage. The fates of cells distributed along the anterior-posterior axis are specifiedearly in fly development.At the sametime' cells are responding to the dorsal-ventral control system'

N i p a mP a t e l

AND VERTEBRATES . C O N T R O LO F B O D Y S E G M E N T A T I O NT H E M E SA N D V A R I A T I O N SI N I N S E C T S

975

Each cell is thus uniquely specifiedalong both axes. If each of the five gap geneswere expressedin its own section of the embryo, at just one concentration, only five cell types could be formed. The actual situation permits far greater diversity among the cells.The amount of eachgap protein variesfrom Iow to high to low along the anterior-posterioraxis, and the expressiondomains of different gap genes overlap. This complexity createscombinations of transcription factors that lead to the creation of many more than five cell types. RemarkablS the next step in Drosophila development generates a repeating pattern of cell types from the rather chaotic non-repearing pattern of gap-gene expressron domains.

stripes,separatedby "interstripes" where that pair-rule gene is not transcribed (Figure 22-27d). Murant embryos that lack the function of a pair-rule gene have their body segments fused together in pair-wise fashion-hence the name of_this class of genes.The expression stripes for each pairrule gene partly overlap with those of other pair-rule gines; so each gene must be responding in a unique way ro gapgene and other earlier regulators.

The transcription of pair-rule genesis controlled by transcription factors encoded by g"p and maternal genes. Becausegap and maternal genesare expressedin broad, nonrepeating bands, the question arises: How can such a non-repeating pattern of gene activities confer a repeating pattern such as the striped expressionof pair-rule genes?To answer this question, we consider the transcription of the euen-skipped(eue) genein stripe 2, which is controlled by the maternally derived Bicoid protein and the gap proteins Hunchback, Kri-ippel,and Giant. All four of thesetranscription factors bind to a clustered set of regulatory sires, or enhancer, located upstream of the eue promoter (Figure 22-28a). Hunchback and Bicoid activatethe transcription of eue in a broad spatial domain, whereas Kriippel and Giant represseue transcrrption, thus creating sharp posterior and anterior boundaries.The combined effectsof theseproteins, each of which has a unique concentration gradient along the anterior-posterior axis, initially demarcatesthe boundaries of stripe2 expression(Figure22-28b). Expression of the other eue stripes also depends on specificenhancers.Each stripe of eue expressionis formed in responseto a different combination of transcriptional regulators acting on a specificenhancer,so the non-repeating distributions of regulators createrepeating patternsof pair-rule gene repression and activation. If even one enhancer is bound by an activating combination of transcriptional

Video: EstablishingEve Expression in Drosophila Embryogenesis ffi (a) eye gene transcription regulation

Stripe2 e nn an c e r Start of transcription G i a n (t { )

B i c o i d( t ) A c t i v a t o r s( t )

!

(b) evestripe2 regulation H u n c h b a c(kt )

G i a n t( 0 )

evestripe2

K r i i p p e(l { )

I

< FfGURE 22-28 Controlol even-skipped(eve)stripe 2 in the Drosophilaembryo.Onlyoneof the eyegenestripesisrepresented. Withineacheuestripe,a segment boundary will laterform Thuseye functiongivesriseto halfthesegment boundaries in the embryo (a)Diagram of the 815-bpenhancer controlling transcrrption of the pair-rule geneeyein stripe2 Thisregulatory regioncontains binding sitesfor BicoidandHunchback proteins, whichactivate thetranscription of eve,andfor Giantand Kruppelproteins, whichrepress its transcription Theenhancer isshownwith allbindingsitesoccupied, but in an embryooccupation of siteswillvarywith position alongthe anterior-posterior axis(b)Concentration gradients of thefour transcription factorsthatregulate evestripe2 Thecoordinated effect (J) andtwo activators of the two repressors (t) determine the precise boundaries of thesecond anterior euestripeOnlyin the orange regionisthe combination of regulators correct for the eyegeneto be transcribed in response to thestripe2 controlelement. Further anterior, Giantturnseyeoff;furtherposterior, the levelof Bicoid activator istoo lowto overcome repression by Kruppel. Expression of otherstripes isregulated independently by othercombinations of transcription factorsthatbindto enhancers not depicted in part(a) [SeeS Smallet al , 1991, Genes& Devel.8:827 |

976

CHAPTER 22

|

THEMOLECULAC R E L LB | O L O G YO F D E V E L O P M E N T

regulators, the presenceof other enhancersin an inactive, "off" state (not bound to a regulator) will not prevent transcription. For instance,in Eve stripe 2, the right combination and amounts of Hunchback and Bicoid createan "on" state that activatestranscription even though other enhancersare present in the inactive state. In each stripe, at least one enhancer is bound by an activating combination of regulators. Note that this systemof genecontrol is flexible and could be used to produce non-repeating patterns of transcription if that was useful to an animal. Similar responsesto gap and maternal proteins govern the striped patterns of transcription of the two other pairrule genes,runt and hairy. Becausethe enhancersof rwnt and hairy respond to different combinations of regulators, the eue,runt, and hairy expressionstripespartly overlap one another, with each stripe for any one gene offset from a stripe for another gene. Subsequently,other pair-rule genes, including fusbi tarazu (ftz) and paired, become active in responseto the Eve, Runt, and Hairy proteins, which are transcription factors, as well as to maternal and gap proteins. The outcome of this transcription-factor cascadeis a pattern of overlapping stripes. The initial pattern of pair-rule stripes,which is not very sharp or precise, is sharpened by autoregulation. The Eve protein, for instance, binds to its own gene and increases transcription in the stripes, a positive autoregulatory loop. This enhancementdoes not occur at the edgesof stripes where the initial Eve protein concentration is low; so the boundary between stripe and interstripe is fine-tuned. The pair-rule genesdirect formation of the embryo's segment boundaries. Since each pair-rule gene is expressedin stripes and each stripe overlaps one segmentboundary, each pair-rule gene contributes to half the segment boundaries. Acting together,all the pair-rule genesform all the segment boundaries and also control other pattern elementswithin each segment.In early embryos each segmentprimordium is about four cells wide along the anterior-posterior axis, which corresponds to the approximate width of pair-rule expressionstripes.With pair-rule genesactive in alternating four-on four-off patterns, the repeat unit is about eight cells. Each cell expressesa combination of transcription factors that can distinguish it from any of the other sevencells in the repeat unit. Under the control of pair-rule proteins and the later-acting segment-polarity genes,the repeating morphology of segmentsbeginsto emerge;it is completedabout 10 hours after fertrlization. As cell-fate determination progressesin the fly embryo, a variety of signaling proteins begin to play a role. Theseinclude Hedgehogand'!7nt, which are encoded by segment-polaritygenesand are produced in stripes, one stripe within each segment, under the control of pair-rule gene products. The broader and earlier-formed stripes of pair-rule gene expressionoverlap in certain regions, and that's where particular combinations of pair-rule transcription factors give rise to the fine pattern of segment-polarity gene stripes. Note that the onset of signal-basedcontrols allows cells to respond to what their neighbors have done and make adjustments.Otherwise parts of the pattern might be missing or duplicated.

From the broad maternal gradient of Bicoid to the singlecell precisionof the segment-polaritygenes'the fly embryo is progressivelysubdivided into repeatingunits. One can readily imagine how changes in stripe-specific enhancers, amounts of particular transcription factors, and the range of signals during evolution could modify the pattern of segmentsin differentorganisms.

VertebrateSegmentationls Controlled by CyclicalExpressionof RegulatoryGenes Now we return to vertebratesto examine how segmentation in these animals compares with that in insects. After the three body axes have been establishedin a vertebrate embryo, dramatic changestake place along all of them. One of the most visible changesis the initiation of a repeating pattern that later gives rise to vertebrae and ribs. This is patterning along the anterior (head) to posterior (tail) axis. In mice and humans, the first sign of the vertebrae appears in the mesoderm that accumulatesunder the primitive streak. The mesoderm forms, you will recall, by an epithelialmesenchymal transition in which cells along the primitive streak cut loose and migrate inside (seeFigure 22-11').On each side of the midline axis, mesoderm composed of loose mesenchymalcellsbeginsto round up to form pairs of spherical epithelia caIIedsomites.Somitesinitially form at the anterior and successivelyappear pair by pair in the posterior direction (Figure 22-29a), giving them value as a way to stageembryos.This striking caseof a mesenchymal-epithelial transition has huge consequencesfor the embryo. From somitescome the vertebraeand ribs, the musclesof the body wall and limbs, and the dermis (inner skin) of the back. Without somites,we would be blobs. The mesodermthat has not yet formed somites is called

from the tail, a retinoic acid signal from the head, and Wnt and Notch signalswithin the presomitic mesoderm.The fgfS gene is expressedin the presomitic mesoderm where it is iorming near the posterior tip of the embryo. Because/gf8 mRNA is unstable, the highest level of FGF8 protein builds

(Figure22-29b). The most remarkable aspectof somite-formation regulation in vertebrates was first discovered in studies of the chicken gene hairyl, which is related to one of the Drosophila pair-rule segmentationgenes.In situ hybridization of developing somites in chick embryos showed that hairyl transcriptsare produced in cycles,with the duration of one cycle corresponding to the time it takes to form one somite (90 minutes in a chick, longer in mammals). A wave

AND VERTEBRATES T :H E M E SA N D V A R I A T I O N Sl N I N S E C T S C O N T R O LO F B O D Y S E G M E N T A T I O N

977

(a) Earlyembryo (5 pairs of somites)

(b)

Somites

Anterior

Late embryo (9 pairs of somites)

FGFSprotein Posterior

Presomitic mesoderm

Growth and maturation

Tailbud

A FIGURE 22-29 Progressive formationof somitesin human embryos.(a)Fivepairsof somites haveformedat theanterior of the earlier embryoon the left.Somiteformationproceeds towardthe posterior, andin the laterembryoon the right,ninepairshaveformed In bothmicrographs, the developing headison the leftandthetailbud on the right (b)Gradients of FGF8, proteinmadein thetail a secreted of hairyl expressionmoves from posterior to anterior in the presomitic (unsegmented)mesoderm. Subsequentinvestigations revealed a rather large number of genesthat undergo cyclesof expression,and all turned out to be related to the Notch or ril/nt signalingpathways. Mutations in either pathway cause drastic defects in somite formation. In humans. for instance, mutations affecting Notch pathway components causeAlagille syndrome and Jarcho-Levin syndrome, both of which are associatedwith malformed vertebrae. For both the Notch and Wnt pathways, feedback loops are established that cause temporal cycling of expression. For example, the hesT gene encodes a transcription factor involved in Notch signal transduction. rWhentranscription of hesTis stimulated by a FGF signal from the posterio; presomitic mesoderm, a burst of HesT protein production occurs (Figure 22-30a). HesT protein, in rurn, controls the expressionof target genesthat contribute to somite formation. BecauseHesT also acts as a repressorof its own gene, binding to its gene and turning it off, the HesT protein accumulates only until it reaches a high enough level to represses hes7 transcription. This negative autoregulation thus limits the duration of hes7 expression.Each part of the presomitic mesoderm does the same thing in turn: hes7

expressionjust as a somite forms, another burst in the cells that will form the subsequentsomite, and so on. If Notch or Wnt feedback loops are blocked, somites are highly abnormal, but the details of how both pathways control shaping of the somites are not known. 978

'

c H A p r E R2 2

|

bud,andretinoic acidfromthe head,controlsomiteformation from presomitic mesoderm, whichfirstarises in thetailbud Highlevels of FGF8prevent maturation of presomitic cellsintosomites in the posteriolwhereas highlevels of retinoic acidactsto stimulate formation (a)Kohei of somiteslPart Shiota/Congenital Anomaly Research Center, Kyoto University. Part(b)adapted fromA F.Schier; 2004,Nature 427i4031 'We

can now understand the two different strategiesfor controlling the formation of repeating body parts in insects and vertebrates. In Drosophila, the regulation differs for each body segment: different combinations of gap transcription factors, activated in specific regions along the anterior-posterior axis by maternal influences, regulate pairrule gene stripes, and pair-rule transcription factors in turn combine to regulatethe still finer stripesof segment-polarity gene transcription. Each stripe has a distinct regulatory history involving different gap or pair-rule proteins. Thus repeat formation is controlled by spatial differences.In contrast, repeating vertebrate somites are formed by the same regulatory process occurring again and again. A remarkable feedback system creates a cyclical clock that causes the genes responsible for building somites to be expressedin bursts. Thus repeat formation is controlled by temporal (time) differences.

DifferencesBetweenSegmentsAre Controlled by Hox Genes Despite the differencesin how repeating body parts are formed in insectsand vertebrates,the two groups of animals are reunified in employing the same family of genesto crearevariation among the repeats.Theseare the Hox genes,which control differences in cell identities and indeed the identities of whole parts of an animal along the anterior-posterior axis. Thesedifferencesare superimposed upon the underlying reperitive nature of some of the tissues.Hox genesencode highly related transcription factors containing the homeodomain motif (Chapter 7). Indeed,what unifies the whole group of Hox proteins is a similar DNA-binding homeodomain sequence;the proteins have little else in common. The homeodomain sequencesare also the basisfor classifying all the Hox genes.

T H EM o L E c u L A R c E L LB r o L o c y o F D E V E L o p M E N T

(a)Activationof hesTtranscription

(b) Sequentialinductionand autoregulationof hesT Signal Tail

Head

/1

22-30 Controlof cyclicalgene expressionin the A FIGURE is developingsomites.(a)An initialburstof hesZtranscription t r i g g e r ebdy a p o s i t i vsei g n a lp, r o b a b lFy G F SA s H e s Tp r o t e i n( a component) accumulates, it eventually bindsto its Notchpathway Thrsprocess is repeated, once own geneandturnsoff transcription performingsomite,in increasingly posterior regions of the presomitic

end, (b)TheFGF8 to be at the posterlor signalcontinues mesoderm to istriggered presomitic mesoderm moreposterior so progressively programs, to leading NotchandWnt expression cyclical activate of of transcription represent bursts somiteformationTheredcircles andthe components NotchandWnt signaling genesencoding loopsthatshutthemdown feedback negative

Mutations in Hox genesoften causehomeosis-that is, the formation of a body part having the characteristicsnormally found in another part at a different site. For example, some mutant flies develop legs on their headsinsteadof antennae. Lossof function of a particular Hox genein a locationwhere it is normally active leadsto homeosisif a different Hox genebethere;the resultis the formation of cellsand comesderepressed structures characteristic of the derepressedgene. A Hox gene that is abnormally expressedwhere it is normally inactivecan take over and impose its own favorite developmentalpathway on its new location (Figure22-3I).

mosomes is colinear with the order in which they are expressedalong the anterior-posterioraxis (Figure 22-32a). The fly Hox genesare located at two locations on the same chromosome but are effectively one cluster of eight Hox genes.At one end of the cluster are "head" genes,which are transcribed specificallyin the head and are necessaryfor formation of head structures.Next to them are genesactiYeand functional in the thorax. and at the other end of the cluster are abdomen genes.The arrangement reflects evolutionary gene duplications and is retained becausethe genes share regulatory sequencessuch as enhancers(Chapter 7). The expressiondomains of Hox genescan overlap' so the development of a particular body structure can depend upon more than one gene. ln Drosophila, the spatial pattern of Hox-gene transcription is regulatedby maternal, gap' and pair-rule transcription factors. The protein encodedby a particular Hox gene controls the organizationof cellswithin the region in which that Hox geneis expressed.For example,a Hox protein can direct

Organization of Hox Genes Classicalgeneticstudiesin Drosophila led to discoveryof the first Hox genes(e.g.,Aatennapedia and Ultrabithorax). Corresponding genes with similar functions (orthologs) have since been identified in most animal species.Each Hox gene is transcribed in a particular region along the anterior-posterior axis in a remarkable arrangement where the order of genesalong the chro-

Normal the LikeotherHoxgenes, 22-31 Hox-genephenotypes. FIGURE (lJbx)genecontrols of cellswithinthe lJltrabithorax the organization wing actsin preventing lt normally regionin whichit isexpressed. sothatnormalflieshavea singlepairof wings Mutations formation, in Hoxgenesoftenleadto the formationof a bodypartwhereit does

Ubx mutant not normallyexist,In the mutantshown here,the lossof Ubxfunction from the third thoracicsegmentallowswingsto form where normally 1978, IFromE B Lewis, thereare only balancerorganscalledhalteres. 1978, copyright Nafure, permission from by Reprinted Nature276i565 Limited Journals Macmillan l .

5 I N S E C TASN D V E R T E B R A T E 5 S N D V A R I A T I O NI N NH : E M EA CONTROO L F B O D Y S E G M E N T A T I OT

979

( a )D r o s o p h i l a

3'

5' Antennapedia comptex

(b)Mouse

Bithoraxcomolex

Hoxa,chromosome6

3',

5'

Hoxb,chromosome11

Hoxc,chromosome15

Hoxd,chromosome2

FIGURE 22-32 Relationbetween Hox gene clustersin Drosophilaand mammals.(a)ThesingleDrosophrla Hoxcluster i ss p l i ti n t ot w o c h r o m o s o m|aolc a t i o nos n ; eg e n eg r o u pi st h e Antennapedia complex andtheotheristhe Bithorax complex. The genesthatcontrolheadformation areat the 3' endof thecluster (yellow/red shades), thosethatcontrolformation of theabdomen areat the 5' end(blue/green shades), andtheonesin-between controlthoracic (purple), structures asillustrated in thefly drawing, whrchshowswherethe differentgenesareexpressed (b)The arrangement of genesin the mouseandhumanHoxgeneclusters issimilar to that in Drosophila, buttherearefourclusters andeach of themis missing someof thesetof genesForexample, theclass 1 Hoxgenesof miceandhumans aresimilar to the /abgeneof Drosophila, basedon encodedproteinsequences Thereisa class1

or preventthe local production of a secretedsignalingprotein, cell-surfacereceptor,or transcription factor that is neededto build an appendageon a particular body segment.Drosophila Hox proteins control the transcription of target geneswhose encodedproteins determinethe diversemorphologiesof body segments.Much remains to be learned about how morphology is controlled by thesetarget genes,but some rargersencodepowerful Y/nt and TGFB signals.The associationof Hox proteins with their binding siteson DNA is assistedby cofactors that bind to both Hox proteins and DNA, adding specificity and affinity ro rheseinteracrions. How about vertebrates?In contrast to flies, which have a single Hox gene cluster, mammals have four copies of the Hox cluster, a-d, located on different chromosomes(Figure 22-32b). \Within each cluster, the genesare numbered 1, at the head end, to 13, at the tail end. The different copiesof a particular gene (e.g.,Hox4) in the four clustersare closer to each other in sequencethan to Hox genesof another numerical class. Although the mammalian Hox genes are clearly related to the fly Hox genes,there has been an expansion of fly abd-type genesin vertebrates(Hox classes8-13). 'Vfithin eachcluster some geneshave beenlost, evidently becausethe other two or three copies are sufficient. Experiments with 980

.

c H A p r E R2 2 |

genein Hoxa, b, andd clusters but not in thec clusterEvidently threeisenough.In contrast, the class4 geneis represented in all of the mammalian clusters. Anotherdifference frominsectto mammal isthat mammals have"extra"versions genes(class of the posterior 9 andup)that correspond to the abd-typegenesin flies The drawings of the fly andmouseembryoindicate whereHoxgenesare transcribed, fromwhichit canbe seenthat the orderhasbeen preserved duringthe roughlyhalfbillionyearssincetheyhada commonancestorThedrawings area simpilfication, sincein many cases a geneisexpressed with a sharpanteriorboundary aswellas a gradedpatterngoingtowardthe tail,andexpression patterns varybetweendifferenttissues[Adapted fromL Wolpert et al, 2001, Principles of Development, 2nded, Oxford University Press, Box4-4al

mouse "knockouts" missing one or more Hox genesfrom one numerical class show considerableredundancy among the 39 total genes. Evolution of Hox Gene Clusters Hox clusters are the most dramatic example of the conservation of gene groupings acrossa wide range of animals. Their organization is so striking that they serve as useful tools for studying evolution. The single Hox cluster in Drosophila is represented four times in mammals (seeFigure 22-32). Comparisons of the genome sequencesof a variety of vertebrates have revealed that the copies of the Hox clusters are far from perfect. During evolution, some specieshave lost one or more Hox genes;in other species,Hox geneshave become duplicated within a cluster. The transitions between different clusters and losses and gains of individual Hox genes among present-day organisms allow ancestral forms to be deduced (Figure 22-33). For example, in the time sincefrogs and humans had a common ancestor, about 370 million years ago, frogs have lost Hox genesb13 and d12. As more studies are done of how Hox genescontrol body morphology, it will be increasingly possible to relate evolurionary changesin Hox genesto pattern formation in the embryos

T H EM o L E c u L A R C E L LB t o L o G yo F D E V E L o e M E N T

Aa Ab Ba Bb

Aa++-rlHlll+ Ab Ba Bb Ca

Cb Da

cb Da

Db Spotted green pufferfish

Zebrafish

Lossof 11genes

Lossof 7 genes

-296 Mya

Aa Ab Ba Bb

-|..flIHl|I|I+

A B

6

c-.. n

Freshwaterfish (Bichir) Loss of at least 1 gene

cb

cc

Da Db

Dflrl|ll-----l|*

D+-r-l|lr------ll+

Human

Westernclawedfrog

Hypotheticalancestor Lossof 31 genes -420 Mya

Genome duplication

-370 Mya

AfrHrHl|ill+ B+-Hl|llrrrl+

Loss of 2 genes

L...,'..'.....|-lr..Ji..Ji..@

D+-flilr-+}-+ Coelacanth A B

Lossof 1 gene -410 Mya

Lossof 1 gene

D Shark

-450 Mya

Lossof 5 genes

A tlffi

Loss of at least 1 gene

-528 Mya A B

c-----

D Hypothetical ancestor

Lossof 3 genes

c-.. Hypotheticalancestor

+ Exsistinggene sequenceknown + Hypotheticalgene sequencecurrentlyunknown + Describedpseudogene

22-33 Evolutionof vertebrateHox clusters.Genome FIGURE projects organisms, fromdifferent in whichallgenesaresequenced a sw e l la sf o c u s e sd t u d i eosf H o xg e n e sa, r ep r o v i d i nign s i g hitn t o TheHoxclusters shownherein of the Hoxclusters. the evolution of the darkgreen,blue,yellow,and redarebasedon sequencing a ryg a n i s mC g e n o m eosf t h e i n d i c a t epdr e s e n t - d o s .o m p a r i n g that the copiesof the from a varietyof vertebrates reveals sequences oneor moregenes In somespecies, Hoxclusters arefar fromperfect: withina havebeenlost,whiletn others,geneshavebeenduplicated conservation of DNAsequence Since thefossilrecordandstudies cluster. shown the relationships betweenthe organisms haveestablished

of Hoxgenesin to deducethe arrangement here,it is possible (genes an reconstruct and light colors) in ancestors hypothetical in the evolutionof the Hox outlineof the eventsthat happened in time(Mya: millionyearsago) distances Theapproximate clusters in red areindicated hada commonancestor sinceanypairof species not every here, so represented are type.Of course,not all ancestors As morestudiesaredoneof how Hoxgenes steocanbe deduced. possible to relate it will be increasingly controlbodymorphology, in Hoxgenesto patternformationin the changes evolutionary 2005,Trends andA Meyer, fromS Hoegg theycontrol.[Adapted embryos Genet21:.441 l

they control. This genomics approach to exploring evolution will yield more detailed biological histories as more genomesare sequenced.

trol target genes,and they use cofactors of the sametypes used by flies. Mutations affecting some of thesecofactors have been implicated in human cancer. As in flies, Hox-gene expressiondomains in early vertebrate embryos respond to seemingly invisible boundaries that correspond later to transitions between morphologically distinct repeating body units. Each Hox gene is expressedin some somites but not others (Figure 22-34)-They are expressedfirst in presomitic mesoderm,where they control the morphology of the vertebraethat will form later. Vertebrae are classified into groups according to their morphology and position along the anterior-posterior axis.

Functions of Vertebrate Hox Proteins VertebrateHox proteins control the different morphologies of vertebrae,of repeatedsegmentsof the hindbrain, and of the digits of the limbs. Mutations affecting some of the most posterior Hox genesin humans cause inherited syndromes that involve polydactyly (extra fingers or toes) and syndactyly (fused fingers or toes). Particular Hox genesare active in many other tissuestoo. As in flies, mammalian Hox proteins often act in combination to con-

AND VERTEBRATES T :H E M E SA N D V A R I A T I O N Sl N I N S E C T S C O N T R O LO F B O D Y S E G M E N T A T I O N

981

Wild type | tz

T13

FIGURE 22-34 Expression of the mouseHoxcl| gene in somites.Theanteriorboundary (arrow)is of Hoxcl0expression clearly visible in this9-day-old embryolFrom M Carapuco eral, 2005, Genes & Devel19:21161

The effectsof Hox mutations on vertebral developmenthave been studied with engineeredmouse mutants. In one of the most striking experiments, mice were produced that lack functional Hoxa10, HoxcL0, and Hoxd10 genes(thereis no Hoxb10). These mice exhibited a dramatic transformation of vertebral patterns. Lumbar and even sacral vertebrae, which normally have no ribs, developed with varying degreesof partial ribs (Figure 22-35). Probably evenmore Hox geneswould have to be changedfor a complete transformation. The inference is that the normal role of Hox10 transcription factors is to prevent rib development.

Hoxl0rnutant (lacksall Hox10a,c, and d genes)

T13

T12

Mutant L3

Hox-GeneExpressionls Maintainedby a Variety of Mechanisms 'Sfhen

Hox genes are turned on, their transcription must continue to maintain cell properties in specificlocations. As in the caseof the pair-rule gene euen-skipped,the regulatory regions of some Hox genescontain binding sitesfor their encoded proteins. Thus Hox proteins can help to maintain their own expressionthrough many cell generationsusing an autoregulatory loop. Another mechanism for maintaining normal patterns of Hox-gene expressionrequires proteins that modulate chromatin structure.Theseproteins are encodedby two classesof genesreferredto as the Trithorax group and Polycomb group. The pattern of Hox-gene expression is initially normal in Polycomb-groupmutants, but eventuallyHox-gene rranscnption is derepressedin placeswhere the genesshould be inactive. The result is multiple homeotic transformations,indicating that the normal function of Polycomb proteins is to keep Hox genesin a transcriptionally inactive state.The resultsof immunohistologicaland biochemicalstudieshave shown that Polycomb proteins bind to multiple chromosomal locations and form large complexescontaining different proteins of the Polycomb group. The current view is that the transientrepression of genesset up by patterning proteins earlier in development is "locked in" by Polycomb proteins. This stable polycomb-dependent repression may result from the ability of these proteins to assemble inactive chromatin structures (Chapter 7). Polycomb complexes contain many proteins,

982

CHAPTER 22

|

EXPERf MENTALFIGURE 22-35 Hoxl0 genesregulate vertebrashape.Separate knockoutmicewereconstructed, each l a c k i n g a p o r t ioof nt h e H o x l 0 a , c , odr g e n e E a c h otfh e s e genesis on a differentchromosomeIn eachcase,heterozygous micesurvived because theyhavea second,wild-type,copyof the gene.Themicewerecrossed to construct a strainheterozygous f o r m u t a t i o nast a l lt h r e eg e n e t i cl o c i C r o s s i ntgh e s em i c e t o g e t h egr e n e r a t esdo m eh o m o z y g o umsu t a n e t m b r y oIsa c k i n g b o t hc o p i e so f a l lt h r e eH o x 7 0g e n e sS. k e l e t o nf rso m 1 8 . 5d a y mutantembryos andwild-typeembryoswereisolatedand stained to revealthe detailsof the skeletons. Thesetop-viewand crosss e c t i o n ad li a g r a m sb,a s e do n t h e s t a i n e d s k e l e t o n isl l, u s t r a t eh e results. In wild-typemice,the thoracic(T)vertebrae haveribs, w i t h t h e m o s tp o s t e r i orri bo n T 1 3 .L u m b a(rL )v e r t e b r a(eb l u e (redbracket)do not haveribs. bracket) and sacral(S)vertebrate Mutantsthat arehomozygous for all threeHoxl0 geneshaveribs o n l u m b avr e r t e b r a(ee g , L 3 )t h a tw o u l dn o r m a l lhy a v eb e e n r i b l e s sa,n de v e no n s o m es a c r arli b s( e g , S 2 ) T . h ei n s e t s h o w crosssections of differentvertebrae and ribsto revealtheirshapes i n t h a td i m e n s i o n F.r o mt h e s er e s u l tw s e c a ni n f e rt h a t H o x 1 0 p r o t e i nas r en o r m a l lrye q u i r etdo s u p p r e srsi b f o r m a t i o o nn the d e v e l o p i nl g u m b a ar n ds a c r avl e r t e b r a e S.i n c et h e H o x l 0 g e n e s a r en o r m a l ley x p r e s s er ndt h i sr e g i o no f t h e e m b r y ot,h i s c o n c l u s i omna k e s e n s el f a n yo n eH o x l 0 g e n ei sf u n c t i o n i n g , t h e e x t r ar i b sa r en o t s e e no, r o n l yr a r e l ys e e n s, o t h eH o x l 0 g e n e sh a v ea t l e a s p t a r t i a l lrye d u n d a nf ut n c t i o n s[ A . d a p t ef rdo m D M W e l l i k a n d M R C a p e c c h i ,2 0 0 3 , S c i e n c e3 0 1 : 3 6 3 l

THEMOLECULAC R E L LB | O L O G YO F D E V E L O P M E N T

six stamensin whorl 3, and two carpels containing ovaries in including histone deacetylases, and appear to inactivatetranwhorl 4 (Figure 22-36a). These organs grow from a collection scription by modifying histonesto promote genesilencing. 'Whereas of undifferentiated, morphologically indistinguishablecells Polycomb proteins repressexpressionof certain called the floral meristem.As cellswithin the center of the floral Hox genes,proteins encoded by the Trithorax group of meristem divide, four concentric rings of primordia form segenes are necessaryfor maintaining expression of Hox quentially. The outer-ring primordium, which gives rise to the genes.Like Polycomb proteins, Trithorax proteins bind to sepals,forms first, followed by the primordium giving rise to the multiple chromosomal sites and form large multiprotein petals,then the stamenand carpel primordia. complexes,somewith a massof =2 x 106Da, about half the size of a ribosome. Some Trithorax-group proteins are hoFloral Organ-ldentity Genes Genetic studieshave shown mologous to the yeast S\fVSNF proteins, which are crucial that normal flower developmentrequires three classesof floral for transcriptional activation of many yeastgenes.Trithorax organ-identity genes, designatedA, B' and C genes.Mutations proteins stimulate gene expressionby selectivelyremodeling in these genesproduce phenotypesequivalent to those associthe chromatin structure of certain loci to a transcriptionally ated with homeotic mutations in flies and mammals;that is, one activeform (seeFigure7-43).The core of eachcomplexis an part of the body is replacedby another. In plants lacking all A, ATPase,often of the Brm classof proteins. There is evidence B, and C function, the floral organs develop as leaves(Figure that many or most genesrequire such complexes for tran22-36b,left). scription to take place. The loss-of-function mutations that led to the identification Many regulators of Hox-gene expressionhave been imof the A, B, and C gene classesare summarizedin Figure plicated in leukemia. Chromosomal translocationsthat fuse the genes encoding these regulators to novel sequences, 22-36b (rigbt\. On the basis of the various homeotic phenotypes observed,scientistsproposed a model to explain how the sometimes causing a gene encoding a chimeric protein to three classesof genescontrol floral-organ identity. According form, are frequently found in leukemia patients. Such futo this ABC model for specifying floral organs, class A genes sions, for instance, can create oncogenesthat cause white specify sepalidentity in whorl 1 and do not require either class blood cellsto grow uncontrollably(seeFigure 25-20l.Hox B or class C genesto do so. Similarly,class C genesspecify genes are active in blood cells, though Hox functions in those cells are incompletely understood. Homeotic genes-that is, geneslike the Hox genesthat control development of whole parts of the body-are also important in plant development,as we shall seenow. Again the homeotic genessuperimposea set of variations on an underlying repeat pattern. also postulatesthat A genesrepressC genesin whorls 1' andZ and, conversely C genesrepressA genesin whorls 3 and 4. t e q u i r e sS p a t i a l l y F l o w e rD e v e l o p m e nR To determineif the actual expressionpatterns of classA' RegulatedProductionof TranscriptionFactors B, and C genesare consistent with this model, researchers cloned these genesand assessedthe expression patterns of Gn It may seema long jump from animal segmentationto pattern their mRNAs in the four whorls in wild-type Arabidopsis plants, molecules that control in terms of the but @ plants and in loss-of-function mutants (Figure 22-37a, b). formation, many principles are similar. Like vertebraeor insect Consistent with the ABC model, A genesare expressedin segments,flowers have repeating parts. The basic mechanisms whorls 1. and 2, B genesin whorls 2 and 3, and C genesin controlling development in plants are much like those in whorls 3 and 4. Furthermore, in class A mutants' class C Drosophila: differential production of transcription factors, genesare also expressedin organ primordia of whorls 1 and controlled in spaceand time, specifiescell identities.Our un2; similarly, in class C mutants' class A genes are also exderstanding of cell-identity control in plants benefited greatly pressedin whorls 3 and 4. Thesefindings are consistentwith from the choice of Arabidopsisthaliana as a model organism. the homeotic transformations observedin thesemutants. This plant hasmany of the sameadvantagesas fliesand worms To test whether these patterns of expression are funcfor use as a model system:It is easyto groq mutants can be tionally important, scientists produced transgemc Araobtained, and transgenicorganismscan be made. We will focus bidopsis plants in which floral organ-identity genes were on certain transcription-control mechanismsregulating the expressedin inappropriate whorls. For instance, the introformation of cell identity in flowers. These mechanismsare duition of a transgenecarrying classB geneslinked to an strikingly similar to those controlling cell-type and anteriorA-classpromoter leads to the ubiquitous expressionof class posterior regionalspecificationin yeastand animals.I B genesin all whorls (Figure 22-37c). In such transgenics' whorl 1, now under the control of classA and B genes'develFloral Organs A flower comprises four different organs ops into petalsinsteadof sepals;likewise,whorl 4, under the called sepals,petals,stamens,and carpels,which are arranged control of both classB and classC genes,givesrise to stamens in concentric circles called whorls. Whorl 1 is the outermost; instead of carpels. These results support the functional imwhorl 4, the innermost. Arabidopsis has a completeset of floral portance of the ABC model for specifyingfloral identity' organs,including four sepalsin whorl 1, four petalsin whorl 2,

AND VERTEBRATES T :H E M E SA N D V A R I A T I O N Sl N I N S E C T S C O N T R O LO F B O D Y S E G M E N T A T I O N

983

Wild-typefloral organs S e p a l s( w h o r l 1 ) Petals (whorl 2) Stamens (whorl 3) Carpels ( w h o r l4 )

Loss-of-function homeoticmutations Whorl 1 Wild type

T

C l a s sA mutants

I

ClassB mutants

T

I

C l a s sC mutants

I

l

I

T

Sequencingof floral organ-identity genesrevealed that many encodeproteins belongingto the MADS family of transcription factors,which form homo- and hetero-dimers.Thus floral-organ identity may be specified by a combinatorial mechanismin which differencesin the activities of different (a) Wildtype wl

w2

4

z

I

homo- and heterodimericforms of various A, B, and C pro, teins regulate the expression of subordinate downstream genesnecessaryfor the formation of the different cell types in each organ. Other MADS transcription factors function in cell-typespecificationin yeast and muscle (Chapter 21).

(b) Lossof function

W3

wl

W4

< FfGURE22-36 Floralorgans and the effects of mutationsin organ-identitygenes.(a)Flowers of wild-typeArabidopsis thalianahavefour sepals in whorl1,four petalsin whorl2, sixstamens in whorl3, andtwo carpels in whorl4 Thefloral o r g a n sa r ef o u n di n c o n c e n t rw i ch o r l sa sd i a grammedat right.(b)lnArabidopsrs with mutations in allthreeclasses of floralorgan-identity genes, thefourfloralorgansaretransformed intoleaf(/eft)Phenotypic likestructures analysis of mutants identified threeclasses of genesthat control specification of floralorgansin Arabidopsis (right).Class A mutations affectorganidentityin w h o r l s1 a n d2 : s e p a l(sg r e e nb)e c o m ce a r p e l s ( p u r p l ea)n dp e t a l s( o r a n g eb)e c o m e stamens (red)Class B mutations causetransformation of whorls2 and3: petalsbecome sepals andstamens become carpels. In classC mutations, whorls3 and4 aretransformed: stamens becomepetals andcarpels become sepals[See D Wiegel andE M lvleyerowitz, 1994,Cell78:203l

w2

w3 W4

(c) B-genetransgenic

w1 w2

w3 W4

A_ se

pe

st

ca

pe

pe

st

st

B-

(se pe EXPERIMENTAL FtcURE22-37 Expression patternsof class A, B, and C genessupportthe ABCmodel of floral organ specification.Depictedherearethe observed patternsof expression thefloralorgan-identity genesin wild-type, mutant,andtransgenic Arabidopsis. Coloredbarsrepresent the A, B,and C mRNAsin each 984

CHAPTER 22

|

pe

se

whorl(W1,W2, W3, W4) Theobserved floralorganin eachwhorl is indicated asfollows:sepal: se;petals: pe;stamens: st; andcarpels: ca Seetextfor discussion D Wiegel andE M [See Meyerowitz, 1994,Cell78:203, andB A Krizek andE M Meyerowitz, 1996, Development 122:11 l

T H E M O L E C U L AC R E L LB t O L O G yO F D E V E L O P M E N T

s86

t N l t A d o ] 3 n l c ' l v u n l N , r . r u v rN r N o t I V ) H t ) l d sl d A . L - r r 3 )

'sareld Suorlt oql uI lurot ol sued dpog asnecsaua8 [ 6 - ya r n b r l ' a u o 1 s 6 u r n r 1 - 1 1 r q r n q 3 ' papuz'tr6o1olLqw1 'uosrel uror,t pardepyl uewnH'L661 eprlq f 11 xoH ur suoIlEtnIAJ'sutalord ulletuoJl{f, 8utlce-errrlrsod 'st)alopqUlq puesnoues eurds 6urpnpur iuanbar]-oo]-lle lo {1auen pue -a,rrle8auJo uorlf,e aqr .{g peureruleluoJEsuJalled uors e o] pealarnsol)oqnl leJnauut s])ala6 a7 {ep uo (etodotneu -sardxa.rraqrlsodfi anssttduetu ut lle sauaSxog 'd3o1oqd sraqn] oql llueLudo;anep -rotu enssrlpuE lepne))pualreloq]le polelduro) lernou IIel lorluor urnl uI lEql soueSto salrollEq oql +opuoppoq lela]ueunqSop7 Aep]noqele sasol)oqnllp.rnau alelnSa.rleqt sJolJEI uolldlJf,suEJlepoJue seue8xoH I aql ]e arodornau oqf urJaposau aq] ur6urul.ro+ /{lanrsse-r6ord leruer) 'stcnpo.rdeueS-xoH 'or.rJr] ole salruros eues oq] ]V oqnl lernouoql 6ururol 'spue dq pareln8ar are eerqeuel;o sadeqseql uI seluero;JlPeqr qloq preMo]speerordpue luaurdole^ep lelo+1oslep t Z ]noqe]e ofuqLua aqi +ololua)oq] ul sul6aqanssr] ol paurlsap 'aldruexa rcJ,'k€-TZ ernSICaes)seueSxog dq Prllorluor lelnouor.lro)aq erll 3uo1esluauSas 1o suralled r.l.uopopa eqt1o6urp1o1 abelssrql1eelnrnou oqt palle)'o{rquua eJEsrxe JorJelsod-JolJeluE (suEunq 'satg u1 I oi ,,ponouJoJ,, or.llleanar uaoqseqauerquiaLU f rloruLup eq1'sburnnerp luereJtrPeql (s]Eruluereqlo duetu puE aseq]ul 'o{lqua ueunq aq} ut uot}elnrnoN8E-ZZlunglJ V '6t-ZZ ern8rgees) alodol

nou

JOUOISOd

e r o o o rn o u loueluv

.\

s{ep 97

-'Sf

arooornou leruerJ sAeP77

p l o Jl e r n a N anoor6;ernap sAep 97 plol lernaN u o r u u l el o e D p a1 n 3 alelo lernaN

si{ep 51 pua llef

puapeeH

'$t-ZZ ern8r4)uorlelnrneupaylerssacord e 'ru.rapotcarorratsod Jo srxe Ierluol eqr 3uo1eSurployslrels odrqua aql (os op o1 'uersds snolreu E e>letuot seq odrqtuo aql .,ltoN 'sllel rureposau leudqrursrrrr trul-asool eroru to sseruE Surqcr,upues'slaags leqaqrrdedla8rel ere urepopuo a.re-rq3r.rot r1rl puE pue Lurepolrerq1 'Jlgetrorap.{1rsea '>lleq ol luo.r; '1e1 ot peeq-sexe aaJql slr pup 'sradel ru.ra8 oarql serl odrqrua Ipturue or{l (uortelnrrse8 ;o pua aql ,,tg

luaudola^aolernaN {;reg ur uor}e)r}r)ods edl(1-;;e1 'slPrurueur suorl '\q9f.-72 -31nru xoH ar{l Jo luersruruer uorlErurojsuErl v 'sasseylaerql a,rn8r4aes)dola,rapsro,violJuer{l reqler sarr.eal 'saua8K4luapt uo3,to-1otoL,f IIp ur suortetnru qlr.e\srueld u1 to sessEIJaergr dq peporua srotrc1 uondrnsuel SCVW jo suorlrp paurquol aqr fq pellorluor ere sledaspue 'sledrer 'suarupls 'sleled Je.{\ol1 ;o su.re]led pue sadeqs er{I r

uorlEruJoJelrtuos uI Pe^loIuI sauaSeIJl Jo uorlElnSeJolnE a,rne8aupue pel Surdola.tapeqr luo{ sleu8rs934 dpears 'SIXEJouelsod uo spuadepuotsserdxaaua81o u.ratledsII{J -rorrelue aqr 3uo1e6aq se LIJnssauo8Jo uorsserdxalerrlcdc aql dq pellorluor sr uolleluroJ etltuos 'selerqalre,t u1 r 'GZ-ZZ aJnBrC aes)suor8a: rot.ratsodarolu uI dllerluanbasuoql pue Peeq eql JEeuts.rr;'selrtuos pallel turoPosalrlJo slrolg SurtBad -er uortEruroJeqr qrrrnsurSaguotlBluaurSasolprqaue^ I Jo 'saueBdtrrelod-tueuBes 'stxe ateln8a.rol aulqluor sutalord aln.r-rtedd1re1rut5 tsJnJfo ;orrelsod-rorJetueaql 3uo1euortrsod utBlrac P ut slurot suralord de8 ;o uorleulqtuol ladord eql eJaq.4a adurs qceE '$Z-ZZ ern8rg aes)suralordaueS-de8Jo suorl -eurqruoJ rr;nads dq punoq uagltr uorldtJfsueJl aIE IIJE leql sJaruEl{uadq pallorluor ore paddt4s-uanasE I{3ns saua8alnr-.rted uorsserdxa su.relledpadrrrs oql r 1o 1o 'sde,la.qredSuqeu8rslurlX pue Soqa8paH aqr to stuauoduor oPorue seua8 .{}rrelod -luaru8aslsrolfeJ uorldtrcsuerl epofue sauaSalnr-rred pue de8 aq1 '(1Z-ZZ ern8rg ees) saua8dlrrelod-lueur8asd1leu -lJ pue 'saue8alnr-.rteduaqr 'lsrr; pessardxeale saue8drg 'srxe JorJolsod-rot.ralue aqr 3uo1eslueru8asorur o.,fiquradg eqt apr^rpqnsor dllerluanbaslcB saua81o sesself,oerIJI I 'GZ-7Zpue €Z-ZZsern8rgees)odrqruaeqt raqloru eqr trg ardcoo ur serrlerurudse IeIlIuI eql ol esIJse^,rt8 's1eu8rs aqr ur dlleornaruudsBpaceld uralold PUEVNU e^rteruJoJur;o sed& olu soleJ8elurodrgua oql uI uoIlIS -od .relncrt.reddue ur IIOJP snI{J '3uru.ral]BdIerlue^-lesroP slorluor reqtoue fo,Lrqua ,{g eql 1o uolleaue8ro rorralsod -rorJolue slorluoJ suralord pue saua8;o rualsds ouo r 'suaSoqdroturo's.roleln8erpape.r8 sE uorlJun' uec sutalord peleJJasPue sJolf,e}uotldtrlsuB.rl '(yZ-ZZ qtoq 'unrtdru.{s e sr od.rgrued1.reeaqr esn€Jeg aas)arel.rnseql uo teeqs e uI sllar 0009 rnoqe qrr.vro,t;q -ruo d1.reo',o\1qdoso.tqu1 t esrJ se,rr8 uE ol luerudole.rap 'sluaru8asdpoq 'sleutue dueu ut tJesurpue eerqetJoi rno dq PelPlIPuI se uorlBtuJoJuJeuEdto eJnlEeIuoluuloJ € sI uollelueurSa5 I selerqel.lan pue spasul ul suollerJpnpue sauaql:uolleluauba5 fpog ]o lol]uo)

I N l l A d o ' 1 3 A 3 l0o

90]ot8

'1'll)

uv]n)310tAtHl

I

zz ulldvH)

986

urrapolcale|.uraprdf

' 1 0 0 2 ' ] l o M u a o q r sg p u e lLltilZZ ulg ptaq ) sPlol I t-r.!or+ paldepe(q)ued ]loMuaoq)sf 9 pue sPlof i{sapno: sqdetbot:ru 191-1.; etnbrl 1 1'1o 'ssard ' pa puz'luawdolatag proJXO p saldouu4 ' prldeppsourMerp LOjZ'P ]a lodloM I uJor+ (e)Uedl uriapoDaleuroprdoeqi ]o uolsuedxo aleltput sMollv (pat; turod lelalelotpauJ e O u r qu e r p e uol l 6 u t sp u e ( e n ; q s) l u r o da O u r q aqnl oM] utqltnns11et leralelosJop lerlaqlrdaotneu lernaN yo 6ur6penn esnet sluaute6uelleaj ( 1 ur o d ] u a u e lU o l ) tt u ' u o t l enl .ni o u 1 o l e p o L l -a6utq)a^tleladoo)aq] ul (q) uorlelnlnau l u r o d a E u r q Ierolelosroc

1o se6e1saules aq] ]e sofuqLrla))tq) ruoqssqdet6ol)tLU uojl)ola 6uruuet5 lt o^oqe sasn]pue ansstlleJnouaq] q]lM {lrnurluot slr sasolsrulepldeaql 'uorlelnrneu6uunC ur>1s ur lueLl6rdarll uJJo]1eq1say{toueleLl pue 'ua1s{ssnonJauleJaqdlod'sauoqlel)p} oql ]o lsouj 'sa^leA ]ieaq o] olnquluo) lllMsllo) asaql aqn] lplnauaql uot,r rienne lsaJ)le.rnou - Ier;aqlrde ele;6ru.rpue uotltsuel] ;eu.i{qtuaseLu ue o6iapun uotsn+Jtoaits aql leau (par)sllo) ullol lo uorlelndodV (panuopst salep_/oq) eql q)rqM uror+)ptoqlolou a)jtl-poroql uiroj o]

lsaJc

proqcoloN l u r o de b u r q uerpolA

uorlrsuer]lerlaqlrdo-leu^q)uasaLu e saobtepun uJroposoLu 6ur{llapunoq} }o }.rpd aqnl leJnau a q l o ) e u r o ] a s n i s p l o , l p l n a ua q ] { ; ; e n l u a n e urrapopuf pue 'ploJ o1surOeq(aleldlelnou)uunrptor.uud urroposol l lernauaqI sllol (ue])leuteprdapue (po.lpup onlq)lernauenritedsotdolur peprnrp ri;;erlrur urrapolce urapo]ra oql Moqs aq] uo s6urnner6 uol 1e; ;euraprd3 'I)rq) aql ur uotlelnlnoN 5€-zz lungll >

sodrqua 3ur11nsar e{J. &r-rrne VNUIuT IIp Suupururrladya,rrrra; -to snqr 'peletnru selrr'sy11g orf,nu sessef,ordreqr aurdzuaaql tacrq qcrq,u ur qsrlerqez qlr.4aserpnls tuory oruer uorlplnrneu ur svNurr.u to lueue^lo^ur eql roJ efuopr^g '(tz-g pue gz-g sorn8rgoas)uortelsuel Suurqrqurro uortrunJ VNUtu Burllorr -uoc dg uolsso.rdxaeua8 eruangur uer .(sy51 sygg tpr{t rroqs -U1ur)sVNg orJrru uo spuedaposle uorleruroJeqnt IErnaN. 'Oloq.r\E sEanssrleql bunuroJ or IIInJ -esn Olngrrtuot ot uorteuro;ur qSnoua e^eq ot sEr.lIIaJ qrEg 'sarntf,nrlsrruodrqua a3:e1;o saSueqr adeqspezrueSroaqt ol otnqrrtuof,uet sller lenpr^pur ur sa8ueqrmoq;o alduexa realc E sr srrlf 'Qet-ZZ ern8rg)plot or raaqsleurrepotf,oaqt Bursnec 'pedeqs-a8pe^.\ sllat eqr a>leru s11ac areld Ierneu etuos ur slueru 'sller Surtedlfrtred -EIIlorrIIu uolalo{sol Jo luoura8uerreau Jo -dc eqr ur sa8ueqcpazrueSrodg8rq uory sllnsar eteld IeJnau

(e)

oqtJo SuploC 'aQntpmau ar{tJoSuruurgeqeqr srsIqL'F6t-ZZ arn8r4) uodueJ poderls-n e a>letuol pre,&\ursploy (are1dp.rneu eqr) teeqs lEr.uropotroer{l Jo uortrod e 'aurds erp auroJog IILI\ teg.ry\Suolv 'srruraprdeurro; dllenrua^e IIr,r\ lerp sller Suqueg r{tr^\ Suole sllar Ieurrepopo Jo Feqs eql ul poletol aJEsllor IEr -nau o.nrDedso.rd aql 'sluela d.roleln8a.rJo sarJesIJEXaar{l JOAO -ter{lA.'slleJurelreJ ur uorsserdxey4yrqgSurrua,ra:dpue raq.rea Surlce fg setpt Iernau ernpur deu osle Suqeu8lslu,4N.tdwg Sursoddodq sarel IIar Ierneu oJnpur uer snroqro) pue ,ur33o51 'urproq3 .uorlrnpur sE qJns td6g 1o slsruo8elue speraq^. 'elrru pue suerqrqdue uor; Iernou slua^ord t.Itt[fl legt s1\oqs 'elep o1 aJueprlorsaqeqf'onssrl Ierneue^nf,odsordsEourlpru oqt Suop slyacateu8rsappoolsJepunllrood IIns orp rrqr qeu8r5

p r o ) leur dspue u r e J ga q l l o u o t l e u r o J s u r 6 a g u o l l e l n l n e N

had proper axis formation but failed to undergo neurulation. To demonstratethat miRNAs, and not someother moleculeaffected by Dicer, are responsiblefor the problem, scientistsattempted to rescuethe mutant fish embryos by injecting them with a prominent classof preformed miRNA duplexes.This succeeded to a remarkabledegree,restoringmuch of the defective neural development.k will be fascinating to learn in the future how miRNAs control neural tube morphogenesrs.

(a) Gradedinductionof differentcell types in the neuraltube b y S h h a n d B M Ps i g n a l s Dorsal Epidermis

Roof plate

Neural tube

Sensory n e ur o n s V1 neurons

S i g n a lG r a d i e n t sa n d T r a n s c r i p t i o F nactors SpecifyCellTypesin the NeuralTube and Somites

V2 neurons Somites

As we've seenalready,developmentalsignalscan act in relay fashion,a sort of bucket brigadein which eachcell receivesone signal and passeson another,or in graded fashion, in which differentconcentrationsof one signalinducedifferentcell fates (seeFigure22-13). Gradedsignals,or morphogens,are important in specifyingcell fatesin differentparts of rhe neural tube: motor neuronsin the ventral part, a variety of interneuronsin the lateral parts, and sensoryneuronsin the dorsal part. The differentcell typescan be distinguished,prior to morphological differentiation,by the proteinsthat they produce. Graded concentrationsof Sonic hedgehog(Shh),a vertebrate equivalent of Drosophila Hedgehog, determine the fatesof at leastfour cell types in the chick ventral neural tube. These cells are found at different positions along the dorsoventral axis in the following order from ventral to dorsal: floor-plate cells,motor neurons,V2 interneurons,and V1 interneurons.During development,Shh is initially produced at high levels in the notochord, which directly contacts the ventral-mostregion of the neural tube (Figure22-40a). The Shh from the notochord induces the most ventral neuraltube cells to form floor-plate cells, a type of non-neuronal glial cell (Chapter 23). Floor-platecells also produce Shh, forming a Shh-signalingcenter in the ventral-most region of the neural tube. Antibodies to Shh protein block the formation of the different ventral neural-tube cells in the chick, and thesecell types fail to form in mice homozygousfor mutations in the Sonic hedgehog(Shh) gene. To determine whether Shh-triggered induction of ventral neural-tubecellsis through a gradedor a relay mechanism,scientistsadded different concentrationsof Shh to chick neuraltube explants.In the absenceof Shh,no ventral cellsformed. In the presenceof very high concentrationsof Shh, floor-plate cellsformed; whereas,at a slightly lower concentrarion,motor 'When neuronsformed. the levelof Shh was decreasedanother twofold, only V2 neurons formed. And, finallg only V1 neurons developedwhen the Shh concentrationwas decreasedanother twofold. Thesedata stronglysuggestthat in the developing neural tube different cell types are formed in responseto a ventral -+ dorsal gradient of Shh, though exactly when in developmentthe signalhas its impact is unknown. The accumulating evidencefor gradientsdoesnot rule out additional relay signalsthat may yet be discovered. Cell fates in the dorsal region of the neural tube are determined by BMP proteins (e.g., BMP4 and BMPT), which belong to the TGFB family. Recall that TGFB-type signals

Motor neurons

F l o o rp l a t e

Notochord

( b ) R e s p o n s eos f n e u r a l - t u b cee l l st o g r a d e dS h h a n d B M P s i g n a l s a l o n gd o r s a l - v e n t r aalx i s PaxT

Dbxl

Dbx2

Nkx6.1 N kx6.1

lrx3

Pax6

Nkx2.2

FIGURE 22-40 Regulationof neural-cellfate in vertebrates. (Shh)secreted (a)Sonichedgehog induces by cellsin the notochord Shh, floor-plate Thefloorplate,in turn,produces development cell that induces additional whichformsa ventral-+ dorsalgradient (TGFPtype signals) secreted fatesIn the dorsalregion,BMPproteins fromroofplatecells. ectoderm cellsandsubsequently fromoverlying (b)Therelative by the of ShhandBMPareindicated concentrations by gradientsCellfatesin the neuraltubecanbe detected colored production factorsshown of allthetranscription the differential levels of Shhinduce between thegradientsHighto moderate of theNkx22 andNkx6/ genes(J) but blockproduction expression at thetop (T) Evenlow factorsindicated of thefivetranscription of Dbxl andPax7,bothgenes levelsof Shhcanblockexpression comingfromcellsin by BMPsignals ispromoted whoseexpression the dorsalneuraltube TheborderbetweenPax6andNkx22, and by mutually betweenDbx2andNkx61, isfurthersharpened the repressive interactions; eachproteinturnsoff the geneencoding cellfates of allthisisa setof different otherproteinTheoutcome (e g , motorneurons neurons) alongthe dorsal-ventral or sensory factors, a uniqueblendof transcription axisEachcelltypecontains of manyothergenesthat controlexpression whichpresumably T.M Jessell, 2000, properties on cellsthattype [See conferdistinctive Rev. Nature Genet'l:2Ol

CELL.TYPS E P E C I F I C A T I OI N E A R L YN E U R A LD E V E L O P M E N T

987

and their antagonists are critical in determining dorsal cell fatesin early frog embryos.A Drosophila TGFB signalcalled Dpp also functions in determiningdorsal cell fatesin early fly embryos.Indeed,TGFB signalingappearsto be an evolutionarily ancient regulator of dorsoventral patterning. In vertebrate embryos, BMP proteins secretedfrom ectoderm cells overlying the dorsal side of the neural tube promote the formation of dorsal cells such as sensory neurons (seeFigure 22-40a). Thus cells in the neural tube sensemultiple signals that originate at opposite positions on the dorsoventral axis. By integrating the signals from both origins, each cell embarks on a particular course of differentiation. The gradients of Shh and TGFB spreading out from the ventral and dorsal sides of the neural tube activate or repress the production of particular transcription factors in neural-tube cells at different positions along the dorsal-ventral axis (Figure 22-40b). When thesetranscription factors are first made, the boundariesbenveenthem are fuzzy,butsomeofthe boundaries sharpendue to mutually repressivecross-regulation. Similar mechanismsdetermine cell fates in the somites, which give rise to body wall muscle (myotome), the inner layer of skin called the dermis (dermatome), and the vertebrae (sclerotomel. These different cell fates are induced by signals from surrounding tissues.For instance, Shh coming from the notochord induces sclerorome.Thus the same signal. Shh. induces motor neurons in the neural tube and bone primordia in the somites. The receiving cells are preprogrammed to respond in distinct ways to the sameinducer dependingon their prior history. Somitesalso receivesignals from other directions, such as Wnt from the dorsal neural tube, that induce different subsetsof cells.

Most Neuronsin the BrainArise in the Innermost NeuralTubeand Migrate Outward The cortex of the brain is a thin sheet of cells organized into half a dozenlayers.This portion of our anatomy-required for the most-advancedthinking abilities-is the source of our greatest pride and feelings of superioriry for better or worse, over other living things. Development of the neural tube, which is initially a singlecell layer thick, into a brain requires both the generation of vast numbers of neq progenitor cells from stem cells and the organization of those new cells into layers. The new cellsform mostly in the subuentricularzone, the inner lining of the neural tube lying closestto the ventricles, which are the fluid-filled cavities inside the brain that arise from the interior of the neural tube. Cells take up their final positions in a simple inside-out order, forming the innermost Iayer first and then, progressively,the outer ones (seeFigure 21,-1,2).Thus neurons born late have to migrate past the older cells that have akeady taken up their stations.The migration of some cells involves inreractions with radial glia, support cells that elongate to span the entire distance from the subventricular zone to the outer layer of the cortex. In addition some cells undergo remarkable tangential migration at right anglesto the ventricle-to-surfaceplane. Scientistshave used timeJapse microscopy to observe the behaviors of migrating neurons. Their movemenr is not 988

C H A P T E R2 2

I

smooth or continuous. Instead, the cells move vigorously for a time, pauseand extend processesin what appearsto be a testing of the waters, and then resumemotion in either the original direction or another. In some casesneurons seemto move over each other; in other casesthey follow the processesof glia cells. Migrating cells are responding to a wide variety of guidance cues in the form of surface molecules and secretedsignals, while internally each cell undergoesdramatic shapechanges. Once neurons reach their final destinations,and often while they are in transit, they form long processesto communicate with other cells: the dendrites, which receive signals, and the axons, which transmit signals,sometimesover distancesgreater than a meter.Ve will discusshow the correct wiring pattern is built, to the extent that it is understood, in Chapter 23.

L a t e r a lI n h i b i t i o nM e d i a t e db y N o t c hS i g n a l i n g CausesEarlyNeuralCellsto BecomeDifferent The development of the nervous system provides examples of an important general mechanism for ensuring that all necessary structuresare built, but not in excessivenumbers. This mechanismcalled lateral inhibition, consistsin essenceof a cell communicating to surrounding cells "I am doing this, so you shouldmake somethingelse."Adjacentcellswith equivalentor near-equivalentpotential are in this way directed toward distinct fates. Genetic analysesof Drosophila neural development first revealed the role of the highly conservedNotch/Delta pathway in lateral inhibition. The Drosophila proteins Notch and Delta are the prototype receptor and ligand, respectively, in this signaling pathway. Both proteins are large transmembrane proteins whose extracellular domains contain multiple EGFlike repeats and binding sites for the other protein. Although Delta is cleavedto make an apparentlysolubleversion of its extracellular domain, findings from studies with genetically mosaic Drosophila have shown that the Delta signal reachesonly adjacentcells. Interaction between Delta and Notch triggers the proteolytic cleavage of Notch, releasing its cytosolic segment, which translocatesto the nucleus and regulates the transcription of specific target genes (seeFigure 16-36). ln particular, Notch signaling activates the transcription of Notch itself and repressesthe transcription of Deha, thereby intensifying the difference betweenthe interacting cells (Figure 22-41a). Notch-mediated signaling can give rise to a sharp boundary between two cell populations or can single out one cell from a cluster of cells (Figure 22-41b1. Notch signaling controls cell fates in most tissuesand has consequencesfor differentiation, proliferation, the creation of cell asymmetry, and apoptosis. Here we describetwo examples of Notch signaling in cell-fate determination. Loss-of-function mutations in the Notch or Deba genes produce a wide spectrum of phenorypes in Drosophila. One consequenceof such mutations in either gene is an increasein the number of neuroblasts in the central nervous system. In Drosophila embryogenesis,a sheet of ectoderm cells becomes divided into two populations of cells:those that move insidethe embryo develop into neuroblasts,which give rise to neurons; those that remain external form the epidermis and cuticle (see

T H E M O L E C U L A RC E L LB I O L O G YO F D E V E L O P M E N T

(a)

Intrinsically biased

Equivalent

< FfGURE 22-41 Amplilication of an initial bias to create lateralinhibition.(a)A differentcelltypesby Notch-mediated equivalent cellsmayariserandomly between two initially difference (/eft)Alternatively, bias(center) interacting cellsmayhavean intrinsic that have received different cells bias(right)Forinstance, or an extrinsic willbe intrinsically biased; those proteins celldivision in an asymmetric (orange) willbe extrinsically biased signals different that havereceived R e g a r d l eosfsh o wt h e s m a liln i t i abl i a sa r i s e sN, o t c hb e c o m e s promoting itsownexpression and predominant in oneof thetwo cells, production of itsligandDeltain thatcell.Intheothercell, repressing of the production Theoutcomeisreinforcement of Deltapredominates (b)Notch-mediated inhibition maycreate lateral smaliinitialdifference. fieldof cells, suchasalongtheedgeof in an initial a sharpboundary a centralcellfroma wing,or distinguish Drosophila the developing precursor establishment asin neural cluster of cells, surrounding

Extrinsically biased

I (b)

et al , 1999, Science234:710] [Adaptedfrom S Artavanis-Tsakonas

Field

Cluster

Ftgwe 21-29). As some of the cells enlargeand then loosen from the ectodermalsheetto becomeneuroblasts,they signalto surrounding cells to prevent their neighbors from becoming neuroblasts-a caseof lateralinhibition. Notch,/Deltasignaling is usedfor this inhibition; in embryoslacking the Notch receptor or its ligand,all the ectodermprecursorcellsbecomeneural. The role of Notch signalingin specifyingneural cell fates has beenstudiedextensivelyin the developingDrosophila peripheral nervoussystem.In flies, various sensoryorgans arise from proneural cell clusters,which produce bHLH transcription factors, such as Achaete and Scute,that promote neural cell fates.In normal development,one cell within a proneural cluster is somehow anointed to becomea sensoryorgctnpre-

cursor (SOP).In the other cellsof a cluster,Notch signaling leadsto the repressionof proneural genes,and so the neural fate is inhibited; thesenonselectedcellsgive rise to epidermis (Figure 22-42). Temperature-sensitivemutations that cause functional loss of either Notch or Delta lead to the development of additional SOPs from a proneural cluster. In contrast, in developingflies that produce a constitutively active form of Notch (i.e., active in the absenceof a ligand), all the cells in a proneural cluster developinto epidermal cells. To assessthe role of the Notch pathway during primary neurogenesisin vertebrates,scientistsinjected mRNA encoding different forms of Notch and Delta tnto Xenopus embryos. Injection of mRNA encoding the constitutively active cytosolic segment of Notch inhibited the formation of neurons. In contrast, injection of mRNA encoding an altered form of Delta that prevents Notch activation led to (b)

(a)

I n d u c t i o no f proneuralcluster

Determination

Differentiation

T] :11

Lower level of Emc

SOP

I

I

Cell fate: SOP

A FIGURE 22-42 Roleof Notch-mediatedlateral inhibition in (SOPs) formationof sensoryorganprecursors in Drosophila. (a)Extracellular signaling molecules andtranscription factors, encoded genes, pattern byearly-patterning controlthe precise spatiotemporal of proneural bHLHproteins suchasAchaete andScute(yellow)Most proteinthat cellswithinthefieldexpress Emc(orange), a related antagonizes Achaete andScute, A smallgroupof cells,a proneural produce proneural cluster, bHLHproteinsTheregionof a proneural fromwhrchan SOPwillformexpresses cluster lowerlevels of Emc, givingthesecellsa biastowardSOPformationInteractions among thesecells,mediated by Notchsignaling, leadsto accumulation of Notch-regulated proteins E(spl) repressor in neighboring cells(blue), restricting SOPformation to a singlecell(green)(b)Initially, achaete (ac)andotherproneural genesaretranscribed in allthecellswithina

andother proneural cluster, asareNotchandDeltaAchaete proneural promoteexpression of Delta.Whenonecell bHLHproteins (/eft),its moreAchaete slightly to produce at randombegins in Notchsignaling to stronger production leading of Deltaincreases, cells,the Notch cells(right)ln the receiving allitsneighboring Su(H), factordesignated pathway activates a transcription signaling genes. F(sp/) expression of E(spl) expression whichin turnstimulates (left)cell,allowinga neural,i,e.SOBfate stayslow in the high-Delta of proteins transcription repress specifically In the rightcell,the E(spl) in Achaete proneural genes resulting decrease The ac andother the initialrandom in Delta,thusamplifying leadsto a decrease of theseinteractions difference amongthe cellsAsa consequence asa SOP;allthe isselected cluster andothers,onecellof a proneural cells intoepidermal anddevelop otherslosetheirneuralpotential

I NNE A R L YN E U R A LD E V E L O P M E N T C E L L - T Y PSEP E C I F I C A T I O

989

the formation of too many neurons. These findings indicate that in vertebrates,as in Drosophila, lateral inhibition mediated by Notch signaling controls neural precursor cell fates. Similarly in the nematode Caenorhabditis elegans, Notch signaling is used during vulva development to form distinct adjacentcell types by lateral inhibition. The Notch signalingpathway is usedduring the formation of many organs and tissues,but not always for lateral inhibition.

11 days

Cell-TypeSpecificationin Early Neural Development r The vertebrate nervous system developsfrom ectoderm cells, which are initially arranged in a sheetformed during gastrulation.Folding of this ectodermalsheet (the neural plate) first forms the neural tube in the processof neurulation (seeFigures 22-38 and 22-39).

13 days

r The notochord, a rod of mesodermlying underneath(ventral to) the neural tube, is a sourceof signalingproteins that induce cell fates in surrounding tissues.For example, Sonic hedgehog(Shh) from the notochord influencesthe fates of nearby cellsin the neural tube, somites,and endoderm.

1 4d a y s

r Shh is a morphogen that directs certain cell fates at high dosesand other cell fates at lower doses.It tends to promote ventral fates, like floorplate. Its influence is tempered by other signals such as BMP, a TGFB-type signal coming from overlying ectoderm that promotes dorsal neural fates (seeFigure 22-40). r The cellsof the neuraltube, which is initially one cell thick, proliferateand form neural precursorcellsthat migrate radially outward to form the layersof the brain and spinalcord. r During normal Drosophild neurogenesis, only some ectodermalcells form neurons;others form other ectodermderivedcellslike skin. The balancebetweencell types is regulated by lateral inhibition involving Notch signaling.In this process,cells that have taken on a neural fate rnstruct surrounding cellsnot to do so (seeFigures22-41 and 22-42).

Growthand Patterningof Limbs Vertebratelimb developmentprovides a beautiful example of how the actions of individual cellscombine to creareDarrern. Vertebratelimbs grow from small "buds" composedof an inner mass of mesodermcells surrounded by a iheath of ectoderm (Figure 22-43). Hindlimbs and forelimbs are obviously related, as are left and right limbs. If the limbs were broken down into their constituent moleculesor cells, the composition of all four would be nearly identical,yer their shapesdiffer in ways that are absolutely crucial to successfullife. Pattern formation-that is, organizing those moleculesand cells into a coherentwhole-is a central goal of studying the molecular cell biology of limb development.Where would birds be with four legs instead of a pair each of legs and wings? Birds have in fact beena prime experimentalanimal for studies on limb development,sincechick embryos can be readily accessedin the egg during the period of limb formatron. 990

CHAPTER 22

I

15 days

^ ,,o;; Limbdevetopmentin the mouse.Limbbuds, whichconsist of an innermass of mesoderm cells andouterlayerof ectoderm, format specific locations on theembryo s flank Outgrowth andpatterning alongthethreeaxesleads to formation of theallthe limbstructures L Wolpert etal, 2001,Principles of Development, 2nd [From ed, Oxford University Press, Figure 10-5l In testing various theories of limb development in the chick, researchershave subjectedembryosto transplantation, injectedthem with signalingproteinsor signal-producingcells, and introduced retrovirusesthat direct geneexpressionin abnormal sites.The continued growth of the animal in the egg then is observed to seethe effect of any particular treatment. Becauseit is relativelyeasyto reduceor eliminatea gene'sfunction in mice, geneticexperimentshave beenusefulin studying limb developmentin theseanimals.The resultsfrom the two experimentalsystemsare largelyin agreement.

H o x G e n e sD e t e r m i n et h e R i g h t P l a c e sf o r L i m b s to Grow The first event in vertebratelimb developmentis determination of where, along the head-tailaxis, limb growth will begin. 'We are not millipedes;decisionsmust be made.In both insects

THEMOLECULAC R E L LB I O L O G YO F D E V E L O P M E N T

( a ) E f f e c to f r e d u c e do r n o F G F 1 0

22-44lhe effectof alteringFGF10 < EXPERf MENTALFIGURE functionon limb developmentin the mouse.(a)Geneknockout partof the fgf10 gene,wereconstructed by mice,lackinga f unctional a few days Thesephotographs of mouseembryos recombination for the fgf10 gene(+/-) are beforebirlhshowthat miceheterozygous impaired limb mice(-/-) haveseverely buthomozygous fairlynormal, mice(+/+) These with that in wild-type compared development growthfactor10 genetic of f ibroblast dataprovethe importance (b)Totest (FGF10), protein, for limbdevelopment signal a secreted limbdevelopment, experiments iscapable of triggering whetherFGF10 embryos areadvantageous weredoneThese withchickembryos operation to growaftera surgical theembryo cancontinue because protein implanted in theflankof weresurgically in FGF10 Beads soaked priorto limbdevelopment, in a region where a mid-stage chickembryo 0, butnotcontrol substances, developFGF'I no limbwouldnormally of a fifthlimb Theyoungchick theformation wasableto induce leg embryo,hasan extra(ectopic) shownhere,froma FGF1O-treated (a)fromMinetal, 1998, isshownIPart Onlyhalfof thechicksskeleton

(b) Effectof ectopicFGF10

Genes& Devel 12:3156 Part(b)from M J Cohn et al , 1995, CeilAO:739l

mesodermand initiates outgrowth of a limb from specificregions of the embryo's flank. Mouse mutants lacking FGF10 develop without limbs (Figure 22-44a). Conversely,implantation of a bead soakedin FGF10 at sitesin the flank of a chick embryo where a limb doesnot normally form causesan extra limb to grow (Figure 22-44b). These results demonstrate the remarkable inductive capabilitiesof FGF. Wnt signaling also plays a role in the initial outgrowth of limb buds.

Normal

and vertebrates,the Hox genescontrol where limbs are made. The mesodermthat givesrise to limb buds, called intermedidte mesoderm,is located adjacent to the somite mesoderm; mesoderm farther from somites is called laterdl plate mesoderm. Expressionof Hox genesin the intermediatemesoderm influencesthe position of limb-bud formation by controlling expressionof genes(e.g., Tbx and Pitxl) that encode other transcription factors in the lateral plate mesoderm.The Tbx and Pitxl proteins,in turn, control production of secretedsignals that are required for limb development. Among these signals are fibroblast growth factor 10 (FGF10), which is secretedfrom cells in the lateral plate

L i m b D e v e l o p m e nD t e p e n d so n I n t e g r a t i o n o f M u l t i p l e E x t r a c e l l u l aSr i g n a lG r a d i e n t s The three axes of a limb bud and developedlimb-anteriorposterior (thumb to little finger), dorsal-ventral(back of hand versuspalm), and proximal-distal (shoulder to fingers)-are shown in Figure 22-45. Early limb developmentis marked by fcrrmationof two important signalingcenters:the apical ectodermal ridge (AER), a region of surfaceectodermat the distal tip of the emerginglimb bud and the zone of polarizing actiutty (ZPA) in mesodermat the posterior end of the bud.

Anterior Mesoderm sells A E Rc e l l s Proximal€

+

Anterior

Distal

Posterior

Posterior Proximal

Limbbud A FfGURE22-45 fhe axes of the limb bud and the hand. A l i m b b u d ( l e f t )a n d f u l l y d e v e l o p e dl i m b ,a h a n d i n t h i s e x a m p l e ( r i g h t ) ,h a st h r e ea x e s :a n t e r i o r - p o s t e r i(ot h r u m bt o l i t t l ef i n g e r ) , p r o x i m a l - d i s t (asl h o u l d etro f i n g e r s )a, n d d o r s a l - v e n t r (abl a c ko f

Distal

h a n dv e r s u sp a l m ) I n t h e d e v e l o p i n lgi m bb u d ,t h e a p i c ael c t o d e r m a l r i d g e( A E R i)s a s o u r c eo f f i b r o b l a sgt r o w t hf a c t o r( F G Fs) i g n a l sa, n d g c t i v i t y( Z P A i)s a s o u r c eo f S o n i ch e d g e h o g t h e z o n eo f p o l a r i z i n a

(shh) GF L I M B S G R O W T HA N D P A T T E R N I NO

991

(b)

(a)

(c)

Fgfs induced by FGF10 _--> FGF10-----+

..+ rroilTerailon

, matnratneo b v, l -adsut pa.raSSrrl sr (17:aldeq3) srsordodedlyentua,tg'sleu8rs seuo8te8rel esoLll.{\or{pue lorluoJ daqt saua8qrlq^\ Jo uoq -sanbaql s8eqpnq qurl aqr;o suorSartrurlsrp ur srotf,€Juorl Surlsrxa-ardJo erntxnu E ol posodxeere sllof,,r,au aqt tlsno -ro8r.nSurzrrorSsr qurq eqr reqr eturt erues arJrtV .tr SururJar -drrcsuBu pxoH Jo uoncnpord eq1-'$V-ZZ ern8r4 aas)s11ac eql uortdulsuert sassarder8ur1eu rorralsod ur aue8 UC 'o -8ts,{.lS 'saua?pxog pue qqS eqt sassa.rdar €IIC peller rore uorldr.rlsuert E 'pnq rolrelue arp uI 'uo peurnl saua8qroq sdaal reqr png rorrolsod aqr ur dool 4ceqpeol a.trtrsode 3ur -urro; 'reqto qcea erep8a.r saue8pxoH eql pue qqs eqjslue^erelel er{rPue u.reuedeql lno 8urq3no.rslue,tedlree aqt rltr^\ (lorluotrleuorldrrf,suertpue SurTEuSrs;o spunor poleader SursnpaureDeaq ol suaas sqrur1or1tur ureDed leur; aq1 'squrl Jo sedeqsalqrcnporderItg8lq (sarcedse urqlr.r,r) pue elrsrnbxo aLIt otur petelsuerl sr suralled lerleds asrcard

' I L6LtZElluauJdop^a1'S0OZ 1 u_ror1 lp 1are6e5;711 pa]oepe sqoe]9 )qow uewnH'€o0z'lp a)ual v I lauaD szLlizl lo aa516ururalledqLUrl antilalopul ]lnsaruel qqs ,Louorssardxa lerleds s e b u e q r] e q ] r a ) u e q u op n q - q u l l lL / L /aSq ] o 1 e 6 e u e p f l l u e t e d d y ++ooq plnoqsaue6eq] aroqMpnq qultllouolue oql +o seoleur qqs ssardxao] puno] oloM a)tLu]uelnu.laq] 'eoptslql q]lM lueueat6e u1 pnq qur!l0r.ltul qrls 1o uorssetdxa 6url1ot1uo> ra)uequaue sutelUo) uorberq>1qslt e)u pue s]lnsalosaql l aq] leqt pe1sa66ns VNC asnoLuuoaMlaq'olol orl] se ol pololal {1ue1rurs 1o uorbatpalruurl orourre, e sMoqsqdet6 LrLolloq go sdorpIirrelrurs eq1 {1;etrleu.retp e r u e n b a sa q ] ' u o r O esr t q ]a p t s l n O ( s M o l e p o i ) s u o t l e l n u al s n o u l p u e ( s M o r e ) ) e l q ) s u o r l e l n u ul e u l n qe q l 6 u r u l e l u o tu o r 6 e tq 1 - ; =

' Vr N Cu p r . u npqu e e q 1u r q 6 r qe l r n bs r{ 1 r r e l r ua. rqr}s} e q }s a } e ) r p u eq16utpold'qdet6do1 uaoMloq o6elua)rad osnour firluapraruanbes 'a)ru oprloal)nu mor+ aql qsl,a)u pue'sueurnq vNc +oseruenbes '{1{pep{1od to; elqrsuodsar eq1pareduorslsrluar)s })e} ur are aql ]r ]no pugol @o1) eua6lrqu7 eq]1o uor6er srq]ursuorlelnLx pe]e)olsruor6ar (qq5)6oqa6paq srql telou.tord uorlulq)[€ e urLl]rM )/uosoql uror](qn) aseqeoauI ]noqepalero;uoroele ul suollelnul o^^lpue asnoLu a^eqol puno]aJoM]lalop reIulrse qllMsurerls relnrtyede q}tMsorlrue} (slrbrp erya){p{pep{;od io Lrro}po}uoqur re)uequa qqs oqr [r1uap; o] Pesnela,l,\ ueunq rno] ]o sroqurot^ uolle^rosuo)a)uanbes[.reuo|1n;one Pue (s]l6lp elya) {;{pep -f;od esnerleql suollelnl Li-zz lungll lvlNlt ,iluldxl v

oq

0001

006

008

002

00t

00t

002

00t

9v"ot

%001 ( c ) %09

%9L

uPurnq sA osno!\l

%001 ( q )

.2 sreoaseqeot+l

(P)

rNlNdollAtc lo

's^,{e,ry\ -aceds ur rer{lo pue turpuedap-aun qJEOJo uorssardxa oql eruenllur pue dlsnoauellnurs >lro,la. lpr{t surolsdsSurleu8rs eary1Jo aruan]Jurarlt ropun uorlEurrurelepaleJJlol se^lo^ur pnq qrrJl eql urqlr.r,rSurureDed'Gv-zz ernSrgaas)8uril; urrep -osarue re^o qleeqs up qlr.4 spnq se uels squr-I r IsruroPolla '>1ueps.o,{rquo eqr suor8ar rrynads uo.r; qt.r.or8 pnq ernpur terlt slpu ;o -8rs uorsserdxaot Surpeal'saue8reqto pue saue8xog ,{q 1o pallortuor dllerlrur sr srxe .,(poqror.ralsod-rouatueaqt 3uo1e suonef,ollq8rr aqr le luaudola,rJp qurll Jo uonenrur ar{I r squrl lo 6ugurap,e6pue qynorg

'sreqto selpnlseprn8or ue8ro ro enssrl ed& auo Io Jo to luaru -dole,reporp tnoge uorteruroJurasn ot stsrtuerJs.{\olle pue eyqecqddedlpeorq ere.rardeqrsrqt ur peqrrrsapruaudolarap rruod.rgrua;o saldnur.rdaqr lng 'eunlo^ sn{t ur srrdot esaql 'sloJluoJ IIe ssnlsrptouuef a1X\ lpluauuoJrlua pue orreua81o eruonlJuregt repun 11e'sassecord3ur3e pue sassaoordalrlera -ua8ararB eJOr1I'urerq eql err^\ ot peau e sr ereql 'azrsdpoq pue ue8.roJo loluoJ er{t pue 'y1eq.raqrooql ruo{ ruoreurp sn to 1€r{ so1eurteqt uals,{s lorluor uortpururretop-xes eqr sr e.req1',(8o1orqIIOJrelnrelour Suueurcse;to tol E ' ' ' srer{lo pue rn8 's8unl 'speuo8 'dauprl 'ra,rr1'arn1elncse,{'seda 'apsnru 'auoq 'ur1s :sonssrlpue sue8roJaqlo dueruSururro; 'euop etrnb lou sr odrqruo oqt rnfl Jo ralteu eql sr orarll

Do]ot8 l'll) uv]n)l'loW lHl-

|

zz ulldvH)

v66

flrlrlre ouoS-xoHto llnsar er{t sE 'setEJqalJa^puE slrosur qlog uI 'sleodar lueurSss -dpoq aql Jo r{lpa uo sernteeJolrlJurlsrp to uorlpJeuoC r '(seterqaua^t)erurl ur Jetlrp rpqt uorssardxa auaS;o solf,drro (sroasur)ecedsur polnqrrlsrp srolf,EJ uorldrnsuerl suorlpurquoJ lueJaJJIpdq uaurtrpstuaur to -8osdpoq Surleadarolur odrqrue dlrea eqr uorsr^rq r Io 'eturteJllE roJ poolq drund pr leqr ue8ro orrlaururdsergSrr-r;a1aql 'ueoq ar{l Jo uorleuroC r 'srxe lq8r.r-qolE pue 'srxe lorretsod -rorretue uE (srxe IErlua^-lPsropE Jo luoruqsrlqErsgI 'sra^,(ey luoragrp ur slloJ uaal\le9 suortJpJeturolrDnpur Jo Jaq -urnu a8rel e uorteJeua8aql pue 'rurepolce pue sruJoposou Jo 'urrapopua aqt .sllef,uals sradelaerql olur uorlelnJlse8pue to or{tIo uonerurotsupr1 r a8e,reelcSur.rnp(ato8(z) 33e pazqrr.rey 'seuosotuoJqJpol\u -ap dlleurared pue dleurereur dg uorssardxeauaS;o sural -red rua.ra;;rpqtr,r\ euoueS ploldlp e orotsor ot uorsnJrrer{l pue sataureS;osaddrruoraJtrpolrnb o,tlt to uorlJnrlsuo3 r :sod.rqrueleturue ;o srsaue8eqt ur slue^e da1 durru pesseulr.ry\ aleq e^\ 'suonJas Surpeoerd oqt uI

p o o g o s ' J e Jo s

s u r a l o r€dt p x o H - L p x o H a q l l p x o qs a p n l ) xqa) r q M' u o r l d u r s u e r l ' aue6-xog ,Loa^eMlaleloql slolluo)U)D oq] pue tpxotlsopnl)ul q)rqM'polelpurse uorssardxe eua6-xo1-1 eq] 1osurelledler]rur s l o r l u oU ) f l l a L l ]' 1 u e u - r d o l eqnuarpl 6 u u n 6 ( 9 1 1 3u) o r O al o r rluo) q u r l { l r e ea q i p u e( U f u o r 6 0lro l } u o )l p r o u aabq 1: s r e : u e q u a D ) onnl{q pel1ot1uo) aq o} pa^arlaq srpnqqultlaq}ursaua6xoHaq}+o uorldr.nsue4 uorlezrlrua+ re4esleprruofuqLue o] ralarS 0 ; 'S 6l '61 'spnqqull {;ree ur uorle;nberqq5 puexoH gt-ZZ lUnglJ V

[ 0[9t:toEa]ua/ls' 'y667'sdueqrsac paldppvl sle6te] slr olent])e o1 uorldutsuetl I uror+ se(€IpxoH €19 ++osurngurelordqqS ]losllauebqq5oq]sellenn ' -t aq1 6 qq5 pellorluo) a) urelotd Iq ale 1eq1sauab1abtel tpxoH sesse;der e 'urelotdgl19luesetdat sa;tto leql rol)e+uorldr.nsuerl a n l q1 q b rol q I l l a Ms ep a s s e l d xeet es e u e bx o Hl e q l o1 n q' s e u e 6 pxolq[1uosnnoqs urerOerp aq1 qqs 1ouorldutsuet] o]elnult]s

'setet IIeJ loluof, o1 dlpaleadarposn ere slool agr pue 'a8re1ool tou sr solnf,olorutuetrodur dlleruaudolarrop'o rr>llooreql'(8CC{ pue 7CCC)tr urelur€tuosle pue (O1SCS) qv'tor8lno pnq-qull otErlrur sleu8rs 4gg {11eug puy '3ur -urelted tr8rp yorluor uaql pue ruroJ auru IIr^\ sqrrlrl areq,ry\ -.ralapsaua8xog dq pepoluo srolf,eJuorldr.rcsuerl',(1re1rur5

'tr.lJls/is snoAJJu Ierluef, aqf dn e>letuIIL{\ lPqt suorneu aqt to uortEreue8lerl -IuI PUE39nl ]etnau aqt etEerJol rurePolle aql 10 3urp10g I

'sexp aerql 3uo1etuarudole^eprreql solrseql Jo uorlloles r Pue Pelelol aq IIIr!\ squll eraq,4a

roFalsod

r Outgrowth of the limb bud along the proximal-distal axis is driven by a FGF signal that emergesfrom the apical ectodermal ridge (AER) at the outermost part of the bud. Patterning of the bud along the anterior-posterioraxis depends upon Sonic hedgehog(Shh) signal produced in the zone of polarizing activity (ZPA) in the posterior bud (seeFigure 22-46). A lil/nt signal directs cells to form ventral cell types. 'Sfnt r Information from FGR Shh, and signals is integrated by cells,so that eachassumesits proper fate and role in the developing limb. r Shh regulatesexpressionof Hox genesin complex overlapping patterns during limb-bud development (seeFigure 22-48). The Hox genes are important for regulating detailed pattern in the limb, for example, of the different digits and the bone, muscle,and nerve they contain.

You have undoubtedly noticed that the sameclassesof signals and transcription factors are used again and again during development of a polarized embryo. There are three reasonsfor this. First, much to the amazementand relief of biologists,the number of different classesof signals and of transcription factors is not too large, perhaps 20 different types of signals for example. For each type of signal, the standard signaltransductionpathway is quite well known (Chapter 15). Variations on the standard are interesting, but it is often fairly safe to predict what will happen when a particular signal (e.g., BMP) reachesa cell. and how to detect and measurethe cell responseto the signal. Second,the protein classesand gene systems involved in development are, to a high degree, conservedin most animals.This conservationhas createda common "molecular genetic" languageamong biologistsworking on the development of many organisms, allowing them to share in discoveryand understanding.Third, the patterns of evolution of development are becoming clear. Understanding how developmentis controlled allows us to understandmuch better how distinct animal forms can arise from mutations that changethe actionsof developmentalregulators.What use is all this work, you might ask. Two answersseemobvious: understanding our origins and using our knowledge of animal developmentto prevent and treat human diseases. Darwin understood the importance of embryology to his theory and wrote extensively on the subject. But the genetic and molecular cell biology mechanismsunderlying evolution were completely unknown in his time. Now we have rich detail about how genescontrol animal form, and a flood of new information is pouring in. New fossils are found regularly of intermediate ancestral forms that link present-day animals. For example, fossils that appear to representcommon ancestersto whales and hippos have been found, as well as the creature Tiktaalik that looks like a fish with scales,but has four legs. Such ancient creaturesprovide clues about how changesin developmental processes-pattern forming processes-underlie evolution. As the networks of interactions that control development of eachparticular tissueare traced,we will be able to

understand how alterations of some components can change animal form and function. Among all the changesthat led to animal diversity-that made bats able to navigate by sonar and moths able to "sniff" single molecules and cheetahsthat can run at 60 mph-were changesthat made us what we are. Genetic damage to any of the developmental regulators are likely to lead to birth defects,cancer,degenerativedisease, and altered resistanceto infection. Indeed,all of theseconditions have been observed, and their relationship to faulty development established.Thus developmentalbiology is a rich sourceof new information about the causesof human disease. Since many proteins work in pathways, linking one developmental regulator to a diseasehas often led to identification of additional human genestied to the same disease.Apart from finding human diseasegenes,there is the very real prospect of applying our knowledge of development to spur tissue regeneration and promote faster, better healing. Regeneration of blood cells from bone marrow transplants that contain hematopoieticstem cellsis now a well-establishedprocedure. There is every likelihood that a flood of new therapies will emerge as manipulation of signals and other developmental proteins becomesmore precise and sophisticated. The ability to transfer knowledge from a wide variety of animal embryos to human biology, a triumph of evolutionary theory and practice, is a foundation for the next stagesof medical advances.

KeyTerms acrosomal reaction 957 apical ectodermalridge

lateral inhibition 988 maternal mRNAs 971

(AER)ee1 950 blastocyst

morphogen 964 morphogenesis969

cleavage950

neurulation 985

cortical reaction9ST

notochord 987 organogenesis951 pair-rule genes976

dosagecompensation system958 epithelial-mesenchymal transitions 950 floral organ-identity genes983 gametes950 gap genes972 gastrulation 951 germ layers 951 genomic imprinting 958 homeosis979 Hox genes978 induction 951

pattern formation 951 segment-polaritygenes 977 sensoryorgan precursor

(soP) e8e

somites977 Spemannorganizer965 syncytium970 transcriptioncascade 974 zoneof polarizingactivity (zPA) 991,

Reviewthe Concepts t. In differentiated cells, only selected genes are tranmost scribed.Yet, except for lymphocytes and erythrocytes' '$7hat evisomatic cells contain the same nuclear genome. denceshows this is true? R E V I E WT H E C O N C E P T S

995

2. Compare and contrast the action of morphogens involved in dorsal-ventral specificationin Xenopus laeuis and Dro soph ila melanogaster. 3. Using in situ hybridization with a probe specific for dorsal mRNA, where in the syncytial Drosophila embryo would you expect dorsal to be expressed?Using immunohistochemistry with an antibody specific for Dorsal protein, where would you expect to detect Dorsal protein? 4. A microarray analysisof wild-type fly embryos and dorsal mutant embryoswould be expectedto yield information on all genesregulated by Dorsal protein. \Why?Other than new genes regulatedby Dorsal, one would expectto seechangesrn regulation of previouslyidentifiedgenes.Expressionof which genes would be increasedor decreasedin dorsal mutants? 5. Deleting the 3' UTR of the bicoid genewould yield what phenotype in a mutant fly? \fhy? 6. How can the group of five gap genesspecify more than five types of cells in Drosophila embryos? 7. At which stage in embryological development do somitesform? $fhat factors affect their development? 8. Sfhat is homeosis?Give an example of a floral homeotic mutation and describethe phenotype of the mutant and the normal function of the wild-type geneproduct. 9. Hox-gene expressioncan be maintained through many cell generations. What molecular mechanismsare involved in this process?

TE Promoter

E1

3'UTR E2

E3

E4

a. Northern blot analysis using a probe complementary to oskar mRNA or to rp49 mRNA was performed on mRNA isolated from egg chambers,which contain nurse cells and the developing oocyte, in wild-type and mutant flies. The results are shown in part (a) of the figure below. Micrographs of two egg chambersthat were subjectedto in situ hybridization with a probe directedagainstos&armRNA are shown in part (b). The egg chamber on the left is from a wild-type female;the egg chamber on the right is from an osAX female. Hybridization appearsas white staining in the micrographs.

^e)

rY \a

s

10. Describe an experiment with Xenopus embryos that demonstratesthe role of the Notch pathway in regulating the formation of neurons in vertebrates. 11. What is the evidencethat a gradient of Sonic hedgehog leads to developmentof different cell types within the chick neural tube? 12. Vhat evidence implicates fibroblast growrh factor 10 (FGF10)in limb development? 13. Synpolydactylyis an inherited human abnormality characterizedby duplicated fingers and toes. !(/hat type of muta'Sfhat tion could cause this abnormality? stage of development would be affected?

Wild type

"S* s.c

s

N^

o-

s"s

,c )\T o-

osk X mutant

Analyze the Data ln Drosophila. maternally produced mRNAs and proteins that determine the body axes are transported from nurse cells into the developingoocyte. During early oogenesis,one of thesematernal mRNAs, oskar,is dispersedthroughout the oocyte and is not translated. By mid-oogenesis,osAar mRNA is transported to the posterior pole of the oocyte where it is translated;the Oskar protein then initiates formation of most abdominal segments and the germJine cells.This developmentalprocessis defective in oskar nonsense (osANS) mutants. To further understand the role of oskar mRNA and irs protein in Drosopbila development,a new mutant (oskX) was generated (seeA. Jennyet a1.,2006,Deuelopment133:2827-2833).In this fly mutant, the oskar genecontains a transposableelement(TE) in its first exon (E1), as diagrammedhere.For simpliciry introns are representedas thin black lines separatingthe exons: 996

CHAPTER 22

I

\What do these data suggestabout the osftX mutation relative to the osftNS mutation? What is the purpose of probing for rp49 mRNA? b. Further study revealedthat femaleshemizygous (only one allele presentin the animal) for osANSlay eggsthat, when fertilized, develop into embryos lacking posterior structures.In contrast, females hemizygous for osAX produce ooyctes that begin to form but then degenerate.S7hatinformation do these observationsprovide about the function of Oskar protein? The following rescue experiments were undertaken. c. Severaltransgenescarrying different domains ofthe oskar gene were constructedand introduced individually into femaleshemizygous for the osAX mutation. The ability of femalesexpressing each transgeneto lay eggswas then monitored. In all transgeneconstructs,the oskar ptomoter was replacedwith a yeast inducible promoter (UAS). As diagrammed in part (a) of the

THEMOLECULAC R E L LB I O L O G YO F D E V E L O P M E N T

figure below, the first transgene construct includes the entire wild-type oskar genewith its three introns (UAS oskWT). For simpliciry introns are not depicted in the drawings. In the second construct, the wild-type oskar gene lacks its three introns (UAS oskAi(1,2,3)).In the third construct,3' untranslatedregion (UTR) of the wild-type oskar geneis replacedwith the 3' UTR from an irrelevant gene (UAS oskK1O). The final construct contains only the 3' UTR of the oskar gene (UAS osk3' UTR). The relative eggJayingability of the transgenicfemalesis shown in part (b). The white bar (*t"t) is a measureof the eggslaid on averageby nontransgenicwild-type females. (a) E1 E2 E3 E4 UAS

3'UTR

1

JAS oskWT r-+

r-+

Z

UAS osk^i(1,2,3) r--+

sro&

r--)

8K

UASoskK10 UASosk3'UTR

(b) 2.00 1.80 1.60 X

r rn

T

'|

t.zo

Ashe, H. L., and J. Briscoe.2006.The interpretationof morphogengradients.Deuelopment133:385-394. Blitz,I. L., G. Andelfinger,and M. E. Horb. 2006. Germ layers to organs:usingXenopus to study "later" development.Semin.Cell Deuel.Biol. 17:1.33-145. Eggan,K., et al.2004. Mice cloned from olfactory sensoryneurons. Natwre 428:4449. Grimm, O. H., and J. B. Gurdon. 2002. Nuclear exclusionof Smad2is a mechanismleadingto loss of competence.Nature Cell Biol. 4:51.9-522. Gurdon, J.8.2006. From nucleartransfer to nuclearreprogramming:the reversalof cell differentiation.Ann' Reu' Cell Deuel' Biol.22:,1-22. Hoegg, S., and A. Meyer. 2005. Hox clustersas modelsfor vertebrategenomeevolution. TrendsGenet.2l:421424. Ingham, P. $7.,and M. Placzek.2006. Orchestratingontogenesis:variations on a theme by sonic hedgehog.Nature Reu.Genet. 7:841-850. Kamath, R. S., et al. 2003. Systematicfunctional analysisof the Caenorhabditis elegansgenome using RNAi. Nature 421:231:237. Kim, S. K., et al. 2001. A geneexpressionmap for Caenorhabditis elegans.Science293 :2087-2092. Kimmel, A. R., and R. A. Firtel. 2004. Breakingsymmetries: regulation of Dictyosteliwa developmentthrough chemoattractant Curr. Opin. Genet.Deuel' and morphogensignal-response. l4(5):540-549. Leptin, M. 2005. Gastrulationmovements:the logic and the nuts and boks. Deuel. Cell 8:305-320. O'Connor, M. B., et aL.2006.ShapingBMP morphogengradients in the Drosophila embryo and pupal wing. Deuelopment t33(2\:183-193. SchierA. F., and \7. S. Talbot. 2005. Molecular geneticsof axis formation in zebrafish.Ann. Reu.Genet.39:561'-61'3. Tomancak,P.,et al. 2002. Systematicdeterminationof patterns of gene expression dttrrng Drosophila embryogenesis.Genome Biol. 8.1-0088. 14. 3 (12):research0O8 Gametogenesis and Fertilization

tr E 1.00 5

Chow, J.C., et al. 2005. Silencingof the mammalian X chromosome.Ann. Reu.GenomicsHuman Genet.6:69-92. Hoodbhoy, T., and J. Dean.2004.Insights into the molecular basis € 0.80 of sperm-eggrecognition in mammals. Reproduction 127:417422. 6 Inoue, N., et al. 2005. The immunoglobulin superfamilyprotein is required for spermto fuse with eggs.Nature 434:234-238. 0.60 Izumo 3; Lee,J. T. 2005. Regulationof X-chromosomecountingby Tsix uJ Science309:768-77 L. and Xite sequences. 0.40 Navarro, P.,et al. 2005. Tsix transcription acrossthe Xist gene alterschromatin confirmation without affectingXlsf transcription: 0.20 imolicationsfor X-chromosomeinactivation. Genes6 Devel t9-.1474-1484. Sado,T., and A. C. Ferguson-Smith.2005. Imprinted X inacti0.00 vation and reprogrammingin the preimplantationmouseembryo. w1118 UAs UAS UAS UAS oskWT oskli(1,2,3) oskK10 osk3'UTR Human Molec. Genet. 1:R59-R64. Trasler,I. M.2006. Gameteimprinting: settingepigeneticpatOffspring from females expressingtransgenes1 or 2 were terns for the next generation.Reprod. Fertil. Deuel- 18:63-69. normal, whereas those arising from females expressing 'Wasserman, 'What P. M., et al. 2004. Egg-sperminteractionsat other infortransgene4 lacked abdominal segments. fertilization in mammals.Eur. J. Obstet. Gynecol.Reprod. Biol. mation can be deduced from these observations about the 1 15S:557-S60.

role of oskar in Drosophila development?

References Highlights of Development Anderson,K. V., and P.W. Ingham. 2003. The transformation of the model organism:a decadeof developmentalgenetics.Nature Genet. 33(Suppll:28 5-293 .

Cell Diversity and Patterning in Early Vertebrate Embryos Freeman,M., and J. B. Gurdon. 2002. Regulatoryprinciplesof developmentalsignaling.Ann. Reu.Cell Deuel.Biol. 18:515-539. Hirokawa, N., et al. 2005. Nodal flow and the generationof left-right asymmetry.Cell 125:3345 ' Olson, E. N. 2006. Generegulatory networks in the evolution and developmentof the heart. Science313:1'922-1927. REFEREN CES

997

Shiratori,H., and H. Hamada. 2006.The left-right axis in the mouse:from origin to morphology.Deuelopment133:2095-2104. Stern,C. D. Evolution of the mechanismsthat establishthe embryonic axes.2006. Curr. Opin. Genet.Deuel. 16:41.3-41,8. Tam, P. P.,D. A. Loebel,and S. S. Tanaka.2006. Building the mousegastrula:signals,asymmetryand lineages.Curr. Opin. Genet. Deuel.16:419-425. Control of Body Segmentation: Themes and Variations in Insects and Vertebrates Akam, M. 1987. The molecular basisfor metamericpattern in the D r osoph ila embryo. D eueIopm ent l0l :1,-22. Andrioli, L. P.,et al. 2002. Anterior repressionof a Drosophila stripe enhancerrequiresthree position-specificmechanisms.Deyelopment 129:49314940. Aulehla,A., and B. G. Herrmann.2004. Segmenration in vertebrates:clock and gradientfinally ioined. Genesbeuel. 18:2060-2067. Carapuco,M., et al. 2005. Hox genesspecifyvertebraltypes in the presomiticmesoderm.Genes(t Deuel. 19:2116-2121. Chang,A. J., and D. Morisato. 2002. Regulationof Easteractivity is required for shapingthe Dorsal gradiint in the Drosophila embryo. D euelopment129:563 5-5645. Chopra,V.S., and R. K. Mishra. 2006. "Mir" aclesin hox gene regulation.Bioessays28:445448. Dale, J. K., et al. 2006. Oscillationsof the snail genesin the presomitic mesodermcoordinatesegmentalpatterning and morphogenesisin vertebratesomitogenesis. Deuel. Cell lO:355-366. Davis, G. K., and N. H. Patel.2002. Short, long, and beyond: molecularand embryologicalapproachesto insec segmentafion. Ann. Reu.Entomol. 47 :669-699. Dubrulle, J., and O. Pourquie.2004. Coupling segmenranonro axis formation. Deuelopmentl3L:5783-5793. Ephrussi,A., and D. St. Johnston.2004. Seeingis believing:the . bicoid morphogengradient matures.Cell 116:1,43-1,52. . Freeman,M., and J. B. Gurdon. 2002. Regulatoryprinciplesof developmentalsignaling.Ann. Reu.Cell Deuel.Biol. 18:51.5-539. Fujioka, M., et al. 1999. Analysisof an even-skippedrescue transgenerevealsboth compositeand discreteneuronal and early blastodermenhancers,and multi-stripe positioning by gap gene repressorgradients.Deuelopment126:2527-2538. Gridley,T. The long and short of it: somiteformation in mice. 2006. D euel.Dyn. 235 :2330-2336. Houchmandzadeh,B., E. Wieschaus,and S. Leibler.2002.Establishmentof developmentalprecisionand proportions in the early Drosophila embryo. Nature 415:79 8-802. Johnstone,O., and P. Lasko. 2001. Translationalresulation and RNA localizattonin Drosophila oocytesand embryos.Ann. Reu. Genet. 35:365406. Lemons,D., and \7. McGinnis. 2006. Genomic evolution of Hox geneclusters.Science313:1918-1922. Morgan, R. 2006. Hox genes:a continuation of embryonic patterning?Trends Genet.22:67-69. Sanson,B. 2001. Generatingpatternsfrom fields of cells:examples from Drosophila segmentation.EMBO Rep. 2:1083-1088. Stathopoulos,A., et al. 2002. Whole-genomeanalysisof dorsalventral patterning in the Drosophila embryo. Cell lll:687-70l. Swalla,B. J.2006. Building divergentbody plans with similar genericpathways.Heredity 97(31:235-243. Takeda,K., T. Kaisho, and S. Akira. 2003. Toll-like receDtors. Ann. Reu.Immunol. 2I:33 5-376. _ Yrng, M., and P. $7.Sternberg.2001. Patern formation during C. elegansvulval induction. Curr. TopicsDeuel. Bio. 5l:1,89-220. Wellik,D. M., and M. R. Capecchi.2003. Hox10 and Hox11 genesare requiredto globally pattern the mammalian skeleton. Science30I:363-367.

998

C H A P T E R2 2

|

Cell-Type Specification in Early Neural Development Colas,J-F.,and G. C. Schoenwolf.2003. Differential expression of two cell adhesionmolecules,Ephrin-A5 and Integrin alpha6, during cranial neurulation in the chick embryo. Deuel. Neurosci. 25:357-365. Cooke, J. E., and C. B. Moens. 2002. Boundary formation in the hindbrain: Eph only it were simple. Trends Neurosci. 25:260-267. Grandbarbe,L., et al. 2003. Delta-Notch signalingcontrols the generationof neurons/gliafrom neural stem cellsin a stepwise process.Deuelopment130:1,39'1,-"1402. Jessell,T. M. 2000. Neuronal specificationin the spinal cord: inductive signalsand transcriptionalcodes.Natwre Reu.Genet. l:20-29. Kriegstein,A. R., and S. C. Noctor. 2004. Patternsof neuronal migration in the embryonic cortex. TrendsNeurosci.2T:392-399. Miao, H., et al. 2001. Activation of EphA receptorryrosine kinase inhibits the Ras/MAPK pathway. Nature Cell Biol. 3:527-530. Santiago,A., and C. A. Erickson.2002. Ephrin-B ligandsplay a dual role in the control of neural crestcell migration. Deuelopment 1293627-3632. Growth and Patterning of Limbs Boulet, A. M., et al. 2004. The rolesof Fgf4 and FgfS in limb bud initiationand outgrowth.Deuel.Biol.273:361-372. Colas,J-F.,and G. C. Schoenwolf.2001. Towards a cellular and molecularunderstandingof neurulation. Deuel. Dyn. 221:117-1,45. Han, M., et al. 2005. Limb regenerationin higher vertebrates: developinga roadmap. Anat. Rec. B Neut Anat.287:1,4-24. Li, C., et al. 2005. FGFR1 funcrion at the earlieststagesof mouselimb developmentplays an indispensablerole in subsequent autopod morphogenesi s. D euelopment 132:475 547 64. Martin, G. 2001. Making a vertebratelimb: new playersenter from the wings. Bioessays23:865-868. Minguillon, C., J. Del Buono, and M. P. Logan. 2005. TbxS and Tbx4 are not sufficientto determinelimb-specificmorphologiesbut have common roles in initiating limb outgrowth. Deuel. Cell 8:75-84. Moon, R. T., et al. 2002.The promise and perils of l(nt signaling through beta-catenin.Science296l.1, 644-1646. Nybakken, K., and N. Perrimon.2002.Hedgehog signaltransduction: recentfindings. Curr. Opin. Genet.Deuel. 12:503-511. Pandur,P.,D. Maurus, and M. Kuhl. 2002. Increasinglycomplex: new playersenter the'Wnt signalingnetwork. Bioessays 24:881.-884. Pires-daSilva, A., and R. J. Sommer.2003. The evolution of signalingpathways in animal development.Natwre Reu.Genet. 4:3949. Rubin, C., et al. 2003. Sprouty fine-tunesEGF signaling through interlinked positive and negativefeedbackloops. Curr. Biol. 13:297-307. Salsi,V., andY. Zappavigna.2006. Hoxdl.3 and Hoxa13 directly control the expressionof the EphAT Ephrin tyrosine kinase receptorin developinglimbs./. Biol. Chem.28l:1992-1999. Tickle, C. 2006. Making digit patternsin the vertebratelimb. Nature Reu.Molec. Cell Biol.7:45-53. Tickle, C. 2003. Patterningsysrems-from one end of the Lim to the other.Deuel. Cell 4:449458. Tickle, C., and A. Munsterberg.2001. Vertebrarelimb development: the early stagesin chick and mouse.Curr. Opin. Genet. Deuel. ll:476-481. Zuniga, A. 2005. Globalizationreachesgeneregulation:the casefor vertebratelimb development.Curr. Opin. Genet.Deuel. 15:403409.

T H E M O L E C U L A RC E L LB | O L O G YO F D E V E L O P M E N T

cLASSTC

EXPERIMENT

22

TO STUDYDEVELOPMENT USINGLETHALMUTATIONS C. Nrlsslein-Volhard and E. Wieschaus,1980, Nature 287:.795

One of the most fascinating questions in developmentalbiology concernsthe proper formation of an embryo. How does a fertilized egg "know" how to form a complex organism? Scientists have puzzled over how to address this question for a long time. In 1980, Christiane Niisslein-Volhard and Eric l7ieschaus first reported studies with the fruit fly Drosophila melanogaster in which a genetic approach was used to addressthis question.

Background To examine complex processessuch as development of an embryo from a fertilized egg, biologists often collect mutant organisms that differ from the normal (wild-type) organism. To apply this genetic approach to developmental biology, geneticists first look for mutant organisms that display an obvious defectin overall formation. Early work uncovereda number of genesinvolved in the development of the fruit fIy Drosophila melanogaster. ln the first genesexamined, the mutations resulted in the birth of flies with obvious physical defects, such as the presence of an extra set of wings. Becausethis approach relied on examining viable flies with physical malformations, it missed many developmentally important genesthat, when mutated, result in the death of the fly embryo. In the late 1.970s.Niisslein-Volhard and Wieschausbegan their pioneering work on the developmentof Drosophila embryos. They sought to identify as many genesin the developmentalprocess as possible by looking for genesthat, when mutated. resulted in the death of the embryonic fly. Their work unveiled severalkey genesactive in the early development of not only Drosophila, but higher organismsas well.

progeny would be heterozygous for any mutations on the chromosomes Geneticists develop systematic methreceived from the father. The hetthey ods, known as genetic screens, to erozygoteswere then bred as separate searchfor mutations that affect biologlines, in essenceisolating eachnew muical processes.Niisslein-Volhard and tation in a separate fly stock. Flies $Tieschaushad to consider severalprewithin each stock then were crossed vious observations on Drosophila dewith each other to generate homozyvelopment when they designed their gous embryos. If the mutation affected screen.First, they knew that genesexa gene needed for embryogenesis,the pressed in the egg, called maternalhomozygous embryos would die but effect genes,as well as genesexpressed could be examined for phenotype. The after fertrlization in the developing emmutant gene, however, would not be bryo, called zygotically actiue genes, lost as it could be recoveredin the hetcontrolled the early developmentof an erozygousflies of that same stock. embryo. They choseto focus on isolatUsing this screen,Niisslein-Volhard ing mutations that affect the zygotiWieschausamasseda large collecand cally active genes.Second,they had to of mutant flies. The next step was tion consider that the Drosophila genome the various mutants to speto assign is diploid, which means that the progbasedon their phenotype. cific classes, eny receive a copy of each gene from focused on the segmentation of They '$Thereas both parents. Scientistshad previously all mutants in the larvae. demonstrated that DrosoPhila renecessarilydisplayed the screen their quired only a single wild-type copy of phenotype of embryonic lethality they most genesin order to develop into a differed greatly in their segmentation viable fly. This made it likely that redefects. To classify these defects, cessivemutations in developmentally Niisslein-Volhard and l7ieschaus exactivegeneswould not resultin embryamined the larvae under the microonic death, the phenotypic screenused scope. They compared the body Patby Ntisslein-Volhardand'$Tieschaus. tern of a wild-type viable larva to those Therefore, they had to breed mutant of the embryonic lethal mutants. By Drosophila to obtain flies that were these patterns, they uncovcomparing homozygous for the mutations of inered three classesof genes that affect terest. segmentation, which they called gap, The overall mutation rate in a natpair-rule, and segment-PolaritY. urally occurring population is quite Gap mutants are missing uP to eight low If a geneticistwere to search for segmentsfrom the overall body which mutants in a natural population, he or results in a smaller body due to the she would have to examine a large death of some of the embryo cells.Three number of individuals. To circumvent mutants-knirps, hunchback, and the this difficulty, Niisslein-Volhard and '!Tieschaus previously characterized Krilppel-felI induced mutations in a into this class,as shown in the accomDrosophila population at the onset of panying photographs. The next class the screen by feeding a mutagenic of mutants, the pair-rule mutants' had chemicalto male flies and mating them deletions of alternating segmentsof to a genetically defined population of body, which caused roughly halfthe wild-type female flies in a process normal-size larval bodies to form. Six known as a geneticcross.The resulting

The Experiment

U S I N GL E T H A LM U T A T I O N ST O S T U D YD E V E L O P M E N T

999

< FIGURE 1 A viable, wild-type larva(Normal) iscompared with threelarvaethat havemutationsin the gap genesKr1ppel, hunchback, or knirps. Thoracic segments arelabeled T1-T3,whereas abdominal segments aredesignated A1-A8.Gapmutants are missing entiresegments fromthe bodyplan,asillustrated by the labeled segments on the left.lFrom C Nusslein-Volhard andE Wieschaus, 1980,Nature 287:7951

Normal

Kriippel

hunchback

previously uncharacterizedmutantspaired, euen-skipped, odd-skipped, odd-paired, fushi tarazu, dnd runtwere placed in this class.The final set of mutants, the segment-polarity mutants, had the same overall number of segments as wild-type larvae. But a part of the body partern within each segment was deleted in each mutant. The deleted porrion of the pattern within each segmentwas replaced by a mirror image of the portion that remained. Niisslein-Volhard and rWieschaus'sinitial screen uncovered six mutants of this class, three of which-goo seberry, hedgehog, and patched-had not been previously observed.

Discussion In the first publishedreportof their screen,Niisslein-Volhard and'sfieschaus described 15 mutations that affected segmentation.Of these,only five were

1000

C H A P T E R2 2

|

Knirps

in previously identified genes. Ifhen they completed the study-often referred to as the Heidelberg screensthey had identified 139 different genes that, when mutated, resultedin embryonic death. These mutants fell into 17 different classes including the ones described here. Together these genes act in a field of equivalent cells and direct each cell to take on a fate appropriate to its position within the field. In this way some cellsform segmentanterior, some make bristles, some make sensory organs, and so forth. Genes that control these sorts of organizing cell-fate decisions are called patternformation genes. As molecular techniques evolved, scientistscloned many of these genesand characterizedtheir gene products. These mutants formed the base for the next quarter century of research into the development of Drosopbila. Moreover, many vertebrate pattern-formation genes are close homologs of the corresponding

T H E M O L E C U L A RC E L LB t O L O G YO F D E V E L O P M E N T

fly genes, and use similar molecular mechanismsto organize cells, tissues, and organs. Thus the work of Niisslein-Volhard and \Tieschaus greatly advancedthe study of development in all vertebrates,including ourselves. The majority of genesidentified by Niisslein-Volhard and Wieschaus encode transcription factors, but their screen also uncovered genesencoding signaling molecules, receptors, enzymes, adhesion molecules,cytoskeleton proteins, and some proteins whose functions remain unknown. Mammalian genes related to some of the Drosophila genes uncovered by Niisslein-Volhard and Wieschaussubsequentlywere found to be important in human diseasessuch as cancer and birth defects.In 1995, the Nobel Foundation awarded its prize for Physiology and Medicine to Niisslein-Volhard and \flieschaus for their pioneering work.

CHAPTER t

t

NERVECELLS

Theconvolutedsurfaceof the cerebrumand(lowerright) Unlimited] the cerebeilum[O RalphHutchingstuisuals

as the pinnahen humanswish to view themselves cle of evolution,the assertionis usuallyrelatedto the brain. since manv of our other abilities fare poorly in comparisonwith other animals.The complexityof the human brain is staggeringand seemsadequateto account for its amazing abilities.In the 1.3-kg adult human brain (78 percentof which is water!), there are about 1011 nervecells,calledneurons.The number of human brain neurons is comparableto the estimated1011starsin our galaxn the Milky Way. The neurons in one human bratn are conthe iunction pointswheretwo nectedby some1014synapses, or more neurons communicate.\X/ith 6.5 x 10o people on r h ee a r t h ,t h e r ea r ea b o u t6 . 5 x 1 0 2 3h u m a ns y n a p s ei sn e x istence,which is also about the total number of starsin the universe. . . so far as we know. Using our neurons,we can keep searchingfor more. Right this moment you are vigorously employing neurons to detectand interpret visual information. Neurons gobble ATP, made exclusively from glucose, at a tremendous rate. Although the brain is only about 2 percent of the body's mass,it usesabout 20 percentof the body's restingenergy. This extensivebrain energyis usedto drive electricalsignaling along neurons, which are often elongated cells, and chemicalsignalingbetweenthem. The electricalpulsesthat travel along neuronsare called action potentials,and information is encodedas the frequencyat which action potentials are fired. Owing to the speedof electricaltransmission, neuronsare champion signaltransducers,much faster than cellsthat secretehormonesor developmentalsignalingproteins. The rapidity of neural signalingmakes it possibleto handle large amounts of information quickly. The amazing

complexity of our neural network makes possiblesophisticated perception, analysis,and response,and forms the cellular machinery underlying instinct, learning, memory' and emoilon. In this chapter we will focus on neurobiology at the cell and molecular level. We will start by looking at the general architectureof neurons and at how they carry signals(Figure 23-1). Next. we will look at ion flow, channelproteins,and membrane properties:how electricalpulses move rapidly along neurons. Third, we will examine communication between neurons: electrical signals traveling along a cell must be translated into a chemical pulse between cells and then back into an electrical signal in the receiving cells. In the

OUTLINE 23Jl

N e u r o n sa n d G l i a :B u i l d i n gB l o c k so f the NervousSYstem

23.2

Voltage-Gatedlon Channelsand the Propagationof Action Potentials i n N e r v eC e l l s

1006

Communicationat SYnaPses

1018

73.3

, earing, , e e l i n gH : e e i n gF 2 3 . 4 S e n s a t i o n aCl e l l sS T a s t i n ga, n d S m e l l i n g

1027

ControllingAxon The Pathto Success: Growth and Targeting

1040

23.5

1001

A

)

l!, (f)

(h)

FIGURE 23-1 lllustrationsof the nervoussystemand nerve cellsby Ramony Cajal(1852-1934). (a)Sensory andmotor nervous systems of a worm(A : sensory cellsof the skin,C : motor cellswith crossed processes, G : terminalramifications of a motor neuronon a muscle)(b)Cross sectron of a spinalcord (c)Sectron throughtheopticlobeof a chameleon (numbers indicate layers from d e e pt o s u p e r f i c iAa ,l ;C ,D : S h e p h esr d c r o o kc e l l s () d ,e )C e l l si n the nuclear layerof the kittensuperior (f)Chiasm (crossinq colliculus

place) andcentralprojection (c : crossed of humanvisualpathways bundleof theopticnerve, d : largeuncrossed bundle,Rv: projection of the mentalimage-thearrow-ontovisualareasof the cerebral cortex)(g)Neurons in thefusrform layerof the human motorcortex(A,E : pyramidal cells,a : axon)(h)Motorneurons (a : terminal terminating on rabbitmuscles arborization of an axon, 4 : pointwheremyelinsheathends,n : branchof a nerve).

Neuronsand Glia:BuildingBlocks of the NervousSystem

high-throughput signal processingand analysis.rWerefer to the processingas "thinking," and molecular cell biology is at the heart of it. 1002

.

c H A p r E R2 3

|

NERVE cELLs

In this sectionwe take an initial look at the structure of neurons and how they propagateelectricaland chemical signals. Neurons are distinguished by their elongated, asymmetric shape,by their highly localized proteins and organelles,and most of all by a set of proteins rhat controls the flow of ions across the plasma membrane. Becauseone neuron can respond to the inputs from multiple neurons,generateelectrical

signals,and transmit the signalsto multiple neurons, a nervous system has considerablepowers of signal analysis' For example, a neuron might pass on a signal only if it receives five simultaneousactivating signalsfrom input neurons.The receiving neuron measuresboth the total amownt of incoming signal and whether the five signalsare roughly synchronous.Input from one neuron to another can be either excitatory-combining with other signals to trigger electrical transduction in the receiving cell-or inhibitory, discouraging such transmission.Thus the properties and connections of individual neurons set the stage for integration and re'$7e will begin by looking at how finement of information. signals are receivedand sent, and in subsequentparts of the chapter we will look at the molecular details of the machinery involved.

I n f o r m a t i o nF l o w sT h r o u g hN e u r o n sf r o m Dendritesto Axons Neurons arise from roughly sphericalnewroblastprecursors. Newly born neurons can migrate long distanceswhile still in the form of simple round cells before growing into dramatically elongatedcells. Fully differentiated neurons take many forms, but generally share certain key features (seeFigure 23-1). The nucleusis found in a rounded part of the cell called the cell body (Figure 23-21. Branching cell processes called dendrites (from the Greek for "treelike") are found at one end, and are the main structure where signals are receivedfrom other neurons via synapses.Incoming signalsare also receivedat synapsesthat form on neuronal cell bodies. Neurons often have extremely long dendrites with complex branches,particularly in the central nervous system.This allows them to form synapseswith, and receivesignalsfrom' a large number of other neurons-up to tens of thousands. Thus the converging dendritic branchesallow signals from many cells to be receivedand integrated by a single neuron.

'When

a neuron is first differentiating' the end of the cell opposite the dendrites undergoesdramatic outgrowth to form a long extended arm called the axon, which is essentially a transmissionwire. The growth of axons must be controlled so that proper connections are formed, a processcalled axon guidancethat is discussedin Section23.5. The diametersof rr"ry from just a micrometer in certain nerves of the hu"*orr, man brain to a millimeter in the giant fiber of the squid' Axons can be metersin length (in giraffe necks,for example),and

next neuron. The asymmetryof the neuron' with dendritesat one end and axon termini at the other, is indicative of the unidirectional flow of information from dendritesto axons'

they have other important roles. Much current work is devoted to learning how glia build the myelin insulators that control neuronal electricaltransmission,provide growth factors and other signals to neurons' receivesignals from neurons, and influence the formation of synapses.

lnformation Moves as Pulsesof lon Flow C a l l e dA c t i o n P o t e n t i a l s Nerve cells are members of a class of excitable cells, which also includesmusclecells,cellsin the pancreas,and someothers. The term indicatesthat the cells can build up a voltage acrosstheir plasma membranes,the membranepotential and

( a )M u l t i p o l ai nr t e r n e u r o n Axon terminal

Dendrite I

I

D i r e c t i o no f a c t i o no o t e n t i a l

hillock Muscle

(b)Motorneuron C e l lb o d y -i

Nodes of Ranvier Dendrite

Myelin sheath

D i r e c t i o no f a c t i o np o t e n t i a l

Axon terminal

23-2 Typicalmorphologyof < FIGURE two typesof mammalianneurons.Action potentials arisein the axonhillockandare towardtheaxonterminus(a)A conducted branched hasprofusely interneuron multipolar at synapses signals whichreceive dendrites, Small otherneurons. hundred with several by inputsin the imparted changes voltage cansumto giveriseto the more dendrites whichstartsin the actionpotential, massive A singlelongaxonthat branches hillock. to signals transmits at itsterminus laterally (b) innervating neuron A motor otherneurons hasa singlelongaxon celltypically a muscle fromthe cellbodyto the effector extending s ,n c e l ll.n m a m m a l i amno t o rn e u r o n a all covers usually myelin of sheath insulating of nodes the at except the axon of oarts andthe axonterminalsThemyelin Ranvier of cellscalledg/r,a sheathiscomposed

OFTHENERVOUSYSTEM A N D G L I A :B U I L D I N GB L O C K S NEURONS

1003

that this voltage can be discharged(allowed to come back to zero voltage or even swing to positive) in various ways and for various purposes (Chapter 11). The voltage in a typical neuron, called the resting potential becauseit is the state when no signalis in transit, is establishedby ion pumps in the plasma membrane. The pumps use energy, in the form of ATP, to move positively charged ions out of the cell. The result is a net negativechargeinside the cell compared with the outside environment. A typical resting potential is -60 mV. Neurons have a languageall their own. The signalstake the form of brief local voltagechanges,from negativeinsideto positive,an eventdesignateddepolarization.A powerful surge of depolarizingvoltage change,moving from one end of the neuron to the other, is called an action potential. "Depolarization" is somewhat of a misnomer,sincethe neuron goes from negative inside to neutral to positive inside, which could be accurately described as depolarization followed by the oppositepolarization(FigureZ:-:1. et the peak of an action potential, the membranepotential can be as much as *50 mV (insidepositive),a net changeof =110 mV. As we shall seein greater detail in Section 23.2, the voltage change (which is eventuallyadded to other voltage changesro creare the action potential) beginsat the dendriteend ofthe cell in responseto inputs from other cellsand movesalong the axon to the axon terminus.Action potentialsmove at speedsup to 100

tentials are all or none. Once the threshold to start one is reached,a full firing occurs.The signal information is therefore carried primarily not by the intensity of the action porentials, but by the timing and frequencyof them. Someexcitablecells are not neurons.Muscle contraction is triggered by motor neurons that synapsedirectly with ex, citablemusclecells(seeFigure23-2b).Insulin secretionfrom the beta cells of the pancreasis triggered by neurons.In both casesthe activating event involves an opening of plasma

Axon of presynaptic cell Exocytosisof neurotransmitter

Synaptic vesicle Synapticcleft Postsynaptic cell Receptorsfor neurotransmitter

Directionof f signaling

Axonterminal of presynaptic c el l

negative-insiderestingpotential (repolarization).Theresrora_ tlon processchasesthe action potential down the axon to the terminus,leavingthe neuron ready to signalagain.Action po-

Synapticvesicles Synapticcleft

A c t i o np o t e n t i a l s Dendriteof postsynaptic cell

+50 mV o c q)

o

q) c o

o)

o N

0.5 prm

E

o

-60 mV

Time ----> EXPERIMENTAL FTGURE 23-3 Recordingof an axonal membrane potential over time revealsthe amplitude and frequency of action poentials. An actionpotentialis a suooen, transientdepolarization of the membrane, followedby reporarization to the restingpotentialof about -60 mV Theaxonalmembranepotential can be measured with a smaljelectrode placedinto it (seeFigure1.1_1g) Thisrecording of the axonalmembranepotentialin this neuronshows that it is generating one actionpotentialaboutevery4 milliseconds 1004

.

c H A p r E R2 3

|

NERVE CELLS

A FIGURE 23-4 A chemicalsynapse. (a)A narrowregion-the synaptic cleft-separates the plasma membranes of tne presynaprrc andpostsynaptrc cellsArrivalof actionpotentials at a synapse causes release (redcircles) of neurotransmitters bythepresynaptic cell,their diffusion across thesynaptic cleft,andtheirbindingbyspecific receptors on theplasma membrane of the postsynaptic cell.Generally thesesignals depolarize the postsynaptic (making membrane the potential inside lessnegative), tendingto inducean actionpotential in it (b)Electron micrograph showsa dendrite synapsing wtrnan axon terminal filledwithsynaptic vesicles Inthesynaptic region, theplasma membrane of the presynaptic cellisspecialized for vesicle exocytosis; synaptic vesicles containrng a neurotransmitter areclustered in these regionsTheopposing membrane of the postsynaptic cell(inthiscase, a neuron) contains receptors for the neurotransmitter. (b)fromC [part Raineet al , eds , 1981, BasicNeurochemistry3d ed, Little,Brown, p 32 l

membranechannelsthat causeschangesin the transmembrane flow of ions and in the electricalproperties of the regulatedcells.

T h e N e r v o u sS y s t e mU s e sS i g n a l i n gC i r c u i t s C o m p o s e do f M u l t i P l eN e u r o n s

l n f o r m a t i o nF l o w sB e t w e e nN e u r o n s via Synapses What starts an action potential?Axon termini from one neuron are closelyapposedto dendritesof another,at the junction called a synapse(Figure23-4). The axon termini of the presynaptic cell use exocytosisto releasesmall molecules called neurotransmitters.Neurotransmitters, glutamate or acetylcholine,for example,diffuseacrossthe synapsein about 0.5 ms and bind to receptorson the dendrite of the adiacentneuron. Binding of neurotransmittertriggersopeningor closingof specific ion channelsin the plasmamembraneof postsynapticcell dendrites, leading to changesin the membrane potential at this point. The ensuinglocal depolarization,if large enough' triggers an action potential. Transmissionis unidirectional, from the axon termini of the presynapticcell to dendritesof the postsynapticcell. In some synapses,the effect of the neurotransmittersis to hyperpolarizeand thereforelower the likelihood of an action potential in the postsynapticcell. A single axon in the central nervous system can synapsewith many neurons and induce responsesin all of them simultaneously. Conversely,sometimesmultiple neuronsmust act on the postsynaptic cell roughly synchronouslyto have a strong enough impact to trigger an action potential. Neuronal integration of depolarizingand hyperpolarizingsignalsdeterminesthe likelihood of an actionpotential. Thus neurons employ a combination of extremely fast electricaltransmissionalong the axon with rapid chemical

"-

O u a d r i c e p sm u s c l e

communication between cells' Now we will look at how a chain of neurons, a circuit, can achievea useful function'

In complex multicellularanimals,such as insectsand mammals, various types of neurons form signaling circuits. A sensory neulon reports an event that has happened' like the arrival of a flash of light or the movement of a muscle.A motor neuron carries a signal to a muscle to stimulate its contraction (Figure23-5, and seeFigure23-2b). An interneuron bridges other neurons, sometimesallowing integration or divergenceof signals, sometimes extending the reach of a signal. In a simple type of circuit called a reflex arc interneurons connect multiple sensoryand motor neurons' allowing one sensory neuron to affect multiple motor neurons and one motor neuron to be affected by multiple sensory neurons; in this way interneurons integrate and enhancereflexes.For example, the knee-jerk reflex in humans (seeFigure 23-5) involves a complex reflex arc in which one muscle is stimulated to contract while another is inhibited from contracting. The reflex also sends information to the brain to announcewhat happened.Such circuits allow an organism to respond to a sensoryinput by the coordinated action of setsof musclesthat together achievea single purpose. These simple signaling circuits, however, do not directly explain higher-orderbrain functions such as reasoning,compuiation, and memory development.Typical neurons in the brain receive signals from up to a thousand other neurons and, in turn, can direct chemical signalsto many other neurons. The output of the nervous systemdependson its circuit

-*-.*/ --

S e n s o r yn e u r o n Axon carries cell body i n f o r m a t i o nt o b r a i n S p i n a lc o r d

Sensory -J n e ur o n D o r s a l - r o o tJ ganglion

Motor neuron

Biceps muscle (flexor) Motorneuron a x o nt e r m i n a l

S-

K n e ec a p Motor neuron

Inhibitory I n t e r n e ur o n

23-5 The knee-jerkreflex.A tapof thehammer A FIGURE in the electrical activtty muscle, thustriggering thequadriceps stretches in the traveling neuronTheactionpotential, sensory stretchreceptor srgnals to thebrainsowe are of thetop bluearrowsends direction andalsoto two kindsof cellsin thedorsalawareof whatishappening, in the spinalcord Onecell,a motor that islocated rootganglion (red), muscle stimulates backto thequadriceps neuron thatconnects

yourkneeThe sothatyoukickthe personwho hammered contraction interneuron "excites," inhibitory an or activates, connection second by a activity (black)Theinterneuron hasa dampingeffect,blocking activate thatwould,in othercircumstances, flexormotorneuron(green) Inthisway,relaxation thequadriceps thatopposes muscle the hamstring Thisisa quadrrceps the of to contraction iscoupled of thehamstring decision conscious no requires movement reflexbecause

B L O C K SO F T H E N E R V O U S Y S T E M A N D G L I A :B U I L D I N G NEURONS

'

1005

properties-the wiring, or interconnections, between neurons and the strength of theseinterconnections.Complex aspects of the nervous system, such as vision and consciousness,cannot be understood at the single-celllevel. but onlv a t t h e l e v e lo f n e t w o r k so f n e r v ec e l l si h a t c a n b e s t u d i e db y techniques of systemsanalysis.The nervous sysremls constantly changing;alterations in the number and nature of the interconnections between individual neurons occur, for example, in the formation of new memones.

N e u r o n sa n d G l i a : B u i l d i n g B l o c k so f t h e Nervous System r Neuron are highly asymmetric cells composed of dendritesat one end, a cell body containingthe nucleus,a long a x o n ,a n d a x o n t e r m i n i . r Neurons carry information from one end to the other using pulses of ion flow across the plasma membrane. Branchedcell processes, dendrites,at one end of the cell receive chemical signals from other neurons, triggering ion flow. The electricalsignal moves rapidly ro axon termini at the other end of the cell (seeFigure23-2). r Glial cellsare ten times as abundant as neurons and serve many purposes such as building the insulation that coats neurons and supporting the formation of new synapses. A resting neuron carrying no signal has protern pumps at move ions across the plasma membrane. The movement of ions such as K+ and Na* and Cl crearesa net negative chargeinside the cell. This voltage is called the resting potentialand usuallyis about -60 mV (seeFigure23-3). a stimuluscauseschannelproteinsto open so that ions flow more freely, a strong pulse of voltage changemay down the neuron from dendritesto axon termini. The cell goesfrom being -60 mV insideto +50 mV inside,relative to the extracellular world. This pulse is called an action potential. r The action potential travels down the neuron becausea change in voltage near the dendrites triggers a change in voltage in the cell body, which in rurn does the sameto the proximal and then distal axon, and so forth. r Neurons connect across small gaps called synapses. Sincean action potential cannot jump the gap, atthe axon termini of the presynaptic cell the signal is converted from electricalto chemical to stimulate the postsynapticcell. r Upon stimulation by an action potential, axon termini release,by exocytosis, small packets of chemicals called neurotransmrtters. Neurotransmitters diffuse across the synapseand bind to receptorson the dendriteson the other side of the synapse.Thesereceptorsinitiate a new axon potential in the postsynapticcell (seeFigure 23-4). Neurons form circuits. They may consist of sensoryneuns, interneurons,and motor neurons,as in the knee-ierk response(seeFigure23-5).

r006

C H A P T E R2 3

I

N E R V EC E L L S

Voltage-Gated lon Channelsand the Propagation of ActionPotentialsin NerveCells Action potentials are propagated becausea change in voltage in one part of the cell triggers the opening of channelsin the next section of the cell. Voltage-gated channels therefore lie at the heart of neural transmission(Chapter 11). In this section,we first introduce some of the key properties of action potentials, which move rapidly along the axon. I7e then describe how the voltage-gatedchannels responsiblefor propagating action potentials in neurons operate.Thus electric signals carry information within a nerve cell, while chemical signals, discussedin the next section, carry information from one neuron to another, or from a neuron to a muscle or other target cell.

The Magnitude of the Action potential ls Close to Ep. Operation of the Na*/K* pn-p generatesa high concentration of K+ and a low concentrationof Na+ in the cytosol,relative to those in the extracellularmedium (Chapter 11). The subsequentoutvvardmovement of K+ ions through nongated K- channels is driven by the K+ concentration gradient (cytosol ) medium), generatingthe restingmembranepotential. The entry of Na* ions into the cytosol from the medium also is thermodynamically favored, driven by the Na+ concentration gradient (medium > cytosol) and the inside-negative membranepotential (seeFigure 11-24). However, mosr Na+ channelsin the plasmamembraneare closedin restingcells,so little inward movement of Na* ions can occur (Figure 23-6a). If enough Na* channelsopen, the resulting influx of Na- ions will more rhan compensatefor the efflux of K+ ions through open resting K+ channels.The result would be a net inward movement of cations, generating an excessof positive charges on the cytosolic face and a corresponding excessof negative charges (owing to the Cl- ions .,left behind" in the extracellular medium after influx of Na* ions) on the extracellular face (Figure 23-6b). In other words, the plasma membrane is depolarized to such an extent that the inside face becomesoositive. The magnitude of the membrane potenrial at the peak of depolarization in an action potential is very closeto the Na+ equilibrium potential Ey^ given by the Nernsr equation (Equation I1.-2), as would be expectedif opening of voltagegated Na* channelsis responsiblefor generatingaction potentials. For example, the measuredpeak value of the action potential for the squid giant axon is 35 mV, which is closeto the calculated value of En1,(55 mV) based on Na+ concentrations of 440 mM outside and 50 mM inside. The relationship befweenthe magnitude of the action potential and the concentrationof Na+ ions inside and outside the cell has been confirmed experimentally.For instance,if the concentration of Na* ions in the solution bathing the squid axon is reduced to one-third of normal, the magnitude of the depolarization is reduced by 40 mV, nearly as predicted.

(a)Restingstate(cytosolicfacenegative) b;-

T i m e :1 m s Closed )

=g.q

,cn

Closed and inactivated

v EA

tro-Jv

>5 234 D i s t a n cael o n ga x o n( m m )

T i m e :2 m s Closed

| +so =S.o

v

R e s t i n gp o t e n t i a l

Eb-50 >6 234 D i s t a n cael o n ga x o n( m m )

the defective protein was a channel. The shaker mutation prevents the mutant channel from opening normally immediately upon depolarization. To test whether the wild-type shakergeneencodeda K* channel,cloned wild-type shaier cDNA was usedas a templateto produceshakermRNA in a cell-free system. Expression of this mRNA in frog oocyres and patch-clamp measurementson the newly synthesized channel protein showed that its functional proDertieswere i d e n t i c a lw i t h t h o s eo f r h e v o l t a g e - g a r eK< J c h a n n e li n r h e neuronal membrane, demonstrating conclusiveiythat the shakergeneencodesthis K+-channelprotein. The ShakerK* channeland most other voltage-gated K* channelsthat have been identified are tetrameric proteins composed of four identical subunits arranged in the membrane around a centralpore. Each subunitis constructedof six membrane-spanning crhelixes,designatedS1-S6,and a p 1010

.

cHAprER 23 |

N E R V cEE L L s

segment(Figure23-10a). The 55 and 56 helicesand the p segmentare structurally and functionally homologous to those in the nongated resting K+ channel discussedearlier (seeFigure 11.-19).The 55 and 56 helicesform the lining of the channel through which the ion travels.The S1-S4 helices act as a voltage sensor(with 54 acting as the primary sensor) and are describedas paddlesowing to the way they protrude from the central complex. The N-terminal "ball" extending into the cytosolfrom S1 is the channel-inactivaring segment. Voltage-gated Na* channels and Ca2* channels are monomericproteinsorganizedinto four homologousdomains, I-IV (Figure 23-10b). Each of these domains is similar to a subunit of a voltage-gatedK* channel.However, in contrast to voltage-gated K+ channels, which have four channel, inactivating segments,the monomeric voltage-gatedchannels have a single channel-inactivatingsegment.Except for this

(a) Voltage-gatedK+channel (tetramer)

Exterior

Cytosol

a/'

lnactivation segment

( b ) V o l t a g e - g a t e dN a * c h a n n e l( m o n o m e r )

minor structural differenceand their varying ion permeabilities, all voltage-gatedion channelsare thought to function in a similar manner and to have evolved from a monomeric ancestral channel protein that contained six transmembranea helices.

V o l t a g e - S e n s i n5g4 c H e l i c e sM o v e i n R e s p o n s e t o M e m b r a n eD e p o l a r i z a t i o n The understandingof channel-proteinbiochemistryis advancing rapidly owing to new crystal structuresfor bacterial and shaker potassiumchannelsand other channels.One method used to obtain crystals of these difficult membrane proteins was to surround them with bound fragmentsof monoclonal antibodies(Fab's;Chapter 24);in other casesthey were crystallized in complexeswith normal protein-bindingpartners. The structuresof the channelsrevealremarkable arrangements of the voltage-sensingdomains, and suggesthow parts of the protein move in order to open the channel. The tetramer has a pore whose walls are formed by helicesS5 and 56 (Figure 23-1la). Outside that core structure four arms, or "paddles," protrude into the surrounding membranel these are the voltage sensors,and they are in minimal contact with the pore. Sensitiveelectric measurementssuggestedthat the opening of a voltage-gatedNa* or K* channel is accompanied by the movement of 12-14 protein-bound positive chargesfrom the cytosolic to the exoplasmic surface of the membrane. The moving part of the protein is composed of helicesS1-S4; 54 accountsfor much of the positivecharge and is therefore the primary voltage sensor,with a positively charged lysine or arginine every third or fourth residue. Arginines in 54 have been measuredmoving as much as 1.5

depictionsof the 23-10 Schematic < FIGURE secondarystructuresof voltage-gatedK+ and (a)Voltage-gated are K* channels Na* channels. eachcontaining subunits, of fouridentical composed andsixmembrane-spanning 600-700aminoacids, of eachsubunit, S1-56.TheN-terminus cthelices, N,formsa globular andlabeled inthecytosol located of the for inactivation ball)essential domain(orange (green) and openchannelTheS5and56 helices (blue)arehomologous to thosein the Psegment but eachsubunit K+ channels, resting nongated o helices. transmembrane fouradditional contains voltageOneof these,54 (red),isthe primary in thisroleby helices o helixandisassisted sensing (b) aremonomers Na* channels Voltage-gated S1-3 s r g a n i z ei nd t o c o n t a i n i n1g8 0 0 - 2 0 0a0m i n oa c i d o (l-lv)thataresimilar domains fourtransmembrane The K* channels. in voltage-gated to the subunits located in the segment, singlechannel-inactivating a lllandlV contains domains cytosol between motif(H;yellow)Voltagehydrophobic conserved overall gatedCa2*channels havea similar also ionchannels structureMostvoltage-gated (F) are not that subunits regulatory contain (a)adapted 1992, fromC Miller, here[Part depicted eral, 1996,Neuron Biol2.573,andH Larsson Curr. 2001, part(b)adapted fromW A Catterall, 16:387; 409:988 Nature l nm as the channelopens,which can be comparedwith the =5nm thicknessof the membrane or the L.2-nm diameterof the cr helix itself. The movement of thesegating charges(or voltage sensors)under the force of the electric field triggersa conformational changein the protein that opensthe channel. Thus the 54 helix is the key part of the voltage sensor,which then moves the S1-S4 helicesacrossmuch of the membrane. The most unusual aspect of the voltage-sensitivechannel structuresis the presenceofcharged groups' e.g.' arginines,in contact with lipid. The location of the voltage sensorhelps to explain earlier experimentswhere a non-voltage-sensitive channel was converted into a voltage-sensingchannel by adding to it voltage-sensingdomains' Such an experiment would seemunlikely to work if the voltage sensorshad to be deeply embeddedin the core stmcture. the imStudieswith mutant ShakerK* channelssupport 'When one or portance of the 54 helix in voltage sensing. more arginine or lysine residuesin the 54 helix of the Shaker K* channel were replaced with neutral or acidic residues' fewer positive chargesthan normal moved acrossthe membrane in responseto a membrane depolarization, indicating that arginine and lysine residuesin the 54 helix do indeed move acrossthe membrane. In other studies,mutant Shaker oroteins in which various 54 residueswere converted to cysteinewere tested for their reactivity with a water-soluble cysteine-modifyingchemical agent that cannot cross the membrane. On the basis of whether the cysteinesreacted with the agent added to one side or the other of the membrane, the results indicated that in the resting state amino acids near the C-terminus of the 54 helix face the cytosol; after the membrane is depolarized,some of these same amino

O F A C T I O NP O T E N T I A L ISN N E R V EC E L L S V O L T A G E - G A T E IDO N C H A N N E L SA N D T H E P R O P A G A T I O N

.

1011

(a)

(b)

Open

(c)

Closed

Exterior

lon-selective pore

c rs u b u n i t

-L Central cavity

Membrane

Cytosol Paddle LateraI window

Bs u b u n i t N-terminus

A FIGURE 23-11 Molecularstructureof a voltage-sensitive nearthe interiorto the exterior of the membrane in response to potassium channel.Thetwo ribbondiagrams showmodels of the depolarization. Since eachoneisattached to an S4-S5linker, each potassium channel in (a)openand(b)closed statesSince themolecule linkeranditsattached S5helixismoved, in turnmoving56 helices, isa tetramer of thesamesubunit, fourcopies of eachhelixarevisible; whichopenstheporeThestructure of theopenmammalian channel the onesfarthestawayareseenonlydimly.Thebrown(S5)andgreen (a)hasbeendeterminedTheclosed-channel structure in (b)is (56)alphahelices spanthemembrane, withtheinterior of thecellat hypothetical, but is basedon observations of a closedbacterial the bottomandexterior at thetop Thepurplespheres potassium-channel (c)Theball-and-chain represent K+ structure. modelfor ions,whichpassthroughtheopenchannel partof the andoccupy inactivation of voltage-gated K* channels in three-dimensional closed channel withoutpassing throughThe56 (green) helices line cutawayviewof the inactive state In additionto thefour crsubunits the pore Notehowthe helices (tanandgray)thatformthechannel, aretlghtlypackedat the bottomin proteins thesechannel havefour (b),closing thechannel sothatthe K* ioncannotpassthrough (purple) regulatory At theN terminus of eachof the B B subunits thedistances betweenS5helices [compare asshownbythe arrows proteins subunit isa smalldomain(purple"ball"on theendof the below(a)and(b)I The54-55linker,locatedin the cytoplasm, connects purple"chain")thatcontrols theopening pore.Inthis of thecentral the54 helix(notshown) to theS5helix(brown). Forclarity, helices illustration S1 theN terminus of onesubunit hasmovedthrougha lateral through54 havebeenomittedfromthemodel;theywouldnormally (a)and(b)fromS B Long, windowto blockthe pore [Parts E B Campbell, beattached to theendof theS4-S5linkerandprotrude fromthe andR MacKinnon, part(c)adapted 2005,Science 309:903-908; fromR molecule "paddles asthevoltage-sensing " These paddles movefrom Aldrich, 2001,Nature 411:643, andM Zhouetal, 200'1, Nature 4'11:657 l acids becomeexposedto the exoplasmic surfaceof the channel. These experiments directly demonstratedmovement of the 54 helix acrossthe membrane, as schematicallydepicted in Figure23-7 for voltage-gatedNa+ channels. The structure of the open form of a mammalian Shaker K* channel has been contrastedwith the closed structure of a crystallizedbacterial K* channel. The results suggesta model for the closing of the channel in responseto movements of the voltage sensorsacross the membrane (Figure 23-11.a,b). In the model, the voltage sensors,composed of helices S1-S4, move in responseto voltage and exert a

1012

C H A P T E R2 3

I

N E R V EC E L L S

torque on a linker helix that connects54 to 55. In the openchannel conformation, the position of the S4-S5 linkers alIows the 55 helicesto lie at about a 45" angle to the plane of the membrane(Figure23-11a, brown helices),and the pore 'lfhen insidehas a'1.2-nm opening. the cell is repolarized and the voltage sensor moves toward the intracellular membrane surface,the S4-S5 linkers are twisted down, toward the inside of the cell. The 55 helicesare consequentlymoved more orthogonal to the plane of the membrane(Figure23-11b, brown helices).This position leavesthe 55 and 56 helicesin closer proximiry squeezingthe channelclosed(Figure23-11a, b; the

double headedarrows indicate spacingbetween adjacent 55 helices).Thus the gate probably is composed of the cytosolfacing ends of the 55 and 55 helices,where the pore is narrowest.

M o v e m e n to f t h e C h a n n e l - l n a c t i v a t i nSge g m e n t into the Open PoreBlockslon Flow An important characteristic of most voltage-gatedchannels is inactivation; that is, soon after opening they close spontaneously forming an inactive channelthat will not reopen until the membraneis repolarized.In the resting state,the positively charged globular balls at the N-termini of the four subunitsin a voltage-gatedK* channelare free in the cytosol. Severalmillisecondsafter the channel is opened by depolarization, one ball moves through an opening (lateral window) between two of the subunits and binds in a hydrophobic pocket in the pore's central cavity, blocking the flow of K* ions (Figure 23-L1,c).After a few milliseconds,the ball is displaced from the pore, and the protein reverts to the closed, resting state.The ball-and-chaindomains in K+ channelsare functionally equivalent to the channel-inactivatingsegment in Na- channels. The experimental results shown in Figure 23-12 demonstrate that inactivation of K* channels dependson the ball domains, occurs after channel opening, and doesnot require the ball domains to be covalently linked to the channel protein. In other experiments,mutant K* channelslacking portions of the =40-residuechain connecting the ball to the S1

helix were expressedin frog oocytes. Patch-clampmeasurements of channel activity showed that the shorter the chain, the more rapid the inactivation, as if a ball attached to a shorter chain can move into the open channel more readily. Conversely,addition of random amino acids to lengthen the normal chain slows channel inactivation. The singlechannel-inactivatingsegmentin voltage-gated Na* channelscontains a conservedhydrophobic motif composed of isoleucine,phenylalanine) methionine, and threonine (seeFigure 23-1,0b).Like the longer ball-and-chain domain in K* channels,this segmentfolds into and blocks the Na*-conducting pore until the membrane is repolarized (see Figure23-71.

MyelinationIncreasesthe Velocity o f l m p u l s eC o n d u c t i o n

As we have seen, action potentials can move down an axon without diminution at speedsup to 1 meter per second. But even such fast speedsare insufficient to permit the complex movementstypical of animals.In humans, for instance.the cell bodies of motor neurons innervating leg musclesare located in the spinal cord, and the axons are about a meter in length. The coordinated muscle contractions required for walking, running, and similar movementswould be impossibleif it took 1 secondfor an action potential to move from the spinal cord down the axon of a motor neuron to a leg muscle. The solution is to wrap cells in insulation that increasesthe rate of movement of an action potential. The insulation is called a myelin sheath (seeFigure 23-2b). The presenceof a myelin sheath around an axon increasesthe velocity of impulse conducM u t a n tS h a k e rK +c h a n n e l tion to 10-100 metersper second.As a result, in a typical human motor neuron, an action potential can travel the Wild-typeShakerK' channel length of a 1-meter-longaxon and stimulate a muscle to contract within 0.01 seconds. In nonmyelinated neurons,the conduction velocity of an action potential is roughly proportional to the diameter of the axon, becausea thicker axon will have a greater number of ions that can diffuse. The human brain is packed with relatively small, myelinated neurons' If the neurons in the hu020406080 man brain were not myelinated' their axonal diameters T i m e( m s ) would have to increaseabout 10,000-fold to achievethe FIGURE 23-12Experiments with a mutantK+ EXPERIMENTAL sameconduction velocitiesas myelinated neurons.Thus verchannellackingthe N-terminalglobulardomainssupportthe tebrate brains, with their densely packed neurons, never K' channel could have evolved without myelin. inactivation model.Thewild-type Shaker ball-and-chain theaminoacidscomposing theN-terminal anda mutantformlacking in Xenopus ballwereexpressed oocytesTheactivityof thechannels Action Potentials"Jump" from Node to Node Whenpatches bythepatch-clamp technique thenwasmonitored opened from-0 to +30 mV thewild-type channel weredepolarized in MyelinatedAxons (redcurve)Themutantchannel opened for =5 msandthenclosed The myelin sheathsurrounding an axon is formed from many curve)Whena chemically normally, butcouldnotclose(green glial cells.Each region of myelin formed by an individual glial faceof the patch, synthesized ballpeptidewasaddedto the cytosolic unmyelinated (bluecurve). This cell is separatedfrom the next region by an openednormally andthenclosed the mutantchannel pm called the in length 1 about membrane of axonal area the channelafterit demonstrated thatthe addedpeptideinactivated ax23-2).The Figure (or see node; simplg Ranuier node of openedandthatthe balldoesnot haveto betetheredto the proteinin extracellular with the contact is in direct membrane onal order to function [FromW N Zagottaet al , 1990,Science250:568]

O F A C T I O NP O T E N T I A L ISN N E R V EC E L L S V O L T A G E - G A T E IDO N C H A N N E L SA N D T H E P R O P A G A T I O N

1013

Action potential ( d e p o l a r i z erde g i o n of membrane) Nar

N o d eo f Ranvier

Myelin sheath

Na*

I

t

E

Na*

A FIGURE 23-13 Conductionof actionpotentialsin myelinatedaxons.Because voltage-gated Na+channels are localized to the axonalmembrane at the nodesof Ranvier, the influx of Na* ionsassociated with an actionpotential canoccuronlyat nodesWhenan actionpotential isgenerated at onenode(stepIl), positive the excess ionsin thecytosol, whichcannotmoveoutward across thesheath, diffuserapidly downtheaxon,causing sufficient depolarization at the nextnode(step[) to inducean action potential at thatnode(stepB) Bythismechanism theaction jumpsfromnodeto nodealonqthe axon potential

fluid only at the nodes. Moreover, all the voltage-gatedNa+ channels and all the Na+/K+ pumps, which maintain the ionic gradientsin the axon, are located in the nodes. As a consequenceof this localization, the inward movement of Na* ions that generatesthe action potential can occur only at the myelin-free nodes (Figure 23-13). The excess cytosolic positive ions generatedat a node during the membrane depolarization associated with an action potential spreadpassivelythrough the axonal cytosol to the next node with very little loss or attenuation, since they cannot cross the myelinated axonal membrane. This causesa depolarization at one node to spread rapidly to the next node, permitting the action potential, in effect, to jump from node to node. The transmission is called sahatory conduction. This phenomenon explains why the conduction velocity of myelinated neurons is about the sameas that of much larger diameter unmyelinated neurons. For instance,a 12-p,m-diameter myelinatedvertebrateaxon and a 600-pm-diameter unmyelinated squid axon both conduct impulsesat L2 mls.

G l i a P r o d u c eM y e l i n S h e a t h sa n d S y n a p s e s Of the four types of glia (three of which are shown in Figure 23-14), two produce myelin sheaths: oligodendrocytes make sheathsfor the central nervous system (CNS), and Schwann cells make them for the peripheral nervous system. Astrocyfes, a third type, are necessaryfor neurons to produce synapsesand use them to communicate with other neurons. The fourth type, microglia, produce survival fac1014

.

c H A p r E R2 3

|

NERVE cELLs

tors for cells and carry out immune functions. These cells participate in inflammatory responsesand constitute a part of the CNS immune system.They can differentiate to form phagocyticcellswith characteristics of macrophages(Chapter 241.Microglia form in the bone marro% are not related by lineageto neurons or to other glia, and will not be discussedfurther. Oligodendrocytes Oligodendrocytes form the spiral myelin sheath around axons of the central nervous system (Figure 23-14c). Each oligodendrocyte provides myelin sheathsto multiple neurons. The major protein constituents are myelin basicprotein (MBP) and proteolipid protein (PLP). MBP, a peripheral membrane protein found in both the CNS and the PNS, has sevenRNA splicing variants that encodedifferent forms of the protein. It is synthesized6y ribosomes locatedin the growing myelin sheath(Figure23-14c),an example of specifictransport of mRNAs to a peripheral cell region. The localizationof MBP mRNA dependson microtubules. Damage to proteins produced by oligodendrocytesunderlies a prevalent human neurological disease,multiple sclerosis (MS). MS is usually characterizedby spasms and weaknessin one or more limbs, bladderdysfunction,local sensorylosses,and visual disturbances.This disorderthe prototype demyelinating disease-is caused by patchy Ioss of myelin in areas of the brain and spinal cord. In MS patients, conduction of action potentials by the demyelinated neurons is slowed, and the Na* channelsspread outward from the nodes, lowering their nodal concentration. The causeof the diseaseis not known but appearsto involve either the body's production of auto-antibodies (antibodies that bind to normal body proteins) that react with MBP or the secretion of proteasesthat destroy myelin proteins. A mouse mutant, shiuerer,has a deletion of much of the MBP gene, Ieading to tremors, convulsions,and early death. Similarly human (Pelizaeus-Merzbacher disease)and mouse mutations in gene the coding for the other major Ui*pylt protein of CNS myelin, PLP, causeloss of oligodendrocytes and inadequatemyelination. I Schwann Cells Schwann cells form myelin sheaths around peripheral nerves.A Schwanncell myelin sheathis a remarkable spiral wrap (seeFigure 23-14b). A long axon can have as many as severalhundred Schwann cells along its length, each contributing insulation to an internode stretch of about 1-1.5 pm of axon. Not all axons are myelinated,for reasonsthat are not known. Mutations in mice that eliminate Schwanncells causethe death of most neurons. In contrast to oligodendrocytes,Schwann cells each dedicate themselvesto one axon. The sheathsare composed of about 70 percent lipid (rich in cholesterol) and 30 percent protein. In the peripheral nervous system the principle protein constituent(-80 percent)of myelin is called protein 0 (Pe),an integral membraneprotein that has immunoglobulin (Ig) domains.MBP is also an abundantcomponent.The extracellular Ig domains bind together the surfaces of

(b) Peripheralnervoussystem neuron

(a) Centralnervoussystem neurons Central nervous system neu rons

Capillary

A$trocyte

Astrocyte

Schwann

Oligodendrocyte

RNA Ribosome

2003,Curr.Biol 13:469,andadaptedfrom D L S h e r m a na n d P B r o p h y , 23-14Threetypesof glia cells.(a)Astrocytes interact A FIGURE withneurons butdo notinsulate them (b)EachSchwann cellinsulates 2005,NatureRev.Neurosci6:683-690,Photos:C o u r t e s yo f V a r s h aS h u k l a f romNIHl andDougField of a singleperipheral nervous system axon (c)A single a section canmyelinate multiple B Stevens, oligodendrocyte CNSaxonslFrom

sequentialwraps around the axon to compact the spiral of myelin sheath (Figure 23-15b). Other proteins play this kind of role in the CNS. In humans, peripheral myelin, like CNS myelin, is a target of autoimmune disease,antibodies forming against P6. The Guillain-Barre syndrome (GBS), also known as acute inflammatory demyelinating polyneuropathy, is one such disease.GBS is the most common causeof rapid-onset paralysis, occurring at a frequency of 10-t. The causeis unknown. The common inherited neurological disorder called Charcot-Marie-Tooth disease,which damagesperipheralmotor and sensorynerve function, is due to overexpressionof the gene that encodesPMP22 protein, another constituentof peripheralnerve myelin. I

Interactions betweenglia and neurons control the placement and spacing of myelin sheaths,and the assemblyof nervetransmission machinery at the nodes of Ranvier. VoltagegatedNa* channelsand Nan/K* pumps' for example,congregate at the nodes of Ranvier through interactions with '$fhile all the details of the node ascytoskeletal proteins. sembly processare not understood, a number of key players have beenidentified. In the PNS, where the processhas been most studied, surface adhesion molecules in the Schwann cell membrane interact with neuronal adhesion molecules. The glial membrane immunoglobulin cell-adhesionrnolecule (lgCAM) called newrofascinl55 contacts two axonal proteins, contactin and contactin-associatedprotein at the edge of the node. Thesecell-cell contact eventscreate boundaries at eachside of the node.

O F A C T I O N P O T E N T I A L ISN N E R V EC E L L S V O L T A G E - G A T E IDO N C H A N N E L SA N D T H E P R O P A G A T I O N

1015

PMP22

(b)

MBP

Plasmamembrane

-l

CVtosolic .Japposrilon

Cytosol

D

Cytosol-

D D

P ee x o p l a s m i c domain P 9t r a n s m e m b r a n e domain P6cytosolic domain

A FIGURE 23-15 Formationand structureof a myelinsheathin the peripheralnervoussystem.(a) At highmagnification the specialized spiralmyelin(My)membrane appears asa series of layers, or lamellae, of phospholipid bilayers wrapped aroundtheaxon(Ax); (b)Closeup viewof threelayers Mit : mitochondrion of the myelin membrane spiralThetwo mostabundant peripheral membrane myelinproteins, PoandPMP22, areproduced onlyby Schwann cells. Theexoplasmic domainof a Poprotein, whichhasan immunoglobulin fold,associates withsimilar domains emanating frompeproteins in theopposite membrane surface, thereby"zippering" together the exoplasmic membrane surfaces in closeapposition Theseinteractions

arestabilized by bindingof a tryptophan residue on the tip of the e x o p l a s mdi o cmain t o l i p i d si n t h eo p p o s i tm e e m b r a nC e lose apposition of the cytosolic facesof the membrane mayresultfrom bindingof the cytosolic tailof eachP0proteinto phospholipids in the opposite membranePMP22mayalsocontribute to membrane protein,remains compactionMyelinbasicprotein(MBP), a cytosolic betweenthe closely apposed membranes asthe cytosolissqueezed (a)@Science out [Part part(b)adapted VU/CRainel/isuals Unlimited; from

The channel proteins and other moleculesthat will accumulate at the node are initially dispersedthrough the axons. Then axonal proteins, including two IgCAMs called NrCAM and neurofascinlSS,as well as ankyrin G (Chapter 17), accumulatewithin the node. The two IgCAMs bind to a single transmembrane domain protein called gliomedin that is expressedin the glial cell. Experimenrsrhat eliminated gliomedin production showed that without it nodes do not form, so it is a key regulator.Ankyrin in the node contactsBIV spectrin,a major constituentof the cytoskeleton, thus tethering the node's protein complex to the cytoskeleton.Na* channelsbecomeassociatedwith neurofascin186, NTCAM, and ankyrin G, firmly trapping the channel where it is needed.As a result of these multiple protein-protein interactions, the concentration of Na* channels is roughly a hundredfold higher in the nodal membrane of myelinated axons than in the axonal membrane of nonmyelinatedneurons.

Astrocytes are critical regulators of the formation of the blood-brain barrier. A mass of blood vesselsin the brain suppliesoxygen and removesC02, and deliversglucose and amino acids, with capillaries within a few micrometers of every cell. These capillaries form the bloodbrain barrier, which prevents, for example, blood-borne circulating neurotransmittersand some drugs from entering the brain. The barrier consistsof a set of tight junctions (Chapter 19) made by the endothelialcells that form the walls of capillaries.Astrocytespromote specialization of theseendothelialcells, making them less permeable ( F i g u r e2 3 - 1 , 6 ) . Many synapsesand dendritesare also surrounded by astrocyte processes.Astrocytesproduce abundant extracellular matrix proteins, some of which are used as guidancecuesby migrating neurons,and a host of growth factors that carry a variety of types of information to neurons. The Ca2*, K*. Na-, and Cl channels,among others, found in the plasma membranesof astrocytesinfluence the concentration of free ions in the extracellular space,thus affecting the membrane potentialsof neurons and of the astrocytesthemselves.Astrocytes also take up glutamate,a neurotransmitter,from extra'When cellular spacesand turn it into glutamine. nearby neurons have fired, glutamate binds to glutamate receprors on astrocytes, enabling the astrocytes to sensethe event. Astrocytes are joined to each other by gap junctions, so changesin ionic composition in any of them are communicated to others, over distancesof hundreds of mrcrons.

Astrocytes The third type of glial cell is the astrocyte, named for its starlike shape (seeFigure 23-14a). These can constitutemore than a third of brain mass and half of the brain's cells. There are two kinds. Proloplasmic astrocytes are in the gray matter (the areasrich in cell bodies);fibrous astrocytes are in the white matter (the areas composed mainly of axonsl it is the myelin that makes it look white). The astrocytesmake long thin processesthat envelop all the brain'sblood vessels. 1016

C H A P T E R2 3

I

N E R V EC E L L S

L S h a p i r oe t a l , 1 9 9 6 , N e u r o n1 7 i 4 3 5 ,a n d E J A r r o y oa n d S S S c h e r e r , 2000, Histochem Cell Biol 113:1 l

< FIGURE 23-16 Astrocytesinteractwith endothelialcellsat the barrieristo of the blood-brain blood-brainbarrier.Thepurpose into cantravelout of the bloodstream controlwhattypesof molecules of thebanierbythe thebrainandviceversaTheformation thebrainis wallsentering cellsthatmakeup bloodvessel endothelial inthe brainareformed astrocytes Capillaries directed bysurrounding to most thatareimpermeable cellswithtightjunctions byendothelial soonlysmallmolecules cellsisblocked, Transport between molecules throughcellscan transported specifically or substances thatcandiffuse andpermeability Theendothelial cellshavetransporters cross the barrier. prevent but selectively thatallowoxygenandC02across characteristics the bloodvessels, surround fromcrossingAstrocytes othersubstances protein signals andsendsecreted cells, withtheendothelial in contact barrier. Unless cellsto producea selective to inducethe endothelial standthebest molecules involved, lipid-soluble carrier thereisa specific thoughtheymaytravelpoorlyin the blood. chanceof gettingacross, by specific channel likeNa+andCl-, aremovedacross Electrolytes, proteins. cellshavelessvesicle endothelial Thebrain's andtransport presumably is sincetransport cells, transport thanmostendothelial bya areensheathed cells(burgundy) Theendothelial moreselective (orange) by on theoutside andcontacted layerof basallamina (tan).Pericytes cellsthat provide processes aremesenchymal astrocyte L R6nnbdck, andE Hansson, N J Abbott, to thecapillaries. support [From 7:41-53]l Neurosci Rev. 2006,Nature

r As the action potential reachesits peak, opening of voltagegated K+ channelspermits efflux of K* ions, which repolarizes and then hyperpolarizes the membrane. As these channelsclose, the membrane returns to its resting potential (seeFigure23-3). r The excesscytosolic cations associatedwith an action potential generatedat one point on an axon spread passivelyto the adjacentsegment,triggeringopeningof voltagegated Na+ channelsand movement of the action potential along the axon.

Voltage-Gated lon Channelsand the Propagation of Action Potentials in Nerve Cells r Action potentials are sudden membrane depolarizations followed by a rapid repolarization. They originate at the axon initial segmentand move down the axon toward the axon terminals, where the electric impulse is transmitted to other cellsvia a synapse(seeFigures23-3 and23-6). r An action potential resultsfrom the sequentialopening and closingof voltage-gatedNa* and K* channelsin the plasma membraneof neuronsand musclecells(seeFigure23-9). r Opening of voltage-gatedNat channels permits influx of Na* ions for about 1 ms, causing a sudden large depolarization of a segmentof the membrane.The channels then closeand becomeunableto open (refractory)for several milliseconds,preventingfurther Na* flow (seeFigure23-7).

r Becauseof the absolute refractory period of the voltagegated Na* channelsand the brief hyperpolarization resulting from K* efflux, the action potential is propagated in one direction only, toward the axon terminus' ' r Voltage-gatedNa and Ca2" channelsare monomeric proteins containing four domains that are structurally and functionally similar to each of the subunits in the t tetramericvoltage-gatedK channels' r Each domain or subunit in voltage-gatedcation channels contains six transmembranea helicesand a nonhelical P segment that forms the ion-selectivirypore (seeFigure23-10). r Opening of voltage-gatedchannelsresults from movement of the positively charged 54 ct helicestoward the extracellular side of the membrane in responseto a depolarization of sufficient magnitude. r Closing and inactivation of voltage-gatedcation channels result from movement of a cytosolic segmentinto the open pore (seeFigure23-11c). r Myelination, which increasesthe rate of impulse conduction up to a hundredfold, permits the close packing of neurons characteristicof vertebrate brains.

O F A C T I O NP O T E N T I A L ISN N E R V EC E L L S V O L T A G E - G A T E IDO N C H A N N E L S AND THE PROPAGATION

1017

r In myelinated neurons, voltage-gatedNa* channels are concentrated at the nodes of Ranvier. Depolarization at one node spreadsrapidly with little attenuation to the next node, so that the action potential jumps from node to node (seeFigure 23-13). r Myelin sheathsare produced by glial cells that wrap themselves in spirals around neurons. Oligodendrocytes produce myelin for the CNS; Schwann cells, for the PNS (seeFigure 23-14). r Astrocytes,a third type of glial cell, wrap their processes around synapsesand blood vessels.Astrocytes secreteproteins that stimulate synapseformation, and also induce the endothelial cells of blood vesselsto oroduce a blood-brain barrier that limits transepithelial flow of subsranceslsee Figure 23-16).

had been stored and upon the frequency of action potentials arriving at the synapse.The duration of signalalso dep e n d s o n h o w r a p i d l y a n y r e m a i n i n gn e u r o t r a n s m i t t e ri s retrieved by the presynaptic cell. Presynaptic cell plasma membranes,as well as glia, contain transporter proteins that pump neurotransmittersacrossthe plasma membrane back into the cell, thus keepingthe extracellularconcentrations of transmitter low. Here we focus first on how synapsesform and how they control the regulated secretion of neurotransmitters in the context of the basic principles of vesicular trafficking outlined in Chapter 14. Next we look at the mechanismsthat limit the duration of the synaptic signal, and how neurotransmirters are receivedand interpretedby the postsynapticcell.

Formationof SynapsesRequiresAssemblyof Presynapticand PostsynapticStructures

Communication at Synapses As we have discussed,electricalpulsestransmit signalsalong neurons, but signals are transmitted between neurons and other excitable cells by chemical signals. Synapsesare the junctions where presynaptic neurons releasethese chemical signals,or neurotransmitters,that act on a postsynaptic target cell (Figure 23-4), which can be another neuron or a muscle or gland cell. Neurotransmitters are small, water-soluble molecules(e.g.,acetylcholine,dopamine).The cell-cell communication at chemical synapsesgoes in one direction: pre- to postsynapticcell. Arrival of an action potential at an axon terminal leads to opening of voltage-sensitiveCa2* channels and an influx of Ca2*, causing a localized rise in the cytosolic Ca2* concentration in the axon terminus. The rise in Caz* in turn rriggers fusion of small (40-50-nm) synaptic vesicles containing neurotransmitters with the plasma membrane, releasingneurotransmittersfrom this presynapticcell into the synaptic cleft, the narrow spaceseparating it from posrsynapticcells. The membrane of the postsynaptic cell is located within approximarely 50 nm of the presynapticmembrane. Neurotransmitter receptors fall into two broad classes: ligand-gated ion channels, which open immediately upon neurotransmitter binding, and G protein-coupled receptors. Neurotransmitter binding to a G protein-coupled receptor inducesthe opening or closing of a separateion-channelprotein over a period of secondsto minutes. These "slow" neurotransmitter receptors are discussedin Chapter 15 along with G protein-coupled receptors that bind different types of ligands and modulate the activity of cytosolic proteins other than ion channels.Here we examine the structure and operation of the nicotinic acetylcholine receptor found at many nerve-musclesynapses.It was the first ligand-gated ion channel to be purified, cloned, and characteized at the molecular level, and provides a paradigm for other neurotransmitter-gatedion channels. The duration of the neurotransmittersignal dependson the amount of transmitter releasedby the presynaptic cell, which in turn depends on the amount of transmitter that 1018

C H A P T E R2 3

I

N E R V EC E L L S

Axons extend from the cell body during development,guided by signalsfrom other cellsalong the way so that the axon rermini will reach the correct location (seeSection23.5). As axons grow, they come into contact with the dendritesof other neurons, and often at such sites synapsesform. In the CNS, synapseswith presynapticspecializationsoccur frequently all along an axon, in contrast to motor neurons, which form synapseswith muscle cellsonly at the axon termini. Neurons cultured in isolation will not lorm synapses very efficiently, but when glia are added, the rate of synapse formation increasessubstantially. Astrocytes and Schwann cells send signals to neurons to stimulate the formation of synapsesand then help to preservethem (Figure 23-17).To

tlcm I EXPERIMENTAL FIGURE 23-17 Signalsfrom astrocyteshave beenshown to inducesynapseformation.lmmunostaining for the presynaptic (red)andthe postsynaptic proteinsynaptotagmin protein (yellow) yieldsfew measured puncta(dotsof stain)in control PSD-95 retinalganglioncellsculturedin the absence (/eft). of astrocytes However, a 3-5-foldincrease in punctaoccurswhenthesecellsare culturedin the presence proteinproduct of astrocytes or theastrocyte : the recombinant (right)(rTSP2 thrombospondin thrombospondin 2 thatwasused).Astrocytes secrete thrombospondin, whichby itselfhas muchthe sameeffecton synapse formationasastrocytes themselves. Scale baris30 pm. [Reproduced withpermission fromK S Christopherson et al . 2005. Cell 12O:421-433 |

discover the signals involved, culture medium in which glia had been incubated was added to neuron cultures, and synapse formation was stimulated. By purifying different substancesfrom that medium it was possibleto identify the signal. Thrombospondin (TSP)protein, a component of extracellular matrix, was found to be the active agent. Confirmation came from mice lacking two tbrombospondin geflesi the mice had only 70 percent of the normal number of synapsesin their brains. TSP probably does not work alone, as it is not as potent in inducing synapsesas are whole glia. Another molecule that appears to account for some of the synapse-inducingactivity of glia is cholesterol, and direct contact betweenglia and neurons may contribute as well. Mutual communication between neurons and the glia that surround them is frequent and complex. The signalsand information they carry is an area of active research.There is even evidencethat neurons form synapseson glia. \fhile glia do not have action potentials, they do have complex arrays of channelsand ion fluxes. At the site of a synapse,the presynapticcell has hundreds to thousands of synaptic vesicles,some docked at the membrane and others waiting in reserve. The releaseinto the synaptic cleft occurs in the actiue zone, a specializedregion of the plasma membrane containing a remarkable assemblage of proteins whose functions include modifying the propertiesof the synapticvesiclesand bringing them into position for docking and fusing with the plasma membrane. Mewed by electron microscopg the active zone has electrondensematerial and fine cytoskeletalfilaments (Figure 23-18). The active zone is assembledgradually, with synaptic vesicles accumulating first, then cytoskeletalelements,and then other proteins. A similarly denseregion of specializedstructures is seenacrossthe synapsein the postsynapticcell, the postsynaptic density (PSD). Cell-adhesion molecules that connect pre- and postsynapticcells keep the active zone and PSD aligned. After releaseof synaptic vesiclesin responsero an action potential,the presynapticneuron retrievessynaptic vesicle membrane proteins by endocytosis both within and outside the active zone. The induction of PSD assemblyhas been extensively studied at the neuromuscular iunction (NMJ). At these synapsesacetylcholineis the neurotransmitter produced by motor neurons, and its receptor,AChR, is produced by the postsynapticcell, which is a muscle cell. Muscle cell precursors,myoblasts,put into culture will spontaneouslyfuse into multinucleate myotubes that look similar to normal muscle cells (Chapter 21.).As myotubes form, AChR is produced and inserted into the plasma membrane of the myotubes, reaching a density of about 1000 receptors/pm2.The AChR is dispersedthrough the membrane, but if neurons are added to the culture, the AChR starts to concentrate at points of contact with the neurons. The neurons cause movement of preexisting AChR and also induce the myotubes to produce additional AChR. The density of receptors in a mature synapsereachesabout 10,000-20,000/p"m2,while elsewhere in the plasma membrane the density is cytosol) powers neurotransmitter import by ligand-specificH*-linked antiporters in the vesiclemembrane. For example, acetylcholine is synthesized from acetyl coenzymeA (acetyl CoA), an intermediate in the degradation of glucose andfatty acids, and choline in a reaction catalyzed by choline acetyltransferase:

Glycine

oC:O O C n r C H r [ O H3N+-CH Glutamate

o tl

cH2-cH2-

NH3r

CH"

CH3-C-S-CoA

o

Choline

?r.

CH3-C-O-CH2-CH2-N*-CHs Acetylcholine

Norepinephrine (derivedfrom tyrosine)

cH-cH2-NH2+-CH3 OH

C h o iln e acetyltransferase

+ HO-CH2-CH2-N+-CH3

Acetyl CoA Dopamine (derivedfrom tyrosine)

t-

CHs

+ CoA-SH

CH:

Synapticvesiclestake up and concentrateacetylcholinefrom the cytosol against a steepconcentration gradient, using an Ht/acetylcholine antiporter in the vesiclemembrane. Curiously, the geneencoding this antiporter is contained entirely within the first intron of the gene encoding choline acetyltransferase,a mechanism conserved throughout evolution for ensuring coordinate expression of these two proteins. Different H+/neurotransmitter antiport proteins are usedfor import of other neurotransmittersinto synaptic vesicles.

Epinephrine (derivedfrom tyrosine)

Ho;Z+cH2-cH2-NH3*

\r1-^,/ H Serotonin, o r S-hydroxytryptamine (derivedfrom tryptophan) HC:C-CHz-CH2-NH3+ N\,/NH

'cH

Histamine (derivedfrom histidine)

o tl

H3N+-CH2-CH2-CH2-C-Oy-Aminobutyric acid, or GABA (derivedfrom glutamate)

1020

C H A P T E R2 3

I

N E R V EC E L L S

SynapticVesiclesLoadedwith Neurotransmitter Are Localizednear the PlasmaMembrane Neurotransmitters are synthesizedby enzymesin the cytosol and then transportedinto synapticvesiclesby transporterproteinsdedicatedto the task. For exampleglutamateis imported into synaptic vesicles by proteins called uesicular glutamate transporters (VGLUTs). VGLUTs are highly specific for glutamate but have rather low substrateaffinity (K- : 1-3mM). The transporters are antiporters, moving glutamate into synaptic vesicleswhile protons move in the other direction. The membrane-potentialgradient that drives the transport processis establishedby a vacuolar-typeMPase (Chapter11). The exocytosisof neurotransmitters from synaptic vesicles involves targeting and fusion events similar to those that lead to releaseof secretedproteins in the secretorypathway (Figure 23-20). However, severalunique featurespermit the very rapid releaseof neurotransmitters in responseto arrival of an action potential at the presynaptic axon terminal. For example, in resting neurons some neurotransmitter-filled synaptic vesicles are "docked" at the plasma membranel others are in reservein the active zone near the plasma membrane at the synaptic cleft.

H+-linked a ntiporter

@ I

Synaptotagmin

Neurotransmitter/ transporter Voltage-gated

U C a 2 +c h a n n e l

I

lmport of neurotransmitter

N a+-neurotransmitter symport protern

VAMP

V-classH* pump

ADP+ P; Movementof vesicle to active zone a

a

.

Cytosol of presynaptic cell

l.

+

-ql

[

S

coated vesicle o

Na*

Plasma membrane Dynamin

g SNARE I comolex

clathrin

\,, Clathrin-

t.'

Synaptic cleft Botulinum -1 toxin

,lt .

Exocytosisof neurotransmitter triggeredby influx ol Ca2+

T

Shibire mutation

'-'E a

Reuptakeof neurotransmitter

23-20 Cyclingof neurotransmitters A FIGURE and of synaptic vesiclesin axon terminals.Mostsynaptic vesicles areformedby endocytic recycling asdepicted hereTheentirecycletypically takes Step[:The uncoated about60 seconds. vesicles employa varietyof (blue)andothertransport (green) proteins antiporters to import (reddots)fromthe cytosol. neurotransmitters StepZ: Synaptic vesicles loadedwith neurotransmitter moveto the activezone StepB: Vesicles dockat definedsiteson the plasmamembrane of prevents a presynaptic cell.Synaptotagmin membrane fusionand release of neurotransmitter Botulinum toxinprevents exocytosis by proteolytically VAMP, cleaving thev-SNARE on vesicles. Synaptotagmin in steps4-E or [, thoughit isstillpresent. doesnot participate For it isnot shown.StepZl: In response simplicity, to a nerveimpulse (action potential), voltage-gated in the plasma Ca2*channels membrane open,allowing an influxof Ca2*fromtheextracellular

in change conformational Ca2*-induced Theresulting medium. with the plasma leadsto fusionof dockedvesicles synaptotagmin cleft intothe synaptic of neurotransmitters andrelease membrane v-SNARE and vesicles containing StepE: Afterclathrin/AP proteins bud inwardandarepinchedoff transporter neurotransmitter process, theylosetheircoatproteinsDynamin in a dynamin-mediated of blockthe re-formation suchasshlbrrein Drosophila mutations At the sametime,Na- symporter leadingto paralysis vesicles, synaptic cleft,whichlimits proteins fromthe synaptic takeup neurotransmitter recharges the cellwith andpartially the durationof the actionpotential creating by endocytosis, are recovered Step6: Vesicles transmitter. vesicles, readyto be refilledandbeginthe cycleanew Unlike uncoated Seethetextfor isnot recycled. acetylcholine mostneurotransmitters, VMurthyandC 133:1237; etal,1996,JCellBiol. details[SeeKTakei andR Jahnetal, 2003,Cell112:519 392:497; I 1998,Nature Stevens,

In addition, the membrane of synaptic vesiclescontains a specialized Caz*-binding protein that sensesthe rise in cytosolic Ca2* after arrival of an action potential, triggering rapid fusion of docked vesicleswith the presynaptic membrane. A highly organized arrangementof cytoskeletalfibers in the axon terminal helps localize synaptic vesiclesin the active zone. The vesiclesthemselvesare linked together by synapsin, a fibrous phosphoprotein associatedwith the cytosolic surface of all synaptic-vesiclemembranes.Filaments of synapsin also radiate from the plasma membrane and bind to vesicleassociatedsynapsin.Theseinteractionsprobably keep synap-

tic vesiclescloseto the part of the plasmamembranefacingthe synapse.Indeed,synapsinknockout mice, although viable,are prone to seizures;during repetitive stimulation of many neurons in such mice, the number of synaptic vesiclesthat fuse with the plasmamembraneis greatly reduced.Thus synapsins are thought to recruit synaptic vesiclesto the active zone' Rab3A, a GTP-binding protein located in the membrane of synaptic vesicles,is also required for targeting of neurotransmitter-filled vesiclesto the active zone of presynaptic cells facing the synaptic cleft. Rab3A knockout mice, like synapsin-deficientmice, exhibit a reduced number of synaptic AT SYNAPSES COMMUNICATION

1021

vesiclesable to fuse with the plasma membrane after repetitive stimulation. The neuron-specificRab3 is similar in sequence and function to other Rab proteins that participate in docking vesicleson particular target membranes in the secretorypathway.

chinery of endocytosis and exocytosis is highly conserved, and is explained extensivelyin Chapter 14.

Influx of Ca2+TriggersRelease of Neurotransmitters

Fusion of synaptic vesicleswith the plasma membrane of axon terminals dependson SNAREs, the same type of proteins that mediatemembrane fusion of other regulatedsecretory vesicles (Figure 23-20). The principal v-SNARE in synaptic vesicles(VAMP) tightly binds syntaxin and SNAP25,the principal I-SNAREs in the plasma membraneof axon terminals, to form four-helix SNARE complexes. After fusion, SNAP proteins and NSF within the axon terminal promote disassociationof VAMP from I-SNAREs, as in the fusion of secretoryvesiclesdepictedpreviously (Figure 14-10).

The exocytosis of neurotransmitters from synaptic vesicles involves vesicle-targetingand fusion events similar to those that occur during the intracellular transport of secretedand plasma-membrane proteins (Chapter 13). Two featurescritical to synapse function differ from other secretory pathways: (1) secretionis tightly coupled to arrival of an action potential at the axon terminus, and (2) synaptic vesiclesare recycled locally to the axon terminus after fusion with the plasma membrane. Figure 23-20 shows the entire cycle whereby synaptic vesiclesare filled with neurotransmitter, releasetheir contents, and are recycled. Depolarization of the plasma membrane cannot, by irself, cause synaptic vesiclesto fuse with the plasma membrane. In order to trigger vesiclefusion, an action potential must be converted into a chemical signal-namely, a localized rise in the cytosolic Ca2+ concentration. The transducers of the electric signals areuoltage-gatedCa2* channelslocalizedto the region of the plasma membrane adjacentto the synaptic vesicles.The membrane depolarization due to arrival of an action potential opens thesechannels,permitting an influx of Ca2* ions from the extracellular medium into the axon terminal. A simple experiment demonstrates the importance of voltage-gatedCa2* channelsin releaseof neurotransmrtters. A preparation of neurons in a Ca2*-containing medium is treated with tetrodotoxin, a drug that blocks voltage-gated Na- channelsand thus preventsconduction of action potentials. As expected,no neurotransmirtersare secretedinto the culture medium. If the axonal membrane then is artificially depolarized by making the medium =100 mM KCI in the presence of extracellular Ca2* , neurotransmitters are releasedfrom the cells becauseof the influx of Ca2* through open voltage-gatedCaz* channels.Indeed, patch-clampiig experiments show that voltage-gatedCa2*channels,like voltage-gatedNa* channels,open transiently upon depolarization of the membrane. Two pools of neurotransmitter-filledsynaptic vesiclesare presentin axon terminals: those docked at the plasma membrane, which can be readily exocytosed,and those ln reserve in the active zone near the plasma membrane. Each rise in Ca2* triggers exocytosisof about 10 percent of the docked vesicles.Membrane proteins unique to synaptic vesiclesthen are specifically internalized by endocytosis,usually via the same types of clathrin-coated vesiclesused to recover other plasma-membraneproteins by other types of cells. After the endocytosedvesicleslose their clathrin coat, they are rapidly refilled with neurotransmitter.The ability of many neurons to fire 50 times a secondis clear evidencethat the recycling of vesiclemembrane proteins occurs quite rapidly. The ma-

1022

C H A P T E R2 3

I

N E R V EC E L L S

A C a l c i u m - B i n d i nPgr o t e i nR e g u l a t e sF u s i o no f SynapticVesicleswith the PlasmaMembrane

Strong evidencefor the role of VAMP in neurotransmitter exocytosisis provided by the mechanism of action of botulinum toxin, a bacterial protein that can cause the paralysis and death characteristicof botulism. a type of food poisoning. The toxin is composedof two polypeptides: One binds to motor neurons that releaseacetylcholine at synapseswith muscle cells, facilitating entry of the other polypeptide, a protease,into the cytosol of the axon terminal. The only protein this proteasecleavesis VAMP (seeFigure 23-201.After the botulinum proteaseentersan axon terminal, synaptic vesiclesthat are not already docked rapidly lose their ability to fuse with the plasma membrane because cleavageof VAMP preventsassemblyof SNARE complexes. The resulting block in acetylcholinereleaseat neuromuscular synapsescausesparalysis. However, vesiclesthat are already docked exhibit remarkable resistanceto the toxin, indicating that SNARE complexes may aheady be in a partially assembled,protease-resistant state when vesicles are docked on the presynapticmembrane. I The signal that triggers exocytosis of docked synaptic vesicles is a rise in the Ca"* concentration in the cytosol near vesiclesfrom EXPERIMENTAL FIGURE 23-23Incoming signalsmust reachthe thresholdpotential to triggeran actionpotentialin a postsynaptic cell.In thisexample, the presynaptic neuronisgenerating aboutone actionpotential every4 milliseconds Arrivalof eachactionpotential at thesynapse causes a smallchangein the membrane potential at the axonhillockof the postsynaptic cell,in this example a depolarization of =5 mV When m u l t i p lset i m u cl ia u s teh em e m b r a noef t h i s postsynaptic cellto become depolarized to the -40 potential, threshold hereapproximately mV an actionpotential isinduced in it

D i r e c t i o no f a c t i o np o t e n t i a l

Dendrite

Cell body

- -S

Axon hillock

\

Postsynaptic \ cell

Electrodeto measure electricpotential

Membranepotentialin the postsynapticcell

-40 mV

Threshold potential

-60 mV

an electricalsynapseis almost instantaneous,on the order of a fraction of a millisecond. The cytoplasm is conrinuous between the cells. In addition, the presynaptic cell (the one sending the signal) does not have to reach a threshold at which it can causean action potential in the postsynapticcell. Instead,any electricalcurrent continuesinto the next cell and causesdepolarizationin proportion to the current. An electricalsynapsemay contain thousandsof gap channels, each composed of two hemichannels,one in each apposed cell. Gap junction channelshave a structure similar to conventional gap junctions (Chapter 19). Each hemichannel is an assemblyof six copies of rhe connexin protein. Since there are about 20 mammalian connexin genes,diversity in channel structure and function can arise from the different protein components. The 1.6-2.}-nm channel itself allows the diffusion of moleculesup to about 1000 Da in sizeand has no trouble at all accommodatingions.

Communication at Synapses r Synapsesare the junctions between a presynapticcell and a postsynapticcell, and consist of small gaps. r Neurotransmitters are released by the presynaptic cell using exocytosis.They diffuse acrossrhe synapseand bind to receptors on the postsynaptic cell, which can be a neuron or a muscle. r Chemical synapsesof this sort are unidirectional (see Figure23-4). r Neurotransmitters (seeFigure 23-19) are stored in hundreds to thousandsof synaptic vesiclesin the axon termini 'S7hen of the presynaptic cell (seeFigure 23-1,8). an action potential arrives there, voltage-sensitiveCa2* channels open and the calcium causessynaptic vesiclesto fuse with

1026

C H A P T E R2 3

I

N E R V EC E L L S

the plasma membrane, releasingneurotransmittermolecules into the synapse(seeFigure 23-20). r Communication between presynaptic and posrsynaptic cellsis abundant as a synapseis being formed. Cell-adhesion moleculeskeepthe cellsaligned.Neurons inducethe accumulation of acetylcholine receptor, for example, in the postsynaptic muscleplasmamembranein the vicinity of a synapse. r Synapticvesiclesfuse to the plasma membrane using cellular machinery that is standard issue for exocytosis, including SNAREs, syntaxin, and SNAP proteins. Synaptotagmin protein is the calcium sensorthat detectsthe action potential-stimulated rise in calcium that leads to synaptic vesiclemembrane fusion (seeFigure 23-20). r Dynamin, an endocytosisprotein, is critical for the formation of new synaptic vesicles,probably to "pinch off" inbound vesicles. r Neurotransmitter receptorsfall into two classes:ligandgated ion channels,which permit ion passagewhen open, and G protein-coupled receptors, which are linked to a separateion channel. r At synapsesimpulses are transmitted by neurotransmitters releasedfrom the axon terminal of the presynapticcell and subsequentlybound to specific receptors on the postsynaptic cell (seeFigure 23-4). r Low-molecular-weightneurotransmitters(e.g., acetylcholine, dopamine, epinephrine) are imported from the cytosol into synaptic vesiclesby H*-linked antiporters. V-class proton pumps maintain the low intravesicular p H t h a t d r i v e s n e u r o r r a n s m i t t e ri m p o r t a g a i n s t a c o n centration gradient. r Arrival of an action potential at a presynaptic axon terminal opensvoltage-gatedCa2* channels,leadingto a localized rise in the cytosolic Ca'* level that triggers exocytosisof

synapticvesicles.Following neurotransmitterrelease,vesicles are formed by endocytosisand recycled (seeFigure 23-20). r Coordinated operation of four gated ion channelsat the synapseof a motor neuron and striatedmusclecell leadsto releaseof acetylcholine from the axon terminal, depolarization of the muscle membrane, generation of an action potential, and then contraction (seeFigure 23-21). r The nicotinic acetylcholine receptor, a ligand-gated cation channel, contains five subunits, each of which has a transmembrane ct helix (M2) that lines the channel (see F i g u r e2 3 - 2 2 ) . r A postsynapticneuron generatesan action potential only when the plasma membrane at the axon hillock is depolarized to the threshold potential by the summation of small depolarizations and hyperpolarizations caused by activation of multiple neuronal receptors(seeFigure 23-23). r Electrical synapsesare direct, gap junction connections between neurons. Electrical synapses,unlike chemical synapsesthat employ neufotfansmitter systems,are extremely fast in signal transmissionand are bidirectional.

Cells:Seeing,Feeling, Sensational and Smelling Hearing,Tasting, Dramatic progress has been made in understandinghow our sensesrecord impressionsof the outside world, and how that information is processedby the brain. In this section we discusscellular and molecular mechanismsand specializednerve cells underlying vision' touch, hearing, taste, and olfaction.

The Eye FeaturesLight-SensitiveNerve Cells Vhile owls would favor hearing, and dogs smell, most humans would choose vision as the sensethat provides the most effective window on the world. Light is fast, about 300,000 km/s, and moves in straight lines, so it is excellent for information transfer. The human eye is a complex structure that gathers light from the environment and focuses it on specializedlight-sensitive nerve cells, which send signalsto the brain, where they are translatedinto an i m a g e( F i g u r e2 3 - 2 4 a ) .

(a)

C i l i a r ym u s c l e Zonular Anterior chamber----rCornea .....-

eupit/,

Vitreous numor

Optic disk

Ir i s

(b)

Interneurons

Horizontal Amacrine cell c el l Axons of g a n g l i o n c e l l s G a n g l i o n In n e r Bipolar Outer plexiform plexiform cell to optic nerve cell layer layer

Light

Photoreceptors Cone

Rod

23-24 Structureof the < FIGURE human eye and three classesof neuronsin the retina.(a)Themain lightpasses of the eye Incoming tissues bythe isfocused throughthe cornea, cells light-sensing lens,andactivates the Theirisrestricts in the retina. located the lensThe amountof lightentering fibers, bythezonular lensissupported andmovedbytheciliarymuscleTheeye cushioning, isfilledwith transparent, fluid Thefoveaisthe location vitreous of cells,and density of the highest the highestsenses consequently imageThereisa blindspot resolution (opticdisc)wherethe opticnerveleaves of cells organization theeye (b)Detailed light in the retinaNotethat incoming hasto passthroughmultiplelayers the of neuronsbeforereaching photoreceptor cells,the rodsandcones cells; includehorizontal Theinterneurons bipolarcells,of whichthereareabouta cells,of dozentypes;andamacrine whichtherearemorethan20 tYPes. ganglion cellscarrythesignal Retinal The to theopticnerve. information arewheremost layers two plexiform (a)adapted aremade.lPart connections andK French, W Burggren, fromD Randall, 5thed, W H 2002,EckertAnimalPhysrology, p 259;part(b)from Freeman andCompany, 2006,An B KolbandI Q Whishaw, lntroductionto Brainand Behaviot2d ed , Worth,p 278l

S E N S A T I O N ACLE L L SS: E E I N GF, E E L I N GH, E A R I N GT, A S T I N GA, N D S M E L L I N G

1027

The human retina (Figure 23-24b) is about 200 pm thick. As we learnedin Chapter 15, it containstwo types of photoreceptors,rods and cones, which are the primary recipientsof visual stimulation.Conesare involved in color vision, while rods are stimulatedby weak light, like moonlight, over a rangeof wavelengths.In contrastto many other sensoryneurons,stimulatedphotoreceptorcellsbecomebyperpolarized, not depolarized. The photoreceptors synapse on layer upon layer of interneuronsthat are innervatedby different combinations of photoreceptor cells. All these signals are processedand interpretedby the part of the brain called the uisual cortex. In each eye we have about 6 million conesand 120 million rods, connectingto 540 million visual cortex cells, so a substantialpart of our nervous systemis devotedto detectingand interpretinglight. Rods detect faint light, as low as a single photon, owing to their sensitivevisual pigment and their ability to amplify a

Pigment epithelium

Free-floating d isks

"" .?%",

Rod outer segment Inner segment

weak signal.Rods have a singlepigment that allows an equal responseto a broad spectrum of wavelengths.Cones (Figure 23-25) come in the three types: red, green, and blue. The brain deducescolor information by comparing the signals from a trio of cone cells,one of eachkind, that sharethe same receptiuefield.The receptivefield of each cell is measuredas an angle with its vertex at the cell. If cells are denselypacked and eachhas a small receptivefield, highly detailedvisual information is collected. If cells are less dense,have large receptive fields, or both, the image will have lower resolution. Rods and conesare packedwith light-absorbingpigments consistingof an opsin protein covalently bound to a small light-sensitivemolecule called 11-cis-retinal.These pigments are arranged in flattened membrane disks in the outer segmentsof rods and cones(Figure23-25; also seeFigure 15-16). Opsin-containingdisks are continuouslyreplaced,completely turning over about every 12 days. Opsins are G protein- FfGURE23-27 fhe influenceof light and dark on on-centerconecellsin image (a)In the dark,a conecellin interpretation. the centerof the receptive fieldisdepolarized, resulting in the release of glutamate G l u t a m a it ne h i b i ttsh eb i p o l acre l lc, a u s i nigt to hyperpolarize andthuspreventing allbut rareactionpotentials Notethatbipolaroffcentercellshavetheopposite response to glutamate(b)Whenlightstrikes the conecell, it becomes hyperpolarized, with a resultant dropin glutamate secretion Freeof the inhibitory effectof glutamate, the bipolarcell depolarizes, andfrequent actionpotentials result(c)Theactivity of a conecellisaffected by surrounding conecellsthroughhorizontal cellsthatconnect cellslaterally lf onlythe centercellisilluminated, the cellwillstimulate t h eb i p o l aar n dc o n s e q u e ntthl yeg a n g l i o n cellslf bothcenterandsurround cellsare i l l u m i n a t et d h ,ec e n t ecr e l l ss i g n at lo t h e bipolarcellwill be inhibitedThesystem is therefore a contrast detector, lookingfor light patterns that illuminate a smallcenterspotbut not thesurrounding retinaHerearethe steps involved: Lightstriking theconecellin the surround of a receptive fieldhyperpolarizes it Il, whichresults in a reduction in the release of glutamate in E This,in turn,results hyperpolarization of the horizontal cell, causing it to release lessinhibitory transmitter to thecenterconecellB Thecentercone cell,in theabsence of inhibition fromsurround c e l l si ,sd e p o l a r i z 4e ,d d e s e n s i t i z i tntgo l i g h t andinducing an increase of glutamate release to the on-center bipolarcell,asin (a) The bipolarcellistherefore g, hyperpolarized resulting in suppression of actionpotentials @ T h es c h e m a st ah o w na r eh i g h l ys i m p l i f i e d , sinceallthe cellscanbe connected to more thanonecellat eachstageof signal transm ission

(a)

we will look at the connectionsof cone photoreceptor cells to bipolar and horizontal interneurons (seeFigure 23-24b). On-center and off-center bipolar cells differ in the types of channel proteins they use, giving opposite responsesto the same glutamate neurotransmitter. For simplicity, here we will focus on bipolar on-centercells. Bipolar and horizontal interneuron cells,like photoreceptor cells, lack voltage-gatedNa* channels,so none of them generateactionpotentials.Instead,the secretionof neurotransmitters from the cells' synaptic termini is controlled by the

(b) Cone cell in center of receptive field

Cone cell in center of receptive field Depolarized-40 mV

Hyperpolarized-65 mV

Inhibitory glutamate released On-center bipolar celI

Glutamate secretion inhibited

Hyperpolarized

Depolarized

Frequent action potentials

Rareaction potentials G a n gl i o n c e l l

----> Optic nerve

@ (c) Cone cell in "surround"of receptive field

Cone cell in "center" of receptive field

E

E

Cone cell is hyperpolarized-65 mV

Center cone cell is depolarized-40 mV

p Glutamate secreilon inhibited

HorizontaI inhibited

E

Light

H o r i z o n t acl e l l E On-center bypolar cell is hyperpolarized

E

Action potentials suppresseo

1030

C H A P T E R2 3

I

N E R V EC E L L S

Cone cell in "surround" of receptive field

degreeof membranepolarization.In the dark, cone cellshave a membranepotential of about -40 mV, which opensvoltagegated Ca2+ channelsand causesthe continuousreleaseof the neurotransminerglutamate (Figure 23-27a). This glutamate, coming from cone cells in the center of a receptivefield, hyperpolarizesthe bipolar neuron that coversthat field, suppressing action potentials. Light striking the center field cone cells hyan inward perpolarizesthem to about -65 mV by sup^pressing flow of Na* and other ions, closingthe Ca'* channelsand reducing emissionof glutamate(Figure23-27b).This depolarizes the bipolar neuron, which in turn depolarizes ganglion cells and triggersaction potentialsto be sentto the brain. On-centerbipolar cellsare most stimulatedif the conecells in the center of the receptivefield are illuminated and the surround cone cells are in the dark. How do bipolar cells detect the light condition in the surround part of the receptive field? The surround input is mediated by horizontal cell interneurons (seeFigure23-24b, greencells).If therels light on a conecell in the surround region of the field, the horizontal cell that is connected to that cone cell becomeshyperpolarized which means it reducesinhibitory transmitterreleaseonto the conecell in the center of the receptivefield. The central cone cell becomesdepolarized,as though it was in the dark, and consequentlythe bipolar cell in the centerof the receptorfield is hyperpolarized (seeFigure23-27c). Ganglion cell action potentialsin the center of the field are suppresseddespite the light falling on the centralconecells.Thus light in the surroundinhibits sensingof the light in the center,a contrast detector. The retina'sprocessingof visual information is just the beginning of a hierarchicalchain of pattern representationand interpretation events.The refined processingof visual information occurs, it must be remembered,by reading trains of action potentials coming from the eye. In the visual cortex, cells are found that are specifically sensitiveto bars of light and dark, and eachcell prefersbars at a certain angle.It is easyto seehow the information that passesthrough retinal ganglion cells, the center-surroundinformation, can be usedto detecta bar of light or dark. If a visual cortex cell is stimulatedby ganglion cells whose visual fields are arrangedin a line, the integratedpattern would be a bar that passesthrough the centersof the individual ganglion cells' center-surroundpatterns (Figure 23-28). Further combinations can lead to recognition of more complex patterns by single cells. Some cells respond to a change of light-on to off or off to on. Others respondto edges,moving spots,or moving bars. Somecellsin the higher levelsof the visual cortex have even been found to recognizea certain face. The discovery of cells that integrate spatial information from cells that have simple receptivefields was describedby Nobel prizewinner David Hubel, who made the discovery with his collaboratorTorstenWiesel: Our first real discoverycameabout as a surprise.For three or four hours, we got absolutelynowhere.Then gradually we beganto elicit some vague and inconsistentresponses by stimulating somewhere in the midperiphery of the 'We retina. were insertingthe glassslidewith its black spot into the slot of the ophthalmoscopewhen suddenly,over the audio monitot the cell went off like a machine gun.

C i r c luar rrouno center-su field receptive

Corticalcell detects the verticalbar

Other cortical c e l l sc a n respondto different patterns

cell A cortical 23-28 Complexpatternrecognition. FIGURE thus ganglion cell"centers," to the sumof multiple responds to cellsrespond barshownOthercortical thevertical detecting of fieldsor combinations receptor of ganglion combinations different patterns. more complex recognize neuronfieldsto cortical After some fussing and fiddling, we found out what was happening.The responsehad nothing to do with the black dot. As the glass slide was inserted,its edge was casting onto the retina a faint but sharp shadow, a straight dark line on a light background.That was what the cell wanted' and it wanted, moreover,in just one narrow range of orientations.This was unheard of. It is hard now to think back and realize iust how free we were from any idea of what cortical cellsmight be doing in an animal'sdaily life'

CellsDetectPain,Heat, Cold, Mechanosensory Touch,and Pressure Our skin, especiallythe skin of our fingers, is expert at collecting sensory information. Our whole body, in fact, has numerous mechanosensorsembedded in its various tissues' These sensorsfrequently make us aware of touch, the positions and movementsof our limbs or head (proprioception), pain, and temperature,though we often go through periods where we ignore the inputs. Mammals use one set of receptor cells to report on touch, and other sets of receptorsfor temperature,heat, and pain' Pain receptors' called nociceptors, respond to mechanicalchange, heat' and certain toxic chemicals (e.g., hot pepper). Genetic insensitivity to pain is

o S E N S A T I o N AcLE L L S 5: E E I N GF, E E L | N GH, E A R I N GT, A S T | N GA, N D S M E L L I N G

1031

Sensoryhomunculus

Motor homunculus

F I G U R2E3 - 2 9H o m u n c u l u T s .h eh o m u n c u l u i ssa m a po f t h e regions of the brain's cortexthatarededicated to parttcular f u n c t i o nS s e n s oa r yn dm o t o rh o m u n c ual ri es h o w nT h ef a c ea n d often due to mutations in a gene,trkA, that encodesa receptor for nervegrowth factor (NGF), a protein mostly studied in different contexts. NGF and other neurotrophins have now been implicated as signalsof pain. Thermal receptors detecttemperaturechanges.Thesecellssteadilysendaction potentials (2-5h) that indicate the currenr remDerature. Each temperaturerange has receptorstuned to rt, so which cellsare firing conveysthe temperature. Connecting from the skin's sensory cells to the brain does not take many synapses.Mechanosensorsin the skin, for example, connect to the medulla, where they pass the signal along to neurons that go to the thalamus. A third neuron goes from there to the sensorycortex. A mere trio of neurons connectsthe periphery to the brain centers.In the cortex the sensory inputs are combined, through interneurons,with proprioceptiveinputs that report the positions of musclesand joints. Presumablythis makesit possible to know what you are feeling and where it must be if that armin that position is feelingit. proprioceprionreceptors take multiple forms. Some of the most studied are m u s c l e s p i n d l e s ,s e n s o r ya s s e m b l a g etsh a t a r e b u r i e d i n musclesto report on how much that muscle is extended. Suchstretchreceptorsare crucial to smooth movementand well-timed responses. Remarkably,the organizationof the body is reflectedin a rrap, or more accuratelyseveralmaps, in the brain. The organization of the cortical neurons that respond to sensory signalsis physicallyrelatedto the spatial origins of the signals.In the brain the sensoryneurons are laid out in a distorted map of the body. The motor neurons are also 1032

.

c H A p r E 2R3 I

N E R VcEE L L s

arranged in a map that can be aligned with the muscles they control. The maps are called the sensoryhomuncwlus and the motor homwncwlus(Figure 23-29). A bomunculus is a "little human," an image of us. The map dimensions are not proportional to the body's dimensions,becausethe homunculi reflect the number of sensory or motor cells rather than the area of the body. The hands and feet are highly representedand take up much more space in the sensorymap.

I n n e r E a rC e l l sD e t e c tS o u n da n d M o t i o n The outer ear captures sound, which moves three tiny bones (ossicles)in the middle ear, which in turn transmit soundinduced motions to the inner ear, or cochlea (Figure 23-30a). The cochlea is shapedlike a snail, with nearly three turns, and indeed the name derives from the Greek word for ,,snail" (cochlos). The cochlea housesthe organ of Corti, the sensory part of the inner ear, which transducesmechanical movement into electricalimpulses.The human organ has about 16,000 hair cellsarrangedin four rows (Figure23-30b, c), attachedto about 30,000 afferent neurons that carry any signalsto the brain. Hair cells produce stereocilia,which are moved by vibrations induced by sound. The oscillatingvibrations alternately bend the stereociliaone way or the other, triggering depolarization eventscalled receptor potentialsin the 10 or so axons associatedwith each hair cell. Theserecepror potentials, which are milder than full action porentials,range up to 25 mV. Hair cells and the neurons they influence are responsive to different sound frequencies.There is a gradient acrossthe

(a)

O u t e re a r

Middle ear

Inner eal

T e m p o r a lb o n e Malleus(attachedto t y m p a n i cm e m b r a n e )

Cochlea Cochlear nerve In c u s

ExternaI auditory canal

Stapes attachedto o v a lw i n d o w

Cochlea

Round window

Apex of cochlea

23-30 Structuresof the ear.(a)Soundenterstheouter a FIGURE to the middleear,wherethreetinybones(themalleus, earandtravels in thetympanic vibration sound-rnduced transfer andstapes) incus, innerear,where in the cochlea the ear to middle the across membrane Thecochlea, signals. to electrical istransduced vibration mechanical of theorganof wouldbe 33 mm long (b)Theinnersurface unwound, electronmicroscopy' asviewedby scanning Corti,foundin the cochlea, (white)wouldbe in contactwith the Thetopsof allthe stereocilia 23-31)Thestereocilia ear(seeFigure the intact in membrane tectorial in a line,whilethethree of the innerhaircells(leftrow)arearranged in V shapes(c).Higher rowsof outerhaircellshavestereocilia Thehaircellsare of outerhaircells. of thestereocilia magnification cellsarecovered support sunounding while cilia, for the smoothexcept K andFrench, Burggren, (a) W Randall, from D adapted microvilli by IPart ed , W H Freemanand Company,p 243 2OO2,EckertAnimal Physiology,5th Instituteof Healthl Parts(b) and (c):Courtesyof BecharaKachar/National

cochleaof frequency sensitivity so that the spatial locations of the cellsthat are stimulatedreflect the frequencycomposition of a sound. Hair cells and neurons at one end of the cochleahear low-frequencysoundsand at the other end high frequencies.This is not becauseof a differencein either hairs o. ,r.uront. The graded sensitivityis due to a tapering tissue called the basilar membrane (Figure 23-31')that respondsto low frequenciesat one end and high frequenciesat the other' Each friquency excites motion in a particular region of the 33-mmlong basilar membrane, which is then locally transferred to nearby hair cells. The orientation of the hair cells, and in particular of their bundles of stereocilia,with respect to the basilar membrane allows sensitivedetection of deflections causedby sound' The polarity of the hair cellsand their cytoskeletonsare key to proper transduction of sound into electricalsignals. Some of the proteins that control the structure of hair cells and stereocilia have been identified through human genetics,tracking genesresponsiblefor deafness'Five g..r., h"u. been implicated in Usher type 1 syndrome, the . S E N S A T I O N AC L E L L SS E E I N GF, E E L I N GH, E A R I N GT, A S T I N GA, N D S M E L L I N G

1033

(a)

bent stereocilia

!

ORGAN OF CORTI

Inner h a i rc e l l s

Outer h a i rc e l l s

B a s i l a rm e m b r a n e deflectedup

most frequent causeof hereditary deafnessand blindnessin humans. The genesencodemyosin VIIa, cadherin 23, protocadherin 15, aPDZ domain protein calledharmonin, and a putative scaffolding protein called Sans. All these proteins are localized within stereociliain auditory hair bundles. Harmonin associateswith both F-actin and with the cadherins implicated in the disease,while myosin VIIa and Sans help to Iocahze harmonin in stereocilia. These discoveries have emergedfrom medical geneticscreeningof patients and are revealingthe key molecular underpinnings of stereocilia and auditory sensing.

Five PrimaryTastesAre Sensedby Subsetsof Cellsin EachTasteBud (b)

T e c t o r i a lm e m b r a n e

(c)

FIGURE23-31 Sterociliamovement. The stereocilia of the innerand outer hair cells(purple)are stimulatedby a sideways movementwith respectto the overhangingtectorialmembrane, which in turn is influencedby oscillating fluid pressure changesin the organof Corti.The fluid pressure in the organoscillates at the frequencyof the incomingsound.(a)As vibrationbegins,the basilar membrane(pink)is forcedupward(indicatedby the arrow)by fluid pressure changes,which translates into a leftwardmovementwlth respectto the tectorialmembrane,thus bendingthe stereocilia to the right (b) At the midpointof the oscillation, theitereocitiarelax (c) When the oscillationgoesthe other way and the basilarmembrane movesdown (indicatedby the arrow),the hair bundlesare movedin the oppositedirectionby the shearingeffectof the tecrorrar membraneThe motionsare repeatedwith eachwave [Adapted from D Randall, W Burggren, and K French, 2002,Eckert Animatphysiology, 5th ed , W H Freeman andCompany, p 24gl 1034

.

c H A p r E R2 3 I

NERVE cELLs

Taste buds are located in bumps called papillae, and each bud has a pore through which fluid carries solutesinside. About 50-100 tastecellsare locatedin eachtaste bud (Figure 23-32a, b). Cells in the tongue and other parts of the mouth are subjectedto a lot of wear and tear,and tastebud cells are continuously replacedby cell divisions in the underlying epithelium. (A taste bud cell in rars has a lifetime of 10 days.) Tastecellsare epithelial cells that show some of the functions of neurons. Reception of a taste signal causescell depolarization and receptor potentials that trigger action potentials;these,in turn causeCa2+ uptake through voltagedependent Ca2* channels and releaseof neurotransmrtters at synapses.Taste cells do not grow axons, instead signaling over short distancesto other neurons. In conrrast to most other sensory systems,there is, as yet, no known topographic representationat any level of the brain thar correspondsto the different rasres. .$7e taste certain chemicals, all hydrophilic, nonvolatile moleculesfloating in saliva. Although all tastesare sensed on all areas of the tongue and there is no topographical taste map of the tongue, selectivecells do respond preferentially to certain tastes. Taste is less demanding of the nervous systemthan olfaction becausefewer types of moleculesare monitored. \il/hat is impressiveis the sensitivity of taste; bitter moleculescan be detectedat concentrations as low as 10 r2 M. There are receptors for salt, sweet, sour, umami (e.g., monosodium glutamate and other amino acids),and bitter (Figure23-32c, d, e, f) in all parts of the tongue.The receptorsare of two different ,,flavors": channel proteins for salt and sour tastesand seven-transmembrane-domainproteins (G protein-coupled receptors) for sweetness,umami. and bitterness. Salt is probably sensedby members of a family of Na* channels called ENaC channels,though definitive proof is lacking; other membersof the family have diversefunctions including neural memory. The influx of Na+ through a channel depolarizesrhe cell. The role of ENaC channelsas salt sensorsis old, since ENaC proteins clearly detect salt in insects. In Drosophila, taste sensors are located in multiple placesincluding the legs,so when the fly stepson somerhing tasty, the proboscis extends to explore it further. However, the ENaC studieswere done using fly Iarvae,which can respond

23-32 A mammaliantaste bud and its receptors. V FfGURE cells (a)Thepinkcellsarethetastecells.These receptor epithelial arriveat the signals contactthe nervecells(yellow)Thechemical of a pairof tastebuds, seenat thetop (b)Photograph microvilli visible in the areclearly cellsThemicrovilli showingthe receptor tastebudon the /eft (c-f)Typesof tastereceptors[Part(a)adapted andBehavtor, to Brain An lntroductton 2006, fromB KolbandI Q Whrshaw Arnoldl 2ded. Worth,p 400:Part(b)fromEdReschke/Peter

(a)

Tastepore Tongue (microvilli) E p i t h e l i acl e l l s

Receptor(taste)cells

Nerve cells

Nervefibers to brain (d) Aminoacid

(c) Salt and acid

M o n o s o d i u mg l u t a m a t e( " u m a m i " )

A c i d s( s o u r )

Salt

Am iloride-sensitive N a *c h a n n e l

a

H +i o n s b l o c k a K +c h a n n e l

H * i o n s p a s st h r o u g h a m i l o r i d e - snsitive e N a *c h a n n e l

a\

G-proteincouPled receptor(GPCR)

H+t-

Na*/o

G protein

Exterior

Cytosol oo

o

( e ) B i t t ecr o m p o u n d s

(f) Sugarsand sweeteners

Bitter D i v a l e nsta l t / q ui n i n e - s e n s i t i v e K *c h a n n e l

Sugars

T

I

T

AdenYlYl cyclase

aa P h o s p h o l i p a sCe

{

{.

{

o K*o oo

@ @ @

I I

P h o s p h o l i p a sCe IP

fAMPI

@

rcMFl

SMELLING S E N S A T I O N ACLE L L SS: E E I N GF, E E L I N GH, E A R I N GT, A S T I N GA' N D

1035

to salt if they have either of their two ENaC proteins.Sour reception is the detectionof H* ions, which ."n -ou. through the samechannelsas Na*. H* may also be senseddue to its interferencewith K* channelsand consequentincreasein intracellular positive charge(i.e.,a depolarizingeffect). Bitter tastes are more diverse than salt and have been found to depend on a diverse family of about 25 genesencoding various T2Rs, taste-receptorproteins with seven

T1R3 fail to detect sugar; it is thought that the actual receptor is a heterodimerof the two. T1R3 appearsto be r...p" tor for both sweet tastesand umami, and that is because it detectssweetswhen combined with T1R2 and umami when it combines with T1R1. Accordingln taste cells express T1R1 or T1R2 but not borh, as otherwisethey would send an ambiguous messageto the brain.

A Plethoraof ReceptorsDetectOdors

The first member of the T2R family to be identified came from human genetics studies that showed an important bitterness-detecrion gene on chromosome5. Multiple T2R types can be expressedin the same taste cell, and about 15 percentof all tastecellsexpressT2Rs. Bitter tastemole_ culesare quite distinctin structure,which probably accounts for the need for the diverse family of T2Rs. Mice that have five amino acid changesin the receptorT2R5 are unable to taste the bitter taste of cycloheximide(a protein synthesis inhibitor, Chapter 8). A dramatic gene regulation swap experiment was done to demonstrate the role of T2R proteins. Mice were engineered to express a bitter taste receptor, a T2R protein, in cells that normally detect sweet tastesthat attract mice. The mice developeda strong attraction for bitter tastes.evidentlv becausethe cellscontinuedto senda ,,goand eat this.' signal even though they were detecting bitter taste. This exferiment demonsrratesthat the specificity of taste cells is diter_ mined within the cells themselves,and that the signals they send are interpreted according to the neural connections made by that classof cells.This in turn implies a highly regulated systemconnecting the different classesof trrle r..eptor cells to specifichigher regions of the brain. One bitter taste is especiallyfamous becauseit is often used in geneticsclassesto teach about human variation. The chemical phenylthiocarbamide (pTC) tastes exceedingly bitter to many people but is tastelessto others. Human sensitivity to PTC can differ by a factor of 16. The inability to detectPTC is inherited as a recessivetrait, meaning that rast_ ing is dominant over nonrasrrng. Sweetand umami tastesare detectedby a protein family relatedto the T2Rs, called T1Rs. When T1R proteins bind an appropriate tastant, they act through G proteins to rrig_ ger the releaseof calcium inside the cell. The three mam_ malian TlRs differ from one another in a small number of

The perceptionof volatile airborne chemicalsimposesdifferent demands than the perceprion of light, sound, touch, or taste. Light is sensedby only four molecules,tuned to different wavelengths.Sound is detectedby mechanicaleffects through hairs that are tuned to different wavelengths.Touch requiresa small number of types of transducers.The senseof taste measuresa small number of substancesdissolvedin water. In contrast to all theseother senses,olfactory systems can discriminateberweenmany hundredsof volatile molecules moving through air. Discriminarion between a large number of chemicals is useful in finding food or a mate, sensingpheromones,and avoiding predators, toxins, and fires. Olfactory receptorscan work with enormous sensitivity. Male moths, for example, can detect single moleculesof the signalssent drifting through the air by females. In order to cope with so many signals,the olfactory system employs a large family of olfactory recepror proteins. Humans have about a thousand olfactory receptor genes,of which about a third are functional (the rest are unproductive pseudogenes), a remarkably largeproportion of the estimated 25,000 human genes.Mice are more efficient, with 1300 genes,of which about 1000 are functional. That means3 percent of the mouse genomeis composedof olfactory receptor genes.Drosophila has about 60 olfactory receptor genes.In this sectionwe will examinehow olfactory receprorgenesare

FIGURE23-33 Sequenceorganization in olfactory receptors. Olfactoryreceptorsare seven-transmembrane-domain G protein_ coupledreceptorproteins.The cyllndersindicatethe extentof alpha helicesthat crossthe membrane.Residues in blackare highlyvariable, and someof thesedifferences accountfor specificinteractions with odorants [FromL BuckandR Axel.j991,Celt65:1j51

1036

.

c H A p r E2R3 |

N E R VcEE L L s

employed, and how the brain can recognizewhich odor has beensensed-the initial stagesof interpretation of our chemical world. Odor moleculesare calledodorants.They have diversechemicalstructures,so olfactory receptorsface some of the same challengesfaced by antibodies-the need to bind and distinguishmany variants of relatively small molecules. Olfactory receptors are seven-transmembrane-domain proteins(Figure23-33).In mammals,olfactoryreceptorsare produced by cells of the nasal epithelium. Thesecells' called olfactory receptor neurons (ORNs), transducethe chemical signal into action potentials(Figure 23-34).ln Drosophila, ORNs are located in the antennae.The ORNs project their axons to the next higher level of the nervous system,which in mammals is located in the olfactory bulb of the brain. The ORN axons synapsewith dendritesfrom proiectton neurons in insects(calledmitral neurons in mammals);thesesynapses occur in the clustersof synaptic structures calledglomerwli. The projection neurons connect to higher olfactory centers in the brain (Figure 23-35). Each ORN producesonly a singletype of odorant receptor. Any electricalsignalfrom that cell will conveyto the brain a simple message:"my odor is binding to my receptors."Receptorsare not always completelymonospecificfor odorants. Somereceptorscan bind more than one kind of molecule,but the moleculesdetectedusually are closelyrelatedin structure. Conversely,some odorants bind to multiple receptors. There are about a million ORNs in the mouse;so on averageeach of the thousand or so olfactory receptor genesis active in a thousand cells.There are about 2000 glomeruli (2 for eachgene),so on averagethe axons from 500 ORNs convergeon eachglomerulus.From there the axons of about 50,000 mitral neurons,about 25 per glomerulus,connectto higher brain centers.Note that in contrast to the visual system, very little signal interpretation and refinement occurs in the sensoryepithelium or even the projection neurons. The initial sensory information is carried to higher parts of the brain without processing,a simple report of what has been detectedwith no further analysisor commentary. The one neuron-one receptorrule extendsto Drosophila. Detailed studieshave been done in larvae,where a simple olfactory systemwith only 21 ORNs usesabout 10-20 olfactory receptorgenes.It appearsthat a uniquereceptoris expressedin one ORN, which sends its proiections to one glomerulus.

pounds or aromatic compounds.The arrangementmay reflect errolutionof new receptorsconcomitantwith a processof subdivision of the olfactory part of the brain. The simple system of having each cell make only one receptortypi also has some impressivedifficulties:(1) Each ....ptot must be able to distinguish a type of odorant molecule or a set of molecules with specificity adequate to the needsof the organism. A receptor stimulated too frequently will probably not be too useful. (2) Each cell must produce one and only one receptor. All the other genes must be turned off. At the same time the collective efforts of all the cells in the nasal epithelium must allow the production of enough different receptorsto give the animal adequatesensory versatility.It doeslittle good to have hundreds of receptors if most of them are never used' but it is a regulatory

cell is receivingwhich odorant so that electricalsignalsfrom the nose can be interpreted. It is often the case that a responseto a particular odor is programmed in the genes,like b.h"uio."l responseto a pheromone. In such casesthe " brain must know which cells are detecting that pheromone' Otherwise the animal might be feeling romantic when it should be running away as fast as possible. The solution to the first problem is the great variability of the olfactory receptor proteins, both within and between

Nematode

lnsect

Vertebrate

ORNs can send either excitatory or inhibitory signalsfrom their axon termini, probably in order to distinguishattractive versusrepulsiveodors. The ORNs project to glomeruli in the antennallobe of the larval brain. The researchbeganwith tests of which odorants bind to which receptors(Figure 23-36a)' Someodorants are detectedby a singlereceptor'some by sev-

Pore Nasal cavitv

Fluid

hAr i -I

Olfactory receptor n e ur o n

Dendrite

Fluid Dendrite

Olfactory receptor n e ur o n

Olfactory receptor n e ur o n

23-34 Structuresof olfactory < FIGURE receptorneurons.Acrossa vastspanof insect, distance-vertebrate, evolutionary neuronshave actoryreceptor nematode-olf exposed forms Eachhasfineprocesses srmilar t o v o l a t i loed o r a n tdsi s s o l v ei ndf l u i d H i g h l y (notshown)in the receptors olfactory specific The the odorants cellsshownare cellssense fromF not drawnto the samescale[Adapted Delcomyn,1998, Foundationsof Neurobiology, W H F r e e m a na n d C o m P a n YP, 3 2 7 l

S E N S A T I O N ACLE L L SS: E E I N GF, E E L I N GH, E A R I N GT, A S T I N GA, N D S M E L L I N G

1037

f \;

Olfactory Receptor

A FIGURE 23-35 Theanatomyof olfactionin mouseand fly. ln boththe mouse(a)andthefly (b),olfactory (ORNs) receptor neurons thatexpress a singletypeof receptor sendtheiraxonsto thesame glomerulus Inthlsfigurethe redandbluecolorsrepresent the neural connections for two distinct expressed receptors Inthe mousethe glomeruli areIocated in theolfactory bulb;in thefly theyarein the

brain ln the glomeruli, the ORNssynapse withprojectionneuronsin the fly,or mitralneuronsin mammalsEachprojection neuron(or mitralneuron) hasitsdendrites in a singleglomerulus, thuscarrying to highercenters of the braininformation abouta particular odorant. T.Komiyama andL Luo,2005,CurrOpinNeurobiol. [From 16:67-73]

species(seeFigure 23-33; the black residuesare highly vari_ able). The solution to the secondproblem, the expressionof a single olfactory receptor gene, has been expltred using transgenicmice, but the mechanism is still not understood. rX/henan engineered olfactory receptor gene is used to pro_ duce an olfactory receptor,other genesare turned off tian_

receptor send their axons to the same glomerulus. Thus all cells that respond to the same odorant send processesto the same destination. This convergenceprocesscould be due to (1) an attractive signal that somehow is specificfor a certain olfactory receptor or (2) to mutual recognition, and subsequent coordinate growth, of axons that have the samereceptor on their surfaceor (3) to a pruning processin which many connectionsare made but only those that share the same olfactory receptorpersist,possiblyregulatedby neuronal activity. Developmentalanalysesshow that ORN axons do not arrive at a glomerular "blank slate." Rather the glomerulushas organizedits projection neurons prior to the arrival of ORN axons. The systemis to some degreehardwired. In mice a crucial clue about the patterning of the olfactory systemcame from the discoverythat olfactory receptorsplay

The third problem, how the systemis wired so the brain can understand which odor has been detected,has been partly answered.First, ORNs that have expressedthe same 1038

.

c H A p r E2R3 |

N E R VcEE L L s

6€0r

9 N t ' 1 ' l l t A sc N V

'9NU-SVI'gNtUVlH '9Nr'llll '9Nll3s

suorlprqr1 'eprsurrlro3 Io up8ro eqt qlr-^a'eelqcoceql sI rolrel -op punos aqJ'(0€-€Z arn8rgaes)punos slretap pue 'peurel

:s']'lt)'lvNolrvsNls

-urEru0q ol esuPleqs^\olle Pue uorlotu sosuosJeerauul eql I '\67-t7 ern -3rg aes)tln?unLuoqpa11er'urerq eql Jo oJetrnsaqr uo dpoq aql;o sdeurot esrre,tr8srolderor asagr.{g peleraue8sleu8rs e q 1 ' a . r n s s e rpdu e ' q c n o l ' p l o c ' t e o q ' u r e d ' ( u o t l d a r o r r d o r d ) uortrsod dpoq rcerapslerutuetuur sllel drosuesoueqrary r 'lgZ-tZ arn8r4ees)plro,tr aqr ;o sa8erurxelduoc dlSurseanure,rt8ot sllel le^el-rorlSqdq eloluls IeIlIuI eI{J r peurqruof,are suJeltedPunoJJns-Jeluef, 'gZ-tZ arn8rgeos)esra,rerIAro punor -.rns roluor pue rq3r1 IrEp to suraDed ur sllal roldecerol 'uterg aqr yo sued .raqSrqof -oqd ;o slos ot arrtsuodssrare purlar aql ruor; sleuSrs.,{rrerqcrqlr 's11aruotlSue8 leurrag '>lrep puE rqSrl Jo suraDpd ureJsrpol Jel{le3otpoteJdJetul eJe sllef rotdaceroloqd aldrrlnur uory sleu8rspeulquro3 r

'ualsls snolreu 'uollres txeu aql uI asoql ssnrslp ily alA. aqt;o sued roqlo uI Pesnsolnf,alouracueprnSuoxe PrPPuPls ol pue roldeJar d.rone;1ou/ro rleql ol qroq puodser suoxe 'urvroul lou sr tuslueqJelu ilnt aql NUO lEql rPelf sI ]t 1nq 'uorleullsapsnpraurolS atuesel{l or peprn8ere .roldaoarewes 'luetu suoxE 5169 aldnpltg 'aoueprn8uoxe aqt Surssa.rdxe -dolalap Surmp 'pue Surpurq luPJoPo :sNUO uI salor o^\l

louaqd1,(q1eLu-7 Q touaqdl,(qtau-Z @ auouaqdotece @ ap{qaqezueq @ 1Iq1aulp loue6na alosrue S ;oueuou-;s) louel3o-€ touBloaqL

o

$so

oo

I louelnqalelece1,(ueost{fi} alelace|r{doJd$

' \ S Z - t Za r n B r l aas)rq8rl e,rrarradol d.reurqcetuorlt sllel roldace.roloqda,tr8 sursQopoqt pue sutsdo peller sluetu8td e.trtrsuas-rq8t1r

auoueldaq-7 @ auorpauegnq-g'7 @ leuexaq-73@

'suornJursoul elrl ezrrelodapueqr reqlpr'azrrelodraddq sllel roldara.roloqd parelnrurlg r

1o-g-uapo-; @ louexaq-p [)

'ft2-tZ ot asuodsar ur sleuSrs ern8rg eas) lq8rl lecr.rl -re1aacnpord ot pelelnrurls ere (ro1ocro;) s11ec ouor aAIIIS -ues sselpue (errq-,npue i{er8 '1ce1qro;) s[e] por ellllsuos fp8tff 'patelol arp sllel roldeceroloqd;o seddro.ryuoreq,l.r 'eurlar aql 'aoe;.rnsaartrsuas-tq8rlE uo rg8rl sncol sadg r

4F13

a1er,{1nq 1,(q1a S alelace|'!uad @ alelecelAq1e$

ooe as9

(q) o p r x o r pu o q r e S p r c ec r u o r o o r d

pue 6ur;;aur5 '6utee5:slla) '6ur1se1 '6ugreag '6u;;aa1 leuollesuas

t 1

'uo,tt;6uno1 aei;o I uosle)uqofpuelaqar)]]ors 'uosue) laqar> IsaUnoJ :(q)Uedgst-Sttigl uunaN'9002 ^ [ U I puP'uoM) 'saurl qltnn paqsep nnol;ari asoql )lleu.roje V SuJorl(p)uedl spunodLUof rlnteuo;O rrleqdrle reaurl osuas autlptlosen;qe riqpaleltput lspunodulor aq o] puelJeltults rol roqloueouoo] ]xaupaien]ts aalqioq]'e;duexa palsa]alaMsuralled rlnrourolg ril;erruaq> are]eqlqueropo6ursues ue oq 1ou1q6tuuotldetxa aue6fuo1:e11o arour;r uotlde>xa uorssardxe qtee(qEytgpue seqsnlnrar-uo16 fuosuas anbrun eql sarloeder spuodsat roidaterqtea auoq]tM]eq] aloN {16uot1s eggrg)uorldatxa q)qM o1slueropoaql ole pole)lputoslv ( )ia 'eztto) sad&urelord sNUOtlrol+suotllalotd uol aq]lo qtea6urrnpord roldarerpale)rpur roldetal aql suornou orl]sale)lpu!qderOoioqd o^rarar ]eq] 11nreuo16 fuope;1o lue^aloraq]]o qleo+ololluo)aq]rapunaue6.rauodat aq1 elqdoso-rg euopsenn Ourddeuaq1 uterq;enrel e 6ursserdxa,iq uruorlerrrrolur fuope11o 1odeu.rleried5(q) spunoduo) 1orlnraLuol6 'e6SJO q)ns'sioqlo pup seaJaqM eg€rO se o1puodsar )rlptrlore 'q19lO 'spunoduor q)ns puodset se ro eZt.lO o1 {lueuttd rrleqdrle 'srolderar e {1uouo pe (e1et,{1nq {uer-u }eq}e1o1toldetate;6urs 1Aq1e ' '(alela)e ' 6 o)sraqloalrqlrn o}elnull}s 1{1uad6 a)stoldetate;dt11nu sesuodsar ropo6uotisale)tputsloppalolo) sluelopoouros]eq]e1o51 oje polsa]slueropo/Z aql pue'dol aqi aplsUataq] uMopu^^or]s eql (e)'ure1s/(s ssorlepalsrla.resutelotdtoldetatfuopel;o]uare++rp f.rope;;o pntel epqdoso,rgeql ut t;n.rauo;6rt;treds ol Pe)etl aq uer sadltl pue slueroposnolreAoI palutl f;;elueur.radxa roldara,rfto1re;;olenpr^rpulg€-EzlUnglJ]VINllAluldxl >

I

louaqdlAq1e4-y loueqdqAqleyl-7 auoueqdolecy

c

o

epAqapqezuag ;ouebne;Aq1e61 alosruv a u o q c u o l( - ) auouexoqolcAC

a

l o u e u o NLlouelco-t l o u e l o a HL-

I I

I

I

I

o I

I

C

m c ot

#

ot o

o

oo il

l o u e l n SLaleloce lAlcO oleloce lAureosl eleleceqAdot4 alelacelAueto9 euouelde;1-7 auorpouelnE-e'z louaxoq-zf lo-€-uelco-t louexeH-I a1e;Icrles;Iq1e61 elerArnq lAqlf alelace lAluad olelace lAqlf (e)

ooco"oo""fauoou"to".do"otoo+to.

eql posneJ osle aurlrqJlo3 .seuot r{l^{or3 grnued dlalerp ,(gI rerdeq3) uorl -euurr tou pp uortJeler uoxe tnq ot pel -ezrreullodep alnqntoJlnu sasneJr.{f,rr.{ \,aurcrqclor :uoxp aql .urlf,e to auoq{leq aql turo} selnqntorf,rtr[ to uortezrreu.,tlod dg parurol ere erpodopl pue erpodqleurelr{tog .sauor qtr.or8 ,UI JO UOI]JEJIPSIUI JO UOI}JPTOJ O] PEI

JArdEg])

UOIIEZIJETU

-,(1odapullf,p sasner r{Jlrllr\ 'g urseleqcotdc leuoc qrmor8 aql to af,ueuelurerupue uorsnrlord .ro; perrnber sr uortezrroudlod urDy 'sdotsaerql eql ol surelsdsraqr; Jo suorlnqrrluot ole8rlsa,r -ul ot pasn uaeq e^pq stuotuela pralalsordr llq${ul reql s8n.rq 'sorue^pe a8peSurpeeyauoc-ql.uor8 aql se dlsnonunuor rnJf,o sdels earql esoql 'uoxe oqt Jo UEr{seqt soruoJeqtr sE euoJ qltro.r8 aql yo Sur,la,orrruolrtf,p eql sr uotlpptlosu@ pue ,uaql s;p8ua auoc qurorS orp sE r{tog ;o Suqlams aqt s luawa7to? -ua 'etpodolr1 erpodrJlarupl pue uorsuJlxo eqt s uotsnuotd '6t-tZ arn8rg)uolleprlosuor Jo pue'tuarua8ro8ua'uorsnrlo.rd :peurtapuoeq a^Er{uorsuetxoauoo-ql,rror8;o soseqdea.rq1 'uoxe ue Sururoraq;o ssacord aql ur sr lr eJer{A\,auoc qulo.r8 erlt Jo >lleq aqt le lJerter pue urnrpodrllor.ueler{r seprsaql to punore o^oru luorJ orp lE pererol ore tpr{t erpodog aqt ,saoue,r -pe euof, tp.tlor8 er{l s€ lpqt .4l.oqssarpntsasdelaurl .uoxe eqt Jo Uer.lser{r0g ilr,vrrer{r purqeq uorSorSur.rodp}E seleal rr tpoq .Buo1urrl IIaJerp tuo.ryde.nese^ou euof,qurror8 aql sy 67 o1dn erpodog aqr 'qrpee.rgur rurl gg1-gl Jo rapro aqr uo llecrddr sI unrpoclr{OurElar{J 'Vt-tZ a;n8rg) alpodol! pa11ersa4rds dreqs aldrrlnru Jo uorsuelxe up lr ruo{ pue ,wntpodqpwo1 0r{l 'lerraleru paueuelJyo peards peorq e :sJrnleal roleru olg are eroqt llerauoS rnq tlaprlr dre,t seuocqlLror8;o sadeqseq1 aJnl)nJls o)uePtng

flosue5 poztJolol4e sl auo) rll^ orD aql 'ruelsds snolJeu aql 8urrr,rr. snql ,qlnor8 to uortJeJrpstr .sarr.radordalqe selq sleu8rsot teql spuodsar auor quto.r8 aq1 ->lrEruorstr ouof aqr a,rr8 seFtelotu erp lErp erotu lnoqp lrq E ,ry\ou{e,vi^\ou pup '06gI ul plua tr ugrueg dq paqr.rosap dlrco.r -rof sE.ry\ auoc qil\or8 eqt to ror^eqeq progeourE eql .surnl Pue slsr^\t slr qSno.rql uoxe SurteSuoleoql speol leqr uorsual -xa IIer e SanBu eqr to rrEaq oql sr ouor qulo.r8 aqI 'lp^rrre uodn slaS.reteql qlr.,r.r. asdeu,{spue azu8ocer uaql pup sta8rel

]ues sr pue M r ,!o,rrapnrlo,d ,;"t::;3::,lJ'tl",J':::;J;ll';

q)rqM LUor]'wntpodtlpuLelaql palle) lpt.laleupauallpl+ peetds 1o peolq p o^eq sauo) qlMorg .sauo) l.ll/vlorg- LE_EZlungll v

s'l'll) SnulN I

tz ulrdvH)

0t0 I

pJE^lol Pueuo ol suoxe raollB ler{l suralsdsacueprnSer{r puE 'suoxe;o qu,ror8 pu€ uouprrrJoJer{l uo snfoJ e.ry\ uorlf,ossrql uI

I'etg-eog:zz-lz

n8nzuy bq2stLuowoipuv OeInod np e.rarurdeollooru pl op sasne^Jeusalnllat sep suorsuedxasa1tuessrered , 0 6 g I , A . U . S. F [ e ] l .deqc -de a n b o d a a l e n b y 519.d,g1 'ssardJnuopery' a2uao sotna pruaruppun N tr, 6 6 6I,. spa ''1e '1er'9691 '1e[e3 puour8r.Z't'W ur le L uotueg'uorlPurlseP slr se rJJElr le 'de,u slr ur sellElsqo JOur Illun sarllsJolulrPlnllol Surc.ro; etuof,relo pue pre.ry\ro] ssord ot elqe sr lr r.{Jrr1.vi ol slueql 'ao.ro; a,rrslndur urel.rar ? qlrlA pue ,stuorualoru proq -aorue prder qlri\ (sseuelrlrsues leJnueqc alrsrnbxaqlr.Lr po,tl.opue'rue.rSur.raDeqro gnlJ Jo uos e se papre8ereq deru auoc qlznor8 aql trrrara;olurod lpuortJunJer{l ruorC

:aroldxa pue rosuas E sr euot qrmor8 aq1 .pu8rs eql tuort de,ne .ro pJe^{ol urnt ot auor q}nor8 aql osner pue 'eprsuruolela4soldc oql eruengur 'auoc quror8 erp lE srotdaJ -er ot purg salnJoloruSuqeu8r5.euooql,llor8 oql palleJ sr uoxe up Jo pue '3ur,no.r8'8urpea1 eqI 'sllet rer{lo tuo{ sleu8rsSursn 'sruolsls acueprnSo1 asuodserur mor8 suoxv .Sunuro;sue.rl sr suoxe pue serlJpuop;oqrrvror8eql .llet punor eldurrsE ,elue -.reeddeur relnrelcadsunsr ueuo rostncerd lyao V lernou 'suoJnouetuos qreap peuuerSord eqr pue ,suorlceuuof, tue Io -reqnxe Surun.rd'sasdeuds;ouorleruroJ.(secuelslp3uo1ralo Jo seunlauros)sanr af,uepm83urmo11o;suoxe Jo qu,ror8 ,uonpru -rot olrrpuappue uoxe'uoqer8rul lec.erf Jo suorneuolul qlel paurrot dl,trou;o uonpurruJolopaqr .q1ecuels ruort uroq eJP suorneu .ry\eu eJoq.ry\ seuozIeurruJa8Jo uoneruro; aqt :sdar p.re -,tesSur,teqse pe.ry\ar^ eg uer luaudolalap IErnaN .perurot orp suorneu ueo.,lltegsuortf,ouuof,or{t ,vroq;o ualqord oql ol urnl A\ou e,vr'sy1acd.rosuespue 'sesdeudslE uorlJnpsueJl IEJrrueqJ 'uorlcnpsuert pu8rs 's11ara,r.leu seuradord eql leJrrtf,ale ;o paquosop3urrre11drlmcrrc xalduroc sr ,suorDun; patue^pe eql ,{leroadsa'uralsdssnolrau oqt Jo suorlf,unJar{rto ueeq aqt rV

6ulle6.rel pue LllMolg uoxv

6ur1;o.r1uo) :ssa))nsol qled oql '9t-tZ ol €t-€Z sam8rgoas)urerq eql ur uorleuusopewESer{l ol suoxp rroql puesaueS.roldacarerupsoql sse.rdxarer{l sNUO 'pasuas ptrtuoql eql Jo arnleu aql sde.nuocdlsnon8rqueun urerq oql ol reqt ruo.rypu8rs e os ,aua8roldeoar Lrorce;1o IIoJ auo dpo puu auo sossa.rdxa uornou.roldace.rdropeJlo auo duy 'seue8 ;o tas e3rc11uate Aqpopolueere,sroldaoarpaldnor-ural -ord g aupJgruatusueJl-uelos aJpqJrq 4.,sroldeca.rluEJopO r 'ftt-tZ arn8rg ees) salspl luareJtfp Da13pot suoneurqruoJ Jrreurporoleq pue -ourorl tuareJJrpur >lJo1$. esaqt pue ,sura1 -ord ureruop-eupJgtuerusuerl-uoles aJeeruos .E^rlpsur pollos -sp sFlrtuar{o lf,alap s.rotderer etsel Jo roqrunu [ptus V r 'punos se palrecradere grrq^\ ,s11ao rreq eql ur slprtuolodrol -dare.releraue8eqrc osaqt sluerualor.ueql .(It-tz orn8rg Jo ees)otltcoanls pe[e] srreq aurJ seloru urnt ur qcrqm ,prng elotu JEe Jouur ol Jatno oql tuory seuoq lerus dq parrJeJ

Bundled

F-actin meshwork ->

Membranous vesicle

. Direction ol mrgrauon

I Leadingedges Direction of \ migration I

23-39 Antibodylabelingreveals FIGURE A EXPERIMENTAL cytoskeletoncomponentsinsideculturedhippocampalgrowth cones.A singlegrowthconeisshownthreetimes,in eachcase tyrosinylated F-actin, with an antibodyfor a differentstructure: labeled (ace-MTs)' The (tyr-MTs), microtubules acetylated and microtubules of the otherthree Notethe relative fourthpanelshowsa composite andthe edgesandperiphery, at the leading lackof mrcrotubules in areconcentrated of actinthereThemicrotubules concentration actin with (although colocalize tyr-MTs some region the central so regionThegrowthconeis paused, in the peripheral bundlespikes F B Gertler,2003,Neuron E W Bentand loop [From the microtubules 40:209-227 l

Engorgement

E

ttf

23-38 Theadvanceof the growth cone.During a FIGURE from protrusion, extendunderpressure filopodia andlamellipodia (ribs)During bundles F-actin meshworks andelongated intracellular andcarry intotheprotrusions microtubules areelongated engorgement, depolyintothem.Duringconsolidation, organelles membrane-bound neckisfollowedbynanowingof merization of actinin thegrowth-cone bundleto formtheaxonshaftA new thecellaroundthe mrcrotubule protrusion axonshaft off thesideof a cylindrical canformby branching z10:209-227 2003,Neuron E W BentandF.B Gertler, | lFrom appearanceof extra outgrowths of lamellipodia and filopodia along the axon shaft, implying that microtubules may normally prevent the assemblyof microfilaments in the axon. The concentration of actin in the periphery and leading edge of the growth cone, and of microtubules in the more central and lagging regions,reflectsthe different roles played by the two (Figure 23-39; see also Chapter 17). Actin is

assembledinto filaments in the leading cone, the filamentous mesh of actin flows backward as the cone advances,and actin is disassembledas the cone transforms into an axon' The transport of actin toward the rear occurs in the filopodia and lamellipodia and is driven by a myosin motor' Note that this is completely different from treadmilling (Chapter 17). Movement of the actin mesh with respectto the growth 'l'-7 cone involves movement of the entire filament at pm,/min, attachedto myosin motors. For a filopodium to advance, the rate of actin polymerization at the leading edge must exceedthe rate of retrograde flow. Actin filaments have beenviewed as major determinantsin turning growth cones'a processthat is crucial for the neuron to respond to guiding signals' Microtubules are also involved, sincemicrotubule-inhibiting drugs prevent turning as well' Microtubules are assembledat the neuron'scentrosomeand transthe advancing growth cone' ported by dynein motors toward 'sfhile traveling' the microtubules with the plus end leading.

C:O N T R O L L I N A GX O N G R O W T HA N D T A R G E T I N G T H E P A T HT O S U C C E S S

o

1041

> FIGURE 23-40 The retinotectalmaps. (a)Thedorsalretinaisconnected to the lateral tectumon theopposite sideof thebrarn, andthe ventralretinaisconnected to medialtectumon theopposite sideSimilarly thetemporal-nasal (T-N) axisof theeyeisreflected in the rostral(R-C) caudal mapof thetectum(b)Themaps of thevisual worldon theretina andthe corresponding tectumareturned90 degrees but otherwise arein register. Thearrowshowshowa patternof lighton the retinaisreproduced asa setof retinal ganglion cellconnections in the tectumThetectumin mammals isreferred to as (SC)[G Lemke lhe superiorcolliculus andM Reber,

(b)

Medialr

2005,Ann Reu CellDev.Eloi 21:551-580l

can undergopolymerizationand depolymerization.A tyrosinylated form of tubulin is preferentiallypresentin more advanced parts of the growth cone, while acetylatedtubulin is enriched in central and lagging parts and in the axon itself (seeFigve 23-39). The roles of such post-translationalmodifications are describedin Chapter 2. An ordered processof tubule assemblyand modification underliesgrowrh-coneadvancement. As we have seen (Chapter !7), actin polymerization is controlled by a startlingly large set of regulatory proteins. More than 20 actin-binding proteins have been found in growth cones,most of which control nucleation or polymerization of actin filaments, or tether the filaments to the membrane. Many of these actin-binding proteins are targets of signal transduction events triggered by axon guidance signals, as we shall seein the next section.

The RetinotectalMap Revealedan Ordered Systemof Axon Connections Researcherslong debatedtwo generalideas about how neurons might get wired. One, the "resonancehypothesis," proposedthat cellsextend axons along pathwaysthat are defined by mechanicalforces.After many paths are taken and connections formed, the ones that work are preservedwhile others are removed.The secondidea, a "tprri1i, pathways hypothesls," suggestedthat axons choosetheir path bv chemicalaffinitg molecules on the growing axons contacting molecules along the way thar provide guidepostsor signals. In 1963 Roger Sperryproposeda version of the specificparhwaysidea called the "chemoaffinity hypothesir.'tH. suggesredthat growth coneswould find their way following molecular cues that form a gradient from start to desdnation,a seminalproposal that could not be properly testedfor decades.Sperry's proposalwas basedon his studiesof how the axons of the retinal ganglion cells, which form the optic nerve, are arranged when they arrive at the optic tectum. The optic tectum is locatedin the roof of the midbrain and is the destinationof retinal ganglion cell axons that grow from the retina. The incoming retinal neuronsform a map on the tectum (the retinotectal map) that reflectsthe arrangementof rods and conesin the retina, and indeedthe visual world outside(Figure23-40).The spatialmap on the retina is in essence copied into the brain. 1042

.

c H A p r E R2 3

|

NERVE cELLs

Sperryperformed experimentswith the frog eye and brain to distinguish the two models, "resonance" versus "chemoaffinity," that describe how axons may accomplish this mapping (Figure 23-47). The frog optic nerve will regenerate if it is severed,and the pattern of regeneration-the route-finding by axons-is revealing about how axons are guided.In the normal arrangement,retinal ganglioncell axons from the ventral part of the eye connectto the medial part of the tectum, while axons from the dorsal part of the eye connect to the lateraltectum (seeFigure 23-41a).For eacheye,the connectionsare made on the opposite side of the brain, left eyeto right brain and right eyeto left brain. Sperrynext added a secondsurgeryto the experiment,rotating one eye 180 degrees,so that ventral and dorsal are reversed(Figure 23-41b). If the resonancehypothesisis correct, i.e., mechanicalforces followed by a functional sorting-out processwere governing regeneration,the visual systemshould end up functioning normally, since the proper connectionswill be establishedand maintained (Figure 23-41c, left). lf there is a chemoaffinity guidance system,then vision should be inverted becausedespite the rotation the ventral axons would find their way to the lateral tectum and the dorsal ones to the medial tectum (Figure23-41c, right).ln this casethe invertedeyewould lead to the frog's having inverted vision: it would see something above and would think it was below (Figure 23-4Id). The results were clear: after regenerationthe frog respondedto a fly passingabove by shooting its tongue down. The axons were originating from abnormal locations yet finding their way to the right connections,so the inverted eye was tricking the frog's brain. The chemoaffinityhypothesiswas affirmed. It seemsa necessary conclusion...thatthe cellsand fibers of the brain and cord must carry some kind of individual identification tags, presumably cytochemical in nature, by which they are distinguished one from another almost, in many regions, to the level of the single neuron. -Roger Sperry, 1963 There is now substantialevidencethat Sperry'sgeneralideas were correct. Nonethelessmuch remainsto be learnedabout how the enormous complexity of the neural circuitry is successfullyassembled.The relativeimportanceof local versus

23-41 Eyerotation experimentstest < EXPERf MENTALFIGURE of the propertiesof axon pathfinding'(a)Normalprojection (L)tectumandventral(V)eyeto fromdorsal(D)eyeto lateral nerves, of two sequential representation medial(M)tectum.(b)Schematic (2) a 180' rotation (1 nerve and of the optic a severing operations,) of Regeneration of the eye(orno rotationin thecontrolexperiment) to occurNotethatin the rotatedleft wasthenallowed the nerves (D),nevertheless still dorsally half,noworiented eye,the dark-shaded (a). half of the light Likewise the in as seen theventralretina, contains ventrally thedorsalretina,isnoworiented eye,whichencompasses is on whichhypothesis (V) (c)Twopossible dependinq outcomes, predicts X: visionisrestored hypothesis correctTheresonance axon Thespecific properconnections functionselects because retinal dorsal inverted because predicts is Y: vision pathway hypothesis affinityspecified, of chemical tectumbecause axonsstillgo to lateral in theventralposition eventhoughthedorsalretinaisnow located (d)Theresults Y; thefrogs visionisinverted supporthypothesis

(a)

Tectum

(b)

T h e r eA r e F o u r F a m i l i e so f A x o n G u i d a n c e Molecules

Optic nerve is severed andeyeis rotated 180'

Specificaxon pathway hypothesis

Resonance hypothesis

X

(d)

Regeneration

Y

Flv

long-rangesignaling,the roles of glia, the influencesof neural electrical activity, and the signal transduction and cytoskeletal changesthat form the responseto signalsare important areas of current research.The lure of this field is substantial,since understandinghow neuronsare wired to one anotherunderlies the working of the brain and at the sametime is important for learninghow to stimulaterepair of damagedneural circuits.

For many years, attempts were made to identify key axon guidance molecules.Approaches included making panels of antibodies against surfacemolecules,culturing neurons and testing extracts for their ability to make growth conesturn, and using geneticsto identify mutants that fail to properly wire the nervous system.All these attempts worked to a degree, but geneticswas the most powerful approach, since it identified previously unknown moleculeswhile at the same time convincingly demonstrating their importance in vivo. 'We can set a high standard for what constitutesa proper guidancemolecule:it must be produced by cellsthat actually guide neurons in vivo, it must be necessaryfor guidance' the guided cells must have sensorsand signal-transductionmaihitl.ry for responding,and the mislocalization of the signal must causecells to turn the wrong way. 1Wewill discusshere four families of proteins (Figure 23-42) that fulfill our criteria and, with their receptors,provide crucial information to growing axons: Ephrins, Semaphorins, Netrins, and two related proteins called Robo and Slit. These proteins have both attractive and repulsive effectson growing axons. Sincethe growth cone is an active sender of signals,as well, the communication is mutual. After the initial connectionsform, the wiring is refined by preservingconnections that work and discarding those that do not contribute to neural function. Many cellsthat fail to make useful connectionsdie bY aPoPtosis. Ephrins The retinotectalmap describedabove is a striking example of the simplicity of the nervous system' which belies the seeminglychaotic mass of neurons in the visual part of the brain. This rather amazingphenomenonis due in part to a remarkable signaling system involving the ephrins, a family of cell-surface signaling proteins, and their receptors' the Ephs' (The word "Eph" comes from the erythropoietin-producing Eepatocellularcarcinomacell line wherethe proteinswere originally found.) Ephs constitute the largest family of receptor tyrosinekinases(RTKs; Chapter 16), with 14 Ephsand 8 ephrins in mice. Although Ephs usually serve as receptors for ephrins,

C:O N T R O L L I N A GX O N G R O W T HA N D T A R G E T I N G T H E P A T HT O S U C C E S S

O

1043

> FIGURE 23-42Families of guidance ( a ) 4 c l a s s e so f l i g a n d s molecules. Fourmajorfamilies of signaling Netrin Slit proteins provide crucial information to direct growingaxonsTheligands (a)areNetrins, Robo/Slit, Semaphorin, andEphrin proteins (b)areasfollows:Netrininteracts Thereceptors Cytosol with itsreceptor DCCin vertebrates; the corresponding but differentreceptor in nematodes iscalledUnc5Bothcontain (lg)domains immunoglobulin (bluecrescents) anda variety of otherdomains asshownThe Slitligandinteracts withthe Roboreceptor, whichalsohaslg domainsTheSemaphorins interact with diverse receptors, generically calledPlexins Someof theinteractions are through"sema"domains (redbars), present in bothligandandreceptor, othersrequire lg domainsEphrin ligands interact with Eph Exterior receptors, although signaling appears to go in bothdirections andin somecases the Ephrins actmorelikereceptors All of the receptors havesingle transmembrane domainsThe NetrinandSlitligands aresecreted andnot membrane associated TheSemaphorin ligands canassociate with membranes to varying degrees-some notat all TheEphrins are membrane-associated Seethetextfor a detailed discussion of thesefourfamiliesln addition to thesefourfamilies of proteins a few others, including thedevelopmental regulators Wnt andShh,contribute guidance additional information Rp Kruget J Aurandt, [From andK-L Cytosol Guan, 2005,Nature Rev. Mol CellBiol6:789-800,

Semaphorins Invertebrate

Ephrins

Vertebrate

+

and B .l Dickson,2002, Science298:1959l

Plexin

Neuropilin

\_]-J

DCC/Unc5 Robo (b) 5 classesof receptors

membranesby GPI links. Ephrin Bs, the other classof ephrins, are transmembraneproteins (seeFigure 23-42). The ephrin proteins affect cell migration, axon guidance,synapsedevelopment, and vascular development,but we will focus on their rolesin guidanceand formation of the retinotectalmap. Ephs and ephrins are distributed in gradients so that advancing axons can recognizeand grow toward appropriate targets. Antibodies against ephrin A5 show a gradient of protein with the highest levels in the anterior. Mice lacking ephrin A5 have guidance defects; axons rhat should have been targetinganterior tectum grow into posterior regions. Further investigationsshowed that cells expressEph receptor proteins in two orthogonal gradients, to control axon 1044

.

C H A P T E2R3

I

NERVE cELLs

Plexins

Eph

guidance along each of the two axes (Figure 23-43).In each axis, the graded amount of receptor in retinal ganglion cells confers differential sensitivity to specific ephrins emitted from the tectum targets.The Ephlt's and ephrin lfs controlling one axis do not cross-reactwith the EphB's and ephrin B's for the other axis. Therefore, axons can "learn" their positions on an XY coordinate system by reading levels of iigand for which they have receptors.To give one example, axons that have EphB2,3, and 4 receptorsare attracted to places where there are high levels of ephrin 81 ligands, so axons originating in ventral retina tend to go to medial tectum. The full situation with all thesegradients is more complex and not yet fully understood.Eph tyrosine kinase signal transduction influencesthe small GTPasesRho. Cdc42. and Rac, thus regulating assemblyof the actin cytoskeleton and controlling guidance of the growth cone. The activation of an Eph receptor may cause arrraction or repulsion of a growth cone, dependingon the cell.

Retina gradients

Tectum gradients

D

EphA5

phB2l83l84

E p h r i nB 1

,fo-ffiffi 6_w@

w

Eph46

EphBl

#k EphA4

E p h r i nB 1

h W

E p h r i n4 2

EphB2/B3

Kry EphAT/A8

EohrinA2lA5 23-43 Gradientsof Ephproteinsform two A FIGURE gradients in the retina orthogonalsignalingsystems.Ephreceptor in the superior colliculus areshownon the /eft;ephringradients (retina) (tectum), on the nght Gradients alongthe nasal-temporal (tectum) gradients in along axes are shown blue; androstral-caudal (retina) (tectum) axesareshown andlateral-medial the dorsal-ventral in red Themorecolor,the moreproteinpresentThewhitearrows (seeFigure 23howthe mapsarerelated indicate andarrowoutlines so for EphA-expressing axons, 40) Ephrin A'sarechemorepellent EphA receptors tendto go fromtheir retinalganglion cellscarrying in the rostral sideof the retinato destinations originsat thetemporal of ephrrnA'sislower tectum,wherethe concentration Semaphorins Semaphorinsare a diversefamily (seeFigure 23-42\. and much remainsto be learnedabout all their effects. They were named for the alphabeticsignalingsystemof flags that was usedto communicateover long distances.Semaphore signalscan spell out any message,but in the nervous system go away.They are semaphorinslargely caffy a singlemessage: potent repellents.The family of two invertebrateand five vertebratesemaphoringlycoproteins(Figure23-42) includessome that are secretedand somethat are membrane-bound.This impliesthat someof them act on adjacentcells,while othershave a longer reach. Motor, sensory,olfactory, and hippocampal neuronscan be repulsedby semaphorinsignals.Semaphorins transmembind to receptorscalledplexins that are single-pass proteins capapicture is receptor proteins. The overall of brane ble of acting as scaffolds both inside the cell and outside, assemblingprotein complexesand modifying their activities. How exactlythis modifiesgrowth-coneadvancementis an area of current research;at leastpart of the answeris differentialadhesionto cellson one sideof the srowth cone versusthe other. Netrins Netrins are secretedproteinsrelatedto laminin (see Figure 23-42). They were discoveredin geneticscreenslook-

N e t r i n2

Netrin 1

Vertebrate spinal cord, crosssection

Unc6/netrin Nematode C. elegans, cross section

23-44 Evolutionaryconservationof netrin signaling. FIGURE with cellbodiesin neurons spinalcord,commissural Inthevertebrate by attracted growaxonstowardtheventralmtdline, dorsalregions followa similar neurons sensory C. elegans' Intherirrorm netrinsignals. followthe whilemotorneurons pathtowardventralUnc6/Netrin, axonsarealso vertebrate Certain pathawayfromUnc6/Netrin opposite that in wormsviamutations werediscovered by netrinsNetrins repelled as neurons, andin vertebrates bysensory alteredthe pathfinding pathfinding of commissural thatcouldinfluence molecules secreted cordexPlants in spinal neurons ing for misrouting of neurons in C. elegans,the nematode worm for which every cell and every neuron has been identified. About 30 geneswere found, three of which affecteddorsal-ventralrouting of the sensoryand muscleneurons (Figure 23-44\: unc-6, which encodesa netrin protein; wnc-40, which encodesthe netrin receptor (called DCC in mammals); and wnc-S,which encodesa secondtype of netrin receptor.Unc6lNetrin mutations affected both dorsally directed and ventrally directedaxon extensions.Vertebratenetrins were found in studiesof how commissural (crossing)neurons find their way through the spinalcord (seeFigute23-44). Theseneurons emerge from dorsal regions of the spinal cord and extend around the periphery of the cord toward the ventral midline. To test for the presenceof guidance molecules,parts of the spinal cord were cultured either separatelyor together. When the most dorsal part was cultured near a ventral part' axons grew out toward the ventral tissues.No axon outgrowth was observedwhen the two parts were cultured separately.Extracts from the ventral part had the same activitg stimulating axon outgrowth from dorsal tissue. A heroic protein purification, starting with about 20,000 embryonic chick brains, succeededin identifying two proteins that were potent chemoattractant signals.Both were netrins. Netrin 1 is highly expressedin the spinal cord floor plate' the most ventral part. Further proof of the role of netrins came from mouse gene knockouts, in which the commissural neurons were unable to properly find their way (Figure 23-45). Netrins guide axons to the ventral midline in nematodes, flies, and vertebrates,an example of evolutionary conservation of protein function over more than half a billion years. A puzzle remained.The worm version of the protein appearedto have two functions: attracting the axons of sensory neurons to grow toward the ventral midline and driving the axons of motor neurons ,Luay from the ventral midline' The simplest possibility was that netrin is attractive to some axons and repellent to others.Indeed evidencequickly emerged

GX O N G R O W T HA N D T A R G E T I N G T H E P A T H T O S U C C E S SC:O N T R O L L I N A

.

1045

Netrin-l-

Wildtype

EXPERIMENTAL FTGURE 23-45 Mouse netrin-/- mutants havecommissural neuronguidancedefects.(a)Wild-type tracing (red)thatoriginate of commissural neurons (top)andgrow dorsally towardandacross (greenarrowheads) theventralmidline underthe influence of netrinproduced (floorplate)cells(b) by ventralmidline Homozygous netrin-/- mousemutantManycommissural neurons wanderoff track(greenarrows) beforereaching theventralregions, andothers(greenarrowheads) turn instead of crossing theventral midline. of Marc Tessier-Lavigne, [Courtesy Genentech Inc] that certain vertebrate neurons found netrin repulsive. Genetic analysesin worms showed that Unc-40 receptor is required for attraction by netrin, while Unc-5 receptor in combination with Unc-40 is required for repulsion by netrin. Another puzzleremained.If the axonsof commissuralneurons are attracted by netrin coming from the ventral midline, how do the axons continue to grow after they have crossedthe midline? One would expecrthem to turn around and go right back. The solution to this puzzle awaited the discovery of still other key playersin axon guidance,Slit, Robo, and Comm. Robo and Slit Guidance Molecules The path of growing axons in the insect nerve cord is reminiscent of a subway map (Figure 23-46a). Geneticsallowed the discoveryof guidance genesand proteins that affect the pathfinding process,changing the map. A large set of random mutations was introduced into the Drosophila genome to generatelethal mutations. The mutationscould be carriedin heterozygotes, and when the heterozygotesof eachline were crossed,a quarter of their progeny were homozygous for the newly induced mutation. These progeny were stained to show the embryonic nerve cord, the equivalent of our spinal cord, which in the wild type looks like a ladder (Figure23-46bl.In lines of flies where the mutarion was in a genenecessary for axon guidance,defectsin the nerve cord could be seen.Among the genesidentifiedin this manner were three, slit, roundabout (robo), and commisswreless (comm). They defined yet another set of critically important and evolutionarilyconservedaxon guidancemolecules. Slit is a secretedprotein (seeFigure 23-42) made by midline glia. The Slit receptor is Robo, a single-passtransmembrane protein with only a short sequencein the cytoplasm and fibronectin and immunoglobulin domains on the outside of the plasma membrane (see Figure 23-42). The Robo/Slit complex is a chemorepellentinteraction. The presence of Slit in the midline servesto repel axons containing Robo receptors,thereby ensuring that ipsilateral (same-side) neurons do not crossto the opposite side. In loss-of-function 1046

.

c H A p r E R2 3 |

NERVE cELLs

s/it mutants, or in double mutants that lack two Drosophila robo genes,axons go to the midline but can never leave (Figure 23-46c, d). This can be explained by the fact that, in the absenceof Robo receptor or its ligand, the chemorepellent Robo/Slit interaction at the midline does not occur. This raisesthe question of how an axon that needsto cross the midline is first attracted to it and then repelled from it. An axon that will cross the midline does produce Robo protein and should be repelled by Slit, but the axon is initially refractory to the Slit signal becausethe Robo protein is trapped in the Golgi network by a Golgi protein called Commissureless (Comm) and neverreachesthe cell membrane.Once the axon reaches the midline, Comm becomes inactive and Robo reachesthe surfaceagain. The newly accessiblereceptor allows a responseto midline Slit, and the axon grows away from the midline on the far side.Loss of comm function allows excessive Robo to reach the surface,so no axons cross the midline (Figure 23-46e).The expressionof comm is normally regulatedso that it is "off" in cellswhose axons are supposedto remain in only the left or right longitudinal axon tract, and is "on" in cellswhose axons must crossto the other side. None of these protein guidance systems is dedicated solely to the nervoussystem;in fact, all of them are employed in other tissuesfor various purposes.Indeed most of the special attributes of neural cells appear to be more or lessexaggerated versions of processescommon to many or all cells. That is apparent ( 1 ) in the polarization of neurons from dendrite to axon, which employs cell-asymmetryproteins, (2) in the neuronal intracellular organelle-transportsystems,which depend on variations of endo- and exocytosis, (3) in outgrowths of axons and dendrites, which have features of chemotaxis,and (4) in the use of channelproteins to control ion flow. Neurons are a variation on familiar cell biology themes,a variation with enormous functional Dower.

DevelopmentalRegulatorsAlso GuideAxons In Chapter 22 we becamefamiliar with a set of secretedsignaling proteins that control cell fates and, in some cases,cell division. Sincethesewere discoveredfor their roles in development and differentiation, they were not initially suspects in the hunt for axon guidancemolecules.Yet it turns out that at least three cell fate regulators, Hedgehog (specifically Sonic hedgehog,Shh), BMP, and Wnt proteins, can also be axon guidancemolecules.This is in a sensereassuring,as the sum total of complexity of the other guidancemoleculesstill seemsrather low compared with the challenge of routing millions of axons on complex paths. In the developingspinal cord (seeFigure 23-44), even in the absenceof Netrin 1 some commissuralaxons still extend toward the ventral midline. Another protein, Shh, made in the floor plate, accountsfor that remaining guidanceactivity. Proof of its role came from the discovery that (1) cultured cellsthat secreteShh reorient commissuralaxons in tissueexplants, (2) isolatedneuronsin culture turn toward a sourceof pure Shh protein, and (3) mutants affecting Shh signal transduction interfere with axon pathfinding. Thus commissural axons are guided toward the ventral midline by both Netrin

LVol

9Nll_l9uvl_oNV HIMOU9 NOXV 9N|]'IOUINO) :SSl))nS Ot HtVd lHL

'uorlJaJrpJregt ur Sur,rorudeel or auoc qt.,llor8eqt elelnturls sluelreJllv 'delre urnl ot lr esnef, ro 'Surcue,tpBdols ol auor qluor8 oqt osn€r saf,roJe^rslndag 'uorala>1soiloalnqnl -ortllu pue ultf,e er{r Surruln8ar dllrarrp dq auor qr.,no.r8 aqt rrolJe solnroloru erueprnC 'aurloqrldtacp sE r{Jns srel -lrrusueJloJnaudq polcenle ag uel oJnllnf ur seuoJ qu,rorS 'passncsrpalpq a^\ sleu8rsuralo.rdoqt ol uortrppp uI 'uoxp oql Puolxa ol de,vr.qJrq. ]noqE suorsrfsPe{eu ol Pooqroq -q8raus1rSurqo.rd'reroldxe uE oJuesseur sr auor qra,ror8aq1

urnl ol auo) qlMor9 eql osne) saln)alow a)ueplnD uoxv '6V-tZ ern8rg aas)urntcet cndo eqt pre^\ol suornau uor13 -ue8 leurlo.r1o qr-nror8aqr Surprn8ur pello^ur osle ere sleu 'suorneu -8rs lu1tr leJnssruruoJ;o uorler8ru JorJetueeql ur srraJepsosnEf,uortlnpsuerl leu8rslu16 rltrm JJuoJeJJetur puE 'sleu8rslul1 ol polJ€rlle oq uer ernllnl ur suornaN 'peaq

oqr reau lunoIUE rsaq8rq aqr qtlv\ 'aleld roog egnl lBrneu eqt ur luelpe.r8 B ut passa.rdxesI lI 'Pe^lo^ur st relnotlred uI hiul\|elull atuEs0tl1lE soxEqloq fnoqB suoIsIJePO>lEIu ol e^eq sllel uaq,l.rteyd ur sI sIxP qJlrl.&\lnoqe PesnJuoJ Surue8 ruo{ sller lue.tard deur qu.tor8 A-CI snsra^ d-V roJ uolsds Surleu8rstlurtslp e to esn eq1 'Sururelred4-y uI alor tuetrodrur ue f,e1dsleu8rs urelord-PalerJoslur6. ler{l .l.roqs aJru pup'allqdosotq'suuBa1a'J uI seIJeAotrsIC'uoISuauIP '(1er or pBOI{)stxe srql ur erueprn8 tnoqe u,tou{ sI ssel1nq rraql PuIt lsnu suoxe aqr 8uole ,(ezlr, (4-y) ror.larsod-.rotratuB ,(uern 'tte1 q '(LV-7T ern8rg) Pearl eql PrE^ ol .{ldreqs urnl daqt 'lr ssorJ pue euIIPIu lerlue^ eql qleal suorneu lerns -srruruor suoxe eql reUE'pror leurds8urdo1a.l,ap aqr u1 Jo 'sdryg.,(q ,.8urqsnd,,PUEqqs PUPI uuloN 'srreJeP dq .,3uq1nd,,ot puodsa.rsuorneu Iernssruluof snLIJ arueprn8 uoxe IEJnssItuIuoJeAEqor PunoJ aro,rlsaua841aqg urPual JOJluPlnlu elll/t{ 'suoJneuIeJnssruuor lader suotSe.t 'qqs PUEI Iesrop ruo{ SunBut8trosutalotd 41,q9tlasre,tuol

' a u r l p r u r ' a c u ou e q l e J o u ro u r l p l u l oql ssoJcsuoxe Aueyl (oqot) lnoqepunor

eql ssorcranausuoxv (Luuoc) sseletnss!u,aoc

'l! e^eol ro^ou pue aurlprtll eql ]e asoellocsuoxv

'ocuo Aluo pue acuo eurlprLuaql sassorcteql uoxe uP spuos uornau qceS ,,leralelerluoc,, edAl P111Y1

iOLuuneN'866L' le]arabeas \SZV-AOV (a-q)8s ilgz uDuawv)l]lluaPst86L ruPrlseS lALrrorl L\ puPueurpooD (p)l6utpurlqled ftlpulelpojeajaq]ose) s a l p u n qu o x e uoxe+osolnlte+ Sf r.llorl Llleaul uMoqsaresuorlplnui sofuqu.re ; e u r p n l t b u o l aoiq]roi sno6lzou.roq luara11rp eLl])jeLUo1;euedslLl]ul anlqpaulpls +osp.to)o^lauaq](a-))ui outlptu.t areral)nuMe]V uaassrurelledad{1-p;rnn aq}(q)ul Ourpur,tqled uoxe 1nofurer{;redord}ou op }eqlsluelnLu })o}apo} posnoq uer ofuqule elrqdosor6 e ursornssr.rluror aq1 (a-q) 1ourelledpeurels-uMorq 6urpurlqled uoxeradordro,Lperrnber saln)alour {1r1uepr o} posnuaaq eneqses{;eue rrlauabareqnn oql urpa^rasqo ofuqueelrqdosorq }eq}o} {1q6rq srfutrnroaql (anlq) relrLr-rrs sllel,,d,,eql}o suoxpeq16urnnollo} sepazru6oret aq uer (eldlnd),,D,,pa)reulseuotq1,rnorO aqf patpnls ' ' . . . :-, { - " puepazrubo>al oq ue)'ZdpueZVsuoJnau rrlredsaqlelduLexa pueparlrluepr ro3 o,lqua aqllo ozrsebreleq1o] anppaMollo+ oq suoxe rouelsod {;rpeer uersuornou aq}urqynorbuoxe araqnn'leddoqsserb lenpr^rpur jo serpnls urol+pailnllsuor senn ue;6erpaqf salpunq uoxe;eurpnl6uo; ,iq papauuorareq)rqlrn aq] aresalnssr.rtuo) suoxeJoualuv aql 1osOunr,, ,,rappel euoc qyaorb 9 sajnssil..uuro) aLl]Lrlo]puessor)leql suojnouAquo)e]sqledaq] yo snnoqs uler6erp srqf(p)'pJo)aruaupasu! aql ul sluelnu saldurexa arueprnbuoxe u! suollelnur1o sadflouaqd W-EZ]Un9lJ >

suoxe d 6u11ce1uoc erpodop;pue auoc qyrnotb9 o^lau ;elueurEeserlu;

.'

o^rou llelueru e l u e u6asrelu; 6astelu; suoxe ,,.a"^ . d bur^^ollo]

aart {Eerq ol auoJ LIr,^AorB aqr Jo upd s,\\olle dla,rrrelas 11ac ol{t apls teql uo surr8aturqlr./{ ef,uareJralur tltuelrruoruo3 Jo '1eu3rsaqt tsoreauouor arJl eprsaql uo dllerlue.re;a.rd para8 Jo -3ll sl uolalalsoldc eql yo dlguassesrper{J 'GV-tZ arn8rg) urlle Jo uortezt.rerudlodapaqr Sursnel dq sauor qr.r6or$yo asdelloc aqt otelnrurtsstuayladerraqlo pue sur,roqdeure5 'tuetrPJue

s'l'll) IAUIN I

tz urrdvH)

8r0t

[9zl-9lLiLDsornaN ^a a.tnleN'ggg7'6uat17 0 [ puezal]rogy\ 1 Llorll ptpodoltiaq] ]o ouo ur +ze) ]o lsrnq oq] oloN lorluor leurolurue se so^rospue a^t]tsuos - + z e ) l o u s ta { p p e 5 - e r n o 1 q } : + z e l l e l n l l o ) e r } uutt s e b u e q ta ^ r a s q o o] posnsr (t-onlJ)e,{penrlrsues-e) (s) 'aur} tolo ouo) q}rv\or6e ul oq ue) saxnll *ze) gv-Ez lun9ll lvtNlhlluldxl Pornseou.f v

-n

uE ot tsesoll euor qr,vro,r8eqt aprs oql uo rncro d11e Jo 'qt.^aor8 -reua8sasearrul [*zeJ] ]Ero-I leuorlrarrpo)uen]Jur uer unrrlpf, ur sa8ueqcellqns erou pue Ielol a.royq'qt.u.or8 'selurr '[*,BJ] -lno ole]nturJs upr lllercedse pa8uolord ro; 'leraua8 ur rlrJnJu siqtq qtrnor8lno 1eqo13Surrnpat os 'suretord Surretsanbas-E3 -ul pue 'slauueqr 'sdund *ze)

c fl o

'Tl

A

'uen9 [ 008-681:9 lol8toN tp) ^aAarn]eN'SOOZ t- ) pue ']puprnv!o6n.r) urorll seupale^t])e r ic u oq]lepunLujo+ ]o o)uonllut '(gV-€Z xuieurrplnllo)erlxo oql ol sluaLuq)epp uubelurpue')eUo^t])e a.rn8rg) € ueqt sselJo +o puotos Jo suortreeqt ot 3ur.&o 0 ) u 0 n l l uorq ]r o p u np u e l x e r p o d r l l o uolLell,l , U O la, ,q tu o a l t q M u e o n spor.radJeAoJnJJo uorleJlueJuoJ ur sa8ueqctrlg111erql *.rJ (uorle^r])e oqg q6notql)urso,{ulolte pospor)ul 'sror^Eqagauoo-quto.r8rra11edpuorts }o uot})el}uo) uer seseelet*zeJ '(uotle^t]lput pue'solnqnlol)tul seuo] onp) ',,Go1orq +ouotleztltqelsap IIel relnlolou a.trtelSaturur rualqordSurleu 'llo)srq] uubeluruJoJ] ouo)qiar'ot6 aq]]o ]uoLUq)elep eqt 1o,,1q0u,, -nsEJ 'efuo e ruatsds arueprn8euo uer1leJorrrol spuods le uo'sasne) sleubrs eq1lo fu1au.ru.r{se eqf auo)qunot6aq}utn}oi -er euoJ egr ,/l\oq puElsJapunot luelrodur d1.re1 qr-r,roi8 s{enaq}ed .relnllale.t}ut puestoldateturxeld q6notq}})e Cteuasa)tl -nottred sI lI aauoc gl.Lror8 eqt rreJ;e srot1pJecueprnfi eql s11ar er16 uo surroqdeutes lo V€eLUaS a1r;suuoqdeLles alqn;o5fenne 'aprsreqto aqt uo esdellocrreqt Sursnec.{1asra.,r. IIe op .^ oH urnl ol seuorqyvrolbesnersurlotldeures6t-€ZlunglJ V -uoJ Jo Alsno-ro8r,reJou puetxe ol ro elgets eroru eg ot auor eqt Jo oprseuo uo (qloq ro) erpodrlleuel .ro erpodoyr; Sursner dq paqsrlduoJre sr euoJ rlt,l.r.or?e p Sururnl xtJleur relnlloseJu:l

'tOOZ ' [ 886 t-t86 L:20€a)ual)S le 1a enolnAsln{1 y Lr.rorl]

VT euros

rouoluearou.rare]eq] sleubrs1u711 o1 puodsarqloB qied lerolel oroure 0)el ]eq] suorneu(por)pue outlptLU aq] o] osol) pualxo]eq] suornou(uaar6){q ue1e1sqled}uolo}}tpaq} 6utmor1s }nq'q}pd aLues oq] +o Mar^,,>1ooq-uedg,, (q) pol ut pale)tputuolnou lernssrr.uulo) e;o qled aLl]qllM uMoqssrofuqule1erplo-{ep-g1e io pro>leurds aql (e) 'pJo) ;eurdsaql u! suoJnau leJnsstruruo)1o uorle6uo;e rouagsod-lor;olueoql lolluo) s;eu6rslu6 4t-€Z lUngll V

JOTJAISOd o

/ JOTJAIUV

a u r l p r ulre r l u o n

louolsod

(q)

le.rluan

-' ,i'

alelo rooll

loua]

uoinau lernssil.Ilr.Llo3

(e)

lesroc

6V0l

lunInJ IHI uol lntl)ldsutd

Surroldxe ur sorup^pu d.reurp:oelxe dq palalpred ueeq e,leq uralsds snolJau aqr;o dSolorg ilel aqr ur sotup^pp er{I 'uorle.reueBar Surn -ultuor r olle ol rlnpp er{l ur tsrsradoruospue tllerrrur er13 pue suoJneuulJoj slleJ tuJls 'uoDf,elo:d eunuur apr,ro.rd pue 'uotlerutol asdeu,,{solelnrurls 'uorlelnsur apr,rord er13 'sa;rlesruaglsuorneu aqt puodag 'etspl pue '11eus'punos 'uted 'qcnol 'rq8rl o1 elrlrsuas sllec drosuas oppru a^eq surato.rdreqlg 'sasdeudsssorce uorlrnpsuerl leJruaql Jo speeueqt or pordepeuooq ser{srso]dcoxopue srsoldcopua 'slerluolod uorlce rsel dlqrpa.rcur 1o ualsds IESJe^runor{J to uolssllusuErlIEuoIlserIPIun^/rollEuoql Pue slrrtualod Suusa.releeJf,slouupqoporeS-e8ello^pup -pue8rl 'sllal to sortrodord lrrlrele oql Jo uorlpln8ar eql o] pardppp eruof,eq eAPq suorlEJluaf,uofuor Jo loJluol trlelsoouoq JoJ puE selnf,olou tllogElour 1o trodxa ro t.rodur ro; pe,r1o,radlqe ..{la,rrlrurlsrptsoW 'sllor uaa,r\l -go.rd 1rq1 suroto.rd IeuuErll -eg slJeluor elqrsrelor pue e^rlf,elosapr,ro.rdselnf,olou uorsoqpeIIeJ-llo:) 'suB8ropup sonssrtreqlo .{urru to tuetu -do1a,lapoqt ur pasn osle ore leql sleu8rseruos Surpnlcur 'slo8ret rreql ot suoxe Surpuotxaaprn8 suatsds 3ur1eu8r5 'sassarord puelxe pue aler8lur or sllal ,\4.ollesJolory .uorl -38 to sotrsrodo.rd rreqt ol salnJeloruoJf,Erupue sa11eue3.ro elotu lorluoc drlrelod pue sossoJordlrodsuerl 'surel -ted peqouerq xalduroo dlsnourroua e^Erl srorlto pue 3uo1 releru e eJe oruos tegl os (s11ec aql sadeqsuolole>lsotdo er{I 'suornou Jo speou lerrads aql ol perdepe auoreq aleq-uortrunt IIel 1o suatsds IESroArunaql-sureto:d pue selleuu8ro.vroquoosolpq ol1 'plro^/\^. oqt qrr.Aa of,eJrelur rno sE a^JOSleql sllal e^rou oql Io soruadord elqelreuer aql or uollJnporlur up pepr^ord a,req a,n ratdeqc srqr uI

'GV-tZ a.rn8rgeas).(lquesse 1ela1e>lsoldc lorr -uor leu elrl sosEdlc lErll llsurs 'o uonp^rlJe lprluoraJJrp pue 'sarlr,rrlceaseleqdsoqdpue aseur>1 ur saSueqc(urnrcleo ur saxnlJesnef,UEJleu8rs acueprn8e;o Surpurqeql 'euol qr.uor8 oql Jo auerqr.uoueqt uo srotfpJ arueprn8to srlelJo 'urnt ol euoc qlzrro.rSaql Ipool Jo llnser oql sr 3uru.rn1 esnec 1{ltcarrpurJo .{lrcerrp 'saynralouracueprn8 uoxv r '(w-tZ ern8rgaas)s3oqa3pe11 pue's1ur1tr 'sdwg :suorlrunJ ocueprn8 aprrr.o.rd s.rolBln8arleluerudo -la^ep sE pertuuopr1s:r;suralo.rdpolenes spuDl oorrll r to 'gV-tZ orn8rg aas) uror; leme mo.r8dlruanb lr -esqns pue 'aurlpnu eql pJe.{\ol ,ra.or8ol suoxe s,r\olle leql qlrr^\s E rrrrot deqr .raqra3o1'areJrns IIOJer{r saqf,par oqog rel{req^.slorluof, teql uralord r31og e sr ruruoJ olrrl.4 'roldece.roqog uo stre teqt 'suretord leu8rspeteroose sr lrls IuIuoS Pue 'llls 'oqog SurpuIJ ol pel suearrs rllaueC r 'ProJ lEurqs aleJqalJe^aql ur eurlpru IPJlua^ eql PJP.ry\ol suoxE lPrns -sllutuoJ aprn8sur:1ep 'GV-tZ arn8rg eas)sleuSrsazrrslndar aq osle UEJsurJleurnq 'JJCI se qJns srotdara.rur.rlaudrreo lpqt seuor qr,t.or8 ot olrlJerile dlensn are sleu8rsurJteN r

'GV-tZ arn8rg oas)seuooqr.tror8 uo lJeJla a,trslndare e^Eq 'sroldacer urxald JIOqI uo Sutlce 's1eu8rsurroqdeureg r 'uJalsds 'sexe puoSoquo o.ryuoqt Io Ieuorsuourp-o.ryqe Sururrol qrEOur lro,!\ surotord qdg yo sessellruereJtlq 'lgy-97 am -3rg eas)deru lelcalouller aql lJnJlsuoJ daqr se suoxe uotl3 -ue8 leuner SurprnSut luel.rodrut e.resqdg pue surrqdg r 'PaAJaSUOJ u00q

aaeq 'eoueprn8 qlvror8 uox€ euIIpIu lertue^ a4t1 'luaur -do1a,rapur suoltf,unJcr;rcadsrlogt to etuos uoAOpue 'uotl -n1o,reSurrnp pe^rosuoJdlq8t.{ uoeq a^eq saIIIIueJeql IN 'suortJunJ.rreqtdyrruaprdlasrca.rdot tFtIJJIp lI a>lEIrIuoqo serlrrueJutalord osot{l to sregluetu oldrrpru JqI 'surtuo3 pue 'soqog 'slry5pue lsroldooe.rf,f,C pue sulrlou lsurxold lsqdg pue surrgde :(ZV-7Z arn8rg aas) pue sur.roqdetuas partnuepruooq e^pq salnf,oloruecueprn81o suelsds Jnod r 'souof,I{l,ltor8 dq uesoqr qred aqr af,uenltulxIrlPIu relnllof,ertxe eqt pue suorneu raqlo pu€ seuor qlmor8 Surcue,rpeJo serpJrns oql uee^ laq Surpurq 1o sglSua.rls ruorourq 'sleu8rs utalo.rd af,EJrnspu€ pelortos Jo dlatrerr e dq papr.ro:dere sanceql'fiV-tT, o.rn8rgaas)uotleluetro Ierlua^-lEsroP Pue lereftl-lelPeul ol se Lurotul leql senl eoueprn8 .&\ollot unlJol ol Euller uror; 8ur.tlo.r8 suoxy r 'runlJal aql ur duru eqr qrr^\ pou8rlesI pullor eqr;o deru oqr r?qt os JOpJolerleds uleluleru tunlJol ol EuIloJlteuuoJ lerlr slloo uorlSue8Ipullor eql'rcV-€Z ern8rg aas)deru B se Paluas -arde.r sr pue 'urerq oql Jo lred lensu B 'urnlcol aql ol peuJer sr Euner eql 8ul{rJts rq8rl;o 1nodr1yerledsaqJ r 'Gt-tZ a.rn8rg aqr qrl^\ dn deol ol 'ssacord aas) auoc qvrro.r8Surcuezr,pe srql Surrnp puelxa ol anulluor selnqnlorrlry 'passud seq auoc qt.tro.r3agl reUE sturoJIEI{I uoxp eql lE selgtuesseslp pue euof, qluo.rS eqr 1o dlr Sur,nor8 oql le sluoruell; otul selgruesspurry '(gg-g7 a.rn8rgaas)sluaueltt UIIJEralrogs Jo EOse ur pesrelutul solnqnlorrltu to elPunq lerluof, E Jo stsrsuof,auof qtry\oJ8aqt sadeqsreql uolalolsordr aq1 I 'uralsdssnolJeu eql ur Sut.rtm;o ureued IeIlIuI eql to lueuruJelap roleu e eroJeraql ete pue uoxe SutlrorS aqf aprn8 seuos qyr\orC r ' orpodolt] pellet suoISn.rlo.rd e1q:a8uIJIIerus a,teqdaql '(n-f.Z arn8rg ae5)uoxe ue Jo puo Suvrro.r8aql tu otpodtllawol p?Iw suet pauoDelJer€ seuor t{l^\orC I 6ulle6leI pue r.llMorg uoxy 6ut;;oJtuo) :sso)lns ol qled aql

'asdeloc ol auof,qvrtor8 eql SuISnef,_orotoJor{l 1{1ques -se urlJE aJnPaJPUEot{u alE^IlJeuI leql soals}o sauasE uo spuadep uorslnde.rauoc-qulor8 ruapuedap-urrqdg'uorsual -xa suopupqe euoJ qr.uor8 JI{t erall,t selnqnlorf,ru }o ssol E ol pPOIol s.reeddeullfe Jo sso-I 'xIrlELuJElnllalerlxe luo{

salnlelou roldof,orelset or{t lalset .rnospue tlps roJ selnrelou roldaror eqt Jo suorlf,unt pue sarnlrnrts eqf aredruo3 'y1 ilsEJluoJ lfelep ol uorler.uroJur11ooroldacerotoqd ale.rSolursllet asoql op ^{oH (uorlf,OleplsPrtuor IEnsrAur pa^lo^ur rr" r.tt$ r{Jlr{l( '€I

euof, Io porp Jouorezrrelod.raddq or "";il"#ffilJl11

-drosqe rq8rl ruorJ epef,sef,srr.{rur sdats relncaloru oqt eurl -lno 'urEJqeql ol uonpruJoJur IEnsrAto uorssrusuEJleql 01 speal dllenlue,Loqtlq^^ 'uottout ur les sI sluala Jo epel 'zI -sec e '1q3r1dg parelnuns sr IIar euof ro por ? uoql( lsrqt suoddns ef,uepr^ereql1 isoll .II -rsal crldeuds Surlcdca.rur unueudp Jo olor aqr sr leql( qlenuafod uollf,? uE oleraua8ol lou Jo reqleq,ry\ auruJelep ol sleuSrsaseql ater8atur uorneu oql soop ^\oH (pe^refal o.resleuSrsr{JnsqJrq,ry\ le uoJnau oql Jo uorsuelxa er{lJo orugueqt sr tpqlN 's1eu3rsd.rolrqrqurpue ,{.rote}rcxaa1d -rllnru elrefar 'ure.lq eql ur esoql dlrelnrrged 'suo.rne11.6-; iPaOos tearS qcns le ereld elpl ot ssarord srqt ^\olle susruer{Jeru leqlN 'stu I u€r{t ssel ur suaddeg srr{I 'euerqr.uaucrldeu -dsa.rdeqr qtr.ry\asnl ,{yprde.rsolJrso^rrrdeuds 's11acpalel .6 -nurls ur yertueloduorlf,Eue Jo IE^rrrEaql Surzrollog

s'l'll) 3nu3N I

g 00L puueqr petEB-o8etlo^ gJ0J selrrsa^Jlldeuds 969J esdeuds g00J uorneu drosuas ,LqL sile) uuE.,\^qrs ,L1f uon)npuoc drolelles 8Z0L sPot TVgy drat letJelounor ,00L rrorlezrreloder 19g y p ornd d.rolcB.rJe.r zg0J salrrsso pygy sa$totpuapo8rlo

EZ U]IdVH)

090t

t00J qteeqsurladur 900JuoJnouroloru €20L puueq) pereS-pue8q g00Juornourolur g totdeter drolrgrqur 79 S zg0Jsnlnlunuror.l 0t0L eucoquror8 796JrprauoyS 976Srcldacerdrolelnxa 6Jg; srsordropua ,00L vo\ezr.relodop g00JsellrPuoP gz]L seuor z€jL eelqror

9gg y s totdeter droreylo

&rurgeorueqc /g]L s]IdetoPo 7791ssegrod.(q ;96; srordarnou 6Z0LPunottns-raluef, €00Luoxe Vygysetltonse ,00L ]Pquelrd uortce

t00J rsElqornou

g00Jolueprn8uoxe

900J sraDrursuerlorneu z00L uorneu 6J0J uonJunI relnrsnuo.rneu

sulal Ao)

.SOSCIEUAS

alrsnru-elrou dlllrlcu aseralsa eurloqrldtare le peseerlep Jo prpord 'esdeuds uorlelrlf,E alrsnr.u.ro; secuenbasuor eqt aql 'uatsds snolrou oqr paJJp le pesealeJJollrrusueJloJnauuoururol E sr ourloqJldtary .g Surlearl;o qo[ raDoq e Surop puE urerq aqr 8ur l€qt sesEasrp .OI -puelsropunro1rodsord Surtrcxaue'ueddeq ol sarue^pr esoql JO rcl:el e ,{q rar,rueg sepou uae,ryrtoq Suneds aql Sursearr lradxa ol uospeJ,{:arrasr araql dysnoauellnrurssuoJneuJo to -ur uorlre ot saouanbasuotoql srogunu o8rel 1o lo 'suo.lneuepurs;o sertrlrlfe eqt alelndrueru to uorte8edord lerlualod llrperd 'uox€ eql 3uo1erar,rue1 to sopou te sdurnd *)/*EN ol sdemraDeqto luarudola,raperlr qtr.ry\pourquor'olrse,turuou pue slouueqJ *eN pareS-a8er1o,r pue e^rsplur 'spoqlaru SurSururur sluaruolordurr ,(g peqs4d ;o Surratsnlr sasnef,uorl -eurladyrqluorteurlodtu sr ler{lN 'uoxe ue 3uole uorte8edord -ruorf,p oq or dla4t sl sHI 'strnrrrr pqop rnoqe uoueuroJur .L asrcordaroru qtr,r\ dSolorg IIer relnJaloru eql lJOuuoJ o1 sde,vr lerlualod uortre ;o drrcola,r eqt sespeJJuruorleuryedy4 i^poq IIel eql Pr?^ o1 (6sPrE^\ Surpur; arrnbar 1pr, drouraru yo SurpuetsrapunIInJ y dSolorq -IrEq,, Surle,rerl urorl elrou e sluala.rd leqlX ,9 IIel relnJalou;o sanbruqoalaqr qSnorql elqeqceorddearuoreq 1eu8rs 'uoxe uP u./I\oPsle^eJl se esPeJf,ep seLIsuornau Jo sragurnu slgEltreJlur a IoAur ol luaas plno^ lr reqr uralqord Surleuosel E snr{I 's1eccrtdeudslsodpue -ard eql l(usaop lerluatod uortJe ue 1o qr8uarrs erlr dr{^{ ureldxE .S ur qrog 'sasdeu,{srolle teql sa8uegr rEInJOIotueqt re peprrrp isleuueqr uor paleS-a8pllo^rerllo o1 dldde drqsuorlel eJeserPnlslueJJn) 'sorJor.uaru -aJ uollJunJ-eJnlJuls srql seop ,ry\oH'slauuer{Juor eql asolJ Jo eJuEJnpuapue tuauqsrlqslse oql er1ropunueqo sasdeu,(s;o qr8uarls pur reqrunu ogt ur pue uado ol sutatord aqr ;o sued rer{to qtr,{i lrpJelur surElu 'pertporu oJEsuornau Surlsrxa(pea1su1 'suoJnou,treu -op Sursuas-e8etlorr eql r{Jrq^. ur de.,llaqr rseSSnsslouuer{f, sa8ueq3 Sururo; uo puedap lou seop d.roruou sosef lsour u1 droruour uol unISSElod ;o sernlcnrls IEls.{JJer{t A/rorlureldxE n Jo srusruEr{Jauolur qf,JEosalto eeJEaqt ur ueeq seq luouolrJxa i IEIlueloo lsalea.r8oqt Jo ouros tSolorq IIar relnralou ;o lurodpuels uorlf,E up to uorteraue8egr ur pe^lo^ur sleuupr{J*eN o} 'uorleruJoJur eqr uoJc IPnsrAJo SurssacordPunoJJns-Jalualer{l parldde puuoq) pa7a3-a8u71o4 rurot aqr sr ,{qr5 .po^lo^ur JoJaJOqueaseAEr{e,ryr sE'sJssef,Jns alqelou uJeq aAEqeJaqllnq uor eqt pue srseqrelnreloru 3ur.,{lrapun oql qJEaroJ equJS 's,vr.an aql qlrm raqnSol s1\\orlpqop eqt Suuq o1 11oc-e13urs .t. -acl 'lPrtuolod uollre uE Jo sasEqdaerqt aql aurpN rlnrIJJIp sr lI 'soporfela pauasur Sursnorurt p lE sllof,a,r.e;e 3ur islleJ lEruru€ur peureturPur -^rosqo ouop erp sraqro dlrarlcBleJrnf,olopqop Surrcereppue 'ller er{t oprslno qlr.,r,rpered lertualod Surlse.roql sr ,la,oH suoJnouJo suorlpu ot spuesnoqlSur.trasqo'sarSolouqcel8ur -ruoJ eprsur .Z -3eur a,trse,ruruougtr.&\euop aJEsluorur.redxaeurog 'sesuodsar Aru 09 - sr uornau e yo lerlualod Surlseraq1 sno^reu eql pue 'l0rtuoo lPuoruJor{peleFSa.r'stJurlsurJo ef,upt iluels,4.s to IEuorloua (loJluof, str€d raqto pue urerq agt ur sllor -rJer1w'serloruoru elor or.{tsr }Er.{lN .I Jol ler13 ;o Jo IeAarJ}eJpu€ luourqsqqplse -oru roJ srusrueqraru >lrpqpoe;'tq8noqrlerrrdleue'uorleurro;ur drosuas1o uorlela.rd.relurJr{l lno serJJEJd-rlrnc.rrcleJneu ./r\or{

sldo)uo) aqt ^^a!^aU

for sweetness,bitterness,and umami; and odor receptor molecules. 15. How did Roger Sperry'sexperimentsin frogs distinguish betweenthe resonancehypothesisand the chemoaffinity hypothesisfor how neurons becomewired?

Analyze the Data Olfaction occurs when volatile compounds bind to specific odorant receptors. In mammals, each olfactory receptor neuron in the olfactory nasal epithelium expressesa single type of odorant receptor.These odorant receptorsconstitute a large multigene family (>1000 members)of related proteins. Binding of odorant induces a signaling cascade that is mediatedvia a G protein, Goo15. Recentstudiessuggestthat there are a small number of olfactory sensoryneurons in the nasal epithelium that expressmembers of the trace-amineassociatedreceptor (TAAR) family, chemoreceptorsthat are G protein-coupled receptors(GPCRs)but are unrelated to classicalodorant receptors (see Liberles and Buck, 2006, Natwre 442:645-650). The mouse genomeencodes15 TAAR geneswhile the human genome encodes6. a. In order to examine the expressionpattern of different TAARs in the olfactory nasal epithelium, researchers localized TAAR RNA by in situ hybridization in pairwise combinations. All possiblepairwise combinations of the 15 mouse TAARs were examined. A typical example of the results obtained is shown in the top set of panels in the figure below in which TAAR6 and TAART have been localized with fluorescent probes in the nasal epithelium of mouse. The Taar6 probe was labeledwith a green fluor, the TAART probe with a red fluor. The lower set of panels shows localization of mouse odorant receptor 28 (MOR28; green), a classical odorant receptor, and TAAR6 (red). Each stained patch in the images is the staining pattern of an individual olfactory neuron. The "merge" panels show the two other imagessuperimposed.\What do thesedata suggestabout expressionpatterns of the TAARs?

phosphatase (SEAP) under control of a cAMP-responsive element. The cells are then exposed to various amines, as shown in the following figure, and SEAP activity in the medium is determined.The figure shows data for some representativeTAARs (m : mouse,h : human). What do these data reveal about TAARs? lfhat does the SEAP activity assay reveal about the signaling pathway utilized by chemoreception involving TAARs? 400

400

uJ a

BCDEFGH mTAAR3

AB

CDEFG mTAAR4

AB

CDEFGH mTAARTf

(L UJ

U)

AB

CDEFGH mTAAR5

G

uJ

a

BCDEF hTAARI

BCDEFG No receptor

A No testcompound

E N-Methylpiperidine

B B-phenytethylar,n_"(rl=,^rr,

F Cyclohexylamine

a) \-N-

Ho_,.\ c ryramine D Trimethylamin"

b. A number of cell lines that produce neither classical odorant receptors nor TAARs have each been transfected with the geneencodinga different TAAR. The cellshave also been cotransfectedwith a gene encoding secretedalkaline

G 2-Methylbutvlamine LZ\.^NH, I

-il-

H lsoamylamine

,\,^NH,

c. In a third set of studies,SEAPactivity was measured in cells expressingmouse TAAR5 (mTAARS) following exposure of the cells to diluted urine derived from two strains of mice or from humans, as indicated on the graphs below. Mice reach puberty at about one month of age. What do these data suggest may be a biological function for the TAARS neurons in mice? Vhat additional studies would you conduct to support your hypothesis? A N A L y 7 ET H E D A T A

o

1051

n -mTAAR5 ffi +mTAARS

n -mTAARS il +mTAARS

.2 o uJ

o

19 23 29 41 46 A g e o f m a l em i c e (days)

References Neurons and Glia: Building Blocks of the Nervous System Allen, N. J., and B. A. Barres.2005. Signalingbetweenglia and neurons:focus on synapticplasticity.Curr. Opin. Neurobiol. 15:542-548. Eshed,Y., et al. 2005. Giiomedin mediaresSchwanncell-asn interaction and the molecularassemblyof the nodesof Ranvier. Neuron 47:215-229. Jessen,K. R., and R. Mirsky. 2005. The origin and development of glial cellsin peripheralnerves.Nature Reu.Neurosci.6(91:671-682. Parker,R. J., and V. J. Auld. 2005. Rolesof glia in the Drosophila nervoussystem.Semin. Cell Deu. Biol. 17(1):66-77. Ram6n y Cajal, S. 1911. Histology of the NeruowsSystemof Man and Vertebrates(trans.N. Swansonand L. \7. Swanson.1995, Oxford UniversityPress). Salzer,J. L. 2003. Polarizeddomains of myelinatedaxons. Neuron. 402297-31.8. Seifert,G., K. Schilling,and C. Steinhauser. 2005. Asrrocyte dysfunctionin neurologicaldisorders:a molecularperspective. N ature Reu.N ewrosci. 7 (31:194-20 6. Sherman,D. L., and P.J. Brophy.2005. Mechanrsmsof axon ensheathmentand myelin growrh. Nature Reu.Neurosci. 619),:683-690. Stevens,B. 2003. Glia: much more than the neuron'sside-kick. Curr. B iol. 13:R4 69 -R47 2. Voltage-Gated lon Channels and the Propagation of Action Potentials in Nerve Cells Aldrich, R. W. 2001. Fifty yearsof inactivation.Nature 4lt:643-644. Armstrong, C., and B. Hille. 1998. Voltage-gatedion channels and electricalexcitability.N euron 20:371,-380. Brunger,A. T. 2005. Srructureand function of SNARE and SNARE-interactingproteins.Quart. Reu.B iophys. 38(1):147 . Cannon, S. C. 2006. Pathomechanisms in channelopathiesof skeletalmuscleand brain. Ann. Reu.Neurosci.29z387-415. Catterall,\f. A. 2000. From ionic currentsto molecularmechanisms:the structureand function of voltage-gatedsodium channels. Neuron 26:1.3-25. Catterall,\7. A. 2000. Strucrureand regulation of voltage-gated Car" * channels. Ann. Reu.Cell Deu. Biol. 16:521.-555 . Catterall,W. A. 2001. A 3D view of sodium channels.Nature 409988-989. Clapham,D.1999. Unlockingfamily secrets: K+ channeltransmembranedomains. Cell 97:,547-5 50.

1052

C H A P T E R2 3

I

N E R V EC E L L S

'1,999.lon Cooper,E. C., and L. Y. Jan. channelgenesand human neurologicaldisease:recentprogress,prospects,and challenges. Proc. Nat'l Acad. Sci.USA96:47594766. del Camino, D., and G. Yellen.2001. Tight stericclosureat the intracellularactivationgate of a voltage-gatedK( + ) channel.Neuron 322649-656. Doyle, D. A., et al. 1998. The structureof the potassium channel:molecularbasisof K- conductionand selectivity.Science 280:69-77. Dutzler, R., et al. 2002. X-ray structureof a CIC chloride channel at 3.0 A revealsthe molecularbasisof anion selectivity.Nature 415287-294. Gulbis,J. M., et al. 2000. Structureof the cytoplasmicbeta subunit-T1assemblyof voltage-dependent K* channels.Science 289:L23-L27. Hanaoka, K., et al. 2000. Co-assemblyof polycystin-1and -2 producesunique cation-permeablecurrents.Nature 408:990-994. Hille, B. 2001..Ion Channelsof ExcitableMembranes,3d ed. SinauerAssociates. Jan, Y. N., and L. Y. Jan. 200I. Dendrites. GenesDeu. r5:2627-2641. Long, S. B., E. B. Campbell,and R. MacKinnon. 2005. Crystal structureof a mammalian voltage-dependent Shakerfamily K* -903. channel.Science.309(5736)2897 Long, S. 8., E. B. Campbell,and R. MacKinnon. 2005. Voltage sensorof Kv1.2: structural basisof electromechanical coupling. Science.309(5736l: 903-908. Miller, C. 2000. Ion channelsurprises:prokaryotesdo it again! Neuron 2527-9. Montell, C., L. Birnbaumer,and V. Flockerzi.2002. The TRP channels,a remarkably functional famlly. Cell 108:595-598. Neher,E. 1992.lon channelsfor communicationbetweenand within cells.Nobel Lecturereprinted in Neuron 8:605-612 and Science256:498-502. Neher,E., and B. Sakmann.1992. The patch clamp technique. Sci.Am.266(31:28-35. Nichols, C., and A. Lopatin. 1,997.lnward rectifier potassium channels.Ann. Reu.Physiol. 59:L71,-1,92. Sato, C., et al. 2001. The voltage-sensitive sodium channelis a -1,05 1. bell-shapedmoleculewith severalcavities.Natwre 409:'1,047 Shi, N., S. Ye, A. Alam. L. Chen, and Y. Jiang.2006. Atomic structureof a Na'- and K*-conducting channel.Nature 440(70831:570-s74. Yi, B. A., and L. Y. Jan. 2000. Taking apart the gating of voltagegated K* channels.Neuron 27:423-425. Yi, B. A., et a|.2001,.Controlling potassiumchannelactivities: interplay betweenthe membraneand intracellular iactors.Proc. Nat'l Acad. Sci. USA 98:1101,6-1,1,023. Zhou, M., et aI.2001. Potassiumchannelreceptorsite for the inactivation gate and quaternary amine inhibitors. Nature 4ll:657-661. Zhou, Y., et al. 2001. Chemistryof ion coordination and hydration revealedby a K+ channel-Fabcomplex at 2 A resolution. Nature 414:4348. Communication at Synapses Amara, S. G., and M. J. Kuhar. 1993. Neurotransmittertransporters: recentprogress.Ann. Reu.Neurosci. 16:73-93. Bajjalieh,S. M., and R. H. Scheller.1995. The biochemistryof neurotransmittersecretion.J. Biol. Chem.270:1,971-1974. Bamji, S. X. 2005. Cadherins:actin wirh the cytoskeletonto form synapses.Neuron 4721,7 5-1,78. Betz,'W.,and J. Angleson.1,998.The synapticvesiclecycle.Ann. Reu.Physiol. 602347-364.

Brejc,K., et al. 2001. Crystal structureof an ACh-binding protein revealsthe ligand-bindingdomain of nicotinic receptors.Nature 4ll:269-276. Fatt, P.,and B. Katz 1952. Spontaneoussubthresholdactivity at motor nerveendings.J Physiol. 117:709-128. Fernandez,J. M. 1,997.Cellular and molecularmechanicsby atomic force microscopy:capturing the exocytoticfusion pore in vivo? Proc. Nat'l Acad. Scl. USA 94:9-10. Ikeda, S. R. 2001. Signaltransduction.Calcium channels-link locally,act globally.Science294:31,8-319. Jan, L. Y., and C. F. Stevens.2000. Signalingmechanisms:a decadeof signaling.Curr. Opin. Neurobiol. 10':625-630. Karlin, A. 2002. Emergingstructureof the nicotinic acetylcholine receptors.Nature Reu.Neurosci. 3:1.02-1.1.4. Kavanaugh,M. P. 1998. Neurotransmittertransport: modelsin flux. Proc. Nat'l Acad. Sci.USA 95:1,2737-1,2738. Klann. E.. and T. E. Dever.2004. Biochemicalmechanismsfor translationalregulationin synapticplasticity.Nature Reu.Neurosci. 5(1.2\:931.-942. Kummer, T. T., T. Misgeld, and J. R. Sanes.2005. Assemblyof the postsynapticmembraneat the neuromuscularjunction: paradigm lost. Cwrr.Opin. Neurobiol. 16(1,\:74-82. Lin, R. C., and R. H. Scheller.2000. Mechanismsof synaptic vesicleexocytosis.Ann. Reu.Cell Deu. Biol. 16':1,949. Neher,E. 1998.Vesiclepools and Ca2' microdomains:new tools for understandingtheir roles in neurotransmitterrelease. Neuron 20:389-399. Reith, M., ed. 1997. NeurotransmitterTransporters:Structure, Function, and Regulatioz.Humana Press. Sakmann.B. 1992.Elementarystepsin synaptictransmission revealedby currentsthrough singleion channels.Nobel Lecture reprinted in EMBO J. 17:2002-20L6 and Science256:503-51,2. Sosinsky,G. E., and B. J. Nicholson.2005. Structuralorganization of gap junction channels.Biochim.Biophys.Acta 17II(2\:99-1,25. Sudhof,T. C. 1995. The synapticvesiclecycle:a cascadeof protein-proteininteractions.Nature 37 5:645-653. Ule, J., and R. B. Darnell. 2005. RNA binding proteins and the regulationof neuronal synapticplasticity.Curr. Opin. Neurobiol. 1 6 (1 ) : 1 0 2 - 1 0 . Usdin, T. B., et al. 1995. Molecular biology of the vesicularACh ff ansporter. Trends Neurosci. 18:21,8-224. White-Grindley,E., and K. Si. 2005. RISC-y memories.Cel/ 124(1\':23-60. Ziv. N. E.. and C. C. Garner.2004. Cellularand molecularmechanismsof presynapticassembly.Nature Reu.Neurosci.5(5):385-399. Sensational Cells: Seeing, Feeling, Hearing, Tasting, and 5melling Buck, L., and R. Axel. 1991. A novel multigenefamily may encodeodorant receptors:a molecular basisfor odor recognition. 5-1.87. Cell 65(1.)21.7 Cohen-Cory,S., and B. Lom. 2004. Neurotrophic regulation of retinal ganglion cell synapticconnectivity:from axons and dendrites to synapses.lnt'l J. Deu. Biol.48(8-91:947-956. Eatock, R. A., and K. M. Hurley. 2003. Functional development of hair cells. Cun Top. Deu. Biol. 57:389448. Esteve,P.,and P. Bovolenta.2006.Secretedinducersin vertebrate eye development:more functions for old morphogens.Curr. Opin. -1.9. Neur o bi ol. 76(1.\21.3 Gillespie,P. G., and R. G. Walker. 2001. Molecular basisof mechanosensory fransduction.Natwre 413(68 52):194-202.

Hoon, M. A., et al. 1,999.Putativemammalian tastereceptors:a class of taste-specificGPCRs with distinct topographic selectivity. Cell 96(41:541-551. Hudspeth,A. J. 2005. How the ear'sworks work: mechanoelectrical transductionand amplification by hair cells. ComptesRendus 5 5-162. Biol. 328(2\21. Lin, S. Y., and D. P. Corey.2005. TRP channelsin mechanosensation. Cur r. O p in. N euro bi o l. 15 (3):3 5 0-3 57 . Marin, E. C., et al. 2002. Representationof the glomerular olfactory map in the Drosophila brain. Cell 109(21243-255 ' McKemy, D. D,'W M. Neuhausser,and D. Julius. 2002. Identification of a cold receptorrevealsa generalrole for TRP channelsin n. Natur e 476252-5 8. thermosensatio Mombaerts, P. 1999. Molecular biology of odorant receptorsin vertebrates.Ann. Reu.Neurosci.22:487-509. Nelson, G., et al. 2001. Mammalian sweettastereceptors.Cel/ 106(3):381-390. Zhang, X, and S. Firestein.2002.The olfactory receptorgene superfamilyof the mouse.Nature Neurosci. 5(21:124-733. The Path to Success:Controlling Axon Growth and Targeting Chilton, J. K. 2005. Molecular mechanismsof axon guidance. Deu. Biol. 292(11:13-24. Dickson,B.J.and G.F.Gilestro.2005. Regulationof commissural axon pathfinding by slit and its robo receptors.Ann. Reu.Cell Deu. Biol.22:65L-75. Gallo, G., and P. C. Letourneau.2004. Regulationof growth cone actin filaments by guidancecues.J. Neurobiol. 58(I):92-102Gomez,T.,M. and J. Q. Zheng. 2006.The molecular basisfor calcium-dependentaxon pathfinding. Nature Reu.Neurosct.

7(21:1.1.5-r25. Hedgecock,E. M., J. G. Culotti, and D. H. Hall' 1990. The unc-5, unc-6, and unc-40 genesguide circumferentialmigrations of pioneeraxons and mesodermalcellson the epidermisin C. elegans. Neuron 4(1):61-85. Hilliard, M. A., and C.I. Bargmann.2006. I(nt signalsand frizzled activity orient anterior-posterior axon outgrowth in C. elegans. D eu. Cell 10(3\:379-390. Hindges,R, et al. 2002. EphB forward signalingcontrols directional branch extensionand arborization required for dorsal-ventral retinotopic mapping. Neuron 35(31:475487 . Jin, M., et al. 2005. Ca2*-dependentregulationof rho GTPases triggersturning of nervegrowth cones./. Neurosci.25(9):2338-2347. Kidd, T., K. S. Bland, and C. S. Goodman. 1999' Slit is the midline repeflent for the robo receptor in Drosophild- Cell 96(61:785-794. Kruger,R. P.,J. Aurandt, and K.-L. Guan. 2005. Semaphorins command cellsto move. Nature Reu.Mol. Cell Biol.6(10):789-800' Lemke, G., and M. Reber.2005. Retinotectal mapping: new insights from molecular genetics.Ann. Reu.Cell Deu' Biol. 21:551-580. Li, W., et al. 2004. Activation of FAK and Src are receptorproximal eventsrequiredfor netrin signaling.Nature Neurosci. 7 (11\:1213-7221. Marquardt, T., et al. 2005. CoexpressedEphA receptorsand ephrin-A ligandsmediateopposingactionson growth cone navigation from distinct membranedomains. Cell l2t(11:127-139. Mclaughlin, T., and D. D. O'Leary' 2005. Molecular gradientsand developmentof retinotopic maps.Ann. Reu.Newrosci.28327-355. Pan, C. L., et al. 2006. Multiple Wnts and frizzled receptors regulateanteriorly directedcell and growth cone migrations in -377 . Caenorhabditiselegans.D eu.Cell. lO(3)2367

REFEREN CE5

1053

Schmitt,A.M., et al.2006.I7ncRyk signallingmediatesmediallateral retinotectal topographic mapping. Nature 439(70721:31-37. SerafiniT., et al. 1996. Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cel/ 87(6\:1.00L-1.014. Sperry,R. \/. 1963. Chemoaffinity in the orderly growth of nerve fiber patternsand connections.Proc. Nat'l Acad. Sci. IISA 5O:703-710. Tessier-Lavigne, M., et al. 1988. Chemotropicguidanceof developing axons in the mammalian central nervous system.Nature. 3366201.\2775-778.

1054

C H A P T E R2 3

I

N E R V EC E L L S

Walter,J., et al. 1987. Recognitionof position-specificproperties of tectal cell membranes by retinal axons in vitro. Deuel. 10l(4):685-696. 'Wen,Z., and J. Q. Zheng.2006. Directional guidance of nerve growth cones.Curr. Opin. Neurobiol. 16(1):52-58. Xie, Y., et al. 2005. Phosphatidylinositol transfer protein-alpha in netrin-1-induced PLC signalling and neurite outgrowth. Nature Cell BioL 7 (ll):1124-1132. 2o1,Y.2006. Navigating the anterior-posterioraxis with Wnts. Neuron 49:787-789.

CHAPTER

IMMUNOLOGY

Dendritic cellsin the skinhaveclassll MHC molecules on their Thoseshownherewereengineered surface. to express a classll green [Courtesy MHC-GFP fusionprotein,whichfluoresces M BoesandH L Ploeoh I

I mmunity is a stateof protection againstthe harmful effects I of exposureto pathogens.Host defensecan take many difI ferent forms, and all successfulpathogens have found ways to disarm the immune systemor manipulate it to their own advantage. Host-pathogen interactions are therefore an evolutionary work in progress.This explains why we continue to be assaultedby pathogenicviruses,bacteria,and parasites.The prevalenceof infectious diseasesillustratesthe imperfectionsof host defense.But killing its host is not necessarilyadvantageousto a pathogen becausecomplete elimination of the host would immediately remove the reservoir in which the pathogenreplicatesor survives.An immune system that could produce perfect sterilizing immunity would yield a world without pathogens,an outcome clearly at variance with life as we know it. Rather, the co-evolution of pathogensand their hosts allows pathogens,which have relatively short generationtimes, to continue to evolve sophisticated countermeasures,againstwhich the host must respond by adjusting,if not improving, its defenses.Sophisticateddefensecomesat a price: An immune systemcapableof dealing with a massively diverse collection of rapidly evolving pathogensmay mount an attack on the host organism'sown cells and tissues,a phenomenoncalled autoimmunity. In this chapter we deal mostly with the vertebrate immune system,with particular emphasison those molecules, cell types, and pathways that uniquely distinguish the immune system from other types of cell and tissues. Host defense comprises three layers: (1) mechanical/chemical defenses,(2) innate immunity, and (3) adaptive immunity (Figure 24-1). Mechanical and chemical defensesoperate continuously. Innate immune responses,which involve cells

and molecules present at all times, are rapidly activated (minutes to hours), but their ability to distinguish among many different pathogensis somewhat limited. In contrast, adaptive immune responsestake several days to develop fully and are highly specific;that is, they can distinguish between closelyrelated pathogensbasedon very small molecular differencesin structure. The manner in which antigens-any material that can evoke an immune response-are recognizedand how these and cell foreign materials are eliminated involve molecular'We begin biological principles unique to the immune system. this chapter with a brief sketch of the organization of the

OUTLINE 24.'l

Overview of Host Defenses

and Function Structure 24.2 lmmunoglobulins:

1057 1063

Generationof Antibody Diversity l evelopment a n d B - C e lD

1069

24.4

The MHC and Antigen Presentation

1076

24.5

T Cells,T-CellReceptors,and T-Cell DeveloPment

24.6

Cells Collaborationof lmmune-System in the Adaptive ResPonse

24.3

1055

Chemical defenses

M i n u t e st o h o u r s

Adaptive immunity

Innate immunity

lnnate immunity

Adaptive immunity

Adaptive immunity

A FIGURE 24-1 Thethree layersof vertebrateimmune defenses. Left;Mechanical defenses consist of epithelia andskin. Chemical defenses include the low pHof the gastric environment andantibacterial enzymes in tearfluid.Thesebarriers provide protection contrnuous against invadersPathogens mustphysically ([) to infectthe host Middle.Pathogens breachthesedefenses that havebreached (Z) are the mechanical andchemical defenses handled by cellsandmolecules (blue), of the innateimmunesystem w h i c hi n c l u d epsh a g o c y tci ce l l s( n e u t r o p h i dl se, n d r i t icce l l s , macrophages), naturalkiller(NK)cells,complement proretns, ano (lL-1,lL-6).Innatedefenses certainrnterleukins areactivated within

minutes to hoursof infection. Rrght:Pathogens thatarenotcleared bythe innateimmunesystem aredealtwith by theadaptive immune (B), in particular system B andT lymphocytes Fullactivation of adaptive immunity requires daysTheproducts of an innateresponse (4). Likewise, maypotentiate an ensuing adaptive response the products of an adaptive immuneresponse, including antibodies (Y-shaped icons), mayfacilitate functioning of the innateimmune (S) Several system products celltypesandsecreted straddle the fencebetween the innateandadaptive immunesystems, andserve to connectthesetwo layersof hostdefense

mammalian immune system,introducing the essentialplayers of innate and adaptive immunity and describing inflammation, a localized response ro injury or infection that leadsto the activation of immune-systemcellsand their recruitment to the affected site. In the next two secrions,we discuss the structure and function of antibody (or immunoglobulin) molecules,which bind to specificmolecular features on antigens, and how variability in antibody structure contributes to specific recognition of antigens. The enormous diversity of antigensthat can be recognized by the immune system finds its explanation in unique rearrangementsof the genetic material in B and T lymphocytes, commonly called B cells and T cells, which are the white blood cells that carry out anrigen-specificrecognition. These gene rearrangements not only control the specificity of antigen receptors on lymphocytes but also determine cell-fate decisionsin the course of lymphocyte development.

Although the mechanismsthat give rise to antigen-specific receptors on B and T cells are very similar, the manner in which these receptors recognize antigen is very different. The receptors on B cells can interact with intact antigens directly, but the receptors on T cells cannot. Instead, as describedin Section24.4,the receptorson T cells recognize cleaved (processed)forms of antigen, presentedon the surface of target cells by glycoproteins encoded by the major histocompatibility complex (MHC). How MHC-encoded glycoproteins display these processedantigens is important for our understanding of how immune responsesare initiated. MHC-encoded glycoproteins also help determine the developmentalfate of T cells so that an organism'sown cells and tissues(self antigens)normally do not evoke an immune response,whereasforeign antigensdo. We concludethe chapter with an integrated view of the immune responseto a pathogen, highlighting the collaboration between different immune-systemcellsthat is requiredfor an effectiveresponse.

1056

.

cHAprER 24 |

tMMUNolocy

Overviewof HostDefenses Becausethe immune systemevolved to deal with pathogens, we begin our overview of host defensesby examining where rypical pathogensare found and where they replicate.Then we introduce basic concepts of innate and adaptive immunity, including some of the key cellular and molecular players.

PathogensEnterthe Body Through Different Routesand Replicateat Different Sites Pathogensaffect alI life forms capable of independentreplication. Two general classesof pathogen, viruses and bacteria, have fundamentally different modes of propagation. 'S(ith the exception of polymerases involved in copying geneticmaterial, virusesgenerallylack the necessarymachinery to synthesizetheir component parts and therefore are completely dependenton host cells for their propagation. In contrast, most bacteria are metabolically autonomous and do not rely on their host for replication, which allows them to be grown in the laboratory in the appropriate culture media. (An exception is those bacteriathat can replicateonly within a mammalian host cell.) Bacteria can cause disease becausethey possessvirulence factors that act on the host's metabolism and physiology. Parasitic organisms also can causedisease.With increasinglycomplex life cyclessuch as those of the protozoa that cause sleeping sickness (trypanosomes)or malaria (Plasmodiumspecies)(seeFigure 1-4), the pathogen'scountermeasuresalso become increasingly complex. Bacteria,protozoa, and fungi-especially thosethat can causediseasein animals-often are called microbes. Exposure to pathogensoccurs via different routes. The skin itself has a surface areaof =20 sq. ft.; the epithelial surfacesthat line the airways, gastrointestinaltract, and genital tract present an evenmore formidablesurfaceareaof =4000 sq. ft. All these surfacesare continuously exposedto viruses and bacteria in the environment. Foodborne pathogens and sexually transmitted agentstarget the epithelia to which they are exposed. The sneezeof a flu-infected individual releasesmillions of virus particles in aerosolizedform, ready for inhalation by the next person to be infected. Rupture of the skin, even if only by minor abrasions, or of the epithelial barriers that protect the underlying tissues,provides an easy route of entry for pathogens,which then gain accessto a rich source of nutrients (for bacteria) and to the cells required for their replication(viruses). Replicationof virusesis strictly confined to the cytoplasm or nucleusof host cells, where protein synthesisand replication of the viral geneticmaterial occur.Virusesspreadto other cells either as free virus particles (virions) or by cell-to-cell spread.Many bacteriacan replicatein the intercellularspace, but someare specializedto invadehost cellsand survivethere. Such intracellular bacteria reside either in membranedelimited vesiclesthrough which they enter cells by endocytosis or phagocytosisor in the cpoplasm if they escapefrom these vesicles.An effective host defensesystem, therefore, needsto be capableof eliminatingnot only cell-freevirusesand free-living bacteria but also cells that harbor thesepathogens.

LeukocytesCirculateThroughoutthe Body and TakeUp Residencein Tissuesand Lymph Nodes 'With

the exception of erythrocytes,few cells in the courseof their assignedfunction cover such distancesas do the cells that provide immunity. The mammalian circulation servesas the necessarytransport vehicle for erythrocytes,Ieukocytes, and platelets.Although erythrocytesnever leave the circulation (their oxygen-carrying function does not require it), leukocytes(white blood cells) use the circulation exclusively for transport and may leave and re-enter the circulation in the course of their tasks. The immune system is an interconnectedsystem of vessels,organs, and cells, divided into primary and secondary lymphoid structures(Figure 24-21.Primary lymphoid organs -the sites at which lymphocytes (the subset of leukocytes that includes B and T cells) are generatedand acquire their Lymph nodes (filteringof lymphand maturationof white blood cells) Thoracic duct (discharges lymph into blood)

Lymphvessels (conveylymph) Thymus (T-cell maturation) Spleen (lymphocyte maturation and filtering of lymph)

Bone marrow (B-cell development, T-cell precursors)

24-2 The circulatoryand lymphaticsystems.Positive A FfGURE for lossof exertedbythe pumpingheartisresponsible arterialpressure (red)intothe interstitial spaces of thetissues, liouidfromthecirculation of andcandispose to nutrients sothat allcellsof the bodyhaveaccess threetimesthat fluid,whosevolumeisroughly waste.Thisinterstitial in theform to thecirculation isreturned of allbloodin thecirculation, structures anatomical throughspecialized of lymph,whichpasses wherelymphocytes organs, calledlymphnodes.Theprimarylymphoid (B precursors) andthe T-cell cells, marrow generated, the bone are are involves the of an immuneresponse thymus(Tcells)Theinitiation (lymphnodes, spleen) organs lymphoid secondary O V E R V I E WO F H O S TD E F E N S E S

1057

functional properties-include the thymus, where T cells are generated,and the bone marrow, where B cellsare generated. Adaptive immune responses,which require functionally competent lymphocytes are initiated in secondary lymphoid orgaas including lymph nodes and the spleen.All of the lymphoid organs are populated by cells of hematopoieticorigin (seeFigure 21-15), generatedin the fetal liver and throughout life in the bone marrow. The total number of lymphocytes in a young adult male is estimatedto be 500 x 10v, roughly 15 percent of which are found in the spleen,40 percent in the other secondarylymphoid organs (tonsils,lymph nodes), 10 percent in the thymus, and 10 percenr in the bone marrow; the remainder are circulating in the bloodstream. Leukocytesmust leavethe bloodstreamand enrertissuesto perform their functions.Vertebrateblood vesselsallow the escape of fluid from the circulation, driven by the positive arrerial pressureexertedby the pumping heart.This fluid contains not only nutrients but also proteins that carry out defensive functions. To maintain homeostasis,the fluid that leavesthe circulation must ultimately return and does so in the form of lymph, via lymphatic vessels.The total volume of lymph is up

to three times the total blood volume. At their most distal ends, Iymphatic vesselsare open to collect the interstitial fluid that bathes the cells in tissues.The lymphatic vesselsmerge into larger collecting vessels,which deliver lymph to lymph nodes. A lymph node consistsof a capsule,organized into areas that are defined by the cell types that inhabit them. Blood vessels entering a lymph node deliver B and T cells to it. The lymph that arrives in a lymph node carries cellsthat have encountered ("sampled") antigen,as well as soluble antigens,from the tissue drained by that particular afferent lymphatic vessel.In the lymph node, the cells and moleculesrequired for the adaptive immune responseinteract, respond to the newly acquired antigenic information, and then execute the necessaryeffector functionsto rid the body of the pathogen(Figure24-3). Lymph nodes can be thought of as filters in which antigenic information gathered from distal sitesthroughout the body is collected and displayed to the immune system in a form suitable to evoke an appropriate response.All the relevant steps that lead to lymphocyte activation take place in Iymphoid organs.Cells that have receivedproper insrructions to becomefunctionally active leavethe lymph node via efferent

Antigen-laden d e n d r i t i cc e l l I

B c e l l b i n d ss o l u b l ea n t i g e n and movesto follicle

B - c e l fl o l l i c l e s Afferent lymphatic vessel

E

M a t u r e Ta n d B c e l l s are deliveredvia the c i r c u l a t i o na n d t a k e u o r e s i d e n c ei n lymphnodes

!

Activationof T cell by antigenladen,activated d e n d r i t i cc e l l ; activatedTcells may re-enter circulation

Blood vessels @ Efferent lymphatic vessel FIGURE24-3 Initiation of the adaptive immune responsein lymph nodes. Recognition of antigenby B and T cells(lymphocytes) locatedin lymph nodesinitiatesan adaptiveimmuneresponse Lymphocytes leavethe circulationand take up residence in lymph nodes(Il) Lymphcarriesantigenin two forms-soluble antigenand antigen-ladendendriticcells;both are deliveredto lvmph nodesvia a f f e r e n tl y m p h a t i c(s4 , B ) . S o l u b l ea n t i g e ni s r e c o g n i z e b dy B c e l l s

1058

CHAPTER 24

I

IMMUNOLOGY

ActivatedTcells interact w i t h B c e l l s ,l e a d i n gt o B-celldifferentiationand antibody production

( 4 ) , a n da n t i g e n - l a d de en n d r i t icce l l sp r e s e natn t i g e n to T cells (E) Productive interactions betweenT andB cells(El) allowB cells to moveintofollicles anddifferentiate intoplasma cells,which p r o d u c lea r g ea m o u n tosf s e c r e t ei d m m u n o g l o b u l(i a nn s tibodies) Efferent lymphatic vessels returnlymphfromthe lymphnodeto the circulation

lymphatic vesselsthat ultimately drain into the circulation. Such activated cells recirculate through the bloodstream and-now ready for action-may reach a location where they again leave the circulation, move into tissues,and seek out pathogenicinvadersor destroy virus-infectedcells. The exit of lymphocytes and other leukocytes from the circulation, recruitment of these cells to sites of infection, processingof antigenic information, and return of immunesystem cells to the circulation are all carefully regulated processes that involve specificcell-adhesionevents,chemotactic cues,and the traversalof endothelialbarriers,as we discuss Iater.

M e c h a n i c aal n d C h e m i c aB l o u n d a r i e sF o r m a FirstLayerof DefenseAgainst Pathogens As noted already,mechanicaland chemicaldefensesform the first line of host defenseagainstpathogens(seeFigure 24-1). Mechanical defensesinclude the skin, epithelia, and arthropod exoskeleton,which are barriers that can be breachedonly by mechanicaldamage or through specificchemo-enzymatic attack. Chemicaldefensesinclude not only the low pH found in gastric secretionsbut also enzymessuch as lysozyme, found in tear fluid, which can attack microbes directly. The importance of mechanical defenses,which operate continuously,are immediatelyobvious in the caseof burn victims: When the integrity of the epidermisand dermis is compromised,the rich sourceof nutrients in the underlyingtissues is exposed,and airborne bacteria or otherwise harmlessbacteria found on the skin can multiply unchecked,ultimately overwhelming the host. Viruses and bacteria have also evolved strategiesto breach the integrity of these physical barriers. Enveloped viruses such as HIV, rabies virus, and influenza virus possessmembrane proteins endowed with fusogenicproperties.Following adhesionof a virion to the surface of the cell to be infected, direct fusion of the viral envelope with the host cell's membrane results in delivery of the viral genetic material into the host cytoplasm, where it is now available for transcription, translation, and replication (see Figures4-47 and 4-49). Certain pathogenicbacteria (e.9., S. aureus)secretecollagenasesthat compromisethe integrity of connectivetissueand so facilitate entry of the bacteria.

I n n a t el m m u n i t y P r o v i d e sa S e c o n dL i n e of DefenseAfter Mechanicaland Chemical BarriersAre Crossed The innate immune system is activated once the mechanical and chemical defenseshave failed, and the presenceof an invader is sensed(seeFigure24-1,).Theinnate immune system comprises cells and molecules that are immediately available for responding to pathogens.Phagorytes,cells that ingest and destroy pathogens, are widespread throughout tissues and epithelia and can be recruited to sitesof infection. Severalsoluble proteins presentconstitutively in the blood, or produced in responseto infection or inflammation, also contribute to innate defense.Animals that lack an adaptiveimmune system,suchas insects.rely exclusivelvon innate defensesto combat infections.

Phagocytes and Antigen'Presenting Cells The innate immune systemincludesmacrophages'neutrophils, and dendritic cells. All of these cells are phagocytic and come equipped with Toll-like receptors (TLRs). Members of this family of cell-surface proteins detect broad patterns of pathogen-specificmarkers and thus are key sensorsfor detecting the presenceof viral or bacterial invaders. Engagement of Toll-like receptors is important in eliciting effector molecules,including antimicrobial peptides. Dendritic cells and macrophageswhose Toll-like receptors have detected pathogens also function as antigen-presentingcells (APCs) by displaying processedforeign materials to antigen-specific T cells.The structure and function of Toll-like receptorsand their role in activating dendritic cells are describedin detail in Section24.6. Complement System Another important component of the innate immune system is complement, a collection of constitutive serum proteins that can bind directly to microbial or fungal surfaces.This binding activatesa proteolytic cascadethat culminates in formation of pore-forming proteins constituting the membrane attack complex, which is capable of permeabilizing the pathogen's protective membrane (Figure 24-4). The complement cascadeis conceptually similar to the blood-clotting cascade,with amplification of the reaction at each successivestageof activation' At least three distinct pathways can activate complement. The classical pathluay requiresthe presenceof antibodies produced in the course of an adaptive responseand bound to the surface of the microbe. Many microbial surfacesdirectly activate complement via the abernatiuepathway. Finally, pathogensthat contain mannose-rich cell walls activate complement through the mannose-binding lectin pathway. The bound lectin then triggers activation of two mannose-binding lectin-associatedproteases,MASP-1 and MASP-2, which allow activation of the downstream componentsof the complementcascade. In the course of complement activation, the C3 and C4 complement proteins occupy a specialrole. These abundant serum proteins are synthesizedas precursorsthat contain an internal, strained thioester linkage between a cysteineand a glutamate residue in close proximity' This thioester linkage becomeshighly reactive upon proteolytic activation of C3 and C4 by their respectiveupstream partners. The activated thioester bond can react with primary amines or hydroxyls in close proximitS yielding a covalent bond linking C3 or C4 with a protein or carbohydrate close-by.If no such reactants are available, the thioester bond is simply hydrolyzed. This mode of action ensuresthat C3 and C4 fragments will be covalently depositedonly on antigen-antibody complexes in close proximity. Regardlessof the pathway of complement activation engaged,activated C3 unleashesthe terminal components of the complement cascade,C5 through C9, culminating in formation of the membrane attack complex, which inserts itself into most biological membranes and renders them small permeable. The resulting loss of electrolytes and 'Whenever cell. solutes leads to lysis and death of the target O V E R V I EO WF H O S TD E F E N S E S.

1059

Mannose-binding lectin (MBL) pathway

Classical pathway

Alternative pathway

Targetpathogencell surface

+ B o u n db y : Antibodies

'\/

-\ll4 -/

,

\-

rrx

B o u n db y : Mannose-bindi l e c t ni

rVr \'1' ' -n-

Recruitment of: c1q r

MASP1

complement is activated, the membrane attack complex is formed and results in death of the cell onto which the complex is deposited. The direct microbicidal effect of a fully activated complement cascadeis an important protective function. All three complement activation pathways also generate the C3a and 5a cleavagefragmenrs,which bind to G protein-coupled receptorsand function as chemoattractants for neutrophils and other cells involved in inflammation (see below). All three pathways also result in the covalent decoration of the structures targeted by complement activation with fragments of C3. Phagocytic cells make use of these C3-derived tags to recognize,ingest, and destroy the decorated particles,a processtermed opsonization.

MASP2

Natural Killer (NK) Cells In addition to bacterialinvaders, the innate immune system also defends against viruses. \(hen the presenceof a virus-infected cell is detected, yet other cell types of the innate immune system becomeactive, seek out the virus-infected targets, and kill them. For instance,many virus-infected cells produce type I interferons, which are good at activating natural killer (NK) cells. Acti, vated NK cells not only afford direct protection by eliminating the factory of new virus particles, but they also secreteinterferon "y (IFN-1), which is essentialfor orchestrating many aspectsof anti-viral defenses(Figure 24-5). Recognition by NK cellsinvolves severalclassesof receptors, capable of delivering either stimulatory (promoting cell killing) or inhibitory signals. The interferons are classified as cytokines, small, secretedproteins that help regulate im'We mune responsesin a variety of ways. will encounter

t*

Anti-viral defense

Neutrophils IFN-y

Surfaceof targetcell (pathogen or antibodydecorated hostcell) FIGURE 24-4 Threepathwaysof complementactivation. Theclassical pathwayinvolves theformation of antibody-antigen complexes, whichrecruit thecomplement component C1q,leading to activation of C1r andC1s.Thiscomplex, in turnactivates C4andC2, whichthenconvert C3to itsactive form Inthe mannose-binding lectinpathway, mannose-rich structures foundon thesurface of many pathogens arerecognized by mannose-binding lectin, an interaction that results in activation of two serineproteases, MASP-1 andMASp-2 Thealternative pathway requires deposition of a special formof the serumproteinC3,a majorcomplement component, ontoa microbial surfaceSubsequent activation of C3 involves factorsB,D andB foundin serum.Eachof theactivation pathways isorganized asa cascade of proteases in whichthedownstream component isitselfa protease Amplification of activity occurs with eachsuccessive step All threepathways converge on C3,whichtriggers formation of the membrane attackcomplex, leading to destruction of targetcellsThe smallfragments of C3andC5 generated in thecourse of complement activation attractneutrophils, phagocytic cellsthatcankillbacteria at shortrangeor uponingestion 1060

.

cHAprER 24 |

tMMUNoLoGy

I nh i bi t s activation

@ Killing (perforin/ granzyme)

FIGURE 24-5 Naturalkillercells.Natural killer(NK)cellsare an important source of the cytokine interferon f (lFN-r)andcankill virus-infected andcancerous cellsby meansof perforins. Theseporeformingproteins allowaccess of serineproteases calledgranzymes to thecytoplasm of the cellaboutto be killed.Granzymes alsocan inrtiate (Chapter apoptosis throughactrvation of caspases 21)

Bacterium

other cytokines and discusssome of their receptorsas the chapterprogresses.

I n f l a m m a t i o nl s a C o m p l e xR e s p o n s teo I n j u r y Both Innate and Adaptive That Encompasses lmmunity

Dendriticcell

\Vhen a vascularizedtissueis injured, the stereotypicalresponse that follows is inflammation. Damage may be a simple paper cut or result from infection with a pathogen. Inflammation, or the inflammatory response,is characterized 6y four classical signs:redness,swelling, heat, and pain. These signs are caused by increasedleakinessof blood vessels(vasodilation),the attraction of cells to the site of damage, and the production of soluble mediators responsiblefor the sensationof heat and pain. Inflammation has immediate protective value through the activation of the cell types and soluble products that together mount the innate immune response.Further, inflammation createsa local environment conduciveto the initiation of the adaptive immune response.However, if not properly controlled, inflammation can also be a major causeof tissuedamage. Figure24-6 depictsthe key playersin the inflammatory responseto bacterialpathogensand the subsequentinitiation of an adaptive immune response.Tissue-residentdendritic cells sensethe presenceof pathogensvia their Toll-like receptors (TLRs) and respond to them by releasingsoluble mediators such as cytokines and chemokines;the latter act as chemoattractants for immune-system cells. Neutrophils, a second important cell type in the inflammatory response,leave the circulation and migrate to wherevertissueinjury or infection has occurred in responseto various soluble mediators produced upon tissue damage. Neutrophils, which constitute almost half of all circulating leukocytes,are phagocytic, directly ingestingand destroyingpathogenicbacteria.They also can interact with a wide variety of pathogen-derivedmacromolecules via their Toll-like receptors. Activation of these receptors allows neutrophils to produce cytokines and chemokines; the latter can attract more leukocytesneutrophils,macrophages,and ultimately lymphocytes(T and B cells)-to the area.Activatedneutrophilscan releasebacteriadestroyingenzymes(e.g.,lysozymeand proteases)as well as small peptides with microbicidal activity, collectively called defensins.Activated neutrophilsalso turn on the enzymesthat generatesuperoxideanion and other reactiveoxygen species (seeChapter 12, p. 502), which can kill microbes at short range.Another cell type contributing to the inflammatory reent mast cells.When activatedby a varisponseis tissue-resid ety of physical or chemical stimuli, mast cells releasehistamine, a mediator that increasesvascular permeability and thereby facilitatesaccessto the site of plasma proteins (e.g., complement)that can act againstthe invading pathogen. A very important early responseto infection or injury is activation of a variety of plasma proteases,including the proteins of the complement cascadediscussedabove (seeFigure 24-4l.The peptidesproduced during activation of theseproteasespossesschemoattractant activity, responsible for attracting neutrophils to the site of tissuedamage.They further induce production of proinflammatory cytokines such as

Neutrophil

|lKcelr-Q

z t tr /)

/w

Antibodies

\n (,

{)r Plasma cell

24-6 Interplayof innateand adaptiveimmune FIGURE breaches againsta bacterialpathogen.Oncea bacterium responses is exposed the bacterium defenses, andchemical thehostsmechanical aswellasto cellsthat cascade, of thecomplement to components (E) Various protection, suchasneutrophils conferimmediate to a contribute induced by tissuedamage mediators inflammatory of the bacterium Localdestruction response. inflammatory localized viathe whicharedelivered antigens, of bacterial in the release results cells lymphnode(E). Dendritic to thedraining lymphatics afferent in response to becomemigratory antigenat thesiteof infection, acquire products, andmoveto the lymphnode,wheretheyactivate microbial and T cellsproliferate T cells(B) Inthelymphnode,antigen-stimulated (4), cells help B to the ability including functions, effector acquire their someof whichmaymoveto the bonemarrowandcomplete of the immune intoplasma cellsthere(E). In laterstages differentiation to anttgenassistance additional T cellsprovrde activated response, antigen-specific plasma secrete yield that cells B cells to experienced produced asa at a highrate(step6) Antibodies antibodies with actin synergy to bacteria of the initialexposure consequence (Z), shouldit persist, or afford the infection to eliminate complement to thesamepathogen in thecaseof re-exposure rapidprotection interleukin 1. and 6 (IL-1 and IL-6). The recruitment of neutrophils also dependson an increasein vascularpermeability, controlled in part by lipid mediators (e.g.,prostaglandinsand leukotrienes)that are derived from phospholipids and fatty O F H O S TD E F E N S E S . OVERVIEW

1061

acids. All of theseeventsoccur rapidl5 starting within minutes of injury. A failure to resolvethe causeof this immediate responsemay result in chronic inflammation, in which cells of the adaptive immune systemplay an important role. 'When the pathogen burden at the site of tissuedamageis high, it may exceed the capacity of innate defensemechanisms to deal with them. Moreover, some pathogens have acquired,in the courseof evolution, tools to disableor bypass innate immune defenses.In such situations, the adaptive immune responseis required to control the infection. This adaptive responsedependson specializedcells that straddle the interface between adaptive and innate immunity, including antigen-presentingcells such as macrophagesand dendritic cells,which are capableof acquiring intact pathogensand of killing them upon ingestion.Theseantigen-presenting cells,in particular dendritic cells, can initiate an adaprive immune responseby deliveringnewly acquiredpathogen-derivedantigensto secondarylymphoid organs (seeFigure 24-6).

Adaptive lmmunity,the Third Line of Defense, ExhibitsSpecificity Lymphocytes bearing antigen-specificreceptors are the key cells responsiblefor adaptive immuniry. An early indication of the specificnature of adaptiveresponses camewith the discovery of antibodies, key effector moleculesof adaptive immuniry, by Emil von Behringand ShibasaburoKitasato in 1905. They observedthat when serum (the straw colored liquid that separates from cellular debris upon completion of the blood clotting process) from guinea pigs immunized with a sublethal dose of the deadly diphtheria toxin was transferred to animals never before exposed to the bacterium, the recipient animals were protected against a lethal dose of the same bacterium, which kills its host by production of a toxin (Figure 24-7). Transfer of serum from animals never exposedto diphtheria toxin failed to protect, and protection was limited to the microbe used as the sourceof toxin with which the animal that

Podcast: TheDiscovery of nntibodiesff Diohtheriatoxin Heatedserum fails to kill bacteria

N a i v ea n i m a l 6'\

*f**-+ 6/' Heated serum

I

l m m u n es e r u m kills bacteria

Serum from toxin-exposed animal

Serum from n a i v ea n i m a l

s.&

Serum

d,\

@dp

*dt\ Challengewith virulent bacteria

Dead bacteria

Heatedserum

-+

*. {, * u ,u* *'@

+

Dead bacteria

Freshserum from n a i v ea n i m a l

II

+ Freshserum contains "complement" destroyed by heating

A n i m a ld i e s

A n i m a ls u r v i v e sc h a l l e n g e

EXPERIMENTAL FTGURE 24-7 The existenceof antibody in serumfrom infectedanimalswas demonstratedby von Behringand Kitasato.Exposure of animals to a sublethal dose of diphtheria toxin(orthe bacteria thatproduce it)elicitsin their seruma substance that protects against a subsequent challenge with a lethaldoseof thetoxin(orthe bacteria thatproduce it) The protective effectof thisserumsubstance canbe transferred from an animalthat has beenexposedto the pathogento a naive (nonexposed) animal.When the serumrecipientis subsequently

1062

CHAPTER 24

I

IMMUNOLOGY

exposed to a lethaldoseof the bacteria, theanimalsurvives. This effectisspecific for the pathogen usedto elicitthe response. Serum (antibody) thuscontains a transferable substance thatprotects against the harmfuleffects pathogenSerumharvested of a virulent fromtheseanimals, saidto be immune, displays bactericidal activity rnvitro.Heating of immuneserumdestroys itsbactericidal actrvrty Additionof freshnonheated serumfroma naiveanimalrestores the bactericidal activity of heatedimmuneserum.Serumthuscontains anothersubstance thatcomplements the activitv of antibodies.

servedas the serum donor was immunized. This experiment demonstratesspecificity-that is, the ability to distinguish between two closely related substancesof the same class. Such specificity is a hallmark of the adaptive immune system. Even proteins that differ by a single amino acid may be distinguishedby immunological means. From these experiments, von Behring inferred the existenceof corpuscles( "Antikorper" ), or antibodies,as the factor responsiblefor protection. The antibody-containing (immune) sera not only afforded protection in vivo, they also killed microbesin the test tube. Heating the immune serato 56" C destroyed this killing activity, but it was restored by the addition of unheatedfresh serumfrom naive animals (i.e.,animals never exposed to the microbe). This finding suggestedthat a second factor, now called complement, acts in synergy with antibodies to kill bacteria. We now know that von Behring's antibodies are serum proteins referred to as immunoglobulins and that complementis actually a seriesof proteases(seeFigure 24-4l.Immunoglobulins can neutralizenot only bacterial toxins, but also harmful agents such as viruses, by directly binding to them in a manner that prevents the virus from attaching itself to host cells.In the samevein, antibodiesraised against snake venoms can be administeredto the victims of snakebites to protect them from intoxication: The anti-snake venom antibodies bind to the venom, keep it from binding to its targets in the host, and in so doing neutralize it. Antibodies can thus have immediate orotective effects.

Structure lmmunoglobulins: and Function Immunoglobulins, produced by B cells,are the best-understood molecules that confer adaptive immunity. In this section we describe the overall structural organization of immunoglobulins, their structural diversiry and how they bind to antigens.

l m m u n o g l o b u l i n sH a v ea C o n s e r v e dS t r u c t u r e Consistingof Heavyand Light Chains Like complement, immunoglobulins are abundant serum proteins that can be classifiedin terms of their structural and functional properties. Fractionation of antisera, basedon their functional activity (e.g.,killing of microbes,binding of antigen), led to the identification of the immunoglobulins as the classof serum proteins responsiblefor antibody activity. Immunoglobulins are composed of two identical heauy (H) chains, covalently attached to two identical light (L) chains (Figute 24-8). Structure of an antibodY molecule

L i g h tc h a i n

Light chain

Disulfidebonds Heavychain

Overview of Host Defenses r Mechanical and chemical defensesprovide protection against most pathogens.This protection is immediate and little specificity.Innate and adaptive continuous, yet possesses immunity provide defensesagainstpathogensthat breach the boundaries(seeFigure24-1). body'smechanicaVchemical

vy chain

Carbohydrate

./ Papaindigestion F(ab): monovalent

F(ab): monovalent

Pepsin digestion F(ab'12: bivalent

r The circulatory and lymphatic systems distribute the molecular and cellular playersin innate and adaptiveimmunity throughout the body (seeFigure24-2). r Innate immunity is mediated by the complement system (seeFigure 24-4) and severaltypes of leukocytes,the most important of which are neutrophils and other phagocytic cells such as macrophagesand dendritic cells.The cells and molecules of innate immunity are deployed rapidly (minutes to hours). Molecular patterns diagnostic of the presenceof pathogenscan be recognizedby Toll-like receptors' but the specificity of recognition is modest. r Adaptive immunity is mediated by T and B lymphocytes. Thesecells require days for full activation and deployment, but they can distinguish between closely related antigens. This specificity of antigen recognition is the key distinguishingfeatureof adaptiveimmunity. r Innate and adaptive immunity act in a mutually synergistic fashion. Inflammation, an early responseto tissueinjury or infection, involves a seriesof eventsthat combines elements of innate and adaptive immunity (seeFigure 24-6).

Fc

24-8 The basicstructureof an immunoglobulin A FIGURE alsoknownas areserumproteins molecule,Antibodies structures Theyaretwo-foldsymmetrical immunoglobulins light heavychainsandtwo identical of two identical composed yields fragments with proteases of antibodies Fragmentation chains. papainyields Theprotease capacity. thatretainantigen-binding yieldsbivalent pepsin protease the and fragments, F(ab) monovalent butthis TheFcfragmentis unableto bindantigen, fragments. F(ab')z properties hasotherfunctional portionof the intactmolecule S:T R U C T U RAEN D F U N C T I O N IMMUNOGLOBULINS

1063

PentamericlgM is stabilizedby an additional polypeptide,the J chain

*€i = Disulfidebond

J chain

Tail piece

lgM

< FIGURE 24-9 lmmunoglobulin isotypes. Thedifferent classes of immunoglobulins, called isotypes, maybedistinguished biochemically andby immunological techniques In mouse andhumans therearetwo light-chain isotypes (rlerupue elrs Surpurq-ua8rlueeql olur slralord leqt-(9,111) g uor8ar alqerre,t.reddq-dool e ;o ued sruro; 'uorsrreldruryeuorloun[ ecuanbasuoJe sE pe]eraue8'luroI ;o esre^rp dn8tq aqr tpql s,vroqsurerlJ rq8rl aqr Jo ernltnns Ieuorsueurp-eeJr{leql ;o uortcadsul'uorsrcardruryeuorlrunl ruo{ osle tnq (slueuSeseue8[ pup A ]o a8esnler.roleurquror eqr tuor; dluo tou sasrr€oroJoroqt.{trsre^rpuregc-rq8r1 'srsaqludsureqc-rq8ryqrr,n alqrredruoJsr leqt eruerl Surpee.r e ur sllnseJsuortJeeJuorleurquloJeJeeJrltur euo ,{1ug 'perlrp -e.rd aq touupf, nnpord fA aql to oruprJSurpearpue aruanb -as eqt 'aurquoler tuaur8as e pue E re^euor{lN'sluro[ 8ur f A -pol aqt seprloelrnu lE Jo ssol pue uorlrppe oqt ruorJ lred ur Surtlnsoruospatdtut puorlcunl sr ssacordtuarua8uer.rear aql ur luarer{ul 'aua8ureqo-tq3qleuououn; e Surleraue8'tlCUX pue AI ase8rlylqq dq.raqraSorpere8qare spueaql'passorord spue Surpoc aqt ueeq pue pauado a^eq surdrreqer{t eluo 'sJprtoJlJnu Jo IeAoruJJur Surtlnsa.r allecrrdloaycnuoxapelleue aq .,{eu osle r{lrq,tr 'secuanbas papuens-apursuorls Surlean'eqlearq ot puat elnlelour VNC eqt spue eqr Allecrrrauruds pauado sr urd.rreqoqt tr uo^a Jo 'rea.a,uo11 'uortBtuJoturSurpoc leurSrroeqr IIp surele: urdrreq e 1o Suruadoculaurufg 'suolSar Surpoc I aqr pue A orp or dllenba dldde salrrlrqrssodaseql 'uor8ar Surpoc 1eur8r.ro eql ruort saprtoallnu Jo Jelotuer aql ur Surrlnsa.r'>petle crtdlo -elf,nuoxa dq pa,roua.r aq deru Sueqra,roeql {lazrrreurarly 'uottsenb ut luatu8as eua8 agr;o uor8ar Surpoc leur8r.roaql wqt 'sapnoapnu-d palyec'sepuoelrnu IEJO ;o ued tou ere,l.r. -^as uourppe eqt ur sllnsar eserarudlod Jo VNC dq Sueqra,ro srr{r ur 3ur11lg'pateraua8sr ecuanbeseuo-rpurledpopuerls Jo -a18urs't.roqs e 'crrlorurudsesr urdrreq e Suruado ;1 ;o 'surd.rreqaqr Suruedo ;o lno sarrrer 'eseur4urelord ruepuadap-yNq Jo lrunqns crtdle -]eJ er{l serrnbar uorlfunl osoq,r\'snualry uralotd aqJ '(E pup U 'SyVZ arn8rg aas)dlecrueurudse ro dllerr.nauurds rncco deu Suruado srqt :ssacordsrql ur dars da1 E sr spua Surpocaqt le surdrreqaqi to Suruadoag1 'paurot aq ot stueu -3as eqr ;o uorlrunf eql ]E petperc sr drrlrqer.rp^Ieuortrppe srgl 'saruenbasurlnqopounurrur Jo drlllqelte,r aqr Surpued -xa JoJ suEaru ]euourppe apr,,rorduouBurqruoJarJo esJnof, eql uI palEOJf, selelpoluJoluleql ;o Surssacord'uro[ o] sluaru -8es aua8 pr" [ A to uonralos ruopupr aqr .{q perea.rcdrlllqe -rrea acuonbasaql ot uonrppp ul uogslrerdr.ul leuolllunf dcuanr;apounrutureJelesruo{ Jet;ns uonrunJ aua8gyg ur strelep qrr.r,ra1doa4'sl]eJg to af,uosqe alalduor aql ot speeldcuanr;ap5yg Apuanbasuocfruatudo -la^op Iler-g roJ Ierluessesr sselo.rdtuaura8ue;.rearaqr trto1 -aq paqrrlsep sy 'stuaue8uerrearaue8rrtetuos dllllqlssod Jo eqt saterell1qosutalord OVU Jo srseqtudseql ur sllaloq

lpql os teqreSor xeldruoc slql ploq sutalord 08n) pue 0/n) eqt epnpw leqt saxaldtuor urolord 'sSSU aqt Jo spua eqt lp s>leorqpuens-olqnop pue 'spue Sutpoc01rr1oql Jo r{lee roJ urd.rreqpesolc dlrualg^or e SuttBarc'puerls d.retuaureldruoc aql uo IrEDe rrlrqdoapnu p selnraxeuaqt dno.r8HO- petearl d1,uauqceg '(e1nr;areds dq tZtZt peller-os aql) lueue8uerre -ar ad& srql ur e8e8uouer ql3ual tuereulp yo sreredsqlr.r,r 1o sSSU reureuou-.raureldeqssassodlegt stuaru8esaue8 d1u6 '559 tueretpe slr pue eruonbesSurpor r{lBO drepunoq ne to -xe aql lp lnl pepuerls-elSulsB aleru ueql seseulquoJororlJ uotssn)stp lo] ]xa]aa5 atelde)p]lou saopuotltppe leuor]rppp uor6ar-p auesaq]{q rnrto slueu.rOes ]eq]]derxeusrueq)our f pue (E) ureq)-lq6r; slueLua0uerreou suorOer burpor n C puen aql lo ulor1peleraue6 slueuOes oM]aql surol asebrl 11 VNC oseol]nuoxo ue{q pesr>xa aqrieuro (E) oserou^lod V5C,iq urpallr}ere parredun suor6el Ourpor C puen oql Llloq]o spuooql lp s6ueqrano {1;e>r1euo1ne aql Qla/'p) 6ueqreno rruuorpurled e solporr 6uruado parredun ia>uenbes ruleururlse uropuer +osaprloal)nu 1o(ano1lal) 'Qq6U'gt)surdrreq peuedo bueq.rano ue 6urlereua6 {1;eruleu.rLuIs o1rauueu]uopueoepur-oleloLUol e ursoprioal)nu sppeeseralsuel] '(E) lueu.rOes n aq}ro} uMoqsse ;{prloepnu{xoap leurLurol Illerulauuu.r,ise ro 'lueubesf oql roj uMoqsse '(fl) {lletulaLlu{s 'peuedo (E) req]1e ere surdrreq oq1 SSIe qtrMftepunoqqleale upal)e puepua6urpotqtee1euldtreq pasolt )earqpapuerls-alqnop 'spuells {l1ualenor e 6urlearr fuelueulaldu.ror aq}))e}}esdnot6pgpuese:uanbas 6urpo:aqtuaaMlaq sauepunoq ,€ oar+oqf (E) SSSU aq]]e sln)papuerls-a16urs saleu xe;duLol oq] ZDVU/i9VU 'poplo] puereqle6o]asoppeurof eq o1slueu6as eq16ur6uuq (E) sr yp6 aurl-ura6 eq1'suorbe-r burpor6 puen lo Oururol ur leuorla;ep sluanl()) umoqsoieVNCoq]+ospuellsqlog xoldu,ro) eseutqulo)al pa6ueleeq feu paurol {11er1eds ZSVUpup [9VUeq]{q pezr;rqe1s aq o1suorber 6urpo> onniMoq]o lopoulletrleqlodA;1 (q) puells (do})ouresaLl]uo peeluaqMn q)ealo pua,Eaq]le puno+osoq] puefueluauLa;duio) o1lelleredrlue oreC jo pua,g aq]le satuanbes reueldaqpue.rouleuou aq1laredsdq-gZe q]tMSSU reltuts e puo,Srreq]]e ssassod aurquro)ar ue)n e q)rllMqltmslueu.r6es 6 aq]]o q)el roLueuou e puelaledsdq-Zt e laueldaqe 1opesodr-uot panJesuo> (SSU) o)uenbas uorleurquo)ar e slsluaur6as euebI ;euOrs eue6urlnqolbounuur lle]o puo,€ aq]]V sluer-u6as ,Louorleurquo)al )lleuros urpa^lo^ur siuourolo VNCaql]o uorle)ole) (qVfVZ 'ureqr-{neeq ernOrg ees)snlol'ureq)-l(.16rl aql ur ate ]ou ]nq luesard slueu6as elduexa 6 luaul6as C e pue]uauubas 4 e 1obururofsprdep srql'6ururolleuollalapernslueu6eseue6ur;nqo;60untuurl 1o lueura6ue.uear lo ruslueq)a4 St-tz lungll >

salpllol B 0utoolo^op aql '(E) o^r})npolduou stosleuotleurqLxo)or fC o] n puo)as '(E) all] 'ra^oMoH spae)ord o^tpnpord sl luaurdolo^ap +r lle)-Buer.l] puo)esaq]11([) aOuetreet uorleurquro)ar leut er.uosoujolq] puo)as aql '(uopo)dolseleudotddeut uese)npollul'o l) anrltnpotduou sruorleurqurofer ol all] speetord lsrU fC luauudo;enep n 11 11er-g '(E) pup'saseo) srlueule6ueleat lueua6ueleerJaqlin,L enrltnpotd

A9o]oNnnnt

I

tz ulrdvH)

zL0l

^lMauoqi auouo ]uaLu6os stqlJLlaurosou-tojq) rc paoueireoj q]rmsa6uerJear uaqlluouJ6os oLll6ur^JJe) n V sn)olureq)-^Aeoq qloquo ro auouo '(u) ]srl+rn))o slueuJeouerJear somosoLuor]r . ^. ^-,-,i-^ -, (9 puee)soLuosou..rorLl) snobolouroq uo .,e I w+u ps+J,uev 6utpo) sn)olureq)-^^eaq aq]]o sardo)oM]oql a)uanbas luosord aq ]snurleql sluour6as aua6f pue uor6or-n e uro] o] pabuelieal '6 oql 'sn)ol sapnl)ur snlol ureq)-I^peq urlnqol6ounLuLur 1 e1dr1lnu )rleuros 9l-tz lunglJ V ureq)-^^eeq eql +o uorleurquro)er

'serp l l a cg 6 u r d o ; e n e p eql pue opeursr ureLlcnneoq leuotlcunJou 'eAl -lcnporduousr os;e luauebuellesl 16-71 puocas11 p

ffi 'spoecoJd lueudo;anep ri oql llac-Epue possordxeaq uec ureqc-A^eoq uaLll'alrlcnpord sr lueue6uelleol fq-n puocasll E

'(q) oLuosoutolqcpuocosaql u o r n c c ou e c l u e u e O u e l J e oul o q ] ' ( l u e s a t du o p o c dols; enrlcnpo.rduou sr gueuebuelleel f6-n lsltlJl

E

'euuosor.uorqc sno6ol -ouroq eq] uo srnccolueuabue;leeJoN sposc -ord ]ueLUdolo^op lloc-g pue pesseldxeeq uec u r e q cA n e a qr l ; e u o r l c u ne; ' e A t l c n p o l ds t ( e )a u l o s -ourorr.lc auo uo ]uotle6uelleer ;; 'r-uopuerI1e6te1 sr lueueoue:reer fq-n roJ1uer.u-des n lo uorlcalos A

'lueurebuelea.r 1-6 o6lepun sourosotuor r..lc sno6o;outoqqloq Allensn' (soutI pal) luourabuereor f-C tol A;r.uopue.r palcalosale uorle.rn6r;uo e cu r ; - u r r e a6 q l u r s l u e u t 6 a sI p u e q

!

,s

,e

,9q

,t

dcfvcA

]ueuebuereel antlcnpold=

'sluarua8ue.r.rear [q-n pue f-q;o asrnoca'qu1'@1-y7 IeurlrJrot ern8rg) lueueSuerreer fC-A dq pa.r,royloy sr luaue8uer.rear 'sruarua8uerreeJ rllpn 3ur1.re1s '1srrysa8ue.rreals^,(e.{\le f-q [-C snf,olursr{r-d^Eer.{ aqt 'tuaudolo,tap ]lrr-g Jo esrnor eqt uI 'sluOruaEueJJeaJ UTELIJ -rq8rl roJ eloqe peqrrrsapsusrupr{rorueutesaqt Er^ paecord stueureSuPrrEOr ureqJ-,{^eeH'alnrroru€uou-raruefieq€U ZI eqt Jo ^\er^ ur pe^\olle sr SururoIC or C rou f ot A l]errp raql -rau lele^\oH 'stueru8es[q pa8uer.rea.r .,(pearleo] stueru8es A pup'stuaur8as I ot urof upf s]uetuSasC lerll r{f,nsoJe sSSUasoqtur sqfuel rareds oql 'SSUelrsrnboreql qtr.ry\pue

'((e)Sf -lZ ,S rreql le paddrnbadlreprursare stueru8asI eqt ern8rCaas)tuauSas C r{reeto pue /t agl pue pua /S:eql lE puB dreluerualdruocuI PunoJosle uorle.rnBrJuor1a11e.redrrue are sSSU esor{I 'VNC ureqc-rq8rl ul (sSSU)saf,uenbesIeu -3rs uorleurqruorer eqt ot r?lltuls 'VNC.raceds ^,(qpareredas saruenbasJerueuoupuE Jelueldoqpe^JesuoJere aragr'y51q urer{r-d^eequr tueur8asaua8 n qlee Jo Pue ,€ erlr lV '1Hr1)uor8a; alqErrE^ureqc-d,teaqaqr Surporue aruenbospeSuerrea.re selpJaue8luau8es I pt" 'C 'A E Jo uolleurqulofeJrrleluos 'GVy-VZ arn8r4 eas) slueuSes (dllsre^lP) q eldrrlnru oslt

deoxynucleotidyltransferase(TdT) may add nucleotides to free 3' OH ends of DNA in a template-independentfashion. Up to a dozen or so nucleotides,called the N-region, may be added, generatingadditional sequencediversity at the junctions wheneverD-J and V-DJ rearrangementsoccur (seeFigure 24-1,5, step 6 ). Only one in three rearrangementsyields the proper reading frame for the rearrangedVDJ sequence.If the rearrangementyields a sequenceencoding a functional protein, it is called productive. Although the heavy-chainIocus is present on two homologous chromosomes,only one productive rearrangementis permitted, as discussedbelow. An enhancerlocated downstream of the cluster of J segments and upstream of the p, constant-regionsegmentactivatestranscription from the promoter at the 5' end of the rearranged VDJ sequence(seeFigure 24-1.6). Splicing of the primary transcript produced from the rearranged heavychain gene generatesa functional mRNA encoding the p heavy chain. For both immunoglobulin heavy- and lightchain genes, somatic recombination places the promoters upstream of the V segmentswithin functional reach of the enhancersnecessaryto allow transcription, so that only rearranged VJ and VDJ sequences,and not the V segments that remain in the germ-line configuration, are transcribed.

S o m a t i cH y p e r m u t a t i o nA l l o w s t h e G e n e r a t i o n and Selectionof Antibodieswith lmproved Aff inities In addition to the diversity createdby somatic recombination and junctional imprecision,antigen-activatedB cells can undergo somatic hypermutation. Upon receipt of proper additional signals,most of which are provided by T cells,expression of activation-induceddeaminase(AID) is turned on. This enzymedeaminatescytosineresiduesto uracil. lfhen a B cell that carriesthis lesion replicates,it may place an adenineon the complementarystrand, thus generatinga G-to-A transition (seeFigure 4-35). Alternatively,the uracil may be excised by DNA glycosylaseto yield an abasicsite.Theseabasicsites, when copied,give rise to possibletransitionsaswell as a transversion,unlessthe nucleotideoppositethe gap is chosento be the original G that paired with the cytosinetarget.Mutations thus accumulatewith every successiveround of B-cell division, yielding numerous mutations in the rearrangedVJ and VDJ segments.Many of these mutations are deleterious,in that they reducethe affinity of the encodedantibody for antigen, but some improve the encoded antibody's affinity for antigen. B cells carrying affinity-increasingmutations have a selectiveadvantagewhen they competefor the limited amount of antigenthat evokesclonal selection(seeFigure 24-11').The net result is generationof a B-cell population whose antibodies, as a rule, show a higher affinity for the antigen. In the course of an immune responseor upon repeated immunization, the antibody responseexhibits affinity maturation, an increasein the averageaffinity of antibodies for antigen, as the result of somatic hypermutation. Antibodies produced during this phase of the immune responsedisplay affinities for antigen in the nanomolar (or better) range. For reasonsthat are not understood. the activity of activation-

induced deaminaseis focused mostly on rearrangedVJ and VDJ segments,and this targetingmay thereforerequire active transcription. The entire processof somatic hypermutation is strictly antigen dependent and shows an absolute requirement for interactionsbetweenthe B cell and certain T cells.

B-CellDevelopmentRequiresInput from a Pre-BCell Receptor As we have seen,B cells destinedto make immunoglobulins must rearrange the necessarygene segmentsto assemblea functional heavy-and light-chain gene.Theserearrangements occur in a carefully ordered sequenceduring developmentof a B cell, startingwith heavy-chainrearrangements.Moreover, the rearrangedheavy-chainis first usedto build a membranebound receptor that executesa cell fate decisionnecessaryto drive further B-cell development(and antibody synthesis). Successfulrearrangementof V, D, and J segmentsin the heavy-chain locus allows synthesisof a p chain. B cells at this stageof developmentare calledpre-B cells,as they have not yet completed assemblyof a functional light-chain gene and therefore cannot engagein antigen recognition. The newly rearrangedheavy-chaingene encodesthe p polypeptide, which becomespart of a signaling receptor whose expressionis essentialfor B-cell developmentto proceed in orderly fashion. The p chain made at this stage of B cell development is a membrane-bound version. Following engagementwith antigen, the B cell switchesto producing soluble, secretedimmunoglobulins from the sametranscription unit, as we describebelow. In pre-B cells,newly made p chains form a complex with so-calledsurrogate light chains, composed of two subunits, no \5 and VpreB (Figure24-1'7).The p chain itself possesses cytoplasmic tail and is therefore incapable of recruiting cytoplasmic components for the purpose of signal transduction. Instead, early B cells expresstwo auxiliary transmembrane proteins, called Igct and IgB, each of which carries in its cytoplasmic tail an immunoreceptor tyrosine-basedactivation motif, or ITAM. The entire complex including Igct and IgB constitutes the pre-B cell receptor (pre-BCR). Engagement of this receptor by suitable signals results in recruitment and activation of a Src family tyrosine kinase, which phosphorylates tyrosine residues in the ITAMs. In their phosphorylated form, ITAMs recruit other molecules essentialfor signal transduction (seebelow). Becauseno functional light chains are yet part of this receptor,it is incapable of antigenrecognition' The pre-B cell receptor has severalimportant functions. First, it shuts off expressionof the RAG recombinases,so rearrangement of the other (allelic) heavy-chainlocus cannot occur. This phenomenon, called allelic exclusion, ensures that only one of the two available copies of the heavy-chain locus will be rearrangedand thus expressed.Second,because of the associationof the pre-B cell receptor with Igct and IgB, the receptor becomesa functional signal-transductionunit. The pre-BCR also initiates cell proliferation and so expands the numbers of those B cellsthat have undergoneproductive D-J and V-DJ recombination.

O G E N E R A T I OO NF A N T I B O D YD I V E R S I TAYN D B - C E L LD E V E L O P M E N T

1073

,_

Pre_BCR______]

],.5

'gro,tsl Exterior

E Expressionof VpreB and 1,5 turned off

,.i i:

t,a

E Light-chain rearrangement

Cytosol

E

Assembly with surrogate l i g h tc h a i n s

A FIGURE 24-17 Structureof the pre-Bcell receptorandits role in B-celldevelopment.Successful rearrangement of V,D, and j heavy-chain genesegments allowssynthesis of membrane-bound p heavychainsin the endoplasmic (ER) reticulum of a pre-Bcell At this stage,no light-chain generearrangement hasoccurred. Newlymade p chains assemble with surrogate lightchains, composed of L5 and VpreB, to yieldthe pre-Bcellreceptor, (tr) Thisreceptor pre-BCR proliferation drives of thoseB cellsthatcarryit lt alsosuppresses rearrangement of the heavy-chain locuson theotherchromosome

andso mediates allelic exclusion. In thecourse of proliferation, the synthesis of L5 andVpreBisshutoff ([), resulting in "dilution"of theavailable surrogate lightchains andreduced expression of the pre-BCR As a result,rearrangement of the light-chain locican (E) lf thisrearrangement proceed isproductive, the B cellcan synthesize lightchains andcomplete assembly of the B-cellreceptor (BCR), comprising a membrane-bound lgMandassociated lgo and lgB TheB cellisnow responsive to antigen-specific stimulation.

In the course of this expansion, expressionof the surrogate light-chain subunits, VpreB and },5, is turned off. The progressive dilution of VpreB and \5 with every successivecell division allows reinitiation of expressionof the RAG enzymes,which now rarget the rcor L light-chain locus for recombination. A productive V-J rearrangemenr also shuts off rearrangementof the allelic locus (allelic exclusion). Upon completion of a successfulV-J light-chain rearrangement,the B cell can make both p, heavy chains and r or I light chains, and assemblethem into a functional B-cell receptor (BCR), which can recognizeanrigen (seeFigure 24-17). Once a B cell expressesa complete BCR on its cell surface, alI subsequentstepsin B-cell activation and differentiation involve recognition of the antigen for which that BCR is specific.The BCR not only plays a role in driving B-cell proliferation upon successfulencounter with antigen, but also functions as a device for ingesting antigen, an essentialstep that allows the B cell to process the acquired antigen and convert it into a signal that sendsout a call for assistanceby T lymphocytes.This antigen-presentationfunction of B cells is describedin later sections.

During an Adaptive Response,B CellsSwitch f r o m M a k i n g M e m b r a n e - B o u nldg t o M a k i n g Secretedlg

1074

.

C H A P T E2R4 I

IMMUNoLoGY

As just described, theB-cellrecepror(BCR),a membranebound IgM, provides a B cell with the ability to recognizea particular antigen, an event that triggersclonal selectionand proliferation of that B cell, thus increasingthe number of B cells specific for the antigen (see Figure 24-11). However, key functions of immunoglobulins, such as neutralization of antigen or killing of bacteria, require that theseproducts be releasedby the B cell, so that they may accumulatein the extracellular environment and act at a distance from the site where they were produced. The choice between the synthesisof membrane-bound versus secretedimmunoglobulin is made during processing of the heavy-chain primary transcript. As shown in Figure 24-18, the p locus conrainstwo exons (TM1 and TM2) that together encode a C-terminal domain that anchors IgM in the plasma membrane. One polyadenylation site is found upstream of these exons; a second polyadenylation site is presentdownstream.If the downstreampoly(A) siteis chosen,

.}l Primary transcript

TM1 TM2

Cp4 lts

5

I Enhancer

Poly (A) sites

( a )P o l y a d e n y l a t i o n at upstreamsite

mRNA

!.,/

i I

fT

-.i

(b) Polyadenylation at downstreamsite

AAAAAAA

]_@AAAAAAAA

J SecretedlgM

J

M e m b r a nleg M A FIGURE 24-18 Synthesis of secretedand membranelgM. The primary organization of the p heavy-chain isshownat the transcript top:C,,4istheexonencoding thefourthp constant-region domain, p, isa codingsequence uniquefor secreted lgM;TM1andTM2are exonsthatspecify the transmembrane domainof the p chain Whethersecreted or membrane-bound lgM ismadedepends on whichpoly(A) siteisselected duringprocessing of the primary

(a)lf the upstream poly(A) mRNA siteisused,the resulting transcript formof the p thesecreted includes the entireC.4 exonandspecifies poly(A) siteisused,a splicedonorsitein chain(b)lf the downstream yielding exons, to the transmembrane splicing the Cp4 exonallows formof the p chain. the membrane-bound a mRNAthatencodes forms generate andmembrane-bound secreted mechanisms Similar SS: signalsequence of otherlg isotypes

then further processingyields a mRNA encoding the membrane-bound form of p. (As describedabove, this choice is necessaryfor formation of the B-cell receptor,which includesmembrane-boundIgM.) If the upstreampoly(A) site is chosen, processing yields the secretedversion of the p chain. Similar arrangementsare found for the other Ig constant-regiongene segments,each of which can specify either a membrane-boundor a secretedheavy chain. In the courseof B-cell differentiation, the B cell acquiresthe capacity to switch from the synthesisof exclusivelymembrane immunoglobulin to the synthesisof secretedimmunoglobulin. Terminally differentiated B cells, called plasma cells, are devoted almost exclusivelyto the synthesisof secretedantibodies (seeFigure 24-6). Plasma cells synthesizeand secreteseveral thousand antibody moleculesper second.It is this ramped up production of secretedantibodies that underlies the effective-

nessof the adaptive immune responsein eliminating pathogens. The protective value of antibodies is proportional to the concentration at which they are present in the circulation' Indeed' circulating antibody levelsare often used as the key parameter to determinewhether vaccination againsta particular pathogen has beensuccessful.The ability of plasma cellsto establishadequate antibody levelsis a function of their ability to secretelarge amounts of immunoglobulins and so requiresa massiveexpansion of the endoplasmicreticulum, a hallmark of plasma cells.

P

VDJ

6

Y3

Factorsrequired for classswitching:

VDJ

p

B CellsCan Switch the lsotyPeof l m m u n o g l o b u l i nT h e y M a k e In the immunoglobulin heavy-chainlocus, the exons that encode the p, chain lie immediately downstream of the rearranged VDJ exon (Figure 24-19, /op). This is followed by

Yl

^'12

t

ct

C D 4 Tc e l l s tL-4 AID

r

- b l o o di m m u n o g l o b u l i n lgG12 24-19 Classswitchrecombination in the A FIGURE locus.Class switchrecombinatron immunoglobulinheavy-chain (colored involves switchsites,whicharerepetitive sequences circles) genesRecombination of the heavy-chain constant-region upstream (AlD),assistance byT cells, requires activation-induced deaminase (e q., lL-4)produced T cellsRecombination andcytokines by certain

lgA - secretionacrossepithelia

of theswitchsiteupstream of DNAbetween thesegment eliminates Class occurs switching to which region constant and the exons [.i, for with thesamespecificity generates molecules antibody switching the original mounted B cell that lgM-bearing antigenasthatof the and constant-regions heavy-chain butwith different response, thereforedifferenteffectorf unctions

Y N D B - C E L LD E V E L O P M E N T O F A N T I B O D YD I V E R S I T A GENERATION

1075

exons that specify the 6 chain. Transcription of a newly rearranged immunoglobulin heavy-chainlocus yields a single primary transcript that includes the p and 6 constant regions. Splicing of this large transcript determineswhether a p chain or a 6 chain will be produced.Downstream of the p/E combination are the exons that together encodeall of the other heavy-chainisotypes.Upstream of each cluster of exons (with the exception of the 6 locus) encodingthe different isotypes are repetitive sequences(switch sites) that are recombination prone, presumablybecauseof their repetitive nature. Becauseeach B cell necessarilystarts out with surface IgM, recombination involving thesesites,if it occurs, results in a classswitch from IgM to one of the other isotypes located downstream in the array of constant-regiongenes(see Figure 24-19). The intervening DNA is deleted. In the course of its differentiation, a B cell can switch sequentially. Importantly, the light chain is not affectedby this process,nor is the rearrangedVDJ segmentwith which the B cell started out on this pathway. Classswitch recombination thus generatesantibodies with different constant regions, but of identical antigenic specificity.Each immunoglobulin isotype is characterizedby its own unique consrantregion. As discussedpreviously,these constant regions determine the functional properties of the various isotypes. Class switch recombination is absolutely dependenton rhe activity of activation-induceddeaminase(AID) and the presenceof antigen and T cells. Somatic hypermutation and classswitch recombination occur concurrently,and their combined effect allows fine tuning of the adaptive immune responsewith respect to the affinity of the antibodies produced and the effector functions called for.

Generation of Antibody Diversity and B-Cell Development r Functional antibody-encodinggenesare generatedby somatic rearrangementof multiple DNA segmentsat the heavy-chainand light-chain loci. Theserearrangemenrslnvolve V and J segmentsfor immunoglobulin light chains, and V, D, and J segmentsfor immunoglobulin heavy chains (seeFigure 24-14). r Rearrangementof the V and J, as well as of the V, D, and J gene segmentsis controlled by conservedrecombination signalsequences (RSSs),composedof heptamersand nonamers separatedby 12- or 23-bp spacers(seeFigure 2415). Only those segmentsthar have spacersof different length can rearrangesuccessfully. r The molecular machinery that carries out the rearrangement processincludes recombinases(RAG1 and RAG2) made only by lymphocytes and numerous other proteins that participate in nonhomologous end yoining of DNA moleculesin other cell types as well. r Antibody diversity is created by the random selectionof Ig genesegmentsto be recombined (combinatorial yoining) and by the ability of the heavy and light chains produced from rearranged Ig genesto associatewith many different 1076

CHAPTER 24

I

IMMUNOLOGY

light chains and heavy chains, respectively(combinatorial association). r Junctional imprecision generatesadditional antibody diversity at the joints of the gene segmentsjoined during somatic recombination. r Further antibody diversity arisesafter B cells encounter antigen as a consequenceof somatic hypermutation, which can lead to the selection and proliferation of B cells producing high-affinity antibodies, a process termed affinity maturatlon. r During B-cell development,heavy-chaingenesare rearrangedfirst, leading to expressionof the pre-B cell receptor. Subsequentrearrangementof light-chain genesresults in assembly of an IgM membrane-bound B-cell receptor (seeFigure24-17). r Only one of the allelic copies of the heavy-chain locus and of the light-chain locus is rearranged(allelic exclusion), ensuring that a B cell expressesIg with a single antigenic specificity. r Polyadenylationof different poly(A) sitesin an Ig primary transcript determineswhether the membrane-boundor secretedform of an antibody is produced (seeFigure 24-18). r During an immune response,classswitchingallows B cells to adjust the effectorfunctions of the immunoglobulinsproduced but retain their specificityfor antigen (Figure 24-19).

W

rhe MHCand Antigenpresentation

Antibodies can recognizeantigenwithout the involvement of any third-party molecules;the presenceof antigen and antibody is sufficient for their interaction. Although antibodies contribute to the elimination of bacterial and viral pathogens,it is often necessaryto destroy also the infected cellsthat serveas a sourceof new virus particles.This task is carried out by T cellswith cytotoxic activity. Thesecytotoxic T cells make use of antigen-specificreceptors whose genes are generatedby mechanismsanalogous to those used by B cells to generateimmunoglobulin genes.However, antigen recognition is accomplishedvery differently by T cells than by B cells.The antigen-specificreceptorson T cells rccognize short snippets of protein antigens,presentedto thesereceptors by membrane glycoproteins encoded by the maior histocompatibility complex (MHC). Various anrigen-presenting cells, in the course of their normal activity, digest pathogen-derived(and self) proteins and then "post" these protein snippets (peptides)to their cell surfacein a physical complex with an MHC protein. T cells can inspect these complexes, and if they detect a pathogen-derivedpeptide, the T cells take appropriate action, which may include killing the cell that carries the MHC-peptide complex. MHC proteins, which commonly are called MHC molecules,also facilitate communication between T cells and B cells.B cellsdo not usuallyengagein production of secreted antibodies unless they receive assistancefrom another subset of T cells, called helper T cells. These T cells also use

antigen-specificreceptors to recognize MHC-peptide complexes.In this section,we describethe MHC and the proteins it encodes,and then examine how theseMHC moleculesare involvedin antigenrecognition.

The MHC Determinesthe Ability of Two U n r e l a t e dI n d i v i d u a l so f t h e S a m eS p e c i e s to Accept or RejectGrafts The major histocompatibility complex was discovered,as its name implies, as the genetic locus that controls acceptance or rejection of grafts. At a time when tissueculture had not yet been developed to the stage where tumor-derived cell lines could be propagated in the laboratorS investigatorsrelied on serial passagein vivo of tumor tissue.It was quickly observed that a tumor that arose spontaneously in one

inbred strain of mice could be propagated successfullyin the strain in which it arose, but not in a geneticallydistinct line of mice. Geneticanalysissoon showed that a singlemajor locus was responsiblefor this behavior. Similarly, transplantation of healthy skin was feasible within the same strain of mice, but not when the recipient was of a geneticallydistinct background. Genetic analysis of transplant rejection likewise identified a single major locus that controlled acceptance or rejection, which is an immune reaction. As we now knoq all vertebratesthat possessan adaptive immune system have a geneticregion that correspondsto the major histocompatibility complex as originally defined in the mouse' An important step in discovering the functions of the MHC was development of mice strains congenic for the MHC. Congenic strains are genetically identical except for the locus or geneticregion of interest. Figure 24-20 outlines

With eachsuccessivebackcross to StrainA, the geneticcontribution of StrainA is increased.

At eachsuccessivegenerationafter the Fr, checkwhether skin from offspiing shows rapid rejectionby a o u r e S t r a i nA a n i m a l . lf yes, offspringpossessesan MHC that is non-A,thereforeit must be MHCB.

By continuingto selectfor the desiredtrait (MHC B, allotypic m a r k e r )a . c o n g e n i cl i n e o f m i c ei s o b t a i n e d .

+ + 20 Generations +

EXPERIMENTAL FIGURE 24-20 Micecongenicfor the major histocompatibility(MHC)are generatedby crossingtwo histo-incompatible strains.Strain A andstrainB,whichrejecteach grafts,aresaidto be histo-incompatible other's anddifferat their MHC.The(A x B)F1progeny acceptgraftsfromeitherparental (e.g, strain.Bybackcrossing F1miceto oneof the parental strains strainA) for manygenerations, thecontribution of strainA to the geneticmaterial of the resulting offspring will increase Breeding is

Breedto homozygosity for MHC B bv brother/sister m a t i n g .S t r a i ni s g e n e t i c a l l y 'A' with the exceptionof MHC which is "Bl'

the MHC thathaveretained performed suchthatonlythoseanimals of the strainB MHCis of strainB areusedfor breedingThepresence Onlyif a skingraftontoa strainA recipient. by performing assessed will inbredmiceof strainA rejectthe the B-typeMHCispresent for the B-typeMHC andassays graft Byperforming suchbackcrosses thatis a strainof miceisobtained for 20 or moregenerations, the MHCof strainB A in itsgeneticmakeup,yetretains essentially for the MHC. Thesemicearesaidto beconqenic THE MHC AND ANTIGENPRESENTATION

1077

( a ) M o u s eM H C ( H - 2c o m p l e x ) H-2K

l-A

t-E

H-2D

L

F3qP ( b ) H u m a nM H C ( H L Ac o m p l e x ) HLA-DO

HLA-DR

HLA-B

HLA-C HLA-A

*fl ep

organization and gene content show considerablevariation betweenspecies. The human fetus may also be considereda graft: The fetus sharesonly half of its genetic material with the mother, the other half being contributed by the father. Antigens encoded by this paternal contribution may differ sufficiently from their maternal counterparts to elicit an immune responsein the mother. In the course of pregnancy,fetal cells that slough off into the maternal circulation stimulate the maternal immune system to mount an antibody response against these paternal antigens. The antibodies recognize structures encoded by the human MHC. The fetus itself is spared rejection becauseof the specialized organtzation of the placenta, which prevents initiation of an immune responseby the mother againstfetal tissue.

The Killing Activity of CytotoxicT Cellsls Antigen Specificand MHC Restricted

Clearly the function of MHC moleculesis not to prevent the exchangeof surgical grafts. MHC moleculesplay an essenprotein protein il tial role in the recognition ofvirus-infected cells by cytotoxic T cells; these cells also are called cytotoxic T lymphocytes A FfGURE 24-21 Organizationof the major histocompatibitity (CTLs). In virus-infectedcells,MHC moleculesinteract with complexin miceand humans.Themajorlociaredepicted with protein fragments derived from viral pathogensand display schematic diagrams proteins of theirencoded belowClassI MHC these on the cell surface where cytotoxic T cells, charged proteins arecomposed of a MHC-encoded single-pass transmembrane with eliminating the infection, can recognizethem. glycoprotein in noncovalent association with a smallsubunit, called Mice that have recoveredfrom a particular virus infecB 2 - m i c r o g l o b uwl ihni ,c hi sn o te n c o d eidn t h eM H Ca n di sn o t tion are a ready sourceof cytotoxic T cellsthat can recognize membrane bound Classll MHCproteins consist of two nonidentical and kill target cells infected with the same virus. The rasingle-pass transmembrane glycoproteins, bothof whichareencoded dioactivechromium (51Cr)releaseassaycan be used to deby the MHC tect the presenceof cytotoxic T cells in single-cell suspensions prepared from the spleenof an animal that has cleared how mice strains congenic for the MHC can be generated. an infection (Figure24-22a).If T cells are obtained from a Congenic strains are essentialtools for assigning complex mouse that successfullycleared an infection with influenza immunological functions to a particular locus, such as the virus, cytotoxic activity is observedagainstinfluenza-infected MHC. As long as it is possibleto selectfor a particularphetarget cells,but not againstuninfectedcontrols (Figure24-22b1. notypic trait in the form of an allelic marker (e.g.,graft reMoreover, the influenza-specificcytotoxic T cells will not jection in the case of the MHC), congenic strains may be kill target cells infected with a different virus, such as vesicproducedfor other loci. ular stomatitis virus. Cytotoxic T cells can even discriminate In the mouse, the genetic region that encodesthe antibetweencloselyrelated strains of influenza virus, and can do gensresponsiblefor a strong graft rejection is called the H-2 so with pinpoint precision: Differences of a single amino complex (Figure 24-21a). The initial characterizationof the acid in the viral antigen may suffice to avoid recognition and MHC was followed by an appreciation of the genetic comkilling by cytotoxic T cells. These experiments show that plexity of this region. After coarsemapping by standard gecytotoxic T cells are truly antigen-specificand do not simply netic means (recombination within the MHC), the complete recognizesome attribute that is shared by all virus-infected nucleotidesequenceof the entire MHC was determined.The cells, regardlessof the identity of the virus. typical mammalian MHC contains dozens of genes,many In this example, it is assumedthat the T cells harvested encoding proteins of immunological relevance. from an influenza-immunemouse are assayedon influenzaIn humans, the discoveryof the MHC relied on the charinfected target cells derived from the identical strain of acterizationof antiseraproduced in patients who underwent mouse (strain a). However, if target cells from a completely multiple blood transfusions:Antigens expressedon the surunrelated strain of mouse (strain b) are infected with the face of the geneticallynonidentical donor cells provoked an same strain of influenza and used as targets,the cytotoxic T immune responsein the recipient. The predominant target cells from the strain a mouse are unable to kill the infected antigens recognized by these antisera are encoded by the strain b target cells (seeFigure 24-22b, E vs. E). It is therehuman MHC, a genetic region also referred to as the HLA fore not sufficient that the antigen (an influenza-derivedprocomplex (Figure 24-Z1b). All vertebrateMHCs encode a tein) is present;recognition by cytotoxic T cells is restricted highly homologous set of proteins, although the details of by strain-specificelements.By making use of MHC congenic

Class I

Class ll frp

MHC

10 7 8

CHAPTER 24

MHC

I

*4tt.,

IMMUNOLOGY

(b)

Target c el l

I Spleen

lnfect mouseawith virus X +

X XX

- K i l l e r Tc e l l s .--.-',' Single-cell -r' Labeled suspension target cell

J

/\

HarvestTcells

Virus-infected targetcell Control K i l l e r Tc e l l

Il crlu

-)pod r o q l o u eL ! o r ]a l a l l pa u o q s r n 6 u r l s r] e p q ] s a l n ) o l o uIJs s e l )u r dllr5lcads,, rJrns o,l.rt 'e?etat'e uO 'PVZ-VZ a-rn8rg)anprsar suorlrsod(sonprsat loqlue) po^lasuolale ]eqi sanptsalaatql jo oMl 'q]6uol poe ourup relnrrlred ter{t strJ .{peau leqr Jlnrelou JHIN epnl)urpue 'tutur.l0l aq] uotlepoLULxo))e radotd etrnbat ul ]o 'sureqcaprs aqt ur ra>pode dq pareporuruof,resr r{lrq.^A sanprsai (e) 6;-g a6e;eneuo oJesaln)aloLlJ Jo qcee I ssel)ol pulq leq] saprldad 'uotl)ulsor ourrueto reqrrrnullprus E to supau ,(q qalc Surpurq-aprr prre )H4 pue 6urpurq epqda6 ?Z-tZ lunglJ V -dad eql otur peror{lue sr aprldad aqt tor{rrnC 'rurrurolaqt lE sdnorSldxoqrec pue ourrur peS;eqreql eteporuruotreteqt sto slelcoo ->1codotur pr{rnt oq ueJ aprrdader{r spueaqr teqr os (sprf,e 6 u r p u r qa p r l d e d Jo lueloJjtpqlt/!^ ounup 0I or 8 dllensn'qr8ueypexrJreqter Jo aprrdade se.rrnba.r saln3alourJHtA Jo alnralou:)H14J sselc dq e Surpurq-aprrded;o epou oql'(etZ s l u e r r e ^c t l a l l v I 'spua -y7 an?;rg aes)spurg eprtded e ur qlrq,r\ qroq tE pesolr 'qa1ce ateerr serrleq oqr 'saJr1eqD o,,ral pup er{r dpurol laaqs $ dq paddol pareald-fl papuu.rrs-tg8ro up uoddns sure1ll raeqs -op eseql 'surptuop a{rT-8I lerurxord-auerqrueruo^.1 slee^or salnroloru :)Htr'\JI ssslt Jo arnlrnrls l€uorsuaurp,eorqr er{J '(ronpord snrol )Z-H aql to slu€rre^tr1elleolu ruesardor "y7 ''3'a) snnpord rqalp er{l dlrluapr eqr alou -H Pue q)Z-H to -ep ol posnsr tdrrcsradns 'lrol qz-Hpue V )Z-H oip,{q pepor 'esnoru aqr u1 .(srrnpo.rd -ua ere selnlelotu :)HI\l I ssplr eqr lrtallE v-vlH lfurlsrp oarrtluesardargzv-vlH pu€ zv-v-IH ''3'a) snrol relncrlred reqt roJ xr+InsI€f,rrarunue dq perpuglsep sr elelleenbrun arn8rgaas)rlol pue g Wel'\Z-VT J-V-IH -y-IH'V-V'IS aqr dq peporuaere sueunq ur selnrelotu )HW I ssplJer.lJ'sueunq ur porJuurprsr:npord Jrlollerluusp 0002 re^o qtr^ 'u,rou>1dltua.r.rnrcrqdrourdlod tsoru oql Suoure are slalsoo 'lce1 u1 'r;er8 eql uo luasa.rd CHW eql dg papoouasaua8eql solnJelotuJHtr^Jrouop orll Jo sernlpayenbrun SurqsrnSurlsrp ,ryer8e stre[ ;o algeder eg tsnru ruelsls eunlururs,luardrcareql -ar luardnar e :(rusrqdroudlod rrreua8)uorterrel potrrer{ur ;1 ol algernqullE 'uotlEus.,tIerntrnrls Jror{tsr uorlcaler rler8 1o stogreleqt aJEselnJalou tpqt uonou aql ur lnqdul :)HW 'sllol crpads-ua8rtup uortrorap eql rot slool I Jo tueuodrur euoteq a^pq pue sarnpecord dSolouqrar y51q tueurqruoJardg pernpord .4ou er€ suratord asaqt ,w.ro; lf,Elur ur selnlolotu :)HI\l I sspll aql ;o uorlrod relnllof,Ertxa aqt sesealerqcrqm'ureded qlr,u uorlse8rpdg salloolnal uetu -nq uror; parlr.rnddlleur8rrg 'ureruop 31 aar; E ot erntfnls ur spuodsa:.rotpue lHI tr eql dq peporua tou sr trunqns urlnqolSo.rrnu-7$ .ra11eus eql'lHW aqr dq papocue(0I-tI a-rn8rgaes)uratordoc,{18euerqurau 1 addr e sr trunqns ra8rel aq1 'seprrdaddlod o.ra.r'o tsrsuor pue dlrureyredns31 aqr or 3uo1agsolnralou:)HW I ssell salnlalot4 )HlA I ssel)

sP suorlJunJre{rEru gc3 eqJ 'Gvz-vz arnSrg)ornlrelrqlrE OJsJrnsruaroJJrpJregr asnEJOqselnf,olotuJHW parEIOr Jo -un qlr.\\ lteratur lou ,a1nre se ,a;o;eraqr il^\ elelle IHW I ssep relnJrtred auo qtr,^A]J€Jaturol pau8rsaprordarar 11ec-1 'roldeJer pue V IIel-J agt qlrm tretuor 1o slurod eql aJuer.{ elnrelou IHW eqt eJEJJnseqt tloJJE senprsercrqd_rou Jo -l1od asaqr'raqtrng .Surpurqaprrdad drplJlrads eql arueq Jo puE ro>llod Surpurg-aprrdadeqt;o ornlfetrqf,re eg] ourruretep eroteroql senprseresoql .Ualr Surpurq-eprrdadeqr punore

'GZ-V7 arn8rg) roldaoa.r IIel-J er{rpue sapndadqtr.&\suorl -Jerelur Jreql eJEse slcedsa.r dueru ur Jelrurs dllBrntrnrls e.re osle selnlalotu :)HW Jo sessslJqlog 'sanadsrrues er{l Jo slp -nprlrpur Suorue stuerJpl rr1a11e dueur ,sr ter{l lttqd.totu ]srxe -K1od (yq3vqere salnrelotu :)HI\l II sspp pue I sselr rpofl

toldo)eu lla)-I eql ql!^ l)eralul pue s u a o r t u ve p r l d o dp u t g s o l n ) a l o N) H l l

eqr dn 3o1cor lou s€ os palotuer dlprde-rag lsnu suralo.rd Sururetuor-rorreqrns 'soruouog1err.l.dq par;neds esoql pue suratord u.v\os.ller rsoq aqr rpoq lroJJesrsaqludsurelord ur sa>lelsruasaql '(3urp1o;pedelepro .redordrur'styrqs arue.r;'spne ourr.uu1o uorle.rod.rorursrur) srorra uror; 3ur -re;Jns.ro dlarnleruaJdpoleuruuol Suraqsureqf,oprrdaddlod pelertrurdl,trau1o tuerrad 0€-0I qrl^\ 'ssecordeuord-rorra uE sr 'uorlplrldar ylrlq a1t1un'stsaqludsulaloJd 'suralord auerqr.uouse IIO^\se suralo.rdorlosoldclprr^ Jo srsaqluds eqt sepn]rur srql 'suorrr^ ,r\Ju rot sllolq Surppnq eql eru -raua8ol srsaqludsuratord uo dlar sasnrrn 'alets petleJ lsoq -ur eql qtrm snoruduoudsdllensn sr uo8rlue uortrsrnbce 1o 'uorlca;ur snrrl e Jo esEJeqt u1 :ua8uuy Jo uotusmbty tr :dats qcea Surrnp JnJf,oteql stuele eql seqrJlsapuorssnf,srp 3ur.to11o1eq1 'eydurexouE se IIel patoeJur-snrrlE Sursn.,{e,n -qred 3gyn I sselJeqr ur sdarsxrs eqt sezrreruruns g7-y7anftg

suo6r luv )rlosol^)sluosard{ennq1e6 )HlN I ssel) ',(ervrqred 'suortceso,l.rllxou r{lee to slretopJelnJaloruaql eqrJJsapa,ryr aql uI 'elelrns IIOr orll uo elnlolou :)Ht{ popeol-aprrded oqr ;o .{eldsrp (9) pue 'alncaloru IHW e or aprtdad ;o 3ur -pulq (S) 'salnralourIHW or saprrdad;od.rearlap(y) 'srsdloar -ord (g) 'uoucnrlsep roJ ua8rlueeql 3ur33er(7) 'ua8rrue;o uorlrsrnbru(1) :sdemqtedo.rtr aqr Surreduor ur Intosn erp reqt sdels elarrsrp xrs olur pepl^lp aq .{eru sde,tqred y1pue I sselr eqt qtoq ur uorteluaserd pue Surssocordue8rtuv 'suaSoqled aruasardaql ;o ro; pada,rrnsaq ol peeu leql stueutJedurocoqt Jo IIE eldrurs uorlelueserd pue Surssacordua8rtue ;o sde.vrqredII ssell pue I sspp aqt 'reqla8o1 'elnlosqe sueatu ou dq sr 're,taltoq 'uorlcurlsrp srql 'llol Surluasard-ue8rtueagt eplstno ruor1 parrnbce slerratetu uo pareluer sr demqted IHIN II ssplr sutelord;o uorteluasard eqr pup ';yasrr11ecaqr dq pozrsoqtu.{s uo .(ylueuruoperd sasnooydeLr.qledIHI\I I ssell erlJ 'uotlpluasatd puo Sutssacotdua8tlua s€ ol perreteJsI uolllu -3orer 1ar-1 roJ e]getrnsturoJ E olur poueluor sr ua8rlueqctqzur dq demqted er{J 'slloJ 1 dq pezruSorer eg uer terlt ruroJ E errnbcet€ql slleJaseqr ur tr deldsrpuaql pue 'lr ssero.rd'uaSrtue ouoq qlog 'sa8eqdorceru sr lI 'sllel g pue 'pa.trrop-1r\orrEr.u pue sllal lrlrrpuop opnllur s11eoSurluesard-ua8nueleuots -solord's11arSurluasard-ua8rtue leuorsse;ordJo tuaue^lo^ -ur aql tnoqlr,tr ploJun louuer 's11ar1 rxotoldc pue .radlaq to uorteJeuo8oqr pue sorpogrtueJo uoll)npord aqr sepnyc 'asuodsa.r -ur r{f,rr{./!t'esuodsol aunruurr elrrdepe IntssaJJnsy e Jo eruof,lno Ienluaaa eqt seururrelepreqr dars de>1eql st ruar -sdsaunrururer{l retua slerretetuu8ra.ro;qcrqm dq ssacordaq1

a)elrns llo) orll ol Palsodpue sl)npord )HlN r{}uvlpoxoldruo) orv s}ueurEerl ura}o.td rlrFlM {q ssero,r6oq} sl uo!}elueserduo6t}uv 'uonruSoca.r uo8rtuB roJ selnlalour :)HW II ssEIf,sesn roldarar-or 7q3 eqr sreegreqr IIer f ernlpu duy 'salnrolotu :)HW II ssplJuo

A90]oNnu\u1llI

tz ull-dvH)

280r

roldecer-oc tclf, el{l'roldoool sarnlee}pa^rosuof sazruS0ra.r -oJ 8CJ rraql PUEselnfalo{uf,HW I ssEll Jot .!\Es0^'\sv 'sauosopua alel ur dn pue dltroleu lsp^ eql lnq 'pazqeurelut aq Leu deqr qcrqm lrro{ or€trns IIOr el{r o1 lr{8prrs POllorIP are saxaldruoct([do) eqt Jo otuos 'sutelord auerquolu IsIu -ososdluo punoJ dyuourruocsleu8rslu^elrlar.ro SurlaSrelleur -osopuJaqr ot turoJuorflsnor,rqotou op PuEIIPI rrr.useldot -dc 11oqr dq parrrec ere uoISroAIPsfi1lroJ elqrsuodsarsleu8rs aql '(I-tI ern8rg eas)4rorueu 131o5-suotlaql le seruososdl pue setuosopuaol pouollp sI PuE derrrqred.{rolarcesoql srel -ue xalduor aqr'palquasse st xaldruoc t(Udo) oqr aruo 'rJOIl (luaur8es Surpurq-aprrdedlUtN II ssell eql sardnroo l{fltl. a dI-Il aql PellEr II to qlreDs e saAIo^uIraulPoreleq glJ eql 't(lldD) :saprrdeddlod auru 1o pu? rI uaa,vrlequorlrBralul aql 'olqtuessesrolulPoro slsrsuorsnql rnpord dlqruasseIEUI;eql -teq dro DHW II sselr aql qlil{,ry\oluo ernllnrls JIroLuIJl e Surturo; dq srseqludsolqIHW II ssell 1o sa8uls,{1rra aqr ur elor de4 e sdeld(r1)ureqclu€IrE^uIaql'(6I-tI e.rn8rgaas) 'uteql luelrelul eqr uraro.rdor.,(pouerqlrIaul I edft e POIIEJ euoredeqc€ Jo sueetudq peqsrldtuof,f,esI Suna8let srql 'runlntrteJ ortuseldop -ua eqt ur stsoqlu.{srlogt raryesuollef,o] esoql ol dgecr;neds 'sallaue8ro asaql pala8rer er€ selnf,elou SHI I 11sselr pue ur areld e{Et olnlaloru f,HW II ssell Pue aprrdadE uoo^\loq suorlJEreluroql 'souososdl pue setuosoPuout dllueunuop -a.rdpeteroua8seprrdadlueserd ol Pe^lo^e selnJalourJHW II sselJ?rroloqpessnf,slpsy roldaror IIer-I eql qll.^asonprser lJeluor eqr tlaJte reql susrqdrourdlod el{l Jo osnereq osle rnq 'selncelourlllelle aqr yo drrcrloads Surpurq-eprrdadaqr 'alnraloru lllefle tuoraJ ur eruoralJlp el{l Jo osneraqdluo lou -JrpE gtr.4hlJErolul 'a1nl e se 'lou III^/I\aFlololu JHIN II ssEll relncrlred € qtr,ry\lf,EJelul ol pau8rsapJoldef,oJIIaJ-I Y 'strnpord lllells luarelpp Suoruera;;tp d1e 'rya1cSurpurq -nsn llr.ry\ dtrcrltcadsSurpurq-eprrdadeqr reqr os -aprrdedeql punore Pue uI senPlsarre1;e dlureur sursrqdroru -d1od3gy,q il ssel3 'aprrdedPunoq el{l Jo sruole urBI{r-uIPuI qlr^\ alnrolour :)HW Oql 'o sulEtlf ePIs uaa^uaq slfEluoJ se I]a,lt se 'suteql aprs aprrdad f,IJIJOdsalepoluulorre 'rI uro{ reqt srelcod seAIoAuISurpurqaprrdadto aPou eql epnrlord reqr seprrdad.ra8uol;o Surpurq eqr suoddns snql 'r1ap aqr Surpurg-aPuded pue spue qroq te uodo st UOItrsIqI o ]o uorlrnrlsuol oqt ot dpnbe olnglrluof, sllunqns fl pue (selnraloru 'GtZ-VZ ern8r4 aas)secqaq aqt :)HW II sself,roC rc o^{t pue laoqs fl papue.ns-lq8reur ;o pasoduoc uorlrod Surpurq-aprrdade rroddns sulstuop o{ll-3l leurrxord-euerg -Iuelu o,{\I :selnf,elou :)HW I ssell Jo leqr solquesar solnJe -loru :)Htr\trII ssEIt1o uSrsepIeuolsueturp-rrrtll llseq eql 'ursrqdrour.{yod .noqs crtauaB 'seFlelotu selnloloru il ssell Jo sllungns $ pue rc eqr qloq I sselr Jo trungns e8rel eqr e1I-l'fi7-VZ a.rn8tgaas)salncalour f,HW II sselJePof,uarPql Ilol IEro^assulEluol JHW u€Il?ur -ueru lecrddr eq1 dlure;redns 31 aqt ot 8uo1eqPUE sulol -ordordp ouerquleu 1 edft qloq are selnreloruJHW II ssell yo ($ pue n) srrunqnso.{$ eqI saln)alolN fHl l ll ssel) 'tr sreaq reqt IIOI J eJnlerudue ;o dlnrllceds uollf,Irls -or atll (lll-llol ,.3.e)s.ro1 -daca.r rr;rcadsuou Jo saddl lere,rasssa.rdxeosle sllel lrtrrp '(1Z-VZ ern8rg) pesorlco8eqd -uop pup sa8eqdorceyrg ueqr pue 'surlnqolSounruurr;o uoruod l{ aqt ro sluauoduoc luerualduor roJ srotdaf,ororelJns-llal ,{q pazru8ocar,s11ec

sltelaplo] lxal aas a)elns llo) oql le soln)aloulf HIA ll ssel) papeolaptldod1o{e;dsr6:pl de15 uralotdeuotadeqta)ll-)Hnl ll ssel)e ,nlC jo a)ue]srsse oq] qltM peqsr;du.ro>te sr 6urpeoleprlda4:B de15{ennq}edtrfi>opue alluo oql 6uole uebrlueio uMop)ealq:rfiloalotd ;o spnpoLdoq+ol pasooxoole saln)oloulf H/! ll ssel)leql 6uunsua,saulosopuo ,{1teepue ,sar-uosos,{1 'sourosopuo a1e;sla6tel fuanrlap stq-L.uteq)(r1)lueuenulpalet)osseaql ur paureluo)sleu6rs1o sueeu.r Aq sluauueduo> ;euosos,i4er_uosopua

u r6 u r p e o ; eprge; p

f,IlIrPuaPpue sa8eqdorf,eruol pelo8rpl ere sluauodtuor luoruelduor uretrer pup serpogquedq palBrorap suaSoqlud 'uorlezruosdo;o sserord eqr u1 'sa8eqdo.rrBru dq dpuep -rga d.re.r. pelsa8ureq uEf,seltruud xet€l ue^g :pa.rrnbo.r sdeztrplou eJEesaql ,suorlreralur pue8rl-JoldafoJtrJrJ -ods,,(qpelenrur eq deur srsoldco8eqdq8noqlly .alorued Bur -uloJur eql elppourruof,Jeol uoleyelsoilc paseq-urlle eql Jo Surlapoua.relrsuatxa se^lo^ur ,s11ar peap stueur.uorpu€ to 'sesnJh 'er.reloegsE r{Jns'sFrreleru alelnf,rupd 1o uorlso8ur

,saln)aloul o] pa.ra^rlop ale 'sltunqns lteq]uloj+ oql ut palqulosse ul pueleutosopua )Hl/\ ll sself:fl de15slueuueduotleuosos{1 ul sesealord snorren {q uMopua)olqsruabllueaql :fl da}S.sls{1oa1oLd ro,ruaOrlue oqtsaJedald puesoL.uosopua sar-uosos{; }o }ueuluoltnuo puegd mo;eq1o1ue6llue;oaLnsodx3 Ouonper :A dels.srsofitopue to srso1li>ourd {q suebrlue elelnrrpeduou pue,slso1{to6eqd ,iq patrnbteatesue6rlue olelnlrped:I da15.uorleluesetdpue ougsserold ueErlue1o{ennqled)H4 ll ssel) gZ-tZlungH v xe;du.roc t6;o9ern

uodsue{ EE

3H1A ll sselc

o6eqdpaztletreds lla) lloql uo {g's;;ar r;y{ro6eqd peleldsrpg/"t1se qtns sroldaral)l Jo sueaLu [q uebrlue paztuosdo]o uol]e]uaso6 LZ-VZ:lun9lJ V tc3 e!^ ppdgl uorlegueserd S H l Al l s s e l cp u e u orleluesard-sso.tc I s s e l ce l n s l l o cI o ] s u a 6 r l u el e t J a l c e q Jo uotletuosold E

rrloedsuebrlue nnou puepal)ulsoj111141 srdleqstql lla)€ aql 01dlaqaptnord pa{eldsrp eq}ro1rr,toads sllalI tCf a)e+Jns uerxeldu-ro> llo)oq}}e utelotdaqi ete'ua6t1ue eprlded-1gy1 sexeldiror ll sseltsepa{eldstp 'xelduLor saptlde; oq]ulor+palelaqtl 6urpnpur aunururt ;o s1ueu6er1 oi palontlop pa{orlsep sr}r araqMs}uauruedu.ro> leuososriyleuosopuo to aql 61ete,Llns aunu.lLl..tt srsllnsoJ uaq]puepazrleurolur ]eq1xeldu.tot 'ue6t1ue 'uorlerluaruo) 'srolderer nnol ]e ]uesard 1tuane llo)I raq] o] purqsllo)B 'slla)g ltq uorgelueserd ua6tluy BZ-1Z]Un9lJ V

sluoluoc lo aseolou urnuolceqlo uorlcnrlsep relnllocerlul tr

uoIqse] cr;rcads-uabrlue u r 1 ; e cg o 1d l e q

tr 3HlA ll sselc Aq paluesard edollda l l o cf p u e

poAollsap 'xa;dtuo3

pozrIeuraluI xelduro3

srsolAcobeqd solelnu rls g,lca enrlcy

tr

d

d Ul.cl ol ulnllalceq spurq 'p6leroc'ep-901 [

uabrlue sarnldec

u e O t l u eP t d t l =

61acelrng tr

aprldodpalculsol SHlA ll ssel3=

ua6oqled pezruosd6

eprldedpalctllsel f,HtA I sselS=

ue6tlue utalold -

uncl J o l o o c a . lc l =

I ffi lH iri

ffiru

180I

NOIIVIN3S]Ud N]9IJ.NV CNV )HIA ]HI

salnf,alou il ssEIJseereq^,sler.rateu rqosotdr .{pueunuopard elclr.uessolnJoloru I ssplJ :aprlded.ro; stueurtredtuor JelnllaJ -EJIUI AIdUES ]UAJA'JTP SOINJAIOU:)HW II SSEI]PUE I SSEI) I 'uonf,rJlseJ

.srlnrelou f,HW pollet sr uoueruouor{dstql :)HW lllel -lp aql euo dluo qrr-,rl, ol pau8rsap :otderer 11ac lrpratur Jo 'oprldad -I p qlr^4.lcee.rdllensn 10u IIr./v\ Iptrluepr eql purq ol are,ry\ deql JI ueira 'alncayoru:)HW up Jo sluerre^ lrlalle ruarolJrp sngl 'ua8rlue roJ -roldorar IIal-J aqr dq palJel -uoJ sanprserapnlf,urosle senprserlrlallv .GZ_VZarn8rg aas) l;e1c Surpurq-aprrdadaqr Jo orntlelrqlre eqt aur;Op lpql sanprsorapnlJur raqloue ruorJ elell? auo qsrn8url -srp rpr{t sef,uareJJrpoqt esnpreq .saprldad Jo slos tueret -IP pulq salntelotu :)HW Jo sluerr?Arrlallp lueratJrc r

'rc.T-vz

orn8rg aas)seprrdadtuoraJtrp dlarrerropr.&\e Burpurqro; Jo pazqenadsuor8ar p sapnlourpue relrrrrrssr soFteloru JHW II sselJ pu? sselryo ernlf,nrls pue uorlezrur8ro aq1 r I 'sllal g pue ,sa8eqdo.rreru,s11ac trrtupuepsE qrns sllar Surtuosard-uaSrtue Ieuorsselordor dla8rel paur; -uof, sr solnJeloru :)HW II ssell Jo uorssardxeeqt seereq^4. 's11oepetealf,nu lsou uo punoJ arp salnJelour IHW I sspl3 'sller J uo srotdacarcgrceds-ua8rlueot ruaqr ,(eldsrp pue saprrdadpurq 01 sr stcnpord lHtr trJo uonrunJ aql r .02-VZ arn8rgees)uorl -elndod paJqlno uE ur suorlerJel Jrlelle dueur ur Burrrnlro 'rrqdroulyod ltq8tq ore suralo.rdasoql .selnlalourIHIAtr 11 sspll pue 1 sselc 'suralordoodp euergruotu1 edft 1o sosselr .roleur otg sepof,ue.sqer8 ;o uortcalor ro acuetdaoceroJ elqrsuodsaruor8ar crlaua8eql se pare^orsrp ,311yq oq1 r

uolleluaserdua6lluy pue )HW aql

'seur4oldcpue stua8e lerqorJnu ot arnsodxedq palelnpou ag upr deqr tnq-eurrr oqr [p Buruaddeq-elrlntrtsuor erp sdatse,toqeoql 's11ac Surluaserd-ua8nueleuorssa;ord.rog 's-roldaoare>lrl-lloJrrerlr dq parcerapsr qlrq,ry\.(541)aprreqccesdlododq lerretceq sE qJns 'sleu8rsot asuodserur uorlplrlJp rraql Burmollo; se8eqdo.rcetupue sllel lrlrrpuop ur peruequa ar? ef,eJrnseql

-xe euerqrueurSurlrurryepeqr ol serpogrplnorsa^rllnu eql Jo solJrse^Ieuralur aqt Jo luaurlrnrreu .Gt_Vt a.rn8rgaes) (sarpoqro) saurosopueJelnrrselrllnur opnlJur qrrqm .sluau -lreduror Ieuosopua etel ul dylsorupezrlelol are saxoydruoc apuded-3gyg ssplJ pelEroua8 dlmau :acoJtngya3 eg1 il aqt u saxalduoS ap4ja4- JHW II ssa13 ,(,o1ds1q g Jo

uI se^rr a1q qrr.&\, ers^r.*r;tl:rtl, j : j:ffi tEg pareurrse

::

oplrdod-f,Htr\tril ssep3ur11nsa: oql .WC qlr.tr xelduoc

eql Jo uortf,?relurdq paSpolsrpeq touuEJ tr reqr punoq dlgers os aprrdade sa.nnbcuelnf,elorrrII sselJaqr prun ,7rgq dq ,,8urlrpa,,rer{iln} ot algrldeosnsse^lesuaql are sexald .sapndadpulq or -ruoc apndad-lHry II sselr peurroJd1zrro51 elqeun sr lr 'salncelourII ssell ol r€purrs d.ra,rdl1e.rn1cnlspue paporue-JHy41sr urelo:d We orll q8noqrly .141q,auo.radeqo e qtr^{ xalduroc dI'IldD or{tJo uorDeralur qSnorql pe^ou -ar st lueruSos dI'I) eq1 demqred rrilcopua aql ur lezr.ord leql suonrpuoJ orll repun >lf,plreorldloalord pue 3ulp1o;un ol luElsrserosle ere se^lasuaql solnf,elolu:)HW II ssEIJoql 'IleDB ru.{1oato.rdol tuelsrsarsl dl-If, 'r;e1eSurpurq-aprr -dod .luou 3gy,g il ssell eqr ur pa8pol dlurrr; sr tr esnerag -3os4113 er{rpolleJ uortrod Ilerus p Jo uorldacxa oql qrr^ rI Jo lp^ouar ur Sulllnsa.r'xolduor E(ldo) agl uo osle lle saprldadolur tueqt apurSappue sua8rluepozrleuraluruo IJE lEgl saseelordeues oql 'lI dq perdnDo sr elnrololu il ssEll eqr uI ryelt Sulpurg-aprrdadoql esnecagaprrdadSurpurq ;o alqedeoursr stuaurileduof Ieruosopueol pera^rlepxelduoc t(tIdo) eq1-:sap?apw il sspl) o1sap4Qa4Jo Surputg E 'sluaurt.reduof, lEr.uos -opue ot xalduoc t(Udo) sselr aql il 1o dra,ulapsarnsuapue ({) uteqo tuprrelur eql uo ruepuadapsr leql dels Surtros e dq slseqtudsorqJo esrnoJ aqr ur paqsrldruof,Jesr srql .loou .aro;; ol salnlalotu ssplt seprrdad^\olle pue ol sr f,HW II -o.raql'luaurarrnbaralos aql 'eueJquau E errnb Jo Ies.rorre.rl -er tou seop saplelour sspll ol eprrdad;o dre,rrlap :)HW II aql 'se^lesueqt seFleloru IHW II sspll erlt sB aoeds lecr8ol -odot euBs eql ur oprsorsrsdloalo-rddg p"r"rc,ra8 seprrded aql OsnEJag'satuosopuoetel ol papeJrp OJerunlnfrleJ Jrru -seldopuaeql ur pezrseqrudssapcelou sselr rsour JHW II :salnlapw tpqr sspl) o1 sap4Qa1 [,tant1aq IIeraU il Jo V 'sasBelord .Burssar leurosos,{1 Jo srolrgrqur op se -ord ue8rtue oraJretur glr.&\ sdurnd uolord ssEIJ-A Jo _&r,trt -Jp or{l tlqryul tegt srua8etpuenbesuo3 .seruosos,(l uqlr.n,r 11d crprceeqt le dleurldo olerado sasealordleruososdlaq1 'selnf,aloru f,HW II sselr ol purq uel tuql etuos Burpnlcur 'pacnpord sr sluaru8er; aprrdad;o o8ue.ropl^\ V .srsdloalord ol olnqrrluol deu osle 'aseelo.rdopueoryrcods-aul8eredse se qcns 'saseelord.reqrg 'soseelo.rdlduedsB ro aurelsdo.raqlra eJEqJrr{^.'sursdeqlecsE ot paJJete;dlezrllcallor,sesealord yeurososdl1o las e3.re1 e dq tno perrrer sr dezrrqred311yrq il sselr eql ur suralordyo uorlepet?aq :xsK1oa1ot1 E 'stsdloalordroJ sua8rlueeqt sereda.rdlueruuorrlue Surcnperpue gd ^\ol Jo uortle pourq -ruor or{I '&NCI or e.rnsodxedg alqrcnpurosplJnparorr{l e dq osp pezllew sr surelo.rdJplnllaterua lueur azrpqels reql spuoq aprJlnsp Jo uorpnpeg .a8ue.rrelouqlnu agl ur sluolezrrnba Surcnpa.r uorlpJluaJuoc e Bururelle saurososdl Jo qlr.tr'Surcnpar sr sluerulredurocyeurososdyyeuosopue eql ur lueruuorrlua aql tequng .sa8pr.rq uorlezrlrqplsep rles Jo ,Hd pue spuoq ueSorpdq;o ernldru g8noqr Jo soruen -xa ot pasodxaueqa ploJun ol puol 11d pDneu lE alqEls suralord '(5-11 ern8rg eas)uorrecrlrprresrr.lt:o; elqrsuods -ar ere seuerqureu leuososdl pue lptuosopuoaqt ur sdrund uolo.rd sspp-A paramod-41y .S.t Hd or dorp deur gd aqr seurososdlpue sawosopuoalEI ul :S.Spup S.9 11duealgaq

uoneurqulorer [-A pue f-C-nlo usluel{rotu aq1 '(urcqc io U]J lureqc rq8rl 31 ''3'a) uonuurgruof,erf-n dq pacnpord lrungns e pue (ureqcd UCf lureqr dreaq 3y ''3'e) uonrurgruocarI -C-A Jo lrnpord eqt sr teql trungns E ul€luoJ uolleurqulorer rrletuos dq pore;oueBsroldaco.rcr;roads-ue8rruE IIE dllenrrrn

s e u e Du r l n q o l 6 o u n u u l o l a r v s e u a gu ) I relluls Jouuen P ur pebueJreou 'strlolu ';erurpouroq Popuoq I IVII eal{l sureluof,ulpl{r ) qcea pue -aprJlnsrp olul se xalduror oql e pater8etursI uIeL{J Uf,I-€Al 'sWVII orp ur senplsar aursordl ) eql ;o uoueldroqdsoqd uodn patrnrf,ar oq ,{eu salnralour roldepe qcrqn q8norgr 'ureurop 1IVII uE suretuof, gf,po uI uIEIuop cluseldotlc aqr pup 'sureruop urlngopountulul ol snoSoyouroqsI sllun -qns €CO eqt Jo uretuop Ieuretxo aq1 'sexelduoc e,! pue sg plard or ureqr g egl uo ,{"oqr grt.Lrrorulp luale^oJuou e sluroJ urpqr s aq1 ('xelduol tql eql Jo sllungns paleu8rsepd1re1 -rurs eql qlr.^apesnJuoraq ol lou ore stlunqns g pue l" 431 aq1) 'sureqc ) pue 'e 'g '/" to pasodruocsutelo:d euerqtueul 'peals 'xalduror fCIl aql qrr^\ setelfosseUJJ aql Io tes E -uI 'uortrnpsuerl srolreJ rrlosotdc ;o uI teql lslsse leu8rs eql to sllEl luerulruf,Or r olle o1 lroqs ool Oresllunqns ull rrtuseldotdr aq1 'rueu8as euerquausueJl E PUE uleruoP uor8er-tuelsuoc e dq pe^'ollot 'utetuop elgEIrE^ e sI eraql pua leurturer-N aqr le :urpqolSounlrrlrIl ue ;o uorrrod (qe)4 eql ol dtrrelrtursleJnlrnrls sl\oqs strunqnseseqlJo eJnllnrls eq1 rred g,! e ro 'rred $o up JaIllIayo pesoduror are srotdar -er eql'aue8 pa8uerreer dlleotleruose dq sI qllq^/r POPoJue 'sltunqns urarordocdlSo,ry\l pasodtuocq ry G7-VT Jo Jo qJeo a.rn8rg)roldacor ilor-I aql Jo arntf,nns aqr papletd 'sotpoq -rtue Iuuolf,ouoru crlcads-llol I raqto pue sarpoqrlur ord.{t

-ouolrto osnq8norqr'qceo.rdde lerrtuaqrounfiH;U*rr"

-er rrlPruos eruaPl^O^rogs Plnoqs sllol I arnlElu uI PUP to uorlern8t;uoc aurl-ura8 uI snrol U)I B Jo uortezlue8ro eql 'JnJJo flleluos uI saop lueura8ueJJeal IJEJ ;o uosr.redruofV 'sltunqns 'saqord sE ueqm posn Jeqraq.,r.r UII Ipe^er plnoqs orlrcods elnelnd aqt Surpocueseuolf VNCI 'sJoldef,or11ac-1 -ue8rtue Jo Jaqlunu altstnbor eql aleraua8 ol luetue8ueJJe -eJ frleuros JoJpeou eql sell. uottdurnssePuof,esV 'Joldof,eJ IIar-I eqr 1o uotldtrosep lsrl; E lv eNile or d1p;ssaccnsPesn eJerAserPoqrrueqrns 'sllel I reqlo qll^'\ lou PuE roldof,er [a] I eql Jo errnos Otll sE Pe^raslEI{l euolJ e(uEsaql luou '(satpoqtl sller grr.^adlenbrun rceor plnoqs salPoqllue qcn5 -ue crddlouop) euop IIar-I rEInlDrcd e ['q passardxeroldac -ar IIer-I e ol anbtun soJnl€eJaql lsure8e solPoqllue osler ol alqrssod aq pFoqs lt ':oldace.rrr;rcods-ua8rluePelnqlrl -srp d11euo1c e sassassod IIOI I qlee teqt uorldrunsseeql uO 'elqeroprsuol oq plno^{ 'sua8nue pelPIOrdlasolc uee^'laq qsrn8urlsrpo1 flrlrge rreqt ue,lr8 'sroldecar IIOJ-I Io ftt1tqe -rJEAoql tetll uollou atlt se^t rsJIJaql 'suotldurnsseo.al uo pes€qpetepronlese,t\roldoJor IIer-I oqr Jo .t.trntt..rlX*UrO elerrdorddBel{r qrl/!\ pexeldurocselnf,eloru3g1,q ,rrruSoru

A90-roNnnvU I

tz ulldvH)

880r

'uollrlluorotllP rlot{l uI sllof, ua8nue ro1 roldacar IIOJ-I oql elarles ol ro sllatrla8rel Surreaq g lsrssElltlvr reqr seur>1oldc -ua8rlue1r1 or dlrcedecaqt errnboepue eleralllord srotdecar rrlrcads-uaSrluresaql;o lueruaSe8ueq8nolqt PeleArlJEuaeq 'uotsued o^pq tpqr sllel I'sJoldef,eJ IIaJ-I rIOI{1esn slleJ1 os -xa leuolf, rIOI{l ot peel reqr sleu8rsetrnPsuerluaqr pue uo8 -rtue ezruSorerol roldef,er IIOJ-goql azllrln sllol g sE I{trnry

ullnqoloounuul ue lo uoluod (qe)l eql salquasaurolde)au lle)-I eql lo ornl)nrls aql 'uorlrunt JIaI{tol Surprome roleu aql af,nporlul pue dolerrapsller J ^\oq sllal f Jo sessplf, aqursePIIErlsel6'(dltunruurtolne "a'I) senssllu.{\o s.lsoqeql r{rr^{sllet I paterauo8dl.vrau;o dlt,tttrear rrqdorlseler dl1erl -uatod pIoAEol sB os 'elou8r ol suollrulquoc eptrdad-3g1tq

'suolleulquoc aPrrdad-3g1nq 'uotlcnpsuerl ozruSooa.rsexalduoc eseql ,l.roq pue leu8rs ro1 daql .troq 'uou IerluessesurarordocdlSauerqtuotu qtrm dn rrrd -ruSorar rr;rcads-uaSrlu?elelpetu letll sllunqns aql eqlrlsop 'sllunqns UII oql plord reqr sluaura8uerreereue8 Ilpqs elN uo ruepuedep .{llculs Jrteuos aql Jo uolralduroc 1n;ssacf,ns 'saue8uqnqolSounurrut osle sI sllet I ;o luaudola^aP eqJ e8uettaat ot slleJ g dq posn esoql ol FlIluoPI uolleulqruorar rnptuos to stusluel{leur dq sllunqns (UCf) rotderal 11or-1 slloJ I 's.roldecarclpads egl Sulpocua seua8aql a8uB:.rea-r -ua8nueasaqleleroue8o1 'surpqolSounuurr ol PelEIerllprnl -f,nrls sroldeJeJ erll 'soFtrelour >lsEr slql qlI.4A ore Pelsnrlue sardcoqdu'(11 Jo suel aql q8norqr ue8tluB azruSocer IHW

luaudolo^ec lla)-I pue

'sJolda)au lle)-l'slla) L EM

'92-vzPUe sT-vz sernSr4 aes) ecr;rns IIetr aql uo solnrolou :)HW PaPEol -aprrded eqr Jo deldstp (g) Pue lelncalour SHIN eql ol aprrdad1o Surpurq (9) lsalncelourf,HIAtror saprldad1o d.re rot ua8tlue eqr SurB -^llep (t) lsrsdloelord (g) fuorrcn-rrseP -7et (Z)'ua8rtue 1o uotltstnbre (1) :sdarsslerlslP xls olul papl^lp eq upf uotleluaserd pue SurssecorduaSrluy I 'esuooseJ 'selnc aunruull uE Jo asrnor ot{l ur PalEInPou eq uec led -alou JHW lue^eler oql ssardxereqr sllol ut '{lsnonutluoc 'uolleluase.rd pue Sutssacord ue8 seierodo ssarord sIqI -uur sE ol Parraler st sexaldurocaprrdad-3914 pa'{e1dst -aJEJJnsolur PalJaAuoJ Pue seprrdad olul Passef,oJ 'parrnboe a.re sue8rlue utelord qllqr ,(q ssacord eq1 'srsoldoopue .ro'srsoldcoutd'srs PalEIPetu-Joldeoar -or,boSeqd dq pezqeuralur slelJalerurelnllef,erlxe aldrues

680r

INItAdO'llAlc'lt3)-I

cNV',sUotdt)lu'l'll)-l's't'llt

t

'sJotdeJOJ ,snourlouasr seua8 enbrun 0r0I poeJxeol paleurlsa UlJ. to tuouaSuerreer rrlpruos dq pafearc dfrsra,rlp er{I

slueuoes euag r pue ,c h uaa^laq suoll)unf e q l u ! p e p o ) u l s e n p l s a ue l q e t r e nl r a q l l o {uey1 q}!/v\asro^lo AJsnoJV sJo}do)eullo)-I 'luetudole,rep -g qloq IIel-I pue stua,rard snqt dcuercrJapCVU y .saue8 roldaoer-ue8rlue aqt tuatua8uer.reeraql uo lpcr.rls sr seilcoqd ruapuadep Jo -ru,(1 lueudola,rap 's1lec roJ ,ry\olaqpeqrrJsepaq ;o 1 IIr^\ sp pue 's11ao g rot ueeso ErIo^ sy .saue8Uf,I to tuerua8ue.rre -ar apnlcard 'saseurqruorer ,snteredde CVU aqt sE qcns uorlpurquof,al or.{tJo sluauodurocLa>1aql ur sarf,uerf,rJaq 'selrs

alncalotrj

c H tJl l s s e l S

eseqt tE puno; dluoturuoc suoSoqled]sur€Bpesuoteprsoq ur olor e ,le1d d1a1rypue (ur4s aqr ,lceJl lelrua8 aqr 3urur1 runrleqtrdaeqr '.3.a) solrslef,rruoleuelrurlsrp uo ur auoq ol peuurerSo.rd ore sllof, I gl" aql .sue8nue pldll Jo uonptue -sard agr ur pozrlercedssr rlrn{,/vr,alncaloru eql Burzru ICI -8orar yo alqeder aruosare srotdacargl, Burssardxosllol J aqr Suoury 'suorlrunJ tf,urtsrp qlrm sa8eaurleleredes paJa -prsuor ere roldaf,or gl" agr ssardxerer{r osogr pue roldef,or dic or{r ssordxo reqr slleJ I 'uortalep dq rsol ,trou ,snco1 g eql to uollezrllln sapnlcard tuaua8uBr.reeJroJ snf,ol rc ,snco1g pesodreluroql uorsrl UIJ eqr Jo orrorlr e os pue to -xa alaldruoc ur stlnser uorlBzrue8ro srql .sntol rc UJI eql urqtrlv\peppaque sr sntol S UII ogt .osnou pue uEunq aqr ur 'ptrq1 'saua8931 pe8uerrear IIE ur tuosord aq uec saPrtoolrnuN eroJoreqlpue 'eSuerrearsaueSuJJ oqr eturl eql olnf,€ sr (1p1) le esera;sue.rrldprloalcnudxoap leurut -ral erudzueaqt 'puora5 .pe.aolle arB sluaure8uprreor CI-oj -C ter{r r{rns sr sleu8rs uorleurgruof,er ar{r Jo uorlezrue8;o aql (tsrr{ 'lrol eql Jo luetue?uetrcat pue uorlBzrue8 UII -lo 3r{l OzrJalJerEr.{J seJnlEal,,(qlrozrralou Jeqrunu v to -6t_VZ arn8rg) aynr.racedsdq ntZt aqr sdaqouorleurqruorarpue .(sSSU) sacuonbes leu8rs uorlEurqtuoJoJ aqr JoJ lueruarrnbar elnlosqE ue sr araql 'srurotrv pue hI asr8rl y1qq ,tlf,Ux 'trunqns cndlerec ,0gn) ,0/n) ,Z-CVU ,I-CVU )d-VNC :.,{.reurqceruSururo[-pua snoBolouroquou er{t Bursoduroc suralordtuauoduoc eql serrnberpue ,saue8ulpqol8ounur 1u -tur rot paqrrJsep lprll ol Ief,rluapr sr rf,ol uf,J oqr roJ

aprldadvH

(q)

I LL95:6t Ogw]'OooZ,a)rauuaH I uopaseq 1q;ue6]{qdet6o;1e1sfur,iet_x ,iq pauru.lalap sexaldulotaprlded-1g171 ll sselle o] punoqtoldatat lla)-Ieq] alnpnlls(q) sl1et6urluesald_ua0t1ue uo ,ilanrpedsal ]o 'soln)alou l)Hn ll ssel)pue)HN I ssel)+osolnleolporuesuof qllmuorperatur MolleqlrqM'(uaarO lqbr;)tCf pue(anlq]q6ll)gC) .uorssatdxe siolda:ar-otoq] Ll]rlln saleoosse taqyn; roldetal eLl_L lla)-t a)e+rns ro] patrnbar srxe;duot e jo uotlerrLro] aqf €O)-dDUll lln] s;eu6rs aq]MolleoI (L€-VZ atn6l1ees)xaldurot ;o uotpnpsuerl 961 ,uorleulquro:e.r oq] qlrmoletfosse ]snuls]tunqns flo eq1 {;anrpedsat f-C-Apue;-4 {q parnpotdeteq:rqan,qtunqnsd pueloaq},suteq) onnl pesoduo:sl uabllueloj (UfJ toldetarllo)1 oq1(e).s.rogdare.r 1o -o) sl! pue lolderel lle)-I oql +oernpnlts 6Z-tZ lunDll >

u/!\oPlrs Po^losuoeq seq oJnlf,nrlse qJrq^ ro} ,{11euo8erp sroldeca.rIIOJ-I eqt ;o duey,q'selnrolotu f,HW or{l Jo salll -eq lo eql Sunoetuoc ZUCI pup IUel qtr.n 'xeldruol oql ;o uon.rod eprrdad lelual eqt qrr^\ opetu ere uot8a.r€UC:) eql ur slJeluor elrsuelxe tsoru aql lnq 'xalduor aprrdad -IHIAI eqt qtl.Aasloop roldaler IIer-I aqr -{\oq ur dltltqe -rrEA^\oqs sarnlrnrls osorlJ 'Pe^los ueag e.teq sexeldruoc eprrdad-3gry II sselr ro aprrdad-3Hry I sselr ol punoq srotdaca.rIIer-I Jo roqrunu E Jo sarnrrnrls letsdrc aq1

A9o]oNnnNl

I

lz ulravul

060r

'asuodsar ue ounlulul Jo esrnol aql Sur.lnpsepoq -rlue Surqtou lrgqxe Jo uoIleJntEIUdrrurge eql ol luale^rnba sroldarar IIar-J snlJl pue 'uorlelntu.reddqcrleuos o8rePun lou '(71-y7 en op seua8UIJ el{l 'seue8utlnqolSounurrutaIIFn -3rg aes)suqnqopounu{ul Jo SUCO eql Jo reql sal{rletuleql 'pessnl suor8arA eqt ur .&qrqerre,r;oaar8ape sI llnser leu aql -srp.{pearp ePlloelrnu-N pue uotstrardurtleuot}ounl uouIPpE eql oP se 'uotlnqtrluor luelrodrul uE so>l€tu Jo srusrueqJaru sluau8as aua8 f pue '(I 'n luara;1p 1o a8esnlelroleulqtuo)

'slueubes I eq1 suotOar luelsuoloMl ele q)lqM+ouleollsuMop 6urpo:ua ra)uequo: qu3laruanbas leu6ts 'sluaubas lolsnl)e sapnl)ul pue'sluaur6as n ]o C ]o lolsnlle uoxa: SSsluauOas n ]o Ja]snl) lelanes f +oiaqurnua6rele pueslueLu6as aln6r1ees)l:o; urlnqolbounuut snro;ureq>-fl lfl aq1(4a:.)'(Vl-VZ srsn)olureq)-D e 1opesoduror lou stsn)ol U)I aq1Qq6ty)peruasqo 'penno;;e eql a;drlutldul sl l)olU)1 +ouollezlue6lo 6ututol ]o ]eLllo] te;rtuts dUft eql ur Oururol f-n por6 6ututof fq-nosle1nq lUnDlJ V eq11oiuaurabuette 'l)ol U)l lo uolleulquo)eJPueuotlezrue6lgOE-?Z uor}eutqulo)al 1-6sr{1uoiou }eLl}qrnsstsleuOrs

-c)O+ uortelsue{t I

-!-A wvflfTI

olncelouuclpalquossv

uorretsue4 t

wvflIl

'oor lonss ( 6 u r c r t dosu) r s s a c oy. rpdg

I

cA :--_

rdrdrdod^\s {VNUur)ypg re6uasseyg

ypg 6ursseco.rd (6urc1ds)

I

,e@-{H---!flflfl-il--l*,s ortonss oc

1

I ,s drdord^ bs

,e zdc

uorldtcsue,l I or Lon

ldlrcsuerl y;19 lretuu6

uorldt.tcsue.tl 1

ldc

SS

d1;d6udn/

6ututol1q-71 I 6 u t u t o tp - 1 : u o r l e u r q u o c ocl r l e u o s 6 u t u t o Ip - 6 :uolleulqulocal clleulos

(o0r-09-)?o I

uondsronis

qurd zdc

zio

zflr

rdo udnss tdnss

ldr

r0,r

vNo ou!lura9 uleqcg ucl

ureqcn ucl

across the peptide-binding portion of the MHC-peptide complex. As a result, the T-cell receptor makes extensive contacts with the peptide cargo as well as with the cr helicesof the MHC molecule to which it binds. The positions at which allelic MHC molecules differ from one another frequently involve residuesthat directly contact the T-cell receptor, thus precluding tight binding of the "wrong" allele. Amino acid differencesthat distinguish one MHC allele from an other also affect the architecture of the peptidebinding cleft. Even if the MHC residuesthat interact directly with the T-cell receptor were shared by two MHC allelic molecules,their peptide-binding specificity is likely to differ becauseof amino acid differencesin the peptide-binding cleft. Consequently,the TCR contact residuesprovided by bound peptide and essentialfor stable interaction with a T-cell receptor would be absent from the "wrong" MHCpeptidecombination. A productive interactionwith the T cell receptor is then unlikely to occur.

S i g n a l i n gv i a A n t i g e n - S p e c i f iRce c e p t o r s TriggersProliferationand Differentiation ofTandBCells Both B-cell and T-cell receptorsfor antigen transducesignals by means of proteins associatedwith the antigen-specific portions of the receptor (i.e., Ig heavy and light chains for the BCR; c and B chains for the TCR). The cytosolic portions of the antigen-specificreceptors themselvesare very short, do not protrude much beyond the cytosolic leaflet of the plasma membrane, and are incapable of recruitment of downstream signaling molecules. Instead, as discussed previously,the antigen-specificreceptorson T and B cells associatewith auxiliary subunits that contain ITAMs (immunoreceptor tyrosine based activation motifs). Engagement of antigen-specificreceptorsby ligand initiates a series of receptor-proximal events:kinase activation, modification of ITAMs, and subsequentrecruitment of adapter molecules that serve as scaffolds for recruitment of yet other downstream signaling molecules. As outlined in Figure 24-31, engagementof antigenspecific receptors activatesSrc family tyrosine kinases (e.g., Lck in CD4 T cells;Lyn and Fyn in B cells).Thesekinasesare found in closeproximity to or physically associatedwith the antigen receptor. The active Src kinases phosphorylate the ITAMs in the antigen receptors' auxiliary subunits. In their phosphorylated forms, these ITAMs recruit and activate non-Src family tyrosine kinases(ZAP-70 in T cells, Syk in B cells) as well as other adapter molecules.Such recruitment and activation involves phosphoinositide-specificphospholipase C" and PI-3 kinases. Subsequentdownstream events parallel those discussedin Chapter 1.6 for signaling from receptor tyrosine kinases.Ultimately signaling via antigenspecificreceptorsinitiates transcription programs that determine the fate of the activated lymphocyte: proliferation and differentiation.

T cells depend critically on the cytokine interleukin 2 (lL-2) for clonal expansion. Following antigen stimulation of a T cell, one of the first genesto be turned on is that for IL-2.The T cell respondsto its own initial burst of IL-2 and proceedsto make more IL-2, an example of autocrine stimulation and part of a positive feedback loop. An important transcription factor required for the induction of IL-2 synthesis is the NF-AT protein (nuclear factor of activated T cells). This protein is sequesteredin the cytoplasm in phosphorylated form and cannot enter the nucleus unless it is dephosphorylatedfirst. The phosphataseresponsibleis calcineurin, a Ca2* -activated enzyme. The rise in cytosolic Ca2* leading to activation of calcineurin results from mobilization of ER-resident Caz+ stores triggered by hydrolysis of PIP2 and the concomitant generation of IP3 (seeFigure 1 5 - 3 0 ,s t e p sZ - 4 ) . The immunosuppressant drug cyclosporine inhibits calcineurin activity through formation of a cyclosporinecyclophilin complex, which binds and inhibits calcineurin. If dephosphorylation of NF-AT is suppressed,NF-AT cannot enter the nucleus and participate in the up-regulation of transcription of the lL-2 gene. This precludesexpansion of antigen-stimulatedT cells and so leads to immunosuppression, arguably the single most important intervention that contributes to successfulorgan transplantation. Although the successof transplantation varies with the organ used, the availability of strong immunosuppressantssuch as cyclosporine has expanded enormously the possibilities of clinical transplantation. I

M H CM o l e c u l e s T C e l l sC a p a b l eo f R e c o g n i z i n g of Positive a Process DevelopThrough and NegativeSelection The rearrangementof the gene segmentsthat are assembled into a functional T-cell receptor is a stochastic event' completed on the part of the T cell without any prior knowledge of the MHC molecules with which these T-cell receptors must ultimately interact. Similar to somatic recombination of Ig heavy-chainloci in B cells,the first TCR gene segmentsto rearrange are the TCRP D and J elements, followed by joining of a V segment to the newly recombined DJ. At this stage of T-cell development' productive rearrangement allows the synthesis of the TCR B chain, which is incorporated into the pre-TCR through association with the pre-T cr subunit. This pre-TCR fulfills a function strictly analogous to that of the pre-BCR in B-cell development: It allows expansion of pre-T cells that successfully underwent rearrangement, and it imposes allelic exclusion to ensure that, as a rule, a single functional TCRB subunit is generatedfor a given T cell and its descendants.After the expansion phase of pre-T cells is complete' rearrangement of the TCRcr locus is initiated, ultimately leading to the generation of T cells with a fully assembledTCR ctB receptor.

SN , D T - C E L LD E V E L O P M E N T T C E L L 5T , - C E L LR E C E P T O R A

10 9 1

Antigen

I B i n d i n go f l i g a n d to receptor activates Src kinases(Lck, Fyn, Lyn)

I Src kinases phosphorylateITAMs

\,.,

T PhosphorylatedITAMs recruitnon-Srckinases via SH2 domain (ZAP-70, Syk), which are activated by Lck,Fyn, Lyn

sH2 domain

SH2 domain

\r"n

I Activated non-Src kinasesrecruitand phosphorylate multiple adaptor proteins(LAI SLP65) Signaling pathways activated (Ras,Jnk, PKC,NFAT)

A FIGURE 24-31 Signaltransduction from the T-cellreceptor (TCR)and B-cellreceptor(BCR).Thesignal-transduction pathways usedby the antigen-specific receptors of T cells(/eft) and B cells(right)areconceptually similar. Theinitialstagesare

1092

o

CHAPTE R | 24

tMMUNoLoGy

depictedin thisfigure;downstream signaling eventsleadto changes in geneexpression that resultin proliferation and differentiation of the antigen-stimulated lymphocytes. Seetext for furtherdiscussion.

€60r

l _ N I r A d o ] 3 n I c ' l ' l I ) -cI N v ' s u o i d I ) l u ' l ' l l f - t _ ' s ' l - l l ) l _

'uot1)aps oqr pelle] sl slql iln-I Jo ppow [,11prrto 'uorlfe]es eruoflno aqt aurr.uJatep ,,ie1dsrp uo suorlEurql'uol Jo aprrdad-;1as-:)HI,^{ tuereurp oqt sar8raueSurpurqto uoneru Jo -tuns :uorqseJ aql a^rlrppe ur sleu8rs stardratur rotdacarIIar-I eqr teqr alqeqo:d dlq8rq rr se>leruuonJOIOS Suro8rapuns11ac I ,o eleJrns oqt uo pedeldsrpsexalduor )HiN-eprrded to dtraueSoralaqeqf 'petrelesd1a.,r.rtrsod sr'srsoldode elerlrur ol tnq '1e,rrn.rns IIer I aqt ladurr plnoznrteqr ploqsan{l er{r.,raoleg ^\olle ol Suorts .{yruarcrl;ns sleu8rs sa,rrac.rad rotderar E leqt qrrm paddrnbeIIal I .{uy 's11ac I a^rrear-Jlesdltrazr.o to errol -redar ylar-1 aqr a8rnd ot solras pue uorlJolase,rrle8aupal1er sl slrJl 'srsordode,{q arp or patf,nnsur ere sllo) I aseql uaql 'roldala; IIel-I eqr 1o SurraSSrrt,^ olle ot luorrrJJnsploqsen{l e sessed.rns suorleurquol epqdod-Jlas raqr.unu qlns 3HW to aqr ;1 'drrunrururotneosneJ plnof, pue eArlJeor-JIOS uorlru -ltap dq eq plno.\\ 'sue8ro proqdrudl ur eruaprsar lereqdrrad dn a1e1 pue snru.{ql eqt e^eel ot pe,,r,rollp yr ,s1]acI aseql 'aprrdad-Jlos;elnrrlred E r.{1r.4^ paxeldruoc JHW-iles rot tr} Da;rad e.^Aot{slerlt srotderer qtr.lapa.4^opua slloJI are eruartxe

uorlent])p loq] lo+uorlrubotar uebrlue uo luepuedap li1pu1s ereselAroqdu{1 atnlepl ua6rlue ezru6otet (Ulj 'l)S) sloldeta_r 'sa}ts rrloads-ua6r1ue palqulesse I11n1 pue {1uo })ut}stp }p le)tulo}eue rn))o uorlerluol0++tp pue ayboqdLlll UfI aq].ro+ (per lueuudolenap ] q 6 r lu) r e q ])oU ) _ L : U )oBq ]l o +( o n l ql q 6 r l u ) r e q ]l q O rbl l : r o l d o ) e r r r l o e d s - u e 6 ral uqei] o ] r u n q n6su r u r e u taeqt ]6 u t p o ) ueau e 6e q 1 'eseqd uorssaldxe uorsuedxa oqi uodn 1osur6aq ]o uorlaldulot aq]to1(uaat6,e6ueto) 911-erdaq]rol (anlq)n1-atd1g3g-etd 91 suorlcunl JolcoJ+e lo acueullollod

uabtlue ubrelo3

uo!lelluala#tp larroyedel loldaca.r lo uorlcolos lroldacaj uabrlue;o uorla;duo3

reqlo aqt lv 'alP PUPsrolderer IIer-I Pelsrauar ^l.4aeuer{l er^ sleu8rslelrlrns elrec.radot IreJIIr,r\ sllrr I qJnS 'sselesnare IHW-JIOS oBEBuetouupr teql srotdaf,oJrltr^\ uroq sllol I 'pelErqrlEJ eg lsnu srotdaf,orIIaJ-I to tes ]Ertruraql qlrq,{\ uo ateldural or{l olnlnsuof selnJelou:)HW II sselr pue l ssell 01 paxald -tuor saprldad-11es Jo suorteurgruof,esoql'suralord-Jlas ruort po^uap saprrdodqlrm pardnrco dyr.resseceu ore selnf, 'ere1dalel slurlr uouf,Jloseseql erorl^\ -elour JHW-JIas IIE 'snrudql eql ur ler{l puu 'sJssJford a,rnnlrtsuof, eJE uorlpl 'srrnpord -uasa.rdpup Surssarordua8rlue ler{r IIeteU 31114 tsoq eqt qll.ry\tf,€retur dla,rrlcnpord louuec qcrq.u 1o drr -roleru lsp,l er.{t's.loldacer [e]-J Jo los p sefnpo.rdoruanbes -uof, e se pa.rapua8uedtrlqerre,r snoruJoueeql pue ssacord luaruagueJJearaua8 oql Jo eJnleu tuopueJ aql iJntJo uel selnJelou :)Hw-ilas r{1r^/\uorlterolur a^rlfnpord e teqt os padegs slleJ I 1o a.rrot.rada.r SurS.rerue d1.,rteu aqf sr .,1aoH 'sllol g Pue I Jo luau -dolerap eql ur sdolssnoSoleueoqt salerlsnlI Z€-VZe.rnSrg puegerd4:se1{>oqduI; arn}pLU uo puno;stoldatattr,Loads-ue6r1ue aql urol+]uesqepuetoldaratq)eaoi enbrunsirunqns apnl)ul :rlteds-ue6r1ue etrnbet 931-ardpueg3g-ardaLllqlo6 uorlru6o:at ay{toqdu,{1 )tlalle lou sooplueLudo;enap lo eseqdslql uotsnl)xo pue {llnlssettns luaura6uelJeel luaMlopun }eq}slla);o uorsuedxa :suor])un+ Jelrurs anlasstoldatet11et 1-ardpueg-ardaq1 (911-ard) ureqr$ pe6ueleer ^lmauoq]ro (Ufg-ald)uteqlrl pebueleet{;,ueu eq]iaqlla;opesodu.tor sroldarar {q peln>exa aresuorst)op ole}llo) 'luaudo;enap lle)-g pue llo)-I lo uosr,redwol ZE-?Zlungll V

uotssordxo rordecer ualque-at4 ../

t' .'t

.,' .r't

a't

u e b r l u e; ; a g

Aroqduad

uolsueoxa.PUe uollernlel! Alle:l

sluaAo roleyl

luepuadep ue6gluy

oN

el!s crurolPuv

snu;Aqtro A/\oJJe[!auo€

e b e a u r_l 1

:+ e 6 e e u rg; \--#

uc8 agAcoqduAl rolcaJlo pslerluataJJtq

egAcoqdul; orntel l

u38-ard

egAcoqdui{1 ornleutul

ay{coqduAl-er3 ey{coqdurll-or6

lloc ruols

drolelnurts eql oleurruop d1a1erurl1n q8norqr III,Ia tvlll paprrrordsleuSrsdrolrqrqur orlr 'gZCJ Jo rpq] ueql taqSrq sr suralord 981 pu€ 08Cl eql roJ tV-IIl Jo drrurJJeoql osnel -efl '98C1 pue 08Cl ;o Surpurq ro; 8ZCII qlr.^asotodruor 'uorleltlle uodn dluo polnpur sr sllal I uo uorsseJdxaesoq.r,r 'uralord eql 'se]nrelou .,(rolelnrurtsof,erues drel tV'IIl eseqtJo uortruSocaruodn yeu8rsdrolrqrqur .ro SurlenuoDeue apr,r.ordlpql sJoldef,ersse.rdxa osle 'pste,rrloEeJuo 's11ec 1 'G.t-VZ ern8rg) uouelure IInJ roJ parrnbe.r ere r{rrrl,a ;o 11e'rotdacar IIaJ-I pa8e8uaer{l ruo{ ateuprue leqr sleu8rsqtr.rt azr8reudsSZCI €r^ para^r1apsleu8rsaq1 '(sUft) sroldarar el{-llol rreql Jo }uaureSe8uedq aldurexa ro; 'sleu8rsdrotelnrurls redord oql po^raf,arse^losuaql e^Er{ sylerSurtuasa.rd-uaSrlue asaqt ueq^/rpareF8ar-dn sl uorsse.rd 'stJeralur -xe rreql IIar I allr q]q,{\ r{rr^\sylaoSurtuaserd-ua8 -uue eLIluo 98CIl pue 08C) r{lr.,la slreretul 8ZC) 'aldurexa eqt sr elnralotu 8ZCl oqt qln{^^ ;o 'srotdara.r u.vrou>l-tsaq ef,plrnsrraql uo d;rer s1lar1'sleu8rs drol rueraJlrpIpreAOS -Elnrurlsof o,tracrado1 'sleu8rs^,(.rolelnurtsoJspaau Jaqun1 l l J J J J q r : l u r r r r J ; n s . l o us r s l q t t n q ' u o r l B , t r . l ol eo l ' 9 3 1 a g r 'roldarar cr;neds-ua8rtuerreqt er,r leu8rse arrnbar sller I IIV

9o]oNnnnt

I

tz u3J-dvH)

v60L

'saue8lue.t aseql ssa.rdxaol ernlle € ol ppel gg1y ul slJOJeC -elar eql yo uottdt.rcsuurlaqt Surteln8ardpcarrp;o palcadsns d1epr.,r,r sr lI ln9 'u.ttou4 lou sr slql saqstldruoroeSUIV 1v\oH 'sue8nue rr;rcads-ansstllJrns Jo sllal lerlaqrrdaIo lesqns E ur uorssardxas.aolle (toreln8er eunrululolne)AU1y Pe]]Prror -re; e te,ra.l,rog'dro8eler slql llt dlsnor,rqolou oP 'rualsdssno -^reu aqt ur rlleer{surladruaql to stuouoduroceql ro searcuBd Jql Jo sllJr fl aqr ur ullnsur sE qrns ruolqseJrrlrreds-ansstt ur passardxaere t€ql sulalord 'saxalduoc epttdad-39ytr se snudql eql ur .{larenbepepatuaserdarsr uo8rlue-;1as erBrrdorddeaqr;r ,{1uosrsotdodedq go pa1lq ere sllol J

osuoosor ,o uorleururJo] '8ZOCselaouoc -lno ?vll3

tr

uorle^rl)vllnl rol leu615lo sad^I o^ l ailnbau slla) I 'Polrles dlarrruatou sr IIOI 1 (palorrrserIHW II sselr) yq3 ro IIal 1 (patrrnsar )HhI I sselc)gq3 E eruoJaqol patrnrlsul sI uroq.4^eue ^\oH 'selnralou JHW II ssell IIer +SCltcJ ro sselr azruSora.r I IIr.4^ I]el I e reqrer{^ seunurelapssardxa ot (8Cl ro rotdof,er-oJ rllrrl,l.r to erlorlr eIlI 'snur tql) -dqr egr ur saterparuretur letuaudola^ep se dluo punoJ arp pue sller (+ gCCl,CC) a,rrtrsodrJqnop prllpr ere sllor eseql 'xaldruor sp'8C) pue tCI:) €CI-U)J leuonf,unJp sE IIe,4^ sassardxateqr erdrour.,(qre sr tuau.rdolr^epIIJr-J ur olerpau -ralur da1 V '8Cl pup tql srotdarer-ocaqr;o uorlrsrnbre yenper8oqr qtr.&\luoprf,urof,srnrro luarua8uerreorUJI 'eseesrp a8euep anssrt Sursner pee.rdsepr.r,r pue 'sasuodselaunrururolne f,etm Surrapyr.r,req E.,rtollsslEnpIA 1o -rpur osoqt'aluanbosuocE sv 's[o) I e^r]reer-1osdllerruelod Jo uorlpunurle or peel reqr snurdqr eqt ur suortfnrtsur to tes 'gA1y ssardxatou IInJ eql elrerer or IrE sller 1 Surdolanap op oq.l.rslenpr^rpuru1 'snudql eql ur sua8rluerr;roeds-enssrl

lloc l_ lo uorleralrloro pue uorle^rlcv

tr

(Zteu6ts)CdV uo9803'08C3 q]t/\^ slcelolul

l l o cl u o E z o J

a

poleurLrlal srAllenluane 'gzcl uorle^r])e oql lla)-I ]o ]eq] ueq]relear6sl ggcl pueOgcl ro+ o] eqtasne)o8(fl) esuodsat tvll) ]o l{lrurge llo)-fotl]]o uot]tqtqul '9961 pue 'a)elrns burpeal 08Cl spulqtvl1f llo)-jeq]o16utnou.t paseol)ur uorssoidxo o] speal'urnlul 'uorle^rl)e JaUV(E) tvll) +o 8Z) ^q 98C) pueOSCf;o lueua6e6ue llnl uorle^rpe lln]smolle pueroldererllo)-1aq] ern; leub15 qloq+ouorsrnold ern7 1eu615 IlMauaq] aq1 (rr6raue) enrsuodsarun souro)oq 1 pe6e6ue 11er 'peprnord sruorlelnurlso) (E) q)rqM'llo) soln]rlsuo) ]ou 7 leubr5 ]l aLlluo (98Cf '08C1)selnrolotu 6urluaserd-ue6rlue aql ]o ale+lns qllM6uole'([) I 1eu615 so]n]r]suol fuo1elnur1sor 1ouorlru6o>er qrrqnn'rolderarllal-f eq] {q xaldu.ror aprlded-3;1141 ue 1ouotltu6otet uorlenrlle oqf 'uorleururol so^lo^ur llo)-I+olepou;euOrs-oM] pue uorlenrpello)-I ur pallonurs;eu6t5€€-tZlungll >

(r teu6!S) g 3 l e r n1 e u 6 r g

tr

s60I

lNItAdo'llA30'l'l3)-I cNV'suoldl)lu'l'll)-l_'s'l'l3)

I

(E) qlpopllo)por.rrurer6ord alpr]rul qrrqm'sesedse) ole^tl)eseut{zuet6 eql 'use1do1,{t aq}ut atuo (U) a)e+rns pazrleulelut uaeqoneqsaln)oloul urropad lla)aq] LUol+ oql rauesller1e6re1 1osluau.ryedLuo) leuosopuo io a)ellnsaq]le osle]nq 's;1er oqi ]o o)e+rns eql te ,i1uo ele 1e6re1 ]ou l)e ol po^atlaq suuo|od (El)soloduuopadaq1qbnotqllalua]eqi sasealotd euues

pue'qJospe aresaur{zuerO Iaql qtrqnn o}uosauelquleu.r aq}ursarod puesuuo,ued ulo,Lsuuol;adseuu{zuerO sopnllutsa;nuet6 aseql erese;nuer6 trxolol{t}o s}ua}uo) eql io lueluo)oqt (E) pasealar peluo>]q6l_L q)tLlM olut']1a1t rrldeulse ]o uotleu.uoJ oq] u! s]lnsol q]lnn s1;ar si)pluo) rr;oads-uabrlue uutols1;at 1a6re1 1qbr1 1 lxoloy{t '(U) eq] uoriruOorel uod6's;;arl rrxoloy{r[q lla)1abre1 1o 6ut;;r1;;ar pelerporu-oru Azuet6pue -uuolrad t€-tz :lgngll V (patelor-oulososAl) e;nuerbcrxolo1A3 uuolrad aLUAZu er g

lpc 1c;xop$X

f\e

cHhl I sselS

tr@' qleoO

llec leorel

I

uorleAllce esedse3

\-

\ \\__/ et

@

uo srotdacarrr;pads or Sulpulg dq op or reqrrrsaldcoqdudl lf,nrlsul surelord poterresllpurs eseql 'seur>1otdr acnpord ans -str progdtu.,tlur slleJ proqdrudpou pue serdcoqdrudlduelaq

s l l e ) o u n u u l l e q l o o l s l e u o l so p t n o r d leql sout{o}^) }o felly ue a)npold slla) I '(g-77 o;n8rg aas)sle8rel rlaqr III{ o1 sarudzue-r8 pue surrolrad uo dlar asr.{\a{rlpue dtr .u1!\ou>l - rlre f,rxotoldclraxe os]esllet rallDl lou llrls lernlpN sr Uall f,rldeudssrgl olur sur.royradpue saudzuerS;o eseala.r uodn pa11r4Suraq sprore IIal I aqr ,{\oH .ller la8rer pue IIaJI llxololdc aql uaa,vrloqpeurot Uall eqt olur pesealererE sluoluoJ Jrer{rpuE salnuerBfrxololdJ er{l (uo8rlueot Joldof,aJ '11a0 IIel-I aqt 1o Surpurquodn I rrxolotdl oqt eprsurporols 'salnue.r8crxolotdr orur pa8eped ere saurdzuer8pue surrol -re4 '(srsoldode)qreap IIOI petuurprSord;o qred E uo to8rel aqr ledord os pup sasedsecropeJJOolelrlf,e teql saseatordaur -res ere serudzue.rg'urro;.radd9 parerauagsa;od agl er,rd1a1r1 '11ac aql Jatue ta8rel ot parunsard eJE pup ot peJe^r1aper€ saurdzuerg'rpeep IIel ot setngrrtuoc,se1n1os raqlo pue IIEr.us saldlorrcalaJo ssol or sppolgcrq.r.terrreg dtlllqeaurradlcelur

ue Jo uortrnrtsep eql 'r{lptle .{aqr qcrq,u ol sauerqr.ueur ur ruu 0Z o1 dn;o sorod uroJ 'xalduroc ltpue auerqrueu aqt SursodruorepErsEJluaualduroo arll Jo stuauoduoc yeuturel eqt ol dSolouroq trqrqxo qcrq.n 'sur.rolred,'G7-VZ ern8rg) sacu{zupt8puu suttoJnd'dllecrlsr8raudslr€ ler{t surelord ;o sesselro,r\l se^lo^ur s-II) dq 3r1[) rilsruegf,etueql Jo '1r srEeq1€gl ylacla8rel aqr illl ot IIer J gq3 peurrd dyredorde A\olle ol serrJtnsxalduror eprtdad-3gry e18ursy :dlr,trtrsuaselrsrnbxa glr^/\ os op pup suorlpurquoc eprrdad-3gyrg alerrdordde aqr .(eldsrp rerlt sllor ie8ret IIr>l deql 'selncelou :)HW I sselJ dq uorlruSocer rrer.{lur pelJrJlser eJp pue Je>lJerrr uralordordlS gql eql d.rrec dlleraua8 '(s113) sel,bot{d -tudl J crrdlordopell€l osle 's11ar orxolotdr 'uaase,r.arrrsy 1

6 u l l l l > rt o l p o z r l e t ) a d S orv pue rolda)ar-o) gC) aql Atre) slla) I )rxolot^) 'asuodsa.r IIJI-1 e Jo uorlernp pue snlets uortelrlJr er{l SurlloJluof, ,o supeu tuelJodrur ue epr.Lord pue drorrqrqur ro drolelnurls eg snqt uef, salnralou drol -Elnturlso3 '8ZCC ;o ruaura8e8uotuory e^rrop tpqt sleu8rs

A9o]oNnv\llll I

tz ulrdvH)

960r

'uorlcnpord dpoq 'spue8rl rrer{l qlr./( surtf,eles orerpl.qoqrpl dltsour ere r1Jrq.,ra Jo uonJnpo-rd aql Surrelrpe; dlgerou 's11a: pe1lecsrofdaJorof,etrnsJo suortf,€rolursorrnberteql ssaro.rd -nue ur elor tuelrodurr ue leld ssoleqleuous11ec;u1 E 'u./\,|op1!\ols]SJIJlsnru lr 'enssrl ur uorlJetur {,tolutuwayfut se osle ol parreJaU 'asuodsardtoteuueJ; elrs e lno a Io -ur up olelntulls pue se8eqdo.rceuolEAIlrPueo ',{"5111 }o uorl {ees ro apou qdurdl E ur eJuoprserdn e1e1pue urnrlaqtopuo -onpord q8no.rgr's11ec1u1 '0I-'II Pue V--il P uorlrnpo.rd aql esra^Erl o1 e{co4na1 E roc 'sef,rot rEor{sJruEudporpdq 'dNI pue ,t uora;.ralul eqr .{q pezrralrerpqf,'s11ac q8rq ol pasodxa ere pue paeds q8rq tE os op slasse^poolq 7H1 PUE Jo uorlrnpord eql dq pezrnt)ereqc 's11ecIHJ se Poltlssell ere q8norql pup uorseqpe ler{l saltro>lna-I'uorter8nu ]a^prt ilel sller I tql osoql 'slasqnsIIOI I tql .relnottreddq pacnp IIel Jo uortpln8ar agl ur dars IertuJSSJuE sp uorlJun1 reqr -ord e.rBsaur>1oldorcqto '7-11 acnpord UPJ sllotr I Pele^ sroldaJor peldnoc-urato.rd are sroldaoal eurlouor{J 3 'sau,4outaq)f.to1otuu.taylu, -rlle IIe sEereIJlN'serlrado.rdleuollrunJ rIOI{1PuP aonpo.rd sp ol palra]er a.rea8euep enssrtpup uorleturuelJurto solrs ol satdcolnal laqr sourlordc aqr uo peseq PeuItoP ueeg snql o^sq sllel 'tuotuuorllue droletuurelJul uB Surlrnrcer ;o asodrnd eql a^res tegt seur>lorueql esoql I tql 1o saddr eldrllnptq 'saut4oLuaqz )rlp$oawoq sE ol Jo luoruqsrlqelseeg] ol otnqlJtuooteql seur4oilc aqt Suttarc PerreJerosls erE sourloluoql (luoudolo^ap -esuoufun; .roleruslr se seq IIOI J tql 1o ad,(l Puolos V qfns proqdurdl leturou ;o ued se Jnr)o stuatu 'd1oqsapr,to.rdrr qrrq,tr ot -elorrr esaqt osnpfeg 'sardcoqdudl IIOr g eIJrPue Jo sresperrnber oqt r{rr.la sB 'sautloldr sue8ro proqdrudl;o uorrelndod eqr or elnqrrtuoJ suorlprS IIel J tql eql ueo^\teqtruluof, lJerIP se IIo.4A 's11er radlaq 'sue8.ro -rtu eseql proqdudl ur of,uoprsar dn alet uonel pue 1 Jo uorleJraspu? uollfnpold aqr qrog sa.rrnber -nf,rrf, dq or{t e^E3l ol sa/coqdudl sour{oueqr euos porrJer 'uotlcun; srgl 'solpoqlrue,{1rur;;s-q8rqalaroas rno lferrp 'uorl€urlsaP sllet eruseld otul uollelluorottrP rraqt aprn8 Pue sllel g reqt 1o3r3l rror{1ol lJodsuErl roJ r.uEerlsPoolqaqt olur horreur euoq ol eruelslsseapuord ol sI sllef,I tC) Jo uolllunt roleu ag1 eq] ur (pelerauaSa.redaqr araq.Lrruo.r; seldoolnal yo uone8r sralrelN a)elrns lo uolssoldxl pue -^pu aqt ,\ olle ot pasnsr Jpol slr.lJ','(rrxalduorlea.r8 senr 1o uorl)npordoullol^) llaql uo Pesegsossel) f,rlf,elowar{r apol e Surteraua8;o drrlrqrs to lerroleurquor ro]en oelql olul pepl^lo elv sllo) I ?c) -sod aqf sol€erf,srql 'seur>lolueglfuareJJrp lere^espurg uEl ;otdacer a18urse pue 'roldarar euo ueql aJoru ol purq deu 'RVt-gtarn8rlaes) eur>loueqJ auo 'sroldJf,JJ our{ouer{f, uJzop E uerll oJoru 'syy[ uorlepe.r8epleuroseelo.rdroJ ulaql la8rel pue pue seurToruaqlllurlsrp 0t dlereuJrxorddeere araql 1o ruro; 'saul>louoqf,dq palorruol ere sdelsSur4cgtert eseql potp^rlre ol{t or pulg 'saut4oldc dg pacnput sallesueql ere IIE 'tearu ot sllel g pue slleJ rot pue 's11or t)ente ot sllel suralo.rdasagl 'suletord Sf,OS ro 'Sutleu8tseurloldr Jo sros I 1 -sarddnsare de.r,rqledIVIS eql Jo Iorluor aql rapun sauaS trluPueP ro} sI soPouqdu.{y ulqtl^\ Suqrrgen al,(r Ielluesso -oqdurdy;o uorleln8ar eq1 'a8euep dueur aqt Suoury '(mer,ta.r4crnb ro; q1-91 arn8tg aas) 91 elrs eql ol slgdortnau to ratdeq3 uI pequlsep sr de,vrqledIVJSiXVI aql q8norql srol slJBrlte lEr{t (8-lI 'eurlouraqc e acnpo.rdsrselqorqrJlueprser 'srnrro a8euep enssrtuer.l/(\'sourlotueqJ ruroJ erlt ur senf, -daoa.reur4oldodq uorrcnpsuerlleu8rsJo tusruPqJotueql 'o rapnrlul orp qrl^\ Isop PUEarero;r1ord.(1pr rrlretor.uor.lJtrue sller duepo8 ol ereq,4aserdco4nel11ar -der uaql sllal I drourau esarlJ 'srnJJo ue8rlue ol ornsodxa seurlowaqr (puer{ ror{lo arlr uO 'suortfunt roDaJJOpezrJen -er ueq^{ uodn pe11ereq .,{eurleqr sllel pacuarradxa-ua8rlue -adsarrnbre ol sailcoqdrudl s.tro11e ]eqt tuerSord leuortdrrls 's71acKtotuaw -uert e Suuoqa ,tg op or req.vrsaldroqdurdl lo wro! orp uI sllol I Jo esuEu 1al sur>lnalratul arP r.{rrq^/\\ -alur€ru eql uI slor e ,(e1dSI-'II PuE /-'II qlog 'srosrnrald seullouaq) Aq pepr^ordsen) pellfiuruof, Iuort sllef fl Pue J to luotuoole^OpJoJ lPlluossesl )rl)eloureq) ol esuodsauur e^olN sal^)olne-l uI sllof, oqt ho.rreru euoq lrtuorls dq parnpord'(t-1) 1 ut>1n41 -Jelul'uollptntureddq uoll€ulgtuoJeJ LIJII^\s flleluos PUE '(sue8nue ol sllof, g Pele^Ilre sernP alerelqord ol pup sselc o8rapun -ur g or dleq 8urpr.tordut paztlerceds sllo) (lr.o1eq passncsrp) ;1esol asuodsereunrururuE Jo of,uasqeaql) ecueralol Sururet -urEru sI qlII{^/\\'(l-tt) t ul>lnelralul dq pernpord sllal ur erE sllar a^rlJEer-Jlas dlertuatod puE lueuodr.ur tql J I 'slloJ 'sllel reqro I POIB^I])P;ouotsuedxaIEuolJ so^IrPPUe J ,o dlr^rlf,e eqt urprlser sllel J droleln8ar eser{J dq uorlrnpord aur4oldc uo tlotte a,rrssarddnse Surl.raxa Jolret r{I,ry\or8(8unce-;1as)autrcolne uE se slfe T--il'pe]€I dq sasuodsarounrururelenuaDeuec deql 'silal I ["to1o1n8at -nrurls ere slleJ I uaq.t\ PJJnPordsaurlotdr lsrlJ aql Jo auo pe[p]er' s[e] I rCC to rosqns pe.ra,rocsrp-{iliX1r:a* sr pue rotJet ql,nor8 ilolI se PaIJrluePIse.rrt(7-11)Z ullnepel -u1 'eldurexa pezrnt)ereq)-1am dlrepctl.red e Sureq roldocer (salluellulls IeJnlfnJls qlrm s.roldarJJJlEu Z-urlnolJelul oql 'pezt -3oc dq peztuSoca.r eJPsullnelralul PelEIerdllernlcnn5 -rotJErEqJdlrepcaloru PUEpeIJIluoPIueaq a^Pq sul1na4ralul uo dlrrerur.rd LZ $eel lE :suDlnelrefulpollsJ are saldro>1na1 '(s11acg) dlt,rrlre lrp ro lq parnpord erE lel{l saur>1old3 '(s1Jar Surlanas-dpoqltuero '(s1ae1 7q3) drr,rrpe radleq 1 gq3) dfrrrrfcecrxololdc lrexa ol fpear 1ac ropaJJoue olul elP -rruera#Ip ro ale.raJr1o.rd reqlle ot erdcoqdru,(1egl s^\olle legr urerSordluuotldtnsueJl e SuIlBIlIuIPuEeJPJrnserdroqdrudl eql f,Il'Iuos PuE uoll'ulqluof,al qf,lr^\s ss'Ir Joi 11tt g tqi sareda.rdos pue 'aseurureeppefnpur-uorlelrlf,E Jo uorllnp -ur roJ alqrsuodsarsr sllor g uo ('I6yCO) pue8rl gtql r{1ry\ sllel I pete^rlrp uo parnpur uralord gtCC aql uae/\{toquorl 's11ac -rerolur dq saur4ordoJo uorl eqt pue pacnpord tCf, 1 -eurquor aq1 '(-rolaq passncsrp) saddlosr931 pue 1931 agl ot uorleurgruofar qrlr,ry\ssselr e^lo^ur leql sasuodsJrIIeJ-g ur elor ue deld'V--IIJo uorlrnpo.rdq8norqt's11ac lu€trodur '€DBt pue 1931 sE rlrns serpogrlue8urxr1-tuauralduror ZHI

L6OL

S S N O d S I U3 A U _ d V C Vl H l _ N t S ' l ' l l ) t A 3 I _ S A S - l N n W W l Ot N O | J - V U O 8 v ' l ' t O )

'seurlouer{J stueuodruor er{r dueur areqs srotdorar e{ll-llol aql dq ruJot eql ur sleu8rs Jrlf,Blorueqcq8no.rql to to pa8e8uesdemgredSuqeuSrsoql 'uorrJnpsuerl alqeue (uorle.r8rur oB ot oJer{,{\pelJnJlsur arp sllal I r leu8rs 11ac) ol suratord raldepe luorulrufar er{l roJ algrsuodserureu to d.raqdrredeqr o1 snurlql -op e sureluoc sroldecar eIII-lloI ;o uortrod cruseldoldc oql ruory pauodxa ere pup (uorlcalesa,rrlrsod)ernteru ol aq1 'uorlruSocerpue8rl ur pe^lo^ur aq ol pa^orleq urpu pa,ry\ollpere saxalduror )Hry-epqdad ro1 ,tr1u5teeterpeu -op relnllererrxe pedeqs-elllrsB u.ro; steada.reseql's1aaQ -retur a Eq tpql esoqt !(uonceleserrne8eu)orp ol palrnrlsur -a.t q2t,t-aux?na1 p pasodruor ureurop relnllof,Erlxo uE ssas are tuaurdololap Surrnp poJotunoJuo saxelduroc a111rJ -sod srotderar o>lrl-llo1 -oprrdadqrrm dpuorls ool lfpralur terll sllal 'sleu8rs IIp pue Jlasrl IIoI arnpn.tls U-lI 1 le.trrr -rns Jo >lrpl roJ arp IIp te salnJelou 3g1z,g,iue azruSolarol 'salEJqaua^ur sP IIaA s3 rpqr sllal I Surdolarraq'solntroloru:)HW II sselr r{tr^\ IIpt slfosur ur asuodsa.r eunruturolpuur uB SurroSSrrlyo alqedeo aruofag IIr^/rreqr asoqt ltuau lf,erelur lsnu sller I tcl se pazruSocar.trou e.resroldacar poteler tnq tlJ lrnrJ eql -do1a,taprraql Jo esrnoJaqt ur salnf,aloruIHW I sspp qtr^ ur Sururanedlertua^osJopur alor luelrodrur slr Jo osnef,oq lJPrelur lsntu sller I 8c3 auof,Oq ol peunsop sllat I I pere^oJsrpse,ttIIoI a1tqdosotq'ueql uea,ryueq,,t3o1oruoq 'lueua8uerreor rpql sllol dUlI lue.^arepund11n;ssarcns IEuoltrunt pue IErnlf,nrls aqt to osnEf,oq11o1 uratord upqdosotq eql reUE parueu ere suralo.rdasaql ',(1rrue; osoqr Jo uouera;r1o.rdpue uorsnlf,xerrlalle soterpau UJI - a r d a q l ' a 3 g - a : d a q l o l r p l r u s ' f t t - V z e r n 8 r ge a s )a u e 8 (g11) ioldarer olrl-llol or{r Jo sraqruetuq8norqr sosnrrl puE Erralreq1o acuese.rd oql lrorep s11ec Surlueserd-ua8rlue pa8uerreerun ue dq peporue lrungns n 931-a.rd pezrlerf, -ads e sapnl)ur oslr gtlq,l\ 'Ull-erd eqr ur palerodrocur 'sller Surluasard leuorsse;ord rer{lo pue sllol trlrrpueq sr ler{l trunqns d leuortrury e Sulpooue 'lsrry sa8uerree.r -uo8uue leuolssa;ord osoqr qrrl4. lf,eluof, pro^E ol JerrJpg 'luorudole,tep slql ser{Jeorqtpqt uaSoqled p roJ elqrssodurrd11en1.rrzr.sn)ol d Uf,I eql ilol-I Jo esrnof, orp uI r 'fit-VZ e.rnSrgaes)uorlcnp lr selerrr siln suoqn8uo7 peyet sJlel Jurrpuep 'o Iro^{ -lou p ur>lseql uI 'rntf,o or dla>lrltsoru sr suaSoqled qrrm lJeluoJ eJoq.r (lcerl lelrua8 'lteil IpurlseluroJlseg(sdBA{ -rre) eryaqrrdetnoq8norql peuo4rsod are sllel Surtuasord -ua8rluy's11ao Surluaserd-ua8rlue leuolssa;ordJo uorlp^rlr€ qSno:qr relncrtred ur 'asuodsel ounrururaarldepBJadoJd e rot rsoq uprlerutuerueql sorederdoslp tr lnq ,repe,ruraql 'il or puodsar Jo uorleultulla rlarlp sepnlf,urasuodsarslqJ pue rape^ur E ef,uosordaqr roarapdlarerpeu IErqorf,rr.u Jo -ul ol &rlrqe aqt sI srsualep eteuur ;o rred tuelrodrur uV

sure$ed teln)ololuoJ)eNpanueo-ua6oqledlo {1a;le1 e snre)JodsJo}de)euel!-l-1ol 'treretur sedft 11actue^oler oql l\\orl PUEPOlEArlJeaJEslueluolosnorJEA0s0gl A{oq eqrJfsap e,r\ uorlras srgt q dlrunurul e,rrtdepeJo pedse tupuodurr dra,r e ero;erar{l sl dtlunwur e,rrrdepepup eleuur Jo sluau -oduroc uoo.,rrlaqdeld.raluraqJ '(SU-II) sroldecar e>l{-ilol .rreqtSursnsrape^urDalep q3tq^ ,s11ac crtr.rpuepsEr.{lnssllel ,u.rnl Surluasard-ua8rtue Ieuorsselorddq pole^rlle aq ,t1uouEJ ur 's11acI asoql 'slleJ I petp^ntp uror; dleq errnba.rdaql -selpoqrlue drrurJte-q8rq uorpnpord ro; satrsrnba:ard Jo -uorlPlnruJeddq pue uorleurqtuoJoJ r{f,lr^ s ss?lf, alnJexe ,s11arg;o ol sllef,g roc 'sllal Sunuasard-ueSrrue pue .s11ac 1 oruosardaql sarrnbotasuodse.r aunruurreandepealtlJeJle uV

osuodsaua^tldepv aql u! sile) uals^S-aunuul lo uotteloqello) W droqdrradeql ur pup sue8ro proqdurdl urqtr^ qrog ,8urryr1 -;ert eldoo>1ne1 uorleln8er asrcerds.{\ollp.,{1rue1 rotdecar ;o ourlotuaqlour>lotuoql eqr 1o drrxalduot aql 'seurlouoqf, ;o Surpurq rroqr Jo srurel ur dtlnrsnuord oruos ,roqs ter1l srolderer peydnoc-urelordg are saur{ouer{f,ro; srotdaceA

-sue.rl trnrJer strtrVII leu8rsur peAIo^urselnf,eloru.,{rossacce asaql 'paleldroqdsoqd uoq,vr.lsureuop WVII eeJr.llro ouo pet rrurseldoldc slr ur sorJJEJxaldruoc €(If, or{t to trunqns qf,EE 'uorlrnpsuerl ro; perrnbe.rsr qlrq^, 'xaldurot leu8rs gq3 drossacle eql sepnpur rotdere.r ger-1 a1a1druof, V r 'sol^roqdr.u^I aulolag ol pourlsap J sllol osoqt ur ^{1uopue snu.{ql eql ur sJnf,Joseua8931 '(gg-97 e.rn8rgeas) s11arg ur saua831 Jo tuatue8ueueag JoJpeur;epesoqt se salnJer.uesaql sdeqo ;o luaura8ueJJpoJ lueua8ue.r.rearrregl !(ureqc fl) sluaru8asI pue 'q 'n ;o pue (ulrqo o) sluaru8esI pre n Jo uon€urquof,ar f,ueruos dq pelereue8 ore slrunqns Uf,J eql Surpocue seuoC r

'saurloldr Jo afrnos tuelrodurr up ere pue slloJ g ol dlaq eprzrordsller I 7q3 fs11ec 1 orxololdr ere sllel I gCf, :lrulrslP dlleuorFun; aJpsllal I Jo sessEllasaql '7q3 dr.rec selnf,alou sselJ esn reqr osor{t!gq3 d;rec sluaur II :)HW -elo uorlf,rrlser sE salnfelour )HW I sssll asn leql slleJ I

'Gz-vz

arn8rg aas)gq3 pup t(If, s.rotdacer-orurarordoc.{p aql;o uorsse.rdxa aql uo pospqsessEIJroleru o.ul tseal l€ ur JnJJo sllol I 'slrunqns g pue l" ro strunqns d pue m 1o Surlsrsuoc suralo:d f,rraurp are sroldaca.r1ec-1 cr;roads-ua8rtue oql r luaudola^aq llef-I pue'sroldo)og llo)-I'slla) I

'0t -gt arn8rgeas) uotlpspltp.uxasEu.,tn,ou>l ssarord e dg lasse,L poolq oql trxa ,r\ou deur ailcolnel aql 'albo>1nel oqr Jo rserre r.urrtsesneJpue pue8rl s1rro1 urr8elur er{rJo ,lt1urJJeur aseerJurue ur sllnser a8ueqcsrql 'e8ueqr lpuorler.uroJuoce oS.repuno1 a/co>1na1 eqt dq PerrrEf,surrSalurs./\^ollereqr 1eu8rse srlllle roldarar eurloueqf, aql Jo uorte,r.rtce'our>louleql tpql .ro; rotdacor e sassessod e/co4na1 aqt tr puE 'xr.llBurrelnllor€rlxe eql ol 'arnlEu ur PeqrosPel{llurrxo.rd uI puno} ore seur>louerlJJI

lLSE:60€ ' peldepe(q)]ledl aJn]lnlls uteLUop atua/)S'EO0Z le ]a aoqf 1 Luory . t e l n l l 0 ) e r l xEaU l l u p u l n q0 q ] + o o r n ] ) n l l s ( q ) A e M q l e dB v - l N o q l o l e ^ l ] ) eo 1 > v u l p u e 9 9 6 , 4 6 ]1t n r ) a ro s l e ' s u r e u r o 6 purpurq - p u e O lrl t e q l } e U [ - l l a q ] u t o r ] ] ) u r ] s r pe l r n b{ 1 ; e r n 1 : n r 1q sO n o q l 'stoldeter a ) l l l l o l ( E g 9 1 e r n O r le a s ){ e n n q l e dB ) i - J No q } } o ' ( ; y g 1 ) a s e u r lp o l e r ) o s s rpo l d a t e r 'uorlenrlte l - u r ) n o l . r o l uatq l I q s M o l l eu r n ] u r p u e r o l d e > e rp a l e ^ t l ) eo q ] o 1 p a ] t n r l e rs r g g 6 I y 1 u r e l o r dr a l d e p eo q 1 ( d ) V g 1 , - 1 1 u )r e l o r d{ r o s s a t t eU t - l l o q } ; o l u e u a 6 e 6 u e - o r{ q r o l d a : a r s } ro } s p u r q( t - l l ) 1 .u r ) n o l r o } u l (911)rolderer o)ll-lloI pue -roldetet L-"llaql ulor+uotl)npsue.t] 'srolda)ar alll-llol S € - ? Zl U n g l l V ; e u b r s1 o u o s u e d u u o (3e )

(q)

)VUI

uorlenrlce 8x-lN )VUI

,/\

8804W reloepv

loso$3

roualxS

Q r-rr ult

dcv-u L-11

dcv tut-'lt u[--tl

(e)

ADO]ONnl/\ltAt I

tz ull-dvH)

860I

'snelonu eql ol useldor^,{raql ruort uollefolsuert ro} 'rolre; uortdr.rcsuerte 'fl)r-CN a^rlre Io eseelorot Sulpeel Xeld orur eruoJ soseur{ uearlsu.4^oplpre^Js '(qgVUt) 9 rolJe! Paw -rrosse roldarer-451 sateldroqdsoqd yygl ter;y '(aseutl peterrosse-rotderer1 ur>1na1.ratut) )VUI to uoItEAItrE PUP 'ggqd141 uralord ntdepu Surpurq eqt s.4\olle urnl uI qcrq.,r,r, eqt to tueulrnner ol speal srotderer a>IITJIoIUEITELUITIEIU Jo 'St-VZ arn8rgur u^\olls sV ape)se) tuerua8e8ue leu6ts U-lI 'suaSoqled '1err,t 'leualreg ;o acuasardoqr roJ f,ItsouBEIP le8un; pue eJElpql solnlaloluoJJeu;o .{tatrerre ;o uortruSof,oJal{l s.4aol -le su-II u€rleurtuEruJo las IIry aql snqJ 'svNu poPuErls -a13ursurelral 1o aruaserdeql ol spuodser1411 1(11eutg '€ull Jo uorle^llre or psal sllel PelJalul-snrl^ OluosuI lue 'dlreltruts'VNC -sard papuels-elqnop salnralou IEIq VNU 'pareldqrauun dq pele^Ilf,Esl -orrrru Sururetuoc-3d3 6U-IJ'uorterrJrpou slqt s>IJEIdllereua8 VNC lelqorf,Itu seerell.{\ 'sapnoalcnurp Cdl dueru te pareldqrerusl VNC uelletutueyt{ 'eprsal sroldeear esall] eJor{,^a sluetuueduroJ leurosoPuoeql 'sptre urqtr.4\ror4lerlnq 'ace;rns IIer oql lE lou os op ,{aq1 lrelf,nu pa,trrap-ueSoqled ecuasard eqr esuas-6dll ;o (tU-Il-srordarar a1q-11o1 les puore]rYrr., pue '/UlI to IIar aqr rE srnrro sruauodurocadole,ruaIelretleq 11r;o Lotr -ruSorag 'ur11a3egroJ SU-II pue lsapudadodryroy 9/Zp'IL ZIL;g-IL ,o rlrolurporeleq laprreqcresdlododll roJ 7U-IJ 'alduexa ro1 :s.toldaceJlJullslp dq pesuesst acuasard.rtagl tprarrp u^ oqs eg or suleluar srotdalar J{ll-lloJ ol selnl -aloruorreu esagt Surpurg rrarrp q8noqily 'seprldadod11 to lelratreq1o slrunqns)sutlla8eg'(541) Ierrelrpqpue'(e11a8eg saprreqccesdlododrysE I{Jns elralJeq ;o adolo,lua IIel eqr ur punoJ salnraloworJetuJpnlJuI srotdocereIII-lloI dq paau -8ocar slcnpo-rdlerqorcrur aq1 'srordaror o>lll-llol sserdxa osle slrgdonna5l 'se8egdorf,Elupue slleJ tlllrpuoP Jo uoll -plrlre eqr rot Ielrnrl sI uoltrunt rlel{t tnq 'sod.,{1 11ar;o fta -rrEA€ dg passardxeare srotdaca.resel{f 'slrnPo.rdlerqolorur snorrel dq patelrrce eq uEJ leqr srordarar e>lll-llol uPITEru -rupruuozop e dleteurxordde a.rea.raq1 sull lo t$tslenlq '(Sg-gt a.rn8rgoes)sleunueu ur derrrqredg)-JN el{t ol uollrsoduroo lernlf,nrls pue uorl -erado dezrrqledsrql 'uorrducs flq8rq sr ur snoSolouroq s1r -uerl ot JIC ulelord eqr smo11e arll ratue pue snelrnu elprlrul 'uratord snllEJ eql to uolrEPerSapleruoseelord IE^ourersl1 'uiaqr uor; Surleu8rs luapuedap-urlrnbrqneqt sr dals da1 y eql pue rotdara.r ,{q parenrlceere t€ql srotfe1 uottdt.tlsuB.tl a{ll-llol aql uaa^[eq pasodra]ur seseul>lIueerlsu^{op olel -rtre ]pr{t suterord ratdepe Jo selrasE to suearudq snleredde eql le roldac leuorldrrcsuerraql qlI.^AseleJlunulluol ef,EJJns -er pete^rlre aq1'saprrdadlerqorcrturtueepoJueteqt seuaS ;o uorldrrcsuerl ellt slor]uor .{lereurrln leql apecsec8ut -leu8rs p serlseeluniloI Jo uolt€Altre 'dg aqr u1 'apqdoso.r.q uo ^,(erdreqr r8un; to II€^\ [a] erlr yo stueuoduror dq pare -rlrur uorsreluoc crldloelord e;o rcnpord aqr tlesrl 'a1zleed5 'pue8rl eq1utatord oyqdosotQ slrpralul go1 stl qtl.,r.r 'Gt-VZ ern8tg) 1-11 aur4oldceqt roJ srotdacer,{g pasn asoql sE (sauoclno pue)

660r

tSNOdSlU I A U . d V C Vl H r N t S ' t ' ' , l tr)A l I S S - l N n n n t l o N o i l _ v u o 8 v ] ' l o )

sleturuPleluerurJodxaJo uorlpzrunuur dg pesreJdluoluruof ore eseur>lE]o ruroJ perel.{roqdsogdaql azruSorarteql sepoq -rtuy 'saprtdadro suralorduo suorl€Jrtrpou JplnfrlJedSurzru -3oca.r;o elqrdec sllor o1 dllenba sarlddeldaruoc srgl fl 'uorterluereJJrp JsrnoJaralduoo str unr or Jo ilel g or{tJot dressoceudlaq eqr aprlord sllel I uer rotdef,orIIOo-Jslr er,rxelduoc apudad-3g7,q II ssell srqtJo uorlruSocarq8norqr dlug .,tlnruaSounururrraql o,rordur ol surelo.rdJOIJrpfol pote8nluooorp sarpoqrluetrrrla ol pesn saprrdadcrraqluds 'uoseor srr.{lroJ 'd1ag 'xelduror p lac-1 ro} IIpl se sa^rosqJrq,r\ epqded-JHl4tril ssell E sE pelueserdaq or repro ur elgelrp^€ selnrelotu :)HW II ssell rrlelle aqr ol purq pue 'srsdloeto.rd pue srsol,{ropuoalrlrns'roldare.r llel-g eqt dq ueasadolrdeeqt urptuof,rsnru daql :Errelrrf,lera^osIIIJInJtsntu saprrdadqon5 'asuodser dpogque lrruqle-q8rq E Jo uortptorla ur sue8rluese d1p;sseccns posneq or saprldad'alduexaroy'ot azrsrunrurunu p dq.rl sI ereql sureldxe uorlruSocar palurl to ldaruoc aq1 'Peleurur 0q uEf, uorlPDuoreJJlp llol-fl Intssof,f,ns 'pr)rll d11eors,(qd are-adotrda IIer-I E-apndad peluasord -II sselreqt pue edolrda Iler-g eqt se 3uo1sV'aFraloru JHW Il sspltre qtr^\ palerrossp ef,elrns erp uo padeldsrp /pl"* -rr1n aprrdad eqt tuory tf,unsrp arrnb aq deu rotdacor 11ac -g aql dq pazruSoraradolrde oql reqr erog '(99-y7 a.rn8rg) xaldruoc aprrdad-lHry II sselt e Jo urot eql ur dlag 11ac-1 rot IIEI e otur ua8uue parrnbce-glg eql troluof, snqr sllol g pecuarradxo-ue8rluy',{eznqredf,HI I il ssell eql erl uorl -eluaserdro1 lr sserord pue xelduoc eunrururoql ezrleuJelur s.roldeoarllal-fl Jrer{ter,rua8rluearrnbcereqt sllal g :oldacar g uo s.roldec sp olros qrrqzrr's11eo I]er-g eqt ro; s.roldaoer-of, -er tuarualdruoc;o tuoua8e8ua q8no.rqtsllal fl Jo uorlplrlfe aql ut rotredns oJe sua8rlue perJrporu-tuauralduro3'apou qdurdl Surure.rpogl ol srrler{drudlaqr Br^ pere^rloposle oJe qcrg.tr 'suratord ler.ralceqJo osEOIOr tuetrrrrof,uof,pue 3ur1p1 Ierroppq ur stlnsor epeJsprluerualduoo lll Jo uort€Artre 'uorlonrlsap enssrt dq parueduorJp sr uorlf,oJurarlr JI lerol 'sua8rluesp elJesupf,lpqt slrnpord lerqorJru Jo esealaraqr dq parueduroccesr qurror8 IerralJeg '(9-y7 en3r.g ees) scneqd -rud1ruerage eqr g8norqr apou qdudl eql soqf,Eeruo8uue elqnlos 'sllel rrlrceds-ua8rtuepele^rlre ;o aruasard aqt I pue roldecar oqr e8e8ueot ua8rlue Jo arrnos e sarrnb ]leJ-g 'snq1 's11eo ruo{ eJuelsrssee.rrnber -er uoll€AItJE I llel-fl sllol g 'suaSotlledSurzrle.rlnautE pue ua8rlue Surpurqlr .ral -leq orp qrrqrrr 'sorpoqrruedlrurge-q8rq elereua8ot rapro u1

slle) I Pue g uee/uueguorlProqello) saJrnbeu s a r p o q t l u v{ l r u l g y - q 6 r H } o u o r l ) n p o l d 'dpoq eqr ur erer{,,taasle suorlJun} rolloJte otnf,oxo ol epou qdrudl 'uorlelnfJo 'ern1e1nf,sp^ el{l e^Eel pue eql 're1ua eql lJeluof ol JepJour .ro 's11ol r{rp Jolunof,ua Jreqt JoJ uorte.redardur fl 'apou qdurdl aqr e^otu q8norqr elnoru ore sllet J pole^nJp pue eql 'llal crxolordr a,ruce dlny E ol rrxololdc rosJnJ 1 1 IIal 'selncoloru -ard e uor; dolarrep,(eru JHW I sspll 1er I gql e ErAsJnJJo tuotue8e8ue1 'addrouaqd lyac radleq e ro droleru -r,rjeuuluE sPrE^/rolezrJ€lod Ilr.^AIIaJ I tcl e rJrpeq^ aurrurol -ep uortf,eer Sururrrd sqt esrnor erp ur polnpord sour>1oldr 'o eq1 'SuuerruorejJlp puB SurtereJrlord dq puodsarqrrq,tr'q1ar1 crpads-ue8rtue a8e8ue sllar Jrlupuep uapel-ue8rruy

'pele^rtf,€0q ol sllel I aqr rot uerSord uollelluaraJJrpredo.rdeql dn SuIDesuI pue 'luesard dpuepunqe tsrsseler{t paonpord are saur>1otdo ere selnfaloru drolelnurrtsoc'sexalduror 3gy4-eprrdad 'lloJ o^reu e Sutlelnoe I Jo rurot eqr ur padeldsrpsr uaSrtuy yo algedec,(1p1sr lpqt etets e uI opou qdudl Sururerpagl ol uorter8nu slr ur stlnser snql uaSoqled e qtr.,r,r 11acSurluasard 'f1e.tqoa;ye p -ua8rtue leuorssa;ord Jo tf,etuor IeItIuI er{I erotu sller J atp^Ilf,e ot rueql ^\o[e llr,tt qrqm '93q3 pue 0gCJ selnleloru drolelnrurlsof, eql to uolssardxe eleln8er -dn s11acSurluasard-ua8rlue leuorssalord pete^qf,E tlleutg 'ef,etrns IIaJ er{r ot sluaulreduoc Surpeol eql tuort saxald -uoc reJsuel ol{l sesearrul pue ss€l] aprlded-IHl I 11 Jo 'sasealord leruososdlTler.uosopua ;o dtrzrrtceeqt saleln8er-dn 'uollrsrnbceuaSrlue elEJsll sefnpeJ'aldruexo.ro; '11erorlrrp Jo -uep pelp^rtf,EuV 'saul{ouraqr dq paprrrordere senJ IEuoIl -Jorrpaqt eragm 'apou qdudl Suturerpeql Jo uonlarlp oql uI atBr8rurot upls pu€ unlprlsqns Surpunorrnseql uort r{lerap eqt Surcnpur daql :a11ou aluoraq ol sllol Suttuasard-ua8rlue 'pale,rrlce e{ll-lloI aql 'suaSoqled sllar aseql uo srordeoa.r eJE 'surarord-;1asruort po^rrep sepndad ;o aruase.rd oqr uI qrr^\ pePsol selnfalour:)HI I il sselr Pue I ssell af,PJrnsrloql 'srsotdropue re deldsrpdaqt 'suaSoqled;o eruasqeeql ur pue snonurtuor ur aBBBues11acSurluasa.rd-ua8rlue leuorsseloJd

slto) 6urlueserd-ua6lluv+o uolle^!l)v ol speo-lsrolde)eu aI!'l-llol +o lueua6e6ul 'eoe1d rsrr;ogtut sU-II oqr to luaua8eSua ot pol teql lua8e snollJaJul egr rq8g ol perrnbar srrlsrrelrpreqr leuortf,un; eql errnbce daql are11 'etertuaro1lrp sllel qllqA\ ur tueruuorrlueoJrrur enblun I aqt pue IIel JrrrJp e aleaJJasuodsa.rur secnpord lr saur>1o1.,(r 'puodsor sllor rurrpuop -uop E ,o uonelrlre to rporu eq1 rr{r r{Jrrllr\ot sonf,JDf,EtoueqJJqt Pue 'padeldsrpselnlelou ere rns aqt 'pecnpo.rdsaurlolb aql Jo surrel ur slleJ Jrlrrp 'urnt -uap pale^rtce ;o sde.tqled uorlelluereJJlpeql adeqs ur 'asaq1 'pele^nf€ eq III^a ter{t sU'IJ eqr }o uraued aqr sles poratunorue eqoJf,rruaql;o dlrluapl eql 'llel lrlrrpuop PalE^IllE ot{] ro} addfouar{danbtun P seleerr snqt luaue8e8 -ue dq apyord aur4oilo oqr pue suralord ecey parnpur U'II 'UlI pa8e8uar.{rearod 'slleJ lnrJp -rns Jo uorleurquor erp -uap dq saurqotdc Jo tas relnlrtred e 1o uorlcnpord slorluof, 'asuodser U-II perp^rrre qreg Ielrluopr oqt o>lodosrotdoror oIII-lloI petelrlre IIe roN 'saldcoqdrudl o^rpu qtr^\ tlerel -ur deu daql areqm 'sepou qdurfl Sururerp aqr or uaSoqred E Jotunof,ue daqr aragm uror; aler8rru ol sllel oltlrp -uop s,&\o[eSurleu8rsU-II 'slla] I e^reu Jo uoue^rtle IInJ ro; suralord eJetrns aqr 'salnralour .(rolelnrurl luelrodrur -soJ Jo uortcnpo.rd Jo uorlBln8er-dn eql osle lnq saur>1oldc dpo tou opnlJur sesuodsa.reseql 's11acSurlueserd-uo8rtue rog 'asra^rp alrnb ore Suryeu8rsU-IJ or sesuodser11a3 'Surleu8rs U-II ol asuodsorur uo pouJnl osle sl 'slca;;a IEJrArlueqlr^\ surolord l1eurs'suora;letur J ed,{r ;o uorssa.rdxg,'TI-T pue (JNJ) rolret srsorf,eurorunl ro1 saua8eql se IIa./!\se 'uorleururelJurol etnqrJtuoJr{llr{1u\'9-lI pue I-lI Surporuaasoqrapnlrur saua8re8rerasaql '(Sg-Sf arn8rg aas) soue8 loSre] snouel solelrtf,e g)'-JN eror1,l.r

uortelnu-ilad^q )tlerr"tos ut o6e6uapuesad^losr urlnqol6ounuruJ loqlool q)]tMs 'lOtCl-OtC) ssel)ue)seut>|o1rir eieudordde aql pue suoipelalut +o urlo]aq] ul sllal radlaqy61 pole^r])e uot; sleuOrs so^tolol l ]eLlllla) ue saLuo)aq s|1a> I V :EE da151|areuse;d6urlaDes-y161 l tadlaq t C ) u l l o , s u o r l f n r l saul er u d o r d d e a q ] s o ^ t a ) al lla] e) gqyl : ! [ d a 1 5 'uotlelnutad{q pue )r}euros uor}eurquloroj q)}tMs ssel) aier}tut o} po)npur-uorle^r1re) (eseuteueap fuaurq:eutrler-u{zua aq1uo 6urutnl puepele^lpe{;;n16utuLoraq sllo)B ro} alrsrnberetd e '(1gyq1)pueflrl

A90l0NnnNt

I

tz ulrdvH)

0 0 [[

palP^rpe aler]rur ^llualsrsrad are}eq}sllal1 otcf aq]jo uorssardxo :EEldals llo)B eq]lo e)ef nsoql uo poleldspsexoldtxo) )Hl^-oprldad a6e6ua slla)I pale^r])V:![ de15 rlonorql slle)E pa)uar.iadxa-ua6t1ue sllo)llLpuap uaqMsin))ouorle^rl)e slla)1o] ue6rlue luesard llo)-1 'pazrleuraiur pue'passarord st uabtluy :E da15 slla)1o] palueserd arrnbre (slle)B 's)C)slla)ourluasord-uoorlue uaorlue leuorsselord :Edo]S slla)I]ouor]e^r])eluanbasqnspueIquorltsrnbr pepeol-uaor1ue {q ua6r}uv(lq6lU)(slC)slla))r}upuap ;o sueau; 'sarpoqr}ue uorpnpordeql o}e!}!u!o} (.l;a7) slla)I }o uor}e^r})V +o parrnbersllo)g pue I uaa/vUeq uor}eroqello)g€-tz lungll V

?--ll* uollceJolul

Aqpue o?cc--totoc uorlcerolur SHtA-u3I

u 6 n o r u ru o cc u r m r slceralu!lloc I gl

lloc eurseld

Yt H'#

n6l pue solarcas

uortcPrelur SHlA-u3I q 6 n o r qlll o cI q l r m slcerolur llocI E

pele^rlce lloc l_

ecellns uo soxotoLUoc eprldad-39141 11sselc sAeldsrpue ua6rlue sessecoldllac g E

l l a c1 ?OCol ue6rlue sluesoropue sesseco.rdCO E

u a b r r ue s e r r n b c et t d ce E epou qdu.rA; ol senoLu pue ue6rlue

serrnbce 36 tr

oo

( u c 8o l s p u r q ) a d o l r d ;e; e cg

- uebtluy

(Uf,IAqpezru6oce.r 'cHl l o]spurq) edolrde1;ac I ue6rluy

t 0 tt

l s N o d s l u l n [ d v q v ] H 1 N r s ' l ' l l ) l l 3 t - s ^ s - 3 N n r \ i ni o r Noll-vuo8v'l'lo)

'dltuere-r oseql:lerJeleurJrteu led 'snorlreJuruoueJpsollrued e>lrl-snJr^ lttun Jo >lsrJeql esn€f,eqPonultuof,slpse,4 osn slr (suratord a,tr1 -aB slr qSnoqrly snll^orlod pJlenuerle sE,\\ E SP pJsn JuTJJB^ prsdef,snrl^ egt to pesoduol Jo pro^ep lnq '(OCS furrang-atletule3elltceg) eqt sasn€tr palerauaSare salcrtredelrl-snrrl 'JeJuElIErr^Jersosnef,lerll PesnsI seseoslP 'asel rapel addtorasp '9I AdH snrr,reuollrdedueunq eql roC 'serpoqnue leqt unrJelf,eqoodtueql to ulsJls Pal?nuelle ue 'n1; lsure8e asn aqt uJ 'slsolnJJeqnlpue 'sdunru 'selseau Surzrle.rtneu saurJf,E^ eseqt !(91-g arn8tg aas)ururlnlS trf,rle 'uotloalo.rd -3euaq pue eseprunuerneusuretord adola,rueegr yo dlrsour ur eJEseurJr€^ pelpnueltp aAI-I IInJ uleluletu ol parrnberueryoare (suotlcalutJelsoog)suoIlPzluntululPalEecl pasodruocere seurffel nlJ pasn dyuoruruoceql 'snJr^ g srl -er pue 'e8e Suoue.nppqtl.laeuerrrderu s1a,ta1 ,,tpoqrrueaseql -rlBdaq eqt qtr.AA suortroJurSurlua.terdur Intsserrnsuaaq seq 'sarpoqrlue slelel o,ttlcalord IIJIIo uBf,seuIf,f,EA pelenuoll€ of,rnos qreordde st{I 'uorleurrrel rot pasn ueSrtue aql to Jo 'a,rr1eqt qrr.r,re8ua11egre tsure8euorlcato.rd3uuse1 aqr ;o sued lueuodruor a^rl qrns 'ruetsdseunluull a.,r,rtdepe ]ualn.rrl eqt I]E Surttnrca; dq te,La,uo11'11elr surotdrudsou sesner pJoJJeot luerfrJJnssr srql seseJureueJ u1 ',{lruntuul tlfl]o e sesnel ueSoqredeql Jo uolsre^ ro aseosrpaqr uuoJ euo dluo 'snJr,LJo runrJelJeg ol pasn sr sluauodtuof str P]lu to Io 'PoolsrePun SursnuEql Jer{lEu 'eurJ luslnJr^ € Jo surerts pelenuelte a.,rr1 ]le.{\ lou sI tlsll{,4AroJ srs€grel Polenualle eqJ -nrelou eq|'uoq?nuailo ol peel ueuo I]r.4^IetuIuEol IEITTIUE -rE^ lrungns E peller sr eurrrel 1o eddr -roferu.ragtoaqJ 'sueSoqlsdlualnJln 'uorle]qruof ruorJ Jo aJntlnJ enssll uI aSessed1erra5 eJoruJo sluelJel pelenueue a II Jo pasoduor aq deu sautc Sur.reauare orlod roJ ]EOJrelrrurs e e^erqre ol sldruarry 'eurff,€A 'oseesrp -JEA ol E up snorlfeJur PeJI uBr satSelerlsIeJe^oS InJSSeJJns Jo uorteururrlJer{t;o oldruexeqfns tsJIJ 'uorlEur)fPA pasnueaq aqr 'xod1leusaleJrp€rodlarelduocol d11n;ssamns Inlsseflns to sJolEf -rpur poo8 are (ettalreq) srra;;a IPPrlIgorrIIu,{\oqslEI{l ro €louc^ uaSoqreduerunl{ aql a,Leg'xod11eusroJ alqrsuodsa.r (sasnrr,t)uaSoqredeqt ozllerlnau uer leql salpogllue asr€r jo e^rleleJasolf e 'snrr,t xodltof eqt uo paseq souIJJ€A (sesetduetu ut 'afror-lJ eur:)le^ aql 1nq 'are daql se In}sseJfnssE ol .{rrlrqeeLIl Jo 'Q2-y7 an se pesn sr snrr,rorlod pa114tpuarrnJ 'trJaueqaqr paq8ra,u. ere saurrrel qcns .{q,t u.{\ou>lsde.ulelou sI lI -lno snrrlorlod aqr Jo sureJtslualnJrl aroru 'o arueS.rarua-ar -3rg) uaSoqredrer{r Jo uolsra^ }uelnrl^ aql qlll a8uolleqr e tsureSeuottcalord Surpr.tord;o esodrnd el{l Jo} asuodse.r eunrurul ue IIJIIa uer legl lnq snonf,ouul eg ol pau8rsap pueA1>prnb puodsar o1peuudstutals{s {16uor1s eJe seulJfe1 'souIJfEA a.re saldnut.rd 1err3o1 slprreteul o1pesodxe sr{lluanbesqns -ounruurr aunL!Lur enrldepe eql 'ueOoq1ed eql dlqen8ry 1eq1 1o uorterrldde tuel.rodut lsour 6utsnel e +l aseasrp uosradpeleuo)pn luetrltubrs ,LosLuoldr,u{s sueooqled lo ^lalJen e 'ua6oqled 6urruo11o+ o] slledsel e o] arnsodxe ]eql lnoq]tM1nq lerlrur ul relrurrs asuodser ounutulue sofnpurour))enlnlssal)ns ou..ros lsure6v fllunulut; onl])a]oJd ]l)llf soul))en ptdererou.t o} puesotpoqt}ue V sllar_Lrrxoloy{r+ouor}e^r})e +o parunouord of speolsnlnoutesaql orourpueprderarou.r uorlrnpord aresulelordasaql dluey rotdacarCNI-CNI eqrJosraqrueul pue'sMollo+ (sll)) aql sreol)l{llenluana o1ernsodxe-aX uorl)olur -I0tcl 'sllar J uo PUEsllel g uo ulalord0tcJ O^lo^ulsr]el aql splloMse 'serpoqrlue sl1er 1 rrxololb+ouorle^r]le ;o uotlrnpotd -uof sself,_ qJlr^\s eseql 'uolleulqluoJeJ PuPuorletnru.raddq asaql suoralrolut aleuur+oued eresesuodsar aLlf sasuolop I ed{t 'sostl f,rlPruosolEIlIuI 01 slJPluof eJInDeJsllel g 'soul>lol^tr Ilel-lleJ pueslla)()N) lall!)lernieu+ouorle^t])e sapnl)ut 1ouorl:npord 'uaSrtue ol ernsodxe paleadel ro ueSrlue 'esuodsar 1o snorl)o+ur sapr;red leltntlue ol uorlrpPB u1 ,Losraqunuaqi uoqMuaas aruatsrsradaqt sertnba.r(sarpoqnuedrrurl;e-q8q Jo uollJeles aql'uorl)alur ler^ e lo asrno)ou.rll l,€-tz lunDlJ v lel]rur pue uorteroua8agr ro; dresserau)uotlelnur.reddqlrleluos pue arnsodxe-ag ernsodxa;et1tu1 sr pacnpord sar ssel3 'ytr31sde,r,rle saddtosrreqto ol Surqctr.Lrs 'sllJJ g lroruau euloreq ragesAe6 ,o'tcoJur lerrn -poqDue I PUPePISe | Jo e E,^ lsrlt ar{I 'ele nLzL0t89iz0 eruseldolul rlerluarJ#rp urJql to auos las are sraqlo !s11ac -ra;r1orduerJrsllar fl oser{I '(t--II ''3'a) IIer I Pal?^uceeqr .{q pacnpordsaurloldr ro; srotdocarPue selnlalou Lrolelnutlsoc s.{eldsrposle IIar g aql 'llar g oIJr dq pa,(eldsrpsaxalduroc aprrdad-3gry II sselr oql Jo sueeru dq 1ar g peruarredxa -ue8rtue uB totdacar IIar-I sll et,t 'setltluapl iletr I ar{I 'aprrdadpe^Irep-relrr€J PUEelnrelotu ;o alqeder srol IHW II ssEIJJo xaldruoc aql SurzruSoca.r ro; -derar qlr.r,rpeddrnbesller I tql dq papuord 'd1eq11ae-1 sselJsqt '1ec g ilpl eqt seluoraq xaldruoo aprrdad-3gytr [ 'ller g reqt dq peu eql 'o alelrns eql le padeldsrpdpedord il euo lsEellE -rer uef 01 ssEll eql lPql selnfalotu PuIq II :)HW 'utalord raurer eql eq plnoqs a.raqrsapndedasaqrSuotuy Jo srsdloetordleluosoPue,tq sapuded;o las xaldruor e saleraua8 pue tarrrec pue aprrdad pareldroqdsogd uoaruaq xaldruor aqt sazrleurolur'lsaralur;o eprrdadaql uo ells peleldroqdsoqd 'urelord rolrrEr aqt saztuSorarIIer g cr;rcadsdlelerrdorddeuy aqr qtt,Lr e or pele8nluoc'uotlsanbur apudad pareld.roqdsoqd

ADO]ONnWU\t I

tZ U3ldVH)

zotL

'r.{rrEeser qlog eJPlpql souIJrE^.la.ou eq1 'lcedur roleu IetItsolorqIIer f,rseq to uorlB.reueS .dSolounurrurrrseq Bur e o^Er{ol asruo.rd qJreaserlerrSolounuur Jo seare IpraAaS Io EereSurpuedxadlprder E sr srql ;o -puelsrepun rno dlaq IIrl\\ uorlf,unJeunrururtsor{ Jot saqord se sursrue8ro cruaSoqled parlrpout dllecrtaua8 Jo asn eql 'uaSoqled pup (sernlf,nrts qloq auroue8 fsor{ pue socuanb -as aruouaS;o Surpuetsrepunpeseerf,urrno urort lrJaueq IIr^4, 1(ltunurruto,Lrtdepepue oleuur r{toq ur eroJralursuaSoqted 'seurJf,p^pesEglrunqns pue l€oloqled Sursnec tnoqlr^\ tlfrl{,{. ur sdem dueu eql pue ,sosuodsarounurrur err.rldeps esuodseJeunlulur a,rrlcelordp eIo J uEJ rlJrql .er.ralleqro ,(e1dra1ur eqr;o Surpuptsrapun Pu€ oteuul uee,ryuoq sasnJr^ e^rl OJEseurf,Jp^to sruJotuo(utuoJ lsour PslenuaDP TB, ,, 'sourllE^ Opnlf,urs[e] g pup uee,4alag uoDEroqEIIoJ -noderaqtroJ uontunt erdooqdudl uonepdrueru a^TIJOIOS OqI I ;o ot roop eql uado os pue .uorlcun; aldcoqdudl Surpuelsrep 'o ldaf,uor 1ec€olounurur or{t;o suorterqdde luBltodurl r -un ur dlaq 'sllar -I0tcl sauoqts uorlerrJrsselJpalo.rduq .suorlf,un; uo sllor g uo O'cf, pu€ IIr,4^ I trurtsrp r{tr^ uorJo 'slasqns ardcoqdrudl uorl se^lo^ur srql 'uorleurqruof,eJ gJtr,ry\s sselt pue uorlelnru Ieuortrppp Jo -ErrJrtu3pr^{olle ol onurluor sloot crtauo8paurJar,roTr"o, -rodfq rrteuros elerlrur ot arrnbo.rsllel g lf,Eluor IIOJ-llot 's11ao pole^rlfr dg pecnpo.rdseur>1o1,(o ot uortrppp uI r 1

lueuodrur a.re'uaql ol ef,ueJalololnpur ol Jo (slupldsueJl aseql ol sesuodsa.rssorddns ot suEor.urrrau dlluanbasuoJ 'sllar r.uels snoSolorarag 'o asn aqr rrurl III^\ reqr rorleJ P sI SOAIIEAIJOP lleJ-uals patueldsuerrqcns uSraro;se azru80 -f,er ol ,{rrlrqe s.rualsdsaunurruraq1 .suorldo lpra^es}o euo st (tuetled aqr ueql raqro lenpr^rpur uE urory parrrrap,.a.r) slleJ uats snoSoloralaqto uortelupldsuerl .(sarrnlurproc 'dqdo.rrsfp ,aseasrp ..8.a) relnJsnu s.uosur>1re4 luurds pue seseosrp eql lueruleerl ur dderegr rot sllot uels asn ol Io ^^.orl pup sllor ruats Surpuets:epun yo ur sof,ue^pe qlr/N '(surrr{uepue,srso_re1cs aldrllnu,sataqerp ''3'a) sosposrp auntuurolnp teeJl Jo luelerd or setdroqd I ad, r -urdl a,trlcear-y1es SurlorluoJ pup Surleyndruuurroy sar8aterls ^\eu eur;Opdlaq ppoqs SurpuelsrepunsF{I .asresrpaunur (peureturpru -Iulolne Jo losuo arll l3 ualorg dlareullrln puE 'peterauo8 sl sua8rtue-1las ot otuprolot .ry\oq puetsropun ,1caqt lsnu olN ';oo.rd-1oo;sr tueql auou ur sal.{r rnq Jo -oqdrufl e^rtreer-Jlesqrns Surdaa4roJ srusrueq)au Iere^es elpq saleJqelJa^ 'eseesrpauntu[uro]ne ol JoIJEJ Burlnqr.rl ,s.roldacereArlJ€oJ-JIes;o -uoJ JolEr.ueql sr qJlr{,^a uorlereueS eqt :ecrrd E tE seruor dflllq"lren ssellrurl dlreau Jo srotdar -o.rrryneds-ue8rlueatBa.rcol seldloqdrull ;o llrlrqe aq1 'rlnurls crlrcadsdq pa4o,re sasuodsa.r erp Iopou pue aJnsearudlale.rncceol pue ,suorlrpuoc droleroqel pourtap .rapun sad& ruaratlrp ,paur;ep-11e,tr IIer uae^Jeg suorltBretureroldxa o1 6uorl0npsuErl leuSrsJo slrElep dpnls ol qrrr{^4,ur Iapou elrtJerlle ue saldcoqdudl sa4eru IIEJI Srql '3JnllnJ anssrl ur Jor^Er.leqcr;rcads addl-enssrl pue -llel ruro;rad ol anunuof, reqt sllal d.rerurrdse parpnts aq uBtr reqr sadft IIar ,{\aJ erll Suotup a;e sardcoqdurll '>lro,4^ saurf,ful qcns dq,tr pue eurJf,E^ In1ssaJf,nse ;o uorlrsod -tuor oql;o Surpuelsropunpolretoperoru p spuer.uopeletullr d.rotelnSarluerrnl eql'a3pe1.,rtou>1 lecr8olounuur pelretap to eruosqp eql ur pedolelep ere^\ saurJf,el Intssetrns lsoru eql Jo auos q8noqr ua^E .paou taluun srqt o^los ol sldof, -uoo leorSolounrururse Ile,vi se lerr8olorg 11aoelerod.rocur lsnur grrposag 'alqplre^p dltua.r.rncerp sourJrel InJssaJf,ns ou rlrrr{./r\ro; allenuue sqreap suorlFu ,ro; alqrsuodsar Jo qree (s.ra11>1 rofeu yo selduexa ooJr1l aJEprreleru pue ,srso1 -nfroqnt luelsrsar-8nrp (uorloeyur AIH :eJuptrJru8rsIelanos pue lerrlrerd snoruroue;o leo8 e sureruero^rllaJJepue oJES

'\St-VZ ern8rgaas)de.u,qred IHIN II ssep aqt pr^ uorteluesard pue Surssecordue8rtue dg pa,vrol1o;,srs -oldcopua parprporu-U)g er,r ua8rlue Surzrleu.raturdq sexold -uor aprlded-3Hlr'{ ruE^eleraql elBreuaSqlac g asaql .sller fl Jo ereJrnseqr uo saxaldruocaprtded-IHIAI n sseloezruSocar qctq,vl's11ac pote^rpe dq qlar g or peprao.rdsr dlaq rr;rcads I -ua8rluy 'sy1oc euseyd eruoJogor urprSord uouenuarelJrpIInJ rlaq] etnf,exaor sllal J pale^rllp to ofuetsrsseerrnber sllar g r 'asuodsar IIer-I E to uonerlrur .ro; parrnbarsalncalourdrol -Elnr.urlsoJ uorssa.rdxapue saxaldruocaprldad-lHl ;o trJo deldsrprreql sosEarruroslEsller trtupuep to uorle^qf,V .sllef, 'sapou qdudl ot o^oru I qry^ rolunoJue rraqt Jot dpea.r pue droler8rtu euoleq s]lel lrlrrpuep .uone,rrlce uodn r 'Gt-VZ a.rn8rg eos) saurlordr droteuuegur srsaqludseqr epnllur slndlno asoq.ra. l(e,rr ;o -qred Surlpu8rsg>r-g51eql selelqfe sroldaca.raIII-[oI ]o luaura8e8ug 'sesnJrl pue ?uetreg dq pernpord selntelotu -orJer.u.roycr;rcadsdlpeorq eJBsroldareJ eseql .sroldace.r e{ll-llol rraqt ol pere^rlap sleu8rsIo suuer.udq uortezrrtce arrnba.rsllel trtrrpuop sE r.{lns sllac Surlueserd-ua8rluy r osuodsau eunurutl a^rtdepv aql ur slla) uals^S-eunuul lo uolleroqello)

'alqElre^parE sourrJp^ I InJssetf,ns ou dlluarrnc qtrq^ JoJ seseesrpot saldrcur.rdesoql puatxe pue seurrre^ Surlsrxa;o u8lsap eql uo a,rordurror olqlssod eq PFoqs lr 'saleredo tuetsdsaunrurureqr ^^oq ;o Surpuets -ropun etelduol eror.u e q8norql .(SCrylruff ,er.releur) lrlpualqoJd sSnrp ;o uortnqrrlsrp opEtu e^eq suorlrpuof, f,r(uouof,oorros erar{^\.ro(snrr,reloqg) alqelre^eare sarderaql elgeltns JOL{loou qllq.ry\JoJ seseesrptsure8?seurtf,e^SuDnp -ord lr polurperEsuoJJeluorrn] 'sasEesrp elqecrunuuoc 8ur -teJrppreur slool elqEprurrotore seurlf,el palnqrrlsrp dyaprzrr pue pelnpord lldeaqc ,a-nrlrodsrad qlleeq cryqnde ruorC 'ratuef,;o ad& relncrt.red srql slue,tardleql eurJJeAe eldurexa lsrrJ eql ,luaued sE r.{Jnurse sdeq ;o 0g -;ad lg suortelndod alqrrdecsnsur raf,uef, lerrlrer Jo eJuaprJ -uI ogt oJnpal ol pel)edxe sr suetunq ur osn JoJposuef,rlA\ou ourf,Jel AdH aqJ. 'uorJrl ltelur eql Jnurur slcadsardueru ur

€0t t

.

Vl-vo lHr lz lVNV

r{rr.i!\ owos'elord :lfltJtrffil paxrJueqrpu' roirqrqur

'de.vrqtedpolf,Irtser IHIAI I sselJeql PIAuollEl -uaserdpue Sutssaoordue8rtuBut sdelsxls aql aqlrlseq 'ZI

oql ur srnor.{o^u prlos)acuosqe.ro (earncpaqsep)acuasa.rd isuoDf,unJrrel{l are lEqlN (alnlolour roJ patpqnlur;o apdqepleruroJ (o,rrnoperrop)[pa111l] IHW Jo sself,l{oea ssa.rdxasller to sPul>lrBr{/)N'solnrelotu qtr,,\ir paxr; dlererpauur uaqr pup eprrdad'I)E{NIIS oqt sernttnrts aql a.redruo:) 'II ssplr pue ssel] Jo I 11 to IHIAI uorlerluaJuoJqglq e ol pesodxaera^.sllar g :3 qderg lesuodserdpoqrrue oql Jo uoq -Erntptu f,truqle serlJapuntusruer{retulplrtueqlolq leqA. '0I pappe sllac # I sOl r0l isad&osr raqto oqt;o ,{ue ol salPoqrlueyq313ut -cnpo.rdIuo4 I{JII^ s sself,P oSrepunsalPoqllueoP -/\^.oH '6 (poJJnJfo sEr{lualue8uerrearuteqc-drreaq -N a,rrlrnpord p eJuo 11ecg-erd e ut slueur8aseue8ureqc-d,reaq urlnqolSounuurr;o tuarua8uertaerratprn;slua,rerdteqr16'8

orl*

xi5

og o o(cl

pappesllacI #

p o p p Ps l l a cI # s0!

vjl f

l

r N

-N

o

0t3

otlH x5

o o

= a

oQ o

oz+

vc3

(q)

(e)

'uIlunqlP^o qrr-v'palerodoJ]f,olouoqt PUE(eur1paqsep) aurosealordaql Jo rotrqrqurue ro (aur1pryos)sasealordaurotsdcleuososdl1o rotrqrqur uE r{1r.ry\ poleqnJur lsrrt erat\ sllal g :g qde;3 'uortelndod IIel-I leuoll srqr dq paztuSotet rou (eur1paqsep) uretord Iorluor E r.llr.{\.ro (aurl prlos) uttu -nglu^o rltr^\ roqtro era,ry\ sllal fl :y qderg petprodolJole 'sde,vr. snoue^ ur peteerl ueeg peq teqr sllef,I eseql re\e z--lI Jo uorloJf,es sller fl qtr.ry\paxru are,ry\ aqt ^\oqs ,\\oloq sqder8 ar{J 'petplntuus auoraq sller J eql 'selnreloru )HW Jo txoluor oqr ur'I)E{N115 sazruSorar .{1err;neds reql uorrelndod 1ao-1 Ieuoll e qlr.4apoxru are sller fl eseql uorll1 '(suorlBnorqqe rarral-epqs) -UECNIIS str aruanbos or{t qtr^{ aprrded p sr s}fnpord a8e.,r.eo1c 'pa,teap sr urungle^o aq1 'sardcoqdrudlg dreur Jo ouo pue -rrd esnoru 1o ruseldordf,oqt oiur uorlB.rodorlraledq pernp -oJlur eq uEf urrunqlE^o 'efupllro^JnsounruullJoJ peluos -ard are IIar e Jo useldoldc aql ur sue8rlueu3ra.ro1ror.{topue (s33aua4crqr ruorl urolord e) urunqle,ro ^,oq puetsrepunoI

Pleo aql az^leuv tuorle^IlJE ller-g uI po^lo^ -uI sllal 1 redleq pue sllet Sutluaserd-ua8tlueare ^,\oH '9I 'seua8 3gyrq II ssEIJto solellprelnrrr.redqtr/!\ potelf,osse arp soseesrpaunrururolne polerperu 1ec-1 dq;n ureldxE '91 tuo{ sllar e^Ippor-1essluairerdreq16 'y1 isnudqr ogt 3urrr.ee1 I 'demqredpelrlrlsar 3HW II ss€lr erll EIAuollel -uasardpue Surssacordua8rlue ur sdels xrs oqt eqlrrseq '€I

tpeqlrrsuErtate saua8n 's11arg u1 'L pa8ue.rreerdpo reqr sarnsulrusluel{f,atuleqlt lssaco.rdsrql '9 ur serpoqrlup'o elor otlt sI reqlN ;uotleztuosdo sI lerllN iS06I ul uelsds lueualduroo eqr pue solPoq 'S -nup rolorslp or Surrqag uo^ Ilurg Pal oluapl^e leql( '^E^/rqtecl O^rlEuJallEOql ol uorl -e^ore tuauralduroc;o deznqredIerIssEIJaql aredruo3 'V '>peltp palord teql lelqorJltu tuory senssllleurelur 't, sosueJepIBrILuaqJpue lelluerlleur .loleur aqr dJIluePI ',(poq aql lnoq8no.rgr suollrunJ 'Z rreql uroJred daql se satdcolnal to esrnof, eql ole{ 'sesnrr^padole,tua(q) PuB snatnp snccoco1Kqdwg ol urelsds ]o surpJts rruaSoqled (e) :e8erue.nPeulvro rleql eunurlur s(lsoq Jraql alelndrueur Jo uJesIP uec suaSoqled 'I 3urmo11o;eql to LIJea LIrIg,tt ut sde.n eql eglrlseq

slda)uo) aql /v\ol^au g991srsoldrsuen 6SOI (sUft) srordarareIII-lloJ 880I (UlI) roldacar11ec-1 9S0I sllerI 690I uoDPulquofor

f,Il€tuos

9961 saddrost g50I suDlnalrorur I90I uoIlEruIuEItuI €90I suqnqolSountuur 0g0I sller 1 radlaq g9g1 rordacerrg adorrde

9961 g961sueS.ro 6S0I sllar rIlIrPueP proqdudldrepuoces 9L1I lpr l orxototdr 1gg1sue8ro proqdrudl.,(reurrrd 6961 saur4ordc 6961 ruauraldruoc €/0I sllereruseld 69g1uorlezruosdo 9991 f,rcaqt uollrelos Ieuolr I90I souDlouroql 1991spqdorrnau 0g0I slleruN)rellPl Isrnleu t/0I (U)g) rotdeoar11ar-g 9S0I sller g 960I sllel droueru g991 drtunrululolne 9/0I (IHIAI) xeldruoo flrlrgrleduooolsrqro [eru 7391 uonrluasard pue Surssecordua8rlue 6gg1sa8eqdorreru g961 ua8rrue 1gg1 sai.{coqdrudl €/0I uollernftw f,truqle I/0I uorsllerdrurleuorlcunl

sural ^e)

' SZ-StgLl' lounuul'1'uorrnqrrrsrp anssrl,uortBtrtuenb ',,(3o1oqdro7,g 'I 'af,rur sue8ro progdudl yeraqdrradur adfi 1o 'uqo) .V .Z pue ,.W.U .uEururalsIIar Ia^ou E Jo uorl€rrJrtuepl'L002 ,85-LI':II€ 'puwalul' ..{lrunururarr.rldepe ot aleuur 3ur lotqontry strQoa'ttn3 -lelsuErl :sllar lrlrrpueq '9002 'rrurueH.H pup ..W 'U .ueruurats 'oJ Pue ,llojJow rnN'au02o\ .S ,qrnu5 4lps aqi pup otlod :uns aqi 3u4ua1o4'055I auef 'sJJPun€s 'pa yil 'saun)p1't002'urrtsuaro .V .lA, ,.V .S ,urlrold pue 'lS9I-SVgItLn€'paw '[ '13u7.N.rurf,rel 91 adh snrrleruol 'ZO0Z.1e ,'y .1 -1rded uerunq e tlsrnoy ra Jo lerrl prllorluor V ' :rue8 €.VIT-I€llt6Z1'pay1' dxg'1,{lnruaSounruurroJlu?urruralap .le le ,.) ,f1s.lrraleg -rlue auo ueqt erorrr lueruarrnba: eyj-. ;o 696I 'V9Z-SZZt6I f.yunwu1 '9 urlnrlralur pue uoraJralur ad,& q8norgr uorterturreJJrp 1 IIef, Etrrselcl af,npursller rrlrrpurp prordrerusel4.€O0Z'p 1a,.9 ,o8ef

',rn.,*,'r77:drrq' erpadopdrug rrou,rur"u'Jffi Ulli:1'Atott '5S8I-SS8I:9€€ 'paw '['l8uf 'N 'uerpTn{f, ur ?ruourrref,:elnllaroredaq;oe]ueprrureql .1651 .dnorg dpn15 pue u?,.'rrBIul uorteur)rel g srtrledeq l?srr^run eluotedrH pooqpllq) uelt\reJer{I roJ .le la ,.H-W ,3ueq3 '65-Z5:L87'u4V 'r:5 'uets,{seunruurreqt ture 3uo1 aq1 .Z0OZ-[,nea.raqrueg Jo 'JurJreA SCIV ue roy stradsordaql uo sararduorurdoJo uortJalloJ '€t8-f 'PaW amwN't002 'of,uarf,s v 08:6 AIH Jo s.real67 asuodseu aunurull a^rldepv oql ut slla) uals^S-aunuul lo uolleloqello) 's L6-t.L6tSS€ 'paq' rrlnaderaql{Bolorq ['l8ug'N'sa8ualleqr pue,1er]ualod :uorlelnrrrrlsor11er-I.9002 .spqqv .) .V pue ,'H .y ,ad.req5 '857-€tT.tl 'lounLuutl 'na[ 'uuv .uor]elnrunsor IJerJ Jo ualsls 1g7g7q3 '966I 'euolsanlg'V 'f pue .seun1e.r{r .-I ..f .q irnoqcsual I 'LLg-699.991ia3 .saldroqdu,(1 pue g ernleu 1 ou a^eq atrur luerf,rJap-I-DVX.255I .lE ra .A .sua?quol^J 'SZg-ZT.gt6SZatuarcg.sauaBsuerr UJI leuonf,ury dq arnu tuarf,rJap_Z -CVU ul luarudola,rap uorterolsaU.t.661 .V ta ,1 ,re>1urq5 I JJar Jo 'gtZ-ZtZt1Zt ampN.saue8:otdacar llel-I Etaqpue _ eqdle euunu lq .{rnrpads ro raJsue{ .9g6I .V re ..2 .rrqtueq '9VL-TtLtt€€. atnpN 'sat,{roudql + g + tql arnleruuou to uortalap sellolur ef,rur lruaSsuerlJoldef,JJ-ll3J-Iur alu?JJIoI .gg5I 'le ta ,.4 .zrrolarsry

490'toNnwnt

|

L't:tt ff 3.;llf #ilH '99t6rt.tz

?z utldvH)

VOLL

't8r-rLltl9 ila)'alnor rrldropua eqt slf,esrelursolnf,JloruJ ss?ll tou tnq 11ss?lJ '066I '1era ''f '[ ,saftaap IHW Jo de,rqred rrleqludsorq)qL ' 6€-t t':V9€ anPN' IEQ-V111 ua8rtue,{rlpqrredurocorsqII ssEI] uprunq arp Jo arnlJnJlsJ?uorsuerurp-aeryL'%6l'lE la ''H .f ,uzrrorg 'ZlS-9OS:67,t atnpN'ZV-V-IH'uaBrtue dlqrqrleduor -olsrq ssBItruerunq er{l ernlcnrls 'Lg6I 'Ie 1e('f 'ueurlro[g I Jo 4 uorlelueseJd uabrluv pue )HW eql '8t0I-g€0I:65 'I-gVU 'eue8Surrearlceuoneurqruorar //a:) f(A)A ar{I'68dI 'rrorurlleg'C pue'regurueo'V'W ''g 'q ,zleqo5 '€9S-€SS:Z0I 'auf,zua Surrrpay51X e 11a3 1er3ua1od ((q1y) aseurueapaurprldc pef,npur-uortp^rlrearrnbaruorlelnuraddq '0002 'lp ''nl 'nslerueln6,J lJ Pu? uorleurquoJJJqJlllylsssell 'It-tZB:6g 'ruaurdola^rp 11a3 IIer B ut uratord S ?pgruelto elor lerrtuf, V '266I 'l? la ''(I 'elnruetry ' gzttTv:o . s €.at4 o51 aua? ureql ntu uqnqolSounururaqt Jo uoxe aueJqrueueqr to uondusrp pala8rel,,(gesnorutuerJr1ep-llet gV '166I'l? te ,.CI,ernruelry

'0Ls-wsttz

'lounwu.tl'naY'uuv'snJol ureqtrdrreaguqnqopounrururar{l uorl?u l? '9OOZ'V ,.q ,8unf -rqruorerf(A)n re lo lorluof, pue rusru?rlrel4l 'Ztg€-Bzg€tt '!?S'pnv 'l.tpN'ro.r4 'suor8er luplsuoo L VSn pue JIqpu?^ ro; Surpor saue8uqnqolSounrurur;oluaura8uerrear f,rleurosroJ rruepr^g' 9 151' eirl'e?auol'Spue,.1q .rurnzo11 lla)-g pue {iltanrq fpoqrluy }o uot}eleuag luaudole^eq

'L66-I96:BV 'uaq)otg 'nad 'uuv 'suqnqolSounrurur ernl to '6L5T'Ie[Iod '['U pue ''111'1 .lazury -f,nrrs Jeuorsuaurp-aerql 's0t-Ig€:g .tounuul 'na['uuv'uorlruSotar aJEJJnsJIef, Jot sur€tuop-flnuelradns uqngolSounrurul rr{I'9951 delueg 'N'V pue'g 'y (surer1yr,16 uotpunl

pue aJnl)nlls :sutlnqol6ounulurl

(1551 ,.1ounwu.t1 .02€l_6ItI , LItltftl)g7 'loyU ul palur-rdeg'slerurueut dlrunruur snuetat pue dtrunuurt erraql -qdlp tusru€qraruaql '069I 'oreselr) 'S pue ,'i1 ,Surrqaguoa to ' .txa[ .uuv talsoSouolauto11q dosotq enL- L6g:s7' lountuwl 'LO1Z 'uvernllog .[ pue ,.g (errreuel Jo esueroprsoq eql 'L€L-6OL:8I'lounwul 'na[ 'uuv 'sroldacarog pue ,luauraldurol.sar '0002 .g ,ueudag -poqrlu? en sasuodser lpoqrrue Jo uorlelngag ' Ogg-sLg:z' Iounuwl atupN (lrunuurr parlnbre pu? eleuur 3ur>1uqsuralo:d lerrtrrc :sJoldetaJ .) ..S,ErlIV rIII-lloJ 'l002 'oqsre) I pue 'EpeIef rt1so,{r;1

sasuelac lsoH lo ,vtatrue^o

'snrudqroqlJouorrunllerr'olounu.*u:ru'ri-::i'\?,1;r::;""' luaudola^aollo)-I pue ,srolda)aulle)-I ,slla)I

'2.0L-I1L:SVZatnwN.rualsls trauaSollenuas ro rraua8uds? un{lr.&{ srtrSuruaruorroqtrn.,(coqdrudlur llrorxololfl palErpeurllel I orlrl uI Jo uorlf,rrtsed'nL6I'drreqoq .l A pue ,.14i.g ,ya3eu:a4ur7

pattrr;su'r *,,'.p'"-rffi -uJpassa;dxe

eql 1o uorlruSolerIIaJI rrxo1o1,{3-Vg5I .V tr ,'U .V .puosu.rol

'punuwl '/tay'uuv .srolderaror pue ,seprrdad,sCHIrrIpulq sUlI .-I .U ,.C '141,qdlopna ^\oH '9002 'uoslrlN .V .I pue6plerluers 'ILL-I9L:gLila).selnrelou I sselr IHW uo paluasard _ saprrdad;ouorleraue8eqr pu? suralo.rdIIeJlsotu;o uouepe.r8ap eql >lrolq auosealo.rdorll srorrqrcJrl.V66I .le le ..T .) ,{f,oU Jo ' .stuarutredruor 9L9- 699t6V€.arnw N leuososdl ol uodsuerl ro1 xalduror l31og aqr ur salnlelou I ss?ll CHW ruo{ .166I .V la ,.f .4 .srala4 salnrelou II ss€Jtr CHW ;o uorle8a:8a5

se)ueJelau irreJJape>lrpruE srl\ogsg lueuuodxa ur rolrqrq 'polarcas -ur aqt Jo of,uasarder{] sEereq.&\ z--llJo lunou? eql uo lceJJeou pErl 3 lueuuodxo ur Jotrgrqur auospeloJd aql .J Jo eruesqp ro af,uesordor.{rrBql >lurql nod op lqlN tsllal f eleudordde eql dq uo4ruSorar ro1 paluosordsr IIeJ fl aqr;o urseldorr(cagl ur urrunqle^o qrrr1,trdq demqred ,(1a1qrsour eqr sr rpqrN iselnf,alou f,HW II ssep ro JHIN I sselr rltr^{ poxelduoJ peluesoJdoq ol dleTl erour eprlded url'ungle^o er{r sI isrotrgrqur euosealo.rdeqr pue Ieurosos.(l eqr to asn eqt ruou po^rrap sr uonEruroJur rEq/N .q iz-ll SurrorleserE sllef fl eql ro sller I eqr reqr .v d1e41rr sI ipolarres Z--II sl suonrpuor ler{.l.r, rapun

90rr

stNt9 Nt]n80]90Nnnwl lo INSy\39NVUUV3 ) [Uv t ^ o s : ] N o l y { o ) 1 8 s l N 3 9 0 A L

aql Jo Puo ,t aql ParElosrueql eH 'aua3 urerlf, )r eql paurptuoc stuaru8er; VNC palerudas eqr to qllq^\ Suucalap roJ aqord e pecnpord e,vr.e8euol 'VNU* ) arrlue aqr 3ur1aqe1dlelnceorper dg 'plrqdq VNC-VNU e Sururro; puerls sil{l grra aapuqdq ueo lr 'paqrrcsue.rl sr lr qJrr{1!\ruort vNC 0q1 Jo PuErls ouo ol drelueuralduror sr VNU E arurs 'v pallel 'ureqc lq8rl 31 ;o addr .roleur eqt Surpocuo VNUUI aql slleJ rournl Ilal-g aqr urou Pelelosr lsrr; e,tre3 -euo1 'srsdleue srr{l u-ro;red o1 's11ec rruodrgura pue sllel g qloq ur luaurSBrJOIUES oql uo Paprsor saua83 pue n oqr tr aurr.urelapol suorl -leg esaqtezdleueppol ueqr eH VNC 'sazrssnorJ€rr sacerd VNC Jo suou ;o -cer; dueur peq e.tre8auol '.to51 'acr1s r,llEeruo{ vNC Oql POlBIosrPUPsaJrls 'txo51 'ezrs IIEurs olur 1aBaqr lnl eq rreqt uo peseq poleredas ara,u sluaur -8e.r; eqr 'a3.reqcarr,rle8eu IIEre o u€ serrref,VNC IIB ef,urs'ozrspue e8reqc Jo srseqer{l uo seFlolouorq sele:edas qcrq.vr.'srseroqdo.lrcele1eBesore8e dq sluaur8er; VNC esaqr peleredes uaqt all 'sluaur8erye8re1,{ueu otur ue{orq se^4,VNC aql 'snq1 'souroua8uerlpru -rrrer.uur dlarer dlalrleler sJnf,Joleql ecuenbase sazruSocarqorq.r '771ueg erudzua uorlf,rJtsoJoqr qrr^\ persa8rp uaql sE^. VNC rnuoue8 eq1 'dpoqn -ue 1o ed.{r erues eql acnpo.rdqlftl,^aJo 'sllal ror.unl II3 ilel-g Jo aurl P Pesnaq 'srsdleueeqr,tlqdurrs oI 'sllar asnou fl uro{ pue sod.rquraasnoru uro{ VNC cruoue8 Sunelosr dq ue8aq ag 'slueru8e.r; VNC eql tlelep ol uorlezrptrqdq VNC-VNU pup uorlso8rpaurdzua-uorlcrJlsoJ uonpurq Jo -ruof e Sursnpelsrxe seluaraJJrpqlns Jr aasol tno los aq'snq1's[e] g Surlarces -31ruor; VNC eql pu€ silel cruodrqrua ruou VNC aurl-ura8 pe8uerrear -un ueo,ry\laq soluorattrp OlqelJeloPeq (sJnf,f,o plnoqs eJoql tuaua8ue.rree;

'arouraqlrnd 'sllel cruodrgrue uror; sureluof, auroueSogl rpql pelsaSSnss8ur ;r -purJ osarll 'uraDed JEIIIUISE petlgrqxe qlao g Suuoroas-81 ;o uonenuaragtp 3ut -rnp ueddeqlsnu lr'slnJlo saua83 pue sureqJ d,reeq lue.ra;Jlp Jo soouenbes eq1 'uor8er (n) alqerrer eql palural A eqr to lueua8uerrBoJ JI teql pauos -par aH 'saua8 alerBdes o,ryudq papoc eJO,{\snql PuE algsrrE^ d1q3rq aram -ue eql s?aJOqA,\'uorSor suor8er eqr JaqFrlia ore^,\ suor8ar pue IEunuJOl-N A C snql Pue (J) ruelsuol eql peurrol 310^4. Surururralap roJ qceordde roglou? J€purs d.rorra.ra.nsureqJ rq8q ruoreJJlp pasrlop ene8auol 'senbruqral d3o -1orqrelnrolou suor8er leurturet-) eqr rel{r punoJ dlmeu uo 8ur pedolezrap ;o -merp 'pealsq 'alqrseeJdllerruqcal lou deql 'sureqc .{,reaq pue rq8tl 31 lera -les paruanbas uoql slslueqJ ulolord sEru.suor8a.r) puE n Surpocuaseue8 'saua8 31 ol pelo^ap eq euroua8 eql 'xeldruoc erotu aqr;o SurddptuTEIITuIS qrnru oJEseruoue8uprlerrnuelu asneJeg ;o uorilod e3rc1eleql parrnber IIns sls -eqroddq stql 'papaauseuaS;o raqlunu ta,Lezrroll'paddeur aq ot seua8IslJelreg oqt Surcnpar teq^ eruos q8noqlly atuospa^\ollepeq'satrscgrcadst? VNC aAEOIIlEgl saurdzue'sasealcnuopua 'surer{J rq8rl pue dleeq ;o suolteulq -ruoJ luaraJJlp uro{ pallnser dltsra,rlp uoltf,rrlser ;o dra,rocsrpeq1 'etuoue8 dpoqrrue reqr pasodord sragrreeser oql ur slurod ruero;tlp te perelol eruos 'sureqo (1) tq8q IErIluapI o1!u ,(1e41rsour era,vr. saua8 pue oqr ueql A ) 'luaura8ue.rreal lue/\{Jepun saue8 uqn pu€ sureqJ (g) d,r.eeql?f,Iluepr o^{l -qolSounrurur teql pezrleerE^{eBauoI :sureqf,eprrdad,{1odJnoI to pasoduroc Jr sE^{ tI sr alnJoloru 31 ue reqr u.4d,oqs lualulradxfl eql 'tsrr4 dlrsraarpdpoqrrue uolterouo8 Jo oql tnoqe sonlJ etuos peptaord salnr -a1our31 to arnlJnrts eql uo qJTEOSaU 'alqrssodlou sB^{ lapour aua8 'auef,s -o^\l la^ou eql Jo lsor e^IlluuJP P senbru oqt uo eruec soue83ur.zA1eue.ro; -qlal InJre^{od ororu ltlun 5a,re.Lrop1 'suor8a.r luelsuof, 31 papocue saue8 to reqr.unu llerus e dpo lrqr Surrrroqs sarpnls uonezpugdq VNC uro{ arueJ 'uleql Iapou sqr ;o; uoddns rcallpq (epltdeddlod rq8q ro d,reoq e raqtlo e13urse otur potelsuel pue poquJs -uEJl se.ry\uaql qlrq^&\ 'aua8 a18uts e ruroJ ol auoua8 eql ur pe8urueor oJa.4(\ ,{\oqeuos aua83 pue A e reqr persaSSns Deuuaflpue radrq'uorlcalgo slql PIo^e o1 'oplrdoddlod apurs e sapocue aue8 qrpa lpql eydrcuudpeluounroP-ilO1\.aql '1ecr3o1 POIEIoI^rr Pauoas psodord srrp q8noqlly 'ureqc rq8rl qrea pu€ ureql dleeq qceaopof,ue'aua83 auo pue aue8 n euo'seua8 elsrPdoso1\,!leql pesodord 'lN '596I uI Deuuag 'f pue ra,,irq 'seua8 n 1o dno.rB.ra8rel qf,nr.ue pue saue8J 'o roqurnu Ileus €

iZg€tEL VSn

PunorE{rEg 'patereua8 sr dlrsre,ltp dpoqtrur .^aoli Jo ,(ratsdtu eql Surllos roJ uouePunol eqt prel'lstSolorq rrlnroloul e'e,tre8auol nunsns 'sOLGI dlree aqr ur Sutuur8 -aq salPnrsuI 'POautqSrtu lenptrrtput uE sarpoqnuealqrssodeqr IF POPof,ua saue8 (31) uqnqolSounurul IenPI^IPUI r€qr dloIIFn lI opelu uelsds eunlutul eqr dq pecnpord dilenualod selPoq -rluP luoJettrP Jo Jequnu snotuJoua aql tng 'auroue8 oql ur PaPolua eJe.{'\. .ry\oqoruos'suratold IIe aI['sutalord e.trl -ralo.rd esaqr{1rea13 'etulreJll€ uI Parel -unof,ue sesnrr^ PUE elrolf,Eq ruereJllP 1o f,etrc lse^ erp JJo pre^,\ ol PoPeeu 'serpoqrtue palleJ 'surlnqolSounur -ur Surrq8r;-ueSoqred Jo oPnlIlFuI aql oteraue8 plnoc dpoq aqr moq .ro4 paJopuol\ slsrSolounururr'sapecep

'pS 'pQV 'L]€N 2ord 'gL'l 'eMebauol 's pue runzoH 'N

srNtDNlln8ol9oNnl^|nlJo

lNllAlgNVUUVlU )lrVlAOS :lNO ltAO)]8 SlNl9 oMr L

L Nlll

iz

luldxl

)lssv'l)

saua6) puen aqt qloqsureluo) ]Pq] auosplat^ snL{} uotlsaOtp 6uruentalur ]uourOel+ /Ht!e€ eltso6eneal) ou qirM'luerefpe atesaue6 pue aq]]eq]os pa6uelleal stVNC ) n oql 'sllalg u1 aue6I eq16urureluo) auopueaue6I aq16urureluot euo 'sluaurbe;] paztslualalJtpoM] sotnpold(smolJe par)ser-u{zue 's;1at uor])ulsor oq] qllMabe^pol) truofuqr-ua ul .sllnsaJ , t /HLUe€ lenne6euol l.llr^ lualstsuo) st leql sllo) g pue sllal )tuofrqua >r1o urel6erp)tleuaq)S Z Jgngll > ul VNOureqr-1qbr;

.1861 uI ourlrpory dSolorsdqd pue ro; ezrrd 'Iro,t\ srr{l roc IOqoN eqt P3^rarer 0q 'd.rerrocsrp IpluetuepunJ s,emeBeuol Jo uollEpunoJeqr uo slser srusruEqJeru aseql Surpuelsrapun rno tuerrnr to 'ursrueSJoSurpenurdue ot IJEaJol .{JES -soJausarpoqr.luB arrotlada; asla,r Jo -lp eql oteerf, ot uatsds aunuurr oql ,{\ollerpqt suouprJtlprrqlo Jo ,{teuB,re oS.rapunsaua83y 'lueuro8uer.rearf,rleru -os ol uorlrppe uy 'sasodrndraqto trns 01 lI relle pue uollEturolur lEqr eTEl sllef, etuos lerll JeelJ arugJeq lr ,uorl -EtuJoJuI otlauaS etues eql poureluoJ ,{poq eqr ur 11ec f.ra,releqt pa^or1oqse^.\ tr aruo eroql( 'dSolounuur se lla.,r.r. se scrloua8petcedurr sSurpurl asaql

(LU3)NOlt-VU9llA 0t9

A90]oNnwnt

I

tz ull_dvH)

,L

)f,

)n

9 0 tI

,5

vNc iloc-s

)o

)n

,9

VNOcruo^rqurf

'dllectlBruos JnJfo lsnru ruotuaBuEJJE -.,{rqruaeql Jo suorlreq ruan}Jtp ol'llt -er ar1t a,rord pue saua8 uorSar-1 or pazrprrqdq agord VNU r{fuol-llnt leqt euolJ 01 uo lue,r\ e.vr.e8auol'lr JO^\suE aql'3ur1yar arorrrua A'(1 ern8r4)ypq ot sanbruqcel relnJelou 1n;.ra,vrod 1o Ilal-g pue rruodrqure uorJ pe^rrep rualqo.rde sem suourp{ luerqJrp or pazrprrqlq aqo.rd luorudolarrap8ur1re,vr.e dlrsra,,llpdpoqrrue Jo uortereue8 r,{J cr;rcads-3 orlr rpqt puno' eH 'eqo.rd uorssnf,sro poleqElrar.poeqr ro euo poppp uaqr pup spurJls a18ursotur suortr€rt or{lJo r1Jea ul VNCI eql PernleuoP lsrrl 0H 's11ac cruodrgtuepup slleJ g uort pourelqo slueur8er;VNC aql areduroo ot dpeer se,t\ e,v\e8euol-euolE J roJ rr;rcadsouo pue eua83 + A peurquor or{r roJ cr;rceds euo-pueq ur saqo.rd esJllt qrrlil'ureqr v Jr{t Jo uor8ar tuptsuof, eql Surpocuasacuanbasy51q erlr dluo eqord puo pFo.^a lralep reqr -ras e Sulplerd'rl pelaqel pue VNUru >l

'(7 a.rn8rg)ureqr rq8tl >r eJllue aqt Surpocue oouenb -es VNC al3urs e olur pa8uerreer ete suor8arJ pue 4 eqt Surpocuasaua8 eleredes 's11acg Jo uorleruroJ oql 8ur 'ruaql -rnP leql paPnlJuol e,r,ru3auo1 uee^uoq sarl lHrueg roJ elrs aSearalc E lPql puE pJlJouuof lou JJp sJuaS I pup A orp ter{} SurtseSSns'y11qcruo

I6(9t.tLVSn !)S pDV t,teN )ord'9161'e,mebeuolpueturnzoH paidepvl Luol+ srsetoqdotpele S N ;a6 asore6e{q peleredesVNC lla)-g.rotruofuqluapatsaOrp }o suot}teJ}o} 'uor6e.r aql sapo)ua]eql puo ,E eql lo] pue () + 4) aue6) ollue I

leb1o urouog

Jsr

le6 ;o do1

,t

r0

'seqord or1lro] )Ur)ods oql ])tdopse^ln) VNUpalaqel ;o uorlezrpuqlq asaql'slla)g ;o lueurdo;anep 6ulrnppe6uelleatere suteq) lq6r; u 1o suor6el()) luelsuo) pue (A) slqerre^aql 6utpo)ua seue6aql 1eq1Eurnnoqs sllnsar;elueuuadx3 t lunglJ V leb 1o r.uolloE

la6 Jo ooI

(urc)Nolt_vu9l1 0t I

Jsr

I

3

euab;opua,tr--r auo6 olotlM -

eueb;opuo,tr-ir ausb oloqM -

ypq o{rqtu3

vNo ilec I

Ljt I

6 E l L r a ) u e ) u r s a u o gi e ) e l a l e ) p u e s u a o o u t ) r e ) s ' s z

suraloJd 6ul1ou-ror4-qlMorg ut suotlelnlAtrueOorug €'SZ

LZLI

slorluo)al)^)-lla) pue 6urlrqrqul-qlmol+ g o s s o l 6 u r s n e ls u o t l e l n n l , ' S z -

vELl

5 0 tI

I'sz

ra)ue) +olesuo aql pueslle) ror,lnl

z'sz

ralue) +osrse8 )rleuo9eql

I NtlInO Pue slerruaql urEtJefaPnlJurrlf,q.{\'lueuIuorr^ueeql ur Parel 'suo8ounrBo -unoJue sof,uelsqns ol ernsodxa s.eurla;q e 3ur -Jnp Jsrre dluorutuor JeJUEJ suorlelntu ruo{ sllnseJ leql 'y51q pa8eruepSuureda; ur aredrcrlredteql suratold aporue sreqto lsrsoldode ,{q qteap IIer ro (alrfr 11aoaqr q8no.rql uorssarSordpue olur .{r1ua ''a'r) qrrrq 11ecareln8er dlaq reql suratord aporue sessp]f, eerqt eseqtur saueSogl Jo dupry'rJJuEJot pcel pup Iortuor qf.nror8a8eruepteqt suortelnu Surpnlcur-aler peseeJlur u e l e s u o l l e t n l ul e u o l t l p p pa r r n b . r se l l J J' p J t E ^ l t r e uar . r e, { a q : ueq.ra,leuoua8 aqr Jo trry8erur eqt tretord .,(yleurou seua8 re{etere3 rrruer or pelurl uoqo osle arB saua8reIpleJEJ pa1lec saua8 Jo ssEIr 'pazrlerreds arotu 'p.rrqt V 'uors -r^rp IIe) aterrdo;ddBur.^aolleuaql ete^rlrpur lerll suorlplnu os 'qt,r,ro;3ureJtseJfyleu:ou saua8rossa:ddns--roun1'tr op 11r,urrnpord a,rrtreraddqe to uorlJnpord ro uorssa:dxaaue8

peseaJf,urJerllIE 'uoltotuo.rd qutor8 ut a,rtlcedlaltsserxa aq ot aua8 eqt esner lEr{l suollElnru dq sauaEoJuoolul pe8ueqc alouo.rd dllerurou seuaSocuo-o]o.r4 a;e daqt lqr,r,ror8 11ec 'seua8rossarddns-rournlpue seua8oJuo-olord:refueJ les Jo -uo eql ur pelerrldurl ueaq e^pg seuaS;osasse]f,Peorg o,tu uI 'sauoru;oq suonelnry '(1-97 ern8rg) sasnrtl soutlletuospue 'slernueqc Sutlouord-rorunt to setuonlJul dq perueduoc -rE ueuo sr leqt e8euep ortaueSot onPere rolusJ Jo seserIIP ro tsoru ol astr a,tt8reqr uoneln8er relnller Jo sassoleqI 'uorsr^rp IIaJ sseJxoesnPJol uorlf,unJ -letu seleJ qluo;8 IEIUJoUesaqt urelulutu legl surslueqlelu eql ueq,l.rsJnJfo JOJueJ 'ustuB8ro ue Jo aIUIIaJIIeJlluo eqt roJ uele ro spor.rad3uo1ro; IeuollrunJ uIEIueruec (suor -nau 's11ac epsnu ueaq 'seldcoredeq''3'a) sllel olgersqlns 'sassaco.rd SurleaqSurrnp ]darxa ale.ra;r1ord lou oP dlleurou 'sensstllppe dueu uI sllal oI{J 'PaLIS Suraqalo;eq ta.r.a.r,roq 'dpt slee^\ t-Z dluo roy eAI Jns dluoruruoc sllol ul{s pue -dsr su pareldar are sllar poolq orlq.,!\ulelrar lpaoelder ere 's11ec pue arp daqr a.rolaqsdep.r,ra;e tsnl .ro; eatl 'aluelsut .ro; 'd8aterts luelsuof, E se Ie.4^aueJ-enssll lerlaqrrdeIeurlselul dlsnonurluoc sJnfJo uouereyrlo:d 11ar'senssrlllnPe etuos uI 'azrs ur qt.,rror8Jo eter eql Pue 'azrsdpoq rlnpe Surqrear leqt selerqteep pue I{}rIq 11a3'sluu8rsqleap pue'sleu aurrrrralap -3rs Suurqrqur-qulor8 'sleu8ts qulor8 ol asuodser.ur qleep pue qurg IIel uaa.,tueqelueleq aql elelnEer suJlsAs lorluoJ rrtauaS elerrrlul 'a11 rlnpe lnoq8norql pue luaudola.n'ap 'sllel Jo uorterallord pue qulof eql Iorr Ierurou Surrnq -uor .,{11ensn ]Eq] slusluEtlJelueql Jo sernllPJ01 OnPsI reJuEJ :ea.{ qrea reruEr yo orp aldoed 000'00I rlree Jo OSi'\ selels PJlrufI pup 00I uoo^$eq'apr,up1ror1X'rcef,r1tea tI eql uI sqlPap el{l Jo qfJlJ-euolnoqE sesneJ JJJU€-

( ]]ors Maquen puP 'org [uoqluy risapnol) uotsl]xapueuolpa]op 'serpe6 uJeS io Aluoaq1 po})a}apale sose)ltnau000'09L pue sr}uau}ear1 ,{Uea purouelou.i uror+atpaldoad0OO'8tlnoqe leaI q)eaaprMplloM {latlene ul slouln} asaq]:sanssl];o oq uel saselselaLu Ielp,L 'lueubtleul .rtoq]utoll 6utnou.t Mau poaso] ur)soq] ut saltsleut6tro 6utlnpord )tlouPlauurtnolq oresroulnleujouPlansuolsnl)ul 's11al enlsenur eurouelaul aurospue rollnu)rep lleulsqltM{ueu ulsoa aq} 6ut,{|repu61 eredo] eq] 1eunrleqlrdeleutou ,{lant1e1ar 'ut)sueulnLlul rouln}eu.louelat\ pautP}s pue ut1{xoleueq q}titn

U1)NV)

U]IdVH)

uel sessef,ord Surlrqrqur-q1,Lror8 pue Sunouord-qr,u,o.r8qroq Surlca;1esuorletnlu ,uoq llefep ur aulupxe suorltos 3ur,,lto1 -loJ aql 'suorlptnu petrrequr rrteruos uea^\tJq leld.rarur pue oqr pup 's11ac .reruer Jo sf,rtsrrJtJereqo enbrun eqt ot peal leqt saBuEqJorlaueS;o saddr leraua8 eql reprsuoJarrr'lxag 'srsaueSoruo ;o sserord datsrtlnu oqt aqrrtsep pue sller rorrrnt;o serlredordaqt eJnporlur lsrrj e.,\a tardeqc srql ul 'eseesrpaql tsure8euortfales dreuorlnlo,raJo {lel aqr 3urrra11a.r sdegredosle tnq sa^rlueunq 3uo1dlSursearcur Surrcagerued ur 'uoruluol sr JeJuEf,oS 'sseronsa.r.rlcnpord -er ur elor rassole sdeld eroyereqrpup ,uortrnpordar;o a8e aqt JeUp dlruenbar; tsoru sJnJJo ref,uef,'aroruraqtrng 'sno ,se8ueqrrrlaue8 -ra8uep erour dla,rrsserSord aruocaq ueJ pue ral{trnJ o8repun 'tuepunqe erour eruolaq d1>1rrnb alerelrlo;d feql slla3 '1ue,4^ tou op a,Lr'asecstql ur 'qrrg,u sllat Jo JoAeJ ur uorlfa]3s 1ry;a.r,r.od e 'seurta;r1rno Surrnp qluorS perellp JoJpalselpue pazrue8elnru'acuasse ur 'aJEsllel to sroqrunu a8nq 're,ralrroH'uorlelnu a18urse dq pa.raSSrrr a.ra,usrsaua8 -Irolunl parudurot refupf, 1o dcuanb JI eq Plno.,t lI teq,4^qrr.,r,r -eJJ aqr sJel\ol osle suortelnu aldrrlnru Jot tuarueJrnbaroq1 'aJrlur retel do1a,r.ap srafueJ tsoru os ,Jorunle ruJo1ot parrnb -eJere lpqt suortelnruoldrrlnu aql etElnuntce ol perrnbaraq ,,(erus.readduery :arueJ ur aloJ tueuodLur ue sleld arurl 'sesrreJOJuef ad.{ratupseql Jaqleq.lAees pue de.ra.qred ;o etues eqt ;o sluauodtuor ror.{toretle ol alqrssod sr lr eseJ ..3.e) sqt q '(.(earqled Surleu8rs rplnllarelur up ,{e,vrqred p JelnJelou relncrued E epoJua ot ul.\ou>l sr lueuoduroJ Jo eua8paratle agl ueq,^A elqrssod sr srsdleue parerrtsrqdos arour y 'aua3 pateleJ-rof,uer e,trtetnd e Surtetnru spaJJa eql aas ,o ot eseosrpaqr slepourasnotu uorlJnJlsuoJ eql pue .uorl Jo Jo -elnru relnJrrred aqr e^eq orntlnr ur sllet ;o sert.rado.rd terll qr.uorSeqt Io slsel tournl yo eddr e rJlr,l.r. paterJossesr oSuEqJ rrtaua8 aqt qrrq.^aqlr.r,rdcuanber; eqr ;o suosrreduor Ief, -rSoloruaprda :saqceorddealdnynru doldua dllensn suorte8 -rlsalur r{tns 'luole eprsIUeAJIJJJT ue Jo ,lorunl aqt Jot esneJ Surlnqrrluoc e sr aua8peJetleuE Jer{tor{.,r uJeal ol lupuodrur st tt dlluenbesqns'slloJ rorunl ur perolle d11euor1e1nu are teqt saua8oror.uJo euo Surdtrluaprdq sur8aqueuo JaJuEfJo ad^(trBlnrrlred e;o suortepunoJrrlauafi aqt uo rlJrpasag 'slesse^poolq Jo qra,rorSeql Suurnds I.uort sllrr rorunt dols leql srolleJ srsauaSor8ue-rtuE (t) pue (enssrtale.rlauadol posnere leq] seseatord >lJolqeseqllsaudzuacr1d1o llec;eruec 'uorsr,rrp -elord ;o srolrqrqur (7) IIar ror.unlurprtserleqt srol -reJ e^rtprelrlord-rtue (1) arnpo.rd daql II tuelsrsar eror.uore daql 's.rape.nur aqt troddns o1 dlaq aseql erurs ,arntBlnlsB,r .rvr.eu rnorSdppear pue srotreJ qr.rorS acnpo.rd,{agry1elgpreu -ln^ lsoru ore 're,Lr1 pue 's1asso,r. poolq ,euoq se qlns ,{r"llB Japun senssrl 'snora8ueplsour aJE sllel olrselur ,a1rlory '(qr,nor8 pue Iesse^poolq ;o sracnpur)srolre; srsaue8or8ue srolJeJ qlr,rorS ernpord sllel rounl rr{t prletrlrf,pJsr srspl Jr -selew 'enssrtpape^urJr.{tpue eqt qtoq Jo 1larSurzrsetseletu ernleu eql uo Surpuadap'uropue;uou sr sanssrl^\ou Jo uors -e,tul 'sdals dueru qtr.Lr, ssecord xaldruoo E sr srsElsElew 'sJorunlpezrsplsElelu 3ur,ra.or3-tse1,a_lrse,rur ol anp aJe sr1l€apJef,ueJlsolN .srselseleruperuJel ssoco.rdB .s.rorunl d.repuorasurroy degl eJoll.,lrsetrsA\euol a]er8ru rournl d.rerurrd oqt ruo{ slleJ saseJeruos uJ .Jorunl e olur s,uo;8 slleJto JuolJ eql dllenlua^g 'ua8dxo ulelqo ol ornlplnrse,r;o gl.Lror8ar{l olel

U3)NV)

I

SZ USIdVH)

80tI

-nurls pup erleqlrda oql sE r.{lns Iprurou rrlo{ odefsa or dt111qe 'aSelue,rpeuE ruorlt a,tr8teqt sarlrado.rdreqlo a;rnbce osp slloJ eql 'suorlelnrupuonrppe.ro; llrunuoddo ue Surtearf,(slurprts -ar qtmo.r8 leturou sadecseteqt adlt 1ar Surrerellord dlprder arou dle,rrssatSotde selperf, saue8 aldrrptu ut suorletntu Io sarrese tllerrddi erow roJuer Jo lesuo eqt or peal aua8a18urs e ur uorlptnu saop ta,ralroq Xye;eg 'slloo poretl€ Jo euoll E ol asrr Sur,tr8's1lerratq8nep eqt ot oSeuep eqt lrusuert IIrl\\ IIe) srr.ll uorsrlrp '11acorleuroseuo ur dluo srnJJo o8euep crtau Jo -eBeqr ue^g 'seuo8 rredarpue 8ur,{docaql ur srorra dq ro Jr Jo suaSourlrer dq pe.rarleare saua8JaUposrJpsJefueJlsory 'tuatu -uoJr^uaaql puE sf,rlauoS uee,i!\leq leldrelur ue sr'srsauaSuoru -n7 to stsauaSo)uopelp)'ssaco.rd Sururro;-racuetaqr snql 'JaJUef, Osnef,o1 suorlElnrupelrJequr r{lrl\ eurquoJ upJ suorlelnru f,rleruos'drqsraulred alrlfnJls -ep E uI 'eturl eruoslE rnf,ro llr.ry\reruer ier{t dtr1rgpqordaql eseeJfur'aur1ure8 aql ur perJJpf,oJEr{f,rrl.{\'suorlelnru palr -JOquIuIEtJal'1se;1uof, uI 'uorlpJaua8lxau oql ol uo passed l o u J J Es u o u p l n t uJ J etJr l l B t u o p s u u ' s 1 1 :a 8ql ur lou r url-u.raa 's11ac rrleruos dllsotu ur JnJJo suorlptnu Sursnec-Jarue] 'seuoSof,uo alEArlfEJo seue8.rossarddns-.rorunl olelrlJeur leql suortetnu Surlca.rrocruo4 slleJ tua,te.rdsorudzuarreder-y11q Surpooue soue8 eqt se r{fns saua8 ra>leteJeJur suorlelnru uorlJunJ-Jo-ssol snql 'uoneretrlord pue qr.r,ro.r8 11ac8uq1o.rr 'saue8 -uoJ ur duuru ur suorlel IEf,rlrJJere rcql osoql SupnlJur -nru etelntunrtp VNCI ur spuaue>loJqJo (sdp8'sJoJJa.rredar ol drryrqeeqr tsol e^Eqteql silel 'uonero;r1ordIIal trqrqur.{llcarrp rou op saurdzua.rreda-r-ygqq8nogrly 'elelnrunrre daql suorl 'stualsdsrreder -Elnru Jo requrnu e8rel aqr ureldxadeu qrrq.u 'uorlsrpEr lelor^?rlln vNC erour ro auo ItrEI sllef raJupr lsow '6requrary1 pueueqeupHr.!or+ paidepv] sarpadotd VU I LStOOt il;2'9667 C porolle relnlla) osaq]ul ]lnsal]pr1]so6ueq) trleue6eq]outuexo oM reldeqrsrq]ul rn))osebueqr osoq]Joauos{1uouoqMesueslouln} 'elaldLuol snote6uep ssel ad{louaqdle:uet ant})nl}sap oq}o} 1sou.r asuenrO ol 'anssr] urq]tM 6urnnotO louln]stq]utalaquMoqsse lelrlJou 'pala]le aresoryedold lelnll0)lelueulepunj xrs'srsauabouotel 6uun6 ro)ue) esne)leql s;1erur sa6ueq)lo lvro!rue^Ot-SZ lunglJ V lerlualod

srsolooo e Jouorse^f

pue srselseletrJ uorse^uranssll

s;eubrsqlmor6 ur AcuarcUJns-lloS

60tt

u 3 ) N V ) l O L l s N o 3 H r c N V S ' l ' l l )u o n n l

slla)la^tlleulrou'6ututels I unerg[ +oAsouno)] '11eus -]qOll'raOrel uorba.r e 6urpe^ur sllo) rorun] 6ururels->1rep Jo 6urnnoqs (e)urJoLuni oqi +ouor])ase;o qder6ottru.r 1q611 )o seo,re

s l l o cl e u r r o N

v (q) sosseurouJn]oql orela^ll aq] ]o olellns aq] uo suolsnlloJo srlouln] 6un1trlelseleu e q)tqltltul lonll ueulnq o]rqMaqf 6utnnorO aq1 (e) 'anssl}la^!l leurou 6utpenut e 1o{boloqdroulsso.r6 rounl e lo s^ a!^ >ldorso.ngurpue ssorg Z-SZlunglJ V

(q)

sllar aql elrq^\ srounl leuortrppeSurpaospue Surpeards'enssrt dq.reauape^ur ol drllqe rrrgl sr s11errueu8rleruJo rrtsrral -rereqr da4 y '(s11ac poolg orq^\ Jo rorunt e 'enue1nal:rqdr ''3'e) '1eu -oqdull f,ruorr{r are.r leurou eqt tE erp or y1e;pue -rou uegt dlprder erou opr^rp pue ,r,ror8dlensn '(7-97 e-rn8rg) JeJuEf,ro tournt E SursodruoJslleJ (lseJluof,uI lupu8rpur 'euorurorJ guror8 Jo uorlrnpoJd.ra.Losasnerrount ^,(;etrntrd u8rueqe uoq,la. JnJJouef 'eldurexaroy 'taa1pue 'spueq'peaq;o qt^,\or8ra^o aqr tleSetuorf,V 'seuoruroq e{rl soJuelsqnse^rtre dl1ec€olorq to slunoue sseJxaeleJresdaqr ;1 ro suortrunJ ]Erurou qlr.&\sere;rolur>llnq rerqs reqt ;r dluo suelqo.rd 'uoa8rns e rc1 le?rcl IeJrpeu snorreseruooeqsJounl u8ruag dseaue lr se1erupue Jotunt u8rueqe Jo luatxJ eqt seteeurlep dllensn alnsdecsnorqrJy 'aleur8rrodagl eraq.r,r. senssrteqt ol 's11arrount u8ruaq deal .raqlaSor pazrlerol 's11ac lerurou a1r1 sanssrlPIoq reql selnJalou uorsaqpP-lleleql 'sllel Ieurou 'e+1 'alquasor deru dlasop uorlruny srorunl u8ruaq 3ur pue -soduor sllel eqJ Torunl unls u8rueq E (sue,l.t. sr aldruexaue lu8ruaq srorunl qJns IIpr elN 'ezrs]lerusJo pue 'pazrlecolere deqt esneraq lsor{ rreqt ol lsrr eplrl asod lsolu tnq 'slenprl -rpur replo ur dllercadsatruanba.r; tparS qtr,A asrre srorunJ

pe or dsue) pue a^rsenul erv slle) rotunl )rlelseloru 'urerq aql ur addl roleru eqt etnlrtsuoJ 11ac tegt sller 1er13agr Jo srorunl ere spu4olsplqorlSpue 's11at 'sl]J) I€rneu ruo{ pe^uop eq uer srorunt urerq lueu8rlery eurseldpue saldcoqdu,(1;o srotunt prlos ere 'Eruorles tueu8rl -Eru addt .raqloue'sawoqdruKlaql ( ,(1yruireadde or poolq Jo s.luarled e esnpf ueo sllof,f,ruolnel Jo uorleretrlo.rda.r.rsseru ar{l :,,poolg elrq.4^,,ro} >loarc aql tuor} Pe^rrepsr pxuLa4nal aueu aq1) 'sasseruprlos eJe sJournl Jar{to lsoru seeJeq.^a 'poolq aqt ur sllar 'seurof.r€s sselr Ienpr^rpurse ,Lror8 Jo e'sptl,ua4nalaq1 '(ruacrad 06 ueqt a.roru)rounr lueu8rleru '(sros:noa.rd ;o eddr uouIIUoJlsou eql n1 Aqa:c spuouIJJEJ enssrlelrtJeuuoJ pue 'poolq 'alcsnur)ruJaposauruoJt e^rrep

deqt JI soruo)lospue '(erleqrrdeIernau pue uols) LuJePollaro daqr (unrlaqrrdern8) rurapopuese qrns erlaqtrdauor; e.,r.rrap eJBsJolunl :ueu8qeyg ']Ins Pa,laol ]t sgtaourilo, sp poIJISSEII -loJ seLIsroulnl ]o SuItuEuot{l PuE'ut3t.toto enssrlcruo.'(-rqua uelto are sllel rreql ol Surproroe IEluIuE IELuroN PoIJISSEII 'sonssrtSurpuno.rrns uolselur pue qr,r,ror8or pardepe 1an Jo 'sassarSord dlSursearcut'ue8ro leturouqe uE eluoJeq sJolunl ra)upr sV roruru oqt olul slesse^poolq Jo qltor8 rgl rlnPul or Suqeu8rsdq os op uar;o daql dlddns poolq e ulelqo lsnul p ueql oJotuot ltor8 ol os '(pe,trels-ua8dxo) sJorunl'ezts11erus 'leuotuull aq p.'r,r dua lxoQt("q dllectlsrralrerel{J ere sroIUnI -3ord str pup IIel ratuer aqr rnq '11ecSurprlrp dptder '1eru.rou 'lurod Xeru e ot dn yo addr relncrged E alqueser IIer roluef, V 'xtlleu re1 rorunt aql peerdspue le,LreeAoIUol esool 8ur>1ea-rg -nllef,erxa oql ro sllal Sutpunorrns ot luotuqrelle rreql a8ueqr uoIsr IP uar;o deql 'arp ppoqs deqr uaq,LraAIIol anulluol PUE 'leu8rs Sunnpul Ieural IIeJtf,rrlsarteql spu8rsesuesol pr; daql -xa ue arrnbal lou seop teql alEJeJITord ol e.,rr.rp e artnbce s11ec rerue] '(1-g7 a.lnSrgaas)addrouaqdracuecIInJol{l Sutrre;uoc 3ur,vrol ,(lareurllnpue slorluoJ qr.r,ror8 leutrou Jpe^J 01 s11ar -1e 's11ac;osaruadordlplueluepunJlere^esralle stsauaSocuo erlrepun reql sa8ueqccrlaua8oqJ roturu e olut .Lror8q1 dla4ll eroruslleJo>lEur rpql sertradordSurppeeuo qJea'sdalsaldrllnur sellolur dpotutuoo IIer raf,uef,e olur IIar Ieurou E tuory a8ueql eq1 'srsaua8rrotunl1o sserord leraua8 eIJr Pus sl]er IEIlIrou ruo4 rueLltqsrn8urtsrpleqr sllol roturu Jo setuadord eql rePrs -uor e,tr taruBc to srseqctteua8oqr IIEToPul SurutruexaeJoJOfl

ra)ue)+o lasuo oql pue slla) rorunl 'suolslrrtP 'stsauaSocuoor IIal aJoul s/!\olle PeelusJ teqt arudzuaeseJetuolele Jo uollelrlJe el{l ro seuaSralel -arEJJo ssol eql dq 'sursrueqraturteda.rVNC ul u^ oP>leerg E .4Aoqpue suaSoutfJetto elor eqt Jo uolssnfslp P qlrl{ .rardeqoeqr epnlruor olN 'uollsrrJrlord 1ar ssof,xeuI llnsor

.SIIA] JOUJNI OIUI SUITUJOJSUEJ]

EIqEdEtr OJP SIIOJESAqI

JO pue (slleJruols Jo uoupquoreurppu€ uoperaJrlorddq pereld -ar.{llenurtuocare deqt'tno leJ,la.ro arp daqr sV 'eprruptou op 'sller rosrnJJrd.rraqlreqtEr dlensn sllel pelenueragrp.{11n9 lnq 'sller pelerluareJJrpeqf tou ere srorunt eql elertrurleqf sllol aqJ 'ur{s eqt uroJ leql sllar pezrurtero>lpup 'aurtsaturilerus egt aurl terll slloo a,rrld.rosqe's11acpoolq elrq.{\ }sotu pue setdrorr{ldre se qJns sller peleuuerellp Surpr.Lrpuou pasoduol Jo ^,{lureru senssrlur JnJJo uet JeJueJ'ssalaqrauop 'stlnpe ur aJEJaJE slloJ r^reu puu rlrsnru Jo srounl dq.r,r. sr qcrq,n taruec afnpur daqr'(s11ar alrsnu pue suorneu''3'a) slao 3ur tou op d11e;aue8 -pr^rpuouur rnrro suortetntuqJns ueqlfi's1lacduaSorddueu or uo passedsr uorletntueqt t€qt os sllal Surpr,rrpur JnJJolsntu .{aql tacuec efnpur ot suortetnturruaSoluo tsoru Jot repJo uI

s l l o ) 6 u r l e r o l r l o t du l o l e u t 6 u O^ l l e n s n s r e ) u e ) 'drueu8rleu e asou8erp ol posnsI uorl)os enssrlIeruJouosr,lJar{loup ur sllal 3urpe,,r,ur 1o aruesardoql 'eJntrnrtspezrlerradsa1Dr1 tr1a.trrelar pue ,s11ac 'ryoayonu 'or]Br Jrlolnu;o druanbar; posearfur uu lueu[uo;d (sller3ur.u.or8 useldoilr-ot-snolfnu e 'sr g8rq dlprde-r;o tEql sJrlsrretfPrEqreql dllensn s11ac trqrqxe lueuSrleru'enssrlcrlrl -ads e u1 'slleJ rorunt u8ruaqro slleJ lerurou ueql pelertuereJ 'uorteururexerrdocsorcnu ,,{q -JIp IIea ssaldllensn are daql sllor uro;y paqstn8urlsrp rq uruo upr sller rerue3 Ieurou 'srsetsplatu setplntunsdlr,nrtreJoqp pas€eJJur srql pue 's11ac ur uorlJEJluorursodtuTurtJp seloruoJdteql uroloJd e sepofuaqrrq.tr'aue8lo{U oqr sse;dxaro,ro ot punot uoeq a^Eq sller rount 'eruelsur roJ'UI rardeq3) uolala>lsoldr unre aqt oteln8ar reql sasedlD IIEtus raqto pue orlu SurpocuaseuaS;o uorssardxaaql ur sa8uerJJruoJJtlnser UPJsuorleJatleOsaql 'srselselarupuE uorleruJotIIaJ-Jounl Surrnp uolala4soldc eql ur rncco sa8ueqc rrlserp ,sural -ord ere;rns-llar ur sa8ueqc luelrodrur ol uortrppe uJ '(tt-SZ arnSrg)s11ac tueu8rleu dg epeu suratordeteJrns luerJel ro .tAeuello^ur uaUo drueu8rleruerlrepunreql s.relel enssrlto s8urssorcaldnpru aqJ 'enssrtSurdlrapuneql otur lr qSno.rqrro ssorre aterSru pue lrelyrdec e Surury[e] Ier1ar4top -ua ue ol ererlpe ueqt tsntu srorunt ,{\au paas IIr,4^tEr{r sllar 'poolq eqt Suuatua rount pue leur8rroeqt Surdersaot uortrp -pE uI :Ioturu f,rlelsPlaul e ruJoJpue enssrlJar{toue ezruolot ot a^r^rnsrounl "{repuoras dreurrrdaql edersareqr sllal 000,0I ur upqt re,4^oJ 'poolq oqt relue IIr^4.sllal rorunl euros lnq I 'sater8atulsrpeuerqueu tuaruaseqeql sV '(qE-SZ arn8rg) slleJ rolrlnl dq uorte:teuad str Surunollpsnqt ,auerquau tuaurospqeql Surrsa8rpdq srsetselaurseloruord ,sarlrlrlte asealo.rdrerJlor{lr,&\raqlaSol Xtr,rrlceuruseld paseerJul.uru -se1daseatorda.Lrtreeql ot uaSourruselduretord runras aql sue^uor leqt (rol€^ure uaSou[useld) uraford E e]arf,essller rount duBtrJ 'euErqureu aqr 3uo1ealB.r8nu,{eu s11arseseo etuos ur lnq 'ezrselselarupue tr ale.lleuadol eueJqureulueru -eseqeqt eper8aptsntu sllor eql '(qt-SZ arn8rg)uoreya>lsoldc ullre u€ yo ,,tlguessepezlletol aqr dq paurrol ..,unrpodope,t -ur,, ue pallpf uorsnrrord 11ace Sursn xrrleru eql et€rl -euad uer daql 'euerqurau tueuaseq pue xrJteru relnllolpJl -xo eql ot uorlplar xelduroo p a^eq sller rarueJ '(61 .raldeq3) slssse^Poolq Jo slloJIErlogropueeql spunorrns oslp puE sllel

u3)NV)

|

SZUlrdvH)

0tLt

p1 uyd6.iln)'SOOa lOgE:tt.1o7g ' paldepyl suolpun,L ;e1arqrn6euelu.rotl lptluasso leutou 6ur>po1q pue uorsenur eql 1osluauodLuot ]noqlrmssa:ordsrselseloul 1a6te1 ]eqt sOnrpdo;anepo] srro)upr 0urlqbrtro1adoq au6 erpodopenur eq] io uolalo)sleulaluraql urro]]eql sluouelrjolur url)e 1o{lquLasse slolluo)(1 ; reldeq3)dSVM-N erpodopenur {q pesnsuro}old()) ,,,^i ^ i^ ,,-!^ ^. qlPo pue e on uooo o] soseoloro roqlo saseololoollelout xllleul arnpordqrqm ,,'erpodopenur,, poolqpue 6urpuelxe {q l1enn lossan xuleLu Jelnlla)erlxa aql olejlouoo slla)eulour)leJ (q) Lr,leoJlspoolq pue'slleMlasso^ aq],roluo aq]sLuro] ]eq1slla)lerloqlopua 1ote{e; poolqaql 1y (nno|1a{) eq] elerlaued,ieq]slassen saoeqdorteuj riq palor)os oq ue) q)rqM'(lDl) rope, LllmolO seqrnss;euOrs leur.raprde {q pa1rer1le poolq q)eolo} sroqti oq ue) sllar ra)ue) oql slossa^ oq} (f!fl) xuleurlelnllo)erixo 6ursn'euerqutaLu aql ))e]]e ]uauloseq puerournlureuroq]aneolslla)tatue3 alduexaueseslla)eLuout)Je) 'srselseloLu ursdels]slrl(e)'stselsela4€-SZlungll V lseerq6ursn

6C u r l a p o u e ta u e l q u J a W Z - u r u e [ o ] d e u ^ s ' g l r v ' Z - u r L U e u:A u r s ^ l o p e n u're s e r d e s ' d t l l - t I W ' Z d I A ll : s e s e o l o r d u r s u e l ' u r l l x e d ' u r l n c u r n ' s u u 6 o: luuol r s e q p V ' ) V l ' c r s ' d v g o q u 0 6 [ d ' ! ) c N ' e ? c p C: s . r o l d e p e 7 6 u r ; e u b r g tdvl lV'nJ)d u r J e t ' u r l r l o c ' d l M ' x e l d u r oec / Z d r v ' d S V M - N ' u r l J e u o C: u o r 1 e ; n 6 aulr l c y

erpodope^Ut 1o slueuodLuo31c;

urnrpooope^ul

urlcv

dsvM-N4 u!tl}oc y 1 3 3q O n o r q u l orsenul

t43l uo uor1er6114;

(q)

e r p o d o p e n uAr q e u e l u a u e s e qJ o u o r l e p e r

raq!'nlcl uo 6urlerbrr.u lloceurourcjeS

slassa^poolq pue urorl pasnJJrp sebeqdorceu

r..uorJ palarceslg3
lerutErF de,r,rB ur petelnru eruof,oqdlatrur;apur tou tnq por.radp JoJ se^lasuaqlarnpordeJot Arlrqp eqt r^prl dyleruroulprll sllar rorruaSord1(pse1'QV-SZ arn8rg) sllof,or{trru uror; sleu8rsSururBlsnsSur.rrnbarre8uol ou 'q$lor8 JoJluerfrJ -tns-1esoruof,oqsllar tuels'oueuef,sroqloup ul'QV-SZ arn8rg) u,vr.orreqt dlerurou lou sr teqt eqf,ruE ur Surlera;qord pue 3ur -ArAJnsol lsnlpe pue olelnru uEJ sllef ruels JOf,uE)e,rrlcedsord aqr 'l1a,rneu.rarry'(qt-SZ arn8rg) sllet ruots roouef,Sunuoraq as€Jlsro^^ oql ur 'sa,rlesureqlef,nporda.rp.vr sllar uets r{rrq,{\ ur luouuoJrlua ra8rel E eleaJf,uel 'uortelnru dq aldruexe ro1 's11araqcruur se8ueq3 'puedxa pue aleur8rroupl sllat reruer qf,rq.^ur sorrpuarsrnot pue (eV-SZern8rg)arlrruE uo acuapued 'sroqlo upql ororu -ep lleJ-tuetslerurou srrdap 7-97 arn8rg qfrvror8IIaJ r.uolsrorunl Jo IIOf,rorrrnt ol o^rfnpuor aq drru s11ac Surroqq8raueuo5 's8urpunorrns rrer{l ol Surproccea^Eqoq deu s11ar ruetsrorunl'(17;ardeq3) aqrru elq€lmse Surddncco e q rdal aq ueo Jo onur^ dq alersSurtBrlueratJrpuou'8urpr,rp sllrJ uels se lsnf'tuoruuorrlue relnoEredE ur sallosuoql pult daqr raqrag,l uo Surpuadepra^\ols Jo JelsEJmor8 deu s11ac rount 'prrq1 'sauo snora8uep .{11ee.r eqr eq deu sllal er{r to dlrrourur e (puoce5'11erSurlerlrura13urs€ tuo{ paleur8rrodeql 's11ac uroyrun ;o dn aperu lou oJEsJorunl 'lsrrg 'slurod Jr uele luelrodut aarqr lq8rlq8rg sllar ruels rarupr qtr^/\ sllnsar er{I

'slleJ lrlrrolEru eqr tuou JOJJIP leql sllef,Luolse^eq I to sJorunl yo seddrdueu lroq reop rcd lou sI lI 'sllal Journr IFq e^IpeJJeoJolu E eq deru selpoq 8una8.relurqt ddereqr JOJUEJ -nue ro s8n.rpqlr.r,{1eor;rceds sllar esorlt Suna8rel ueqr pue sllal urels rournl Sur,qrruoPrlEr{l lsaSSnssSurpur;oseql 'srotu -nt ruroJ ot Ierluelod snora8uepoql to lsotu ureql qll^\ pelrrel reqt sllol Jo sselcdltrounu e Io uollerltlluopl PO^^o[eosp slleJ Jefuuf, lspeJg ueunq uo suralord oJ€trns 'quro.r8 Jotunl uels 01 sllef, ar{r Jo lsor oqr ueqr ,(ltpqe :etee.r8dlqe.laptsuoceleq sllar eseqlpue'8€LQJ PellEr ro>lrpru3 >Pel sllel Oqlto luol -rad 9 uegr ro^.\ateror{1\\'euroladu aldrrpur uetunq Jo} eppru ueeq e^Eq s8urpur;JEIIIUIS'srolunl poesol olqEun areA\sllaJ rounr €tICJ-uou Jo spuusnoqtdueu seereqa 'eJllu pellrd -ap ur sJorunl ,vreu3ut1re1sut luelod ere llyecrSolounrurur afeJrnseqr uo ttrql pJIIel ua8uueuE t{tr^\ sllJf,Jolunl UIEJq ueunq porpunr{ ma; e 'aldruexo roC 'lou op gJItlA{ pue rolunl ^\eu E paosor dr1lge eql o^pq ilol Jo sesseltqctqm leorra.r'aotur qrrzvrdylensn'stsel uorlBlueldsuerl '(6 .lerdeq3 aas '53y4) Jeuos IIaJ palelllf,E-lueJsaronll e Sursndpnsn 'sra4.reurare; -rns tuorellrp ^rrcJ teqt rournl e tuort sllet Jo sesself,lua -regrp d1.rnd ot sr sllaf,uols raf,uer d;puapr ol .,(emauo 'lenualod alrlerrlder pelIuII eroru qlr^ IIer E PUEIIal ruals rournl reqloue :sralq8nep o,,r,rteonpo.rdol aPIAIPsllel ruals Jorunl 's11ecurals IeruJou o{Il 'sluelulEeJl Jef,uPf,IluP qlr-,n dorlsap ot luetrodrul tsotu oqr pup snore8uep tsoru eqt eJe'esJnor;o tarrey eq1 'qr.rrrorSsnoJefue) anulluoJ ueJ sJer{loalq,lt Surpr,rrpessel IIr,/\\sllar eruosureql Suourepue 'es.ra,trpeJesllar Jo(unl leql sI ldacuoc eql 'Jounl zrteuB 8ut -pees algeder sllef,rounl dluo aqr ore esoqt 'st leql !s11ar to ruels u,ry\oJleql eAEq ol readde Jotunl ;o sed,{roruos lseol 'slloJ ruals 1e 'uorlrppe q IeLuJouruoq esIJPdeu s.rorunl

e og ue) sllo) uals re)ue) uorlelndodA1r,rou!61 'ertue>lnal;osedlr snoIrEAor Prcl uEf sllof rolruaSord rolrueSo.rd'Surlecrldar ur suoltetnruqtrnS's11ar crlarodoteurer{ 'leu8rs 'palertua.ral;rp osle Sutlouord uI rnJJo uer eJotu Ilrls tnq -qluo.r8 oterrdo.rddeutue eleraua8ro srsoldode1ua,r.erd leql seuo s? qcns 'suorlrlntu cruaSocuoduey,q'uoIlEIluaJaJJIP to o8rapun rouuef,dge.reua8pue ,&nedec e,ul sessacord Ipurrou -era;r1ord leurrouqp u€ eAEqsuollplnlu aseqt parmbf,Ea^€q teqr sllal 'sller rof,uet olul uaql Suturro;suer1,(lyenluale 'alelnurnrle uel VNC Jlaqtur suollelntu cruaSocuo'urstuB8ro up Jo otrl eql ro^o dllenutluor epl^Ip uEtrslloJ LuelsesnEJOg ' ^olaq passnJslp'slPz Lua$routnl Oresllel I{rns lsrorunl zrreuoteraua8pue dlqeyyorruorunaPI^IPor &lllqe ogr qlr^ s[o] uleuer,(po aq deur areqt Jounl E urllll.ry\dpepur5 '^\orrsru dq sdervrqrrd ot snoSoleue srserodolBruaq auoq ur 's1acpeap pue pe8eSurceldar'sanssttosaqtuI saddr 1oc eqr 1o dueu ro I]e ol osrJa,rr8asr,le4y senssllJOI{loPue 'euoq 'ur1s te,rr1'aurlsatuleqt uI sllef,luets;o suorlelndod.'GYrc arn8rg) ^\orreur euoq oqt uI sllef,uats (SunuroJ-poolq)crlarodoleruaq ruort paqslualdar dgenulluof, oJP pue sueds atII rroqs '(7-17 e^pq sllar poolq peteuuareulP.{uetu 'eouelsutrog arn8rg)ursrueSroue Jo eJrIorlr ro; anssrtrelnrlued € elrreueS -ar upf, leqr sllet Surterluo.ragrP ol esu orr.r8pue so^lesuaqt etenledredqroq sllal urelsleql PeurealeM'IZ rardeq3 u1

'uorr rosuesueSdxoup dq 'ulnl ul 'pellorluor sr d1r^rr1ce 1-g111 -ereuoSdIV roJ uorleldroqdsoqdanrleprxou€r{l reqler srsdloo -dp or Sururnr dq ue8dxo ,4A,ol ot rdepe or sller rounl dlaq osle deu snqt laseuaSorpdqep elelrel se qrns sarudzuarr/1oo 1-9111 -lp are asaqr Suoury 'qrznor8 rorunl Jo drilqeqord oqr rloJ 'saua8roqto -rE uEf, rllrr{^\ 0t lnoqp pue auaSgggn Jo ^{uBtu eqt;o uortdr.rrsuerlsarnpurpue ol spurqueqt pue ua8dxo,to1 E'( l-llH) Jo suourpuorut pJtE^ltJesrter.{llolJEJuondrrlsuB.rt rotreJ olqrlnpur-erxodlqdq pererparusr erxoddqaq1 leu8rs 'dtlllgearured 'uorrer8lur 'qr.uo.r8 II€.^alasse^pue IIel pue le^r^rns ylec (11u-nr lasse^ poolq) lerleqropua se gJns

U])NV)

I

SZ USIdVH)

zltl

qr.LrorSIOSSa^ poolq ;o slcedse tuoJeJlrpalrln8er 'seseurl aurso.rdlere qJrq,&\'s.roldelel{DAA eq1 'Sgtuur t > [7Od] uerl.vi srnJro leqr ueS,txo JoJ sllol Jo uorte^J€ls 'erxoddq dq parnpur eq upr uorssa:dxaCCAA 'saua8ureto,rd-roldace.r -CCEA oorr{1pue saua843gn e^rt arp ereqt sueLr-n};:r* ;o ssacordeql setplrlrf,Eosle lesso^poolq rueoe[pEuE Jo J]uJ -sard aq1 'rnf,f,o llr.,rasuorletnru In1rurer{Ipuortrppe reqr ,{rr -lrqeqord eqt aseerfursnqr pue ezrsur es€erJurol tr 3urm.o11e ':o[unl Sur,ra.or8 eq] qsrJnou slassel poolq A{eN 'sarlradord rrueSor8ue o^Er{ IIE 'slorunl dueu dq pelenes ar€ r{lrq^{

(^^ouar-]las touueJ) sllac urals JscuecJo Aua6o.r6

lloc orlcrua^rleurollv l l a co q c r N lloc ulals lacueC ri.G} llec urels leLuroN

s;gecrolrue6ord 6urAl ldue-1rsuer1

' ; ; e cr o l r u a 6 o r de ruor; 6ursuesllac urolsracueC

' s n o u r o u o le n- l l o c s r l e M o u e . r - J lpause ' ] u o p u a o a p u l -aqcruaurocoqslloc urals leuJjou urorJosoreleql sllac uralsracueS

uorlecrlder ur lerlualod peuJurer6oJd aurlcap eql urUrqS

.,J

I lltltvzL paldppvlUotleulo] lollnl puea)iell uror+ ila)'9OOZ rournl ol peal ue) lorluot qlrrnot6 ]o ssolstql leMauol -l;es1oApadord aql slla)lolruebotdantb 11et-ue1s ]eq] asue{eur suorlelnyl(a) {ua6otd true6uoutnluou (o) raq+ pue slla)Lrals6urrrnauet-;1es 6urureluo> srounl 6ur1lnsal eq_Llemauar-]las snoulouolne -1;ero6rapun^oql ]pq] qtns luepuedeput-oqltu ' u o r s u e d x rar a q l 6 u r n n o ; 1a eq c r u au.ro)oqo] uaq] alqeuosllol LUols lo)ue) ut suotlel0llp e ol loepe sllac [rJalsleurrou ]uaroJJrp rrleuebrdero )rlauaD(p) Iuobotd truabrloulnluou LUOrI ASOreleql sllac uralsrocueS raL{} pue sll0) ulo}stalue) }o oln}xil.uI aJesJoLUn} ]uellnsoraqf srseiseloLu ,Losols ]e qlnnorO 11a>-uals lalue) ole]tlt)e1 {eu ro sanssl} uotseAul aq} -, le)ol +o srql s;euOrs q]rM ]lnsarplno) Lusrupq)oLu leMauar-]las uueqieprnordo1 (ueat6)lep) slla)aq)tu a^tleulolle riq peurelsns oq ol ulaq] elqeue(aldtnd)sllal uols ro)ue) ul suorleJollv()) sllo)eqltu aql ut suotletalle

>rleuab{q uenupeq rieu s1letoqltu }o uorsuedxe 0ql slla)ul0]sleul.rouLUor]uasue0^eqleq1 (oldrnd)slla)Luolsra)ue) jo uorsuedxe 6urpuodsa-uot e sMolle(uaer6eled)oq)tu llo)-Lralseq] lo uorsuedx3 (q) uorsuedxo uo suot])ulsarluapuedep llo)-LJJals -eq>ru6urluanr-lnlo{q sllar uiois ulo-, leLUrou esue{eu sroLun}qlrqM ur s{enne;qrssod (a) }uosoldo.r pue '(p) '()) '(q)slauedsourl)apsllettolrueoordaq] 1o Iloede> uorleralr;otd 'uotst^tp qltM eq] Ll)ea slla) lla) 'aq)tueq1r-uot1 LualsureLual lou op Ieql os leu6rs 'eOuero'>1urd) e a^ra)or1ouop (mo;1aA s1lettolue6otd eseql (>1utd) alerluora]+lp uaql pue elerellord o] uo ob leql sllo)burrnpotdou.l] ouJesoq] ]e oltqM '(Mor.re pe^Jn)) so^losuloql a>npotdetol (onlq)slle) uralsleLulou6urlqeuasleuOrs eprnotd(ueer6iqbrl)s1ler oq)ru oql 'sanssrlleulrouul slla)eqtru 6ulltoddns pue slla)u.t0]sleurou ueaMlaqsuot])eJalul (e) .slle) uels ro)ue);o sur6r.roolqtssod ?-SZ:lgngll >

la

i.:

(p) 'slla3Lua]s leullou ruorl osore]eql sllac urols racue3 ] o u o r s u e o x oa q ] s l r L U r oaoq c t u aql ]o uorsueoxf llac urals le|',l'lrou

q 5

(c)

(q)

'uotsuedxeraql slr.rtrloqcruaql uo slloc tuels leuuou;o ecuapuedapaq1 l e ! l u e l o du o r l e c r l d aur r o u r l c a pp a u r u l e r 6 o J d

u o l l e ! l u a r e l J r]po a e r 6 a p6 u l s e a l c u ;

(t\ +-

@

@@

ped {;eso1r ,(lrapro slla)tI€ leurroN(e)'slle) €I€ Paurolsuell Pue leulou uoaMlaq pue leuollezlue6roeql ;eene.t se)uara#rp ;er;6o;oqd.rour sqde;bonru uorpola6uruuer5S-SZlungll'MNlhllgldxl V

U])NV)

I

SZU]IdVHf

vttL

g1E Jo uorl€ru porrnbor erp fllpnsn suortetntu oldrtlnur 's11ec -roJsuert lrueSocuo aql dq pelerlsnllr pue rorlreo polou sV

a)ueprnf lo sourl Iera^as[q peyoddn5 sl u o r l ) n p u lr a ) u e ) l o l a p o w l ! q - l l l n n v 'ruaql saua8urvros(leurrueaqt Suru.rnl tsure8e oJo.ry\ SsSnJrlSnO.raSuep JSJr{rtEql pur} ot 3ur1t:els sE,{i lr 'pa.ralocsrplsrrJ se,vr' srqt ueqrN 'crueSoruoag ot perarle pup atuoua8 rsoq eqt ruorl papelrq are^r teqt saueSoruo-olo.rd tuou PeArJeP uouo aJEslErurueur sJorunl esnBJlEqt sesnJr^ dq parrrec saueSocuoeql'spt sp qf,ns 'euaSocuo-olord e 'aua8 Jplnllal Ieurou E tuoJ; sasrre auaSocuo uy 'aua8 -oJuo uE se ol poJJeJaJ sr slerurueur JeJuEJSuronpurJo eJnl -lno ur s11ac Surturotsuerl Jo alqeder uratotd e sapolue teql 'osu,tsv qons 'aua8duy 's11errolruaSord rorunl arll ouo ur Jo uorlplnru rrtetuos p Jo llnsal eqt sE esrJelsnu tr snqt :VNC uetung IEuJJouur lou tnq 'sJeruef,Jor{lopue 'lrlearcued 'rep -pe1g'uo1oo u€runq ur puno1 sr eua8 sot tsoru luplnu V 'sllof,rounl otur 's11ao dreurrrd perntlnr tselqorqrJ Ieurou tou uorlf,oJsuerl rnq 's11ertJE ruroJsup.rtuec aue8 osot eq7r{lr1\^. 'uoseal srqt JoC 'suralo.rdoruo Jeqlo ro urolord sp1 elrl -oe d1e,rrln1r1suof, e aJnpord daqr uaq.r,rdluo s11ac Jor.unlu,4olq -iln1orut ore sller €I€ leuoturureseqJ'(q 19-6 ern Peuro;suel -3rg aas)op touuef,sller lerurou qcrqlr'stuarrtnu qtr^r parlddns pue pernlp dlecrpor.rad1rernllnt ur arurlpelrurlun ue;o; .vr,or3 uef, sllal qrn5 'alcdc IIer eqt ;o srotelnSorere qcrq,vr.'saue8 euo8rapun tSd rc ldv6Ld eql ur suonelnu uorttrunJ-Jo-ssol a^Eqsller parnrlnl'ta,ra,,noq'ernllnc d.reurrd p ur sllaf,e>lr1 tI€ -un 'stselqolquesnou ro (ler 'uerunq erntlnl (qso4).{reur Jo -rrd e ur slleJ esneJot luerJrllnslou sr lpruJou Jo uorlpruJolsueJt utalord Jo uorlplrlf,EalrlnlusuoJ pue uollJnpord aq1 aspu 'uorlrunt 3ur1eu8rsstr olplrtte ot parrnber dylerurou sauoruror.{eql to eluesqp eqt ur uele snelf,nu aqr ot leu8rs Surtouro.rd-ql.rto.r8e Surpuas'olets alrtle eqt ur setplnrunfJe eroJereql pue d1.r,r,o1s dra,L41g punoq sazllorpdq uralord l l a )r o u o pr a ) u e )r a p p e l lqe u r b u o oql uror]ralsueJ] s1leturguasatd s,ienn;e sry56 VNC^q paulJolsue.i] uaoqseqeueOotuo 1oerardpouol)aql leq] lo (pouol){1e1e1du.ro> aq]]r)sllo)r.u.rolsuel] ue)VNCebeqdeq1leq]loqlta6urnnoqs {q panordaq ue)}lnsarpeltedxasrql aua6otuoburLrLtolsuetl aql 11e ro 1oyed ureluo)plnoqsa6eqd6urzrpuq{q eql aqotdn/y ue qltM seztpuqAq ueLUnq sa^ro)ar ebeqdeq1{puo11ebeqdoualteq +eqt VNC olurpouol)uaqlsrlla)asnouJ pal)alsuetl Itepuotase uloll VNC ,

-,,^A^-,

lu+uldql dudooruoo^l]le oq] suteluo)osle]eq] llol e ut on puo o1 peuaddeqlsnflnq'uotleurroJsuel] llo) uo llo,+o ou seqlelll VNC ueunq srVNC snot]tlua^pe aqt sllo)osnoLxol Jalsupr]fuepuo>as ,iq VruCueurnqsnor]rlue^pe uiol] peletedassl auaOotuoeq] pue 'palelosrsr sllo)osnoLupeullolsuel]]o sn)o+lptltutoq] uol] vNc saruanbesn/y palle)setuenbesy56 enrlrgader {qteau aneqsaueb 'uotlpLllolsuell upurnq aq] ua)e1 sr a6eluenpe lsotu ]eql ilel +o ro; e;qrsuodset auaOoluoaq] ouolt oI sllal paLulolsuel] +o 'ouol) io 'sn)o+ e ul.ro+pue {1;eutouqeeptntpo} uotlltule ut llo) auo ]noqe sasne)sllo) Ef€ esnoul+o a.rn]ln)e o] le)ue) loppelqueulnll e uor] VNC +o uor]rppv'eueForuo ose,,oql;o 6uruo;r Jeln)eloul pue uorle)rlttuepr slrruled lle) la)ue) ueu.tnqe uoJl VNC qtl^ sllo) osnour +o uotleulolsue4 g-sz lun9ll'lvlNll lluldxl >

,1OC 3 uOI ebeqdelel4 |

yp6 crtuoue',rrrr"= |

sllacosnou ^", -.,o;.u"rl t 'vNo perlxl I sl l a c por..urolsuerlun 6uoue 6urnnor6slloctlt/HlN pourrolsuerlJosncol

sraa^z rorarnlrnc I

sllac Jo[!nl ueLUnquroi] vNc

auaFo)uo sBJaql Jo uotleltilluap5 :lselpod

sllt

ulf NV) lo r3sNo lHr oNV s'l'll) uownl

.uot]lnpul l S g V t Gi lVD ' L 8 i l ' l e l a u u r sI a o s l ro)ue) ur suorlPlnulaldt]lnu s]la#a a^tlpledoo)aq] alellsuouJop lo ,{;teelts1;nsetaq1 saua6sueliq}oq ute}uo)leql slarlle) oseJ qlrM sraulel:z{u ;o ssott e lo {ua6otd aq} ut se ;1enn se seuaosuetl ]o aslno) aut] all] osetrc ilw.raqlre burfuteta>rutur srsaueOuouinl snnoqsqderb aql saua6suellaq] +o uorssatdxalano aq] a)uall pue relouord Al-nlN aq] sale^tpe lirueuberd qllM palet)osseuotlelnultls (All nl) snlt^ .iotun] leuouroq aqf raloruord)t]Deds-]seorq rleuureur esnou aq] Iq uenupsem saue6suet]aq] lo qlpl .uolDnpul re)ue) ur suollelnu a;dr1nu lo alnleu enrleredool aql smotls sauo6suerl rrua6oluo oml Jo auo Jeqlla 6ug{.r.rere)tu aletua} ul eruerBadde lounl +o s)l}autl eql g-gz lunglj MNllNlu:ldxl V (s^ep)aOV 002

09r

00r

09

0

0

0z; 3 0nl o nqo a o

089

d

001

rorunt;o dcualel 3uo1aqr leqr lsaSSnsosle daql'seueSof,uo aldrrpu srJeJlerrlsr8raudsagt ozrseqdruasluaurrredxoqJnS to '(g-97 ern8rg).racuecot qrunrrns sleruruu pup dlprder a.rour IIE I{f,nluaslJesroulnl'.rns"d pue cdy,qqfoq acnpord sllel dreru 're,ra,roq 'pessorcarB srrue8suetl -ulerrr IIB ler{l qJns zt'sot pue )KLu-r eqr ueql1 's,iep 991 ra,ro dcuercg;aluac.radgg lnoqp glrrA pue d1.,ra,o1s llrts tnq rorlree srorunl sasnef euolp

spuaL't661 I L o L : ;6a u a 9 g uLorllabelo ulqltJe60l lalzur)> pueuralsqa6o4 aq1snstanpel1o;d sra)uapr)ur oq]]eql aloN'uot])npur Ja)ue) lenuue]o ulr.l]t.le6ol oq] qllMlualslsuo) sra6eqlrnn a)uapt)ut aql llr.l-!]lnur lo lapouJ pa)reuaq1'a6elo uotpunl e se soseol)u!sla)ue) urasear)ur ueunq e)uep!)urorll /-sz lunglJ]vlNlntutdx:l v lo (srea^)abv 08 0L 09

09

0n

0t

0z

s'o 5 I

,.

a

a a ^O

a

--E

o o

o

s n 6 eq d o s 3

093 o)

s e e r c u e da urnlcau ulls qceurols , alelsord

00t3

urelord zrnsEd luetnur egf to uorlcnPo:d tlreprurS 'tueu -8r1eur euof,oq uralo.rdrd14 aqr ecnpo.rdraloteqr sl1ac,fueu -tuetu aql to uorloe{ olnurru e dluo dl.reap lecrruu.e1e dpo ur ueql pue 'sdep 00I JauB dluo srounl sasnsJauaSsue.rl t{.tu-t eql ';1aslr dg 'ouoSoJuo uE olul aueSoJuo-olord (uorlduJsueB c[tu-t dn urnl oql Surueluof, leql suollelnru oruaSoruo portnuepl dlsnorlard sf,Irulruc[,ru-t 1o uoIldIJJS -uerl peuetqSreH'anssrllsEOJqur to cKut-t 1o uorssard zL|spt. -xore^o ol Surpeel'srolelnSarJrlrfods-anssrlPUEsle^oleuoIu -roq snouaSopuadq pornPur sr reloruord aql 'snrr^orlar E ruor] raf,uErluo/rotoruord cr;rcads-11acdreurueur e Io Iorr -uoJ Japun esef,r{Jpeur 'aueSoouo-oloJdctan-t aqt rc (osa.t aql raql to uorsrel auo) aueSoruo lueurruop(aldurpxe e[Asot luetnru -ra drrec lrql epetu uoeq olreq ef,Iur Jo{ 'Jatruef, 8uI -snef ur aleJadoof,uec sauaSoruo suorlBurqurorJo dlarre,\ Jo 'oorrucruaSsue.rlruo{ seuoJ uollfnpul JoIrInl ro1 pe.rrnb V -er ere suorlelnru eldrrlnur leql oJuepr^e lf,eJlp erotr i 'a8e qlr.u dllecrrserposeorculpaapul saoProJuEJueurnr{ Jo saddtdueurIo e)ueprfur eqr lnoqs 297 enfug ur plep aql sV 'o8Jerues]laf,Jaruef,snora8ueplsou eql sE elelnrunf,f,Elsn{u tsuorlrlnur Jo .,tslrq,, !-g rsaSSnsselelullse luoJJnJ rlp, uI 'auo lueu8rleru e olur IIer IEruJouE lJeAuoJol poJrnboJoJO.&\ uortplnru ouo dluo;r a3e;o tuapuedaputaq plnoa rafueJ Jo saddl lsotu Jo eouoprJureqr ueqr'eurrlaJrl E Suunp tuelsuoJ dlq8nor sr uorlelnru to eler eqr reqr Surunssy 'JnJJool suoll -e1nur eqr roJ sopelep o{El uer lI asnpraq a1dr1lnu peJrnbar 'puooa5'a Ilf€uI a8e qlrzn eseeJf,urplnoqs afuaplf,ul JOJUEf, y leurared aql ro Ieureleu eql ragtlo t{ll1\^slleJJo pasoduor eq uef, sJounl luaJotlrc 'etuosotuoJql x O IIJEUIatlles eql 3^Er{rorunl s(uEruo^\e ruou sllol Oqr'rlPJ uI 'pole^IllEul x reglo eql Jo euo qtl^\ sllal Jo xltu e;o pasoduroJeq plno^. 'pererr Ir 'rolruaSord al8urs e luory eslre rou plp Jolunl p JI -rtfpur x Jeqlo aql o^eq JapulplueJeqr pue 'pale,rrlluur X auo eleq sller eql JIeq icresour cttaua8 E sI ueIuol[ V 'SaluosouIoJtll o1t\leql X Jo euo SelEAIlf,euI IIa) qJEOOIEuIOJ ue{unr{ E }o eJrl IEreJ eqt Sur.rnp reql IIelaU 'rollueSord al3urs e urorJ po^rrap erp sllaf, erp IIE letll uolllrperd eqr slroddns srournl uurunq IEnpr^IpuI uror1 slleJ ;o stsdleue crleualsl5 'uoruruor uI suolt?Jelye crtaua8 oruos lseol 'tsrtg le urpluof plnoqs rorunl uarrrSe ur sllel aqr 11e 'suorlrrperd alqetsot dlsea olrl so>lerulepotu slrlJ 'JeJuPfrllelselalu Jo >lrerullpqeql (setlsrel{to lp saruoloJrerq8nep qsrlqetsopue poolq oqt olut edersaol ,(uoSordsll lrolle PFol\ sllal aseql 'suollElnlu earql eseqle^eq Jo euo ur uorlelnru IPuorlrpPBuv plno,ry\qrq^4. Jo qcee 'sl1erto ssPtuE luroJ plno^\ duaSord slr pue 'tueruuolritueoJf,IluJorunl aql dq pesodur slulerls -uof oruorralo pup sJOI{loeql mor8lno ol lI Arolle plno^\ Jorunl sn{l unllrl\ IIal E uI uollelntu pJn{r E irounl uSruaq p Ileurs urJo; pue dlqelorluooun erotu mor8 ol sluePuetsep str ^ olle plno^\ lerll uorlelnru puores e o8rapun uaql plno.v\ s11ocduaSord eql Jo euo 'a?etuetrpe quvro.r8rq8rls e lt arrt8 '11arurels e sdeqrad '110ceuo uI uollElnlu plno^/rA. v :sref,upf 'uoll 11eor dldde lou detu ro deu gttq.u'ot.reuef,saql sI areH -elndod a3rc1e uI slpurlue pnpt,rlput Jo uortf,alesaql aTlun lou uorlrelos Ieuoll Jo ssocorde dq asrresref,uef,(,,lsaDgeql 'lapou ((llq-Illntu,, srqr ol 3ur Jo Ip rAJns,,.ro) dreuortnyoira -prorJv 'auo lueu8qru p olul 11ecdpoq IPLurouE lreluor ol

(paururalap 'sllull urelref, urglr.Aa durru oq srorunl plnol ro; srsouSordoq1'ddocsorf,rtupue Sururelsdg pessasse uaeq e^eq sllar IEruJoupue rorunt ;o sarlradord eqr dlleuorrrpe{

U])NV)

I

SZ U]IdVH)

9ttt

'sddlod urro; pue leurrou ueqr raq8rq eter e le olera;r1ordsuonutnru JdV ro1sno8dzoruoqq1a3'a1c.{o[a] aql lC oqr uroq uortrsuerr er{l roJ porrnbor sauaS ;o aseqdS oql ot

'cd7rqyo slla) rounl uaorvuag dueru;o uorssardxasernpur terp rotreJ uoDdt.rosuBDe so)uarelllo allqns lea^au ue) surelled uolssoJdxf+o sts^leuvfelleolrlgl ypq 'suorlSlnru

aldrrlnu e^lo^ur r1f,Eareruer ;o saddr dueu leqt realc 'pau.rea1 sr Suraquuoudluo sr dlye.raue8 retuef, ol ser1dde lr tng 'rJlupl Iepour srr{l qJrr.{^{or aarSap JrlJ Jo apou lrq-rlFru egr aldurexe tuellerxe ue sopr.tordEruourrrpf,uoloJ ;o 'sllar reruer Jo {Jer.ulleqe sr dlrlrqetsur rrlouoC 'srsouaSocuo ol luu^alal are aseglJo .4et e ,(po d1a4l ,fue,rq8noqr '11acqcea ur suorleralle rrteua8 000'II lnoqp a,Leqsddlod Sursrre dllecrperods leql aterrpur selerurlsa luof,eU 'llnPsse luElsuoJ repun aJe trorremoqtsauroue8rng 'srsaua8r.rounlol JerJJeqalrlf,oua ue dlSuruoos 'Sursrrdrns tueeslsrrt lE deur uorssarSordJefupf uolor JoJ papeeusuorl -Etntu requnu oqI 'eddroueqdtueu8rleu eqr rot perrnbar Jo arp qf,rql{ 'srsetsetatupup uorselur Surpnpur 'sa8els :aley 'luaurdololop Jor.unl eqt alotuord suorlelnu Jeqlo sgeJeq,Lr ql,nor8 JaJuof,or reedde suorl ;o a8elsdlrea ue te sa8etue,r.pe -elnu esoql ouros 'r.uroJol raruer ar{r rot papeeuOrp Jo IIal etups eql ur suonetnru eldrrlnru rpqt Surqsrlqersa-(saua8;o dpruel sot aqt;o euo) sr;-y euaSotuo lupuruop oql ur uorl -elnu (uolllury-;o-ure8) Sutle.l,tlreue pue 'aua8 snot.ralsdtu 1ad-seeqt ur pue 'ttd pue )rIV srossarddnsrorunl eqt ur suorl -elnul uorlf,unJ-Jo-ssol-saua8 rno; IIp ur suorlelntu sureluol

dlerauags,ruourf,rpr uolor ueunq r"rrrJJll#,lllrX*o*r,

olEArtJEpue saruanbasVNC rrloads ol purq or dllllqe sll qsl 'uorlprurot ror.untssorddns -loge suorlelnru g9d 1yed11ent-r11 sseleqleuouuec uorldrnsueJt etelrlJp ol olgeun sr lerll urol -ord ggd luetnru e 'eldtuexa roC 'reelf,unsureruoJuorltrunt Sursserddns-Jorunlstr ot lup OIeJlso(u sr uaqt Jo qJrql\ 'oluef,souos pue 'suotlcunJ seq uratord eql Iere^es €Sd 'luouerulod ro 'slcaJep IIer ur Surtlnser IIOI to uorlf,er -.ror 3urmo11e X:e:odruel aq uer tserre arll 'tg ur y51q pa8e -urep r{lr.,t.t. sller slserreteql lorluoJ turodlraqr eqr rot lerlues '(EIl 'S€-02 a.rn8rg)aseqd -sorosuase sr urolord €Sd eqI g Surrelua urort VNC pa8eruep gtrlr\ sller slua,ra-rdqcrqzrr 'lurod4cagc IC ar{t sr ]ortuor alcdc-ylecJo ornleeJ leJrlrrr y 'srsotdode ro lserrp aycdo-11ac olur oB ot e]gnoJt ur slloJ sosneJ €Sd p .(lr.r,rlrv :o1e1n3or leuortdr:rsuerl asuodsar -sseJlsde1 e saporua qfrr{,4^'eua8 lossarddns-.rourn1ggd eqt uI suorlelnur dr.reosrorunl ueunq IIe Jo JIeq tnogv 'sluela rrteue8 Surtngrrluoc Ipuonrppe aq deu oraqt 'a.rnlordaqr slrEd dlurel.recorp arar.{pelsrl ;o lerJnrf, 'EtuourfJeJtueu8rlpru E ..slrq,, JnoJ aqr olrql)N to uorlerrrJoJ luanbesuor aqr pue uorteln8e.rlerurou Jo ssol lenper8 eqr ur sllnsor 'aue8 Egd eql Jo uorle^rtJpurdq pa^.\olloJ,(urrrou>1 led_tou sr aua8 tue^aler eql) uor8er leruosoruorr{f,rplnJrt 'G-SZ arn8rgees)euouepe n8lr-l -wd e 1o ssol leuouetnry e Suturo; 'uorqse; pelloJluofun eror.u uo O ue ur epr^rp dueSord 'eue8 s1r su eql uorlplnr.u SurlB,rrlcB uE orurl srql Jo 'uotlelnru Jarltou€sao8repunddyode ur sllef,erp auo to JI

uorlcnpo.rdaler.rdorddeur01 speal snqt urelord JdV leuorpun; 'aua8 cKu,t-t erp SurpnloursaueSocuo-olord 1o Jo of,uosq?aq1 uorssardxaSune.rrre uo;y (91 Jerdpql) ,(e,vrqreduorpnpsuerl qeu8rsrul( eqr Surluo,ra.rddq os seop urotord 34y eq1 'e1cdc aql srrqq ilol oqt q8norql sller Jo sed& ureuec yo uorsse.rSo.rd -ur lpqt uralord p sapof,uo34y'saua8 rossarddns-:oumllsoru a>1r1(leurrou uorpunJ ol urelord 34y q8noua ssardxaaua8 34y addl-p11,rauo qlr.&\sllof,osnef,eqr.uro;ol sddlod rot uorlel -nu SuneaDcBurue ,fu:ec aua837y orlt Jo sololleqroq puE lsnur 'peJJnJf,ouortelnu pur8r.ro 'aua8 rossarddns-roruntE sl O.Iy aqt qlrq^\ ur ilar eql to sJuolJ a.re,{aqrsnqt fuorle,rufeur Jo ssol slr ur llnsor leql ouo837y eql ur suorlelnu o^{-tro ouo etueseql 'adlrouaqd aturs eql ur llns ureluor ddlod e ur sllef,aqt Jo tso1zrg '5-97 ern8rg -ar deru suorlelnu Jo suorleurquoJ tuorotlrp snql ur panrdep repJo eql ur ueql sa.rrnbcero suorlelnru rolpl aql 'lle^. uolol oqr 11esarrnbcetale,troq tacuec uolor drerreroN Jo oprsu aql uo (squlo.l3 snorecuece.rd) sddlod;o uorteurro; ur 3ur -tlnser 'aua8 (rlor srsodllod snoteruouepp))dV p IEuorDunJ to uoloJ ur dals rsrr; aqt dlqerre,rul ssol so^lo^ursrseuaSourcleJ '(6-97 orn8rg) lapou lrg-Dlnr.uagr roJ lroddns Suo.rls Surprrro.rd'rapro paur;ap-lla^\ e ur rnf,Jo dluoruruoc teql suorlPlnru serJosE ruo{ sosrJEJof,uBf,uolol lBql ,{\oqs sor Jo -pnts snorournN 'porlrruepreq ol saSrls Prr3010qd.roueqr Jo r{Jeeur JnfJo lpql suorlplntu Surzrtolle'uoa8rns e dq parelosr oq upf,-seurourJreJ pue 'seurouapeu8ruaq 'sddlod-seSets olerpourelur eseql'se8rls pcrSoloqdrou pazrrelf,EJpr{lJle{\ 'trurlsrp 'raourJ so uoloJ sr uorldecxe q8norqr Io O qcrqm uV 'peurrurerapdpseaeq louueJ uorssarSo.rd Jo e8els lcexa slr 'eulrl ouo te dluo poAJOSqo sr Jorunl orll eJurs1nq 'sreruer uetunq dueru ;o seldures a.rnd dlrle; urelqo uec suoe3.rn5 'uorlJnPur rof,uEr to Iepou lrq-rllnur eql roJ 0l3P ol OJuaP -r,ra SurlladuoJ tsoru oql epr,ro.rdroJuer uolor uo serpnrs

sJo)ue) uolo) ur po)e{ e8 ue) suorlelnru)rue6o)uo e^rssa))ns 'aua8rossa.rddns-rournl E Jo ssol qlr^\ eueSocuo ue Sururqruoo .(q suorleruJoJsuerlIIaJ paJntlnl Jo sol€J JOf,uEJof,uer{uool algrssodosle sr lr 'seuaSoruo;o suorteurquoJ are pouorluau saydurexaaqt alrqlN 'uorlpturoJ -suerl o8ropun sllaf aqr pue pezrlerlnou are sasuodsoJJelnl -1aca,trle8auasoql '11eroruesaqt ur passerdxaare sauaSocuo 01V\laql ueq^L'nunsauas PernJ€lsr rlJrq^,\'apFlp ra3uo1 ou ueJ feql ereqrn atels e otur oB or sller sefnpur euole 'srsoldode ol slselqoJq qspt peleLDJe1o uorssardxaJelopue -rt ezrtrsuasoslE lnq uortBreJrlordof,npur auole c[,tu-t 10 s1e -,raypareln8araq 'sller oqt uJorsupJl ol oleredoor saua8 o.vr1 eql laqleSol palletsueJt uaq,!\ spaJer{,v\(uorleurroysue.rl cruaSocuoJot tuerJrJ;nstou sr cs?l pelplnf,B to t[u.t-t nrqra qrrzrr(srselqorqlJ€I€ porurotsueJtdlerlred loz) srseyqorqr; 'elduexa JoC 'slleJ peJnllnJ ur uees Ierurou Jo uorlJeJsue.rt eq uEf, souaSocuoueo,ttlJq stJeJJOo,rrturadoocrelrrurs 'suorletnru Jrteluos leuortrppe e.rrnbceol paau eql ol onp sr 'ef,rurcrua8sue.rl-ayqnop eqr ur uale 'uorleurro;

LIIL

ulfNv) lo rlsNo 3Hr oNV s'l'll) uoy!nr

,{eldraluraql Suncagarsa8ueqrJo apelspJe o8rapun rorunt B 'slue^e 'rofuEJ uolof, Jo sllor agl fa^e^,\oq lErtruraseql Jsuv 's11ecrournt otur sllar Jo esef,oqt ur sE lpr.urouJo uorletu -JoJSuEJlasnel lpql suorlplnur 'o sorJasJo uorlelnru Surlerl -rur ay8ursaqr d;rruaprueJ sorpnlscrlaua8'ueese,r.auvr sy

'ddereqr pue srsouSord lnoqe suors -rfap alBJnf,f,epue lrySuruEeurlE aATJJE ol pue stsaueStroru -nl PuPlsrePunol qroq elqErlsaPerP sllal 10 satuadord eql uJaJSrpol sde.,lrJolleq pup 'lualuoc uorleuJoJul perluJrlspq euolp sllor;o aouereeddeagt Je^e.ry\og'/Soyorstgrlaqt IuoU

lossaA poolE Jnccool srselselau, 6urmo;;e'slassp ao nolq ope^ursllecJournf

aArseAul

seEueqcraqlg

uoloclo lleM

uolocJo uourn'l

raqlo ol speelds) soztselselatx recuecaql

+ I

I

sdolo^ap Pr.lrourcJec 1ueu6r;euy

I I

I (/! aLlosoLuorqc) ouoo rosselddns-.rour nl ggd 1o sso'1

t

s M o r O( u 6 r u o q ) Pr.r.rouape lll sselcv

I

't66t'ralzur) pue [ [0]:6tauagspuilf ) I urals;a6o1 r!ol]paldepvl (8|, ourosor.uorqc) le)ue)oneqol ptes I 336;o uor6e.r stlot!nl e qlns qltMluotlede :salts luelstp ur aueblossarddns pueslossa^ poolqaql uloJ+ ]e aleJa]rloJo ltxa -rouJn]lo sso'l ol slla)rourn]aq]lrurJao suotlelnuJ leuot]tppv '^poq aq] ur sa]rs raqlo oi uoq] olnqulsrp I poolqolurpealossll0)roLunl llr^^]eql slassa^ I aurosonssr] aq]spunoJJns (zl, omosourorqc) leq] aue.lqLxaLx eua6ocuo aqlape^ur,{ue6otd aqlpue'ept^tp ]uouraseq seJ-)llo uorle^rlcv o] sonurluo) Jot!n]e ll0)slr..llJossaloons sesanros {lrurtrn ur6urqleuuos 1eq1 }nq la)ue) o1padsarqlran eua6lueuoduraqi pe] ul sraua6reln:ryed srqlleq]leall]ousr11eueb ut polalap))fOoq]sapnl)ul 0a)ue)lepa.rolo) g e [rJoJ] sasrJe oLuosotrjoJq) leLl]uorlalop l uo aua6rossarddns-toutnl eq]]o ssol aql suorlelnu rnol11e 6urt{tte: lueuOrleuL 11et ar^,-' '^A --A ^"^ pue c6 d+erdudu-[su uo 8l aulosoruolq) aua6perliueprun ue-seua6tossetddns-toutnl oM]]o ssolpueuralo.rd seuanlppIlenrlnlrlsuot aueO (g aurosourorqc) e uorssardxe 016urpeol suotlelnul lo losserddns-.touln] d,{;odto 's11at tor-un1 u6rueq luanbesqn5 ,dy lo sso'l pazrlelol e 6urulol 'lou op slla) +osseuJ qbnoqle'apr^rp 6urpunorns o] lla)aq]sesnpl a;6urs e ureua6rosselddns-routnl ;qarlerlaqlrda \ fdv or.]]uruorlplnLu V'stseqrrlaue6s1r pue Ja)ue)leparolo) ueurnqlo stselselaul pue lueudole^ap oql 6-5ZlgnglJ >

sMorb(u6rueq) eurouopell sselcv

t

sanor6.rounl snoracuecoJo ' u b r u a qy

uolocaql lle/'l^ uo stlJol (ql^ orb l l e u s )d l l o d y

stecuorocreurroN

]aqapezrlv I sos:Eotarn]pN'0002'le v v urorll(saldules g pa}et}uara#tp {er6)s1lar aloulol leltLl.ls srraqloaLll1(seldLues e6uero) sraluofleurutro6 ursail:oqduIl g peierluata;4rpun Ilanrle;at o1 relrurssrdnor6aug (Z pue t eeq ))plq)sdnorO onnlolutlle](sa;dues anlq)rep)euloqdu{;;;arg ebrelasn#tpq}rMs}uarled uuol;saldues aql leql snnoqs (u:er6erp ao.rt) Luer6orpuap 6ur11nsar aq1 suralted pednor6olaMosleuer6erpaq11o uorsserdxa JelrLurs ralll o16urpro:>e 'slla) po}e^r}re do1aq16uo1e seldLrLes eq1 {;enrpadser I lla)]uoraJJrp puesllo)I ura^rpeerelq6uaql uo sleqanlqpuepetaql,iq pole)tpul sauag(do11eleq anlq)rep)slla)euoqdu,i;1;atg a6te;esngrp to (do11ereq)urd)slla)poinlln)pauuolsupll seq)ns'sllo)6ur1eta1;ord ura^r1re erelq6uaql uo leq uaet6eq]Iq paletrpur saua6'alduexa lol uorlezrpuqlq ltaq]o16urptottepadnot6 1osutapedleltLuts erann saua6aq1 alrsoddo aql sale)tpu! ueet6asualur :aua6]eql lolo) patasualuluV saldues ;o uorldursuerl;o lanalq6rqe sale)rput eyboqdu{ snoue^aq}ur}sotuoq}stegrpuorssatdxa asoqMsauo papolase {|uouJor1 aq] 'sauab aq] elepeql sapnl)ul ]as OOO'8|, ]o uuetOetp lalsnl)aq1'euaOe16urs e lo; elppsuteluo)Mol leluozilot.l q)eopue'({a1alduesaas)a;duesa/:oqduIl te;ntryede to; elppaql sureluo)utlnlo) le)tya^qlea'r-uet6erp lalsnl)stqlu;('srs{leue {erreor>rr.u lo} 0€-Epue6Z-5satn6r3 ea5)sa{roqdu,{; 1ouorldrr>sap pue seldeues 1ueu6r;eu leLulou 96 aql +oq:eaurseue6 1o ;elueuLrradxa aql poul.llalapVNUpaper]xa 000'8[ lnoqe]o sla^aluor]dutsuet] aql1osrsrileue {ereor:rujy56 euloqdulAl 1osad^]aalq]q]tMquarled tuol]paurelqo sa/roqduIl lueubrleuuol] pueuotietluare#tp ]o so6els papellxaaraMVNUrillo luora#rpie sayi>oqduli; leullouLiror+ salduLe5'seuoqduf; re;rurrs A;;errdfilouaqd estmreqlouaamlaq qsrn6upsgp uer srs{leuefelreo.olu VNO^q peutura}epsuJaped uogssardxe auebur so)uora#tq Ot-SZlUngll lVINl4luldxl >

slla3I poolq 6urlsau I saurl llac paulolsueJl .:i. sllac I pale^rlce/6u!lsauI sllac I poolq pale^llcv slrsuo]/sapouLlou^l leuJoN rx sllac I Jaluacleurura9 lI

euoqdu^l

crlselqoqdu^l euoqdu^l

euoqdu^l

seilcoqdull leuroN

ur)Nvf

I sz ulfdvH)

8ttt

sad^l llo3

o

oluorqC gW i?lnclllol

*fdb

llac g a6rel asnlfro I

seytcoqdull lueuDrlelA

'raruer -rrd eqt seuroqdudy lsrsouSordraueq e eler1uorlprtueret;lp1o sa8ets Jo sluala Surlertrur aqt ;o saddr auros JoJ lsarlreeaql ur sotdcoqduldlfl reqt ot rplrurs sr uorssa.rdxe lsEal lE 'leqt dtrlrqrssodeql saslEroslE SIL{I 'rllslsEtaru 8ur Jo -tuoJag p aue8 asoq,r\ seuroqdudl 'ad.,{rreqro aql qlr.{\ esoql uEql Jo drrlrgeqord .ralBer8 a^eq leql srorunl d.rBrurrd ro8uol qJnu pe^r^rns ElEp derreororur oqr dq pour;ap addl lJrruapr ot elqrssod eq .{eu lI IEI{I SurlseSSns's.roulnl crl -elselotu;o yecrddreroru srrtsrrelrereqr e^Eq ol punot uoaq Jorunl euo qlr^\ sluerlpd 'sJournl 1o saddr orrrr aql qsrn8 -uIlsIP plnol lEqr PunoJ ora^\ Prrolrrt olqrsr^ .ro 1ecr3010qd ser{ srounl d.rerur.rdprlos ]o lesqns e l(ySurlsaralurorotrAl 'uorsserdxo -roru oN 'ht-SZ arn8rg) uorssardxo eue8 su.raped.rror{l yo aua8 uJOltEd aqr dq sJorunl f,rlelseleru uoJt ;o ,{q paqstnSurlslp sdnor8 oe\r sluarled polea^ar algeqsrn8urrslp aq ueUo d.rBrurrd uec sJournl tlSursr.rd lueretJrp 'sosposrp 'serlredord -rns Iuory seruoqdudl srs.{1eue derreo.rcr141 aldrrlnru rounl uorlEurruexo polrslep aror.u loN ;o Jo 'lce; ut '09 ol (seluof, pelcadsns ueoq Suoy seq osposrp eqt os 3ul,vro11u sr ,(Solouqcat {€rreorJrur VNCI Jo rue^pp egl 'sapou -lno elqprre^ d1{8lq eler1 stuertrd porco;;y qdudl ('anbruqcarsryt to uoueueldxa ur lnoq8norgl sardoogdrudl g a8rel dllerurouqe ;o acuasard ro; gt-S pue 6Z-S sern8rgae5)'1ara1rrleue8 relnf,elou aqt lE paur;apag or sed,bouaqdxalduroc Suulnurad 'dlsnoauellnru -rs seuo8 spuesnoql suol uorssardxeeqt eurr.urelep to Jo ;o 'uorsso.rdxeeua8 uec srs,(1eue derreorolru VNC Jo suJenpd 'sgac yo arue.reedde (slloJ oql tuorJ pallelap eq uer deql aqr tuort pazruSocareq tou deru saoue:aJlrpaseql q8nogryy 'suorlelnu ro uorlelnru Surlerlrur erueseql uorl asr.redeql 'tuara;;rp alrnb auoreq upl slleJ rorunt 'tlnsar e Jr uo^a sV 'aprstno uro.r; sleu8rs pue slua^o Surlerlrur aql uee^{leq

:ii:il:T ;:fi'::," ;;T::lTfl#l1 HH,:ilJ 1'l; H

'srsslsPlaurJoJ ^JES -seJeuoJe tpql suorlelnr.uJaqtJnt sarJese 8ur-rrnbce.rou to -nt dreurr.rdor{l urqlr.r sllel Jo tesqns erer e Jo of,uo8rarue r q r J o o r r e u e r s e r 1 t r u o r ; p e q s r n 8 u r l s r pe q u p l s r s J q l o d -dq tl,{I 'srselsrleur pJEA{olasJnof,E las dEuJ.rournl d.reru

6ttl

u l ) N V ) l o s t s v Sf t r l N l 9 1 H ]

tV'uorletnu uotl?unJ-Jo-urp8B so^lolur dleraua8 euaSocuo up olur auaSoluo-olo.rde 1o tuorlelrlle ro 6uorsreluo3 'sJolJet uorldrrcsuBrletuos pue 'sutelord (1e,tr.r.rns-1yer) crlotdode-rlue'srotdeloJJror.{tpue selnrelotuyeu8rsSurlouord-qunor8 opoJuasauaSoouo-otord .raqrg'leu8rs Surlourord-qrmor8 pallorluolun ro olrsserxo ue saptrrordulelord papoJueesoq,{\'eueSocuoue sr spl ruoJJ pe^rrop aue8 osa.t tuelnru aqt luralord uorlJnpsuerl-leu8rs relnllaJerlur uE seporueteqt eueSof,uo-olorde sr dlsnorlard pessnrsrpeua3 sp,t eql 6olduexa roC 'rarueJ ro; lueuodrur sernleeJreqto Jo uorlera;r10.rd11acelouord strnpord eddr -PIL{\ osoq./$.(sauoSocuo-olord('a'r) saua8 re1n11ar lErurou ruorJ pe^rrep eJE,,lao1 e rnq IIE 'soueSoluo u,ry\ou{dueru aqr 'sleturueur JOJuefeJnpurol .ro 'suorlelafle JO IIOJJoqlo qlr,r{ uortpurqruoJ ur dllensn 'orntlnr ur sllet ruroJsuprl or alqp urato.rdE sepof,uoteqr aua8 due sr auaSoruo uE ter{r IIEtaU

'suJellEduorsserd -xe rror{l ur serlrrElrrurs uo pesEqluerudola,rap;o sa8etsurel -rer tE slleJ Isturou;o sed.{rcr;roadsol POleloJaq ol reedde sllal rorunl aruos 'EIJalIrJ IEuoIlIpBJr dq alqeqsrn8urlsrp -uI uorsse.rdxeaua8 sllel rotunl Jo soddl uee.ryUoq aJE fPqf dJrruaPruEJ srs.{lpuPde.r.reorcturvNC I ur sof,uaraJJrP ' ( 6 - 9 7a n -3rg aas)sauaSof,uo-olo:dpue soue8rossarddns-rourntcutc -adsur suorlelnru qlra polplf,osspeJEdluoruruooleql sa8els lerrSoloqdrou llultslp q8norqr sdolarrapref,uet uolo3 r 'oJllu ouoSJossaJddns-Jo{u Jor.un] uo suollElnlu ur uorleruJoJ -ru sauaSsuerl cruaSocuo PuP Jo rloue e,rrl?radooceql puE 'a8e Surcue,rpeqlrl\ sJOf,uEJ uelunq Jo eJuoprf,ureql uI esEeJJ -ur pe^rasqo or{t loturu uelrS B ruo{ slleJ Jo &IouoSotuoq rrlaua8 eql qllry\luelslsuof,sr toluec esnet ol PopaeueJesuoll -e1nu aldrrlnur lpr{l sesodordqrrq.tr '1apou llq-nFru er{I r

soue6o)uOolur saua6())uo-oloJd 'slleJ rorunl qlrrn sarlradordutelrac oJPI{ssllel PaurroJ uaAuo) suorlelnlNuotl)unl-lo-urpg -su€4 r{rns '(9-97 an?rg aas) uolt€IuroJsuurl o8roPun 'saua8 dllelueudoye,rap ref,uEf uee/\.\teq uorl tuetrodrur pue -EIaJaqr Jeprsuof,pue sJJJupf,reJnrrtredJoJ 4su er{l oseeJ3ur suorlelntu pelrJaquratuosdq.u ureldxa osle olN'JaJuEJesneJ uer sasnJrl urpuol .t\oq JJs pue rru:3oruo oJE ler{l suorl (uorlces sql uI -etnu Jo soddr lerauo8 aql Jeprsuof e,ryr '(6-97 ern8rg aas)uorlras snor,ta.rd aqr ur peqrrtsapserlradord11ao-rounr Jo unrtcads IInt eql ol eslr orrtSol suorl€lnu rossarddns-rount qlr^a ourqruof sauaSoouo'lorunt e or Surpeal sluele yo quLor8 'urof, erres erlt seJEJ o,l.l.t are ;ardeq3 Jo sorrosagl uI 97 Jo ur pessnrsrpapdc 11ac aql Surllorluor roJ sassaf,o.rd alecrrtur orft pue dcueu8rleursnql 'sur8r.roruareJJrpa^Er1luqt rorunt ueunq Jo spur{ IeJO^esur punot sr urelord uorlrnpsuerl -leu8rs polplrlJe Jaqto Jo sEU elrlre dlarrrlnlllsuof,E (esr^\ -a41 'epdr IIel aqr q8norqr sllal or{r a^rrp reql suralord ro; Surpor seuaSur suorlelnruSurlelrlce ro (eltdJ gar aql;o a8ers IC eql q8norqr uorssarSordlf,rrtserdlleurrou leql suralord eroru ro euo Jot Surpor seua8eqt ur suorlelntu Surle,rrlreur oAEr{sraf,uertsoru 'aldruexeroC 'pa8euep uoaq spq VNC ;r ro dllcerrorur parrnrro seq dals snor,ra.rde ;r alcdc 11eo eqr q8norqr ssarSords(lleJ e dots ol slurodlcaqr aycde-11ar snoIJEAlP lle dlletuJoustrnpord esoq,4seuegur suorlelntu Surlerrlreur e^Eq srounl uerunq 11edllenr.rrn '(II-SZ arn -3rg) uorleralrlo.rdpue qulor8 IIar Iorruol dlag teqr surer -ord;o spurl , ueu eporue seua8eseql 'uortrnpur reruer ur solor ,(a1 deld-saua8 releta:et pue '(34y ''3'e) seue8 rosserddns-rournl'(sa.t''3'e) sauaSocuo-olord-seue8 'palou oleq a^\ sV Jo sesselJpeoJq eaJqr ur suonetnlu

VNC llol-rounl qlt,Lrpalf,eJsuErtsllel pornllnf, uIElraJ r 'ssElueSrpl B (sIS poolq .,ltouto uolleluJot aql ol ,tor8 ol JapJour 's1assa,r -auaSor8uearrnbar srorunl drepuoras pue dreluud qrofl I 'sad& 11arpeleltuaroJ -Jrp erntPtu arotu ol u€ql sllel rosrnlard Surpr,rrpol aluPlg -ruesor eroru rEOq sllof, JarueJ 'slleJ Sullsra;r1ord uro.r; esrJeueuo pup s[e] tuols IUoU asrre deur slleJ JaJUE) r '(g-97arn8r4) xrrtEru rElnl]arErxa Surpunorrnseqr aperSapqotq,tt'sasuel -ord atarces uelto srount Jllelsetery 'stsplsopLu PeIIe) ssarord e 'ql.tror8 to seerp drepuoras qsllgersa or dpoq aql q8norqr Surpeerdspue senssltJo seuePunoqeqr euIJ -ep ler{r seuErqruou luauesEq eql qSnorqr 8ur>1eerquel -;o 'sanssrlSutpunorrns epelul selulleluos sllef JaJUP) I 'vNC aurl-uJreaaql ur porrrBJlou oJepue sllof,f,rtetuosuI Jnf,f,osuolletnlu f,IueSofuolsory I '(srsordode)qreep IIar uerSord dlyeurou teqt sleu8rsot luelslser ere pue sllor Ieurou Jo uorlereyrlordroJ perrnber srolf,et Suuouord-q1rn'or8eql ;o etuos tseol le Jo oluasge eqr ur dldrrlnu ueJ sllal raJueJ I

rorunrrueu'rleruol osrraar' uec^p.q ,q, l[t:t(Ji] ;'ll lso14 dSolorq ilol relnrelotu Jo slf,adsedueru uo Sulqrnol 5or^eqeq relnllel uI uollerreqe lelueuepunJ E sI relue3 r

re)ueJ lo lesuo aql pue slla) rounl

ro)ue) +osrseg)rlouag eql 'IBIIUOlOd

rrl€lseleru ro a,rrlecrlderpalrurl qlr. a sllel Jo ssew e Suoue slleJ Luels JoLUntsnoro8uep Jo uorlefrJrluapr ,r,ro11e deu seJuarotlrposaql'sortsrralf,erpr{Juorsserdxeeua8 a,rrlrurl -srpqlr.^ perJrruapreq u€J sller slesqns'srorunl ulqrllA. I Jo

|'sllor Jorunt ;o sarl.redordeqt olur stqSrsureprrrord ol oslP PUE 'slueruleorl lnoqe suolsllop peuJoJur 3ur.tro1le'srsou8rtp PuE uorleflltsselo arrordur or d1e4t1eJE srotunl Jel{lo to 'suJO11Ed uotssa.rdxaaua8 eql 1o sasdleue ..tse.rnleu3rs,,lo relrurs 'srsouSord esro,t\ e a,req sat.{roqdurdl g ParEIr -uJrJtJrperou Jo teqt ol .lrsolf,sr uotssardxoeua8 asoq.tt

ul)NV)

|

sz utldvH)

jzll

urel0Jd or{1Jo alrs agl le adorsorf,[u rr{3rl eq] ur elqrsr^ sr lEr{l poporuo orlt Jo uorlf,npordre,toot Surpeal'tsrxa sardol sno (USH) uor8e.rSururBlsdlsnoauaSoruoq € ol speel eseJror.u -rournu lBr{1os 'eueSocuo-otorde SurpnlcurlueurSosy5q -roJ eql'sernttnrls e>lrl-eruosoruonlJ-rurru tuapuadapur'11eurs e 1o (uonecqdar ygq lerurouqe ''e'r.)uotlonJtldtuy 'V sElsrxo detu lr;o'oruosoruoJqJ e uo alrsa18ursr lr pezrue8ro dpuepuer ag drru VNC prrerrldnp aql :sr.uroJo&u Jo reqllo eue8eqt ;o uorsserdxoelEudorddpur sesnEJlpr{l a>1el drru.,{leuoue sF{I 'puH oruosJo nayap (ra>lerarec) rredar :alourord lueraurp E oql repun aua8drolrp8er lorluor Jo -VNCI p dldrur suorsel r{tns os 'lorluoc lurodlceqc ol 3ur,vr.o '€. -qr.rror8e s8urrq rcql uotlwolsuptx lpLuosowotq1 3ur1o.bdors ppo.r,r. er{rro parreda.req pFo./r^,tuele uE r{rns ]lal dleu;o11 'sJotunl ur ueesa8ueqocrlaue8uoruruoJe sr (soseq alrlnlrlsuof sr uaryo 'suralord lua.redaqr leql a4rlun ',br,rr1cr Jo -olH to sporpunqSuruuedsuorSa.re dlyensn)uor8eruo,rr8e 1o asoq^\ uralord crraurrqce Surpocuaaua8prrqdq e acnpo.rdol sardoc691 se .{uBu se acnpord ol VNCI }o uortecr;rldrnepezr reglaSol saua8o.ut sesnJler{l uorlwolsupll lpluosowo.tq) 'Z -lerol aql 'pacnpord llletu.rou Jrp laqr aregm sllel ur ro tou slo^ollerurou-ueqr-req8rqtp uorlf,npord or enp sr lcege cruo8 rcnpo.rduretord eurrlredla,trl -oJuo rroql fsuralo.rd -nlllsuof, aqt qlra ere slcnpord ro orrrtcBred.{r{ E ur sllnsoJ eueSocuo-olord leql leurou leJrtuopr uralord esoql/r seueSocuoateraua8srusruer.{Jauo.lA.tJaqlo aql e ur (.rredasega13urse ur a8ueqc ''a'rl uor1o1.nw luxod 'I 'tselluoc u1 'euoSoruo-otordSurpuodsar.roc aqi dq pepoJuo uralord aqt uro{ sJeJJrp :sauaSocuo-olordSurpuodser ue sepof, terJt .,ureloldof,uo,, IEuJou -ue susrueqraru o,e\-tlsrrJ er{t raqrralq pJuroJ auaSocuouy -JoJ aql uro.r;sauaSocuoarnpord uef srusrueqfeu Jnot lseol Jo urerlsajol uor])unJL srql leql (A)suralordlorluoreP,b-;le1,fuooa1er pup'(lll)surotord lo)ue)e)nputue)oslp(e1) urare(nl)s.rolre; uorldr.osuerl uorpnpsuerl-;eu6rs stoldatat;eubrs alenllte '(ll)srolda)ar '(1) 'lle poporuo-snrn sutelord 6ur;eu0rs relnlla)erlxa elouotd1eq1 suraloldo>uo 1eq1 ]ou le^rruns 11a> leu6rs solnrolour puesrsoldode q}Mor6 eloulotd1eq1 enrlre{;lueuruopo} asuanr6{11eraua6 stossetddns loun] opnl)ut(lln) 1nq[ue61seua6o:uo surelord rrloldody sassell laqlo utln))o alouord{1leLurou surelord uorssardxa ro aJni)nJls suotleinLU lo sllo) aq} 1eq1 leql 11e> lltM 1o rournlauo)aqllrMslle)luelnuloql ieqi,ilrlrqeqotd 6ur6ueqr suorlelnlA suralord asaqlJosu.jro+ aql 6utseal)ul luelnu 1ouorssaldxa {;1eer6 [lenrssa>et pue qlrvror6;ler 6u1;1o.r1uor saua6Jossalddns uror]]lnsarue) ra)ue) 'uorlera;1;o,rd 1teseuabesaqiut suot]eln1/\ roLunl{q papo)uoare(ln)suto}old pueuorleraylord tredet-y516 ur aledrrlpedleql suralo.rd;osadfl uo^as !l-SZ lungll V 11et

lsulegordpe1e1tuoqdsoqd) puocos sreOuesseur

-{ (l) alnoelour6u;1eu61g

A

1eu6;s lo srole^!lce papocuo-snrln

IZL L

u l ) N V ) l o s t s v S) t r l N l 9 t H t _

sasnJr^r{Jns 'euaSoluo-oloJd ,ls-f, IErrrJouar{l Jo afueseJd 'aueSocuo aql uI uOAauoIlELuJotSuEJl IIaf eJnpul UEJqcrq.ry\ Sunce dllueuruop E olur lr polreluoJ uaqt aue8 pecnpsuerl aql ur uortetnu luenbasqn5 'euoua8 rroql olur auaSoruo 'SurcnpsueJl ro 'Surle.rod.rocur -olord Jelnllal IEruJou e dq uasrrr e^pq 01 rg8noqr ere sesnJrl Sururro;suertdlatnre rer.ltopue n5g '(:;s-c) ..c,, xgard aql .,tqaue81e.rr,r eqr uort peqsrn8unsrp sr dluouruoc 'oueSoouo-olo.rde 'aua8 .reyn1 'eua8,ls-^ -lar ASU oqr ot perelardlasoyrsr terll Ieturou sq1 eue8 e ureluof, sanods Jerpo pue sua>lf,rr{rurort slleJ Iprurou reql purJ o1 pasr.rdrnsora,tr slsrluens 's9151 otel oqr uI 'uorlfnPur JafuPJJoJ parrnba.rsem 'saue8IErrArer.{loaql lou 'eue8 Lrs-,reqr dpo lPql PelBrlsuoulopASU Io sturoJluBlntu qlr.nrsarPnlsluanb -asqns 'auo8 a.ls-aeql hSU Jo esef,aql ur :auaSoruoue urel -uoJ ASU e{rT sesnJrl Sururro;suerl f,ruoSoJuo'sosn.rrlo.rlol IIe ur tuesardsaueS,,1erulou,,eql ot uoBrppe ul'(6V-V arn8rg aas)auoua8 ilel-rsoq egl olur pale.rodrooursr gcrqm 'y51q olul pequf,sueJl-esJolalsr auroua8 VNU osoq,&\snJrloJleJ E sl (ASU) snrrl er.uof,ressnog leqt pa^\oqs slsrSolorgreln -ralotu rateysreaddue1,g'purrue lsoq olgelrnse otur pelralur uaql,r JeruEJasnef,plnof, snrrl e leqt uorlruSof,oJIerlrur eql ol pel II5I ur Suruur8aqsnoX uolda4 dq sorpnlsSurreeuor4

sauo60)uo-olordrelnlta) alenrl)v ro seueEo)uoureluo) sesnJrn6ulsnel-raruel

llueururop dlecrlaue8 erp sauaSof,uool sauaSofuo-oloJduol -uoo lEgl suorlElnu uorlf,unt-Jo-ure8aql'asrre deql Ja e^{oH 'qr.nror8rorunl ot elnqrJluof, 1 q1 d1a{I ssal arp passo.rdxa dHS1qtou orr leql saua8palJIId -rue ef,urs'seuaSoouourrauSureq JoJ salePlpusf,JeDeqeJeA e^rJ osoql 'passardxedt.{81qosle oJe.4 a,tg i(po rnq 'Pal} -rldure aq ol punol a.ron saue8dryrg 'sderreorf,Iluuo PalPnrs osle eral^ saua8 asoqt Jo sle^al uorsserdxa aqt pue 'saua8 peryrldure roJ poueerrs sBrlt 'suor8ar etuosoruorqc per;r1d -rue ur\ou{ JnoJqlr./\.'auq 11acPtuouIf,JEJ }seaJqV 'uotssa.rd -xe eua8 SurrnsBaurosp dq peltuuaPl eq upf, seuo luelelar (soue8 par;rldureeqr Suoruy eqr roJ seleprpu€rtseSuorlsagl 'slods lorluoc ueql syeu8rsra8uo.rlsa,rr8y51q per;rldrueqtrm stods pue 'y51q cruouo8 ;o stuaur8er; Sururetuoc s.{e.Ire aqord ol pesn sr sllof,JeJuef,ulory VNC rltuouoC 'saouenbes VNC ruepunqe dlpurouqr ro;8ur4ool o^lo^ul slueturradxe asoql (S rardeq3 uI pup areq dysnorrrardpeqrrcsapsde.r.reorc 'uotssa.rdxaeua8 le >lool ueql roqreg -rur Jo uorlrf,rldde eql 'seruosoruorqc suor8er pol;rldure Surpur; ro; qceordde ;o pye,rod e le1Josde:reorlrtu VNC :rournl agl pasnec aue8 qrrq.u Sururruralop uI dals tsrry e 'par;rldure ors saua8qllqrt eururotop or lplrurp eg uef, t1 'saue8dueu SulutBluoouot8 -oJ eruosoruoJt{JE Jo 'eurolselqornau ur par;rldruu eJEqf,Iq./\l. 'LXee roqq8reusll pue aue?c(ul-y1eql se qcns 'saua8 Jo Jegrrnu llpurs e o^lolul deur uotlBcurldure euag

auollVNCa6te;e ql|muotleztpttq{q aq16urure}uor :z{u.r-p eql 'sauosol.lolq)aql nlrsur{q (par)po)reuistttedy eulosoulolq) {;luatsaton;; a.resauol> ol pazrprrq{q urVNCparnteuap VNOpaloqel nlrsut luelsolonl]6ulsnpallalap q)rqMur (11511) uorrezrpuq,iq 'uaasaq ue) y16 rr;rrad5 setuanbas atann lle]eql ose{p an;qe ql1rut e uol] euiolselqolnau peurels aresauosotuolq)oqf ,i;Lurolun lla) (e) suot6al6ututels uerunqe ul (sUSH) aLuosoLr.rorqr {snoaue6oLuog 'adorsongur lepun alqlsn'suJol orvuelel sauosotuortl) aql 1q691 paurelsur suotlerg;t;due VNC Zt-92 lungll lvlNlt lluldxl Y

[ 1 Lp u e ' la ra^olos r$ue) sawosowotl)sauag'OOOZ V s6l!'80€-L6Zi6Z I le r.llorllsaLuosouJoJq) leuilouaq] Joloualutlo a)ef nsoll] qllM poler)osse solnuruJ-alqnop ele)rpur sMo.uvslopporlleuis^ueuraq] aresaulosorloJq) olnurur-alqnop aql :soin]lnJls anlqpueuaat6aq] ql lPrlrJou q) alnuIul-alqnop palIe) aresourosor.uor 0q1 saulosoulol -osureluorleql lla)eurolselqoJnau ueunqe uol; rallnuqOnorql 'USH poq)uuo suorlfas aq] ur aruanbas e to,t6ururels ra4e lelrldo(q) (uaer6) e;qrsrn srUSHuett aluosourorql eqi lo auouO 'aueboluo

' ftt- SZ a.rn8rg)uorleredord lrluosotuorqJ peurpls 'sotuos B raddad l€r{l soruosotuoJl{tIEtuJou aql uroJt aleredas -ouroJqJ ((olnullu,, erxe sesnel osBJJaUPIoqr JuonecrJrldure

'saua8palelar-raruEf, sasselJruoJOttrp Jo 'roJuef, uorlJnpur rot ;o uosrredruof,e rot I-SZ elqel oes Jo luerfrJJnssr selalle o,vr'loqt Jo ouo dpo ur uorletnru 'sr lpql

u,,\\ou] eqt 'sJoLunl ur uorl€JeJrloJdJreql ^\ollp ol ldecxe snrr^ eqt rot uortrunt ou e^eq pue saua8relnllol lerurou 'seuaSotuo 'rer uort ore pa^rrep qf,rg,&\ e{rlufl lerrlorler ',{1a1eun1rog 'euop uoeq -uer ot peel suortlaJul AdH IIe tou Suruaarcspeq patua^rrd uaaq e^Eq plno.tr srltuep esaql Jo erros pue leeA r{rea re)uer IErrAJaruor1 erp 1lrrsoldoad 'tuacrad Jo spuesnoql re^e.^aoH 0/ tnoqe dq arer qreap eqt pernpor a^Eq ot rg8noqr sr 'slaf,uel alqrssodroJ ueerf,s pu€ Jnssu IeJl^lJf aqt aldues ot pJsn sl qlrq/!\ 'JeJrus de4 aq1 :aldeqc eqt ur retel surolordocuo AdH eurue -xa ]leqs e.lr !;ecuer tseaJqpue 3un1rar;e uJruo.4lur JeJuEf ;o ad.,{ruoruluoJ tsour pJrrlt arJl lafuef Iplrlraf, sr uorlf,aJur '(A.IH) AdH Jo eruortno snorrasarou qJnu ,(1lecrpauV sesnrrleruollrdeduerunq Surureluoc-yNq aq1 dq pesnec ere slleJ lerlaqrrdaJo srorunt u8ruaqreqto pue strB,t dueur 'olduexa ro{ 'sller perJeJulrrrro}suerl dyluaueruradqllqrv\ 'sauaSoluo eJoru Jo euo sureluof 'auoua8 VNC IEJrAaq1 Ilet-rsoqeql olur VNC IErr^rreql ate.r8alursasnrrl y51q rrua8 -ocuo '(g .raldeq3 aas)s11ar lpurrue tJeJurleql sesnrr^VNC tsotu elrlufl 'cruaSocuoare osle sesnrr^ VNCI n aJ V .scIV sasnEltEr{l 'sesnJrl snJIAnIH or{t uo ssarSo;d peleJalJJfe ralel qJrr.{1\{ -oJler Surpuetsrepunpatecrlsrqdoseql ur pue sauaSoruo ]o rplnllal ;o dra.tocsrpaqt ur qtog 11o pted raruer uerunq JoJ lopour E sE sesnJrlorterSurdpntsur tuerutse,ture8nq eqt '(,\-IJH) snrr,r.euoqdrudyeruo>lnel IIel J uerunq sr sJorunt uerunq esnet ot u,&\ouI snJrlorleJ dluo aqr q8noqt -ly 'JarueJ esnef sesnJrloJterr{f,rq.4dq usrueqcer.uJolpur

uorleldqleru 'uorlelntu lurod 'uorlalap dq asrry

AAISSE]EJ

,{llecrrauaB eJe suorlelnw

alelnlUnJf,E ol suorlElntu ,\\olle suorlelnlU uorlJunJ-Jo-sso'I

ul)NV) | szurravx:

.

zzll

oql dlqEgord sr uorle,trlre euaSocuo-otord leuoruesur 'snq1 'snrrl erxof,Jpssnog sE gf,ns sasurlorlar SururBluol -aueSoouo u€ql uorutuoJ oJoru r.lfnu eJe sesnJrloJlaJ Surlre-lrroys'suorlelndod esnoru pue prrq lernlru uJ 'luepr^e seruoraq rounl pa8pall-11nyE oroJeqrnJf,o o t J ^ e q s u o D B l n | ' I ll e u o l r r P p Ep u e ' l u e ^ a a J e J ' t u o p u e J e sr (t{,tu-t ''3'a) ouaSocuo-olordJelnllel e reeu uorter8 'sy1ac -elul :suosporo.l.rlroJ d1mo1s lre sesnrll 3ur1ce-.uo15 Jo uorleratrlo.rdprde.rdlleurrouqe sasneJr,(7rg-oIo uorl -rnpordre,to terlreo palou sE lurelord cd14-cecnpordre,ro slleJ osor{J 'erre8){,1u. aql rpau peuesur sl VNC leJrlorlar aql '(A-IV) snrrl srso{nal uer^E dq pasnec srorunt urorJ slleJ eql ur 'aydruexaJoC 'uorldrJf,sueJlsu Surlelnurrls dqeraqr 'eua8 re1n11arfq.reau E ro; retoruord ro raruer{ -ua ue sE lfp uel VNC paleJ8elurur secuanb IEJTAoJIaJ -as (UIl) teoder 3uo1 aq1 'uorssardxe slr 8ur Ieururet -]P IIf,P JEINIIEJ E IPAUVNCI IIEJ-rSOq PUPEUOSO]UO-OIOrd eql olur Surler8eturlq raoueoesner ol reodde sosnJrloJleJ 'auoSocuoue .ro '3urlf,p-,4aols ,,'drualel-3uo1,, 11y 4ce1daqr :tf,adseJIErf,nJf,ouo ur sasnJrl Surcnpsuerlto osoqr uJoJt 'sasnnno.4atSuticp ra;;rp 'SururoJsuert d14ea,vr ere r{f,n{,la, -tnols esoql seuouaS aq1 's-readJo sr{tuolu porrad e Jo ;o rat;e dluo JOf,uerafnpur sesnJrloJleJcrueSocuolsotu 'lserl -uof uI 'sdep urqlr,tl.srounl sotnpur n5g Surrnpsugrl eql 'auaSocuor.ls-,rluelod or{l sorJrEJauoua8 slr asnefeg 'eueSocuo-otord relnlloJ paf,npsuert e tuort pe^uep aueSof,uoue ureluof, sauoue8 rroqt osnuJaqsasnilnotlat Surtnpsuou pellel ere

saudzue .rredar-ygq

a8eurepy51q lue,rardro .rredeS

saue8re>1elere3

uorlera;r1ord IIaf, urerlser reqr s,{emqred leu8rs;o slueuoduroo 'a8eurep Ieurosourorq7vN( ss3ssElPql IeATAJnS pue uorlerayqord sutalord Iortuot-turod{teqJ 'uorssarSordalcdr 'suralord -llal to srolrqrqur Surtouord-srsordody

uorleldqlaru eArssef,eJ 11arpaleln8arun tuorlelnru dllecrlouaB .&\ollEsuorletntu lurod 'uoneyapdq esrry eJESuortElnw uorllunJ-Jo-sso'I uortecryrydrue 'uotlecolsuzll JErUOSOruoJrlf, tuotlelntu

turod .(q asrry

JEATAJnS

pue uorle;a;r1ord luEurruop lyacpareln8arun dTyecrlauaB .&\ollEsuorlPlnru eJe suorlelnw uorlf,unJ-Jo-urec

SNOIIilN!! l0 Ntctu0

l1'll9 IilVINl^l IO silIuld0ud StIlNls

uorlera;4ord ro Ie^r^rns IIer trqrqul

sauaE .rossa.rddns-roun1

sJotrEJuorldrrcsuerl 'uorleralqord ur tlnser lpr{l sdz.uqled uorlrnpsuz-rt IBu8rs puz SurleuBrs;o sluauodruoc 'surelord crloldode-r1uy

sl3n00ud lNtSl0 sildl{v)(l

NOIIVINUJ l0l3illl

uortereyrlord JO IEATAJnS

IIal eloruord

sauaSocuo-o1or4

slNtcl0 Itl0tISNnl

tvt/[u0N

€ZLL

ul)NV)

lo stsv8 fil_lNt9 lHt

p) _gA/,SAsnoo^zoLuoq e a)npordol po.ilnbor are^uooojd s]rro lla)leurlar ieln)ruede ursuorlelnu.r )rleuios eleredas onnl plrq)e 'eurolselqourlor ul (q) s0l0lle oMl slrrequr )rperods +gUleLurou suorlelnur oM]o] onpuorpun+ euebqy riue6ur1>e1 1;are arnpord1;rnn ololleleuJrou aq]salenr])pur )rleuros sno6{zoreleq e leql llo)leurlar y luaredreqloaql uorl elolle BU]uelnure pue uruorlelnuJ a;6urs ppq)e 'euolselqourlaJ luaredeuouoi+elalle+gUleurloue s]uaqur (lellrLue+) {relrparaq ul (e) salalle oml 0ur^re)slla)LLroi+ _gU}ue}nLU asue]eqlsrot!nlleurlor {q peleuLsroseasrp srrll'euro}selqourleJ ur uorlelnu )lleuos snoaueluods lo alog €l-SZlgngll V eutlol ur sJor,!nl ol asu sanr6 lloc sno6^20u-roH

llas leurlarcrleLuos

uorlelnur crleulos

{-

puz

uorlelnur crlPuJos lsL

€-

crperoos (q) eLuolselqourlar eurlol ur slounl o1esu sanr6 ;;ecsnobAzouroll

lloc leurloj crleuios

uorleln ur crleuros (e) eLuolselqourlor ArelrpoloH

'(t ratdeq3 aes) Y11q;o suor8ar poqrrrsuprtuou uI Punot .(luouuror sr uorleldglau 'sluauela qcn5 'uorldulsuprl rlaql srlqlqq qllq^A Iorluof, Jeqlo ro ratoruord eql uI sanPlserautsoldc;o uoue1.{greur sr sauaB rossarddns-.rorunl SuIlE,rIlJEuI JoJ tusIuEqJOtu Jeqlouv 'uralord leuollrun;uou E Jo uortcnpord ol PEol ro uratord truep uorlcnpo.rd lua,rerd IPI{I suollelnur lutod 'slacuBr luBru Jo suorlalep a.teq seua8 rossarddns-.routnl su) auaSocuo u1 '(uorrras snot,ta.rdaql ur poqlnsop se.'rtr ''e'r 'a1a11e lueurr.uope IEIuJouauo Jo ecuasardeql uI uela qvrro.r8sosnpt leqt uratord ro aue8 E to uolle^Ilx (7) pue 'eua8 'qt,nor8 lorluor ol lcnPord lualrlJtnsul ut 3utl1nsa.r E ddor euo ssol (1) :lueuluoP oq UEJ rossarddns-rorunl Jo ;o 'uaql 'ragrueua'rol seua8.recuBcqrlqr dq sassacordo.vr1 1n; -esn sr tI 'lueultuop sI uolletnlu yo adft srql os 'ad&oueqd Ieurt oqt ro; a^IsIlaP st eua8 aqr ;o .{doc auo to ssol oql 'reJuEOol 'TuanlJnsur-o1daqs auo8 PPal uer ;o Pul{ slql 'q8noua lou saue8o,lu ot{l Jo euo rsn[ 1o ssol aql ese) qf,n{^\ uI 'paruazra.rd sr lrnpord Jo lunolre aql IIEq 'seua3eutos qlrr16 eq IIIrl^uoIlEuIJoJrotunl 'lcnpord utelord to lunotue Iensn aql jlEr{ lnoqe Sunnpord 'aua3 Suqrolt euo ue^e sI areql JI '( tprll superu..rllsseJrr,,lxoluor slLlluI t -SZ elqet) ollsserer ^(llerrleua8uolto eJe saua8rossarddns-rorunl uI suollelnlu uorlrun;-Jo-ssolotueSoruosnql'luaurdola.roP Jolunl ololu -ord ol Jepro uI pelEAItf,PuIro lsol oq lsnru aua8 rossarddns -rorunl p to salalle qtoq (uouera;r1old IIaJ lorluoJ ol saf,IJ -;ns ouaS .rossarddns-rolunlE ;o ddoc auo dlleraua8 acur5 sauaBre4ela.rer 'S ,,(qpaporua iredat VNC ul eledlrlued teql saudzug

dretrparaq aqt elrql\ pue 's.rorunl dueu ol selnqrrtuoJ srsoldodealourord let{l suretord 'V 'ada auo dyuo srra;;e dllensn pue uorlptnru gg 'ra,ra.vr.o1-1 erer sr Eruolselqourtercrperods 'auo Sursoyueqt d1a>1t1 (ggd ''3'a) IEruJouqEere saluosoulorqrJI ro pa8eurepq VNO 't sselrEI sr aua8 gU eqt ;o sardor o,,r,uSutsol osnerafl 'llal Ieu ;r apdc ilol eqt lsarJeleqt sutalord lorluof,-lulodlJeql -rlor elSursE ur uorlelnru frlpuros uorlf,unJ-Jo-ssol e auo8rap (peqctedroldacarSoqo8paqeql'dCCI - u n s p q q l l q . & \ q J p a ' s a l e l l eg U o.tu rrrequr'tsell J o l e r u r o u ''3'a) uorle.ra;qord '(€I-SZ 'eurolselqouner -uor r{tr^ slenpr^pul ur rrperods 11erllqqul teql sleuSrspluaudolerrap ro 'Z sauourroqpetortresroJ sJernPsue.rlleu8rs.ro sroldecaU ern8rg) uralo.rdqU leuoDlunt ou sernpord reqr 1ac e ot oslr Sur,rr8'uorleuJoJ Jorunl ur dets Ierluasseup sr oruosotuoJt{f rar{lo aqt uo auaSgU Ierurou oql Jo uorlelnu ro uolralap eqJ 'sa,{a dlleroue8 .{1.rra srolunl ur a1l ur qroq pup IEuItorl€ra^as dolazrapurrplrql eql '€I eruosor.uorqf, 3o sardoceqr Jo euo uo uorlalap IIEtuse sE ueesseurleuros 'aua8 gg aqr 1o ddor e^rtlolop a13urse lrrequr Eruolselqourtar dretrparaq qrl^a uarplrrlJ eurolselqoullau )rperod5 snsran ftelrparap

'a1cdc 1ar eqr qSno.rqrssarSordelep8ar sdlaq gy dq paporua uralord aql 'rate1ssnosrpo,v\sv 'pertDuepreq ot aua8 rosserddns-rournl 'euol tsrrt aqt 'gU to uoltJunJ Jo ssol dq pesnec st qctqrvr -selqourleJ sr aspf frsself y 'uorssarSordrournl selelrlrf,eJ elolle puoles eqt to uollernlu rllptuos leua8 eql to elalle auo ur uortElnr.uauq-ura8 E lrrar{ur d1e.raue8slenPl^Ipul qlns 'sroJuer ureuef ro; uorttsodsrpa.rddrelrparaq E o^Eq seua8 rossorddns-rorunl ur suorletnru pelrJor.{urqlIA slenpIAIpuI

Islu re)ue) oseaJ)ul uI ns-rou nl u r suollelnlN Pelllol.{ seuog Jossordd

(I) roJ qu pue 91d ''3'e) alcdr [o] eql 1o o8elsrr;roedse'q8norql uors -sarSordllqqul to 'aleln8ar leql surelord relnllalerlul 'I :aAIJ eseqt er€ seue8 rossorddns-rorunl dq papocua suralo-rd'o 'sJeJuef,dueur luaurdolarrap sessplf,aqt Suoue luauruoJd 1o eql o1 olnquluoJ .6se>lEJq ,, esaqlto eJolu Jo euo uI suollelnlu uoucunJ-Jo-ssoT'uolleretqord 1ac llqFlul raqloue ro dert euo ur lpHl sutato.rdaporuo dlleraua8 saua8 .rossarddns-.lourn1

) r u o 6 o ) uoer v s eueg u! suollelnn uo!l)unJ-lo-sso-l JOssetddns-JounI 'uorlrre;r1ord Pue qfznor8 IIOtrelElnrulls ol s,{ezrtsnoIJEAur lfE sllotrpelfetul uI vNC IeJr^ peter8aturruor; pessardxasutalordoruo ag]'lele1 passnJsrpsy 'uortecrlder IerI^ ro; perrnbe; ore Pue euroua8 seuaSoruo Iprr^ eql ;o srred ler8alur are sesnrll VNC Jo

'ref,uer qlr^\ peterJossposle erp dE,4^qled Surleu8rsqH oql ur seue8Jaqto ur suorlplnnJ 'er.uotselqourleJ ur se lsn[ (sosea -srp esaql sasEJ(perrraquruou)crperodspue (perrraqur) Io .aua8 eql Ierlrtupt qtog ere eroqr snql ;o sardocqloq Jo ssol raJJnsdaqr JI (ool (saseasrp asaqrre8 uec eldoed raqlg 'pe8eurep sr alellp Sururetuar eql uaq,la,rnf,Jo uer reqlro :rarueJ urerg pue ur4s dola,ropot .{lrsuadordE a^eq Lzd lo ldoc Surlrom o13ursE lrrer{ur oq.tr e1doa4.aua8-rossorddns -rorunl e t pLQ snql lleu8rs qH ue Jo ef,uasqpeql ur uorl -ereyrlord lyac rrrurad L?jd u! suortetnur uorlJunJ-Jo-sso-I 'Gt-gt arn8rgees)(1rq) Ipeqltecl pellet uratordauerqurau e Surte,rrlceurpup or Sulpulg dq uorsr.l,rpIIar solplnrurls 'Soqa8peqrruo5 'surelo;d qg ueurnq eqr to auo ,run11aqa.rac pue ur{s eqr uI 'uortf,npur rof,uer ur palerrldur dernqled 8urleu3rse 1o alduexa poo8 e sr ,sate; IIat Iorluor ot tuetu -do1e,rap Surrnp dlpereada.r pesn sr qrrq.vr ,Surleu8rsqg 'qluor8 snoJeJueJro olBr.rdo:ddeur8ur -snec 'cruaSoluo oq ot d1a>11 are Surterado[uoJJsrusruuqJeru Surure.rtsar qrns luaaard leqt suouetnW.ftZ raldeq3) sleu -3rs SurledruoJpue 'sra>po1qrotdocar ,slsruo8elueJelnllol -Erlur alqllnpur are sleuSrs yn;renod Jo srJeJ leluarudola,rep -Jeor{t ur SururarJot alqelre^esusruer.{f,our eqr Buoruy .are1d pue arurl rq8u aqr or patlrull sr qlnor8 teql os pareln8ar oq tsnru sleu8rsqrns Jo slle}te eql .srsolnuprder;o duodord eql opnlrur deur qcrq,u,,sateyyeluaudola,rap relntrupd ol sllol trerrp ol posn.{yruenbery a.re(qg) Soqa8peg pue ,dCDI 'lulN sp qcns sleu8rs polerf,eslueurdola,repleurou Bur;nq

sJo)ue) fue61 qlrnn peler)ossvarv lueudole^eo lojluo) ul suollelraqv leql s^e^ qled 6u!leu615 'uorlerper sEqrns sua8elnur letuouuorr^ua ot elglldacsns dlleuorldecxo aq ueJ uortelnru eua8 rossa.rddns-rorunlarrrs -soJeJe qlr.Lruosred e snql .do1a,rapo1JeJuEf,ro; .{ressaceuare osle seua8raqlo Surtre;Jesuortetnu tng lsr uortEtnruJdV ov uaq.{\ue^o'raplo.ro 69 aldoad ro' Eeprpoo8 e sr ddocsouolocdq sddlod ro1 Suruaero5.gg 1o e8e eqt oroJeg raJuef, uolor Surdole^ap ro1 >lsrrpos€orf, -ur dllear8 e e^eq slenpr^rpur qcns '.{cueu8rluruol ssorSord sdllod eseqr Jo eroru ro auo teqt drrpgeqord t{8rq 11r.,n E sr araql ecur5 '(6-97 e.rn8rgaas)sddlod leurlsalursnorof, -uecerd spuesnoqrdola,rapJIellE Jo :)dy auo ur uouelntu aurl-tu-ra8E lrrer{ur oq,ry\slenpr^rpur 'aldruexe ro4 .seue8 rossarddns-.rotunlJeqlo ur suorletnru pelrJaqurqlr,t\ palprJ -osseuaag seq srarupf,rorllo roJ uorlrsodsrparddrelrparaq JElrrurs ta)ue) lseatg pue uolo) lo srulol palrraqul 'suorlelnur srlEtuos Jo llnsor eqr se osrreeseglto lsotu llemqted auresoql Jo sluou -odruoc raqlo 3ur1ca;Jpsuorlplnu ro selellpgU luelnu urel -uor (srorunl leurler rsnl 1qu; srorunl ueunq dueur dlrroqs eesileqs e^\ sV 'Jetuer aqr dolarrepol uosrad e asodsrperdol tuerf,rttnssr ldoc luelnru auo ilrerl lueuruop e sE polrJer{ur sr ptrrolselqounardola,repot {)uapuat aqt snql .luored IEru -rou'reqto Jror{lulory elolle gU leturou e trror{uldaql q8noql uaaa 'tuared pertaJJprretlt se srounl lpurler doye,rapol uorl -rsodsrpardpef,uequaatues egt e^pq olalle tuplntu er{l lrreq -ur oq-a.asoql 'rolazuroH'solallr gU IEuJJouo.,vrlseg lueJpd rar{lo rraqt Jr Ierurou ere alellE I€rurou eql luor.luroq.{i uerp -llql 'JIpq roqlo eql ol alells Ierurou 0q1 puE uerplrr{l rreql (e8ure^e (llr,ry\ uo slpnpr^rp IIpr{ ol alellu luelnu oqt uo sspd -ur Oseql'elsllE gu luslnu auo puE Ierurou ouo ureluof, slloJ urraS rraql esnereg 'uerppll ernpord pup pooqtlnpe [tun o rAJnsueryo Eruolsplqourlardretrperaqqlr^\ uerplrqo .lueu -8r1eru etuoreg daqr a.ro;aq pe^ouer orp srorunl Furler JI 'uorldecuoclsodpa:rnbce suorlelnru flU o^lo^ur Jeod qf,Ee sasef JOf,uef,rer{to 000(00I tnoqe ''S'n aqr ur read ;ad gg1 lnoqr rogtunu ses€JEtuotselqourlar

sztr

u l ) N V ) l o s t s v S) t f t N l 9 l H l

'o^rlJedlluBrJsqEeluoraq sPqeuoSor{1arer{,\\onssrtror{louEur ro uorlEralrlord IIef setotu -ord dlleuuou eueS palJeuE uE erar.{,{\sanssrlur uorlEr.uroJ Jor.unl oJnpur deur sauaSocuo-olord leluerudolarr.apur suorl -BtnW 'uorsrlrp IIef,lou lnq-sdole,rap ilal Jo adl.r req.,n-erey IIer IoJtuor ol sr .rorelnSarpluaurdola^ep egl Jo olor d.rerurrd esnerlou IIr,r suorlslnur aueS;os eql aJegl,\senssrlur sJOruPJ -sorddns-.rournreqr snqJ 'gurror8 urprlsar sdlaq dlleurou eua8 porro;Je orll oJeq,l.rsonssrlur uorleruJo1Joulnl alourord saua8 pluaurdola,rap .rossarddns-Jorunlur suorlelnry Tef,uEf,uoloJ ur suortelnr.uuruapc-El to tuoruo^lo^ur eqt to dra,rocsrpaql ol pol qrrq.,v\'(91 raldeq3 eas)de.vrqredSuqeu8rsrulX oqr;o rred eq ol u1!\oDI A{ou sr 'Eurourf,ref,uolof ol gred agr uo poletnlu aua8 ls.rr; Ief,rlrrJ eqr 'Ody 'aydruexoroC 'roJuef,eql to ses€r aql ror{lo ur saua3.{emqledpuorrrppe to luaua^lo^ur ayqrssod

'saIIJ'sluro.ry\0>lIIstusl ;o suorle8rlse^ulposnloJ s,l.rolleellul Jo 'raruec -ue8ro Iepour LuoJtpaueelSde,tr,qredeqr;o a8pal.r,rou>1 ueunq;o addr E ol POIuq uaeqsrq derrtqredletuaudola,l'epe uaag ur aue8euo af,uo'paapul'Gy-SZ ern8r4)ref,uef,or pe>lul1 a.,r.eqsde.trqted Suqeu8ts asoql Jo lsoru Io slueuoduror 8ut -]trauesuorlBlnrusreodluesor uI 'sensslllFPe uI uorleralrlord IIer pue luerudolalep ctuodrqura Suqlo.lluoc uI salor deld s.rer -deqc raqlo uI peqlrJsePsde,vrqred Surleu8rsaqr;o dueyq ' Pid/ L?14sno8dzoruorl 'lueurdole,rapJoJ ore lEql sllel rolunl eqr dpo sI 1I os Pepoou trpea 01 peel plno,4 uollJun; Irld sr lr erurs 'qreap ryn1 'saue8 rossarddns-roruntraglo Jo reqlunu Jo ssol alaldruoc E roJ oser arll sI sV'IJrd e>lr1sroleln8ar a^Ile8eu lroJte lEI{l suorlelntu allsseJeraJp sJeqto ldlalerrdorddeursaue8la8 -m qH uo uJnl leql saueSoluoeleeJJsuollelnlu qJns oruos

'alalle luelnu ouopueleulouauouleluo)lllMllal luellnsal lnq uZ leulrouoq] 6uuolsoi aulosoluolq) raqurnu eql saurlauros ololleluelnLuoq],Losardo)oMl sureluo) leLlllla)e o)npordup) 'ouo 'uorle6ar6as 'alalle aarq} so^lo)el e seulosoLuolq) adIl {;leraue6 }eLl} asol pue penno1lol aurosotxorq) {q e ad{1-p1rnn llrnn +o }ue}nLU {eu se;e1;e ue ura1e6al6es p uae^^leq }ue}nul )rlol!tA(q) setinp 1;arraiqOnep e q}M ou.rosouroJq) uorleurquro)or V orleJL:€]uerraqe pale)lldnp oql uaql'anl])alep pue 6uueaqsaulosoulolqf eq1 puesalejpa]rurla^eq]eq]slle)io seuol)urpo]ejolo] aq ue]+o leulou {;leltdIloueqd )tlo]tulaql ]o uotleulo+ sre;purds ue]o salnlrnrls xeldu.ror Aueu;aql olutdolenep +l(e) leurrou uel ]nq 'usrueOro e +oolallelue}nulauopue steue6tosserddns-rotunl {11ereueb Ilenrle;at o1leqlallo 6urOeulep o] a^eLl slla)paleltuara+]rpun ]eql 'sauablosselddns-.tounl (HO'l) qrns +o Iproldnaue ,{;1ereua6 sr(uorin}r}suo) aurosor..r.iolr.l) ;1aty leurouauo6ututeluot leuJrouqe) I1rso6{zorelaq}o ssol,o} stuslueq)auo^^l tt-92 lgn9lJ V oq lllMll seull}oLllos leq] aloN olalle]uetnuiaql ro1snoo{zouloq ololleluelnLu alolleluelnu .to;sno6r{zolelag ro;snoblzotuoll olalle luelnlu ro; snoOAzoruo;1

a l o l l el e t r J r o u ro; snoOlzor.uog

/ solc

uorleberOas [:t lueiloqv

s p l l e L u o r qscn o 6 o ; o u o q uoaMloquorleurqurocou

t

uo,r"r;lonp I

u o r l e c r l d nIp ourosouror.{3 |

I o l a l l el u e l n u rr o J sno6Azorele;1

aurosourortlS I alolleluelnurlo' snoOlzoteleg

alalle luelnN

alolle leurroN uorleber6es-sty1 1e1

uorleurquiocarcrloun (q)

'sol'udzuerreder,VNc (saIlIAIlrE pup espur>l sJolelnSor(suralord crloldode Jo -o.rd'sde.vrqledSurleu8rsSurtrqrqur-quror8 ;o slueuod -ruoc 'epdr [o] eql lserrE leqt suratord lortuof,-lurod{reqJ s€ r{f,ns'e1cdo11acaqr q8norqr uorssarSord.vr.o1s dltcarrpur ro dlnarrp leql suretord eporue saua8rosserddns-rown1 r 'dltl, -r.rdo.rddBur pue dlsnonurtuoJ pele^rloe sr aua8 relnlleJ eql Jo uorldtnsupJt leql derrre qrns ur euaSocuo-olorde rBau SurlerSelurdg racuer esnel uer sosnJrloJlalSurtte-lrro15r '(9-97 a;n3rgaas)etuourcrec ropPelq ueunq e tuo{ perplosr sezrrauaSoruo srql .urel -ord uorlrnpsuerl-lBu8rse 'seg to urJoJ e,rrlce dlarrrlnlrls -uor e seporueperJrluopraq ot auaSoruoupunq lsrrt oqJ r

I LsiooLila) '6667'oraqureg V U pueueqeueH C uorll soutlpaqsepqll/\\pale)tpul are sleanqledpallstlqelso ri;Lutr; ssel pal ut uMoqsale sllo)lo)ue) ur polelnu.r0q ol uMou) sauaD lou to aleta;r;otdo] ]uaultLur.uo) e sauro)ual1ofirluapr]eq1qlM 6uo;epue [1r1uepr traql s;1etenr6 JOSUeS '-tg

/

/^rrreuiouqv

ul)NV)

|

9Z UIIdVH)

gztL

'uorssaJoxeJe^o PUP 'uotleoolsuerl aua8 'uorlecrlrldureeuo8 (uorlelnur rurod ,(q JnJJo ueJ srql 'euaSocuouE 01 lr sueluof, eueSocuo-olord e Jo selofle o^u or{] Jo ouo Jo uorletnr.uSurlBlrlce uV r 'fit-SZ arn8r4ees)suralordcnotdode pue'srotre; uorldrrcsuerl'suroloJd uorlf,npsueJt-leu8rs 'sroldaca.rrreql pue surotord Suryeu8rsSurlourord-ql,uor8 erB sauaSocuo-olord ,{q peporua suralord eql Suoruv r 'lruaSocuo a.resaua8 rossarddns-rounluI suorlelnu uorllunJ-Jo-ssololrsseferpue seuaSoJuo-olordur suottBlntuuorlJunJ-;o-ure8lueurruo(l r

ra)ue) lo slseg)llauag aq]

'6urprnrp s,{ennq}ed anut}uot ol Jo optntp o} Joq}aqM ;eluaurdolana6 s]tsa)pu lle)aql sepelet6alur aulo>aq slndur lnoqpuotsr)ap aq] pue'sleu6rs;o sad{}{ueu Iq petuanllur ole 'ro)ue) leurelxa 'a|rfu oLl]pue 'suotlelnu oursner lo ]jeaqeql lla) lojluo)qiMorD -lo)ue) {q pepage st }eq} ft}!n)l!) lla) s t-sz lunglJ v

@

" !?ttr;3i3 l

"1"

tl,.

I

(rser"6 a)

e u p u o q J o l r [ 4< - ;

t, I,

',rii"l ' ' ' l l " - ' .. i\., r '1, ':

f

,;-*+

I *

it

. t ;

,t ll

t z o- z ) o 3 : l

i

!

"

ti

s p e u s ^ * - , r 1 , " - * *-, p , 9 ! d. , 'rl:r "', 'r, ft

,.

,

I

{t)o3:olr^c

t,:

rcl. 3dv

t

6

I de-ysc

t

! (tllcr' 6 a)

nlnS#!19

sJolcej qlMoi6rluV

LZI L

s N t 3 l - o u d9 N i l - o t A o u d - H lo- A u9 Nt sNorrvlnw )1N390)No

'Pele^Ilre 6ur]lnsar aql +o^]r^rlleeseur)aql +ouorle^r])e surPuop lEurelxe orp ol SuIPuIqpue3t1 Illun luer Bqrl uratordoruo -sarnbsr tEqt dlIAIlfE ue 'sureruop lrlosoldJ rleql uI dltzrtlce roldo)allDl oq] ul a^rln]rlsuo) ol 'suosPer uMou)unro]t'speal relnllo)erlxa aq] ssolsasne) ureuop6urpurq-puebrl ]eq]uorlalap eseur{ eutsordl-utalold ltsutllur a^eq sJolJeJql,lro-r8 qrns lo aql 6urleuL dueru ro; sroldef,eJeL{I tarueJ ;o soddr IEro as qlr-4 PalE e nap utalordo)uo V :lq6!Aaseur)anr])e{1enr1n1r1suor 'pue6r; peleler-lglleurouaql +oa)uosqe aq] urua^a'rolda:ar -rfossEueoq aAEqsleuSrsSunoruord-qulorS acnpsuErllerll sasne) rolde)arzroHaql;o uol6alauerqulotu aql ]o uorlezuaurp Surpocua saueSocuo'lse.rluoc u1 elEJrns-llel sroldefar -suer]aq] ur(eurLue1n16 '11 pr)pourure a;6uts e slolle]eq] ol ourle^) 'uoIlEInIUIlsaulrf,olnp PellEl sI slqJ uorlelnury .4a7'srolda)al e)e#ns-llo)apo)ua leql seuaboluo secnpordl?qr IIel aqr uodn slf,€leql rolre1 qlv\or8 pererleun -olord ur suollelnurrgua6oruo spa#l 9l-SZ lunglJ v 'rol +o ue ecnpord or ur8ag sllatrr€qr sI luele uoluluof, aJolu V suleloldocuo roldacar luepuedepu;-pueEgl -dacarggq4 eql ssardxadlleurrou leql sllal;o uortela;rlord etelntulls .{pue.rregeurc '(g9q4) rorlel qlznor8 Pr^IreP -re1are1dto turoJ PaJallEue saPolue qllq^,l 'auaSoluo sls eql 'poJe^orslpuaaq seq 'szs'auaSocuoSutrrncco dllurnreu qfns euo .,{1ug's.Incrodlare; stql ]JeI w }nq TeruEJto tuslu -er{fetu d1a4r1e uraestq8rru 'urelord oql }o uorleralle uP o1 anp 'uralord SurpuSrsSurcnpur-qltlor8 e 1o uorlezrrlrered.{11 aseur)eursor^l -uraloJoantlce AleArlnlrlsuoO gql3 uralo.rdocuo

oururelnIc

uo,raao .ro,,r,n*ri;tT;J: ", | aseurI aursorAl

toloecol - /

onrlceul

srol)el ql/u\or9leulelxl +o a)uesqv aql u! uorlerelrloJdolotuold ue) srolda)au )!ua60)uo 'uorlerayrlo.rd lu€punge saluol Pollorruolun slr ol Sut.,rno -ag uorletnu to uos relnrrlred d.ra,re auo8raPunseq reql IIal 'stsaueSoulfJef, ol Surlnqul aJEJe A\oq ees a,tr asEJLIJEau1 -uoc dqeraql 'uotleruro;suen Pue uotlela;qord 11acalouord uorlrnpord;a^olleqt uI lo suleloJdutel;ar ;o {lt,ttlre a,ttlnl -rlsuor 'paleln8a.runeql uI llnser ler{l suollslntu ^aot{IIeloP 'seue8rossarddns-lorunl eJoruur oultuexa eA\uollJas srqr q uaog o^EL{11-97 arn8r4 ur ro saueSocuo-olordse POTJIIuoPI parcrdapuretord.fuoleln8arIIetrto ssEIJqcea SurporuaseuoC

sulalord 6ul1otuo.l6

-r.ll^or9 u! suollelnlA)luooorug 'PalelnIu loldecer roldecerTreg roldecer393 Tleg sulalordroldacereue6ocuo-o1or6

are saua8 aql uoq,tt eslrr letll srolunl 0q1 Jo ra}f,ereqf, orll uI polraljer ere srnrfo qlmor8 uaLI^ Pu€ eraqm 3ur -re1n8a.r uI salor IPIurou rlaql'(SI-SZ arn8r4 aas)sde.Lrqred Surleu8rssnoIJEAuI uollJunJ leql suralord oPolua sassacord dueytq I leluaudola.tep IELurou elrln8er reqr soua8 'HO'I alqlssodor pelqns JOIrrEloql se^POI ddoc a18urse rala,ry\oH'a8euep VNC snolras lua,la.rdol luerlrtlns dllensn sr ddoc leuollJunt euo uale Sut,leq acuts 'a,rrssefoJdllecuaua8 are seuaSJe>leleJBJ uI suollelnry I

II€ ro tsou uI perelle sr de.vtqled gU eqr 1o luauoduror arqos lslournl Jeqlo duetu pue PuolsPlqoullor uI Polplntu sr 'gy 'pazruSore;aq ol aua8rosserddns-rotunllsrlt aVI r

Surleu8rsrelnllaterlul uE satotuord fyateurrln reqr dea,,rqred Surlerlrurtdlr,trlrBaseull JIeqrJo uoIleAIlJppue uoIlEzIJeIuIP rrer{l ot speel (sy1g) saseur4 autsordl JoldeJer aseqt Jo

' Gt- SZarn8rgaes)uoualepro'Surcualtseua8'uorlelntu'uotl -e8a.r3as-srru aruosoruorqf,'uorteutqruocar f,Itolltu dq (HOf ) 'aua8 dtrso8dzorelaqJo ssol o8repun uer IIaJ JIlEIuos B uI r rossarddns-rournrE roJ sno8dzoralequroq sIEnPI^IPUI ''3'a) seua8 '(f -yCUS puv JdV 'do1a,top Jeqlo dueru ro; os€J eql sr se .rossa.rddns-.rounl .&rlrqegord eqr sosearf, IIr/\\ rerucJ Jo pull cr;roadse reqr -ur dpear8 gU Jo elalle luelnul aJ8urse Jo eluellraqul r 'sJoIunl

oqt Io uorlalap 'eldruexa .ro4 :o1daca.r ctuaSoouo arr.rlce dla,rrlnlnsuor E sernpord uretuop Surpurq-pue8rl relnylar 'suralord la8rel 'sosec -Ertxe raqto u1 aql to qrnu Jo uonelop LuJoJa,tttre dlorrtl str seleldroqdsoqddle,trssof,xe rueartsu.l.rop -nlrlsuof aqt os 'aseurl ourso.rdl-uralorde sr roldecar CNCC 'urpruopJelnllafeJlxeeql ur uouernu lurod p tuory sllnserletll rotderer (fNCC) rolcey rrqdorlornau pe,rrrep-eq8JIToLUIP eirrlce dla,Lrlnlltsuore eonpord Z a4& atsvldoau autDoPua '(4ut'9t-SZ arnfit4) lfulryw Pallel srounl ueunq Xlrepur5 srafuef, osnoru ureuef Jo Jol€rlrur uE sI qflql\ '(eruotselqor -neu ur'a1or u.ltou>1lsJI' sll lo; ,,nou,,)uralordoruo nJN aql olur roldarar (ZroH) Z rotdeJer {CE ueutnq leturou oql slre^ -uof uorlplnur lurod a18urse 'oluelsut ro4 'pue3q to etuas -qE eql ur eArlJEdlo,rrlnlrlsuof,sI Pue sezlreullpleql auo olul 'sasel aruos u1 IsruJOue saSueqruorlelnlu lutod e 'uorleratrloJCl )IU

lvt€€.tgLt ogwl '6661'p nrsaurluplsuof ]a N s aosl odl ]o e)uosqeoq] ut lolda)et eq] 6urie^r])epue 6utzuoultploldatar od3 aq1purqIllerrloeds ggd6 rueurp +o suteuJop auelquauisuelleql auelqLUaLu eutseld aqi ol pazrle)otqloq 'SSdblel^ pue toldatet od3 eql etnpotd snrn 6urr-utol-sntol uee;dseqt ,iq pepa;ursllettollueOotduaqnn rn))o sro)ue),i|;eutol s;;e:rolrueoordprotqlrtauJot1sai{totq1{ta se)nputpue tolda:er oql soztlaultp od3 1o 6urpu16 +o uorieuJro, 'uraloldoruo ,odg ,pue6r; leltn e lo lelnleu aq1 fq .roldere.r (odg) urlerodo.rqgf.re oq] ]o uot]e^lpv 8l-SZ lUnDlJ V

aseu rI

oof 99d6

Jo uortere1rlordaql Surtelnurls dlsnonurtuor pue dlalerrd -orddeur dS '(St-SZ ern8rl) etues aqt ur srotdararodg IIar ate^rtre pue ot pulq ot lrrlrqe alqe>lreruer eql parrnbceseq 1t 'uotlrelut pue Surppnq snJrl ur urato.rd adolelua snrrlorloJ 'sur^ eqt Iprurou p se uorlf,unt louuer ggdS qSnoqllv Jo lra;ya cruaSoJuoeqt ro1 alqrsuodsersr ,ggd8 peurel ,uralord .O-% arn8rg aas)rotdacar -ordp adolarue ACCStuplnru V odg Surpuodsauot eqt pue (odg) urrarodorqrdraerrnber dlernlosqesllat per arnleru olur s.rolrue8ordprorqldro uorl Jo -erluareJJrppuB 'lB^r^rns 'uortera;r10rdoq1 .1eu3rs letuau

'6861, I Sli6 lolg tp) tory le la rorlno)3 aa5l loso1baqi ut puno+ 'euelquleLu sr uraloldo)uo aq} eu:se1d aq} ol paztlelolsr q)tqM )l1 '){ st oseut)uralo;do>uo lerlrou oLl}o)tlun enrl>erilenrlnlrlsuo) 'lueLuOas urso{urodo.rt eq1,iq pazuaLutCurso,iu.rodotl 14 aq1 urelo,rdocuolrl clteru!qC

ur)NV)

|

9Z Uil.dVH)

SZtL

-do1o,r,ep leurou e Surlelndrueurdq ecrtu tlnpe w (srolrue8 -ord prorgrdro rounl e) erua4nelorr{ldre sarnpul Jo fAJ{Si snnn SunutoJ-smoJuaalds pellef, snrr^or1are ,aldurexorod 'sllef, ref,uer paflotul ruo{ snrrl Jo uorlJnpord eqt osee.rrur o1 dlqetunsard tacuer asnet ot s{frrl u,vr.oJreql osn sesnrr1

suteloJdo)uose l)v sJolda)eu rol)el-ql/vlol g lo sJolenrl)v leJtn 'Polelopueaq s€q euerqueu egt ol lr Surtcerrpof,uonbesIEuBrsleuruuat-N eqt ecurs'1osol -dr aqr ur pazrlelol sr uralordoouo I{ a^rlJe ,(lo,rrlnlrlsuoc eql (lsuJtuoruI'fi7 rardeq3) rolteJ r{11\.or8 elrau E spurq leqt aseul{ autsorll roldaJer oJeJrns-lleJe sr uralord 4.r1 'pue8rl ef,uosqear{t ur sureuop eseur{ ar{t Ieturou aq1 Jo Jo uollelllre ol Surpeal'o;ntcn;ts lroJ-pelrol e Surruro; dq tor -dacar 4r1 f,rreturqfoqr Jo uorlpzrJorfirpelelporuuec luaur8as ursodurodorl poteoolsueJleqJ-' (LI-ST ern8rd) ursodruodoJl alrsnruuou sprle our(u€ lrururet-N aql Surporua seruanb Jo -es oqt qlrm roldacal 1{ Ierurou oqt Jo ureruop relnllal -prxe eqt lsou Surpocuosaruanbaseqr pacelda.rteqt uorl Io -pf,o]suerl E Jo llnseJ eqt sp ureloJd crreurrqr lpr.uosoruoJr{f, p sepof,uo ouaSocuo srqJ 'eruourf,Jef uolof, e urory pelel 'auaSoruo -osr sE.{\ qrrq,vr 4.tx.uErunq oqr dg porsrlsnllr sr rol -darar cruaSofuo ue Surleraua8 JoJ rusruer1tatu Jer{louv '(9I raloEq] aes) slleJ uonere;rlord etelnrurts ot ^ ol oot suorlprl leturou to -uef,uoJ'sauoruroq palEIaJpue CCE Jo suorleJtueJuof,,vrol dral;o aluasard eqt ur eteretrlord or pelelntuus ere sllal eql 'llnsar e sV iroldaral ZreH IEruJou e arnpordralo sJOf,uEJ lseeJq uewnq dueru 'oJuelsur rog 'rruaSoruo eq uef, osle )IU lerurou e to uorlrnpordra,ro or Surpeelsuortelnry '(PagtluoPt 1srr1se.traua8 'SnJrAsrsolsElqorqrdra PaJellEer{l to uorsJa^IEJr^ E OJeqA\ uory) ureto.rdocuo gq.rg f,rrorrrrpeqt ol lr slreluor (lq31t,91 -97 ornSrg)roldarar {CE Ipurrou orll Jo urpr.uoprelnllaleJtxe

al)snuluou]o ureLUop aq] q]!M 'aseur>1 aursot{1 leutLuJe]-N rolda:ar e 'uralotd ){ ueLi.tnqleulJouaLl}}o uteuloplelnlla)el}xe oql +o ]solrl ;o luauateldeJ ut sllnsal uotle)olsuell leulosor.lolq) y 'ugegoldo>uoIl1 )uou.lq) pue ,loldare; {4 leulou aq} 'ursofurodol1 leurou ]o selnpnrls uteuoq Zl-gz lgngll V

ursoA.uodoll;o uorDel;eurutlel-11

Cp 3p roldecar l.rl leturoN

N N uret!op aseut)

uteuop auerqulaLusueJl ureurop Jelnl loceJlxf

SZLL

.

S N I 3 I O U d9 N l l O l A O U d - H I AO U D N l S N O I I V I n N ) l N l 9 O ) N o

roJ elrs Sur4rop e opr,rordoslc uer lqv-rtrg '(Zt-gt arn8r4 o1 (unerodorqtdra ''3'a) s.rol aes) sroldeceJ of,€JJns-l1ar -re; qr.uror8 ;o Surpurq dq palezrrlre ere dlleurrou qJIqA\ 'rolce; uortdrrcsuBn SIyIS pue as€ull Tyyf etelrlce uec 'of,uelsutroC 'lqv Jo seleJlsgnsIELuroutou ere sulal Iqv-rlg -ord aseqlJo oruoslsrel lE pue 'sutoto.rduolllnPsueJl-leu8rs relnllafenur duBur ale,rtlce dgareqr pue areldroqdsoqd urc Iqv-rrg ( tt-SZ arn8rg uI u1\{oqsutelordocuo I{ Jlretulql eql to uonE^IIJE pue uollezlrotulP ol rEIIurs sl slql) '(qgz -g7 arn8r4) dlrlnce oseur>lIqV snonuuuof, pue poleln8a.run slrqqxa leqt Jeuerlat E turot aueSoouonp4?q eqr dq popoo -ua surelo.rdoJuof,rrorurql eql 'adeqs ilol pue uotalo>lsol -dc eqr lorluor ot dlueurrrd uollf,unt deu 1r os 'sasselord IIaJ to uorsualxe pue urtf,p snoruetuellJ;o Surqruerq saloru -ord uralord IqV-l FurJou aqJ 'sonradord snora8uep pue ppo seq lr pue 'parnpo.rdsr uretord plrqdq E tEI{l sI uolsnt oql '@OZ-SZarn8rg) u.4r\ou{unsI uolllunJ ;o ecuanbasuoJeuo esoqlr\ 'eue8 nq aql ;o rred qltm 'aseutl eutsordt E soPor -uo qJrq.ry\'aue? yqo-t aqr ;o ued e sesnJleql uollrrolsuerl utalord roldacaruou euosoruoJqJ e dg petereue8st aseur>1 cqosoldc e Surpocuo auaSocuoau6 eseu!) uralord lqv 'GeySZ arn8rg) esereqdsoqd e dq uorle,trlce a.rrnbartou op doql 'st teql ldtrarlcB esEuII enllnlllsuoJ o^er{ reql sutalordoruo rr5 ur porelle ro Sutsstruueryo sI 179 eutso.tl.l 'lrS-J sele^Ilf,e dllerurou audzua aseregdsogdct;tcads e Aq tZS eulsor -droqdsoqd srsdlorpdH '(q 'E6I-S7 ern8tg) snulurrel-J ;o aql ruoJ; senprsorxIS sI qrII{^4.'179 uorlrsod 1e anprsar eursordl eql Jo uorreld.roqdsoqddq POtPItreur sr d11eu -rou sutalo.rdpal€ler pue JrS relnllol;o dlt,rtlceoseul{ oql 'sureruopuortf,EJelulutalord-urelord €HS PUEZHS uIEluoJ 'utalord seseurl esaql 'ureruop ortdleler e ot uoItIPPe u1 lrs-^ eqt or pelpler saspul>leutsordl roldaceruou;o dlnue; e epof,uesaueSocuo-olo.rduerluulueu lq8re lssal lv 'esEul>l eursordl-utalo:d err.tlcedla,ttlnttlsuof, e sapotruo'snltlollel EruoJJEsSnOU IUoJJ l.tS-,t 'parazroJslpJq ol ouaSoruo 'sderrrqledSuryeu8tsrelnllef,erlul lsrrJ eql paepul 1o dra -rre^ e ur sleu8rsaJnpsuerl dlleurou leql seseul>lutelord rrlosoldo apoJue sauaSofuo IEre^aS aseu!) ulolotd ).tS

'dressacauunroldac parellp reqto Surporue sauaSocug -eJ eql ol Surpurq pue8rl dq pareSSrl uoIlEAIlrP ureeJlsdn Surleru 'deltqred slql to l.red lsrrl oql llnf,rlf,-lJoqs suoll -plnru sEU Surle,rrlcy'(SZ-gt ern8rg ees)suralord cr;roads -uouenueroJlp Pue elldc-11acluelrodurt ;o srsaqrudsacnp -ur lEql s.rolfE' uorldtmsuell to Jequrnu e sareldroqdsoqd 'aseulI uagl asEul{ dvw ParP^I}lE OqI dvw ol sesEu -r4 uralord elelpeuJelul o^{l EIA leu8rs aqr slltusu€rl seU '(97-91 ern8rg pale^uf,E te,vrqred egt 1o rred Puores aqr u1 'seg ol eas) urro; punoq-dlC eAIllE aql ol 1r Sulueluot surato.rdraldepe o^\l 3IA PeIrrEf,sl )IU PelEAIllE uE luor; 'saseutl urerord yeu8rse te.vrqred slql Jo ued lsrrl eql uI ;o epef,spf B ol sroldecer POl€ IlJe ruo.t; sleu8rs Sutcnpsuerl '91 rardeq3 uI 1\4.8s o^/r\sV ur luauodruor do>1e sI sPU 'sEIlua>lnel pue'seurolselgoJneu's€tuoutJlef, 'uoloc 'rappelq Surpnleut'slotunl 3un1pue 'ur>1s'dreuruetu uerrrnq;o saddrdueru dq pacnpord are surelo:doluo sEUo Il -ce dla,trlnlrtsuoJ 'alels Punoq-dJC aAIIJEaql ut seg Sututel -ureul snql l.{tt,ltlce esEdID s.uralord eql sef,nPoruollelnlu aldurs srql 'urelordoluo oAIllE dlerrrlnltlsuof,E olul Pouo^ -uor sr urelord leruJou egl 'aruonbass€U eql ur 71 uorlrsod re aurcdlSrot prf,p oulure due solnlllsgns uollelntu lulod e ;r 'relnctlred u1 dlrrrrlcr lueuluoP aroleraql PUEPellorl -uoJun sll or Peel uec uralold s€U uI sa8ueqr to Jequnu e Jo ouo ,{uy 'pezruSotreroq ol souaSocuoIErIAuoulsrlJ 0rll aJe^/rqfrq^\ 'saue? osot eql oJe droSalec slgl uI seueSofuo femqled seg perpnts-lseq aqr Suoruy sluauoduto) 'sllef, rolunl 1o sed& dueur ut eglJfs qlns to seldurexaIereAOS passerdxesI qf,ee.'seuaSocuo -ap elA.'p3rc1 relnlleJ e ol;oldarar PelE^IlrEuE tuort sleu8rs Surcnpsuerl ul PIE sutalo:d Pepolue osoq,tt sauaSocuo -olord ruorJ peAIJaperr saueSoJuoJo raqunu a8rel y

su!elorduoll)nPsueJI-leu6ls aPo)u] soue60)uo Auen eArl)v [1an;1n1;1suo) 'snJIA saddl aruosIsPOIlE dq pasneorac Jo -uef, IEJI ToJtsute8eloalord ol Puno; uaoq seq (11) urarord 'uolleluJotsuerl prsder AdH ue tsute8e eulf,f,el e dlluaceg 'seua8 rossorddns-rounl ltgtq IIaJ ol Surlnqrrluoc JaqlrnJ -ur ol lJp teql surelord Jaqlo lere^esrot sePor osle aruoue8 'uollururotsuerl 'rale1aas Ilaf, dlpnluaira IILvi o^/r\sv AdH eql pu€ uolte^IlfE Joldesal PeulElsnsSutsneo'uorlezrteurrp .rordacerpotelPeu-auourotl sf,IIuIIusIqI'euergluaur eurseld aqr ;o aueld aql ulqll^. sroldacar CC61d eJotu Jo 01vrr8uI -rc3at338 dqe.reqr'gCCa ro; roldecer snoueSopue auo qll^{ 'JeIuIrl Jo xalduroc elqers ? ulro1 u€r eprrdeddlod Sg qleg reurp E sruJo1pue ou€rquletueurseldaql sueds'sprceounue 'Sg pateu8rseputelord snrl^Eluol yy Aluo suleluof, qJlq^a -pded y 'stJE^{IElIueBpue racuec Isf,IAroJsasnef leql snrl^ '(AdH) snrr,reurolltdedueur VNC poilruIsuerl dllenxas eql -nq dq paprrrordsI uouo(uoueqdsrqr;o eldurexareqlouy 'sllel

'lrEJl lueunuoP leluosolnP ue se PalI -Jaqur sr'eurolselqourlar drelrparaq a>lII(sISolEIuoJqIJoJn0u pue'aua8 rossarddns-roruntp sl 'gU o{l 'IdN snql'seruorq -rJoJneu uolleuroJ 01 sPsal alolle Jalpo eql uI uollElntu Jo f,rleuros tuenbasgns le1oge Jg11 tu€tnur a18urse PalIroI{uI a^eg srsotPruorqrtornauqll^/r slenPl^rpu1'uralord eddi-dvc seU p soporue qrlq,vr 'tdN Jo salo[€ qroq ]o ssol dq pasnec sr 'sazr,rou Punorrns lEq] sllol qleeqs eql Io rolunl uSrueq e 'srsoleurorqgornau'aldurexe JoC'sutalord uollf,npsu€Jl-leu -8rsrueerlsu^\opto uolle^Ilf,E s€U Peulelsnsol sPeeldVC to ssol aql'(7E-g arn8rgeas)seA Punoq-dqC elllreul ol sEU punoq-dlD o^Ill3 Io uolsre^uof aqr PUE dlc Io srsdl0rp -dq eleralacoeol sI uollf,unt dVC IEuIrou aI{J '(dVC) uler -ord SurleralaJJ€-esedlCE uI uollrlnlu uollfunJ-Jo-ssolaAIS -sef,eJP r.uog esrJEoslE uPf, uoll€Allf,E sEu eAIlnlIlSuOJ 'PeIlIluePI u03q a^EL{ osle de,rqted eseurl dVtr\tr/sEUi)JU al{i ;o sluauodruor

Surtera;rlord dpueueqe eseql uI suollelnlu rol{rrnJ to llns a8reurasrolluo8 -er e se uollf,etul A4CS reUB slee^\ I€JeAOs -ord prorgr-(JaJo seuolf,1ueu8qe1tr'seldcort{ldraJo sJaqunu 's.rorrueSordprorqldra elrssoJxeto uollelurot sornPul ggd8

eql srlnpord aql (JiW-J pup soC-l uorpnpord to l?turou Jo egt ur asEorfurplot-Os'= ue sef,npur sllel luaJsornbJo tI€ 'aldurexaroC 'lorluof, luarulEorl qrmor8 ur elor lJerrp {CCd .rraql Surtecrpurtrror8 ol polplnurts arp sllar lpr.urouueq^\ pernpord e.re surolord aueSoruo-olord reaycnu lue141 'souaB Surssardet-qgnor8 uortdrrcsuert Burlrqrqurdq .ro surel ;o -o.rd Surtourord-ql.Lr,or8epof,ueleql saua8do1 ;o uortdr.rcs -uerl Sunelrlle dq surotoldocuosE uorlJunJ leql .s.rorce; uorldrrcsuerr se lFuapuadapur lre uer osle un[ pue soC qtog '(9I raldeq3 prre 6T-L arn8rg aas) seue8 dueu ;o sJorueguapue srolourord ur punol aruanbas E ol spurq fiql 'LdV pa1lec';o1ceJuorldrrf,suprl f,uar.urpoJaler{ E uroJ ol olerrosse sarurloruosleql sureloJd aporua saue8oJuo -olord soJ-t puv unl-t aql.srournl uEtung eluos ur pesserd -xeJe^o aq ol punoJ Jelpl pue sasnJrloJleJSururrolsuerl ur parJrluapr aro,tr dllerrrur qllq^\ ,soJ pue unl arc sald -ruexo o.{[ 'srotJe; uor]drrrsueJt JpoJue soua8ocuoluBru lerll SursudJns tou sr lr ,srolJe; uorldrrcsue.rldq perra -xa sr uorsse.rdxaaue8 uo llo''a tf,aJrp lsotu aql aJurs 6t-SZ a:n8rg ees)sderreorrlur VNO qlr.vr.sauaS spuBsnoql Jo ;o uorsse.rdxeeql ur sof,uererJrp qlns OJnsEeru .1v\ou uEf alr terlrea possnJsrpsv .slleJ Jorunl snsral leturou ur pecnpo.rdsVNUlu ruaroJJrp slunorue er{l to Surredruoc dg parnseau ag uef, srqr dllerueurradxg .uors -sardxa euo8 ur sa8ueqcasnec dllrnluarra saue8rossarddns -Jorunl a8eurepro sauaSocuooleeJf,leql suonelnru crue8ocug

uorlelujolsue4 o)npul ue) sJol)eJuolldu)suerl real)nN lo uoll)npotd alerJdorddeul .s8n.rpo^rleuralle I Jo uorlue^ur eql SurtElrssarau,s3nrp Jeqlo qlns pue qrurle{ur ot luElsrsereq ol a^lo^e upr sllaf rorunl lEqt sr a3ua11eqr 3ur -o8uo eug 'surelord oseqr sertrlrtf,paqt Burllorluor ur IJE to .eruoua8ueunq InJOsnaq deru qrurteurrol potelar s8nrp os eqt ur peporua seseur>loursordl 96 erc orar{I .lle^,\sE ,sJotu -nl ,seseesrpesaql 8ur lpurlsalulorlsESto sr.uroJSurpnlcur -lEarl roJ slErrl ur ueeq spq pue srafuEJ ruereJJrp InJssettns ut paterrldur arp tprll soseurl aursordlroqto lera^essrrqlg -ur grurleurl 'sllal Jounl ol enbrun uralord uorlfnpsupJl -leu8rs E ot pele8rer Bn.rpref,upr rsrrJ er{t,I00Z ul VCC aqr dq poao.rddr sE.4^tr .slca;;a eprs atuos arrdsep 1yrq3 Sulrearl ur e^rlf,oJJadlqelreuar sr qrurlprur Burmoqs s1e '(og7-97 -IJl e.rn8rg eas)s11ac Ief,rurlrreryy lpurJouBurreds elrr.{^lsllel .Ihtrl ol dyq8rq sl .dll,rrlcB asrur>l strqrq leqra1 -ur puE alls e^rlfe OsEur>l lqv aql or dprerlp spurq qtrq.ry\ ,grurletul 's0d6I dlree aqr ul -Itr tr1 JoJ tuotuleerr alqrssod

ul)NV)

|

sz UlIdvH)

o

0€t t

(alrlrurlsrp -oluoJqf, .,'alnlEu8rs,,Jo Jo serJes3uo1e;o $Jrt oql dluo sell uorlef,olsuert euosourorqJ -IWf, eq1 .1uar1edaq1 sllr>l ueuo qllqllr' 'errualnay elnJe ot speal (gy ro g.gd ur ''3'e) po-ttq Surdrrec e ur uorlplntu uonJunJ-to-ssolpuo IIeJ -res 'sllel poolq etrg.ry\ roqr.unuaqt ur uorsupdxoue dq V Jo pazrralJerpql'(-IWC) erura{nel snouaSoledurJruorr{f, ueru -nq ;o aseqd lpnrur eqt ur sllnsor euaSoouonp-nq rrJerurqr al{l Jo dll^rtte aql tra.orretu euoq eqr ur IIal crlarodotetueq E ur srnf,f,ouorlef,olsuerrsnlr JI '@OZ-SZarn8rg aas)9951 ur para^oJsrp'au4osoluotqcoryQapopqd lrlsou8erp aqt selpre -ua8 1qo+tq suro; l€qt uorlpf,olsuerl Ieuosourorql aql 'uorlrnpsuprl d[erluet leu8rs3ul1e1nurr1s -od snqr 'ued rcg aqr q8norqr suralord uorlf,npsuerl-pu8rs

'L661' 'Z8S:S8€ 'L66L,uosMed arn]eN arnleN I S6S:SBE le]a nX M pue , arnleN'1661 I osleaas:209:SgE le]a uaq)rs I r.!ol](e)Uedlo^t])e ,{;anr1nlr1suot stsnqlpue/Zg aursor,{} 6urpnpur spt)eoutulegI stsnltneulo)essnog{q papo)uaurelo.rdo:uo leurure]-Jaql 0utsstul ,aseut) :t5-n6ululo;suerlaqt {1nrpeaseull)JSaq}solent})eut aursotll.telnllal lorlloue'1s3{q /ZS aursot{} 1ouorle;fuoqdsoq6 ')lS-^ pue )rS-) jo alnl)nrlsurerrJoc (q).US aursotfiuo eleqdsoqd eq16urnoutat {q palenrlte{;;eutousrsuralotd)lS relnlla) st]t os o]tsantppaseut) ,toIlnrlte aseut)aqf ont])eutI;1elyi1e1et 'sureulop aql 6ulUolslp pue oseut) EHSoqi ut suteJls leuotleul.toluo) salnpututeuopZHSoql ot (LZgJ\-d aursor{loqdsoqd LZS ,))H alnlfnlls ut saseur) )lS lelanas 1oburpurgsleuLueuJ +oeuo lo (e).uoqelnu lua6oluo ue Iq uollentpe leuotsuaultp-oalq1 pue saseutl augsolg)rS lo alnpnrls 6l-5Z lgnglJ V ,n^

dcv

ctS_A

dl_v

\]tJ-/

@:,^?

,nu,f ors-c

snurullal ;Axoqre3

snuruilol ourulv (q)

.;:^il1*l #ffi:#H? ,:;T ili,T'i3",'#*rt El

'ue{et ueeq f,pua4e seq dderoql ol dots puores IntssaJf,ns .sarde-reql ,srsou8erprerlrea reqr '11aq3 esuf, aql u1 ,/,aeu pue Io 'aseesrpeqr;o Sulpuetsrepun ralear8 roJ dtlunr.loddo ue slue -serd asecq)eg.'kZ ratdeq3) saua8 xo11 ;o sroreln8e.rleuorl -dt.rrsuerl telncrlred ur,s.ro1e1n8a.r leuorldr.rcsue.rlBurpor -ua saua8e^lo^ur suorsnJaua8 eqr;o due14 '\Z-SZ arn8rg) prtue{nel Jo sruroJrelnrrt.red or pa>lull suorlerolsuerl or.uos

THS

t€tt

s N E r o u d 9 N t r o t ^ o u d - H l - oAu 9 N I s N o l r v l n W ) l N l 9 o ) N o

'uoIlEr0JII 'Surputq -ord I]ar Iorluor ot uorlpln8erlpuolldlrf,suertpue sdoldurereqr VNC ul suorterJel'uorlercosseutato.rd-utelo.rd >lro.{\teudroleln8a; E luro} sutalord eseqt IIe 'reqte8o1 'uorl -drrcsuen {rolq or dloq reqr saseldtaceap euolslq ur Surrq ol uralord rosserder-oc tuIS eql qlIA >lro1!\lur^[ pue pE]/{ 'saua8 c,try or (1 rordeq3 aas'uorldrnsuert o]€lntulls laS.ret .{11ensn qJlr{lrt) seseraJsuerlldlareeuolsrg Sututeluor saxeld -uor Surd;rpou-ulleruorqc Sutttn.rlardq uorldtnsuerl peJJe saxelduoc uralord rd14 'uotleuJoJ Joulnt ol solnqlrluol IPIJI dtr.Lrtrerd14 altssacxe uI uIeJ ol 'sutatord PeW el€lnrulls reqr s8nrp ro'suralord pery SursnuI lserelul uE ot Pal seq qrrq,ra.'suretord cdry rlqlrlul sutalord pelaq'sutlodc sE l{lns 'uorlereyrlsrd srelulp xery-cdy'q Iorluol legl saua8a1e1n8a.r 'tury pue 'pery 'rd1aqqtl^\ ezlrelulporeteq uer xEIAI ']uW pue 'xeyq 'peytr apnlrut las urato;d eql Jo sragtueturaqlo 'saua8 ta8ret Jo uortdrrrsuprt aql aleln8er dlaleutprooc pue 'VNC or pulq 'suorleutqtuoJsnolre,l uI azIJeuIPuEf, t e q t s u r a l o : dS u r l l e l a t u tJ o t J Sp 1 o u e d s Es t l e r e q t u r a l o : d nddrz xrlaq-doo1-xlleqllseq E seporue eua8t{.tu-c aq1 'uratord c^,(y,q leurou esl^\rerlloeIJtJo uollJnpo.ld g8rq uI sJnflo dlalerrdorddeursesnuJosle 'srorun] uerunq IEJeAas qcrq.r,r'aua8 t[.u aql SututBtuor YNC Jo lueru8ase Jo uoll -erryrldue pozllelol 'snoreouer euoJeq or l]rr eql SuISnel 'passardxadllenurtuor st 'reruequa eua8 dpoqrlue aqr dq 's11ec Surcnpord pareln8ar ,trou 'aue8 cKw pawolsuert eql -dpoqrrue Jo uolteJnl€ru Surrnp JnJJo letll sluauraSue.rreer VNC IpruJoueIJtJo uolleJJegeeJEJe sI uolleJo]suvtl c{'tu-c eql'rc7-SZ arn8rg) sller poolq atrq.u Suonpord-dpoqtrue ur e^rlre dlleurou are qf,rl{,\\ 'seua8 dpogrtue ureqc-d,Leaq orll reau etls E 01 poleJolsuerl st aua8z('ru-c etTt'awoqdut[4 'sluslu s,11t4.tngsE u,/\^ou>lrolunl uElunq eql to sllol uI -Eqf,atutuoJel;rp dq rncco uBl auaSocuouE olul auaSocuo -olord tKtu-t aqt Jo uolsraluo3 'pa^Il-lroqs urelord pue vNuru sol rl{r elPtu lEqr saruanbasrql Jo suollalaprrrauaS eAIoAurauaSoluo ue ot eue8 IEuIJouE tuoJ, sorl-curnr teqr

sa8ueqr oqr ]o eruos 'POJnPurare saua8eql rolte ssol prder dllecrsurrlurere aporuo lagl surat rraql ol Surpeay'elqelsun -o.rd soC-l 'slPc lerurou u1 olyg-r pue sVNUru agl pue 'sla^01 PerBlnberunPUEqtsIqrP pessardxe.{puanba.r;oJe srolreJ uorldrrcsuerl rerllo ro esoql .sJoLunluI 'uoIlISuEJr 5-ol-t9 oqr Jo sruJot ctueSotuo eql tC ar{r q8norqt uotssarSo-rd aloru pue e1c.,{r IIer erlr ;o aseqd -o.rdler{l sutetord Surpocuasaua8;o uotldt.rrsuerl elelnrulls sV 'rarueJ PIoAe odry-c pue soJ-l '67 rerdeq3 uI PJSsnf,sIp 'srnoq ot dlaq 's11arleur.rouut ',(etu letll lleJJOd;olelnSer e '(77-97 atn ,t,r,et E ulqlr^a aullrap sutalord qloq Jo sle^ol eql -3rg) odyq-r Jo eslr pa8uolord arolu e ralsl PUEsoC-f,ro esu 'sauaSoruo-olotd c{t'u pue soJ tuersueJl E sI araql d1lerrrul

qrurleLuI

urolorouorsnll8v-u38 (c)

6urputqalellsqns sl!q!Llu!pup olls oAllce o ] s p u l qq l u l l e u l

alelell lolo louues llos loulnl

| g€zvizg (r)uedl olrsantl)elqvrlg aq] q)reasaa Luor+ rrue)'ZO1Z' 1e1areOeS o t p u n o qq r u r l e u(rrl) / i l t n t p ea s e u ts) l ts ] ! q l q upl u el q v r ) g ] o o ] l s u o l l ) n p s u e l ] - l e uabl rdst i l n u . t a n r i ) eo q ]o ] s p u r qq r u t l e u lsl u t e l o l d uotsnl e st utalo.td salelfuoqdsoqd 1eq1eseurlanrlte{1anr1n1r}suo) l q v - r ) €a q I ( q ) u o r l e ) o l s u eortlu o s o u . r oer qr ql d ; e p e l reqq6l I q poruro+ urelorduotsnlttuabotuootli pue 'ZZpue6 sou.losouiolql eulosot!orqletqdlepeltq6 e LUo.1,t lo sdrleq] +ouotle)olsuer] eq] lo urbug(e)'aseull utelold lqv-.r)g OZ-SZ :lUnglJ V

(q)

uraloJo u o r s n ll 8 v - u c 8

{-

uorlelsuerl /uorlorJcsue{

(e)

euroqdur^l e olut qlMol6 itaq] a)uaq pue sa]I)oqduIl ur ro1:e1uorldutsuerl r{y1 aq11o uorl>npotdteno oi 6urpeal,(Hl) uteq) {neeqIpoqllue eq1 yed ro] aua6 aq1o1luerelpepete;dsr aua6tt{w->aq} ,tl pue ;o uaamlaq uotle)olsueJ]e ]o ]lnsoJe sy .eruoqdru{; 8 sor.rJosoLuolq) s,lltlrng u! uotle)olsuerl leuosoruorq) €z_gzlunglJ v euoqduAl s,ll!Irng +olL cAuJ-c

ur)NV)

|

S ZU t r a v u :

zEll

dlrluapr ol dlrunuoddo ,4eu e sapr,rordsllof, lerurou pue sllol rof,uer ueo.&\taq eJuoraJJrp qrea'1e.reua8 u1 ddereqr^\eu InJro -.rr\ode or ppel uec-urelord lqv Jql Jo uorlrB rBlnf,eloruerlr Jo dre,Loosrp-fJlsrtueqlolq qlrm raqnSot-seleorJ 1r eua8ocuo Ierrlrrr er{r pue eruosoruorr{l eqdlepepq4 aql 1o d.re,tocsrpeqr -scrtaue8 ^\oq selelsnllr rerlrpepeqrrlsopdrols qrurteur aq1

paleaJl sl re)ue) ^ o H 6 u t 6 u e q ) s l f 6 o ; o r g l l o ) r e l n ) o l o ll

na

-8o.rdro ueSortsa;o q8rq E reqtarl^\ euruJalep ol serpoq 1a.ta1 (srsouSerp -rlue qlr.r pelset sr pup oqt {leqr ol ua>lelsr sseru .suorleuruexa (der anssrt ur-Z-I e lo lsdorq e ,,{11ecrdd1 -x) ue-rSourupur eurlnoJSurrnp pasou8erpuaUo are sref,ueJ tseoJfl 'perJJeJaJp suorlelntu ureuef, Jr pes€aJJursr dcuenb -e{ eqf tnq 'um.ou>1un sr ref,upr tseerq Jo esneJer{I 'sqteep JeJuEl s(uaruo,\\to JSnpJluenbe{ lsotu puof,aseql surerueJ t1 pue 'uauol\ roJ Jatuer dypeeplsotu eql sen\ ref,ueJlseoJq tsrelous ueruo.lA ur eseoJlur up tuo{ Burllnsa_r laruec Bunl .e,rrlerl1ed pup elrternr Jo af,ueprturer{t ur esrraqr lrtufl 'sluatutea.rt perJarJe a,req sanbruqrat lSoyorq IIat rpln -ralou ,r\or{ aldruexa poo8 e sapr.ro.rdroruef, tseerg Jo 'sllar JaJueJ dlasrcerd lsn[ ]eBJet eroru teql stuatulperl raruerrlue dola,Lapol srer{f,reese-r dq palroldxe oq uel leql uorleruroJurIeJrlrrr sr sro(unl agr 1o , Bolorq IIel Elnleloru aql yo e8pal.nou>lsngl .qlro.r8 pelloJtuorun rraqt sdols lseal tE ro sllol reJuef,aql lsnl sllDl rer.{llua{ul?ert .ro Bn.rporpads e

$uaDS' L66L I t90 [_650L:81,2 ')oo'l urorll setule)nel]o sspl)letauebaq] +osluelleArelntryed I burletrpursopol Jolo)]o ]es e srlreqt ard q)eo ,lnolegpanjasqo uaoqseq luauabueleer autosoulotq)ou aloqMsase)ol slolal 'sauab uMou)un 1a,ise 6ur1>ege s)ealq oLuosoLuoJr.l) ,,ouoN,,pue ol sJalol,,ulopueu,,sluaue6ueleel aulosou.loJq) lualegrp{q posne)aseastp aq] +osase)1o e6eluettadaql ole)tputsabpenn aql 1o saztsaq1 etula)naltrlItolaIu olnle : lt\V:erula)na; l1{toqdLu{1 aln)e : llv slol)e] uorldu:suetlate surelotduotsn+aql ,lqv 'setu.talnol aln)e asne) pue -o6 saue6oruo 1o uorldetxaoq] q]lM urol leql suotle)olsuerl euosoruorqf Lz-sz lungll v proleAurosranrp'cr1seldsApo1eA61 pz crlselqouot4 tz

cry{couor.uo;al61 pz

crlAco;eAuo.rg cr1selqo1eA4 pz

I LL:g)t'(Ll: LL)t'(Lt:91)r (9j0)l

rtlw-wdN {6:9)}

NV3-)EO

n ava-wdN n avu-Jz1d n avu-1wd

(^:€)l

IIAl

(LZ:91)l

9A3-Snt

./.1

( LL : t ) l

6VXOHA6dNN

( tz:8)l

'+]lZ I lpue6taquearg ,rotrn,,o,tllr3l;:::ll:|.o'3:j I 1,,\jaasl

pepo)uo esoqan 5r(ul-> pue sol-) uoriserdxa elnpur o] srsrolle+ 1o qlnnot6 eq]+oauo sllatluetselnb 1ospa#a]satllea 1oqlnnor6 aql elelnurlsleq] (l9Cd)rolte; qlnnorO panuap-1a1a1e;d a)rl slolle+ sureluo)uunte5'r{y11-rpue soJ-) ,spnpo.rdeua6oruo_o1o.rd orvr} 1o flrnrpe aql ut eseoJ)utpalreu e sp;arI slle) €I€ luersarnb ol ulnroslo uolltppv zz-523un9ll tvlNlwluldxl v slnoH OL

89

salnutlA 06

09

0e

IlAV llec g-ud v

llac 8-ord

|ax87

( 6 tj t t D ' {t t l [ ) ] ' (t r j r ) l

pappe uJnros

suorsnl-77r4/ %9

0

tpsrv

(6tj1t)l

J1H-VZl (6rjrD o/.1 LXgd-VZ] (zz:alt'18: zlt'ft L:alt

3AW gDUcJrLbrt JO

daxJJgtbL

YO 0)

o o

oAlA-3 ^r

JAW ZOWI zlvt Lowl

%n tzz:6D

IIVI LIXOH t1A1

18V'ACg

S,

sol-c

E E II

s N t S t - o u 9d N U - o N o u d - H l M o u 9N l s N o l l - v r n n ) l N l 9 0 ) N o

'rrxol dlqrssodpue a.tnce;Jeureg II.^a teql sarpoquue.ro s8nrp Jo asn oql sotnper pue stuaued 1en 'rorunl rEI -pl^pul Jo lueulport o^nJJJtJarow slturad srql -ncrlred e ;o saruadord eql la8rel tpql stuolulea.pdpogrlue pue s8n.rp1o uorlerrldde aqt Suurolle sI srolunl IEnpI^IpuI Surzrralcereqr.ro1sanbrur{lol Jplnralou to luo^pe aI{I r 'uorteln8ar IIer-rolunl yo Surpuelsrepunoql Surnurluor uo paseq 'sluatulea.rr;o Surla8rel ur sas€eJf,ur roDeq llrts ro; snedsord poo8 are areql 'drrlerroru Jafuef, lsEerq uI uollfnPer IslluElsqns Po,^aollea^BL{sluoueulter eseql 'rount relnrrl.red E Jo esef,relncrlred e .royleuudo eq ot dla>1yare teql stuetulperl Snrp pale8rel dlq8tq;o asn relnlalou aslJerd^r sr srorunl .,(1ree aqr 8ur.tro11e yo srs.{1eue '$Z-SZ ernblg aes)cdry-r to uorlrnpordralo ot Surpeel'aua8 dpoqrlue ue ot asolr petelolsuerl sr t[,tu-t's11aceuroqdudl s,llrlrng u1 'uorleruJo;sueJleJnpur uec c1141 pue 'unf 'sog se qf,nssJol -ce} uorldrDsuEJl JEelrnu to uollrnpord eretrdorddeul r 'qr.r,ro.r3 'slleJluElnu Jo Juoll e olut s,r,rol3llJl Jtll pellortuoJun sesnerurorord .vrauJr{r sererrr legl IIOI poolg e ur luale Sururola.rpue a8elea.rqauosotuoJqJ aJEJV 'sulel -ord uorsn; pue saua8uorsn; .4eu elpeJJlet{l sluatue8uerrear eurosoruorq) ot enp os]Eere elua{nal Jo sosertaqto due141r '62-SZ a.rn8rlees)sJSEU -r{ pareler dq ua,rr.rpere teql srefuel rel{}o ^\aJ B tsute8e ctuorqo 8ut 1ro,u,feru pue (-II\ll) eruralnol snouaSoyadru -teaD ur a^rtreJJosr (1o^eelCro 'qlulleurr) rollqlqul asEur>l 'tcaga rrueSoruo stl .ro1elqrsuodsarst utaiordocuo IqV uV 'aua8 Iqv-rrg aqt 1o dlrrrnf,peseull 1qy pereln8erun eq1 -oruo lqp-nq rrrarrn{f, eql sacnpord}pqr uorlef,olsuertIPIU -osoruorqf € ruorJ sllnser etuosotuort{f,etqdlepelq4 egI r 's11ardueu Jo uonerell]ord lerurouqe salourord aursordt srqr >llEI teql suralordof,uoJJS;o dtr.trtcePelEIn -3erunaql'(6I-SZ ern8rgaas)snutruret-Jeql reau onplser aursordt e ;o uorreld.roqdsoqdappue uorteldroqdsoqd alqrsro^or .{g parep8er sr dlleurrou 'aseur>1eutsor.(l-utel -ord Sunnpsuerl-leu8rsrrlosoldc E (JJSJo d1t,a,t1re aqJ I 'Jtets dqa;aqt Sututetuteur pelelttJE uE ur tt 'dlr,rrlce asedlD slr seJnpeJ 'uor1er1ua.re;Jlp pue uolre -ra;rlord 11acatouo.rd teqt s.temqledSurleu8rsdueur ur urol -o.rd Surcnpsuerrda1 e 'seg uI uolletntu turod el3ursy I 'srotre; glmo.r8 letu -rou SutsnBc Jo aruesqpaql ur Surleu8tsSuttoruord-qr,r,ror8 'suralord uoltrnpsueJl-leu8rs relnlleteJlul eJolu Jo euo to suJJoJarrrlcedlalrtnlrlsuor aonpord slleJ Jotunl lsow I 'sleu8rs lerurou Jo eluasqe aql uI uorlpra;r1ordlac Surlelnurts dqaraqr 'srotre; qurro.r8ro; srotdacerilel-lsorl elplrlJp pue ol pulq uEJ sutato.rdpepoJue-snJlluleue3 r

el{rtffi".iiitttt:X IIerl,rurouq'o] pEarpu' 'aJree..-,ES

-derer rotlB]-I{l^\orB ;o uorlcnpord.re^,r,9'sllar ruroJsuPrl uer leqt uorssardxeaua8 ut sa8ueqcsernPul dlaleutlln u o n e ^ u J e q r n s ' ( / I - S Z p u u 9 I - S Z s e r n S r ge a s )d l r , r t l r e rotdaca.r elllnlllsuoo or ppal spue8rl IEIUrou rlaq] Jo

eruosqe eql uI ezueIrIIPol srolreJ ql.uor8 JoJ s)IU rltu -red lep suollefolsu€Jt IeluosouoJt{f, Jo suorl€lnw I sulaloJd 6ul1ouror6-qil oJg ut suollelnlN )!ue6o)uo

uef,a^\tuaulr'erlto.xli]ilfft:i aasdpearle ur sser'ord

'sller rounl s(]enPI^IPuIue 10 serlredordrelncrlred eqr or 'lsrlJ 'ere paunl 'puocas 'pue sseluJPqPUPe^IlletIo erotu l€qt PorolslunuPe aq ol sSnrp ^\olle III/v\ Jefu?J ;o d3o1o - l q l l r r r e l n r e l o u a q t 1 o a 8 p e l l r o u l p e s e r J l u l' a S e u u p1 e ; a -tplloJ pue dtnrxor Surcnpe.r snql'pecnpe; dlletluetsqnsaq uorl€IP 11r,ndra8rns uarra sdeqred pue .{dereqlotueqJ PUP -et rcJ POeuaqr lEql sr euIfIPOu to Ornlnt erF roJ uorsl^ aql 'o>lIIplno.,!\ouo ueql ssel rEJ 11rsq8noql I 'saler uIIlrIA Jefuel lseeJq uo lf,PduJIe8nq e 3ur 1e,tr,r.rns -^€q sr dSolorqrplnrelou snq1 'ulJlordoluo neN/ZreH eqr lsure8e selpoqllue qrl^\ luoul€orl dq luaJrad 61-9 raqro -ue pernpar sr dlrleuoyq 'sller raJuel eql uo luasa-rdrol -darar euouroq E s^\ot{sreqr EIEPrelnJelou aql Surlroldxe 'uelrxouret sE rlJns luauleorl 3ur1colq-auouroq dq tuer -.rad91-61 .{q parnpar oq uEJ IsIr sII{I'ref,uef, raq ot 3ur -qunrf,ns Jo >lslrrea.,(-61 luacred 0t-0€ e seq (apou qdudl enrlrsod 1 'torunt ruc-7 'p1o s.rued99) ueluo,\\ lstr-a8era,re 'uolleluroJ ue 'tuetuleorr slql IIe elrdsaq IIel Poolq per olEI -nurus or (91 ratdeq3 SodE)urlarodorqldrapue (suotlce;ut adft e) le8un; pue leuelreq slq8r; rrqr IIer Poolq elII{^ Jo uorr€urrot lqdorrnau etoruord 01 (9I rardeq3) {S)-D Joi -ce; ql.tror8 aqt ua,'rr8ere sluerled 'd1aq o1 'dlddns ua3.{xo 'uralsds esnef,PuE 'uotlca;ur Surlsrr rood ol onp ssouIEO.&\ 'dqtedornau eunruull aqr yo qr8uerls eql ernPor uel sII{J 'uotlcnpord pue 'easneu'sso1rreq IIOIPoolq;o uorssarddns 'rena Surpnlour slleJJOapIS Jo Al:ttvtt € esneJ osle daql -.uoq tsylar roruef, Surprzrrpeql III{ or pauSrsapeJe sluour -tpel qsreq aseql 'slua8e ;o saddr ruorettlP earqr qlrm dde -raqlouaql to s>lea^\8 PUEuoIlEIpPJ to s>leo^/r 9 so^lo^ul 'JolleI rrlsouSo.rdas-rarrpe .roleur lueruleaJl luenbasqnseql JoJ sJpou qdu,i1 eql sr qlrq,^a'Joutnl rllelselaul Jo af,uoPIAO uorlrese.rlecr8 Jo uorteulrupxo Pue roulnl oqr 1o (le,roruel) -rns sr dals lsrr; aq1 'dderaqrouel{f, pue 'dderagr uoIlEIPBr idra8rnsJo uolleulqurof, E t{]IA\ PelPoJlsI Jeruef, lseaJg 'zreHIo slunoulE alE -rapou ecnpord terlr sllol (raqlo pue) lseorg Ieurou uo lJaJ -;o due lnoqlr.tr sllor reruer aqr 3u1111dla,tureles luaredde 'paztleurelut eq or rl sasneJ Pue TraH sezlubo)er poolq oql otur polra[uI dpogrrue ueH'TreH arnpord-ra,roterll sref,uPf, e rseerqJo resqnsaql roJ lueulearl ^/\\euIntssalcns^t18ur4rns '7 rordar uaoq spg ZreH roJcr;neds dpoqrrue leuolf,ouou y -er cca soPorue 'ueas arr.eqe/\\ se qllq^l 'rr7511747Fl eua8 -oruo-olord eqt Jo uoll€f,r;r1druero; Pelselosle sr dsdorqaq1 'ouoruroq Surtelnruns-qt.Lror8e Jo sllol rorunl aqr a,rrrdep 'uaJlxolu ol pesneq uer 'roldecarueSorlseeql slrqqut r{rlq^\ -et pe[e] Snrp y 'tuotuleerl aql ur perroldxa sI lI 'luasard st q8rq le pessard roldeoar raqrlo JI 'sllar ref,ueJlseorq uI s1e,ra1 -xe serullaluosore pue 'ql.tro.r8rotunl Surlelnurls ;o olqed -El aJEs;otdarar PloJalsJseqJ 'luJse.ldsr :otda:al ouoJalsJ

'gld p uorlElnrunf,fE ot spEel turod PauoDuaIUsld eIIl uorlf,rrtseraqt qBnorr{1aBessedot ror-rdIe.4^erpt{tr-^A ueBotrry 'lurod uorllrrtseJ eqt lsed uorssarSordsatouo.rd ftr,rrtre aseu 'saxalduror -r>l esor{,{\ yq3-urlrdc aartre d11ecrl.(1e1ec elera -ua8 or pue sreulred rrer{t elquosse qlr,\\ surlJ 9)Cl tXIl 's-rolcey -dc asaql 'suatolrur .ro :e1n11acertxe qu,ror8 dueu dq parnpur sr saua8 urlcdr addr-C uorsserdxa oq1 to 'tutod uortcr-rlsar eql aBBssed gSnorqr alEln8ar uats.{s teqt egl stuaruele Iortuof, Jo IIE are urato.rd .surlrfo adll-q qU eqr pue'(syq3) seseur{ tuapuadap-uqcdc 'kt-OZ ern8rgaas)y51q stl Sunerrlda.r aseqdS eqt Bur pue -Jalueol 'lurod uorlf,rJlseJ dlqrsraaarrr seluoJeq lr PellJLutuol 0gr pallpl 'rC elEIur lurod uretrer e tsed sassarSo.rd e eruo 11ac

) r u o 6 o ) u o a t v a s e q ds o l r g u o l l o6essedpoleln6otun oloruordleql suotlelnn :rJJue)ol JtnqlJluoJro tuaudo;a,r -ep lpruJouqp ot peel uaqo ruals,{ssrql ur suorlptnru ,patced -xa aq rqSru sV 'snoJaJuerSuruoraq uoJJ sllal tua,rard ot suortJunJruatsds epdr-I1acelorJl!\oql .apl^lp ol lou (qlpap Iorluor lseal le Jo l1arperuuerSord errreprfrnslrurruoJ ol Jeql -re palrerrp erp sllet petsorreaql Xlaaneuraryelparrederaq ot aBEruEpyNC eLIrroJ erurl s.l^olletserre sn{I .uorle8er8es eruosoruorqroroJoqz5 ur.ro parerrldar sl VNC rraqt eroJeq patserreere d11eu.rouVNC rraqt ol a8erueppaurelsnse Er{ tpqt sllal'uortrppe uJ 'srsolru Suunp uorsrlrpIIal pue uorte8 -ar8aseuosoruorqJ pue ,eseqd aqr Bur.rnpsrseqrudsy11q 5 (zC pue tg Surrnp qlrto;8 JelnlloJ Jo uorlpurprooc .radord eql sernsseruatsds,{.role1n3a.r slr{J .(tt-02 arn8rg aas) srs -ollu pue 'zC 'S 'IC :seseqdureru JnoJ stsrsuof,qtrq.u .ap Jo -dr lyaoaqr q8norqr uorssarSordrrerJtpue olul slleJyo drrua eql IoJluoJ dlasna-rdsurolord Surtce-o,rrte8au pup -aarlrsod r{1og 'suorlplnur oruaSoluo ro} staSret arur.rda.realcdr 11ac crlodre>1naaqr Surreyn8arrot srusrupqreu xaldruol eql 'uorlrqrqur qr.,vror8dlDI ol a,rrsuodsarunorp ero;areqt pue srotdarer dCCI IeuonlunJ >ltel sllor ref,uer uolor pue pruols€lqouner lsraruer orlea.rrued ueunq ,(ueu ur sJnJJo aua? ypntug eql Jo uortelop 'aldruexa roC .sreJuer ueunq Jo {teue^ E ur punot ueeqpEJ ur e^pq suortelnurqrry.GZ_SZ arn8rg) sllal Jolunl 'o srsElsElarupuE sseue^rse^ureql ol elnqrJluoJ dlqeqord pue uorlpreJrlord 11aoatoruo:d sngl sper.usur ro sroldara.rfl491 raqlre ur suorlEtn(uuorlfun1-Jo-sso-I'xrrleru eqr to uorteper8ep pezdletec-uruseydaqr sernpor qllq^\ '(I-IVd) I rorlqHul rolellrre ueSouruseldpue surelordxrleru rplnllelertxo Surpocuasaua8yo uorssa.rdxe satoruord osle Surleu8tsdgCt 'IC ur lserre ot sllrl sosnprrltglr\ ,(t)Cl) t eseur1 luepuedep-urycdr;o rorlqlqul ue ,gyd Burpocua aua8 aql ;o uorssardxaatoruord ueJ sperus snalrnu eqt ol Surlerolsue-rtrertv '(y-91 ern8rg aas)s.rotceyuorldrrcsue.rl perus rrlosoldr Jo uorle,Lrtf,esaf,npur.rotdarer str or dCCI 'sllac ruelsds eunrurur pue lerlaqrrdelsoru Burpnlc 1o Surpurg -ul (seddl ,eureu slr alrds lyacdueu Jo uorleratrlord slrqrqur -eP '(dCCI) 'alduexa rog rorcet qrzvro.r8 Sururoysuen d 'PePeeuerE daqr uaqlrr sde,vrqred Surlrqrqur-qr,t.or8 dtr.r.rtre yo oqt eseerr -eP teqr suorlelnru er€ .sde,la.qred Surtoruord lefrlrJf se tsnf

U 3 ) N V ) | s ZU l l - d V H ) o

ttLL

-qulor8 Jo dlIAIltr€elerrdo.rddeuresnef,uorlles snorzrerdeql ur pessnfsrPsuorlplnur eqr to rsow 'reJuef ot pEal u?f, efue -leq srqr ldnrsrp leqt suortetnpsdernqred Surlrqrqur-qr.vror8 pue Sunoruord-qlvror8 uae^ueq eluelpq paleyn8ar itqStq 'paunl dlaug e uo spuadap luaudola,rap pue qlra.or8leuroN

slotluo) el)^)-l le) pue 6u tltqtqul-rll/l^olg

lo sso'lbursnelsuollelnw ' lrgO€tZL^ae I sauag'8661le ta enH X aa51aseettur(6113) xuleurrelnllo)et1xa 6urpunolnseql +o uotse^ut pue uorletellold 'uotlelnul CVnS e .rouotlelnultoldeter e .joq]!oo16urnno lla) Ourleu6rs fl391 anrpegejo a)uosqeoq] ul slol)ej uorldutsuetl peurs uotlenr])e sasne)lo])el qlmolbtlueue ,dlDl +o '6ur;eu6rs dlCf +o ssol lo 1ra#J 1Z-SZlungll V ,Lo6urpurg sutalotd xuleu Jelnllocerlxa saperOap leql ssealord lo rollq!qur butpoJue aua6 lo uorldlrcsuerl

stselselau 'acuaq'pue uollepei6ap xr/leu ,elnllaSellxo pssearcu!smolle l-lvd lo uorlcnpoJdpesearcac

uolleJal!loJd sasearcut gld lo uorlcnpotd paseatcao

snalcnN

teubtsdlgf lcolq sroldacai ut suollelnu uo!lcunf-lo-sso'l

(a\

v.) gl9l-

lllll>

A"*qled 6ulleu6ls drcr :uolleuluvsn)oJ

s€il

sl0utNo)

3 ' l ) A ) - ' 1 ' l : t )o N V g N r l l S l H N l - H 1 AO U 9 l O S S O ' I9 N l S n V ) S N O l l v r n w

'aue3 ta8rel aql Jo uorsseJdxour sa8upr{Jesnef IIrl\\ suratord CNS ro aqt Surporua saua8aqt ur suorlelnru 'se8ueqcurleu IIA.S -orr.{rpeterperu-JNS/IIA.S dq pesserdarro pale^rtre dlyeru.rou sr aua8e;1 'uortdrrcsuerl Iorluor teql suralord Surpurq-ygq ol elqrssafJeurro alqrsselresaua8a4erusaxeldruol {NS/IINS 'saruosoalonu seJnlJnJlsJo suorlrsod eql ur sa8uegr Jo Sursnecdg '(1 rardeq3) Suqepourer-urleuorqf,pu€ uonerlt -rpou euolsl{ Iorluoc ueuo pue eserlleq ruapuadap-4Jv ue eror rraql lE olerl saxalduoc uralordrllntu osro^rppue a3.re1 aser{I 'reall dlSurseerourouoreq s?q lorluor leuorldrrcsuerl ur 'xaldruor CNS/IINS aql s€ qrns 'saxaldtuoc Sutlapowal 'soJnlJnJls -utlpwotq) ;o acuelrodur aql sreed luacar uI urlEuroJgf,a,rrssarderdq pecuels oq oslE uBc sauaS;o saddr asaql'rerrazrtoll'seuo8 rossarddns-.rorunl Surle,trlreur dq IoJluoJ quro.r8 auruJepun UEJsuortelnru,{\oq uoesaAEqolN

srolunl ol elnquluo) surelord6ullapouau-ultPruorq) 6uplre;;y suorlelnn uorl)unl-lo-sso-l ireruef ro; a8elsorp tes pup lorluor quvror8leluJou ue^gns ot dressecausr tpqt ile sr lurod uorlJrrlsaraqr q8norqt a8essed Surllorluoc .ro; uratsdsd.rolelnSersrql Jo luauodruoc euo dluo 'qU ad&-pprrturelar dllereue8uoncun; 91d rsoy ]o ssol snql e^pq ro 1q urlcdc ecnpordrerrorerlr sller rount tlasra.tuo3 'ureto.rd 91d leuorrount pue 1q uqodoJo sle^el]pturou etnp -o.rd srotunl dyyeraua8 rl suorlplnru Surle,lrloeur qU glr^ 'luasard ly 'ans 'Jaf,uef, lef,rlraf ur dpo rnJro ol u1!\ou>lsr srql -srt Surrnpord-sn.rrrr alperl ot >lf,rrlIErr^ltseu reqtoue '(AdH) snrr,reurollrdedueurnq dq peporue sr ter{t '19 pateu8rsep 'uratord dq d.rorrgrqur ue;o Surpurg eqr eq uer uoll pereunuqo -lun; uortrppe q'u,{\op SuDB,rqceur 01 qU'suorlBtnru rnqs,(11e 'JeJuefSuttua,r -nluale dem eJe suortfunJ tagioue ro ouo q5 'qg yo reqr qrl,n -a.rdro1 lef,nur os lou sr qU to ssol snqr pue tuppunper sr uortrunJ asoq.r lggd ot polEIeJdlemlon.rrs qtoq ''3'a) suretordror1toof,npord,{1a41tsoru 'ons 'gg1d pue 1g1d 'sanssnosaql'(rappelq puE (tsPerg'3un1;o -srl Isurler e4r1un seruounJeJ''3'a) eq ur relpl asrJeteqt sref,ueruoruruoJeJotu ur punoJ sr osle uorlf,uny eua8 gU Jo ssol ta,ralrropl 'rof,uer to e 'eurotselqourrarpooqplql or peel selellp addl erBr .(1arrr1e1a.r {pEOJIEuaesal,il ;o ,i1tnt1le ' asoqM ale spnpold (aserau{;od VNClo} partnbat VNC 6 e)srsaqluls pelelpaLU-lZl sluanald saueb,iueu+ouotlentpe ;euorldtllsuetl spulqutalold slo})e}uotldtrlsuell pueJZi pa;;n{lenrpa;;ol ^qaraLli ';o.4uorlutod uolplrlsau 9Z-SZlgnglJ V qg peleltuoqdsoqdul srsaqluAsy116 ro] pepoousurololoIo erl ;o lolen tlcv uorldr.rcsu

\--

d d d

FIF t)o3

JZf

oseull a^!lcv

ccAS

)21

qu

\___Y-_/ srsaqluAsVNC lol perrnbersutelo.td;o uorldr.rcsuell Jo rossoldaU

F)"c],\

@2

oseull a^!lceul

'a1cdr 1ec eql q8norqr sllef,e^IrP or sdlaq 1q urlcdr erxe aql lracuec lseeJq uerunrl uI uorutuoc st utotord 1q urpdc aql 'o uolpnpo.rdra,toluElltuoruof, pue aue8 Jg utlcKt eqt yo uonecr;rldluv 'af,rlu ctua8suerl esoql uI pedole,t -ep srorunl lseerq dlentue^e PuP 'uotlele;t1ord:addq lueluap -un sller letrnp eqr dlerrrul 'silol IEllnP dreurureruro; rrlrceds raJuequo ue Jo Iorluof, rapun paceld sBm aua8 yq urycf't arp qfrql\ ur ocrtucrue8sueJlqll.a salpnls Lq um'oqssem utalo.rdoc -uo uE se uorlrunJ u€l IC urlcdr reql (:oqrea PossnlslPs[0] etuoqdrudl s(DrIJng ur uolleJolsuerr 2Kt4-Joql ol snoSoleue sr uouauouaqd srql) 'sPuSrs rplnllof,sJlxe 1o azrrlredsar -rr'epdc ilel eqt lnoq8norqt uorlcnPo;d 1q uqodr pale,tela3ur -snel 'raruequaauaS-dpogltueuE Jo lorluol JaPunsI uotldrrcs -usrl slr teql rlJns Potef,olsuerlst aua8 7q ur1t[t aql (oJuelsul .ro1 'saldcoqdrudl g Sunnpo.rd-dpoqllue Jo srolunl ulelrer 'aldtuexa ro; 'sutlcdc uI 'sJof,uef,u€tunq dueu ut Punot eJE eddr-q oerqr oqr Io ouo '1q urlcdc to sle^al pare^el[ 'G1-OZ arn8rg osle aostSZ-SZa.rn8rg)sleuSrsqr-tror8re1n1 -lafeJtxe rodord oqr Jo aJuesgeol{l uI aseqd5 aqr orur pa11ad -ord are sllel aqt leql qlns de,rtqredsqr to slueuodtuoc eql sesnecleql uorlrlnur crue8 Jo auo to ssol Jo uollf,npoJdra,lo'slsoqluds -oruo uE uleluof, srotunl lso6 y1qq ol ilel eql slrturuo) dlqtsrerreutCZg uroq uollelf,osseslPsll Pue qd Jo 'uolsserSord uorlrldroqdsoqd eraldurol ot{I 5lurleq IIIM saxaldurorJelrturspue {NS/II(S 'uottdtrlsuerl etelrlf,E ot lllrqE potnpal eleq sratuo8rlo qfns leqt patcedxe sr 1r '1or1uocyeuorldr.rrsueJlto stcedsBdueur 'llunqns e^Ilf,aJap euo ur?tuoJ sreuoSrlo lsEO] te eql os ur saxelduoc pe^lo^ur Suryapourar-urleruorrlf rltrlN PUP IIr,,!\ 'uoltlsuprt alcdc IC eqt Surlourordur a,rnce.(y1n; ile dilenlrrl osnereqltr.tuce tsd IIE lsorulp elpSorqeuEf,IIer E l1acSlrl eqr ol apdr 11ac;o uorssardxapeuarqSragot spprl S{NS Jo ssol Jo Suqnqr.rluoc's11er retq8nepot uo possedere sluerua8uerre eql feql puE (SJouJnluel'unqPu€ esnou;o dtrrelrurs uorssard lpr{t 'aleclldar uer yI\Iq pa8e -er -xa-aue8eql pa^\oqs sorpntsaseql 'srorunr oqr ul se8ueqr VNC Pu€ suorlElnur Surtenladrad -ruep Xlredord ateradotou seop .{rolelnSarJelotsrp ol pesn oJel/rserpnlsder.reo.rclu.pa,r1o,t Ioluof, rurod4raqctg ggd aqt 'elolle uaql6 'sJOJuEfueunq;o ruer.rad ueqt JJor! ur rncco oua8 -ur usrueqf,er.u oqr u,lrou{ sem Surqlou arurs 0S Jo Ieuorl -run1 Surureurar or.{t e^er1 tSd or{l ur suor}Etn[uuorllunJ-}o-sso1laqrea peuorlueursV sllot rorunt eqt sespJ tsol ur pue IIE 's11ac 's.rourntpropqeqr dolea.epsalo8dzorataq ralg8nePol uolssltusuErlstr lue,rard tsarrB III'rn *9Jus1_gJus ]o tuar z3 parnpur-ggd 'uorrecrldarslr Surzurollo; -rad gg-91 'acruru1 'srorunt Jeqto pue urerq ruroJ ot uorlrsod pa8eruepsl VNC JI snql 'uorlrsuprl srsolrrulrptu e or Burpeey aerqt porJrruepr sor1; eseqt ur uorssa.rdxere^o trZg eqr to paJ ,g1y ezllrqetsdqaraql pue ereld.roqdsoqd 'sfJe}ePqr.,tlor8 -ta seseur>l aurras leqr eql eseerlul suorlElnu rot I{Jreas lPql V JO UOIIE^IIJE OqT OT SPEOI ,4AOqEUOS SESSAJ1S JEqIO ]AIJV pl1trrul patlnsorq)Iql,t''l7g ssa.rdxere,to ot pJlf,utsuoJ are,4 JO sarg crue8sue{ 'sollJr{1r.4stuerurradxaortaua8ur poro^ofsrp se,u eua8tr27 aql pue surato.rdJNS/I/NS apof,uareqt saue8 eql uea.,\Aloq drgsuorrelaraq1 'a1c,bIIOJeqr qSnotqr uorssarS -ord Surtrqrqurdgaraql 'seue?g7E eql Surssordarur olor E sdeld gg541165leqr tsoSSnsacnu crue8suprlr{tr^\ serpnls,a1d -ruexe roC 'seue8SurlelnSer-guror8 euos apnlJur dlluepr,ra slo8rpl eqt rnq 'ataldurooursr sexeldurocqf,nsreqlo puE CNS /I/A.S dq palep8a.r sauaS laS.rel aqr ;o a8pel,la.ou>lrno

(leeq .ua8dxo .,{qro uonerper.rr l" dg e8euep y11g ,ln.ol pue 'uorlerpeJrrt\.Jo ]elorlerlln se r.lJns,suortenlrsInJssaJls ur dluo 'esuodsar ,pauatq8raqsr urarord g.gd leuortdr.rcsuerl-tsode .srorunl saddroldrrlnru do1a,r Jo lunoue aql eJrrulerurou uI 1o -ep ot uorlrsodsrparde ro; ldarxe dqlleag pue elqer^ d1e8.re1 are ggd 8uqrel arrry .pape.rSap dprder pue elqetsundlerue.rl -xe sI lr esneJeqsllal d.ra,tle luasard Ipturou ur slo^al .la.o1 sr ggd'surelord alcdc-1ecreqto a>lrlun.tou op ggd puorrruny

LELI

s ' ] o u r - N o )3 ' r f ^ ) - ' r ' r : l )o N V 9 N l r s r H N r - H l M o u 9l o s s o r 9 N l s n v ) s N o l l v l n n

'srsolnu ur sJoJJeSurpnlcur'serlrleurouqe due7,q'iZ Jel -deq3 aes)srsoldodese u^\ou>l osle 'qleap gao peuruer8ord ro1 paleu8rsapere sllel dueru lueudole^ep Ierurou Surrnq

seueg JossaJddns-rounIro saue6o)uo -olord se uorl)unl ue) saueg )rloldodv 'qfzuor8Jotunl sluJA -ard ggd qcrq.Llq8norql suporuluetrodur lsou eql eq tlpJ uI dBru srsoldode pue eruerseues'OZ-SZ arn8r4 aas)saudzue rredar-y51q pue suratordrrlotdode-ord ;o uorlrnpord saleln -rurls.rosserddns rorunt asod.rndrllnusrql 'uortrppe uI 'tseJJB epdr-11acSunnpur ol petrurT lou sr ggd Jo dlr^rp€ ar{J 'sllel PJruloJsueler.{t;o uortera;r1o.rd sof,u€quototdacar {CCd eql }o uorlelrtf,E peurptsnssesner qcrq.u.'uralord SE AdH eqr rpqt IIereU 'surelord droteln8ar ]ler ur suorlElntu Io eJuesqE0r{1ur uortPruJotsuEJleJnPur 'sros ot tuarrrJJnserc LApUE9E'raqtaSol Surloy 'dla.rrlcedser -serddnsrorunt qU pue ggd eql llqlqq pue ol purq-/A pue gg-esaqt Jo o,l.rl 'sllel pernrlno ;o dtarrea E ur srsolnu pup uorlpurJotsuerlelqels eJnpur ol drqrge slr ol Jlngrrtuof lpr{l

suratordeerql sapofuaAdH '94 pa1lecurelold (n4g) sn;t.teu -o11rded uetunl{ e dq petrqrqul sI osle f Sd Jo ftr,rtpe eq1 'uorlelperrrot esuodsaruI lsoJretg paonpur-ggdaqr gst -loqe o] q8noua uollenuefuor ggd aql ernper sle^ol ZrUPW rorunl qcns ,{q pecnpo.rdsr g5d leuorrun; pete^elaaql 's11ac q8noql ua.tg 'eue8 t9d leLuroue uleluor ]Pq] srolunl uerung reqto pue setuorres duetu ur peryrldue st aue8 TwpW eq1'uorlf,unJggd ssecxaSurtua^ta.rd dllerurou sdeqred 'g9d qrr.Lr '€Sd dool 4reqpee;droleln8arolne uE uI suollf,unt ZulPtr1tr dq petplnre dlleuortdr.rcsuerltlosll st aua8 Txupw aq] esnef,og 'ggd 'tr uror; dqaraqr Surzrlrqers ZuIpW Punoq sareldsrp 'uollep€.r8ap leruos UIV ro WIV dq €Sd Io uoneld.roqdsol{d -eatordro; g9d SurlaSretsnql'selnreloururlrnbrqnto uoIlIP -pe eqt sazf,letecpue ggd 1o ,&r1rqeSutlezrtlle-uotldrrlsuB.rl eql slrqrqultl'tgd 01 Punoq sl ZIUPWveqy'ZwpW PeIIer uratord e .{q zvrollda4 sr .,{11eturou €Sd to drrrrrFe aq1 'suonetnru tSd lo stJetlaegl Jo eruosa.teqsaua8Z4q) ro WIV eql uI suollelnlu uonrunl-Jo-sso1'snq1 '(EEl 'SE-OZarn8rJ pue 97-ST arn8r{ >lJolqsnql aas)aseqd g aqr q8norql Pue olur uotssa.rSord 'aseqd eql rJlue 01 sllal JoJ S VSZJpI Jo sle^al peseerreq

'srsoldode

'zD

'uotssntstp sase)oLuos a]ouold eql e lo] I rrederyp6 ur eledrrrpedto lxalaq]sas tSd ]o 'seuo)res L9 seueb1ouotssaldxe Ourpolua pue sutaiotd asnel ur ur per;rldLue leql q>rq,u ]sorle ur uorle^rpeur a^rsso)xa sasner,ilqer,unserd 'lt ggd lo uorle[oqdsoqd palenrlre-€Sd 6ur]lruLtad soztlrqPls {1}uenbarl sreua67upy1ueu.rnll aql ggdssarre}ouueflr alaqM 'l{ennr.lled 'snlool)nu puo)as e ul E uollP^ll)eAC) ul alol sll 6ut1lo1qpue pueZurplA oq]ul ll srelsanbas spurqqlrqM'(erru.r ur6Lfvd) 'ulnl 'uorlrppe ^qalaql'VgZ)pl selelfuotldsoqd ut lo1]r 6ur>1leuu uorleperOap oresla^ol u1 gEd uorlezrlrqels ttfvd ^q pollorluol Zurpy\ ]o 'pue palelfuoqdsoqd oolql sr 71q3 E :LDul ]sorteo] 6utpea;sAennqled y11y pasea.r)ur ol Zulpy\sale;fuoqdsoqd 6ursner'lr ale^r])eur 'uollelpPlJr ' snolje^ s.re66uluaql nIV palP^lllv (leaq 41 6 a) sassarls pueuorleurlrnbtqn aurosealo-rd aq],iquorleper6ap luanbesqns Egd 'Egd aseutl qi;tn xelduo: Oursner e LUJo] ue) uro+a^rlres1rururalord7Lup1/1 o1anp a6euep VNC o] esuodsalul pale^l])es! nlv jo I1tnt1>e eq1 'a6euep VNO ol asuodsal u! lsalle'9- 9Z-SZlunglJ V prtql aql €Sdlo loodaqt6url1or1uor sr{ennqied 1o,{e,uLeq}oue sosPolcururololo tgo lo lood

rredarypg

]sorre zg pueLg poutelsnS stsoldodY

\I/

1 c o l qg T l g p t d e g

1 o ^oo o"o o uo;tepetbep I leuroseelold I

6;;) Y

s nl o o l c n u ur perelsenoes 'aArlceu I

V

G)

tI

,o,r"n,rr, I

uorrdrrcsuetl I

pazrtrqels fu?)

-@@

a'G) Y (d,

ET

1

ypg pebeue6

Jaqrunu IEruJouqEuE Surureluor 'proldnaue eJE sllof, Joru -nl uEunq lsow 'srournl ur pa^rosqoare aSeruep VNCI Jo sadft .,tueyqref,uer 'o >lreruller{e sr ltrpqelsur f,rruoueC r 'lca;;a rrueSocuo rppurs e a^eq (asereqdsoqd pue rolf,e1uondrrrsupn NiIId ggd ''3'a) srsotdode aloruord suralord Jo sso-I .srar teql -uef, r0r.{lopue (l'I)) prrue>lnal cnselqoqdtu,tloruorqr qrr.u polerf,osspsr pue IE^rArns11acelerrdorddBur ot peel uEJ (7-pg ''3'a) suratord Juoldode-uue Io uorlJnpordrarrg r '(roldeJeJ {CCd aqt sele^ -nce) gg pup '(qU srrqrqur) :suraro.rd srrqrqur) LA'G.Sd 99 cruaSocuooorr{t soporue (A.IH) snrr,reurollrded ueurnH r 'qUlor8 rolunl ^\ollp ol pelp^llt€ur sr .{e,vrqted asuodsar -ssarls€Sd aqt laqtou? ro dem ouo ur snql'uralord ggd leur -rou ssa.rdxeleql (spruolJes''3'a) sJafuef IEJe^asur sJnJf,o '€Sd 'Zrupw ]o ,{ll.'rrpBol{r srrqrqurdlleur.routeqt uralord E 'sref,uer uerunq Jo uort)npordra,rg ;o luocrad 0S uerll eJor.uur rnolo aue8 oql ur suortelnuruorlJunJ-to-ssol r ttd 'gZ-SZ ern8rg aes)y5iq pa8eurepot asuodso.rur rred 'srsoldode(zC -er I5 pue pue ur lsar.resalouo:d leql VNC .rossa.rddns rorunl E sr uleloJd ggd aq1 r Ipuorlruntrllnu 'uorssardxaeua8 uo e a,Leqdeur uralord lcaga Surssarda.r rossa.rddns-rounl rpalf,nu e qlr-i!\ xalduoc {NS/ll\S aql Jo uorDeJelur'sespf,auJosuI 'sJorrJruJo llerre,t e qlr,4^peler)ossg ere 'lorluor leuorldrnsueD ur saledrcrr:ed qcrqrrr ,xaldruoc Suqapoua.r-uueruollf, {NS/I.,4NSaqr Surrage suorl?lnry r 'sJorunl uelunq ur uaassde.tr -le are serlrlprurougeqJnS .IC arel ur lurod uorttrrlsar oql q8norqr a8essedpetpln8arun ,ater.rdorddeuresner ueo qU pue 91d Surpocuaseua8rossarddns-rount aqr Jo ssol ro Ie urlcdc Surpoluo auoSoouo-olordaqt uorssardxara.,r,6r Jo .ft2-SZ o.rn8rgeas) doueu8qeu;o lueudole,rap pue uorte.reyrlordyyacsotouord 'roleln8a.rql,rror8 e,rrle8auE ,dCCI dq Surleu8rsJo sso-I r slorluo) el)^) -llaf pue 6urgtqlqul-ql^ or9 lo ssol 6u;sne3 suotlelnn

ferEolt euof,aq IILV\slloJ rournl ur dproldneueor SurpeaysDatap leuortrunJ eqt rot srseqreln -f,olotuoql (lsorre elcdr-11eratplpotu pue serlrleurouqp esaql lJalap lpqt suratord eql Surdynuaprur apelu are seruE^pEsV '(E-tr 'S€-0T ern8rg aas) dlradordtul ate8ar8asseurosoru -oJql aql srseur4oldJ yr srsolrru pue [uort lrxe slua,r.erdlurod -Iroql uorteSar8as-atuoso(uoJr{f, orlt puE jsnteredde Jrlolrtu eser{detarueqr or dlredoJd grelle saruosoruorr{cparecrldar 0rlr IIE ssalun eseqdeue olur drtua slua,rard turodlceqc dlqulasse-alpurds aql ly11q rraql palecrldardlaraldruocarrerl saruosoruoJr{tIIp ssalunsrsolnu olur drtua slua,ra.rd dlleru.rou lurodpaqc VNq-pelerrlderun eql ,67 rerdeq3 ur passnf,srp sV 'leuortrunJuou ere sturodlcaqc apdc-1oo uretrof, uaq,tr

r.uroJ seruosoruorqr sregr.unu E Jo l€Iurouq urr^.rlrrJ?r? oo, dylerauaS-seruosotuorr{tJo JOqrunuluEJJaqeue Jo etue

ul)NV)

|

sz uSldvH)

8 E tt

-serd aql 1(ploldneue sr sllal rournt 11ellreau Jo Jnsrralre -reqr dlrlerurouqp raqlouv '(yq urltb pue Ierrrosoruorqf, (Z-pq (lqp-t?q ')[ta-r ''3'a) qroq ro 'uorlecr;rldrue ,uor1 -Bf,olsuprtdq pau;oy ore lpql seueSof,uo;oseldruexa lero -^os perolunocuadpeorle aAEr{alN 'sllof,rounl ur punoqE sarlrlprurouqp leruosouorql reql u.4ou>l uaaq 3uo1 seq r1

slle) rounl ur^ploldnouv ol speal uouo slutod{)aq) el)f)-llo) lo ernllel d1a41arour seruofeq dolarrap ol JeJuEJ -ro; parrnbar suorletnur Jo uorlplnrunl -Jp aql pue polnput eq louuef, srsotdode'lso1sr uorrunl ggd uaqlN 'suonetnur aldllpru olelntunJJeor dlo>lrlare leql sllol 'e8euep Jo uorlpreJrlord sluala.rd 1r VNC ol esuodsa.rcrl -serp e o>lrluaes deur slql allqA. '(92-S7 orn8rg aes)esrurap 4crnb ;roql ot sppal suratord tnoldode-oJd;o uorsserdxa pecnpur-ggd eql (Erxoddq .ro uorlelr.rdep rolce;-qlvror8 se qJns sesseJlsJeqlo snoJerunu Jo a8rurep VNC alrs -uelxe raJJnssllar lsotu uoq/A.'(It-I7 orn8rgaas)xeg sE r.{f,ns suralord f,rtotdode-ord SurpoouaIere^es ere ggd dq parerr -rlre seuogoql Suoruv 'tSd sl sJef,uEJuerunr{ ur pateoqdurr aua8rosserddns-.rournl Jrloldode-oJd uoruruoclsoru oql 'g eseur>lurelord rorrrnl ,o lleJta rrloldode-rtue agl Surseetraplq .rosse.rddns crtoldode-ord e se srJE NEId snql 'sdenqted lera,ras,(q ,g eseu srsotdodustua,rardpue setoruord qcrq.nn le^r^rns 11ec -l{ ularoJd o^Dtp pue eteqdsoqdsr:]-S'V't lolrsourldprleqd -soqd ;o sle^el palu^ale e^Brl asereqdsoqd NAId 3ur4ce1 sllel '(0€-9I a.rn8rgaas) g aseunl urolord Jo uortp^nte ur suortrunJ lprll ro8uessetupuotos e 'ateqdsoqdsrrl-S,t,t lolrsourydprleqdsoqdsetpldrorldsoqdapaua$ srql dq papoc -ua aseteqdsoqdaq1 '91 rerdeq3 ur passnrsrpeua8 ryEg4 aql sr alduexa uV'srossa.rddnsrorunl sE o^Eqaqslsoldode alplnwlls sfnpord uralord esoq,{\ seua8 '.{1esra,ruo3 'SurmorB ruoJJ sller retuef, dols ol popeeu sr lr uoq.{\ ua,te srsoldode slua,rerd suratord popolue rrer.{lJo uorlrnpordra^O .soue8 -oJuo e[uoJaq ol polelnuJ ueeq a^Eq srsoldode 3urle1n8a.r dloa.ne8auur pollo^ur dlpurou eJElsql saueSocuo-olordos ro uazoPJeqlouv'qreep IIes eJnlrsJE o1 elqElnqrrllE OroJ Jo 'sllal rorunl asaql -erel{l OrEsrounl l-I) to lE^r^rns s.4ollp pue srsoldode Ierurou stua,rerdurelord Z-pg, p uorlcnpord -raao olerrdorddeur luetlnser eq1-'(L7-IT arn8rg eas) srsol ,71cq -dode yo ra>lJolq IEJurrr E oq ol .a,ou>l&rou an qtx1n pa11eroua8 E alEArtJEleql suorleJolsuprl IetuosoruoJqJ e^eq sllel'I-I) 'e1ptou op daqr rnq 'Surpr,rrpdlarrlce lou ere lsou pue t1mols etelnrunf,f,eslleJ aql '8urdp ag ppoqs deqr ueq^{ a^rlrns sllef,esn€reqsrnrro (113) enua4nalcrlselgoqd -rud1cruorqc 'aldtuexa ro{ 'tuJoJdeur .rounl e .Surle.retrlord daa4 peelsur pue plnoqs laqr uaq,r erp lou op slleJ JI 'uorlEruJotur to spuDl snorJel oql sateJ8elururatsdsdroleln8er xalduoc e 'erP ol suorlfnJlsur pue oArTol suorlf,nJlsur eAroJaJuBl PUP slle3 'le^r^rns IIOI ornsue ot perrnber sleu8rs qlrm .uor1e (sesecauros 'srsol -nlrs llneJePeql aq 01 sreaddeqreop uI IIec -dode ro88rn uE) osle 'ue8.ro3ur1ro.u e ;o tuaurdolo^apro; pepaeutou sllar to ssorxe Ieturouge u€ pue ,a8eruepy51q

6€lt

U3)NV) NI S]N]9 U])VI.]UV) CNV SNS9ONI)UV)

ur uouprp€r to elor eql 'euerdd(a)ozuaq'tt6l ul ret IEor pue al6 'e8eurepy51q rredar ot dtr1rgeslelalefuI suollelnlu uror; uaSourrrer lerruaqc ernd e Jo uorlef,r;rrnd eqr or pa1 ./!\or{sE IIe,r\ sE tacuec ef,npur or VNCI uo lle sueSourcreo sdesser{rns 'l€rurue orlt ur srorunt rrura}sfs pue Ierol qtoq 'uotlles slt{l uI snorJel qllql\ uI sdem aql e;oydxo ozrt ;o luarudoya^eprot lool pup osnotu € Jo IJeq ar{l uo of,u€ls 'rnrJo uEJ rof,uer 'a8erurp aqt rredar dpuetct;;aol elqeun -qns e dlpareader aq1 ot sr crssep lsel tured tuorurredxa 'sleruru€ur sarpntsyelueurrradxeq8norqt ref,ueJqtr^ pelerl pue suosrod lptueruuorl^ue dg pazrua8eJnluere saruedo.rd 'JafuEJ -osse e>lr1-lletruets gtl^.\ sllat ueql( 'saslr eler uollelntu dlleur8rJo eJe,{\ s]Efrrueqr ql,nor8 leluetuuoJrlug IesEu 's11ec eql seqf,eor oql pue pasrurorduor sr .rrederVNC tl ro r{lr^irPelerrosseaq ol pauoder se,u (occeqol) ;lnus Jo esn ua8elnur lnlreltod e tI euoJralo ag uEJ sp.renSelesasaql eql I6LI ur pup 'JaJupJIploJJspesneJloos ol sdaamsdau -rurgr raI{rJnJ sepr,lord (.-Oi) Surplelgs uor; rarupJ te.re.tro11 to ornsodxa oqr teqt palrodo.rsew r SLLI ur eldwexa Otp qrIA\ PauIqIUofuotleotldal rog 'suosrodlelueuuoJrlue ol enp eJEsJeJueJeruoslseel lE suortelnurJo alEJ,t\ol IELUJou 'eztselsrlalupue 'sansstlSurrogq8reuaperrur{1d lpr{l rEO]l erueJoqlr ssouore^\Eraf,uef,yo sdep lserlreaaqt uI Jo elEJ,trol e -dns poolq e !)eDle',rtor8 ot Jolunl e ro1 pe.rrnbaroJEsuoll€l sJof,ue))!lr)ads ol -nu aldrtlnruJI 'oslv 'sro(unt snora8uepqll^\ palelrosseuoIS pelul-l uoag oAeHsueoour)re)euos -r^rp iler Jo uotlelnSorsturpolnPul-uorlelnlu Jo dlrlrgrssodaqr eql lrxe daql uotsurtpJo sPunor lere^osJal Suonpar 'a1odc11ec 'serudzue relnlloldqperyporu deqrprunrrega -;y dlalrur;apu apl^Ip or dr{lqe eql e^Erllou oP sllel tuels Jo ueoge^Er1 (erourrequnC'srsollru pur uorlecqdar y51q 3ur duaSo:d aql orua8Btnu alDrl a^pq suaSourcreclerrruaqJ lsour 'peapul 'suaSourlrec -rnp porrnrur a8eruepyNC Jo dlrpqrssodoql sarnPorqllr{^/t osle olur slelruoqf, ssalruJpq osr,taJoqlo uJnl upf, 'uorsr,rrp eler ,vro1dlelnelar rrel{l sI sllel tuels rot uoll sarudzuaggy-4 'ra,ta.trog 'dpoq eqt urorJ eq uel pelerlxo Jo -elnu tsurB8euotlcalo:d Jo ruroJ ouo raf,ueJ or PPalPFol daqr reqr os ueql ezrlrqnlosol repro ur 's8n:p rrtnaderaql pue soprJrlJosururpueJ sp qrns 'syecrruaqru8re.rolreloduou lprll suorlelntu l?luaulJloP to uoDelnunocBlua.terd ol stuslu -pqretu leroles doldura dllensn s11ecSurprltp IPturoN 'sllaJ se qcns 'sraluac crlrqdorlrala ppe ol uoltounJ ot (sdnor8 HO Jaf,uEJJO eJJnOSlUenbor; E o.lP'suotst^rp11ardueur lno 8ut dlleurrou saur.{zua0St-d 'sllar ra^rl ur s1a.ta1 q8rq dllenadse -l,trct utat5 drrpqedecrleql qtl^\ '( 17 .rardeq3aos)s11ar lp pue sllol lsou to unlnJrtor rruseldopua eql ur perelol 1o 'sllof,rounl pe sdem ate satut(Zua0Sr-d auoqcol[)'sleanrue uI 'sre]uer rrlgdorr 1o saddreldrrpu uodn -rele rer{to ur lutp ro srounl or dlddns poolq eql qllltl oratJelulteql Jo uorlrnporlur eqr rarye dluo sracnpur rarueJ tuatod 'slloJ lueu8rleur sarderaqlur lsarolu eql or sPPEuelqord sql sp lJE uEJ reqr spunodruor olqnlosur-Jolpl ueryo 'a,trlleer -un eJedlle.raua8sua8outc.tptSurpa-7cauput'1se guot u1 sr saua81o dnor8 e uele 1e lno SutdvrruI e^IpaJteulaq or d1a>11 'uosuel slql JoC 'sprptsntuuaSo.rlrupue'(SWC) atEJIns .ro eue8a13urse pre^,ltolpaparlP dde.roqrouraqJ ldqreup'(SWA) 's11ertueu8qetu elpuoJlnseueqrarul.{qrasepnlf,urueSoulrrer ;o adlr sIL{J 'uoll Jo uollelndod snoeuaSoroloqe to uoIlPIlIroJ -errlder Surrnp palerod;ocur eq ol epnoellnu lf,erroJurue ,{\ollE ot spEOI&rr8alur cnuoue8 uIetuIEIrI ol sllef, rounl 'o &4rgeut ot olnglrluoJ osle ,,srleder,,asoql'(97-7 o-ln aq1 'srsouaSof,uo daql 'pe.rredeJ lou ere sepltoelrnupaIJIpourasaqtJl 'Surrredaseq -3rg ere so^lesuerp stuslu€qfalr rredar-ypq auo.rd-ro.r.la aes) ;o ureued Iprurou er{l uolsrp ol sEos VNC ul saseq{rpour uec (eJotuJOI{unC 'snoJaluetreuof,oq deu aseql Surreeqs11ar euros spunodruocesaql'VN(I ur sruoteue8lxo pue ueSortlu glrrn 8ur -trea: dllecruraqrdg '(spunodurorreqto ur sretuoJIIJIJ-uortrole eql pue 'sls1e1n8e.r apdrlac tf,aJJEeluos 'alelntunccelsql suorl -elnu duetu eql JO 'oler uollelnlu POSeerruIue or speel (7 rar qtr^\ tf,EOrpue tno {ees teql spunoduor) sapqdorrceloelrJf,eer -deqC) srualsdsrredar-y51q aql Sursr:druocseue8re{ElereJ ol dlureur o.retra.a;e dpo ere eror{l qrq^\ 1o'suaSoutc,tptSuticp -pa4e'sarro8ater yerauo8o.4A.t a8euep 'uortrppe uI 'suollef,olsueJlPuE suortucrlldtue IEIuos otur porlrsselloq upf, daql 'sa.rnl -ouroJqf,sEIIO,use 'suotlnlttsqns aseqpue 'suoDalap'suoDlasul -ea; Surlyrun snor,lgo ou qtr^^ seJnlrnrts;o a8uer peorq E e^Erl pue qrrr'ror8de1 ut suotlelnur e1c.{r11ao apnlf,ursaue3,fuole1n8a.r suaSounrec lerrtuaqr sE porJrlueprsoouelsqnsq8noqlly 'sJatuEJlsolu asnecSurdlrePuner{roJBsulol 'slaPotu Jo ]Eruru€ur JefuBf,Of,nPul cruaSoluo eseql -o.rdocuopue suralord.rosserddns-rounrto uolllunllelu or PEel pue sllel urolsuerl or .{rrlqe rreqt ot leuorlrodord dlq8nor 'urrroqs seq uolssnlslp snor.terdJno sv sr sueSoun.reJJo rroJlocrue8etnureql'O-SZ arn8rg aas)s1ler rPql vNC uI suoIlEJOllE 'sauaSocuo-olorduorl seuaS ro 's11ac 'suatsds rreda.ry51q aSeuep ro olur errru ur palueydrurs11ao o>lrl-ref,upr 3ur.uo.r3-rse; 's11ac -of,uo olEeJJ'saua8 rossarddns-rounl Jo uorlJunt eql alnPeJ ot sllol a8ueqc uer suaSourcrpr pornllnl tlt se qcns Jo e.rnsodxa,(q pararle VNC rplnllar teql uorle^rosqo aql tuory leql suonelnlu osnef sueSoutrre3 llef,uer asnef,leql slef,Iueqf seruor sue8Blnruse lrB suaSourJJpJtpql efueprle lseSuoJrs are'sueurnqdq paonpordro IErnlEueq ueJ l{llg^r'suaSoutcre3 eq1 'suo8elnurere oslp suaSourcrecsnql 'a8eurepsrqt.rreder ro)ue) ul ol slroJJe.sllar eql Surrnp VNC olur pernportul srorro el{l sp IIa^ sp esner daqr regl e8eurepVNC eqr ol enp sI raruef, sauagJolelole) pue suaoou!)Je) of,npur ol suaSourcrecleorsdqdpue lerruraqr ;o drnqe aq1

V N G 6 u r O e u e c/ t q r a ) u e ) e ) n p u l s u a o o u t ) J e ) 'sllef,ref,u€f,ur olor str pue 'a8eurepy1qq tsure8e slcalo.rd teql eruoua8 aql to uprpren8 raqloue 'eserauolal pa1ler arudzue yerradsE Jo uorssnf,srpe qtlt\ rardeqc aqr

'oslre ol slar proldneue slIIuJOdsauos -ouorqr ;o uorleser8as-sltu.ro {lqurasse alpurds radord -rul 'VNC palecrlderun rarap dlerurou lPql sturod>paqc '(dueur ool dlensn) seuosoluoJqf, alcdc-11ac Jo Jo ernlleJ

I a]ual)S a]q Jo etpadophul

'Iooz 'ual lolq]aot I I uror]pa]depvl oq] ]e aln)alouJouor^d(e)ozuoq olalduroraq] ut uMoqs oln])nlls 6urr-r11nu aq] ,Loisal aqi sluasetdet pe)npal}o uot}laltpaq1ur se0ueqt loqiu,ts,9, a6telaql {1rrrxo1 ole)rpursMoJiele)tua^ellqm ,{tualodtaieat6 pleMo}suot}eJa}le olefrpursMore leluoztJo; aie;du.rot oq lltMuotlelnulaql pue 'V aql airsoddopouasutoq lllMI e 'pelerrldetsl VNC aq] aultl lxaN .aseq

U])NVf

I

SZ U]IdVH)

0vtl

aqi allsoddo) e ueL{} raq}erv ue}rasuto1aseteullod D peutpoul sosne)'Dp-zN-de)g-tlue-suert-(+)'Unppe 6u11;nsel aq1 VNC ureseqauruen6 e ;o uole zNaql ]e {pteuuud VNCq}lMs}leal}eql settads >tuabelnur Ilqbrq e'eprxode-O 6urxpord tualod 1'6-10lp-g'/ 'suor])eal e urauetrid(e)ozuaq IIpouL 'sau.rlzua ]o sat.tas 957-6 'saurlzue ,ilrelnrrlred tanrl'ue6ougue)pue ueEelnur1uelodelou e og euerltd(e)ozuoq;oEurssaroldrrleut{zug SZ-SZ]Un9lJ V

HO

--.-A-ot

olt I -^)f-o,

reoor vNo

tI

HO

tI I

HO

HO

I

I

oH looueC (

----l.--ot

€- oll I

uollelnl^ uet6un1JosaieJeq1 srseuaOout)le) le)tuJaq) {1peep e ;o a;duexeree;te saprnotd Oullorus el}ete6rJ Jot.ulo+ 'a{oruso))eqol Iq srsaua6out)Je) le)tuaq) /Z-SZlgngll V reoA

o o

0 OL

aa+

a""t

at"t o"d ot"t dd

"""t """t

a.,"'

"-"t

"q 'tu

o

009 000r 009L qleap rocuec 0002 6 u n ;e ; e u r e 1 0092 o o 000t 009t 000t

0z

=6 0t ox 0t x' = 09 = ^ 09 -" ii OL oii 08 06 001

>lslr 0q or po^er1agere suaSourcJerIerrrrreqr q8noqlly '(uorrerper ,\n) rq8rluns qf,ntu ool ot pesodxa slenpr^rpurur (raruec urls) eruoueleruur esperJ -ur aql dq dlruacer eror.upue ,(uorterper Burzruor) rElN II

aleJ qleop rscuec 6 u n 1e ; e 6 ;

o o

uorlor.IJnsuoc 009t e11erebrc elrdecre3 0009

f

lvta

U]fNV) NI S]N39 U])VI-]UV) CNV SN]DONI)UV)

o*f zHN' .r'N' -

lJnppe srua6elnlA

T

N-r.,,-N-

|Y

lil

/'* o/-* ,rxzHN

dlqd-HO/V

N

l a r pu B L u n q a q tu r( v c H ) a u l L U e LUoc crlcAcolelaquoLr-r ,dlqd ] s o L Ua q ]

N ZVLil\J ggy-6 er-uolqcoilc i\q uorleptxg

^l NJ

e r n l e r e d u e lq b t q ]e leour 6ut>1oo3

(q)

eulsouen6Axoeq

sonsstl laqloo] eletbru.r ue)sV)H aq]'slla)renrlurAqureLu rn))osuors.ro^uo) reruer Ogt-deq] q6noq1;y elelsordueLUnq ur poAloAUr eq {eu pues}uapolurseurour)re) uolo)puelseorqsosne) 6qq6rrue6elnusr]pql I)nppeVNCe ulo+ ,{1;errueqr o] VNCuraseqeuruenO e qllMst)eorq)rqM'turo]a^rl)eal gst-d auJorq)o}^ftalpueurnqoqi ul rfuan e o1]r ua^uo)seur{zua auouor!r.!o)]sor.!aqi sr2lqd'eroquMoqsVf H oqI spr)eourure pueourlear) jo seq)nssrosrnrerd uror+ (sVlH)saurure >r1>liloroleq 'leour 'arnleredural par {1;eoedse sad{1luaregrpuao}xrs }noqea}earf pooj ueu.rnLl qOrqo1pesrer urJn)roleql suor])eor le)ruroq)(q) r o ) u e )u e L u nrqo +r o l ) e ]) s u u M o u )e s rp u e' s u o r s J o ^ s uf e i ] 'eue6rosserddns-rournleql soJelnur punoduo> o19 6ursne> Egd srql uorletrlde 6 -uru n pa s e qD p a q r p o ue q l e l r s o d dfo p u e q l {11nq iaLllerV ue uosuro1esereu.r{1od e1n>agout 1eq1 VNCsosne) {llerrueqr e6rele 6ururoJ'VNCul euruen6 e ]o Luote/-N oql qlrnn 'LrLole ua6Ixoup qltms])eolpuoqolqnopporolo) l)eal 011r6urlqeua paz,i;e1er oql urxolelle ul vNc qllM])earue)]r oro+aq uorlelr+rpour 'sua6ouDrer -eu,{zue obrapun}snLU urxo}pl}e 6ur1re-}:errpur 11e a>111 (e)'sue6ou!)re)le)luoLl)oml lo suorl)V OZ-92lungll >

g ulxolellv

uorlecllpour crleurAzu f

lreer svJH 'slaPol'uIEluIuEuI setuoulf,reJlsEarq PUEuolor osnErpuE sua8elnu luelod ar€ rlf,Itl,^A'(sVlU) sautwv ctlc{c -onpq LuJoJIEI{I suoltJeal sesneJ sa:nleredrual ]Ellruorll 'auo8 q8rq re teaur Surlooc 'alourraqlrng t9d eql uI uoll -elnru E sosneJoslE ulxolEllv 'suoIsJOsuEJlI-ol-D seJnPuI 'seurdz pue VNC uI sonplserC ot Pe{uII soluoJequIxolEIIP -ua ralr1 dq uonecr;rpou lerlluot{r ntly 'p67-97 a:n8rg) rao -uer ra^rl soJnpul 'surelS dplotu ur punoJ ollloqeleu le8un; e'urxo1oTfy'poIJIluaPIuoaq seq rolreJ >lslrlnf,-realr e qlrq.&\ JoJ Jef,ueJu?unq roleur dluo er{l lou st racuer 3un1 reJuur lerleqrrda;o eddr e 'euoqoqloseur 'sueSouto ol pe)url dlrealc sr arnsodxa solseqse i(1.reprur5 -Jec 09 usqr eJotu suleluor lr aluls 'saue3 raqlo uI suollel st -nr.uarnpur alorus auore8tr uI slerlluer{r reqlo leql d1e>1{ lI raJuEf uEunq Pue e>lotusallareSlJut uaSoutcrel IEJIIuaql 'snq1 peurJap auo ueo.{\loq uollPlarJof, Suorls e st araql 'luudlooJ sll se^EelueSourcreceI{J 'srotum ;o sad.{rreqlo

'uals,{s asue}ep lerrnn e sr rrBdarVNCI snql 'uorteurrndappue sarcedsua8dxo elrtreer lsnI uor; dep qrea rnJJo VNC er{l ot suorteratle996'67 ueqt eroru '11e0dreae ur tpql palBrrrnseueaqsprltI 'VNC ratlE qJrr{.{\;o11e'slectpe.t (suoll eql ol pue ua8dxosEqfns saroedselrlJeal Jo uorte.raue8 -rear uorleldlle ot 'suorlceeruorleurrndep or enp sr e8eruep aq1 'e8eruep lunorue a3.re1e ete.raua8sassarord VNC Jo 'ua8elnur ro uaSoutcref lpuJalxo due lnoqlr.,lrua,tg IEruJou

,o)ue) ol pee-lue) suals{5 iledeg-vNc }o sso-l 'rac '8uqce1 dlleraua8sr (sertue>1na1 -uBcalelsord pue 'uoyor 'lsee.rq''3'a) sracuecuoturuof Jel{lo pro^E sn dlaq p1no.,r,r teql srotrp; >lsrr leluoruuorl^ue pue .{.rerarpSuru.racuocof,uaprlepJeH 'sJeruef Jourruqlr^\ pelel -orJor uaeq seq sleJrueql Jeqto ot ernsodxg '(q67-97 am -314)srrnppe crua8etnu ruroJ ol seseqeulsouen8dxoepqrun

U:I)NV)

I

SZ U]IdVH)

zvtl

'6Vt60S'savuotlolnw'2002'PIIqrs'c puPuosouoql'T Pue:99t"Ill arnlpN '.1002'srajeurhaoH'[:88/ 'd '.,(uedruo3 pue u€ueerC'H 'lA.''pe p7'uouotq(a4 VNA'266I taleg l pue 8;aqu.roy'V uror; pal]lpontr:salunos

j::y; uErrEAo orr erureup !uote1a1s eql sertrurroJaP Jo Pue llrlIlJaJuI Sulpnlcur serlrlErurouqe Ietuaudole^e(I

JEJUEJ

uEuE/\o Pue lseaJfl

11ao-snourenbs 'enua1ne1prolodruarntv

s]eJrtuer.{J tuPprxo olrlteoJ 'slua8e 8ur4uq-ssorcy51q

uorlEurqIuof,aJ .{g s>1ee;q druororyap snoSoloruoq Z-Vf,Ug pue I-VlUg'rrf,uer ts€orq.{relrPrraH Puerls-elgnoP ;o rredag

uorlEurqruoJaJ snoSolouroq .{q slearq Pu€r1s-0lqnop yo rtedag

9rruaueruoJu€c

uorteurgruof,eJ snoSoloruoq dq slearq

suorleJetl€ atuosoruoJql 'sasetcerBuelel

stueSe Surreldlye p1t1atr

leIJe;'.,oiartrsuasolor{d

Puerls-alqnoP ;o rredaa

S)VAUfl CNVUIS-ETINOC CO UIVdAU sesot€reI Xlr,rrlrsuesoloqd eda pue ur15

sJorunl Jo tuoudolez'ap llreg

suorlelnru lurod 'uorlerpeJJrAn

dre,ro(uoyo3

sua8Blnu IEJruaqf, 'uotletperrt ,111

SNOIIE'IEC QNV

runsoluaruSrd eruJePoJex

rtedal uorsrJxa ePrloellnN

Ja3u3J IeIJeJOIOf, srsoddloduou

rredar qrreusltu vNq

d;elrpereg

.SNOIIUESNI .SNOIIVJ.NW JNIOd CO NOIJNEAEUd

0tI$Jlv tills^s UIVd]U-VNO

sIqI 'srolunl osaql Jo f,Ilsuolf,Preqrqr,ttor8 paleln8arun eqr ol SurlnqrDuor dge.raqr'dgol dq uorrrqrqul gr.,'ror8 ot s l l J Je l e t u s u o l l e l n r uS u r t e , r r l c B uqrf n s : J r I J E J t u p l s r s e J palou sV 'urelord roldarer oqt to uorlrnpord saqsr ]prurou -1oqe aruenbasSurpot-urato:d aql ur UrqseruerJtu€llnsar eqt (ruels.,(srreder-r{rteusru aql dq pexr; tou sr uorletntu eql JI 'seuruapEII ro 6 Sururetuocaruanbase ol uorlptnl'u seoSJepunuelto oluenbas srql 'uorlecrldar Surrnp eseJoru '.r!\orE ur seuruepe -d1od y51q Jo ,,e8eddrls,,Jo asneJag 0I Jo eJuanbase sureluor JotdoJar srr.{lSurpolue aua8 eqJ '(9I raldprlS pue orn8rCoas) roJ rotdal d{CI VZ-SZ -a.r11ad.{f aqt sepoJuerrEdar qrtEr.usnu eluasge aql Jo Io osnef,eqsref,uef,uolor ur patelntu dlruenbarl oue8 euO 'Jredallnoqlr.{\ srsaueSelnuqf,lel'usrlusno -nurtuor SuroSrapunerp sller reruer Iertrureql asnecoqdlqe -urnsard (1ensnueqt dlprder ororu qrnu srotunt pe8pag-1ny ot sddlod uSlueq uorl ssorSordsraruer ar{I raruer uolor to sesef eql to luef,Jadma; e ro} lunoJJE ueql ur suorlelnlu

pue (ualsds Jreder-qftElusrluoql to sluouoduof ePoJue sauo8 JJ.INH 'pelelnru aluoJeg qrPOPPUP qrzvror811ar etEln8arosr,rrar{loro elrdr IIel aqf IoJluoJ ler{l seue8'tuslu -Er.{lerurrPdor srql Jo aJuosqEel{l uI PuE 1{.rautqreurreder -uorsDxe eql Jo sluauodtuoc apocue seueSdx u,t\ou>llq8le 0q1 ua^as 'atPr Ierurou 0q1sel'ullPuEsnoqlE lnoq? l€ JOf to -uEf ur>lsdo1a.,r.ep ol aldoad porJeJlEsasnEf,4y 'uorlera;r1o.rd pue qluor8 11ar8url1o.rruoJuI IEJIIIJJere legl esoqr Surpnlc -ur 'soua8Jeqlo duetu uI suollelnlu elelnlunf,fe ol dlrsuodoJd € e^pq (CCaNH) JeJuEr IelfeJolor srsoddloduou d.rul -rperer{Jo (.IX) Iunsoluetu8ldeurreporaxqrr.Lraldoad treder y5trq radord lnoqrll6 'ft-SZ alqet) sraruer ulelror Suldo -le^ep Jo ,{]rlrgego.rdPeseerJurdlsnourroueuE e^eq ulelord rredar-uorsrlxa.rorrede.r-qtlerusrru IEIJnJJP ePofualeql saua8 ur suorlelnu lrJOr{uIot{,^ sueurnq 'olduexa JoC TOf,u€f,JoJ arEleql ISrJ peseerf,urqlr^\ solElerlor 7 rardeq3 uI pJqIJJSaP suelsds redar-ypq dtrlapr;-q8rqoqr to sso'I 'eSeurepy11q .rredarro lua,to.rdot sI seuo8Je{eleJel Jo eloJ IEurJouaql

EVLI

ul)NV)

Nt slN39 Ul)VllUV)

ONV SNlgoNtfUV)

lsovanE saappv pnN'9ooz 'aueurn/\ d r pupIalreSn s urorjpaldppvl.al)^) g-l-B aql la66u] ue) sursrueq)aul aldtllnuJos 'aselouolalaldue o^eqleq] slla)la)uel ur ua^elsol uouo ale seJauJololsoua6leutLujal+o uotle)t]tldue ue Jo suor]olapleutulla]laqlta a^eq lltMlla) Jolq6nepq)ea pue 'arorlolaloql ]o oseqe6oul! utq]lM{;;ensne.res)earqaq} }o sa}rs aq1 'sleedara1t{t aq} os 'salauola}))el sauosoutotqture6e';;ar relq6nepq)ea ul )ealq lsnu pue loqla6o] peurofete sptletrlolq) oMl aq] 'eseqdeue1ealetedassoJouloJlua) aql uaqM pue ,potlJol sr abpuqV stso]L!6uunp prleu.rolq) lelstsslt qllM asn+o1 ptieulolq) e sMolle'aspleuolal]o a)uasqeaq] ol anp to a6eleatq,iq 'eiauLo;a1 e jo ssol0q1 salnl)nlls auosoluoJq) o1 e6euteptoleu: luanbasuo> tlllM'ol)I) (8-l-B)aOpr.lq-uorsn;-eOeleatq e sta66u1aulosourolq) e ]o pua aq] ulol] aJaulolale lo aluasqe aql .seJaurolallo ssol ruol+ebeuep pue uotsnl leuosoruolq) O€-SZlunglJ V

o

'srsoldodBpa.reSSrrr-ggd ol Surpeel,uratord tSd Jo uouelrlf,e pue uonezrlrgels luonbasuof,rltr^.\,a8euep VNC to pul{ p sE IIar aqr dq pazruSocarsr saror.uolat Buruauoqs elrsuol ;o -xg 'qreop IIer pup (Of.-SZa.rn8rg)suorsnJauosoruorr{rpua -ol-pua 0t sppel .a1r.,tr serer.uolal ssol oroldruo3 gree to 11ar tlll./\\ ueiloqs seJeruolel:raqt 'uorlcunJ asEJarrJolel lsepotu dluo Sur,reqJo tlnsor e sy .aseqdS tatua daql se asereuolal Jo Ia^el .uoy e dluo ecnpord sllaf, trleruos uerunq lsoru lnq 'esereruoletalnpord sllal t1relspue .s11ereury-ru.rafi 's11accruodrquu1 'GV-9 ern8rg aes) sauosoruorr{f,ueru -nq to spue oql olerotop leqt sreadar;o suor8arq{-02 ol -€ eqt urelureru ro uaqtSuelol spuo aurosoruoJr{Jol sleador

CCCVIJ sppe llpaleadar'aleldual VNU uE sureluof,lerll eselduJsuertesreler e 'aspnuopl'(9 raldeq3) olnrolotu VNC pepuerrs-alqnope Jo puo aql eleorldar dleraldruor ol saserarudlodVNC Jo drlllqeul eql-uralqord uollecrlde.r -puo eqt ol uorlnlos eqt epr,ro.rdseJoruoloJ'seterqelrel ur 'acuonbes CCCVII VNC lJorfs e Jo s[ene tuopuel ]o lsrs .seroruoyaa -uo) 'seruosoruorr.lJrpaurl to spua lecrsdqd aql

slla) Jo)ue) lo uorlezlleuourxl ol selnquluo) uolssatdxf eseJouolel :redo.r .roJalelduret eql Surpr^ord sr auosouro.lqc snoSolouoq oqt eraq.&\VNC rreder ol alqpun are suorlrunl VlUg oqr Jo auo 3ur4re1 s11a3'sruars,(srrede.r>leorq-vNq Jo slueuoduof, luelrodr.uT opofua'sJaf,uef, uErJeAopue lseeJquerunq ur pelecrldrursaua8 'Z-V)vg puv ur rcol ouaS L-V)dfl'seuroqdrudl pup sprue>lno1 asal{lJo tueruollo^ur luenber; aql Surureldxe'seuo8rotdaca.r Sur.rnppete IIer I ro uqnqolSoununurrror.{lJo luaure8uu.r.reer -erf, s>learqpuuns-alqnop dq pesner stuaure8uerreeJ VNC ol alqrldaosnsdyrelncn.lederu uralsdsounurruror{f Jo sllol J pue g aql '(7 .rardeq3) apm8 e sE eruosourorqr snoSolour -oq eqt Sursnuo spuadepa8eurepqtns Jo .rredo.raql ueryg :etoruord luare"rp E Jo Iorluof, erlt rapun ouoSdrolplngar -qulor8 e Surrq ro aue8plrqdq e acnpo.rdleqt esoql sp qJns suorleJolsueJl pue sluaura8uerreal leuosouo.rqo sso.r8ol peal uel VNC Jo spueJlselqnop Io Sururofar]f,erroJuresneJ -oq suorsel ara,ras dlrelnJrued arE s>lpeJqpuerrs-alqnoc 'ecuepuedapure8e.roqcuepue uorleruJoJ snroJ rllrl\ 'acuereadde peruJotsusJl e qlrm ,vro.r8 ol uraql pesnpJsllaf asnou ur sruJoJasererudlodluelnrrJer{l 'aldoed luereJJrpur punoJ suorlEtnu Jo o^ l Sulsserdxg to urnJlf,edslpruJoueql ur Jou luorled etupserll ruory onssrlIetu -Jou ur J0r{1r0u suorlEtnru esoqrJo lsour pue .eua3 PunoJOJe,ry\ speururexeero^{ srorunl d Ioa VNC er{l ur suorlernu peq 85: 'srorunl qlr.ry\polerfosseore 58I uaql( d toa VNC ur suorl -ptnru aJueprle 8ur,u,or8sr eJar{I 'slsrxeuonetnr.uelqetr ter{l -rer{ e rreder ogl raUp ,{1uo lr erurs 'uorterpu.rpue sleJr sr leq} tl1etou;r ';o r{tnlu alErpou -ruoql Jo slteuo cruaS0urlrecaqr or ere sualsls .rredarauo.rd-rorrg'suorlelnru Jo uep rg8noqr -rnq 3ur,r,or8e $eI aql ur IIE lE apr^rp or alqe 09 ol sllec Io ro1 sle^al q8q pepeeusr tr esnpf,eqsdeq.rad.sroulnl urel re -ref, ur pessardxere,r,o sr pue pear;oo:d lou seop d toa VNC 'ssarord uorlecrlda.rorue8elnurB drreo leqr pue ,uro; lno -rad ol olgeun erB saserarudlodalBrncce pue lpuorlueluol aroru ueq^\ pesn seuo or{l (lJosoJtsEIJo sesereurdlodaql erp deql 'auou uegt rouaq sr rredar ,,tueueqo asnpf,oqpelerolol a.resaseratudlodqcns dlgeunsar4 'suorsalVNCI Jo seddrrel -ncured qlr.u edor or selrllqpder rcuFslp seq dlrue; oseraru 'sesprorudlodypq ssadt(,q-uorsal -f1od aqr Jo rogruetu r1f,pg pellpr ore esaql 'saseqSurssnuue,re,suortef,lJlpour IEJnueqJ reqlo PUEsrlnPpP VNC ulEluoc reqr saleldurelSursn e1q ;o -edet erc 'Elasotaw{"\odyltO pelleJ euo Surpnlour,sesp.roru -d1od eseqr ourN 'aSeruep Jo VN(I tJorror ol sase:atudlod VNC to dlrurey e ezrlrln susrueqrer.urredar-ypq 11y 'uorrela;r1ord pel lleJ lorl -uorun ot esr^\reqto peel rq8rur reqr e8erueprlleue8 Surlcer -roJ ur rreder qcteursrru aJuplJodruraql ol slsellp Burpul; Jo

,rual Jo se^lesueqtpu ot slleJdq pesn .,(llereua8demqred E -sds eruorqroldc erll Er,n srntfo uollelrtJe trloqelatu 69y-4 'sleurrue uI 'VNCI a8eruep uec daqr eroJeq polp^ul€ eq lsnuJ'ad.(l uoruurof,lsoru oql 'sueSourcrelSurtce-lcarrpuyr 'VNC ul seprloelJnuororu ro euo rJlle daqt ,sr leql lsua8elnure-rBsuaSourlJElIIV 'sueSourlrel .ro ,sluo8e IeJrrueqt pue lecrsdqd snorrul to stlatJe ar{t pue srorre 3ur.{doo uort llnser acuanbas VNCI eql ur sa8ueq3 r

Jo)ue) u! sauogls{e}oJe] pue suo6out)Je)

dderaqrorueqt rot loSrp] alqrssodv eut(zua aqt a>lEr.u pue uorsr^rpIIal pelp -rJqun rot esEJeuolal aJue^elal0q1eleJlsuoruepsalpnls Jo oseql 'esereurolel ssol lq petJJJJesselrre srorunl reqto to etuos 'eserer.uolat ]lel af,[u aq] ]r peJnpar ere ool aseql 'srounl uoyor dolaa.apdlleurrou uorlElnu Cdy uE qtr^/r PUE ef,rry .reJu€f, tsEeJqpue 'uoyoc,Eruourc:etylac-snouenbssu I{Jns sror.untlerlaqrrdato eler pespaJJurue sr aJer{l.luesqe .epl^lp ol un8aq are ggd pue osereruolatqroq ;r ,re,ra.trop1 a^eq tEql sleJ Jo sJrrr.uolJtSuruat.roqs ra^r rrlJ ot osuodsa.r ur pernpur sr srsoldode pe.reSSrrr-ggd asnecoq dlqeuns -a;d'af,nu IEurJouur ueqt as€JouJolat IeuorlJunJe 3ur1ce1 oJnu ur dlruanbarysselsawrl 0Z Jnf,JosueSourlrecIeJrruaqf, ,elduexo Jo uortpurquor e dq paJnpur seurollrded ur>1s roC 'op arrru leturou ueql dyrpear ssal srounl doyalap elrru IInu-espJourolot,suaSourJ;€f,qtr.a poleeJr uaqlN '^rlllueJul PUE'ouusalul 6uorsrlrp pue ur>lse>lr1 saler a.rrnbar q8rq senssrl leqt IIer to to uonaldaP stJetapeql 'relJor{s dllueorlru8rs ruEf,aq PaPnlrur (ql soreulolal3uo1d.ral Jror.{r se arrru og-ol) llnu-asproruolel aql ur reedde ot ueSoq sfletop suorle.rauaS xrs ot rnoJ rau€ ta,ta.rlo11'Sursrrdrnssp,lt elrlre; pue alqerl aJE aseJor.uolal Jo llunqns VNd or{t Jo uortalap e ro; sno8dzouoq etrrtu reqr Surpurl IEI1IuIaql 'raruef ur aspreruole]Jo alor eqt Jo Surpuelsrapunrno pe,tordrurJar.llrn;o^eq seqJeoJdderrlauaC 'uotlBln8arslr puE srsaqtudsereuolel I ;o d.rlrncrrc aqt SurpuelsropunJo eruel.rodur lecrtoe;daqt ^\oqs stlnsor esaql 'srsouSordesroznr e e^eq roqunu ddot q8rq ol oua8 cf,tu-1troqr per;rldrue o^pq lpql sJotunI .sror.unl eser{l Jo sotuoJlnoaqr JoJ a,rrlrrpardosle sr ,aseraruolel uorssard ;o -xa saleln8al JoIJE} uorldrrosuell B ,uratoJd c["ul-61 ler{l 'asuodse.r poo8 e rorpardsle^el.laolalqm .dde to le^el eq1 -JOrIt ot asuodsa.r rood e esEreruolet lcrperd qSrg 1o s1a,ra1 'sllar rotunt ur dtr.trlre as€rauolet dq Jo la^al agt Burssasse pauretraJseeg uef 'ruatsdssnolJeu leraqdrredeqr yo Jount crrlerpede 'eruotselqornouJot srsouSo.rd eq1

ul)NV)

|

sz ulldvH)

wtl

uaqlr 'alduexo roC 'qr.rlor8 IIaJ rof,ueJ qlr^\ erolJolur uer 'ateldurel 'sesere VNU palJlpour e Surdr.reJesoqt sE rlf,ns -ruolol o,rr1e8au-lueuruoc 's{ao.ry\Jnot lnoqe ur ql,v\o.r8 E S P E J O I u I E q ] P O S N E J E S E J J I U O I J I ] S U T E S EV N U

3SUES-IIUE

r{tr.,rtarn}lnJ ur (s11aoE'IOH) sllef, Jor.unl ueunq SurlBa4 'd1as.ra,ruo3'qr8uoloretuolel Sururelureur alrq,l s8urlq -nop 0Z ueqt arotu dq uedse;r1rroqt puelxe uer eurdzuaaql >llEI asr^\rar{to sllel upunq peJnllnJ olur saua8suerl lpr{t Surcnpord-asprerrolet u o r t t n p o r t u y ' s l u e 8 e rrlnadB J o -raql rerupr se uaaq e^Er{ palsoSSns eseroruolat Jo srolrqrq -ur crlrcads pue 'lelrorurur auroJag ol IIOo Jounl E JoJ Iprtuessesr uorssardxeaspJeruolollErll olerlaq sJOqJJEas -a.r duepeserauolat Surrnpord f,q a*1 srql oruorralo 'otel uorlplalrlord prde.rrraql alrdsap'syyac rount tso141 'sleaqaJeJoruolal Jo sroqunu tuare;lrp to stJOJJa er{l sazrrpuruns It-SZ e;n8rg

qleappeuurer6ord a p e ^ op u er i l e l r u r l a pouprt ^ t p o ] u l o q l6 u t M o l l'ea s e r a u l o l a l a)nporduotlosllalratue3 srsoldode puetredatVNC+o6uue66ul o] spealsieadarororuolal enrle:r1det ;o sso;alelduo; ,i1l>edet ol lrurlouoseprnotd arolaiaLl] sorerLrola] aq11oq16ue; eql papore 's1ane; {11enper6 ,{lluenbesuo> srsarourola} nnol,tan 1oqi6ueloq}pue ro Mol sraseraurola]'sll0) ltleulos ul uotst^tp spunol ie ]soLtl lla) ]o pue6uo;satar-lo;a1 alrurlopur {1ler1uessa nnolle deal o1pepaou st}l pueslla)ulelsurluasetdstoseJaulolol araqM'sllol eurl-u.rab stsoltLu q ) e a6 u u n p) u u q ss o u J o s o u l ool q) )u a s q sel t u t p u e , { r e s s a t a u arolaraq] sraseraulolaf (g taldeql y516 aas)spuaaqi 1e,i|1n1 'puetls elerrldar 6ur66el aql +opueaq]]e euoustoleLll ]ouupllr pueraurude serrnbet asetaLu,{;od VNCasne)agpuaeulosoruo;q) q ) e au o s l e a d eat s a q]]o q ) 0 L - g a n e qs l l o )t r u o , i t q u eu e u t n H 'saulosoulolq) spuo0q1uo gDDVI_1 sleadet rilquLasse lo 1o 1o aq] eprn6o1posnsrq)lqM'a1e;du.re1 VNUUoqse ]t q]tMsaulel 'sa.lau.rola] pe;;eteullzua aseJaLuolol aseJawopl e setrnbet oq] leoads 'saLuosotrtorq)spuoaqi uorletr;dou 'uotstltp +o ;o lla) lo spunoJlo requnu oql sllrullI;;eurlouso.teuolello sso'l l€-SZlUnglJ V orourolel suorlelap llaclarueO puea6elee.rq-i{lrpqelsur ourosor.lorqcosle lnq "(gogvll) 'q]^ 016 luolsrsrad Jo ql gg ol dn qleap ; ; a c c r l o l d o d e ' A l ! l l q e l s u !l l a c l u a c s a u a S '(gggvll_) euosourorqc'e1cAc e6puq-uorsnl-ebelealg ouqttmspuf

llos crlPL!os

"(999v.r_r_) pouult-l uorlecrlooJ

le .,!rar8'esnotu p olur palueydsue.Il,eserauolol luElntu ,erorureqt.rng agl 3ur.{rrEJ sllel rorunl uEtunr{ tsEerq 'trloldode oruef,oqsllol eqt ,e.rnt1nc ur sllel reruer lseerq ro alelsord ur passe.rdxa sem (72-g7 ernSrg)luetnru e qsns

pecnpau

llas LUals ro cruoArqLU!

u o r l e c r l d oart l u l l e p u l

'(9cavll-) Joql 0z-€

9TLL

runlnl IHI uol s3^il_)ldsuld

eue8 Jo lue(uernsEe(uslrrured srsdleue der-reorcnu ylrlq 'dlruarrn3 'srsouSord s,tuarled aqt pue sarlrodord qurro.r8 s,llol oql Jo srotelrpur 1n1ra,nodse pJrlrtuJprore sartrlrlre asoq.&\seua8 uo dlrelnrrlred Sursnroy 'saue8 1o spuesnoql eql Surrnseer.u ro; sanbruqcaldq paceld Jo suol ;o uorsso.rdxe 's11ec -er ro peluaru8neeq ddocsorcru flureur peurels lpvr ;o 's1yacrournr alqrssod Surssesse spogroru Jo Ipuorrrperl eql 'sf,rlsualrerer{l [e] ]o sraqunu a8rel rotruotu o] drllq€ punoJ -,la.eurno dq peu.ro;sue.rr3ula9 sr rurf,rpoturrtsou8erq 'pela8rer eq uer s8n;p qrrq,r,r, suratord or dlfarrp slurod te JeJueJ qlr.,lr palprJossesaue8 petelnru Surdyrruapy.uorluol -ralul JoJ serlrunt;oddo aldrflnru luasard Jorunl snora8uep e olur .nor8 ol IIer € roJ rnrf,o lsnru ler{t sa8ueqcaldrrlnu aLII rerueJ srusruer.lf,ou aqr aroldxo ot pasn pue pazruSo Jo -car dlpea; aq uer lr Surrredarro1 sue1s.,(s eqt ur pup VNC pa8etuep Surtrelap roJ slortuor turodlror1r aqr ur stlotap frlauec 'lorluoc ap.,{c-1lac ur sdelsu.4^ou>l uo slf,oJJa rreql rot possosseaq .,r.t,ou uec suaSourcJeJ'eseasrpoql Suueerl pue SuttuerrardJoJsorlrunuoddo lrrausnoruJouepauedo seq eseO -stp crlaua8 e dlleruaruepun; sr raJuer teql uorlruSorer eqJ

'osuodseJ 'slotunl ureuof, tS:d err.rlralordaqf of Sur.Lro to uorleJeua8ol ecuelsrseJr{lr.^apaterJossesr eseJotuolelJo eruasqEor1l 'uorlezrleuorrrrurrraqt ol elngutuoJ leu pue uorterrlda.ry1qq Surrnp souosoruorqr;o Surualroqsslua^ -ard qcrq.Lr'aseraruolalecnpo.rd's11oopelerluarettrp lsoru a>lrpn tng sller r.uotspue s[e] ura8 a4r1's11ecracue3 r 'ft-SZ alqe1-eas)s.racuerureuef, ro; drrpq -IIdOJSNS

POSEaJJUI ue IJII^\ PeIEIJoSSe OJE Seseaslp ueIIJnq

uletraour punoJsassoco.rd rrede.r-ygq ur spolep palrrequJ r 'rerupr aruer.{pu€ pa8eruepor Iorluol a1c.{r-11ar ppol uel teqt etuoua8erll Jo srsaue8etnru Surnurtuor e pue 'arp plnoqs reqr sller ur Jo le^r^rns or{t^\o[p saua8ra>1e]erec suortelnry rnrf,o seopa8euup VNC ueg.r\s[o] Jo qreepeql aloruord Jo (sourosouloJrllor{l Jo dllr8atur aqt urelureru asr/!\ -;eqro 'y111qrredar leqt saurdzuaapof,uesouo8Je>lelerp3 r 'sJounl 3un1uerunq uorlerlrur eql ol to Surlnqrrluoc snqt 'aue8 t9d oql ur suorlelnru Surlelrlceur sesneJ'alorus olloJEBrJ luauodurot e' euat(d(u)ozueg r ;o 'y51q aSeurepol rapro UI SUOIIEf,I]TPOIU JEINIIEJ I{JNSOUE.TINbA.T SNtrCPUESWg SB qrns suaSourc.rec Surlre-lJaJrq'slef,rureqru8ra.ro;snorxou

la86Lt86vsnps pDV ' l,teN )o.td'1002 lp]a url) lA lA r!or+paldppvlosejaulola]]uelnu aqi +o sla^olMol ua^o q]rMo^rpalJaseM q)eoJddestqt stsoldodepaseal)ul pue uorsr^rp aq] 'asnoule llo) pe))olq aselauolo]elelduel-]uelnLU olur palueldursllo)le)ue) ]seaJqut lo 'oln]ln)ut sllo)la)ue) alelsotd ro ]seoJqueuJnqur passetdxa pue 'a6euepy16 uaq41 srsoldode 'aloulola] aql ;o 6urddetuna^tl)o#o 'saulosoulo.lq) 1o 6ursuasllo) aql uotlezlllqelsep sr atuenbasuo) aql aut{zua ]o 1ue1nu eql,iq pazrseqlu{sse>uanbas )iloruola}paJa}leaq} uo elqLuesse o} elqeunoresatuenbas olauiola]leLutoultalotd pue putq]eq1(MollaI) surelordosne)aqanrle6eu-lueuruop e a)tl s])e oselaulolal ]uelnul oql (q) uMoqsere slsaqlu{spue 6urLuud}o spunol oMl (par)saseqfp

oM] q]rMsieadaL reu-g uot]tppe eq1sezIlele:aseJaulolo] leuorlrppe ]o 'slla) possordxa ur ueqM (pol ut u^^oqs) saseq ]uelnursrl.l] V poppe oM] q]rMe1e1due1 oserar.uola] alelduue] VNUpalalleueseq(>lurd) -]uelnuJ julol-]og pappeetuenbas eqt uear6 ur sr 1q6rl leadar1se1e1 eq1 lq6u aq1o1speatotdsrsoqlu^S (ueet6)99y1_19reul-gaqi +o qeader1osauose sraruanbes )rJor..llola] oLlf oseiaulolo] utqllM leLulou poureluo) e1e1dua1 anlq)lep oqf alouiola] e VNUaq]sretuenbas esetauolal lo puaaq]]e VNC+opuplisauouo pale)olumoqssr({erO) qltMalalralutol pasnsem leurop :do1(e) uorpunjeu.r,izue letuJou (aserauolal eleldulal-]uelnu e)aleldurol VNUa^tpalapp suteluo) paraeurOue uy 'qynol6 lle) ra)ue)llqlqul ol leql osereuolal pele;ndrueuoq ue) eserouolalZ€-SZ lungg 1VINllNlUldxl V

olqulasse ]ou op sutalolo alaulolal : a s e J a u l o l o l e 1 e ; d u t a 1 - 1eu1 n; 4 ; olq urosse su ralojd alouolel

joselauolal

letuJoN

:::!:::::::::::::::::::::::::::::::::::::::::::::::::::::::

(q) ]eaoar lu eln ur puosas

,9-3NVVVV333NVV3 |||||tl

SYrrrlecavrrrreSDvircaevr-Le eevil555trffi 55VIT5ccv

]eadar ]u eln LU lsrtJ

,9*3nVWV3f,3nVVO CVLtl-l99CVtlCOCVl-l-99CV1-L999vl-l-CCDV.l-l-999V souo let]llou sql puo^aq sleedal elaulolel luelnlA

.

,9-CnWCf,3nW3 |||||l ,99VIt_999V-Lt-999V1t_CCCVt-t999VI1C99vl_t_99CV srseqluAspue e1e;due1orar!olal leLuloN (e)

U])NV)

I

SZ U3IdVHf

gVLL

(HO-I) Ororu ro euo sexaldutor llqlqxo sllal reJuef rsol'u op ^q1x\ ,TLL {NS/I1S.S Ho-I 99p I Jo '(ffOf) drrso8dzoratoq ssol dtrso8dzoralaq rdacuoo oqr ureldxg '6 Surloe-mo1s sesnil^orler Jo ssol to ;o L L ZZ 'lueultuop 60r r selulelnel Ieuos 60LL seworres -olnp up sp pelrJer{ursr Eruolsplqourlar,{retrpereqlad 'dlazrrs 5gy; saua8ro>leteref, TgSy aueSotuo-olord -sof,eJlf,E seuo8 rossarddns-.rorunlur suorlelnru uorlfun} 60 t r setuoulrrEr 0tr r OluosotuoJrlf, -Jo-ssol :xope.red tueredde eqr ureldxE 'Eruolselqourter Jo erqdlaprlq4 76;y suaSounrct stuJo}o^u esoql uoo^uog uorl)urlsrp PcrSolorueprdeeqr roy yg y euoqdurdl s.rlr4rng gySy uletotd ggd ; srseqcnaua8aql ureldxg 'edo ouo ur dpo poor{tFpe Surrnp gyys euttrdlo)ozueq yy;; aue8otuo srnf,f,odllensn Etuolselqourler snoouetuods epqm'seda qtoq uou)un[stpuou ur uerplrr.{rslreJ}p ,{1e.roua8Eruolsplqourta.r.fuelrpa.reg 'g 5g;S u8yeq ,ZLL 'seuroqdrudlr?lrurs dlpcrddlouaqd uaezruaq J J;; srseuaSor8ue 69;S sua8enut 1\,\oqureldxg 'L qsrn8unsrpuec srsdleueder.reorcrtu ggyJ dproldnaue srselsPFru VNC 0 t, r '9 Ld Puv'unl'7-1cg'so.t f,rf,L urxofille 69;S weu8qeut 'g9d :seue8rossarddns-.rounlJo seuaSoouo-olordsB saue8 aqt d;tssr13isuorlElnr.uuorlrunJ-Jo-ssol 8ur,vro11o; ro uorlrunJ sural fa) -1o-ure8ograpun saue8rossa.rddns-:ournlpur sauoSocuo -olo.rd op 'Surlouro.rd-racuecoruoJoq o1 'seue8 rosso.rddns 'ul?uler leql sellBlsqo 0q1euoJrolo 01 -Jorunl puE sauoSocuo-olord uee,ruaq qsrn8urlsrq '9 >looqsqr to sroprar aqr 1o dueru reqr edoq alN 'llel dlaq 11r,r,r 'srsaqroddqaql surrtuoJ reqt ErEp;o aydruexeup o^rC aqr 1o dSololq rplnlolotu aqt Surroldxaqlreesar Jo saP€JeP 'o eruep lo8e qrr.u dllerluouodxo saseerrursreruer ueunq Jo str;euaqeql dee.rol Suruur8aqere e^\'satlllear Surtunepasaql -rrur teql suortelresqo aql sureldxe srseqroddqreqlX 'S alrdseq 'srer{toroJ Intesn aq lou dBuraddr auo rot Intssof,f,ns (sll3l rselqorq ale tpqt suolluelJelur 'saseesrpesro^rp,(1.{3U;o dno:8 e .ro1 -rydreurrrd parntlnr r.uJole s ta)up) acur5 '3urra;tns snouJoua osnef,Pue leeJl lerurou lou tnq slleJ tI€ urolsue4 aua8 seJe qlr.^auorlf,oJsuEJl uer,,tq1x l4rozrrLesseuorlPr.uJoJsuEJl or tlnJIJtIp ulptuor srof,uer dueu lleuorldecxa sI Elluo>lnel 'PJEqaru sdals lsurB8e(qlurreur) la^aalC qtl^ ssof,Jnseql IIer tI€ egl seoPMoH'sila? tJt pollpr srsElqorqrlesnou 'arnc ourl p qlr.t sdesseuorteuuoJsuerl pornllnJ Sursns11ec dals tsrr; uolluelerd pre.'r,r,ol pup or{l lnq lxeu Jo 1ac IeJIIIJf, 'eJuaIJS Jorunt uetunrJur per;rruJpruJJq e^pq sauaSoruoseg 'V E sr refuef, ;o ,ffio1orgIIel oql Surpuels.lapun PUE '{rgy pue 'Dtrtl72gq :saue? eurrrpeureAIo^uIlsnru sdalsrequn{ reJuEJrsearquelunq Jo 3ur.uo11o;aql to euo ur suorl€tnru uorttrun}-Jo-ure8ssassod sosererrrosur pe^lo^ul utelord e'(7tag) 7 rcTdetatCCE ueru -nq tsureSeselPoqrluPIeuolf,ouotu Jo asn IEf,IurIf,aql qlr^^ uolto sJounl lueu8rleru dueu ,(q,u ureldxe 'uorlerurolur srql sesseJJns.rar;e dllenedse 'edoq tea:8 ;o ef,JnosE oJE sller uo PesEg'lassa^Poolq 3 Jo rulu 00I u[{]r..!\ePrseJtsnl'uanssrl ref,uef,qsrn8urlsrptEI{l sre>lrutuaJeJrns-llellsute8e selPoq E ur sllof,'sluaurarrnballuerJlnu pue ua8l.xo Jo asnpf,ag 't. -rtuv 'sJeruer IE)I^rar II€ Jo srolJrnb-aerqr Peluo^oJdslsol 'slleJ sleu8rs dq reglo ruo.r; qser ur tpHl ouIrrEA reruuJ e 1o lelo.rdde uolleJlslultupy 3ruq Surlsa8rp-euoqe ol polrnrlor oq ueJ deql '8ur1eaq.ro qulor8 SurrnpSurleporua.r ur'alduexa.ro;'sauoqrse8rpreql sller ore Iprapo{ pue luaudole^ap aql or Pel ref,uel lef,I^rer Jo sosef, tsoru ur 9I snrlleulolpded ueurnq Jo luoue^lo^ur eqr;o a8pa slsElf,oolso'slsElJoolsoJo uorlf,unJOql slrqrqur r{Jrr{.{\'JlEu -1,{\oDI^\aN 'elqISEeJ^\ou or€ saqceordde crlcads uIElJef, -oqdsoqdsrq(o) pue 'sonssrtsnorrel to sef,rJleruJelnllelertxo 'lq8rluns Jo olorus se qJns sueSoutcrecol ernsodxe Sutztrut Pue euErqruaru luaruasEg ar{r ot sllsr Jo luorur.{fPlle elE -uru puo.{ag 'lle^ sp (spur4raqto sdeqrodPUErof,ueJ3un1;o 'surr8alur uorlJunJ -rpaurler{l suralord eueJqruou ler8elur Jo ar{r{rolq rpqt serpoqnue(q) tordacer rotplnre ueSourruseld aJueprfur oql eJnper dyluecgru8rsuec'a4ouls ailare8lc reln -rrlred ur 'suesourcrersnollqo Jo oJuEPIoAV'ddereqrol elqP xlrleur }o rolrqrqu ue 'letseru er{rJo pue saseuralo.rdolleraur -.ra;ardpup lerJnrl suletual uotlua.terdlnq 'serdrragl .^aauJoI -rteq (e) :sluerutearlroluef, .u,au3ur.Lro1loJ or1trot elpuorter senuele saprrrordJeruef,1o dSolotq [e] Jrlnlelour eqJ aqr ureldxg 'stsplsppLueurJeq 'srorunl dreurrd ueql rer1ler 'srorunl Surleco;1ns to sueelue se p;edoq >loolor senulluor f,rlelseleu dq pesnecore sr{leoprof,uef,;oluacrad ,{reulN 'T 'ePr^ul pue 'qcellu 'ale.r8rurol srsauaSor8ue 1o uorlelndruery ietuourrrer uoloc lueu8rlru urory sd, 1oduoloc u8rueq dq pesn stuslupqroruoql Jo erour 3ur.{;rluepl uI InJssaJtrns s11ec saqsrn8urlsrptpq.^a(suorlelnu aue8 ol lJodsor rprlN isrorunl eq plnoqs 'srsetselotu'sselold alllsnrlsaP dl.lrlncrued legr lueu8rleu ruo.r;u8ruaq qsrnSurlsrpsJrlsrralf,preqJreqlN 'I 'satlra uo snf,o, V 'luoluleerl InJssalJnsarolu 1\\olle plnoqs -dord lar;o Suuoltuour rolleq uo paseg'srournl Jo uollralop slda)uo) oql /l^a!^ou rerTrEE'stueuleeJl lueroJllP dlaler:dordde uozrt8PUEluoJet -;rp dpcurrsrpse peztuSocereq pPOlsuI]lL\ JBIITUIS drarr.ro leo 'sller sltBrlrod peulter orotu -puapr ^,ou sJorunl sp pe^ael^ to 'uott 'salels €LLL uolf,er]JroJsuErl ueAOsn e,rr811,,rn 11ecJo soJnseeulueuodtul 11e -e^.IeJoIpue'uotlect;tporu'uotlrnpo.rd utalo.rdSutrnsearu (aJnlnJ aql uI 'uotldt.rrsuerl dllecneruelsdsro1 sanbruqral 1g;7 auaS .rossa.rddns-rournl

ZZLL sesnJrloJleJSunnpsuurl

LVll

.

Vl-Vc lHt ]ZA]VNV

J o e r u a s q e r o e t u e s e r d a r l l u r s 8 n . r pr r l n a d e r e q t o u a q l erlt qll{\ paleert ere,\\ sllel 6ygy 'srsoldode tsurefie sller tretord leqt suretord tlrrue; (4y1) srsoldode ;o rorrglq -ur aqt .f, Jo sreqtuaturJtoq erB dvIX puE ur^r^rns

U!}3V

L+eM/LZo

oed

*#*

***il}# #

l- eol ge

duvdlecrerL

{t#t

t#

'IZd '€Sd sle^elaql Surssasse;o esodrnd iuure pup Jo erJtsr lpqlN (euuof,ruJo stlaJJeOqllnoqe Elep eseqltuo{ atnp -ap nod upt terJl)N'srsotdodeSurrnp po^eolr sr teqt uralord e sl ddvd 'sutato.rdpat€Jlpulaqr lsurefieserpoqpueqrr.,npaqord pue penolq ara.nsla8eqt ueql pue ,s1a3SCSuo unr ara^\stlerl -xa 'pes,{1 era.4^ sllef,eql 'aurlolru etuesqe.roaruaserdaql ur Jo 'urceqtotdurec'8nrpcrlnadereqloruer{J ,(e) Ieuourppeup ur pue ued ur se s8nrp aql qrr^\ pateqnf,urare,4^sllol 6tS:V .q oQ

. ^+ \uo^+ d)g$S

.n

-f"

."*";od'oof"od;...-o;o;J.l-e' oi'.*"" ai,".es \o' \o'"".j'

s 0 z -- , c z a

0ro nco 6

i s s r u l n J r s nl e r t u a r o d Jlagl lJetJEeulloJlu seoP,/!\oqpue 'racueo3un1;o tuetuleoJl aqr roJ 1n;asn dllerruarod s8nrp rrlnade.raqrouoqf,eseql are .{qr1X'peuretgo era,4 plep 3ur.lno11o;ar{f .eurtorru Jo af,uasqero acuesardeqr ur s8n.rprrlnade.raqlouor{l eaJql eql .(qteap Jo auo qlr,4^poleerl ro pelEortunreqtroare..!\slloJeql IIal petutuerSord)srsoldode SuroS.rapunere ]pql sllal slrel

-ap qrrq.,lt',(esse 1g51n1 aqr dq peurrupxeann'667111pue 'tZH-IlN'6ygy'saur1 s l l o r : ) I I S N r u a r e J t r pe e r r { I ' e 'vt€g-Tt.t9ttjr 'lrs 'pErV 'cot4 '9997 ''1e la erdn8seq aas) VSn I.tpN aurlofru Jo eJuasqepue ecuesa.rd eqt ur '1oxe1 pue 'urteldsrJ 'eurqetnuo8 s8nrp crtnederer{totuar{roqr qtr^\ peteert ero^\ seurlIIel JerueJ 3un1'aruelsrsarsn{t slloJte eurtoJruJr puets -repun o1 '.{dereqroueqr ot ef,uelsrserpue srsouSo.rd.lood dq pazrraloererlJsr :)'IISN 'srerupJ 3un1 11e;o luar.lad gg roJ stunole r{JIr{A\'(C-I1511) .racuer3un1 11acIlerus-uouJo luarudola.Lapeql ur rotreJ lsrr roleu e sl Surqourse11e:e8r3

elec aqt az^leuv serd (slleJ tuets 8ur.n1o.tur -Eraqt Ierrpaur.ro1asod srrJlseop suroJuol tpr1lNiasereruolel ;o uorssardxedg paroruord sr .raruecJo Jrlsrratf,ErerlJtpr{l1 'l,I ldl,rrre eseraruolorssessodsaddr 11ecueunq qrlr{lA. 'uonrunJ €Sd to ssol osnef,uet euettrd(a)ozueq moq ureldxg 'addroueqd lueu8rleu p ol selnqrrluor uortrunJ tsd Jo ssol qrrr1,{\ ur s.{e,vr. o,^^l etupN 'srorunl uprunq Jo drlroteru oql ur srnrro uouounJ tSd Jo sso-I '9I 'uretord lsoq lo8rel str qlr.r\ stJeJelurr{)EO,4^or{eqrJlsaq ;suralord ]pJrAeerql esorll eJE ol elnglrluoc tegt suralo.rd tEt{/N 'uolteluroJsuprt l]or-tsoq earrJteJnpoJdsurBrtscruaSoqledesorlJ Taf,uEf, IeJT JOTesnEf, ueo (1149) snrr^ €ruollrded uerunq Jo surerts Ipre^as 'SI (srounl cneenued ;o addrouoqdf,nplselatudtq3lq pue uorrrqrqur ql.LrorSJo ssol eqt atourord uorlslnru srql seop ^^oH 'urato.rdtpetus aqt sapof,ueleql eue8eqt ur suorl -Etnru uortrunJ-Jo-ssolssessoduJUo srJJusf,rrteerJued 'tI 'cKul tua8otuo Surteraua8.royusrueqoaur rerllou€ oqrrrseq ireJupr 1o eddr raqloue uerll roqler euoqd -rull e ur c,(,tu tua8otuo ro; SurlBraua8 rusrueqrau tlnseJ relncrt.redarJrsoop dqr11'euroqdurdl s,ltr>1.rng ur aueSoruo t{.ru aqt sacnpord lpr{t tuela aqt aqrrlsac '€I Ieuorlptntu lrruaSotuo :.rs-rr repuer aouaroJJrp srqt seop ^\oH trls-^ pue )$-J lq paporua surelord aq] uae^ teq uoltrullslp Iprnlf,nrts egt sI l€rllN 'ZI ii-CN ur suortelnrrluerll sJeJuEJur punoJ dluoruruor aroru se1 ur suorlelnru ere dqlX 'I-{N pup se6 Surpocua saue8 eql ur suorletnru Surlouord-recueo dg paqrnrred sI teqt luola uolttnpsuetl-leu8ls uoluruoJ JI{r oqlrrsac- 'II 'JOlO0f,eJ

ZreH egt 1o uor8er euerqtuorusuerl oqt urqlr^\ auruetnlS ot aurlel e slJeluoJ ]Eqt uorlelnur turod (r) 'ursodurodort ;o uor8ar leurruret-N er{t r{lr.&\.roldarar 1{ eqr Jo ureur -op relnlloreJfa oqt saceldertpqt uou€f,o1sue;r(q) (ggd8 (e) :rordera.rrolcey-qlzrror8 uralord IErr^ eql ;o uorssa.rdxa aqr lue^eler oqt 'o uollplltf,e ot ppal sluela 3urru.o11oy ^\orJ egrrrseq l.roldaca.rCCA eqr sE qJns srotdaoar rotcr; -qrmor8 euerqruerusuerlrllr.&\pelerJossp.{ll,rrtce rrldleter e.rourJo euo Jo uoIlEA oqf sI feql[ 'sroldaoar JolJeJ-q1./r\oJB -rtre eqt f,q pazlrcnerer{r ore srorunt tuuu8rleru duepg1 cdtlso8dzoretoqJo ssol ot peal aql Jo ernlrpl saop ^ oH lsaue8 rurodloeqc dyqtuesse-alpurds

U])NV)

' ttn)'sroltqtq ourqel!curo9 t 89-8LgtLl'luuo'uxqo '5002 'JtoS'g pue ''V 'eftrc4e7 -ur uo alepdn + ourloclN aurqellcLus9 srsaua8or8ue ' rgg-I99tt 6 ila)]aProq ayt 1ederl aql ralua sur:qde:srolJeJqu,ro:3 pue'srseuaSotSue'stsauaSol ''g 'solnodoruel -nlse4 '866I 'upruIloJ '[ pue 'urnqsS?l) '14tr .00t_6gztz D)up) .nav atrupN'qlzr,'o13e^rsEAur :o; 8ur11eu8rs 11ec:sroldararut:oqdeulas 'oq3ouro3 'W ''1 'ouqosnrl pue rolf,EJ-raDers'Z0OZ a PUE ' VS-6tt€.t.' pua t atnwN'srorunl pr1os.ft eurrrdur 'lE ta ''S {ruertseu:ea srselseteru Jo ernleu8rsJ?lnJalou V'€002

I

SZ U]IdVH)

SVLL

oulloclN

OL 91

'st8-9z8tz

JoJslaBJelIJAoU n)upJ'na[ atntpN ;dde:aql srsauaSor8ue-rluE ('S (rrJeU :sllef,uralscrlarodoteuraer{ pue relnlse1 'ZO0Z'p ta ' 919-ZIgt99 ( puo'I)' paytr'dsolq'['tu.g satuet aletsord 'S'l puu '1'y'uossyg Jo srseqrelnrelou rql'S002 radoo3 ' eruole,{u 9ttZ-ZttZt€OI poolg's11ar a1drl1nurrueSouolcJo uonezrrelrereyJ '1002'lp la 'ZN 'Inslp, { ' I 6II-18III LZ sKoss7or g'sIsElsPiaurPu? uols 'S002 '8rrrrpn1 1 l]ef Joruntur dlr,rrlreorldloalo.rdIBJo'I 't7-rt7,L

s-t , m t-

o zoE VNU!S-dVIX+

s zo3

vNUrs-ur^r^rns I 0s g vNUrs-ur^r^rns

-e^ur

'say 'npv 'rrrel dors b)up) sree.( gy srorunt 1o luarudoJa,rep '855I 'C 'ulel) -Illntu :Pallsr,rar eql eapt sno.ra8ueP .sPInoC ' 0LI- 6SIt Lg ila)', reruer Ieltrerolor Lrelrpa.raqurog suosse'I'9551 'urarslaSo4'gpue ''16 ';4'ralzuty 'tOI-Z1ItL6T,nuans'lAW lo uortelnf,eurlerrq dq addrouaqdrrtseldoauE Jo ssolpeuretsns'Z0OZ'Ie le ''yq 'uref '8SS-8tS:l,I' tolg ilD'urdg ttn2'uors -sa:3o:drounl SurrnpuortrsueJl JoJstuaru lerudqcuasaur-lerlaqrrde '9997 '8nag '11 'tne:; '51 ''y '1,11 'reqn11 -a:rnbar relnlalory pue '0L_LSt00l .re) ilaJ -uef, slrprull€q eql '0002 'Srequral6'V 'U pue ''q 'ueqeue11 Jo 'gl-s ',louo 'utuas 'srs€lselaur I:62 'ZO0Z'f 'ueur11og pue qr-,'ror8rorunl ur srsauaSor8ue ,o elod '9-I:€ n)uoC 'nad atn:zN'pursr^er srsaqrod,,(q ((llospue peas,, ar{l :srselselaurrruef, Jo srsauaSoqred eyl'ZO0Z'[ 'I 'replg ' 'g g€-€g€:01,' lotg'tuonfi dtutg rcqfiH Suttig p1o2'uorssarSordraf,uEJur role.rrdsuoooc rrlsel -qorqrt agl '9002 'qrel( '7pue'a8edallll-I 'E "I ''141'pelqe8g .Lgg-jggt0z,v atapN 'raf,uEtr '2002 'qraa\ 'z ''w ''I 'suossno] PUEuorlEururel]ul Pue 'SIITIIII:VTI 'r^A ilaJ Jo srf,E :ref,u?rpue sllal ruers '9002 'rellnJ 'W pue ''C 'r\l 'e>1:e13 o^4.1 '787-r87tztV atryeN rof,uetrurerq Jo toor rqt rV 't002 g 'ytr 'a1re13

o

VNUIS-dVIXg

+ lvNHrs-lolluoS zvNUrs-lorluoS I lvNurs-lorluoc E e u o Nn

u!lcv urArAJns

dVIX

dUVd

'0689:00r (t002)ysn 'nS'pwvI,tpN')ot1uI unlerrg'885€-€86€:00I

-rrorunl

'l?s 'pnv 'sllef,:eluer lsearq :tua8 vsn l,lpN'rord '€002 'le ra ''1A1'f [e111y arr.rttadso:4 Jo uorlEtrrJrluepr Ja)ue) lo lasuo aql pue slle) Joiunl

'af,uertrS puepeC 'ncuo2

Jo KSolorgaqJ'9002

'y 'A'Sraqura2X uol})npoJlul

sa)uaJalou

ill ol

roud SurIorus trnb or{^4.esoql qlr.,rl.poredruor dderaqlouaqc Surrnp elorus or.{^. sluerled ro; .rerood sr srsouSord aqr 'p dqzvr.royuoueueldxe porSolorq p opr^ord no/ upJ

iuoIlJE aulloflu Jo usru?rlf,au oql lnoqe ls333ns salPnls osoqr oP rEI{lN 'sllal aql Suuolue ylqg Surre;relul ue Surrreq Jo slteJJOlltll -adsuou rot loJluor or peppe sVNU luelalarrl ere T pue 'vNu I svNurs-lorruoS PalloJsuErl ou qll.ry\qrsaP IIar Jo 'svNuls lunoruE er{l selEJrpur..euoN,, POIEJIPuIeqr qrlry\ patlotsu€u ueoq e^pq sllar 6tSV tlrlty uI desse1EN1I E qderS eq1 '.{\oloq slolq urelsal( erll ur Jo stlnser orll s1v\oqs (uI IAJns 'dVIX 'aUVa u^\oqs sE (possossEeJeA\ullfE Pue '(9I rerdeqJ aas) ospur>l €-Id to rorlqlq Jo sle^al aql -ur uE 'Z00V67XI Jo a)uasqe ro eJueserdPUE eurlof,ru

6VtL

sl)Nlulllu

'vs-sttL

'|.auau 'naYatnlpN.sf,rlJuaS usunq ruo{ slqSrsur_rredeJ{eerq pu?rrs-alqnopJo elor l{I .9002-o83e[.V A pue ,.y41.11orsrrq.g

'9€.IA-I9IAtV 'lolg ilQ atnlpN'r{rpop pup aJIJIIer;o .roleln8arB se I-dV 'ZO0Z.tilrey .trtrpue ,'g ,ueqneq5

.ref, uprpuesespdrc-oHu.roorrr:X:;:*r:f :'#?,;1!';i:**

ntuos'(3a4s11)aruorpursreruerrerrarorr"'2I;llJigI",J'^3!, .097_svztl tnuoJ .nadatqeN.pallsl^ar suosrs -paror.laqr pue rreda: qrlerusrry .2002 .laqslC.U pue ,.V .ralnyg .1002 -rJ .C .f snore8uep :suorlerolsuerl aurosourollJ telanoa 'Surqslqnd arnl?N -snuans aJ17 olpadolt(cug u1 ' 0 6 8 LL8t6r Jo ^ 'sua8elnru '1 .g ,re1qcao1pue suaSoun.reJ 'nae sauat laf,urf, ol urrJpopuetuoJt :unrTar{lrda l?luJruuoJrlua.ZO0Z leurlsa} -ur aqt ur Surleu8rslul1'S002 .sre^elJ .H pue ,.O.;;arro8arg ' u n . (" js-"6,9;ti o g t o n 7 q B u r t du, g o p er*qr e d d s,r, @ 'ZZ-nt€ H 2.d 1 h)up) .nay atnwN'dde:aqr :er -uer ur sde.rnqled .7OOZ.[.pre.,nu.,noq 3ur1Jeu8rs 5yg SurraBrel f",rsf.o JriE ,'g ,errle8etr;,1 rurpuaoep-Wlv ar{J .S002 .uels?) .{ .W pu? . . .rnE .sll:o ruelsra3uetr t.1ZI_L6lI:Zt t atuo2 [ .9997 .qdlopnU

'z0s-t.6ttr

'-I .) ur aserrrrroJet pue sareuolal 'no)ste pue,.7.n[ .padola.tep Sntq 'naAatnpN.Snrp raruecrlue pale8.rer .(1e -uortEre '(qrurleur'II,SIJS)re^qC .2002.le te ,.U ,a11r,rapde3 -,1s'uarsdsrreda;-qrlerusr-;'l"?':il'-':;:t!';3r/'i,^:3!,ri""t 'lt t-bT,ttTEn atnwN'srsauaSounreJ ur pue .rredaranssra IJJJulals 't002 'uer.urrg'W .C puule-,rraual .relpeqr?) .S .S ,.V .4 ,.{qreag

j:?^"' IoJsrusruprrreurru",rrr,rf"',i?.?:":l:::#l:it:f;X'Ji,

'jvV-gtVtl|t atn1o57'xedat pue a8euep VNC .t002 .g ,Sreqparrg . .ns .waq)otg 002_g6lt sT, spuaq's$o^reIna ur drrlrqersuraruoua8pue uorleurquola: ,uorlec -rloer uee.rleq sryl-I .0002 .U ,.H ,sezoX_sarolg .rrupolo) pu€ . ./t .,{eurrqred t €_e7.tt taj up) a[ atup N VJUg/ErureuE ruof,ueJeql .t002 .adruo.rg.W pup ..y ,earpuy,q '9LS-V9SISh)uo) 'na[ .atnwN rreda.rygq sreprosrppa]el -ar pue rxnsoluaru8rderurapoJaxur JaJuEC.S002Jo .A .[ ta,rea13 .zgi_LLvt€.sad ta)upJ 'lory 'arntlel r.lJr?esar roJuef,f,rseqrc1 azttd UIVV_uop -uE-I 'V lrr) :rrtrue) pup aserarrrolal'5002 .H .g ,urnqlcelg 'b1z-t6I:I-yZ azaC 'VNC;o rredar uorsrtrxeeprloalr -nu ur uorlruSoca:a8eueq .0002 .pool6..g pue ,.q Alreg 'LM_g1nZ:n€ -say spDV .pnN .n)ue) pue dlqrgelsur auosouorqr 'sarourolal .9002 .oueurnry .[ pue ..y41.5 Aalieg A '807-507t0/. '1org 'luonQ 'du.tg rcqnp Burtdg plo2.sller ruats raf,uer .sorueru.ry pue aseretuolal .5002 .reprerC .lA..) pu" ,.1y'9 ra)ue) u! sJalelaJe) pue sua6oul)Je)

'1otug 'urQg .ttn) .",tourord;orunl 'sarossarddns ,.*rr'ltl;$tt* ereq-CCIto arnleuIenC'5002'{red 'H 'g puE''g 'y 'ueruqreg suleloJd 6u;1oruot6-qynoJg u I suolleln lA )t ua6o)uo

'pwvt,tpN':o.r4'sallorduorssa.rdxa!:r?'*;!?^ht:::"yJ*#{ u?tunr{ snlels aqt Sururpa-rd.I002 .le 1e,.7rq,lser16 Jo letrrurll 'g€s_0€s:slt arnwN .raf,uErtsearqJo eruof,lnoIEtrurTl strrpardSurlryorduorsse.rdxa aur1.Z00Z.1e re ,;[.1 .rael l.ue,r 'ZIVt1ltZ, ila) n)ubJ racuer ur sle.,n .Cpue ..f .3 ,r.req5 Egd pu€ [U eql'Z0OZ.If,nuro3]W -qred

u'runqurs.rolrey orlsou'rru plgtlfj$j'r:!::;#rg . :raf,uer

fl1ure; gg'9002 'olTnrJspry pue ,aur8ra,rol.5 ;.9 ,erqrueo5 n '206-L68:9OV anwN'euueBy(E)14 eqr , to llungns rrlflerer (r4ese5 .OOOZ,.p 3ur1ce1alrrrrur seruourJJetr ta ,1 lerf,eroloJ

'!!s 'srounr ,r"t;{?t#il''E'J':'E - vsn 'wv t,tpN'rord

-ord leuorldrnsueJl er{t ur uorl?relle roqrunu ddor y51qi ;o a1o.r tJerrp ? sleaaa.tsrs.{yeue fer.reortryq .ZO0Z.p le ,.U .f (pe11o4 ,1981_LT.gI:NI 'pnae sauat raf,ueJpu? Suqeu8rs lulA.'0002 7,sr1e1o4

o,asuodsa:r'o,dode,u,''I$?fi2:r2{'r:;;:1.11{',;"'"*0"" 'stzttv

p3 'slsEtsorruoqrrtorodoleruaqpue sasuodser iseris ,rt _a1t{2 rossa:ddnsrounl qU aql '5002 .poaJrEW.J:) pr" ,I .g ,allds '7.LL-89L:6 'lolg 'utdo '.ttnC'uorle:e;qord lP) IIatrJo lortuof, pue rurod ,er1r5_seue14 uortrursrr egl'L66I'8raqura2tr .v .u puB ,.q .1ag '9V01-Ltjttss€, 'paw '['8ug 'N .raf,uer o] peor aqr uo Burlyeq_acuoJseuas parnpur-auaSorug.9667 :adae4 .S .C pue ,.[ .lA,,loory TTef, 'Itz-TTZtI brupJ .nad atnwN:af,upJ ur uorsrJepl?f,rlrJJe :JIJ . -.{r ot lou;o alcfr oI '1002 .prreqreg .W pue ,.1r1,se.rgrun1eytr

'I uon

4u,t{,g n qn

3_u rt

dg p1o2. rru r r,,-'r',f F':lr:::ri ;f, S' H " " .q ,aue1' srsou8erpaqr ro1 de.,nqredgSd Burlroldxg S00Z A "ql.nad .gtl_0€I:€ b)upJ a.mpN.gX dq qreeppu€ eJll ^ '6997 .Bue,6 .I (.N :g (nEr{J - ' 1o uortelnSarpatEurprooJ X p""

.zs6s_Ln6S:€,Ol,ps.pnv .esnou vsn LwN.rord

aql ul eurourJJBJouep? rrleenuedyo uorssar8ord urpJlsuoJ der*gred eqtpue(e91u1)91d qrog.9002.1,ra ;.51{saap:eg 69d-(1;y)61d slorluo) al)^)-lle) pue 6ulllqlqul-r{Mnorglo ssol 6ulsne1suot}elnnl 'tZI-LIItl llal n)uo).ddereqrraruetrur slolrqrquraseur4aursordl :s8nrptreurg'2002.r{rlrTln.y pue ,uouels.e':.X .-t tea.ueq5

L;|t'+! 'Lun er pueI e.rqred H' re)u rzslqu' y:i:g t' ffi

' w n

.9n9I_W9It96Z nuans'urualer_eteg q8norqlBur -yeu8rs rurlX;o slrradpueasrurordeq1-.Z0OZ.lE le ,.I .g ;uoofr '759-7r9tE9 '?S al!'I'low lla)'sseeslppueruludole^apseanuedur 8ur ^ 'I pup ,errqerrey'H ,.f ,neT -leu8rs SogeSpaH'2OOZ.>lorqeH I

to:ttpHBu*dsp1o3'srroareur,o.rr;lr1;ti;?1:'i::";'"#3,{

'S002 .rJlopued -oi ,.5 ,rpuesr:g ref,uef, Jo sepelepo,l[ U A pue . .lotqotndN .1 .uorleur.rs;rorunl urerq ur SLV_ggVtVg_ sde.,nqledSurleu8rsleluaudoleaep Jo rlor erll ,raruej otul Burqd:o7,q 'S002 'E,trg-relsqrelN.f .U .C .I ,U .yq trre8og pue ,relssa;4 n?upJ 'na[ ,mwN.JJf,upf, pu€ luaurdolalap rorrrrdd/.I.1;%I.f"1 .A "r euolselqourlrr eq1*.Z00Z..&\olrpH pu€ ,.W (uossey3

'vtl-vjItv€ ta)upC 'LuotqJ saua1 .seuolaVNCtr parlelas ,{puopue: ;o s.{er -reoJtrrul otuo uorlezlpJrqlq a,rrleredtuor,(qracuer rsearqur seua8 passa.rdxa pue paryJdrueto uorterrtrlueil.ZO0Z.le ra ..f ,4rei3

-eureredlpf, rurrrolsrq-ql,^,ro,r",rrrortfi.-"'Ai:t#ti:t,fff[tr'jjl

-uetruolof, Suryr;o:duorssardxaruaC 't002 'lE ,.C,nrntjag' le Jo

Je)ue) lto slsPg)lleuag egl

' ptg b)up)' utl,,,as's$eu)t 98tt LV.|I' 't002 'l? la ''N'c 'uEtolN -ourJreJI?JIIueI{J Pu?IelueuruoJFua ' s07-96It7,'puac'navahtl -/N 'uorlrauuor{Eerqpepu"rrs-elqnoP VN61eqrPUElrllrqersleur -osoruorqJ'1002reeue)'g pue'sreleurlleoH'f''( (lueg uerr '8L-5VI60S',say'ffiw'esPesIP ueumqpuerred '"I'uosduoql '2002'Pilt{ls'O -arVNC leuolleulquof,a1 Pue

u3)NV) | sz uSrdvH)

0str

'6SZ-OSZtS rournfe rted rossarddns a1crt3p3 iruslu?qf,eru ''fl '[ l(sea'r'r5 -er uorsrtrxe aseqsJ'9002 ',iirygq 'q pue 'tue1 J. 'I60I-t80I:88'uaq)otg 1a3' ['uotr ut -drnsuBr q8norgrsdeqred-rrederVNC pue uorlep8are1c[r11ec '2007';1 'rueruPuns?tuos elor :(1-y3gg) 1 euet reruer lsEerg ' t L6Z-9t 6ZtZValtfficlc alPuom -aBuy'tedatVNq to,tSolorqpue,ftrsnueq3'€002'O 'rareqrs

t-9 'Surssrrusr sardoc l?rurou eqt to auo ro tuasard are seuosoruolr{J eJoruJo auo;o sardor erlxo r1Jrq,&ur sauosoruoJr{J ,o reqrunu ploldp Ieurou aqt uort uorterrrep ,{uy dproldnoue 'uorsaqpe &I-5Ipue 71-51 sern8rg) xrrleru-l]af, eterperu qttt4nt,sawosotusaptluaq pue,uorsaqpe ,sauosowsap pve suorpunl suanqpo Iler-ller olerpatu q)lgr epnlf,ur lsroldocar uorseqpe ro selnJaloru uorsorlppllol Sururel -uoJ er?tJns IIat eqt uo suor8ar pazqercad5 suorlcunl turroqcue (yE-g1 arn8rg) .se1odolpurds eqt pre^lol (ale8ar8es) trede arrotu pue oleredas (1 srsoraruur senSoloruoq parerrldnp ro) sprteurorqJ retsrs eqt qln{.r\ Surrnp e8ets f,rlotrry aseqdeue '126) ue8,ko snoaseS eruesqe arll ur suorlJunJteql Jo ssacord f,r1oqeleruro 'tusrueS.lo,1yace ot Surrro;ag f,rqoJoeue 'tred cqrqdorpi(q e pue rrqoqdorpdq e qtoq seq teql orntJnrls ro elnJolo111 e ol SurrraJeg crqtedrqdue (51-9 ornSrg)'elnteloru e;o dnor8 er{l ol puoq rel VNUI llxorpdq-,E -solSJeue-q8rqE ErApelurl prf,e ounue ue SurlsrsuoJ,srseqtu.(sural 1o -ord ur pesn 'prce ourrue ue Jo urroJ pete^rDv VN1lldJpoururp 'paqJetle sr urer1f,aprs elqerre^ e r{f,rr{.r\ GI-Z pue y-7 sarn8rg) ol'uoqrer n eql 'urote uoqrpr Ierluor E or pelurl ere dnor8 llxoq -JEf, pue dnor8 ourue ue ,surelord Surppnq JoJ sJoruouou eql are ler{l spo€ ourrue eqt u1 'dnor8 ldxoqrec euo pue dnor8 ourure auo lseal te Sururetuoc punoduroc rrueSro uy prJe ourrup (91-y ern8rg) 'y51gu-ard a18urse rrrorJ sVNUur eJnteru luaJeJtlp eroru Jo o.^al Surleraue8 'suorleurguroc tueJettrp ur ragtaSol perqds or? VNUur -ard euo Jo suoxe aql qlrq.lrr.dg ssecor4 turorlds oarteurolle (y-g ern8rg) 'ouoq{tpq eqt ur sdnorS aprue pue ldxoqrer uea.r\tegspuoq uaSorp,(qdg pazqrqels lerrds pepueg-lq8rr p otur paploJ sr sprre ourrue Jo acuonbesreeurl oql rlrrg.{\ ur ernpnrls drepuoras uratord uoruruoJ xqeq (o) eqdp (7-7 arn8rg) 'dnor8 a ro .ureql oprs aqr Surpnpur (eurcdp ur tdacxo) sdnor8 Ierrrueqf,tuereJJrprnot ol pepuoq sl teql ruot€ uoqrer lerluor eql'sprre ourue uI ("J) r.uoreuoqJef, (n) eqdp dtl,trtce s.uralord oqt ur e8ueql e Sursnel ,o1rsdro1e1 -n8ar rr;nads ? ol olnf,e]ou Ilerus e 1o Sulpurq dq pacnpur uratord e Jo ernlrn.tts dreuratenb roTpue drerlral eql ur a8ueq3 ,fuelsolle 'drelsolle lq pereln8ar ere teql sessecordrelnllor pue surarord ol SurrraJe1 Jrrolsollp 'souosoruorgr snoSolouroq uo (snco1)alrs Surp -uodsarror eqt le pelef,ol 'aua8 q)eo Jo selelle o,v\l ur€tuor slleJ ploldlq 'aua8 e Jo srurot e^rleuretle eroru ro o1\\tJo euo eloilp '(euoruror{ ,.3.e) p e alncayoru Iemleu Jo uorlf,unJ 'f,rlaqluds uolto ,e1nrelou y lsruoEe lerrSolorq aql sf,rturru ler{t AJv Jo srsaqtudsaqr or peldnoc Azg pue zOJ or spne lrre; pue sre8ns to rusrToqeloruSurrrnbor-ua8dxg uorleprxo f,rqoJoe 'zO to ef,ueserdaqt ur.ror8 uec teqt ro (zO) ue8dxo snoese8sezrTrln teqt ssacordf,rloqeleu ro'tusrue8ro (11ere or Surrra;eA JrqoJOe

([S]'f -el arn8rg)'sroldaf,er uorseqpproIetu ere surrtelur erlJ 'uorseqpExrrleru-lleJ Surlerpaur dqarerp 'xrrlpur Jelnllef,Ertxe rqt Jo sluruoduror spurg tpql sllar Iprurue Jo au€rqruou euseyd aqt ur urelord rold$or uorseqpp 'asalc(c awlCuapp ftZ-St pue 17-91 sorn8rg) paller osl€ !41y uror; (aWVr) 4ryy cqcdc Jo uorleurroJ sez,(le -ler pue sroldacer ef,etrns-llef,rraqt ol spue8rl ur€tref,yo Surpurq ,(q pate,rrrce sr tegl saru,{zua Iero^as Jo ouo aselJdf, 1i(1,tuepe AIV aes

(41y) areqdsorldpl oursouope

(t-tt olqet:[t] '€-tt arn8rg) A_[V Jo srsdlorp.(q peldnoc aql dq ua^rrp ruerperS Ierrruorlf,orlJele ro luarper8 uorleJluef,uof, slr lsureBB eupJgrueru e ssorf,e elnf,o]otu Ilerus Jo uor uP Jo luoruo^oru PelerPeru-uroloJd godsuerl a.rrlre (17-g ern8rg) 'etBrtsqnspunoq ow uI aSueqJ IpsrrueqJe salourord pue (s)alnralour olPrlsqns E sPurq leqt aulzua ue;o uorSar cgrcad5 olrs oarl)e 'uorldrrosuer satpFrurls leql Jopey uorlducsuerl rryned5

Jolpalpe

(gE-7 arn8rg) 'uortf,eor e lo aier aql sas€or)ur eurrkue ue 1(3raua uorlelrtf,e Jql SurlnpaJ dg 'uortoear Ief,rueqJ E alertrur (ol rarrreq zltreua uorle,rrlce aqt eruof,Jelo) ot parrnber dtreua;o lndur eql 'sleuuer{f, pu? G-tZpue g-E7 sorn8rg) *) *e51 paleS-a8ello^Jo3ur -solf, pue Suruedo e^rtJolosog] tlnser erll se (qlol elJsnru pue suor Jo -nau ''3'a) s]lef, alqetrrxe to auprgruetu eurseld aqr ur pale8edord llrrrrlce lef,rJtf,eleeuou-Jojle 'luarsuert 'prdea prluelod uorpe (g-4 1 arn8rg)'luaruelrJorrnu (s11er osle eos 'uorlJerluof, Surrnp ursodru qtrl\ stf,eJatururlce-g (ut4co-5) eltrsnu u1'@t1co-g)slueu€lrt urlf,p rurot o1 sazrreur,{1od urrot JEIngolS crreruouoru eq1 'surolord Jor{lo /u?ru r{lr^ slre -relur lEql s]lal Jrlo/r€lno ur urelord urlre Ierntf,nrls luePungv 'selnJJIoruoJf,eru lecrSolorq ur sdnorS Jrpn€ .,fieurud aqt are sdnor8 areqdsoqd pue ldxog -rel erlf '(*11) uorord p ateuop upr lpqt punoduoc .(uy ppe (5-71 arn8rg)'salnce -loru Jeqlo pue 'sprorals 'sprJe dDe;;o srsaqtudseqt ur af,Jnosuoq -rer 3 se Pesn sr PUE elJ^J PrJE JrJlrJ orll ur olsrlrf, o1 POJJOtsU€Jl sr dnor8 1&ace aq1 '(VoC) V arulzuaoc or peTurt dnor8 llrace ue SursrrdruoJ alrlogelau algnlos-reteull '11eru5 yo3 l,bare (61-97 ern8rg) 'uetsds snolreu leroqdrradpue urerq eqt uI sasdeuds uoJneu-uoJnou snorJe^ le PUE suoDJun[ JPlnJsnruoJnaua]eJq -alre^ te suorlf,unJ lerll re]]rtusuerrornrN (q3y) euqoqrl&aJp (y1-11 ern8r4) 'souerqrueu Jeln]]al ssoJf,e(saPrldod'suor'sleSns'loratselogr 'sprdrloqdsoqd ''3'a) salnreloru rsrelrp elour ot surelord lrodsuerl aueJqrueu pera-arod-41y s€ uorlJunJ ueuo leql sural -ord auerqureur dyueyredns 3gy ler8etur ;o dnor8 e8rel y

eqrrosaretsq"r lrunqns'rnu todlqurassesrp ",til:!t:?'".iliJ,Ji

r{1r^\ PelErJossedllensn sluetue^olu rPlnf,ololu o3re1 qrlll| dIV Jo dpuuy ase4ay yyy srs.,ilorpdqaldnoc teql suratord;o dnor8 y

'sasaqlueredur palou are slurel peulJepetertsnlll leqr selqa pue sarn8rg 'dresso18srqr ut pourJep oslp or€ uorllulJop p urqtl.&\ sluror poteJplog

AUVSSOl9

srTarprde lsolu serlrapuntErp sluauodruof,xrrleur-J?lnl]eJsrxa IeuIIue '1d) euIruEI lespq Jo lro-4Atauelrt-raaqs urqr V (auuruel pseq (67-91 orn8rg) 'oyorrluacE ot rpllruls dllerntonrls lalquasse erueuoxe aqt SunuJot salngnlorf,Ilu qf,rq^. dpoq pseq ruory urnlletell ro runrlrr p to espq er{l tE erntrnrls 'lErelPloseqaas

Pseq

'turuolo VNq1 ul sJolrel se pesn dlaprr' ore sa8eqd auros'sller l€rrelreq srreJurteqr sn:r,rduy (ateqd) eteqdouaneq (E-1 ernSrg)'selodre{na pue Eaeqf,rp ruort trurtsrp trl1ecrraue8o1dqd'puapoqna pellpr osle lsusrueS:o .{ep-urepour ;o sa8eeurl .{reuorlnlo,re lJurlsrp eerr1legl Jo euo selntrtsuof,leql solodre{oJd to ss"l3 Erraltpq (67-9 1 ern8rg)'ruaure.rour pue eJnlJuls Jraqt ro; ayqrsuodsa-r pue elaEeg pue €r1rf,ur lue -sard suralord pelerf,oss"pue selnqnlorJrru Jo Jlpunfl aruauoxe (gt-8t pre 11-91 sern8rg) dpoq 11acprpl(ot ]purrurat uoxe ruorJ 'uodsue.rt apo8o4at i(1eu -rruJetuoxe pJe^l,otdpoq lac ruorJ srnJlo uodsuerl apotSonluy 'sllJf, e^rau 3uo1e selJrso^ pue selleue8 Jo suoxe ur selnqnloJf,rur lrodsuerl Ieuoxe ;o trodsu€rl patplpeu-utetord rolow -ro

(7-97 e:nfug\'(snulural uoxp eql) pua Surqcuerq '1et '(pp -srp str pre,^aottpoq IIel aqt glr,tr uortounI aql le perereue8 -ualod uorlre) eslndrur rrrlf,ale ue Surlrnpuoc;o elgedec sr leqr dpoq uornou e er{t uor; Surpuetxa ssaco.rd3uo1 uoxE ;o IIar 'euosoruoJr{J xas E upql reqlo eruosoruorqr duy euosolne 'q do,tBorpo,tolno to t'uot8orpotolno ue pallef, sr urlr1 pesodxa eq1 'eldures eql ol rolratap rruorlrale I€uorsuaurp-o.41 ro (uorslnrue) rul; crqderSorogd e Sursodxe dq (]aB rrle.roqdo.rlcelero uorlJes enssrt E ''3'a) eldues e ur selnr -elotu elrtreorper Surzrlensr,t.ro; enbruqcel dqdurtorperolne 'tt ot spuodso.rpue spurq ueqt pue (rotoe; qr.r,ror8''3'a) alncaloru Surleu8rsp sernp -ord 11ace qf,rq.{r ur rusrupqJeru Suryeu8rsol Surrra;eA aurJJolnp (y7-71 en?r.g\ 'xaldu.rocigog pa11ec osle lsrsaqrudsotoqdpue uorleldroqdsoqd o^rtpprxo Surrnp 41y 'auerquau euseld er{} pue 1o srsaqrudsseztrletr.cleqr lprJerf,Eq 'stseldorolgc seuerqruetuplo>1e1,(qr'sruprqrueru ;o ]?rrpuor1rotrru reuur ot punoq 'xeyduror uralord f,rrarurllnry aseqruds 41y (5- 1 1 arn8rg)' d.wnd pa11etdldurrs uerlo ltuarper8 I€rrurerlrorlrele str lsure8e eueJqrueuorg e ssorJealnJe]orullprus ro uor ue yo lrod -suerl elltre oqr ot dIV Jo srs.,{lorpdqsaldnoc pu€ dtr^ute esedlv sEI{ rEI{r utalord euerqueusupJl ,{uV drund para,raod-dlv 'dund paraarod-41y pue asedlv *)/*eN osle ees dS.reuaaar; Jo esEelerqrrrrt ereqdsoqd rrueSrour pup dqv plard or dIV Jo srs -dlorpdq eztlelet tegt sarudzue;o dnor8 e8rel e;o aug asedlv (19-7 ernBrg) 'sassecordrElnller Suurnbar-d8reuoo^rrp ol pesn eq uef, ter{t d8reue ee{ ,o tunoue a8nl B sespelerdJV uI spuog apupdqueoqdsoqd o.ur arlt Jo r{rea ;o srsdlorpdll 'sy1ac ut dtraua eerl Surr.ra;suerl pue Surrnldzc roJ alnf,aloru tuzlrodurr tsotu eqt sI lerlt rprtoelf,nu V (oreqdsoqdrrr-,Seursouope)dIV GZ-IZpue 9Z-IZsanr ''3'a) stuauodtuoc -3r4) '1ae leluered eql uro{ (suratord "VNU* er{l J^rJJeJsllJJ rarq8nep tuJlq1lp lrrer{uresr,trJJrltotnq saua8Jtues o.ru eql qJrq.\\ ur uorsrlrp 1ar duy uorsrarp 11actrrlaurtui(se

AUVSSO'19

z-,

(y-7 arn8lg) 'raglo qJee;o saSeurrroJJIIu eJElel{l sreruosroerels SuDnpord 'sr(ezrr tuara;tlP or\{l ur pe8uerre aq u€f, sPuoq eql'LuoXo uoqro) Pnqz P}IP) osle lsdnor8 IeJIUaqr ro sulol? lue -rorlrp rnoJ ot pepuog ruole uoqrpf, V Luole uoqrer orrlourtu,tse (9E-91 ern8rg) 'srsolru,rSutrnp auosoJlueJ e ruoJt PJP.{\tnoalelP -Er ler{l (sraqr; relsE lerfse) salngnlorf,Ilu Jo posoduof, ernlrnrls 'luplsuoJ runrrgrlrnba eos (?) luersuor Pelelf,osse (g-1 arn8rg) '(errarreqna) prJel)eq ol ueqt selodJelne ol Jepruls eJotu eJE sueaeqrre 'slcedsar owos uI 'suoaoq),M pue pt,tappqaoqz,M Pelle) osle lsursrue8ro .r(ep-urapour;o se8eeurl dreuotlnlona lJuItsIP eerqr oql Jo auo salnlrlsuoJ 1€gl soloifuB{ord Jo sse]J eopqJre (g-11 e;n8rg) 'souerqrueruolqssorr ol'1ore -c.(13se qrns 'solnraloru pa8regcun IIeIus reqto - aeJ e pue role.4^ surrodenbe nd.ollelpqt suralord godsuerl ouprqtuolu to dFue; y (gg-17 arn8rg)'sasedser JOlrOJJaPue JoleIlIuI roJ Jurqf,elu uoIlPAIlJe ue se se^JesPue sleu8rssrsoldode ol esuodserur selquesse leql uralord e 'yydy aurosotdodB u€rlerurueru ;o roureldeq pedeqs-rsrp'a8re1 (eW-tZ pue 'Et-lZ '51-1 sarn8rg)'sesedsecosp aes 'qpap Jo 11atparuwo8old paylecosle !se8ueqclecrSoloqdrou peulJepJIe.^a sarrase pu? stueuoduo) IIer lsolu Jo u^{oPleerq aqr dq pa4reu l;1as -1r s.(ortsap dq 'aseastppue luouldolalap Surrnp sensstl ller e r4Jrr{.l\4. srsoldode or;rcads ur Surrrncco 'sselold pale]ngeJ dllerrreua8 y (g-51 ern8tg) '(trnp 'uaun1 leurtsalur''3'a) eceds uedo ]euralur ue ol Jo dpoq agl to JolJetxe eql or pasodxa sr ef,etrnslerrde aqr 's11aclerleqtrdero osef,eql uI 'erntrnrrs dpoq rer1to ro 'ue8lo ue '11ere ;o (xede) du eqt ol SuIrreJeU pclde

([cE]'E-rr

ern8rg) 'lrodru,{s osle eos 'suortf,arlp atrsoddo ur auerqruotu ]lar e ssoJJe suor ro selnJelou lueJaJJrPorrrl slrodsuen uauodllup) urelord eueJqruaru p r{Jrr{1'tut ilodsuerlor ;o ad,fi y lrodrlue

QZ-VZ pue LZ-VZ sarn8rg)'selnf,elou 'seSeqdor]Eur f,HI I 11sselc sserdxa dlarrlnlrlsuoo (s11acg PUE 'sllel 's11ecrrlrrpuap) sf,dv J .(q pazruSooereq lpuotssalotd uec dagt erer1/rere;rns IIar eqr uo selnf,elotuf,HW II sself,I{lI.{\ uorlerf,osspur seprlded eqr deldsrp pue seprrdod 11eursotur ue8 -rtue up lse8rp uer teqr 11ac,(uy (CaV) ilor Eurluosard-uaSrlue dlr,rrlre rrxololdc Jo uorlelrlrp er{t ro sauDloldc;o uortcnpord dq pamollo; 'asuodsel e,rtlera;t1ord e slnrle ua8rlue ue 's11ar1 ro; lueSrlue eureseql spurg .{11ecr;tceds 's11ec 'asuodsar g rog teql dpoqrrue ]o uorteruroJ slrf,rloue8llue ue ua8rlue eunrulur uE strlrla leql (u8reJot l.lensn) ler.roreurduy (67-y arnBrg) 'ureqr aprrdaddlod Surlror8 eql ol uortrppe JoJ prJe ourure Sutpuodsarroc eqr SuIdJ -rel yNUl aqr su8rle uopoJrtue pue uopoJ e uee.,taleg Surrredeseq 'srsoqluds uralord Surrnq 'VNUtu ue ur uopoJ e o1 ,{reluaruald -ruoJ sr leqt VNUI p ur sapuoelrnu errlll;o aruanba5 uoporllue (61-g ern8rg) 'dpoq aqr ruory ef,uereelJ slr setetrlrJe; pue ua8rlue aru"s eql uo (edorrda) alrs relnrrtred € qlrfi\ slf,pJelurleqt'uatBue ue ot asuodsor ,(poqrrue ur pecnpord dlleurrou '(urlngolSounurur) uretord y '(euourroq ''3'a) elncalour p Iernleu to uortf,unJIef, -r8o1orqaqt s{lolq leql (f,rlaqtudsuaryo'alnrelour y lsruotelue 'uor pa8reqr lle,trre8eu y

uolue

and other organized groups of cells (e.g., muscle), separating them from connective tissue or other cells. (Figures 19-19 andtg-201 base Any compound, often containing nitrogen, that can accept a proton (H-) from an acid. Also, commonly used to denote the purines and pyrimidines in DNA and RNA. base pair Association of two complementary nucleotides in a DNA or RNA molecule stabilized by hydrogen bonding berween their base components. Adenine pairs with thymine or uracil (A.T, A.U) and guanine pairs with cytosine(G.C). (Figure4-3b) basichelix-loop-helix

Seehelix-loop-helix,basic.

Calvin cycle The major metabolic pathway that fixes CO2 into carbohydrates during photosynthesis;also called carbon fixation. It is indirectly dependent on light but can occur both in the dark and light. (Figre 12-44) cancer General term denoting any of various malignant tumors, whose cells grow and divide more rapidly than normal, invade surrounding tissue,and sometimesspread (metastasize)to other sites. capsid The outer proteinaceouscoat of a virus, formed by multiple copies of one or more protein subunits and enclosing the viral nucleic acid.

basolateral Referring to the base (basal) and side (lateral) of a polarized cell, organ, or other body structure. In the case of epithelial cells, the basolateral surface abuts adjacent cells and the underlying basal lamina. (Figure 19-8)

carbohydrate General term for certain polyhydroxyaldehydes, polyhydroxyketones, or compounds derived from these usually having the formula (CH2O)". Primary type of compound used for storing and supplying energy in animal cells. (Figure 2-18)

B cell A lymphocyte that matures in the bone marrow and expressesantigen-specificreceptors (membrane-bound immunoglobulin). After interacting with antigen, a B cell proliferates and differentiates into antibody-secreringplasma cells.

carbon fixation

SeeCalvin cycle.

carcinogen Any chemical or physical agent that can cause cancer when cells or organisms are exposed to it.

B-cell receptor Complex composed of an antigen-specific membrane-boundimmunoglobulin molecule and associatedsignaltransducing Igct and IgB chains. (Figure24-171

caretaker gene Any gene whose encoded protein helps protect the integrity of the genome by participating in the repair of damaged DNA. Loss of function of a caretaker gene leads to increased mutation ratesand promotes carcinogenesis.

benign Referring to a tumor containing cells that closely resemble normal cells. Benign tumors stay in the tissue where they originate but can be harmful due to continued growth. Seealso malignant.

caspases A class of vertebrate protein-degrading enzymes (proteases)that function in apoptosis and work in a cascadewith each type activating the next. (Figures 21.-37 and 2l-38)

beta (B) sheet A flat secondary structure in proteins that is created by hydrogen bonding between the backbone atoms in two different polypeptide chains or segmenrs of a single folded chain. (Figure 3-5)

catabolism Cellular degradation of complex molecules to simpler ones usually accompaniedby the releaseof energy.Anabolism is the reverse process in which energy is used to synthesize complex moleculesfrom simpler ones.

beta (B) turn (Figure3-6)

catalyst A substancethat increasesthe rate of a chemical reaction without undergoing a permanent change in its structure. Enzymes are proteins with catalytic activity, and ribozymes are RNAs that can function as catalysts. (Figure 3-20)

A short U-shaped secondary structure in proteins.

BLAST A widely used computer program for comparing the amino acid sequenceof a protein with the sequencesof known proteins stored in databases.BLAST searchescan provide clues about the structure, function, and evolution of newly discorreredproteins. blastocyst Stageof mammalian embryo composed of :64 cells that have separated into two cell types-rrophectoderm, which will form extra-embryonic tissues,and the inner cell mass,which gives rise to the embryo proper; stagethat implants in the uterine wall and corresponds to the blastula of other animal embryos. (Figure 22-1) buffer A solution of the acid (HA) and base (A ) form of a compound that undergoeslittle change in pH when small quantities of strong acid or base are added at pH values near the compound's pK".

cadherins A family of dimeric cell-adhesion molecules that agg^regatein adherens junctions and desmosomes and mediate Ca"--dependent cell-cell homophilic interactions. (Figure 19-2) calmodulin A sma^llcytosolic regulatory protein that binds four Ca" ions. The Ca'*/calmodulin complex binds ro many proteins, thereby activating or inhibiting them. (Figure 3-31) calorie A unit of heat (thermal energy). One calorie is the amount of heat neededto raise the temperature of 1 gram of water by 1 'C. The kilocalorie (kcal) commonly is used to indicate the energy content of foods and changes in the free energy of a system.

cation

A positively charged ion.

cDNA (complementary DNA) DNA molecule copied from an mRNA molecule by reverse transcriptase and therefore lacking the introns present in the DNA of the genome. cell-adhesion molecules (CAMs) Proteins in the plasma membrane of cells that bind similar proteins on orher cells, thereby mediating cell-cell adhesion. Four major classesof CAMs include the cadherins, IgCAMs, integrins, and selectins. (Figures l9-l and 19-2\ cell cycle Ordered sequenceof events in which a eukaryotic cell duplicates its chromosomes and divides into rwo. The cell cycle normally consists of four phases: G1 before DNA synthesis occurs; S when DNA replication occurs; G2 after DNA synthesis; and M when cell division occurs, yielding two daughter cells. Under certain conditions, cells exit the cell cycle during G1 and remain in the Ge state as nondividing cells. (Figures1-t7 and20-l) cell division Separation of a cell into two daughter cells. In higher eukaryotes, it involves division of the nucleus (mitosis) and of the cytoplasm (cytokinesis); mitosis often is used to refer to both nuclear and cytoplasmic division. cell junctions Specialized regions on the cell surface through which cells are joined to each other or to the extracellular matrix. (Figure 19-9 ; Table 19-2) GLOSSARY

G-3

cell line A population of cultured cells, of plant or animal origin, that has undergone a genetic change allowing the cells to grow indefinitely. (Figure 9-31b) cell strain A population of cultured cells, of plant or animal origin, that has a finite life span and eventually dies, commonly after 25-50 generations.(Figure9-31a) cellulose A structural polysaccharide made of glucose units linked together by B(1 -+ 4) glycosidic bonds. It forms long microfibrils, which are the major component of the cell wall in plants. cell wall A specialized,rigid extracellular matrix that lies next to the plasma membrane, protecting a cell and maintaining its shape; prominent in most fungi, plants, and prokaryotes, but absent in most multicellular animals. (Figure 19-37) centriole Either of nvo cylindrical structures within the centrosome of animal cells and containing nine setsof triplet microtubules; structurally similar to a basal body. (Figure 18-6) centromere DNA sequencerequired for proper segregation of chromosomes during mitosis and meiosis; the region of mitotic chromosomes where the kinetochore forms and that appearsconstricted. (Figures 6-40 and 5-46b) centrosome (cell center) Structure located near the nucleus of animal cells that is the primary microtubule-organizing center (MTOC); it contains a pair of centrioles embedded in a protein matrix and duplicates before mitosis, with each centrosome becoming a spindlepole. (Figures18-6 and 18-35) chaperone Collective term for two types of proteins-rn olecular chaperones and chaperonins-that prevent misfolding of a target protein or actively facilitate proper folding of an incompletely folded target protein, respectively.(Figures 3-t6 and 3-l7l chaperonin

Seechaperone.

checkpoint Any of several points in the eukaryotic cell cycle at which progression of a cell to the next stage can be halted until conditions are suitable. (Figure 20-35) chemical equilibrium The state of a chemical reaction in which the concentration of all products and reactantsis constant because the rates of the forward and reversereactions are equal. chemical potential energy The energy stored in the bonds connecting atoms in molecules. chemiosmosis Processwhereby an electrochemicalproton gradient (pH plus electric potential) acrossa membrane is used to drive an energy-requiring process such as AIP synthesis; also called chemiosmotic coupling. Seeproton-motive force. (Figure 1'2-21 chemokine Any of numerous small, secretedproteins that function as chemotatic cues for leukocytes. chemotaxis Movement of a cell or organism toward or away from certain chemicals. chimera (1) An animal or tissue composed of elements derived {rom genetically distinct individuals; a hybrid. (2) A protein molecule containing segmentsderived from different proteins.

containing membranes (thylakoids) where the light-absorbing reactions of photosynthesisoccur. (Figlue 12-29) cholesterol A lipid containing the four-ring steroid structure with a hydroxyl group on one ring; a component of many eukaryotic membranes and the precursor of steroid hormones, bile acids, and vitamin D. (Figure 10-5c) chromatid One copy of a replicated chromosome, formed during the S phase of the cell cycle, that is joined at the centromere to the other copy; also called sister chromatid. During mitosis, the two chromatids separate,each becoming a chromosome of one of the two daughter cells. (Figure 6-40) chromatin Complex of DNA, histones,and nonhistone proteins from which eukaryotic chromosomes are formed. Condensation of chromatin during mitosis yields the visible metaphasechromosomes. (Figures 6-28 and 6-30) chromatography, liquid Group of biochemical techniques for separating mixtures of molecules (e'g., different proteins) based on their mass (gel fihration chromatography), charge (ionexchangechromatography), or ability to bind specificallyto other molecules (affinity chromatography). (Figure 3-37) chromosome In eukaryotes, the structural unit of the genetic material consisting of a single, linear double-stranded DNA molecule and associatedproteins. In most prokaryotes, a single, circular double-stranded DNA molecule constitutes the bulk of the genetic material. Seealso chromatin and karyotype. cilium (pl. cilia) Short, membrane-enclosedstructure extending from the surface of eukaryotic cells and containing a core bundle of microtubules. Cilia usually occur in groups and beat rhythmically to move a cell (e.g., single-celledorganism) or to move small particles or fluid along a surface (e.g., trachea cells). Seealso axoneme and flagellum. cisterna (pl. cisternae) Flattened membrane-bounded compartment, as found in the Golgi complex and endoplasmic reticulum. citric acid cycle A set of nine coupled reactions occurring in the matrix of the mitochondrion in which acetyl groups are oxidized, generating CO2 and reduced intermediates used to produce ATP; also called Krebs cycle and tricarboxylic acid (TCA) cycle. (Figrre 12:l'01 clathrin A fibrous protein that with the aid of assemblyproteins polymerizes into a lattice-like network at specific regions on the cytosolic side of a membrane, thereby forming a clathrin-coated pit that buds off to form a vesicle. (Figure 14-18; Table 14-18) cleavage In embryogenesis, the series of rapid cell divisions that occurs following fertilization and with little cell growth' producing progressively smaller cells; culminates in formation of the blastocyst in mammals or blastula in other animals. Also used as a synonym for the hydrolysis of molecules. (Figures 22-l and22-8) Large, multiprotein comcleavage/polyadenylation complex pre-mRNA at a 3' poly(A) site of plex that catalyzesthe cleavage (A) to form the residues adenylate of addition initial and the poly(A) tail. (Figure 8-15)

chlorophylls A group of light-absorbing porphyrin pigments that are critical in photosynthesis. (Figure 12-31)

clone (1) A population of genetically identical cells, viruses, or organisms descendedfrom a common ancestor.(2) Multiple identical copies of a gene or DNA fragment generatedand maintained via DNA cloning.

chloroplast A specialized organelle in plant cells that is surrounded by a double membrane and contains internal chlorophyll-

cochlea Snail-shaped structure containing the organ of Corti, the sound-sensingpart of the inner ear. (Figure 23-30)

G-4

GL O S S A R Y

S-9

.

AUVSSO]9 (g-91 arn8rg)'losotdc

eql Pre^aot pelf,orrPeuerqlueu IIel e ro eJeJoI{J are; oqosoilr 'slueuoduror lerelelsotdo elqnlosul pue'sauerquraru'salleue8ro yosolb Surpnlcxaurseldot.(caqr;o eseqd snoonbe perntrutsun ( I - 8 I p u z ' Z - L I ' l - L l s a r n S r g'); 1 a s 'sauosourorgr 'seylaue8Jo tueruelolu -rr PattarlP Jo IIar er{t pue slru.red pue IIaJ aqt ro; rroddns lerntf,nJts pue uollezlue8ro sepr,r 's11ar -o.rd eqr ut puno; eq1 rrrodre>1na;o urseldotdc uotalalsordr 'sluerueyr; olerparuJelur pue'sluourelrJoJJllu'salnqnloJJltu Jo dlreurrrd Surlsrsuor's1uaua1esnoJqIJJo {ro./\^.leN uotolo>1soldr 'sna]Jnu eql eprslno 's11occrlol.relne uI 'tnq euerguaru euseld eqt urqtr^\ peurpluoJ ore r€tp ]lel e to slueluof, snof,sl1 urseldolr(r (y E-71 e:nfi g)'sa11aue3ro crurseldotdr pup snelJnu e qtIA{ qJea 's11arrolq8nep ont aleraue8 ot srsolnu 3ur.trol1o; tuseldoldc eqt to uolsl^rp oI{J srsaurlolr(c

rcr-91pue g-91 sarn8rg)'sde.vrqredSurleu8rsreln]]ar€rlul Suttetttut dqaraql toldarar eqt qrl^\ palElf,ossesoseull ;y[ oqosotdc Jo uoIle I]rP 'euotu or sppol Surpurq pue8rl 'suoJetJetul pue 'sutlnelrolut -roq qfruor8 'urtetodo.rqldJe JoJ osoql Surpnpur sroldecar 3ur -leu8rs ace;rns-llal to sselcroleu Jo rrqru€W roldooer eurlot,{c 'uorlPJeJrlororo uoIlelluereJJIPJIeqr re88r.rt ot s11actuals.(s-eunurrulpue poo]q uo sroldacar JJeJJns 'suore;retut 'SSC-C 'utlatodorgl -ller ol pulq leqt (sur>1na1.retur -.{ra ''3'a) surato.rdpataJJas'11etussnolatunu ;o duy aur4olb pue uorler (eVyZt a:n8rg) 'srseqtudsotoqd -rdser re1n11ac Sutrnp srorrreJ uoJpalt se uollf,un; I{JIrl^{ Jo auos souorqrol,(c surato.rd Surutetuor-aureq 'paroloc yo dnor8 y (79-97 arnBtg) 'suralo.rd ta8ret rr;nads Surrel,troqdsoqd dg apdc 11erctlodre>1 -ne eql;o se8erstuara;Jlp q8norqr uotsserSordraSStrl saxalduror snolre1 'ur1cb e ol punog ueqm dluo e,rrlre .,t11ert ;q3-urlcdr -ldlelel sr l€ql espull urelord y (yq3) eseurl luopuedap-uqc,b 'serudzua eseqt to dtror;neds eterlsgns aql SuruturaloP Pu€ 3utle.,rtlre ,{qa.raqt 'saseur>1luapuadep-urlcdo qrrm sexaldruor uroJ sullr -d3 'e1cdc11arrrtodrelne eqt Jo esrnor aql Surrnp IIeJ Pue eslr suoneJruaf,uoJesoq^\ sutalo.rdpJtElel leJe^Js;o ,(uy urp,tr (tg-St pue '8I-SI '6-5I sarn8rg)'sller rerllo Pue rlf,snluqloorus JEInf,seAur C eseul4 utatord satelllf,E Pue sl]ef, Por uI slauueqf, uonec suado teql roEuassau Puoros V (aWCr) 4y15 crlrIc (Z-St olqet:6-91 arn8rg) 'V eseuDluralord sale^Ilf,eleql 'sroldecer paldnoc-uratord g utelroc to uoIlEIntulls l?uourrorl ol asuodse:ur pernpord 'lotuassaut Puof,esV (aWVr) 4ytgy rqcdc 'seruosoruol{J (61-g ern8tg) 'uolleulquoter osl€ aes peurqruoJeJornpord ot slsoloru Surrnp sprleruorqc leurared pue turssorc Ieuleteru uaellloq Ielraleu rttauo8 1o aSuegcxg rorro (E1-g ern8rg) 'turcqds VNU tJartotr arnsse pue salodre4ne raq8rq ;o sygaur-ard eql ul suoxa elee -ur1epsdlaq reqr sluauodruof,reqto pue surelord g5 Sutputq-y519 xalduor uorlrutocar uoxe-ssorJ Surpnlcur dlquasse a8rel 'uorlf,erelu luel?aof,uou osle ees 9-Z pue 7-7 sarn8tg) 'suortf,ala srred erour ro euo yo Surreqs dg raqraSor selnJo]otu ;o ur surolE aql sPloq leql errot lPlllueql alqers Puoq luaP^otr

'g-11 arn8tg) 'uolrcerlP (I-tt alqeJ.:[C 'gg] (uodnue) elrsoddo ro (rrodurds) oruesoql ut luarper8 uollertuof,uof, str u^\op elnlelotu puotes e to lueuelour ot Suqdnoc ,(q ue,rtrp tuarper8 uolleJluaf,uof e lsuteSe eueJgruetu E ssorf,E alnJelou IIEurs Jo uor uE Jo tueruolotu PelelPau-ulaloJd lrodsuerloc (9-E1 ern8rg) 'pareSuolaSutaq pue etuosoglr eql ol Punoq IIIrs sI urelord luefseu egl se urnlnf,Iler cruseldopua oql olul utelord drol -erf,esp 1o lrodsuerl snoeu€llnuls uon€tolsueJl leuonelsueJlot (t-tt tlqet) 'r31o3 aqr ol unlnf,Ilrr rruseldopua 'de.Lrqreddrolerl aqt ruort suralord alolu seltlsel PerPol-IIdOf, -es aqt ur sa]tlsel trodsuerl lPoJ leql sutalord Jo ssell V IIdOf, ( t-tt etqet)'eeurelslt r31og roqree ot ret€l ruo{ Pue tunlnrllar cruseldopua aqr or r31o9 'deaaqted drolarc aqt tuo{ sutetord elotu selrlsal PalPol-IdOJ -es eqt uI selrlsel trodsuerl leor 1€I{1sutalord Jo sselr Y IaIOf, '(s11acSurpr,trp ur.Sutt apLcal|uot) fuaw -elorrr IIef,rc (s,taEJssa4s ''B'e) uoISaqPEIIaf,uI uollrun; r€ql sllel elf,snuuou ur ursodru pue ultce Jo solPung tr]0""0 elllJerluol 'sl€uErs leuJolxe Jo I?u -ralur.{q pareln8ar tou sI leql (uorle.rrasolllnlllsuof t'8'e) ssarord J?lnlleJ e;o uotterado snonulluof, JI{l Jo olnf,eloru Jelnllef, € Jo ,fur,lrtre ro uortcnpord snonulluof, egl ot Surrre;ag elllnlllsuot (g 1-51 a.rn8rg)'srlerqaur^ ut suotllun( sulxauuoJ deE rurol teqt suralord aueJqrueusuerr 1o dpure; y (3-E ern8tg) 'elnralotu eql uI stuole 0I{l to uoneJol lerreds aqr uror; Surtlnsar suolsuaulP aorql ur elnlaloru -oJf,eul Jaqlo Jo uralord e ;o adeqs asnerd aq1 uorleluJoJuoJ 'euPrqluetu rP]nllef,€ Jo saPlslueroJJIPuo ro od.rque Jo IIel e ;o suotSer lueJeJtIPuI ef,uelsgnse Jo uollerluaJ -uof, aqt uI erueraulP e ASolorq 11acu1 luarpert uoperluoJuof,

rrlauet,., I'uotrunJpueuollelueuolduroc 'VNOr ees

;:iIilil:FH::

(VNqo) y51q dreluauelduror

'uorqse; da>1-pue-po1e ur raqlaSol rr; reql (orerrsqnssll pue eurdzua ue ''3'e) sa]nJalou Suttce;elut o- al uo suot8er Surgrrcs -aq (Z) :raqlo qf,eo qtr-aTsned eseq rce;red luroJ uef, leql spuerls .ro saouanbaspIJe Jlelrnu o.t\l ol turrra;ag (1) ibeluatualdruor ' @-p7 unfu 7l' xaldwoc 4)oilP auotquaut crldloldr el{l Jo uolleruJo, uI saleululnJ leql oPEJseJ3tld1oal -ord e Sutle.ltrce ,(qoreqr 'sare;rns 1e8unl ro lelqorf,Iru or dllf,erlP luarualduroc purq tpqt suralord Iunres elllnlllsuoJ Jo dnort y 'aletrosse doqr qrqr'r qrtm ft-6l elqel!77-51 anfu7) surato.rdaf,pJrns-lleJpue slueuoduroJ relnllaf,Erlxa eql Pue uollng -rrtsrp enssll JIeI{]ul ragrp sad.{rgnssnorelunu eqJ'senssIl elllf,eu -uoJ pue xrJleru Jelnllef,eJlxaaqt;o tueuodtuoc roleu e sI t€t{l ueteloc auqord pue aun.,{13ur gctr uralo.rdocdlSpcrlaq-eldrrr y (e6-g arn8rg)'srolf,EJuotldt.rlsuB.rl urelJar pue sutetord snoJqIJuI Punot dluoruuror isutelord uI seJnl uEJ leql suorSorlecqaq -Jnrts aIrTpoJ'elqets uro; ot aleIf,osse-JIes m crgredrqdure dq pa4reru Jllou lernlrnrls utelord y IIoJ Palrot -uds oullue 1o uolleulruret asnef, Pue sPrJe 'suopor dots e:e earql 'suopor olgrssod t9 osle fsrseqlu.(sutelord Surrnp prce oulrue terlt VNUtu ro VNq uI sePlloelf,nu oarql

(1-y o1qe1)'srseqr .(;rceds rou oP gllrl^'\ eql tO 'rc1dru pe11et J€lnf,Ilred e sor;rcads to ecuanba5 uoPor

fit-ZZ pue E-17sern8rg) 'ruJeposeu pue urepopuo osle aes .sue3.roesursIeurelxe pue 'rualsdssnolJaueql ,sanssrl leruraprdeol asrrsa,rr8lodrqureleru -rueerll stadel lreur:d eJrql eqt ]o lsourrrtno uJapolJe 1o 11eo

(SZ-gI pup y7-91 sarn8rg) 'srsolnu Burrnp tuaua.,loru euosoruorr{J ur alor p ,,(e1d pue ,e11e3eg pue erlrt Jo luerueloru aql :o; alqrsuodsara.re,salyeue8ropue salf,rse,r lrodsuert ueo suraudq 'salnqnlorrrru to pue (_) eql pre.r ot elotu ol srs,{lorplq dIV iq paspalerd8reue aql esn tpql suralord Joloru Jo sselJ V suraudp 'uea4sdn '(de.trqredSuqeu8rs,.3.a) osle ees sdals .rta (t+ ,Z+ pereu8rsap to apef,setE ur rrtpl rnrf,o lzr{l stua^E (Z) are (aprtoalcnup aqrrlsuert rsrr; aqr) uorlrsod I+ eql ruo{ ruearls .dnor8 -u.^Aop seprloaltn5l xo.rpfq-,g y, e qtr.^Apuerls y51q ereld -uel eqt 'uortdrrcsuell Surrnp sa.rotu Jo pue Oql pJp.4ol sr qJrq.^A aseraru.(1odVNU uorlrarrp eqt .aua8 e rog (1) uperlsua\op (E-y ern8rg)'pepuoq-ueSorpdq seseq lretuarualdurot qtr.4irrerllo r{rpe punoJe puno,4 pue Jalleredrtue are spuprts aprloelrnullod o,nr aqr q)rq,lr ur relnllal ro, ernl VNC -Jnrts ,x1eq elqnop leuorsueurp-ael{t uoluurof, rsou eql VNq .,{e.nqtzd e ur tuo ueerlsu^aop ro urearlsdn uleloJd e ot ro tr ot rer{traSurpurq lq uralo:d JErurourr{l Jo uorl -runJ aql sllolq leqt uratord luptnru e Burpocuaelallp ue sr d11ere -ua8 fuorlcun, to ssol e ot JEJnurslJaJte u" sarnpord tnq rauueu lupurtuop E ur stoe tpqt elelle ue ,srrteua8uJ a,rnetau lueurruop (7-g arn8rg) 'uorlf,unJ;o ure8 e uI dyleroua8 tlnser selallE acnpord tueurruop suorletnlAJ.elellE teqt lueuruop E r{tr.,rt pelertosse adltoueqd eqt ot sreJJJosle la,rrssecar sr elelle passerdxauou eqr fato8dzoreler{ e Jo ad,(loueqd aqr ur pessardxaaue8 e yo eJellet"qr ol Burrra1er,scrlaua8uI lueuruop 'urato.rdaqr Jo tser Jql ol drqsuorlelar lerteds elrtJurlsrp e seq uretuop \otr\o1oQo|.e lanl -rnrls drertral ro drepuocas tf,urtsrp e ur pa8uer;e ,qr8ual ur sprf,e ourrue eJoru ro ef: sr ureruop lo.tnl)nus z luralord aql Jo Jrlsr -Jetf,"reqJdtr,rrlcerelntrlred p strqrqxa ureruop .aJnl louor4tunJV -rnrls urpuop leuorsuaurp-eeJr{ls,uralo.rde;o suor8a.rlf,urlsrq 'VNd ro yN61 Jo puerts .raurrd Burtsrxaa.rduorls e,o pue ,t eql ol uoltlarlP ,t FoJ uouecrlder

'po^oru 'uorlPPlxo Jo etrsoooo oql -er sr uo8.(xo ro elnreloru e or poppe sr urote uaSorpdq e uel{^\ srnf,f,o sE e]nJalolu ro ruote ue .(g suortcale Jo urEc uorpnPaJ 'Jor{loue ol auo Iuort luElf,eeJ PaJJa}sueJlaJ€ suoJlJele eJoru Jo uonJEeJ xoPeJ auo qf,rq^.\ ur uorlJeeJ uorlf,nPeJ-uorlEPrxo uv (91-g a.rn8rg)'saurdzuapue VNq perltrnd qll.4aortrl ur lno .rreda.r-yyg Ielalos Suunp .rnlo paureJ Jq uEJ pue srusruer4Joru osle (od,b crSoloqdrour ruereJJIPJo seuosouorqr uao-tuoq ''a'r) uoneurquof,er snoSolouoquou pue uorleurquof,ar snoSolourog 'srs 'sauosoruoJr{t snoSo]ouJorl Jo JeAo Surssorl ol osrr 3ur,lr3 -oreru Surrnp sJnJJo uorleurqruoJer sno3o1ouro11'suollpulgruof, .Llaue,rr3ol pauro[e.rere slueruSerl eql PUEPaAealJJre selnJolou uollpulqluoJeJ VNC Jo saruosoruol{f, rlJrrfl\l. uI ssarord duy 'saJrnos tuereJlrp uror; sluaur8ery ypq 3u1 -uro[ dq or]rl ur peruro; elnreloru VNC IUV VN(I lu?ulqruorer (7-9 arn8rg) 'uorlf,unJ s.aua8aql Jo ssol E ur tlnser dlyeroua8 se]allea^rsseJorecnpord leqt suortetntrJ'se]e]le elrssoler o,r.-t8ur -trtrct (ato8trzouroq)lenpriupur ue;o addrouaqd eql ol sraJeroslp llueserd sr elellp lupuruop eqt ueq,t\ ed,$ouaqd eql ut pessardxe tou sr teql eua8 e;o elel]Eter{t ol Surrra;ar'srtleue8 u1 ealssaJeJ 'sde-tnqred 8ut Ut-gt pue 9y-91 sorn8rg) -leu8rsrelnllererlur Surletlrurdgeragr'utetuop rrlosol.(r s,roldacer eql ur dlr,rrlce eseut4 uratord cgtteds-autsordt sete,rtlceSutputg pue8rl 'srolreJ gtmor8 lueu pue uIInsuI roJ esoqt Surpnlour 'ureruop eueJqruerusuert e18urse qlr.rrrdllensn 'sroldecar ef,eJJns to sselc a8rel € Jo regruew (yag) eseurl aursordl rolderer -IIar

(67-y1 antu1) '(aurosopuodlrea) e]Jrse^ popunoq-eueJqrueu IIErus € ruro; ol eupJqrueu eruseld uorleur8e.rurdg sroldarer ereJrns-llef,cryrcadsor punog sle arlt Jo -rJalpru relnllaJeJtxe srsol,bopua pelerperu-roldacar ;o elerdn 'roldoral Jeelrnu pue roldacor uolsaqpe fi-9l pxe 1-91 sern8rg) osl? aes 'asuodser rplnllel e Surlerlrur dqeraqt totdacar aql ur e8ueqc leuortpruroJuor e sernpul ueuo qlrq^.'(puetq) elnf,elou rplnllef,€Jlxe cr;rcedse sPurq leql snelJnu ro '1osoilc 'auerqureur euseld eqt ur pateJol utelord E setouep .(1uouruo3 'ssecordre1n1 -letrraqto ro 'srsoilcopua 'uorseqpe 'Sutleu8tsllel-llal elerPeruol roldoror elntelouI raqtouE spurq dllerrtrrads reqr uralord duy (91-y arn8rg) 'selue{ Surpeartuare;Jlp oml ut Sutpear dq soptr -dad,{1odtuaroJJrpotul pelplsuerl aq uef, sVNUtu euos 'uopol dots e ol VNUru up uI uopof, tJets uolte]suerl cryrcadsE luorJ sunr teqt (suopor) staldrrt apltoelrnu yo ecuenbeseq1 aurery8urpear -Jeer

'uortf,eor IeJruraqJ e lo efer aql ot sluel luelsuof, a}PJ to suorteJlueJuor eql selE]eJlel{l luelsuof, v

'sroldocer ef,BJrns-llef, ralllo GZ-gt pue 67-91 sarn8rg) euos pue saseuDleursordl roldeoar or Surpurq pue8rl lq Patelrlre lsde.vrqtedSuqeu8rs relnllelerlul uI suoltf,unJ pue .roqrue prdrl

(qOf -E ern8rg) 'suralord (trunqnsrtlnru) crreurrtlnru uI suleqr eptrdaddlod aqr alnlJnJls dreurolenb ;o suorlrsod e^rleler pup Jeqlunu aql

'uortf,€ar (uorreprxo) asJeler er1l .roJIerluelod uorlepxo eqt se u8rs etrsoddo lng epntruSerueues eqt spq g'uorlcear uoltf,npeJ ue^,rr8 e roC 'uorlra]e ue ure8 ot elnl -elou ?;o dcuapual egt Jo aJnseeu e luorttelo ue sure8 elnJa]oru ro ruole uE uer{^\ a8ueqr e8€tlo^ eql (g) prruotod uorpnper

e dq auerqureu etuseld aqr ot pereqral sI tEI{t sutalord qrll-ry\sJo dlrtueyradns as?dl5 el{t Jo requreru f,IJeluouou V uralord seg (t-g elqet) 'selnJelou lerr8olotq uI slaq€l se .,(11er -uarurJedxepesn dluouruor a.re sadoloslolpeJ IeJe^aS'sderep lr se uon€rpEJ slrure leql luolP ue ,o tuJoJ elqElsun adolosrorper

(17-p antugl'(srseqrudsuretord) uorlplsuerl Surteururratdqaraqr'ureqc eprtdeddlod peraldruoc eql Jo aseeleratouord pue VNUrU ur suopor dols azruSora.rleql suralord Ieurosoqrruou ;o seddr o-AuJo auo (gg) rorey oseeler

AUVSSO'tD

0z-9

(gg-g sern8rg)'eruanbaslueror1rpE r{lr.ry\ sVNUur drel ,o teql tou lnq ouaSaqr .{q pepocuavNuru papuerrs-elturs -uarualduroreqt Jo uortpper8epro uonelsuertto uortrqrqurreql -re sernpurleqt VNU pepuerls-elqnop Surpuodserroc e dq euet rr;rceds? Jo uortelrtreur leuortJunC (IVNU) oJuoreJrolurVNU

(y1-7 ern8rg) 'f,not8 g pellel osle lpne ourue qree Jo saruedord relnrrr;ed aql saurrurelapdla8rel leql ruole uoqrer (o) eqdle oql ot paqtet -te dnor8 luenlrlsqns a]qerJel eql (sprJe ourue uI ur"qJ oprs

aprsoelrnuoqrr selerlsqnsse 8ur (11-y arn8rg)'sereqdsoqdrrl -sn puprtsy51g dreruaualduroroqt o{pru 01(puerrsaLoldutala'qt) VNC Jo pu€rls euo sardor leqt ourlzuo uy oseraurdlody11g

'sllaJ Jerllo ot Jo lueuuoJr^uo IeuJOlxe slr ol IIer E Jo asuodsoraql Surlprpetuur pe^lo^ur elnrrloru relnlleJ -€JtuI Jo Je]nllef,erxe due ro; ruJel IpJeueO alnJelou tuqeutrs

(41-g arnSrg)'stsoqluoJet -Jrpo.{\r ur uorte8edord;o alqedeorolJo^ prwseld ropaa ellrnqs gt-6I pue 7-61 sarn8r4) 'suralordocdlSr€lnllarerlxe ur ro sllrr tuerelpe Jo ereJrns aqr uo sprdrloodlSpue surerordordlS ur serta -roru aprJerlJJeso8rlocr;nads qtrl\ suorlJeJelur luapuadep-*zeJ eterpeur ler{t selnrelou uorsaqpe-lleJ Jo ,{1rue; y surlreles 'srsorerupue srsotru Suunp s11atJerq8nep ol saruosoruoJqJ to tuerueldruor lenba ue satnqrJlsrpteqt ssacord aq1 uorlBtartas 'odrqrua dlrea eql ur srxe rorralsodorratue aql 3uole suotalalsordc 1o lrrrelod aqr pue sote1IIel aruanl;ur reqr sualsds Surleu8rs;o stueuodruoo 8ur -porua saueS;o dnor8 e 'oyqdosotq u1 sauat dlrrelod-luaru8as 'llel eql ruort Peseel -er eq ot pourtsap selnralou Sururetuor {ro^rleu rt1o5-suo,4 ayl ruort Pa^rJePelJrse^ Punoq-eueJquaru lleurs alcrsarrfuoloDos (1-91 arn8rg) 'llrl aqt tuog peterf,es,{11enrua,ra suratord pue !suratord euEJqruerueuseld !saruososdlpue'r31og 'unlnrrtar rrruseldopua oql ot pazrlelol surelord euerqruorupue alqnlos 3ur -lros pue Surzrsagluds ro; de.Lr,qredrelnlla3 de,ra.qredfuolortos 'suJnl 'leeqs 'xleq o aql Surpnlcur sernlrnrts reln8a.rotur urer.lJ d pue d aPrtdaddlod e ;o 8urp1o; 1erol 'suralord u1 ornltuls drepuores (5-91 ern8rg) 'uorlf,npsuert leutrs ur suortrunJ teql pue leu8rs r€lnlleJeJlxe ue ;o Surpurq ol esuodsa.rur (sasearcap 'dWCr ro) saseanur uorl€Jluef,uoJ esoq.u (e41 pue 'Cy('*ze) '4yqyc ''3'e) elnrelou relnlla)Ertur llerus V retuasseu puoros 'spuoq a13ursere spuoq uoqreluoqreJ er1l ''3'a) punoduror e ol Surrra;ea pelernlps II€ rlJrr{.{\ur (prce ,bre1 'y51q aruanbos-oldurrs aa5

VN61 arleres

(79-71 en?r.g) 'uorlterluor sraSSrrluorlelnurls elf,snu .,iqparnp -ul *zEJ perols Jo eseeler:suor +zp3 sratsanbesreqr IIeJ alJsnru e ;o useldordc ur seueJgrueuJo )Jo.rtraN runlnJrloJrnuseldocres 'uorlrertuot 67-71 sen?r.g)

(0€-l.I pue Surrnp suetror{s lauo tuarelpe ue

or Isrp Z euo rnor! Surpualxe pue sluaurelrJ (ursodru) Ilrqr pue slueru€lrJ (ur1re) urqr Surddelre,ro 'pazrue8ro ;o pasoduroc e1c -snu (1era1a4s) paterrrs Jo trun Iprnlf,nrls Surleadeg ororuorrps 'apdo ler ea5 aseqd (slsaqru,(s)5 'aseldxoqree areqdsoqdsrq-S'I esolnqu ees

orsrqnJ

(77-y a:nF;.gl'solo.{.re1nereqSrq ur VNUr SS pup 'Sg'S 'S8I 'S8Z :luelrrJJaor uorletuarurpes rreqr dq pareu8rsap uar;g 'saruosoqlJ Jo stuauoduor IEuorlf,unt pue IerntJnJts eJeteqt selnJ -elour VNU e3.re1 (ygg ptuosoqlr) VNUr lerarres;o auo duy (6-8 Pue g-g sern8rg)'auosoacqds oslz aas 'sy51Aru-ardur suoxe yo Sururol turcrlds yNg Pue suolur Jo lE^oturr ur sllnsar reqr sseoord y

'VNUur drelueuraldtuof,-Jpau ro drelueurelduroce ro uorleper8apselerperu reqr (VNUrru ;o uorssordar IeuorlelsuBrt ro y51grs) vNU pepuerrs-a13urs lrorls E rllrl( pelerf,ossexolduroc uralordrllnu e8rel (CSIU) xalduroc turcuays pernpur-VN1 'pererlesr ygau-ord e;o aruenbas eqr qrrq.{ ur SursserordVNU Jo ed,{rlensnun 8urrrpoy51g (11-p an8r.7\'uorldrrcsuerl pup erntrnrtsurtpruorqrSurllortuoc ur pue 'sy11aurJo uonelsuerrpue drrlqersaqtSurllortuorur selor deld sy51a l]erus Jo ,(torre,te fsrsaqlu.{suralord ur selor tueroJ -1rpdeld pup 'VNUr 'VNU* 'seprtoelrnuesogrr;o pasod VNUr -uoc'raudlod papuerts-a13urs l€our.1 (prre rrelf,nuoqlr)VNU 'xoldurocEuroueysparnpul-VN1 ees f,SIU (Ey-71ern8rg) 'o)stqnf,pellel osle lereracllSoqdsoqd-g selnrelou o1r\.1 urroJ Jo or (ereqdsoqdsrq-g'1 asolnqrr)re8nsuoqrpf,-o^rt e ot zOJ Jo uorl 'a1odr -rppe aqt eql ur uouf,eer eql saz(letet ur,rp3 slseld tsrrJ reql -orolqr ur petef,olaur.,{zug aseldxoqrecoreqdsoqdsrq-S(I esoFqu 'srseqlu,{s uralordpue SunrldsVNU ul uorlf,unJ soulzoqra 'dtr,rrlcecrtlletec qlr^^ alnf,elou yNU uy orudzoqrr 'vNur ees vNu leuosoqrr '(srs G.e-, pue 77-y sorn8rg) -aqludsurato.ld)uouelsuerl;o eur8uaagr ltrunqns]leluspue lrun 'suratord upql oJorupue selnf,alou -qnsa8rele olur pozrueSro 0S VNUr tuare;Jrplera^osSursrrdtuocxelduror e8rel y aruosoqrr 'sdNU ruro; rqr ur Jo llol or{tur tueserd are selnreloruVNg rsoyq 'vNU pue surolord;o pesodruocxeld -tuoc due roJ rurol Ierauog xoldruoc (4111a)urerordoapnuoqrr 'vNu ees (vNu) PIrErralrnuoqrr (96-9 ern8r4)'serpnlsate -{uq uErunqur r€lnJololuPOs€q-vNCse e^resuer teql sro{r€ru slenpr^rpuruea.r\tegsef,uereJJrp acuanbes;osed.{rIera OsJo auo ery 'saudzuouorlJrJlseJrelncrlred lq pozruSocersalrsdorlsop Jo elEeJJrpql VNC rnuoueS;oeruenbaseqt ur slenprlrpurSuoue serueretJrq (srusrqdrour,(1od qrtual lueutery uortorrtsar)sd'I{U (y1-9 arn8rg)'ereldurarVNU pepuerls-e18urs e uor; pazrsaqruds sl VNq PePuerts-elgnoP e rlllq./!\ uI uolrJear xalduoc e sazlle -teJ lErlt sesnrrloJtalur punoJ eu:dzug aselduf,sueJlasJeAeJ $g-g a:nfig\'suralordlerr^ roJ sVNUU eqt se se rnuoua8Jar1tJnJ ol esrrse,rr8pue 'snrr,rorde Sunu.roy VNU Ile.,rn .VNC Ipruosoruorrlrrelnller olur peuasur sl VNC IErr^ srql 'vNU eql ;o ddor VNe e Sur4eurrsrr; dq sller ur salerrlderreqr auroue8VNU ue Sururetuoc snrr,rorlodrelne;o edl.1 snJraoJleJ (qg-9arn8rg).uos 'dots -odsueJl osle JaS uondrrcsupJt esJeeJ E se^lolur pu€ elerp -eurrelurVNU u€ .{q pererparu sr aurou:3rqt ur lueruelou esoql\ crlodre4na;oadll uosodsuerlorlar lueuele yll.Iq alqesodsuerl

LZ-'

UVSSO-t9

dq pa,rou ere leql rro3 Jo uetro aql ur sller rrprl Jo suorsnrl -ord pa11ry-lueu€lrJ alrtouuoN (run{rroorals'8urs)erpooarols (7-17an*gl slertualodletueudolaaaptuoreJJrpqrr^aslleJ;arq8nepalereue8 o1 Kllwrualuu.tCsp to IIer urots le]urrpd eql ol Iprrluepr sr lprl -ualod esoq.ry\ sllel rarq8nep o,r\t ot esr.r a,rr8o1 leluarudole,rap Kllotulauu(s epr^rpuef, legl 11erSur.trauar-JlJs V IIOrtuols $7-7enBr.g) 'luelsuoJ '1enbeaJEeoupls sur€rual uorleJtuJ)uoJ str os leql -qns € Jo uorldrunsuoc eler pue uortpruJo! ewJ eqt uerln\ Jo Jo uortrpuor aqt 's.,te.u,qted rrlogetau relnllel uI elels dpeets (71-91 en?;.g) 'sroldoceraur4ol(l ot Surpurgpue8rl3umo1 -1o; eqt ur pele^rtJeeJeteqt srotJeJuorldrrcsuertJo sse]J losordc 'uorldrrsuerl Jo uorle^rtrv pue uorlf,npsuera1eu8r5 IVIS (97-71ern8rg)'s11er rueld ur arerpdqoq;era8erolsdreurrd eqrsTteqt 'slrunesocnlS yo dla,rrs -npxa pasoduoc'eprreqcces.{1od peqruerq'3uo1drerry rlf,rpls ( g g - 9 1e r n -8tg) 'sptdtlrer1to srseqluds se IIa^{se trodrurpue srsaqluds Jo IorerseloqJur pe^lo^ur surelordSurporuasoua8;o uorsserdxa atelntultsueql pup sla^el re]nl]ef,,violol asuodsarur lorotseloqr 'sJolreJuortducs pate^rtf,ear€leqt 'aue.rqrueur UA eWur pazrleJol -ue.rl luepuedep-lorerseloq3(saggUS) suralord turpurq-ggg (11-garn8rg)'EurcrldsVNU rno serrr€rpue y51gur-arde uo salquesseleqt xaldruocuratordoalcnuoqrJ aSJe'I otuosoarqds (q9-61orn8rg)'(seprsoq8ue8'saprsorqoroc) dno.r8peaq ererp,{qogrec ;o (uqo.(uro8urqds) dnor8 peaq parell.roqdsoqde rar{trepue surpqf,uoqJEJorpdq 8uo1o,!u ureluoJl?rll'ourso8urqds ruo4 pa^Irap 'sptdrl auerqwaur;o dnor8 .lo[eyrq prdqoturqds 'surelo.rd cr;neds8urre1.(.roqdsoqd trq apdc I]ar eqr;o aseqdS eql otur 11orcrrodrelneE to eruprtues.re33rrl pue uqr,b t9 e pesod teqt '(yq3) aseuDl luepuedap-uqcdc ;o -uroc'uretordJrrerurporetaq V (rotteyEurtourord-aseqd 5) g45 'ftt-ZZ pue 'Sururelted ZI-ZT.sarn8r4) IErlue^-lesrop ur suorlJunJteql sodrgua uerqrqdue Pue rorratsod-JorJelue .(1reayo eprslpsrop rrlt uo ratueJSuqeu8rg nzruetto uueruads

(61-y1 arn8rg) 'uortrsodde osolr otul souerqlualu 'saxelduroc elgEls dra^ sluroJ auerg teSret pup olf,rse^eql Sur8urrg -ruaru la8rel e uo SEUVNS-I eteuSoc qll^{ olJIseAP uo sgUVNS-^ 'seuEjqtuetu leSJel qll^\ soJJIsJA uOISnt alou JO to uorlJBJelul -ord reqr surolord ou€rqruaru ler8atur Pue lllosoilJ SSUVNS fiZ-07, pue gE-9 ern8rg) 's11ecrelq8nep ol sauosouortlt I€Iratreg Jo uolleSer8es radord 'aseqdeue uI uollere eql ur uorlf,un; sutelord JWS IeIJerJEg 'sutsaqo) pue 'srsolrtu -des rraqt lrlun sprtpruoJtlJJatsIs{uq glII{^\ Surrnp soruosoluoJl{f,esuJPuoJ dlag qrq,,n 'sutsuaPuo) ePnlf,ur dpue; srql Jo sregruory 'slsotllu Surrnp uorte8arSos radord rreqt pup saruosotuorr{l Jo eJnlf,nrls lecrSoloqdrou eqt Sululelureur JoJ Ief,rlrJf, oJe leql sutelord ult€uroJqf, euolslquou ;o dpue; IIEIUSe suralord 31415 isuralord atuosoruoJtlf, Jo eJueuelul€Iu lernlf,nJls (y-91 arn8rg)'sroldacar of,€Jrns-llef, rrer1t ol salnreloru Surleu8rs ;o dpue; (dgCf) $ roree; qr.trort tururro;suerl eql to srequreu ;o Surpurq 3urmo11o; uorleldroqd -soqd dq pelp^rlJe ere lBql srolJe, uotldtrcsueD Jo ssEll sPelus (qg7-g arn8r4) 'VNUttu osl€ aes 'seue8 rr;rcads ;o uotsserdxe lIgIquI dlletuaurrredxe ol peuSrsap aq uer sVNUIs 'yNU Klottqtqut llpus Pue yyly Suuapaw ilputs ro Ltoqs pa11erdlsnorrerr fd1roe;redsrted eseq VNUIS aql qrlg.4a ot SVNU 1a8re1saleelc lPql (f,SIU) xolduroc Sulcuelts Paf,nPuI -VNU up rurot ot sutatord Iereles qll.4t salelJosse VNUIS eql Jo puErts alSurs V 'pua rlJeo te seplloalJnu pepuerrs-a18ulso^\,1qll.{\ ';1eursy 3uo1 saprloepnu tZ-lZ 'y11a pepuerrs-rlqnoP VNUIs 'SANIS IIE rsuElunq uI 'VNCI lnoq€ roJ luno)Je slurlurle n/y to sPrrrll-o.ry\r (8uol dq uerunq lelot Jo ruerrod €I: elnlrlsuol lPql ool-oot 'suosodsuerlorler Jo ssPIJ (stueurelaPesredsrelulrroqs) s9515 .YNC anpps peller oslE :Peqursuerl lou erE Pu€ suollProl letuosolu -oJqJ reqlo le sP lle/r\ sP soJeluolal Pue saJaruorlueJ lE Punot aJe 'rroq5 y5q eruanbas-aldurrs leqt seruanbospaleadar ,(luapuer

aqrif:"llt**X";t:i aqrJodrl'rqeeurrad pot'r osp :euerqluoru

o1 leuoruodord ete.r e te tuerper8 uolleJluaf,uof, sll u.,rroPeueJg -ruew e ssoJJe alnJelour e Jo lueureloru leN uorsryyrp eldurs

'atuanbas ta)uafs (97-gornSrg)'oqordpne rrolf,nu peleqelp ol uollezrpuqdqdq srsarogdorrcela.(q seruanb pal]€J osle lrecueltseql to srl€d osPqPerPunq l€re^esult{]I.ry\seuoS pareredas -as VNC rrpeds Surloatopro; anbruqcal turrrolg ureqlnos dqareqr ;o uortdrrcsuerl ro; perrnbar sutalord to sserle Suqcolq 'uor8a; pozrlef,ol E uI seJnltnJls uIlPttroJLIJ PesuJPuof,to uoll ft-n anel) de.ra,qred -Erurot rJJuells ut acuenbasy crlo.{re1ne setourord reqt VNC rndropua ro ,{rotarcaseql ur euerquerurouop € ruort pnq lagt 'sesuooseJ sp salJrseltrodsuerl.relnrrlredol uralordeql stJeJrpleql uratord e ulqtl.!\ ecuenbas ,{]e^rlplal prJe ourup turuos uoqs V leutrs 'sller

urelstlnpe .rocruodrqua uro.r;3ur1re1s aJnllnf,ur sed^&11ec cryroads Surtereue8roj arnpaf,ord (fNCS) raJsuerlreelf,nuIIer f,rleruos '11arturat E ueql raqlo IIer Ieruruero rueld duy

ilor f,qeruos

Jplnllaf, Jr1rladseJour ro auo ut SurleululnJ PuP roldecar P ol IEU -3rs relnllacerua ue eql Surpurg dq palerrrur ssacord lelluanbos to 'ratpoue olul ruro1 ot sreter .{luouruor l.3o1otq IIaf, uI IEJIIUaqT ro yecrs.(qdeuo luo{ leu8rs e Jo uolsJe^uo3 uorlcnpsuerl pu8rs

(I-€I etqer) 'atuanbas osl€:llel pu8rs pa11et pue apaQad 3ur7.agw1-a4u1in

(II-8 pue eqt urqlrll uorlpJol rr;rcads e ol utelord eql slf,aJIP tetll uratord 'yNlXu-ard Surrrldsur uorlf,unJpue otuosoocqds e ulqtr^\ ecuanbospne oultue rJoqs .{le^IreloJ V acuanbes putrs 6-9 sern8rg) to er{t Jo stuouoduor a.resvNuus e^rc 'snalf,nueql or Pezrl?rol SVNUalgets'lleurslere^esJo auo (ypg realonuleus) yg1us 'snloelJnu eql uI uorleJrtrpouaseqpue Surssecord VNUt ur suouJunJter1l '1yeus;o addr y (y515 reloapnu VNU elqers lleurs)VNUous

(9-91 arn8rg) 'paralduror er€ UE alp olul uorlerolsueJt pue utelord Jql Jo slseqluls areqrrrou€Jqtuetu uE aqt ol xoldruoJ Jruosoglr/uleqf, tuaJseu aql sre^IIePpue urelord drola.rras tuef,seu e ut ecuenbaspuErs gg eql or sPurq legl aprtred (4g5) ollrrred uorlrutocarleuErs urolordoolcnuoqrr cqosotdr y

'lduJsueJl ,fueuud el8urs e otul peqrrJsuert sr teqt 'elrs uorlBunuJet pue elrs (;rers) uorlerlrur ue dq pepunoq 'VNq ur uor8er y lrun uorldrrcsuerl 'secuanbaslroleln8er rroqt ol Surpurq .(q seueBrelnrrtredyo uortdrrcsuerl (srossorder)lrqrqur ro (srole,rrpe) elelnwrts srotrej cltcadg'atrs lrels eqt reau xalduoc uopetlrurard-uorlducsuerl eql ro uortEruJo,ur etedrcrued ,saua81e;o uorldrJ]suerl ro; parrnber 'srotf,EJ rrrodrelne ur uorl lpnuaS's1Iar -drrcsuert atelnSar to elertrur ot parrnba.r'aseraur.{1od VNU ueqr raqlo 'uralord due .ro; rurat IEreueC (ga) rorcey uorldrnsuerl 'aua8 Jplnf,rued e;o uorldrrcsuerl aleln8ar teql seouanbas.(roleln -8er y51q eqt uortar lorluoo-uondrrrsuel ]lE rot urrat e^rrrallo3 'aseraudlod y51g fifV pue gp-y sarn8rg) /q VNU dreluaualdruoJ e Jo srsaqtudsro; eleldurel e se pasn sI alnJe]ou VNe e Jo puerts euo r.{Jrr{1r^. ur ssef,ord uorldrrlsuerl (E1-91 arnSrg)'auerqueruunlnJrlar cnuseldopua aqt otur suretord euerqrueusuer Jo sass€lJsnorJEA Jo uoueluJlro pue uouJesuraqt tJJJrp luauaSue:re pue lequnu 'ecuenbasasoq.Lruralord e urqtrA.stuauSa5 secuanbasrruatodol (gg-y7 arn8rg) 'addr ylar eqr uo Surpuadapsasuodsarsnorrel sef,npurreqr de.rgted 3ur1eu3rsp selertrur Surpurq pue8rl .strn -pord lerqo.rrrur;o dlerre,r B azruSoce:tEgl sJolda)er relnllafpJtur pup aJe;rns-llel Jo sself,e Jo raqruery (911) rordarer e{q1lol (9p-61 ern8rg) .euerq -uaru eurseld eqt;o suor8er lereleloseq pue leorde oqt uee^\teq stuauodruoc eueJquau Jo uorsnJJrp pue sllel uaarr,rtaqseceds eql ur suor pue selnJalou lletus ,{ueur pue solnlaloruoJf,eru Jo uorsnJJrpstue,rardlpqt sller leqeqrrda luerefpe Jo seuprqurau eruseld aql uee^4,taquortcunf l]er-llel ;o eddr y uorlounl lqtrl (67-71 an?r.g) 'suelsdsoloqd pue sluau8rd rrraqlu,{sotoqd aql uretuoc puE sTtpls ur pa8uerre aq uer teqt rseydorolqcE ur sres snouprgruorupeueuelC spro1e1,(ql (e61-g ern8rg) .sureqcaprs uee.trteqsuortreretur luolelof,uou a1dt1lnrulq pazrpqerssr r{r1{1r\'ureqc aprldaddlod e .suralo.rdu1 alnlJn4s ,fuerlrol Jo ruJot leuorsueurp-eerr{tIIpJaAo (9-g a;n8rg) 'eJIIroJ Ierruessesoua8Jo uorlef,rtrtuapr ur InJesndllercedsasr uonelntu;o adlr srql '(arnleradural o,rrssnu -:aduou eql) aJnleradtuetror{toue rE addtouaqd luetnru e 1nq (arnt -zradruel arrrssrturad eql) ernt?Jeduet auo t€ ad.&ouaqd adlr-ppn e saonpord teqt uortetnru v uorlelnu (sl) orrprsues-arnleredruel (79-91 ernSrg)'paralduoc sr (srseu ,asuapuocepsaruosoruorqJ -qordc) tuseldotdc eqt Jo uorsr^rp pue egl (sauosoruorqr pale.ledos Jo sles o,trt aql punore sruJoJ-er adoyaauereelf,nu eqt qlrq,/y\Surrnp aSelsJrlotru leurC eseqdolal $y-9 anfu g)'uorlectldar ylrlq Surrnp sauosoruorrlf, ;o Suruot.roqs slua,rard leqt sserord lercads e 19 paleclda.r ere pue uonBtar8es euosoruoJr{J redord JoJ parrnber ere seJeruolel .acuanb -es ('IgJ) rrreruolet E uoqs ;o sreader uapuel eldrtlnur Sururel -uoJ eruosoruorqc crtodre>1nee ro pue rlf,eo le uor8ag eJeruolel

Gz-vzen

-3rg) 'xalduror tqJ Jrreurtlnu Surcnpsuert,leuSrsaql qlr.,rapoterr -osse pue suorSor pu€ olqerJel tuelsuoJ 3urureluoc uralord aue.rq -ruelusueJt Surpurq-ua8rtup JrJaurpoJ3far{ V roldecar IIet-I OE-VZ pue yg-97 sa;n8rg) '(sllal g ,seurloldc ecnpord ,pelcrrlser to uorlelrtf,e ro; pa;rnber

A U V S S O Il

zz-,

'(sllar rorunl pue IHW II sselote4reur tqf,) sllor TnQaq pue 'palcrrlser 're>1reur pelre;ur-snrr^11r>1 ace;rnsgq3) IHW I sselc sllerL)txotoTd: :sasselo ro[eruo-n]arpereql'solnJeloruf,HW or pexaldurorsaprrdad rrua8rtuepurqreql srotdecercr;rcads-ua8rlue sossardxa pue snurdqreqt ur serntetuteqt et/Joqdu,{l V IIor I (71-7 an?r.g)'selgruesse xelduoc uort€rlrur-uorldrnsuerl eql eJeqA\seua8Surpoc-urelord f,tloIJe{ -na dueru;o ralouord aqt ur aruonbospo^Jesuof, V xoq VIVJ

(c'q6-Sern8rg) 'aua8petelarp Jo erueseql ur uorlelnur Jaqtoue roegecrddr ;o -ouoqd oqt saseeJf,ur trreqrur(s teqt uortelntu V uorlelnru 1eqra1 'seIJJqS luaJOJJIPeJotu Jo o./r\l uI OIuOS -oruoJrlJ e uo repro orues aql ur seueS ro eJueJJnJf,O i(ualuds 'SurleuBrs 'sleu8rs Ilel-lloJ ur Surledrcrtred .,{geraqr Ieurolxe purq deru pue 'uolela>lsoldr aqt r{ll^{ lf,eJetur 'uorsaqpe xrrleru-]lef, ur uortf,unt teqt supl^ltoalord are;rns-llar Jo sself, v suecapuds 'eueJqrueruetuseld a13urs e dg pasopue ruseldotdr to sseru paleo]rnurllnu V unr1lbuz(s (y-E7 arn8rg) 's11ec crldeudstsod pue -e.rdeql 3urlcouuoo suorlrunI det er.t sJnJf,ouorssrrusuerl aslndrur 'osdeuds )t4)ap ue le frollrursup4ornau e dg parcnpuoc sr eslndurr egl 'osdeudsp)tuaq) E tV 'peltrursuert ere saslndrul glH,1a ssone (y1er elf,snru ''3'a) 11ecalgplrf,xe ror1lo Jo uoJneu tuecelpe u€ pue uoJ -neu e Jo lpurruJatuoxe ue uee^\teq uor8er pazrleroad5 osdpuds

( [s e]'g- r r

ernSrg) 'godrlue osle ees 'uortrorrp auos eqt ur auerqruaru IIar P ssoJJe suor ro selnJslor.utuaJeJtrpornl slrodsuen (tauodutcsl urelord eu?Jgrueru e r{Jnl1\^ur ilodsuerloe ;o addr y trodruds

(e6-g ernBrg) 'suretord Surlreretur Surpocuasaua8dyrluaprol pesn dlruanbar; ere suorlelnu rossarddng 'uort€lnru puooes E to rle;ta crd,bouaqd eqt sesJelal ter{l uorlelnu V uorlelnru rossarddns 'dnot7 ptqt e pa]]Er osle lurole rn11ns E ot pepuog dlluele,ror uote uaSorp,iq e;o Surlsrsuof,selnf,elou reqlo pue ourelsdr prJe ourue eqt ur lueserd dnor8 tuentrlsqns V (gg-) dnor8 l,trpdqyps 'uo8dxo Jelnf,elou ro o)Jo, ar'rtoru-uotord e uo puedep lou op teqt suorlf,eer ur sour,(zuacrlosotlc dq pezdprec t4 pue dCV uro{ dJV to uorl?ruroC uor1e1{seqdssqd leirelarerrsqns 'atulzue ue dq pez,(p -leJ uortJeal e ur e8regc e soo8repun reqr olnJOIoW oleJlsqns (ca-91 arn8rg) 'sdno.r8ldxorplq eroru ro auo Sururel -uof, sproJots erc s1rc.,.als'sproJels ere (euorelsoSord pue ueSorlse ''3'a) 'spunodruoc seuourroq .(ue14 lueuodrur patelor pue Iorel -salor{r Surpnlcur suoq.recorpdqSurr-rno; yo dnor8 y sprorals 'puoq elqnop e Sururetuor srlnf,elour surro1suotl lo pue sn eqr apnlJur staLuost )r4awoa7 'uorqspJ e8erur-.rorrrur e ur paBue.rre ale ruole uoqJer lrrlaunu,(se uB ot papuoq sruolp eql (-I PUE C peleu8rsop 'statuost \oil1flo u1 'sluaura8ueue len -eds ur JepJo erues aql ur aJe ruaragrp polurl sruole esoq^^sel lnq -nuroJ Je]nf,eloru sJaruosroeJels l€Jrtuopr qrr,n spunodruoJ olnl .11errreq r{rea r1lr.r\peteroosse ftt-tZpue gE-97 sern8rg) suoxe eql ur uorlezrJelodep3ur.ra88rrt'suorlerqr,r pef,npur-punos

€z-9

AUVSSO't9

-nrurur rprlord ol rep.ro ur asuodsar aunrurur ue ol pau8rsap IJIJIO pue uaSoqredp ruo{ pelrrepuortBrederd snonf,ouur uv eurJJea

$y1an8r.g) 'salodre4naraq8rq ur tueruele leurxo.rd-ralourordro raJu€r{ua u€ ol lual€,rrnbeluorssardxa aua8leurrxeruro; d.ressereu sr lpql seto,fue4na aldrursraqlo pue lseadJo VNC aql ur aruenbas.{rote1 -n8a.rSurpurq-urelord duy (gyn) eruenbesturle.,rrlce rueorlsdn

-r{reru selodrelna pue satodrelord uI selotu dq euoua8 aql e tegt 'uosodsuerl ul tuaserd lueuele y51q olqesodsuerl v vNq (g-9 arn8tg) 'tueruelo yo addr egt uo Surpuadep uslueqJalu elsed-Pue-ddor Jo rusrueqJeru etsed-pue-lnc e ,(q srnoco ieuouaS eql ulqll^\ uoDlsodsuErl elqesodsuerl e Jo luauo^o,^{ luauela vN( (f -g atqet) 'padal Pashdsnlut pue lualuaP VNQ afqou PaIV) osye luorlrsodsuerl ,(q uotttsod A\ou e ot elolu ueJ pue satceds

'rueorlsu^{op ''3'a) sdars E JO slenPr^rpur IIe uI uolleJol leuosouroJrlf, elues Oql uI lue osle aes'(de.uqredSurleu8rs ;o epeJsetr E uI JaIIJBa Jnf,f,o leqt sfueaE(7) ')te'Z-'1- pateu8rsap -sard tou sr l?gl ef,uonbasy11q duy luetuelo ypq alqesodsuert are (aprtoalonu peqrrf,suprt rsrg aql) uorlrsodI + eqt ruorJrueorls 'euErgrueru ]eSreteql qll.&\ uolsnj dq stuat -dn saprloapng 'uorldrrcsuerlSurtnp saloru esererudlody51g ur lpqt ol elrsoddouorlf,errpaql'eua8e rog (1) ueerlsdn qrrq.ry\ 'puoq eldrrrro elqnope sr spuoguogr€J-uoqreJer{t euo Jo qlrr{^r.ur (poe drre;''3'e) punodurotE ot Surrra;aA petprnlesun ([VE]'E-f f arn8rg)'srat.rodrun;o sald -rupxeparpnls-lle.la. erp (surelordIn.1O) sratrodsue.rt esornlSeq1 'uodsupJl pelelrlrf,pJer,r uortertueJuoostr u,r\op eupJg tuarperS -rueruE ssoJJpe]nJelour llerus€ Jo tuetua^oruseterporu(.tayod -run)utalotd euerqruauE qJrq-a ur trodsuerl;o ad.{ty ilodrun 'srseqtu^s dIV Sunrqrqurdqaroqr'slseldorolgrto euerquou proleldqr ro eu€rq -ueru Jeuuraql ssoJJeoJJoJa,rrlour-uolordeql lerJpuoqf,olnu setedtssrp ro (urue8 teqt (louaqdo:lturp-l'Z''3'a) lua8elef,rruerlf, -oruraql uralord eqr ''3'o) ef,uelsqns lernleu ,{uy raldnooun (67-9arnSrg)'uretordre8reregrJo uorlrun, eql ur uorteJatlero 'auososdlaql ol Suruos'eruoseolord eqr dq uorr -eper8ap;ot suralordasaqr3ur33erdqareqt'surelordrelnllef,erlur rar{loot pa1ur1dpualelof,aq uer leql uralordlleursV ulrmbrqn

'raruef, sadft reqlo pue fit-SZ pue 5-97 sarn8rg) ;o letrero -lor Surdolo^opro; lsrr eqt sesearf,ur .{yrear8(f ylUS pue 'JdV 'gU ''3'e) seue8;ossa.rddns-rorunt dueu yo alallptuelnru a13urs e 'rruaSocuosr uonelnruuortf,un;-Jo-ssol e qf,rr{.la ur to aJu€trrer{u1

-uor rraql espelerpu€ elleue8ro rouoP eql ruo{ Jro Sutppng .{q urrot salf,rse1'de,r,lqreddrolenas aql uI uoItJeJIP esJeAeJJo PJEru\ -Jo, aqr ur suralord ,,o8re),, aueJgluotu Pue elqnlos serJreJleql tueruuedruoJ pepunoq-auerqureu IIEurs V elJlse^ lJodsu€Jl 'urelord uodsu€Jl eueJqruou aes uralord ilodsuerl 'urolord eueJqlueru pJtelur oes

uralord eueJqlueusueJl

(4-91 ern8rg) 'pazrsaqtudsSuraq st lI sP uotunl Ug eql sroluo urato.rd drotaroas tueJs?u e gJltl.^aq8norqr runlnJllar crtuseldopue q8no.raqr;o euerqrueu eqt ur xelduor uIOlordIllnIAJ uorolsu?rl (41-y ern8rg) 'VNdur ue uI ef,uonb -as eprtoalf,nu eql trq Peltllads st eruenbes PIre oullue esoq.t\ dlqruassepetelPeru-euosoqrr aqJ uollelsuerl aprrdaddlod e ;o 'alotpaunlut alo$ uorl.ts -upu eql po]ler osle !1aire1d8reua rsaq8q stl le sI rualsds eql ueq^4' uorlJeoJ IeJrrueqJ e Surrnp sluelf,?eJ oql Jo erBls alPls uolllsueJl (tt-W pue 1-y1 sarnttg) 'soruososdlol Jo eJetrns IIaJ eql ol sulaloJd elqnlos Pu€ ousrqlualu drrel luatu -lredruoc r31o3 prsrp-rsou slql uor; Surppnq selrlsa1 'z(er'rqred drolarces aql ur lurod rllueJq roleru e se selJes leql selJlse^pue (NOf) {ro^ lau tE1o5-suo'4 seuerqlueu to lro^.\lou xalduo3 'ouatsuerl e Sutdrrer lerulue Jo lueld due ol SuuJOloU f,ruotsueJl 'suonBreuaB

elrssaf,f,nsot uo pessed sI pu€ IeuIuE ro lueld e olur pelerod slrqrqurtrltcarrpur ro dlloar pue e1c.(c IIar eqt q8norqruorsserSord -rp urelord papoJuaasoq.la, aua8duy aua6 JosseJddns-Jorunl -rocur dlqers pup paJnporrut sr legt euo8 Peuolr V ouatsuerl 'ref,u€Jlseerq SulPnlrut 'taruef, ueur 'lueu8t1eturo uEruoq b-9I pva g-91 sern8rg) aq derulqluor8 ]leJJo sror?ln8arleruroueqrto ssolot enp sesrre -nq ur peteJrldtur ere slueuoduof, uorlJnPsuerl-1eu8ts Sggl ur suortelntrAJ'tI elelnurlls ueql l{r^{or8 llglqul uauo arolu e luorJpa^rrepdllereua8's11ec sseruV rorunl teqr 'llef,e18urs 1o dlue1 Sggl el{r Jo sraquatr J 'sletulue I]e ro lsou uI senssll (g-91arn8rg)'selnqnlorf,rru eqt ruroJot azr Jo IIe^\ lecr.rpurldr lsoru Jo ruerudole,rap eql uI Pesn er€ leql sutalord 3ur1eu3rs -raur.{1od relnqop;o dpue; y uqnqnl tegl surerord1era1a>lsordc ,tyue1 y (dEcr) eleg rolrEJ qrmor8 Eunuro;suerl petarf,es ;o 'luaBP SuIsnef,-Jef,uEJ

'srsoldodBdg o8rapunueqo sl]of,'sleu8rs gcns ..aprcrns,, egt ur fsursrue8ro .re1n11acrr1nru ur sller Jo le^r^rnseql Jo of,uesge ro; parrnbersurato.rd Suqeu8rs snorerunu;o.,{uy rolcel rrqdorl

'VNUrJIreourruE ue Suru.roy 6Z-V pue 61-y sa;n8rg) 'prle ounue relncrtred e ot palurl dlluale,rocseruoJeq VNUr gf,EE'srsaqtudsurelord Surrnp srouop prf,eourue se uorlrunJ lpgl selnf,elou VNU lletus ;o dnor8 y (y5111ro;suerl) VNUI

Jer{lo Jo snJIA ? tllrlr\ luaruleorf dq pocnpur dllensn sellJodord elrl-ref,uEf,r{1r^\IIal e olul I]eJ uellPlurueru .(lPlurou,, e to uols -reluo3 (7) 'uoucaJsuoQalqp$ Paller osle lauoua8 Ilrr-lsoq eq] otul VNC u8rero; e;o uotte:od;orul PuP a{Pldn aql ruort Surrlns -eJ IIef, E ur uollEJelle elqerlJal{'lueu?uIJad (I) uollEruJo}sueJl

'vNur aas vNu reJsuErl

P ot perJ 'VNC fieV'd)'elnrelotu1o;ard13 ern8rg) pornPorl (79-g -rJelsesurpqJ11oe1o]r-1 earqtJo stsrsuoJlsleurue ur palrodsuerl -ur 'arnllnc uI sllar ,{11ensn eql ur seuaS;o uotsserdxedq pe.vro11ol pue perots ere sprf,e.{ue1qrrq.r,lur ruroJ ro[eIAJ eprror,(18rrt otur VN( uglarot to uoIlJnPoJluI JeluJlulredxE uorlteJsueJl 'eprrao,(1trrlaa5 yorer,(ltldrerrr (0I-9 pue 5-9 sarn8rg)'uosodsuerl -orlor osleoo5'uorlrsodsuerl ursrue Pupsrsoqtulsylqq 8ur,tlo,rur

'srsodcoxa pue srsolzb 6t-VZpue g7-g1 sarn8rg) -opue pelerpou-roldacar seulqruof,reql leeqs IErloqllde ue ssorJe se)uelsqns uleuoJ Sutlrodsuert Jo' IuslueqJery stsolfcsuBrl

'JIEIUPE AIEUTO' PUE EIBUI

e uorsnJwo{ Surtlnser proldrp t33apezrlrra; y etot(z to ilel 'srotrq uoudrrcsuer Jnodre1na snorerunu GT,-Lpueq6-Esarn8rg) ur luoserdluor Jurze punorepeplo,sernrJnrts drppuores;opesod -tuor slrlotu PrnDnrls Surpurq-yNq petslar lEre^e5 raturl curz (7y-E an?r.g) 'sllnsarteql slodsolarrsrp Jo ure$ed uorrrp{Jrp aqt Surzdleuepue salnralou par;rrndaqr Jo lelsdrc e q8norqr sder-xSursseddq (sprcef,ralrnupue surelord ,(lrelnrrued)solnf,elouroJf,eru'o eJnpnrtsleuorsueurp-eorqt aql Bur -unuratapro; anbruqcalpasndluouuro3 r(qdertogelsi(rJdur-x (79-91 arnSrg)'stuaru8as eu?rqruarusuert ueAOs qtrznsurolordsselJ-palzzrJ{ eresrotderagraf,ueouo1oc,{llercadsa 'Jef,uef,uerunq ur pelecrldrurare stuauoduroJuorlf,npsuerl-leu8rs lulN ur suortelnry 'slerurueIIe ro lsou ur senssrllsour to luarudo -la^epaqr ur pesn surelord Suqeu8rs palarf,as;o dpure; y rul6, 'tusrueBro ro '1101'upto.td 'auo8e Jo urot luetnuuou ,1euro1q edft ppq (gg-gern8rg)'8ur.1go1qounuut palIl.coslelserpoqrlue peleqel;o asndq petrerepar" ueqt suratordcr;nedspue .euerq -rueruraqto Jo asolnllaf,oJlru e ol peqtepe ere srseroqdorDele .,(qpeteredassuralord qrrq.{t ur anbluqcel turpolq urelsa I

AUVS50'19

vz-9

W-u Puezz,-t.

sarn8rg) 'ouerqruau p ssorf,e selnJolou Jo uodsuert polerpeur -uratord se qcns ssacord Joqlo Jo uorlf,poJ paztrlele>austlzua ue ;o dtrcolal lerurxeru eql seqrrJsep leql JatarueJed xeu1

@y-yentugl 'qrrpeser dSolorqIIer ur pesnl.lepmr:llel tsoq olqrldacsns e ur

dluo atecrldorUEJterll'teoc uralorde ur pasolf,ue (VNC ro VNU) prJe rrelf,nu to SurlsrsuoJ'otrseredrelnlloJpJlurilptus V sruIa .OICTITCd I?JI^ IBNPI^IPUI UV

UOIJIA

Qy-y en?r.g)'suralordocdlS paporuo-sur^suretuof,pue euerqrueul]el-tsoq e uor; Surppng/q po^rropsr !(sasnrr,r serqerpue ezuanl;ur''t'a) sosnrrlauos;o 3ur -rolof, retno aqt Sururroyrodelrqprdlogdsoqd y adole,ruepru (91-gern8rg)'rolro^ elDnqspup rope^ uorssardxa osle ae5'saruoue8 a8eqdorrolreq per;rpourpue sprwseldlprratJeqare srotrol pasn.{luoururoJ '8uruolf,euaS;o asodrnder{rroJ IIOJrsoq E otur VNC crurouo8;o tuau8er; ro VNCI e trttet ol pesnluaur -e1ecrtaua8Surleoqdar dlsnourouolneue {3o1org1ec u1 ropel (61-7 orn8rg)'(salodrp)surore punoJp suorlngrJlsrpuoJlf,elecrrteurru,{se ol tuersu€Jl.11eurs enPuollf,EJelu lueleaotuou uoIlJPJoluIslEe7ltJePueA ]eal( V 'uaSogred eureseql to turoJ luelnrrl e dq a8uelpqr arntn, e lsurete dlru

t-l 'lo 8ur:a88rrl S00I Surp.roca.r JV00I'lo 'dIV lnoqll,!\ 500I g00I 'Jt00I 'ur sasduuds ,;o poads 9191 'tl00r J800r 'L00I-900I'ur slauueqruor unrpos '900I-Sgg1 'ur Suqeu8rs lS00I VI1I'ut uouJnpuof,dro1e1les '600I 'LO1I'ul l.rolre.r;a.r porrad €I0I ]VIOI '7IOI-€rcI'ur SurdurnIepou-ol-apou SIOI 'IVWI'VI1l't 'ur rer,rueg apou 161 to qreeqsurTedwosp aas'ISI0I 'ltlol'gror-tIoI'Pue uotleuqelur

lv70r'szjv€zjr

(uorlre-rtuotalJsnruur ' OVL'Szjl-t Zg1 ''o druanbarl 600I '600I 'to l0I0I 's001-€961dllleuolrrorrp ';o uorle8edord tt00I 'Pue t00I Ierluarodauprqluou 9ZOI'900I'to opnru8eru ploqsarql J7ZOI'SZOI'pueyerluatod ']o etrs s:zoI d.ressarau SZ0I'rol suorlrpuotr 19701'SZOr'800I-1,00t 'ur uouezr:elod.rod,{q oue:qu:u

r ' 1 8'ul 00i t 9 7 0 r ' s z u ' €'JtooI r 0 rr-0r''ool 'lLoor'6oor-Lool uort ezueloda:Tuortezr.relodap auErgruerx

lvzu' nzor'J800r'600I-1,00I

'ur Sursoppuluado lauueqruor g7g1 'uouaurouaqd auou-ro11e se ;o uorle:aua8 SZ0I'lo druanbar; t00I lo uortrurJap uortcy lertualod '8ZL 'uIuIlrV-l0 lSzL Surleapnuurlry 9T,L-€ZL'sureto.rd g51' twotnl aSe,reap ursodur-urlry ';o saddt lglL'9IL I€ L-gZL';o uorlezrueS.ro ut I LVL'9b L-Sl L'uotle:8tru11ar saJnlf,nJlspas€q-tueuJelrJ urrlv h1L' nzL-€zL'paqrue;qun '16€L'0vL-6€L 'utqt rvL rJosrrunqns 8ll lSrL'8rL'Jo arnlrnrls (utrlrads '8zL ul l67L 'to LZL-971 uortrqrqurpaf,npur-urxol'Jo uortezlraurdlod 1 6 IL ' I 8 I L ' \ I L ' ! o d l r r e l o d 1 6 IL ' 8 I L ' J o u o r l e l u e u o ur L'sllaf,elrsnruuou ITnL'7r L-W , .ur ozrsdals JL€L LtL-StL lLtL's€L.Io r&r ytL'Le L'a,rrsselord 'Jo srlPnlsderr lerudo JLtL'LtL-S;L 'ur Surpurqursodur l9€L'St.L M-B€L'uonre:luol alJsnruur J8TL,\€L-LT.L 'Jo o-puPq IaporuruJo^\gJurs^ puEr.l-Je 'ur Lt,L'S€L orte:dlnP 'ur srsdlorpdq 41y l9€L'StL ursody,qosp aas petelporu-ulsodru SbL+e L';o luarua.toru '

'Ie 'to sluauqleDPauerqlualu L-8ZL nt L 9Ig'Pue sulrSalut '8ZL'utrqurg ut l6ZL l57L'87L'utruelryut aliro:ql.{ro ut L' OtL-6ZL'aue.rqruaru JOE 'urYdotts,lPur 1571 LZL-97L';o satpnlsutxol I L Z L ' 9 7 L ' e'1o t ; l l r e qu t srtureudP €ZL-8yL 8ZL'lo 8ur1uq-sso.rr lO€L'l6ZL' (salpunq uI olllf,erluol ZVL-WL 'uorlBr8tur ut 8VL-SrL IIar o8rel ut Itn L' rVL-€.VL'l.rodsue:l I SZL'SZL-VZL'Paqruerq 'ur qlr,ror8pua lenbeun

IOZL'IZL-OZL

lTzL'lrzL

ase41y ISIL'8IL'ur p1o; 'Jo uilo] n LIL '!o $uePunqe LIL ItL_gIL.uttrY LS6' 9S6'uoltrearIeurosorlv J S S 6 ' g S 5 ' g g 6 ' d'dle8aurony elte u o s o r f , V 6911 snrr,r,{ruarcgepounlululu€IxnH osP aas '6g1'auro.rpulslruatrr;opounrurut perlnbry (ur sesEdIV tsv-€.sv +H ;o n Ln' OLV-69V'se11aue3.ro rlnse81o JZ.L7'ZLV'uaurnl UOII?]IJIPIJV 'sulr?rel IIPITV €6l €dZ'sureuop uoIleAIlJE f,IPIJV t/t'sssPlorP^rl PIrv

'tS-€S 'ro] se^rnl uollErul '7ZL-IZL'ur SuqltuPeerl l€S 'ZS-LS 'lo ';o aseqda1e1s-lPeals Hd !75 OZL'!6IL 'ng-Zg (uI suorlf,ee: uoIlEIlossIP IES lI9 L-l8VL' 8bL-9VL'ut SuqeuSts g 11';o,{rqtqts.ra,rar J€S'VS-ZS'sro;;nq u1 zs'(s)Plrv 971'ur sdorsSuttturrT-etu: ';o aseqduolleopnu ut'xaldtuol atnJs-alaeqf,V 575'srseuaSornau l6IL'6IL yZL-7ZL'1o srustueqreru tZ0 I'esErelseuqoqcl,{rary JIvg'Iv9'eTf,snul rEIPref,uI JVZL'VZL-7ZL'uIsuluuo; auqoqcldrary VZ1l' 610I';oldarar 1ZL-6I L'yo sorpnlsleluaurt.radxa 'lo ut OZOI srsaqruds ILZL'9ZL'srsoldroPua ';o oseqduorle8uola J\ZU'OZOI'to arnllnrls 16ll'OZL-6IL elf,snluuI It.z\I'v70l-tz0l'uollf,"rluof, TOZL 'IZL-1ZL'ur uorte;tuJJuolultJp h70r'szjr€zjr IEf,IIIJtr 'uorle8edord uorlle ul yerlualod j Z ZL ' Z ZL - I ZL ' u t u q l g o r 'surToqrl^rarv 6I0I r77/ '97/-77/ 'ut sutalo:dSurdder l I S Z , I g Z , J O S Z , 0 S Z . s e u o lJsor q ILZL'9ZL'luetuatourIelralrequI vy-€v'sqne ourru?Jo 9ZL-eZL'ur sureto;dSutleapnuutlre uoqeldlary WL-OVL,JTZL ' gEy 'eseldxoqreryo3 ldrary ZZL-IZ L'ur sutatoldSutputq-uure 'Jo Jrnlf,nrls l88t 16L I ' l Z L - 6 1L ' 8 I L ' ! o d l g r u a s s e 68V-LBV'tOJ or uoll€Ptxo l 6 7 L ' 8 Z L ' u r u l l l ? - nu I ut ullrv 68n-L8V'srs,{1ord13 It L-9I L'(s)ruaurEIIt '971 'sutalord8ut1uq-sso'rr utlry L8V'0€V'Josuollf,unJ l\€L'l6ZL ggy'stsaqrudsPtre duet ut W L-1V L'uolloeJluof alf,snuuI lSgV'6gV-L8b'apdrptre f,rrllruI IZZL' ZZL-V L'sutolordSurputq-utlry yo3 l,tracy €€l'esPdIV Pale^llrs-urlrv ';o ry srsaqruds 6ZZ-8ZZ'sluaruala 911 'gIL 'ILIL 'LIL ';o a.rnlcnrts tgzb '9ZV'JSZ?'sua8rtuednor8 poolq 6gy lgIL ' 'etllroa.tals ut J1tfi 01II-6711'eua8ocuo 1qo 7gg1 ul'tgCflV ILSV'95 V'uodsuer palerparu-aseddtlJ LZL-92L'Jo uoIrIqlqulPe)nPuI-ulxol ut 9gt-S St'saseesrprtlaua8ur ILZL' 9ZL'stsotlloPua 'uone:8rur saseddrll I1SV'9Sn'pue 11erut JLVL'SnL 'ILVV 'srauodsue:l LSSV'9|V-VSV '8VV uI 3gy Tellalf,eq |LTL'9ZL'luerua^olu '(s)ruauelryurlrY oslo g3g1 'de.ra.rped JHW I sselr uI ;o .{lqurosse s(Iulnd oslp aas a a s ' ! 6 L1 ' Z Z L - 6 I L ' 8 l L ' P u o t t e z r : a r u d l o d ' 's11or 3gy alcsnuuouut 9;V-VSV'BVV\LW'sdurnd,(1rure;radns IZVL'7VL-I7L 'uolwzlTerol tgZV'SZV'adlrPoolqgY vNulu uI 8S€ g9g 'Surssaro:d VNUr ul I L I L ' 8 I L -L I L ' c r : e t u o u o r u 'srs.41o:Pdq ut 4aY 999 \IL-LIL'Jo suuotosl .dlturelasedfv VVV 999 117'1o unol L 'Jgil JLtl'Ltl ILIL'8LL-LIL !o ruro;9 '9€l'Stl'JV€I'€tl'aruosoglr Jo'ells V I LVL'S b L'slselqorql;uI 'SII 'VNCI V 'SII I L I L ' 8 I L - L I L ' ; o t u . t o4; 'LIL '1o uorlnlo,ro JLII,,LIT,.xogY JLIL 'IZZG';Z6-ZZ6 'ur uorldt;rsuerl 'Jo uoIrIuIJaP JtZ6 9Il IVZ6' V76-tZ6'pue sauouo.reqd /I/'Jo uollE^Jesuof, I€26'IZZ6 YIL' LI L';o slunouere1n11er ' 'uotlerStru Ez6-726'Jo uorlerutcads'proldrpTproldeq 11arut 8VL-SVL ITe6 111'1o uttol fl '!I€6'IE6-oe6'uotsr'rtp IIar JuloultudseuI '69I 'saddl oP 8uneur7s11ar

6tor-8v0r'lw\r

'J1nffi'ZVOVO761'auor qrruro;8uoxe ut

IOLI

'salqetuerrPurr ,tq pa^\ollot asoqt lsarn8r;alef,IPul; dq parrrollo;sraglunua8z4 :a1og

X]CINI

XlcNl

.

Z-l

't ,snoSoloruoq 'se sur.r8elur urqo13-o lEL L-T.L IIZZ' IZZ-0ZZ,saue8 919 (seuolsrq 'eurur€l ur 6rZ'057,-lo uorle^JesuoJ t t 7 8 ' I Z Z \ ' Z Z 8 - I Z \ ' u a B e 1 1 o c ' s u ro eqr 028 lEseqJo 'Jo '19 'urore uollEulrureleP m €0I uoqrEf, 39 O Z 8 ' J L O \ ' 1 , 0 8 ' € 0 8 ' s . r o t du eo l er rs a q p v sJrrrrou0g utalo;dur {ralso11y suerar.lpv 1I I8'J0I8' 608'IZ0B'suortrunf JO6'158'95-53'uortelnSe: (rlaqsuereqPY 'uorsnpxa oslp aas 'LbZ-tnZ (;o srslleue a,rrle.reduroc JrlaTIV 9461 7VL-W L' l9I L' 9I L uoItEIsue{ :uorldulsue{ os/Z pelull-X de.nqred4141yqroldara: l00Z' 002'a,\rsserer 'eddrp1r.m a a s ' ! Z I ' 1 1 ' ; o S u r s s e r o r dp u e l l q r u o s s e paldnoc-uratord-gosloaas' ZS9-9t9 991 '0V9'6t9'SuqeuBrs saluanbas prf,e oururv ur'aselcdrldlluepy 169I' 691-L9I'Jo uorle8er8as 'suralo.rdur Jaqrunu (eseurlarelduapy 99 l Lgl' L9I-99I'elrssoler J0l 1g ' s a n p r s a l p r f , po u r u l V suonElnltraas.luelnu (areqdsoqdr.rr 'sr. 'eteqdsoqduleursouepv sad{l eursouape) I7l'p ILI'99I'srusrueS:o proydrp proldequr dIV aas (;o 1 : o d s u e : 1l e q a q l r d a s u e : r IILV'ILV (areqdsoqdouour eursouape) 1 6 9 I '6 9 1 - L 9 l ' ; o u o B e 8 a : 8 a s .67 .yo a:n1rnrls llV'ZV-|, aursouepy l LgI' L9I-99I'lueutruoP dI{V aas'ateqdsoqdouour (areqdsoqdrp ls9 J00Z'002'e,\rssolar I€ruosolne ' 'areqdsoqdrp 5 9 ' J t V ' t V ' J Z V ' € V - Z V ' I V ' Jsou r e qer p r s eursouape) eursouepv J00Z' 007-66I'lueutruop I€urosotnP dCv aas a^rtpler (s)auagosp aag'991'sa1a11y €t'Jo eluEpunqB gIII 'llol srsod,{yod snol€ruouepv 'yo 'ZS-IS'pue sarlrado:d 7y 11dftruqe>11y lZS lW,W .Jo arntrnl]s 'SII-'.II '.xrTeq 29'17y lepd elqnoPur VVL'ur Swtneerlsrrurseldotft JSII gg,';o uolreldroqdsoqd salo,fue>lo:d os10aa|'ae31y ( s ) a s ?ogs / ?a a s ' t I I ' 1 S t ' V V ' e u r u a p y (s)pneourtuy osp aas'rcV'ZV'auruely dlrunururl lasuodsereunrutul ' I S 9 ' 5 9 ' l I V ' 0 V ' l 8 t , , L € . y o s p u o ga9p9n d a d o s l pa a s 't 0 l I - L 6 0 I ' € . 9 0 t - 2 9 0 I J eseur1uralordaas'urerordl>1y 'JI90l ,tgggI ,gg0I i{rlunruur oiutdepy se 107.01'020I-610I'sraurusuprlornau t50I'auIV '99 'snuru:ar-51 SnJIA g5g 'saxayduoc(4y) urarordratdppv ;99 se druarrr;opounurur ueunH osp aas'5SI ,SCIV ilV'0n(s:eurouour J009-r86S'009-855'r/8S '1o tt18rclrl. '58S-9gg'saprsa,r relnralou 99 paleor-ura1o.rd rotdepv 649'ur srueldrruoSsuerl ,99 ,;o a8elurl ;99 179(ur saprtrqreq 1889'889-189 '1989'SB9'saseurleursordt w'lvt'Jo srarxosl arnllncr:3y rotdef,errot 's?ntp 69'lB9'99'!ZV'ZV'rrqoqdo:pft1 lsruo8y ILIS'9I8'sutJ8elut ro; 579 '189 '99 'lZV 'f.n-ZV 'rrlrqdo:plq 69 lwz , t I 8 ' t I 8 ' l Z I 8 ' Z I g ' s u r . r e q p e er o 1 'slrer 'lvT'Pue auotsrq ur z9z-ISZ'tosz suonElnur yNQ IErrpuor{lolr(u (s)urarordrard€py 'SIII 'pue eluapnut .raruel 99 !o uorrellsordlS JSIII 90L \ SOL'S 0 L'1L99'sasealord WV(IV 0I'lelruassa 795'8ur8y 1799,lexped asera:ras-3761 'uera:38y .1,11yqy It'Jo uorlrurJep l0€8'0t8 J\CV 5(V 'suralo:d 16zl 'J6Zl '187.I'6ZI-L7I 'ro; suopoc u r , u r x o r E I J V auPJqrrreu JI7II'I9II':aluel ;o'uorleldry IZV'ZV';o uorrergrssep g; 'dno-r8 ldry €/0I'uorteJnleru,{trur;;y 'uorterryr.rnd '8ur1aqe1 bt-t t' lo uoIlerIJIPotuI€rIIuaql :oldarar ur lrrul;;y 199 9191 trlledornaullod 'sarnrynrylar:o; 'uorlerrJl.rndroldara.r ut 959 Surleuqadruap droleuuregur elnry I€9 '99 'snuru:ar-3 'ur sotpoqnue Ipuoltououl 199 16y sg6-tg6'ur^Erv ')tseg 'ur. uortezrTefol JTl'71 eprsa,r rrldeuls JL6' L6-9 6'177';1,rqdefioteuro-rgrdlurgy IZOI uorlplsue{ osp aas.lZI,Il (;o llqruasse 'eue:quau_etuseld 'auoza,rrtcy Ob-5t'Prre dlt:etuauralduroJ JElnJalour 5191 ;o g;oruole uoqrel lc 'I8-08 'rol (-)) 1g tuelsuof, srTeEqrrry uoosuErl auPjquretr^{ JI8 .ureqr ' S u r p u r q aprldad ol uorrlppe lgEI'LtVStl osp aas 'nVV'1VV'uodsue:l e,rrrrv Z 6 ' I 8 O g ' 9 1 .1ouorlelllrr 'gg 'rqeurlzuo 'sa1rs 1E1 drtut;yy arrrpy t8-I8 l0S 1rng St II'IZ€II,191 1 w'ltt,'s1-t,€'uog.rer .slaPou {rrts-Pue-ll?qur spuog

'lzzrr'Borr'Lorr'ur'3lj*;1iJi",",

(')""r :,:!,' i:ilr,e .;"t

,uolteln8e:ulalo:d ur 96 7SV-ISV'JouorlrurJap ZSn-ISn't:odsue;1 +zeCul urTnPotuTeJ .sn11e3 .ppatnpl779 stsda14p1 605 l1sn'rcV-6VV'uonexelarepsnu ur (;o strunqnsrr/1u1er ilS V'ISb-05 V zsv-I sv'Pue ullnpourlel sdurnduor runnle) .uorsr^ur Zt9 II0I-0I0I'Jo a'rnlrn:ls pa1e3-aBe11oa ,S59'S59'Pale'rado-a:ols .uorlferluor ellsnurur lv70l'v7.0I iS9'Pale3-e4t sl0uueqf,uor IunrfTEJ

t?or',#?lffi#"t

'rcv'zur,errr,s ;;r"r'#ir"!,"n*-'

'0€.s-67s

lSzs 'uorlexg uoqrEr ur .s11arqreaqs alpung

J€t

L1

.

XscNl

l L S 6 ' L S 6 ' u r o r o r6dq 3 (ru3udola^aP t60I IIal-I uI rrxoroldr '(s)llar 'pue s11ar 1 rtxotoldr Losp aas'080I J€8OI 't80I-2801 ':o; de.,nqled JH1^{ I ssell 8Cf, (ruaudole^oP '+8CI)yCC t60I IIer-I ul 'ruorudola^ep ;561 llel-I uI :ad1eq '(s)1er osp aas'080I 'pue s11ar radlaq a 1 'JS80l 1980r 'ro; de.vrqted OHW I ssell '/80I-t80t

tc)

g961 'xalduocgq3

f3,"'.f X;g""lftg3 ne'n-et' *.o.",,lf ggg 'lertualod Joldef,al tualsuEJl €t8'e^uJalasuou e,{lllsuesoueqJelrr ItVg'trg'uorsr,r ul'pore8-4yrJgr

Iezu'nz0r-t70r

'uorlle:luol elf,snru uI'pale8-autloqrldrore slouuBI{Juoll?J g9y ta1-todrlueuolle3 916-VI6'uot1et1u3:a;11pIlaf,-ulelsur ';aruer uoloJ uI 9711 'uollEulurrelep al€J-llet uI t96 lL99'1999';o uotle,rtlre uIUalEJ-g 'seulueioqJelE3 5:29 'J08 '63 'sotts rlr,ileleJ t8-28 ,6II ,y51a cu,{1ere3 E9e

to8

'08 'Pue*'*A

'I8 'Jo srtla8raua JZ8 (s)audzuE osp aas'llg' lg-6L'v9'

6v'oI'srsdlete3

g19,asery*a TZLT

'

il;'11-1ryI's11acSurlera;rlordut ]OVII 'lVlVG€fi 's;acu€f,cr;nadsol 0III-601I';o uotsserSo:d Pa1ut1 '8uqre-loe;tPut s11al.rolruaSold SIII'IIII'ut 5g11 '69Z 'uI rr.uosotuol{oerqdlapeltq4 '8urne-rro:tP Otll 5g11 'lef,Ituaqf, 'tllp17fi IILg'lL9'ut sutalo;docuo ilVlI \1Vfi TZTII l v l l - 6 t I I ' j 8 0 I I ' 8 0 1 I - L g 11 ' s u a S o u t c : e 3 'I€I ((CIl) uIPuroP1eu[u:ar1.{xoq;e3 I-0€II'ut s:olre;uotldlrlsue:l 9T.t'087 sluslueqf,au lt;lseBut lzll-6lIl'Jo JZLV'7LV'uotleltltptle Jluoqr€C 9IIVCTII IZII-6III,IIIII, 397'ase:Pdque 'tl 'LZS-SZS'ut orstqnr rtuo8oruo II-€l II'ur uolteturo;suert ILZ;-ISZS 'JLZS'5ZS-LZS'urde.,tqledt3 lruoSocuo gZlI- LT,II'pue s:otdara-r JSZS (s)auaSorug JL79_TSZS 'LZS-gZg'ur e3 A A S ' O S P .{e.uqled ur,r1e3) I Z I I 6 III'VIIT (ap.b I T , f i _ L L I I , '€Wl'8OII-LOII '8SI 'ut souaSosuo 6zs-v7.s'l€rs lSzslszs' 'eurotselqornau yy1 1 6tll \eseu 'euq-uua3'sAf,Ileuos 9611 'lt|lr 'vzlv€zll 'gE stII 'lrln'rvllr-€lrl'sot JSlfi 'rulod 0ZI I gllr'€sd 'Ug '5g'stsaqludsoloqd ut 'uotlext; uoqJ€C S 8 t - 0 8 t ' s r s d 1 o r d 1 8u t uI l68V'68V-L8V'e\:dr Pne f,Irllr ;o uortcnpo:d'oPtxotP uogreJ rl€flql €t sPuoq ltt'S€-t€';o lV€'J€t.'gg'ltlleruurdse 89'W'n suole uoqJEc stsaqludsoloq4 osp aas'lIS 'slueld ur ';o stsagluds 9y'a?etots l SV ' 9 V - l V ' s a l e r P d q o q : e 3 SZL , N 7L , J Z ZL , € , Z L - 2L7, Z d E J 'sPrsde3 'SgI-tSI '1e:r,T JgSl sasedse3

lzzL't7L-ZZL rlquresseruaru'IrJurlJeur,rHlo:r1"t:i:ff3 l\vl'\iL'z de) de3 ll6-t16's11ac dryVr-dvf, IZLZ'ZLZ'1 77'xaldtstot I67II 6 Z I I - 8 Z . I I ' l z z l l ' 7 Z y V I Z I I ' u Is a s n r l ^

'ZLZ'ILZ '(4y3) urero:drote^Itre atlToqeleJ . ( l t s o 3 . , { . z o r a lJeoqs s o l $ U l '7ZII'ut pgy 'srsl1or.{18 ut I,7II '8'l'ul uollf,unJ-Jo-ssol suollelntu 55:'ursrToqereJ 'S50I 'y6991'uotte,rtrre ITZII IIor-I uI tSd0l ' WI I-1ZII'uI suollelntuPelllarlul VV6-tn6 \OV6' Il6-8E6'srsoldodeut 'ur '0€8-528 ueuornledq TZZII' LOll'ut sauo8.rossa:ddns-rournl J0€8 Jorunl IIII'ul lualuuo;IlueoJf,Iru L78-928'ur ua8ellor 'ul '0€8 'ut uera:33e erxoddq.rorunt tilVUW'50I1 t0€8 'ul qIuIl€uI aBeltue3 0€II lsea:g:o1 srauodsue:1aag'utelo:d €ilVZeU teruer 'ut saleloltlue 'rrloua8 799 IOOZ'007 'ul srollqlqulslsauaSot8ue SJeIJJEC €IlI 'Jo '€86 's1ad:e3 rueul€erl 026 lV86 JVTII 7ZS-y7S'1osrragea,rtlralord 'ttll 'ul :orel ql.,no:8Suturo;sue.rl 'il5 'sPIoueloreC fl JIIS srolrejuotldt:rsuerl €5S rcell'If.II-}tII'uI ' u t '58S-88S'085''uoclsuerlrelnrlsrl uI s 1 1 aEr1 g JtWI'V11t-t111 'ZLS 'Jo uotlelolsue:l dccr zL9-rL9'ul JZLS 'l9Z'uI asereruolet 'WM.nfi rcVfi hLs'lzLs 'V sller ruals IIII'076'uI LS-I LS'uodsuerl reallnu uI yg11 'ur speut5 sulato.rd o3re3 'l,0It 'raruer ul eru:apo.rax u T 17g11'urnsoruaru8td 2.VI1-ZV11'80II 'uPIs 'saua? ral€lalPJ 6'I-8tI t7711 .O€II-5ZII,J97II,SZIT_VTII s6L le]:'lP J O TII '60II 'OIL '289-0g9 'ZL9-IL9 'ut Suqeu8rs 971 1(qredodtuotP:e3 'utdqotprz3 6€Il'lerons 669 'snoleulof,J€s 501I J896'l Lg6' 695- L9 6'srseua8orP:e3 g9y'ut :ouodrtue *zeC PeluIT-urnlPos 5SI-8Sl'Pue sesnrl^orlar J€ZII' VZW-t7 11'eruolselqouilar lrv9 ,1189 SZII-LUI 'Zg9-0g9'ut saseut>1 aulso:dl:oldare: 539'ut suratoldsea I0ZII'6III {q paporuesuralo;d IZII-6III 'ut suorlelnruuolllunl-;o-uleB Ljfi'lo uolllulleP IZII-6111'sauaSoruo ol uolsreluol IZZII'IIII' L}W' 669'ut sauaSoluo-o]o.rd 'W9 'u\ s:oldela: aulToqcldroref,IuI.rEssnlu l Z S g ' Z g 9 ' u r s u t a t o : dp o l e l J o s s e - a s e u l l gg1';o s;ap;ostp uorlf,PJluoS elrsnlN os/? aas 'OlL';o uorlre;luor 'ur sasealord WVCV 991 'al)snlu IEIPJES )JeeH oslo aas OVII-6€II'se uottetpe: 6tII'Jo sre;;a rtua8elnur

strr'9Id

'a1dulnu 9111 Lt,II-ST,II ' tZlt' BVI'uourun;-]o-ssol 'suratordSunouord-qtrtror8ur LZII l z l L - 6 1 [I ' u o t r ] u n t - J o - u r e g 'VZII'9WI 'reluEruolor uI SZII

'lZlII

'VUI'9111

tvrl

'larue: lsearq uI

trlr \|nrr'vur'v)dg lvfir'vrlverrr

'IIII'60II-80y1'ut suoqelnur SIII-IIII';o'ro; laporu lrq-Illnru srollqlqulUOIIU SS€ 'ul ,{dorso.rrlur 0III ,IOIIT JLIII,?IIT ,J60II,0III-60I I'uI slsels?latu I'80I 'euroqdur.{1 5911 'Pue Surlous'3un1 JO7W'17fi Jo ssol ISZII'VZII'ut l1tso3'{zo;alaq 'ul uolse^ulIef,ol 0lII ItII :e^rl EIIueIno'Iaas'erualnel erPodtPe,rut t0IIl'0lII'ut 'Palr;equt VZIYqZW 6ZII 'ut ' uEIunq snrr,reurolltd?d €Z.II-ZZfi .q I-CIH T,III_7III 'ul .{rrlqersurltruoua8 8€II LZII.6III 'S111I'0€.-62'utsuot1e.ralle ctlaua8 ':o1 suollca.rtptlrr"esal ernlnt 1VII-Snfi 9111 Xrzuotrnio,la '€I8-ZI8 'ut ut:aqPer-g J€I8 X,VII , T . V f i - I I I I ' I L C I I ' L E II - 9 EI I '801I 'StI 'ur srrelaPrreda:-Y51q ,8III '6III-9III'ut derreolrlurVNC sts,{1eue y51q ilZn,IZIy_0711,ur uorteuyqdue 'ul uolalalsold) 0III SZII,IZII' 9lll'ul suorlelnlu slselsEls(u ILIII'9IlI'uI 'Parrrsl{ul tzl I iZbII'ZVII'ur slcalaprreder-y51q a,raP JL I I I ' 9 I I I ' 1 o r u e u r d o l'uo1or gg1 OZII'ul suollPf,olsueJlIEIrrosoIuoJI{J 9TII_STII 'ur saxaldruolSuqapoura:-utteruorqr (uI saleloJllue Z0t 026 1oJ.{deraqrouraqr E z l l - z z l l ' 'ur 6s I'l"rl^r0l qirrro;3 11er ilU'0ilI-6011 uoIsI^rP IIal 6IIl'fiII'0III-60II'uI

CD28 in T-cellactivation,1,094-1095 in T-cellkilling, 1095 cD40, 1096, 1.101. cD80,1094,1099 cD86, 1.094,1.099 cD133,1111 ddc mutations,1,70-1,7l,'1.70f, 85 1,-852, 8 52f, 860-862 in cell-cycleregulation,859-853 Cdc2.853t,859-863,86l f Cdc6,879 Cdc138 , 5 3 t ,8 5 1 Cdc1.4,871 Cdcl4 phospharase, 858, 879,886t Cdc20,869-870, 888-889, 889f Cdc25,861-852,8621 Cdc25phosphatase, 861, 862, 883, 886t Cdc25Aphosphatase, 883, 886t Cdc25Cphosphatase, 886t Cdc28,853t,873 Cdc42 in actin filamentassembly, 725,726f in cell migrarion,746-748, 748f-7 5Lf cdhl, 858, 859f, 870, 871,,876 Cdh14phosphatase, 858, 859f CDK-activatingkinase,862, 862.862f,862f CDK inhibitoryproteins,851, 883-884,886t CDK mutant,ATP analog-dependent, 866-867,867f C D K 1 ,8 5 3 t ,8 6 1 ,8 8 1 ,8 8 3 CDK2, 853t, 862-863,863f,881 cDK4, 881, 884, 1134-1135 c D K 5 , 8 8 1 ,8 8 4 ,1 1 3 4 - 1 1 3 5 cDNA (complementary DNA) amplificationof, 189 definitionof, 181 cDNA libraries,1.79-1.82, 1,80f,1,82f screeningof, 1.81.-182, 1,82f CED-3,in apoptosis,939-941,939f CED-4,in apoptosis,939-940,940f CED-9,in apoptosis,939-940,940f Cell(s),1-30 birth of, 906-921..Seealso Cell division blood,2f red.SeeErythrocyte(s) stem,8 synthesisof, cytokinesin, 672-673 white. SeeLeukocyte(s) cancer,1107-1119,1,1,08L \ee also Cancer as chemicalfactories,15-20 cloningof, 9, 9f, 372, 394 cohesionof, 16 competent,951 daughter.Seealso Cell division;Stemcell(s) germ-line,913-91,4 in meiosis,167, 1,68f in mitosis,L67, 1.68f,872, 8721 retroviral,158 from symmetricvs, asymmetriccell division,906-908 deathof, 19-20,20f,88. Seealso Apoptosis development of, 8, 8f diploid1 , .9,1.66,849 diversityoI, l,2f endothelial, in Ieukocyre exrravasation, 837-838 epithelial.SaeEpithelialcell(s) eukaryotic,3, 3f evolutionof, 4, 4f, 6-7,23-25 excitable,1003-1004 muscle,1004-1005 neural,1003-1004 extracellularmatrix of. SeeExtracellular matrlx l-8

.

INDEX

fluorescence-activatedsorting oI, 394-39 5,

395f founder, 908-909 functionsof,15-20 germ,905 divisionof, 1,67,1,681 in oogenesis, 9 53-955, 9 53f primordial,953 germ-line,13-1.4,91.3-91.4, 950 fate of,907-908 segregation of, 953 stem,91-3-9L4 haploid,1.9,1.66,849 horizontal,7027f, 1029, 1030-1031 hybrid,401 immortal,398,398f integrationinto tissue,801-843 lumen of, 410 microclimateoI, L4-15 microscopicappearance of, 2l}Jll moleculesof, 9-1,4 movementof. SeeCell movement/migration necrosisof, 937 nucleusof. SeeNucleus parietal,472,472f pH in, 52 plant,2f elongationof, 378 growth of, 840 properties of, 839-842, 839f plasma,3f in immuneresponse,1,061,1061f, 1075 polarityof, 8, 47L,71,4 cytoskeletonand,7 14-715, 7 l4f postmitotic, 264,849 postsynaptic,1005 precursor,905 presynaptic,1004f, 1005 primary,definitionof, 394 progenitor,905 prokaryotic,structureof,2-3, 3I protein contentof, 11,23 quiescent,781 reproductionof,7-8,7f, 8f, 1.8-1.9,1.81. Seealso Cell cycle;Cell division; Reproduction satellite,cultureof, 396,3971 secretory, in rough endoplasmic reticulum, 376,376f senescent,1115 s h a p eas n d s i z e so f , 7 , 2 f , 1 6 somatic.SeeSomaticcell(s) steady-state reactionsin, 50 stem.SeeStemcell(s) structureof,2-3, 3, 3f,1,6.Seealso Cytoskeleton transformed,397-398, 399f transientamplifying,905 typesof, 2f Cell-adhesion molecules(CAMs), 16, 395, 803-805.Seealso Cell-celladhesion; Cell-matrixadhesion adaptorproteinsfor, 803 in adhesivestructures,833, 834f cadherins,803, 804f. Seealso Cadherins diversityof, 808 domainsof, 803, 804f, 808 evolutionof, 807-808, 807f familiesof, 803, 804f fibronectins,830-833, 831,f,8321 functionsof, 803-804, 804f heterophilicbindingby, 803, 804f homophilicbinding by, 803, 804f Ig superfamily,803,804f, 1.067 immunoglobulinfold in, 1057 immunoglobulin,836-837

integrins,803, 804f,816-81.7,817t. See a/so Integrins intercellular,836-837 isoformsof, 808 laminins,805t,821, 821f,822f in mechanotransduction, 843 neural,836-837 selectins, 803, 804f. Seealso Selectins in signaling,803, 807, 807f, 833-835, 843 in synapticcommunication,1019 synthesisof, 803-804 vascular,835 Cell biology,20-21 Cell-celladhesion,802f, 808-819 adaptorproteinsin, 803 cadherin-mediated, 810-814 calciumionsin, 811, 811f cell-adhesion moleculesin. SeeCelladhesionmolecules(CAMs) cis,803-804,804f disruptionof,806-807 formationof, 803-804, 804f heterotypic,803, 805 homotypic,803 IgCAMs in,835-837 integrinsin,8l6-817 intercellular,804, 804f intracellular,803-804, 804f lateral,803-804,804f in leukocyteextravasation, 837-838, 838f motile, 833 nonmotiie,833 oligosaccharides in, 552 overviewof, 803-808 in plants, 847-842, 842f propertiesof, 804-805 signalingin, 833-835 tightnessof, 804-805 trans,804, 804f Cell colonies,396 Cell cortex,7L6,71.6f Cell cultures,372 adherentcellsin, 396-397 animal-cell,395-396 for artificial tissue,404 moleculesin, 395 cell-adhesion in cell differentiationstudies,396,3971 cell linesfor, 398-400 definitionof, 398 differentiationin, 398-400 immortalized,398, 3981 cell strainsfor,397 clonesin, 372 disadvantages of,400 embryonic stem cell, 91.'1.-91.2, 91.1.f epithelialcellsin, 399400,401f in expressionsystems,1.94-196,195f fibroblastsin, 396, 397f future researchareasfor,404 hybrid, 400-402,402f life spanoI, 396-397, 398 MDCK cellsin, 399400,4011 mediafor, 395-396, 40L, 402f in monoclonalantibodyproduction, 400-402,401.f myoblastsin, 396 nanotechnology for, 404 nonadherentcellsin, 396 primary cellsin, 394,396-397 in protein factories,L94-L96,'1.95f satellite cellsin, 396,397f stagesof, 398f transformedcellsin, 397-398, 399f viral, 155, 156f

6-l

.

XloNl

'l9t tuarudola,raq V8g'1288'€88-I88'leuotldtrsue:l L 9SL-S9L 'gg1'1.rodsue:t alet-lla3 98-6S8 osl' aas' ur L8 paterpau-ursodru v L8-t lrSS-IOSA'f 696-096'uotleutru.ralap ' aqw o sn ['u'tofi qn osozt q) \rr. 'uon:e:tuoc elf,snuur 4 S t16'uI 2Jrolre; quo.r8 Sunu:o;sue:l 9SL-SSL '6L8-7L8'1798 ,JT.LL,bLL-69L rcL8-IeL8 J 0 I 6 ' 0 1 6'luleuruds ' 1 o8 u r u t 1 ]VLL 'ZS8-I 'uodsue:l pesPq-alnqnloJf, La) sail.uoto qttog ut. rru ur 58' aptstna 905 ur srsdlorplq41y l6L8-ltL8 t l 6 - Z I 6 ' 1 9 0 6 ' 1 0 6 - 9 0 G ' s l l eure l s u I '6L8-ZLy'ZS8-IS8'eseqd ut 'ls 'Proqalu€ 5 L-5nL JtI6'nI6-€I6'1606' 606'ut SuqeuSts Jls L '1088'I88-088 'ut slutoduollrlrlsor (s)ruaruelt; utrY t€II t96'806-L06'uo suollf,ulsar gyg'ut saseutlutalord :urlrv osl, aas 'I€.L-glL'uI slualuellJulll? l0I6' lI5-L06'1o uotteln8a.r J8S8'8S8'utuotteurltnbtqndlod I SL-SVL' 91'uonetflwlluoure^orx J89I'L9I'Jo ^\ellro^o IIaC 1988'ul sasereqdsogd srsolrwos// aas'I6L-I8L 3961 luepuodop '1298'€98-198 'JlS8-,SS8 , ! g 9 I , L 9 I , 6 I , J g l . g 1 , s r s o l r uu r -dpoqrlue 1{lrlxotoldr palerperulle) It98 '8S8-SS8'uI rotre, Sullourord-stsolttu 'tt8-€88 'C-€ 116-016'ul VNUIIU Jr€8 (uI '1088'Vg8-6L8'slerurueur ut osp aas'868-768'slsoloruuI srsoretr/{ suelsPuds lZ88 528 'ur 'I88 saue8osuodsor-alel 288 1eg-LZB'ur suerdlSoalo:d I0l6' II6-606'sluBlntu rlruorllJoralequI 'Jo tggg 'ut saseutl ^.\el^ro^o 606-806'ut s1larrePunoJ 808-t08 1988'ul sutalorddrortqtr{ut uorlerlTda.r t7€8'tr€g'€t8-rt8 '7I-ZI'ur 'tLI8' aJnln; suoIpaJIPqJJEesoJ uonerrldo.r 669'JoI VNC VNC osll aas L I 8 - 9 I 8 ' L O 8 ' 9 V L - 9LV' u r s u u 8 a t u t 'e1th 'tt8-0€8 'ur urlrauo:qry 11aaosP Jo salPnls 1 0 9 6 ' 0 9 6 ' u ra 8 e r e a p JZS8-J088 aas' 658-IS8';o sarpnlsleluarut:adxe ut xrneru-Tle] 6III'WII'0III-60W'raruer 6I8-808't208'uorsaqpE 'ur 'apdc 'srsdl saua8esuodsa.r-d1.rea 1gg quror8 J0r6'1606 991 lern ur 11a3 ' 9gg'ur sllaJep uorler8nupuaureloru II 6-906'sun8ala sttp qoLPouaoJur IIeJ aas'uorloruorolIIeJ 1It.6' If.6-0€6'lseadut 6 L 8 - 6 5 8 ' 1 € 9 8 ' t , ' l s e au' {t 186E'86t'pazqel:orurur 658-€S8JZS8 9€6-0€.6';ouorleln8er 00t-85€'ur uorterruaregrp 'snao1 '€S8-5t8 't0S8 'Jo uonlurJeP sn4ouaYur TIf.6 85€ 'aseqd ut ' 5 srrnllnf, oslpaas '86t'sauq t:odsuerl pa.ra.,tod-utsodru 6L8-ZLB'yy1 It6'ul 11a3 il)J sarpnrs €.s8-6V8'Jo.$eIAraAo 796'ut uontqtqulTErelET lg16'LI6-9I6'ur sasn:r,ro:ta;';o 'rtlolttu '0t,6';o sa:ntee; €93-6S8 lerauo8 w6'J016' ll6-L06';o uonelnSar ile 6 898-698'stsolttuut 606-806'ur sllel rePunoJ IV€6-lZ€6 'Jo uoIrIuIJeP 'S uI oyqdosotq ur v88-6L8'slBurur?ur s:05 t,6-l€.6'n\soJouo1atu gtl1-9t6'lacuef, ul 11ar JIt6'O€.6'ut uorleztrelod l0r6 '1606' sexoldruocyq3-uqrd3 806-906'3'lr:latuuldse II6-806'suo8ala sqtpqoq..ouaoJ lo osp aas'JZSS'J0S8'€S8-6t8 osl0aas'LV8 apdr TTOC (s)llel ' 'L9I'87-L7'J8I '6I-8I ';g '3 'uotsr.ttp rrrelsosizaas'J906'906-S06'(s)a8eautl €8 L'ul saxoldruor)C)-uITldJ 11a3 11a3 'ut 'sIg-tIg 'Jo ernlJnrts JL;B'l9sg'8S8-9S8 g uqrdr l € 2 6 ' 1 7 2 6 ' e z 6 - z z 6 ' l s e eul t ttlg sturodlcaqcap,b11o3 906-S06'ut s.rolle;uotldt.rrsue:l 1 9 1 9 ' g 1 gt 1 e a 1 osP aas' 668'1168-1888'158-188 €.96' 80G-L06'uo suotlculsa.t lSIS'SI8'VI8'!o suorlrunJ ' 9 g g' t 8 8 ' I S 8 ' 8 t 8 ' u t s l u t o d l c a q c 626-926'elrsnuruI 10€6-1926' ilLn lLV'leqarPtda 'ut )I]olc olrdrllel 9gg tvz6-1276 J S';793 I 8 ' J t ''rq8rr t8 ' 'grg-vrg'rIIg 'l0Ig '663 gtll-V€II' 6III'668'reruEr uI VZG-IZ6'ut uotlert;tredsaddr-11ar ut 1698 r11g 'srueldur 11ae 1906'906-505'sa8eauq '8S8'ul xaldruorSuttoruord-aseqdeue IIaC 095-6V6'906-S06'13'3'uotletluarejJlP 1gg'ererusapouseld's,r 'IrolJ epdr-11a3 ;o uorlelnSor 9S8 J97OI 'gZOl-SZ0l'sasdeuds lO6L' I6L-06L'slueldut J068'068'r888'888'uotltsod-alpurds lef,rlrele ur '9gg-tS8 '6V8'lg78'gyg '1osaseqd JSgg J688!888'588-888 {lqruasse-alpurds l8r8'6r8-Lr8 'rII8'j0I8'608'!ZO8'J€8€'€8t'91'de8 r888 '888 'aseqd5-u.rlur JSSS'988-t88'tS8-LV8'to .t\allra^o 'a8euteP-YNlq 8t8'Sultra;te suollelnul I58 1888 l0I8'5I8-508'€08 ' srsolrryosi, aas'I6L-I8L 'ut stsoltur 8€ll-9tll' 6III' 668 5e)ueruI I7o8'JrLV' r LV' 0ot-56€.'77E'rytletptda '6t8 'ut aseqdzleur 'etstrr I0S8 668'1068 !98V 't688 '1888'168-888'sturodryaqr oslo aas'868-768 slsolotr/{ alrdr-lla:) 9r8'1rr8'J0I8'0I8-608 , l g 9 l ' 'snao1 '9S8-tS8 'JtS8 ' sndouayut L9I'OgI'!61'61'ut stsotau JSS8 t208' 96L'sP seruosourseprurorl '6V8-LV8'urslua,ta uretu J0S8l8t8 III8.JOI8 l O 6 L ' 0 6 L ' s l u e lu dr srsotrW '0I8-608'1208' 06L-68L'tt 8L' t g L'stsolrtuur 96L'sE saruosousap 'Jo oslo aas'JgS8'6t8 'J8t8 '8t8 ';o aseqd11 uolleulul 1 tI 8 058 'J0Ig '608 'lZOg'sesuorlrunIsuaraqpp J O 6 L ' 0 6 L ' s l u e ludr 143'ur aseqdolar eseqdreluloslv aas'!OS8 I I I 8 't0 I 8 '563 '3ur:oqrue JZS8'ZS8-IS8 'Iz8L 'ul eseqd'ralur 'JBv1'8vg 't '6lg (uI suollelnrua,rtltsuos-a.inleradrual 8L llI8 'J0I8 '608 'J208'suararlPe 'lVS6 'J€96 'ES6'stsauaSolerue8 ur SS6 ZS8-IS8'ctlaua8 €r8'10r8 ' 6 1 8 - 6 0 8 ' € 0 8 ' J 2 0 8 ' 7 1 9 ' s u o u r uyn1[a 3 I 'I88'sleunuetu L L S ' L L 8 ' l s e audt 153'uotsnl11a: 'lErltueqrolq ur JZ88 ts8-zs8 IOZII'lo uorleln8ar (Pue gg11 taouerut'ur sa8ueqr esEul) JOsalPnls tld s69-n69 ' 1 0e s e q dz 9 .UI '0IlI-5011 ':aruecur 6V8'!8V8'r1L'l8r'91 IUVIS ZI,8 IIII 'ul '0S8 'I98 uotleln8a: 6L8-ZL8';o qrr'ror3 11a3 {ls LL8-9L8 !0SS 'lVL8 JL€II J9L8 J S S S ' 8 S S ' s d e sasae: ; 1 1 a 3 'gL8-,Lg 'uI 'L7II-77II 'gg11 taouerut'ur saSueql rotreJBurloruo:daseqd-5 80V-L0n'17'uorteuonrery IIe:) 'setruJos YT,II ur 886 J1LB' LL8-9L1'ur rotrgrqutaseqd-5 ';o lsarre 'Ltll-9€.fi'a8eurep VNq dq ut J SL 8 1096'096'atoB.'(z '8I '1oaseqd19 '6t8 'J8t8 'S'8 ' 19 t8I JgSS BL1-VL8'ut saxaldruotyq3-uqrdr lv L5' It.L6' nL6-€ L6'lBuollElsuerl ut JLLS'LL8'lsead LL8-ZL8'olutd:lua It L6' JZL6' €L6-I L6'leuoDdt:csue:l 'sleuureurut 288-I88 Ygq J9L8' 6L8-gL8'ur uorrerrlda.r ;o uorlelnSar '6t8 'J8t8 'J8I '1oeser;do5 J0S8 L8' 6L8-718'ZS8-rS8'l 6V8 168L}-l€. €96' 806- L06'uI suollf,ulsela,rtssa.rSo;d '1o uotrernp '6V ' J 8 V 8 ' 8 V 8 - L V 8 ' r \ L ' t V r 'a ; os e q d 6y3 uI 5 066-Sg6'luaurdola,rap Ierneu 'IS8 LVq'1LI'JouoIlIuIJeP 1988'LL8-9L8't6S8'8S8 It60r '6t8'ur aseqdzue 'ut 'OSS ' sase8rluralord-urlrnbrqn l0S8 tOSg e60I-160I'ruaurdola,rapar.{roqdudlut 'ur uoIsI^IPlle) oslo .rournl srossa.rddns ru:a8ur 163 €.9G'JL06' 806-LO6's:a.{e1 'leuontlsuerl a a s 6' 6 8 - L V 8 ' 2 L8 - I 8 L ' ! 8 I ' 6 I - 8 I ' e p d c 1 1 e 3 1g3 lI96'Pue uorlef,olIIaf,

xlcNl

.

0l-l

a,rrleprxo S0S'ttgs'ur uoneydroqdsoqd Surpurq 1gg'uorrellroqdsoqda,rrleprxour pue8q-roldaraa osp aas'19-gg'Surpurq ZZS-IT.S'ura8eruepe^rleprxo ur InSV-lZ8b'S8'-Igt'srsd1ord13 'to SZS-VZS suolllunJllToqelalu uorleprxopne drrey 59'rrqo:ae Z6V-I67'ul(uodsueJl suortfeerTelrueqc 1gg'urxuteur uoJtf,Jlaur I8t ,JosuoutunJ .apb prle f,rrlrf,ur gg t3.taua TerlualodIErlu3qO 5/€ lgy (tunuqrlrnba .ur srsorusoruJaql L SS , Z n Z , J L ; Z 9, t 7 , 1 o u o r l n l o , r a 0g-dt lecruraq3 0gt 'Jo (to slopour 8uq1r;-aceds fI VNC relnrrrf, l€J-u.,r\orq J€t 0Is:'|ur PrrPuoqf,olnu 'Jo slepolu '60S ,aSueqrxa l87S-JS7.S J€€ lrrts-puelleq 160S dCV-dIV ul '675-V79 'Jt.Ig 'ZIS 'uorlexrl uoqrer ur spunodruorl?)nuarlJ l08v'08n-6Lv'uoDePrxo f,rqoreEur srsaqlu,,(soroq4 s u e 8 o u r t : eo3s p a a s. ! M l rplnlleC 6Z5- 6LV,srr1a3;eua 'ZIS'IIS .08t ,6LV,6Lt. 'rcnry' osp a a s ' l t I S Wil-071 1'suaSounretTelruraqJ IIP.ry\ II0l , J g L € , g L f . , 6 ,SI S I . S I , 5 . u l s r s a q : u d s spuog osiz aas'gt-Z€'spuoq letruroq3 41y ltreldoslpaas'rceg'ZVg-6tg,slue1d ;o '6III'668 t a c u e r u r 5Lt'I€L . t'(s)rseldo:o1q3 f,rlourso StII-9EII lVtV'VVV'pue a.rnsserd 'llP^\ 1 ; y 9 ' 9 1 9 ' qa d d r 668.]r68-J888 t€l€ IIef, 't 'e ' 11019 19 adAt 168-L88'ISg'gtg'01c.{r-11ar'srurod>paq3 h76-lZZ6'nZ6-I76'lseedut g1g1 (aseaslp qtoof-erreIAl-rorreqo 179 la1dr:l 065-S86'tuerudola,rap Ierneuur tIS'Jo a:nllnrrs 095'JLL'14'suruo;adeg3 626-V76'elf,snruur .SIS-;IS t r e d l e n a d s 6IS'JSIS uorrerrpeds ad&1e3 €ss-zss 'IZVS,M,J9L, surorsdsoloq4 oslpaas'IZS-0ZS,6IS r dr Suqeu8r5aa5'Suqeu8rs L L - SL , 8 u r p 1 ou ;r e t o . u IIal-or-lle) 'l9Is'JsIs'sIs-tIs taluor uorlreo.r ur ef,PJrns-TI3f, IZVS 'ltj:'uonpJoTsueJl '(s)roldaregaas's.roldeca.r ur 6rs arEJJns-lla3 IpuorlelsueJt-lsod 'il5'lt$'saxaldruo: Surrsa,r:eq-rq8r1 ur 779'slsaqtulsoloqdur '09s '6ss-8ss 'uollBr8nu YNL,T9NL,9VL-SVL US-il;,.(q uoud:osqerq8rl r9s J6ss lyarul'suorsaqpearprrsqnsJla) '1o uotlturloP '1:odsue.rl uretordlerrpuollJolI11r ur IIS /5€'sur?rlsIIaJ ,V$ ,pue (sauoroder{3 sproualo.rec fuIS Suqeu8rgaa5 .Surleu8rs11a3 9Le,6VZ.g€I '11Iqdoro1q3 slauuer{J uoI ras'suralo;d leuueq3 619 I0ZII'Jo uorlelnSar'uorlerayrlord 11a3 'uoilIrg 'aru€qc ISVV'gVV'poolq's^ ur .suoraprrolq3 Josoldr 65t 7n8-l'o8'uorleztue8ro -aprroT1{J 'ul ';o druangap,sluar:lnuIIaC 997'ra1:odrlueereuoqrerrq J€V9'€.V9-ZV9'eseralsarpoqdsoqd 4yg9r SSE UOIUr 11e3 jVZ'!o uowe rusruBr{raru'lorrueqdure.rolq3 Jo l€v9 D V L , S.ur n L , u rs d e l s (syouueqt 'tt9'uorsr^ ur uorlec pa1e3-41419c lI8 L' 08L-6LL'ur l:odsue:l Suqeu8rs l$ L-l LVL' gVL-9 VL reyla8ege:1ur'filp,toqutat souoruop[,tuo1q2 lLs9 Jl sL-lgvL' LnL-s v L'to uorlulnSer 'uorreln8a:ap.{o11or ' ul ,Zlq) 169 \St9'Vt9'8uqeu3ts ur gI L_t I L .Jo ..rl3i^ra^o LS9-959'VV9-ZV9 1988'seseurl ?lqc/I]rlf, ursol|w J7r9'W9-7V9'uorle,rrlreursdopoq:ur alf,snuur lSV€.'SVt-rVt.'urtrodsue:l osp aas'SnL-WL'pa.ramod-ursolur LS9' LS9-959'uorlexeyo.r I (apsnurur JEallnuVNUr.u,supl.ual. snowouotrqJ (aregdsoqdouoru aursouenB rqcdr) 4rygr _ 1626,626-gZ6 .suorlernrug1g3 sulelororoloIAIos12aas'9LL-6qL 199'gg 1{lr1e:rq3 955 ,SVL-I7,L,Sg,€9 .ul surotord:otoru rrreurgJ 11g !o uorlrugap,surato.rd 7ZII-ZZII'591':aruer lef,r^raJ 'L07'sererurqS 08t (s)eynqnlo:rr11 osp aas 9I5'se[]ulue^IErqareC '596 'snJaqraD . .ur salnqnlonnu 759'ereruserg3 98G LLL-LSLlt.L-911 ,ur seteloJrlue Z0t JZ9L' I9L'to e.rntln.rls 19VL'9rL'ul Surpber auerqru3ru .ur uorsualxssuerqru3ur uouerrldnP 975 {de.raqlorueq3 L ' t 8 L ' J o l v g L'snL lgn .Jo uoururJoP .Jo sursru€gf,aur ilS L \S L-gl L,srxeroruoq3 0gl JgvL' gt L-sv L ,JI9L ,19L-O9L.saurosortuoJ 'er,boqdudl 9561 A.toreruurel;ur JZgL L60l-960I 'lrr€rsoeruog 950I vzT'u$I.lJorqJoJalaqJrleruoJluaS vLL-6gL.ur sursrurl 'sOurloureq:) ,VNC Jrreruo4uaJ I90I ur 9gl, l9VL' 9, L-SVL'sa1drour1e.ra1 (1sealur It V1I' €V1I-T.;g1'srsaqrod.{q,{rruryyeourog3 It gZ'1792't97-I9z 9VL-SVL,ursur.r8atul 'Jo ernpnrls ftt.'z€-I€'Jo sldaruorda1{rrsruaq3 t9z Sll'';o uotletttur aJJoJ elrlorx-uotro14 !u6d5uB11 uoJlJala Surpurq-419 l 0 L 8 ' 6 9 8 ' P u EV Z d d lgVL' 8, L-9VL'ur suralo.rd oslpaas lt0s .t0S-€0S ,6gy ,srsourso[ueq3 J t 9(pue Z,t9Z.;o ql8uey IISL-lgVL,gb L-7VL,ur srolreJgurorS g5 .e)ueJseururnInuaq) gg s:;oqroreur>1 L-198 L' Jg8 L-99 L 86L-L5L :oJ suorllerrp rlf,tEesarajnln] 'Jt00I 'asdeuds S00I lerrureqJ t9Z'Jo suorrJunJ 7VL-SVL,uruonereua8acro; 'LS-95,u\ 'yo (ur alets uorlrsuerl l0g'6L'lLS uourur;ap urlrauorqr; 197 €€g-6f g '0s 'erers-lPeers . .seraurorlueJ JOS 6Vg'It gz-Jlgz tgz-Igz ur 9nL-Sn L'srselqorqry 'IZt. 'p lrtTtqtr'.n", 6V I9l'salourueC JSLL'fu LL' 9LL-i LL'ur suraudp 'xopa: .1euoz_ater 109'l6s'99-69 It6, V6-t.6 6VL-gVL.yodlrleuorlra;rp 'ur 'Z6e 'sa11oue8.ro sluelreo: 6g lz6t 560I'trurrPuop ,pue JaqrunuJalouJnl '8\v-Lov 'l€6€ '7.6t 1o Ig ggl ,yo uourugap l80v ' jzg .Jgg,Ig-6L,pue slsdleler -'llrsuap y qrnba fi urnr.rq I' L0 9 0I' 5'tuarpe J 19VL,9VL.ur uorsaqpp-ep 'l0S '59 LS-95 \o etet lzSt'z5t'IE6' t 5'lErlueraJJrp Jo slueureJrJ 'n6-26 'uoEe8n;r;lua3 'uora1o1sord3 0S':oJ tuplsuof,ater oslp aas' 6 Je L6L-96L,ISIL 'SV-fl 6t'ul sllnPord stuaurelry It.II,ZII.eruSop lerlua3 L'l9l'9I'ur 1e1a1o1so1dr 'lrrurar{loxa 9S: SLI'(I Ir) ue8rorurluar L6L-961 'tSS 'SS 'rtuo3:axa ' erndaterpunorrns-letuaJ l,S l5Z0l' 6Z0I'sp1ary patEurproof, ISL-SVL'ur sassarord .ruu.rqrlrnba .ur srxPlorlrqf, le l0S, 0S_6b €92'V-dNgf, I sL-gvL lSroug fstrtaS:aua reTnlla)osp aas .ur saurloueqf, esolnJIeC 6yg'eseqruds L60l-960I 'JZg'J0g'Ig-6L,lLS ,09-VS,lo '16t 'zL€.'9V 'aso1n11a3 scrla8raua 0v8 8 JLVL']9VL .)ruraqropua (ur suorsoqpB '9V 9S atElsqns-TIaf, IIS'pue srsagludsoioqd L-gVL j:8t (JouoururJap /S 1SS.SS.:ruo8rapua LVL-9VL.puedlrrelod11ar ,09-5S .ul ssolTure8 uolraTa l09,165 rplnlleJ 5gr-L8V'S8t'5S'uorlerrdsa; lpoqlJel I68V' l9VL' 9VL'ur uorteJolsu?rr ,ro1sluptsuof,uorlerJossrp 'ssorrsa^rt?pIxoreTnlJe) IS-0S Eg5 tuolur{r?ileIIaJ JgtL'9tL'ur uondn.rsrp ,6V.y7g.1o uorlre:rp 9S-SS JZSV'Z8V-t8' It VL' VtL-€.tL'uodsuBrtogrer ur 'uorreldror{dsogd 'Suruop poolq ui 55-39'79, p:1dnor ur 9nl ,pue 1aral-ete.nsqns \-/'*"_*5 0IS 1o:luoc,(roterrdse: J 1 L L ' J 6L9, I L L - 6 9L . u r s u r s a u r 4 oslp aas 'J1g ,09-6L,Lg ,6, ,u! .ur surau,{p stsllelel 0 Is-€0s 9LL-SLL lrodsuetl Ieuoxeur (panu4uotl uorle.r8rurpuaua^owIIoJ 55'cqoqeret J t S , V S - Z S . u rs r q J n q

'JI9V'1gV'ur elroJ a,rrtour-uolo.rd J08t'09'.'519,srsagrudsoroqd ur

I l-l

.

xscNl

'650r ur JVSZ'SSZ-L7Z'oseqdeleur J090I 1898'698-L98'uruotleuntnbtqn 'ul iord3 osp aas'6L9-TL9'1999'stotdetar tZ88 '0sg'Ic J0sg 719'p sadf,t l€S8'Jo uorlerltlssPlr 960I-560I';o uorrrnpord11ac-1 11a3osll aas'tS8 ;o uorreln8er'epdr JVL9'tL9-€.L9'Joernlf,nls 'uorleln8ar 'J0s8 '8s8-6t8 'Surleu8rs apb-11acut g)'-JN ul 961 'ul xoq uorFnrlsep lggg'L9g'858 I 6 1 6 'g I 6 ' : r l a r o d o l e u r e q 'pue ';o xaldruor3734y suotloun; I88 t L9-ZL9 g uqod3 fy uqc,{3 saQ["qttJoaQsosp aas'I90I-090I'(s)aur1orl3 osp aas'l8S 1898 '199 'ggg 'adlr-g 8urddel1osp aas'ygg7'Surddeurrlauo8old3 rfSS '88 '(s)uqrd3 6€I I'ggt-d aruo:qrold3 '66n-86v'ts6v autsouenB rqcb) (areqdsoqdouoru lr}s'aseptxo 'aleqdsoqdououraulsouenSrqtd3 'uodsue.DuoJlrela ur , aruoJqloils dWOr aas y95'Sutta8retutatord lViL' bVL'ur SuttuBarsrtruseldoilt sarol:e1o:4osp aaS'erralreqoued3 IprrpuoqJolruur'ase,{1aruaq',: aruo:qro}t3 66V-86V'aptued3 195'srsordodeul otuo;tlf,old) (19'sruore oxxxx3 39 I S 6 n ' 9 6 V - S 6 V ' p uaeu a q rS6t'lS6V'96V-g6t'uodsuerluort)olrur J59'89'orerr'xxxJ seJnllnr ile) aas 'ssrnllnc (s)etuorqroilD ZI6-116's1yocuals rtuodrque pernrlnC LZL-9ZL,q urseleqroil3 g5; 'sune:a1-or.{3 70L totre; uottdt.rrsuerr(r3) snldnr:elut snuqnJ tssv'gsr-s9v'(urgo) uI'7VTI3 g60I-V60I'uotle,rrlce .rolelnBa.r eueJqueursueJlsIsoJqIJJusdJ 'Lt 'ZZ {e:-y IIaJ-I tqder8o11ersd.t3 g67 ';o salpnrslnopou>1-aua8 lgt 'rSSt uorlf,elaodr) €59 106€' 6ge l(qde.tSoruot 'gSt-S 106€' 688'961 {dorso.rrruruorlra1ao,tu3 S l ' 1 0 0 2 ' 0 O Z ' t 6 6 1 ' s r s o : qr rrl;s d 3 (s)pneounuy osp aas'€l'JZV'auta1s.{3 lv LI's Lvv LI'Pue Surddeuaua8 'ourrodsolol3 1661 J V 6 g ' l e 6 g ' 2 6 8 ' J B 9 t ' E S I ' 0 S I ' r oS, ur ot s s o : 3 '1 gg61'ue8nue'uotleluasard-sso:3 Jsl€'gl€-src'uorlra;ut n1g ur uqrd3 (urr)e 'sutalo:d 'gZL '1€S8 'g 3ut>1uq-ssor3 uI ulTrd) I88 J6ZL uoxo-ssorJ r€S8 'lseeduI !V$'€e€'xaldruor uotltuSocar 'a1ttrtaSpug-ssor3 6E7 l€99, e9g-Z9g,toa.rnlrnrls 'uottettuera;yrP gzt .urTnqnr rselqodurut 976 'ellaJnu ul ggg torrel Sutloruo.rd-stsoltur 8Zt 'IZL-jZL 'ull]e IjzL L9L' pue suteto;dpalelJosse-alngnloJf,Iru uorlPJluaJuo) Its8 'sl?urujeluuI .I?f,r1rJJ eersrr3 ';o uoututlaP J 9 8 n , 9 B V , l'suolllunl 8 L €g,L e 193 ElsIrJ saxalduoc;q3-uqcd3 J98i osp aas'€98-658'J0S8'€S8-8t8 9ZZ'lW'snue.rg'>1ct:3 'ttI'(s)Cl) uottdttcsue:lut saseurltuapuadap-uqr,{3 1869'g69'uotlettrur (;o uorle,rtlre 'uotterluara;;tp rselqodurur J L99'1999'V6Z-t 6Z 976 'uraro.rd 'raruul ggg3 ur 967 S€II 'spuelsrgd3 797 IC uIIrdC 't088 'lg9g 'q uqrd3 l$ €' ZS€.-ISE'utelo.rdSutputq-943 I88 (uoIlP^IIJP uolldtr:sue.rlul 'gd] 90€ t88-888'srollqlqul;q3-uqrl3 / /

in vertebrates, 977-97 8, 982-983 in cell differentiation, 905-906 in cell-type specification in muscle, 925-929 in yeast, 922-923, 922-925, 922f, 9231 chloroplast,3lS chromatin and,256 co-activators fo4 293 in combinatorial regulation, 294-295, 294f,295f cooperative binding of, 294-29 5, 294f,

29sf definition of,270,286 DNA-binding domainsof, 288-296, 289f. Seealso DNA-binding domains DNA-binding motifs of, 69-70,70f, 290-292, 291.f_293f in embryonicstemcells,917-9L2 gap genesas,974-977 general,253,296-297,297f , 298f in preinitiationcomplex,297--299, 297f,298f heterodimeric, 294-295

homeodomain, 291 identification of DNase I footprinting in, 286 electrophoretic mobility shift assay in, 286,287f sequence-specificDNA affinity chromatography in, 286, 287f in vivo transfection assay,288 interaction of, 294-295 mating-type, in cell-type specification,

922 mitochondrial, 318 in myoblast migration, 928 i n n u c l e a r - r e c e p r osr u p e r f a m i l y ,2 9 l .

312-31.3, 3t2t, 373f in oncogenic transformation, 1130-1131, 1,1,32f r e g u l a t i oonf , 3 1 1 - 3 1 3 repressiondomainsfor, 290 repressor, 290,923 in signaling,632-633. Seealso Signaling specific,286 Transcription-initiation complex,72f Pol I, 31,6-317, 317f Pol II, 134, 135f,296-299,297f, 298f, 307-308,308f Pol III, 317, 3l7f Transcriptionunirs,217-21I bacterial,217 eukaryotic, 217-221, 2l9f complex,218,279f mutationstn, 21.8-219,2L9f simple,217-218, 279f s t r u c t u roef , 2 l 9 f insulatorsfor,254 Transcriptionalgenecontrol. Seealso Transcription,initiation of activatorsin, 270, 270f, 27| in bacteria,27l-276 enhancersrn, 274-275, 274( PhoR/PhoBsystemin, 275, 275f RNA polymerasern, 271.-275, 274f o (sigma)factorsin, 271.,273-275, 273t,274f rwo-componentregutatorysystemsln, 274-275, 274f, 275f combinatorial,294-295, 294f, 295f coordinateregulationin, 271 in developmenr, 28-29, 291 enhancersrn, 18, 274-275, 274f, 284J8 5, 285f,295-2e6,296( in eukaryotes,276-281 functionsof,275 future researchareasfor in, 318-319 in geneticprogram execution,276 overviewof, 269-270, 270f post-transcriptional, 323-367. Seealso Post-transcriptional genecontrol repressors tn, 270, 270f, 271. silencersin, 1,8,299-301,,300f Transcripts,processingoI, 123-124, 125f. See a/so RNA processing Transcytosis, 505 of immunoglobulins, 1065-1066, 1,06 5f Transducin,rhodopsinand, 641, 644f Transducingretroviruses,1122 Transepithelialtransport,470-472, 47|f, 472f Transesterification, in splicing,330, 330f, 332, 332f Transfection,1.95-1,97 in secretorypathwaystudies,582 stable,196-1.97,l96f transient,196,196f in yeast,261,-263,262f Transferproteins,in lipid transport,

+55 TransferRNA. SeeIRNA (transferRNA) Transferrin cycle, 611-612, 617f Transferrin recepror{TfR).356. 357F Transformation in cell cultures,398 of tumor cells,1113-1114,1,1,141 chloroplast,242 oncogenic,1.1.1.3-1114, 1114f PI-3 kinaseand,694-695 in plasmidcloning,1.78-779 transformer,alternativesplicingof, 339, 339f Transforminggrowth factor B. SeeTGFB Transfusions, blood types and,426 Transgenes, 209 Transgenicanimals,209, 209f Transgenicplants,470 Transientamplifyingcells,905, 916-91.7, 91.7f,91.8f Transientreceptorpotentialcation channels, 843 Transienttransfection,1.96,196f Transitionstate,56-57, 57f, 79, 80f Translation,11, 72f,74, 1,1,2,113f, 1,27-131, 21,7 in cell-cycleregulation,881-882 973-974,973f, in ceff-faredetermination. 974f chain elongationin, 1.35-137,l36f in chloroplasts,557 codonsin, 127-129, 1,28r,129f concurrentwith transcription,'123 cytoplasmicpolyadenylationin, 351-352, 351.1 delnition oI, 127 eIF2kinasesin, 355-356 G proteinsin, 354-355 initiationof, 127-128,128t, 130f, 1 3 3 - 1 3 51 , 34f,355-356 sitesof, 217 in learningand memory,352 in mitochondria,557 mRNA in, L27, l27I mRNA degradationin, 352-353, 3521 mRNA surveillance in, 357 nonsense suppression in, 138 P bodiesin, 348, 353 polyribosomes in, 138, 139f poly(A) tail lengtheningin, 351-352, 3s1f preinitiationcomplexin, 134 in prokaryotes,123 proofreading in, 131, 1,45,146f,355 in protein targeting,537-538,537f rateof, 133, 138 readingframesin, 128, 1.291 regulationof, 323-325, 3241.Seealso Post-transcriptional genecontrol;RNA processlng cytoplasmic, 351-353 global,353-355 sequence-specific. 356-357.357f repressionof eIF2 kinase-mediated, 355-356 miRNA-mediated,347-3 49, 3 48f RNA-inducedsilencingcomplexin, 348 Rhebprotein in, 354-355, 354f ribosome in, 132-139 rRNA in, 1.27,L27f, L32-1.39,1.35f,1.36f. Seealso Ribosomes of specificmRNAs, 356-357, 357f stepsin, 130f terminationof, L37-1.38,l37f 3' regulatoryelementsin, 351-352, 351f' TOR pathwayin, 353-355,354f INDEX

t-49

Translation (continued) IRNA in, 127,1,27f Translocation(s) chromosomal a n a l y s i so f , 2 5 8 , 2 5 9 f in cancer, 1,1,30,11321 in proto-oncogenes, 1 120 protein. See Protein translocation ribosomal, 1.35-137,136f Translocons, 539-540, 540f Transmembrane proteins. See Membrane proteins, integral (transmembrane) Transmissible spongiform encephalopathy, 77 Transmission electron microscopy, 388-389, 389f T-^-^^l^-.^.:^r r drLrPr4u r4 Lruil

bonemarrow,920 MHC moleculesand, L077 Transport electron,493-503.Seealso Electron transport membrane.SeeMembranetransport transcellular, 471.,471.f transepithelial, 470472, 471.f,472f vesicular,579-606. Seealso Vesicular transport Transportproteins membrane.SeeMembraneffansport proteins nuclear,342-347. Seealso Nuclear transport Transportvesicles,376-377, 375f, 579-606, 580. Seea/soVesicles;Vesiculartransport Transporters,439, 439f, 440, 441443, 441.f, 442f. Seeaiso Membranetransport proterns ABC.447f. 448, 45445 6. 4554 50,45 5t flippasesand, 456, 456f in geneticdiseases, 455456 antiporters,440, 466, 468470. Seealso Antiporters conformationalchangesin, 440 cotransporters, 440, 440t exocytosisof, 1020-1021,1.020f in glucosemetabolism,441.443, 441.f, 442f, 471.,47 tf, 488f, 489491, 490f, 545f,547. Seealso under GLUT as multipassintegralmembrane proteins,547 G L U T ,4 4 1 4 4 3 , 4 4 l f , 4 4 2 f, 4 7 l , 4 7 l f . Seealso under GLUT for neurotransmitters, 1020-1022,1021.f phosphate,in MP-ADP exchange,509, 509f symporters,440, 456470, 471 in transepithelial transport,471.,471f uniporters,440, 441.-443,441.f,442f Transposable elements.SeeMobile DNA elements Transposases, 228-229 Transposition,226-227 Ac elementstn,228-229 of Alu elements,234 in bacteria,227-229, 227fJ29f cut-and-copymechanismin, 227 cut-and-paste mechanism\n, 227, 228, 228f,229 definitionof,226 of Ds elements,228-229 in eukaryotes,229-234 integrase in,230,233 of IS elements,222-228, 227-228, 228f of LINEs, 230-234, 232f, 233f P elementsin, 229 retrotransponsin, 227, 229-23 4 reversetranscriptionin, 230-234, 230f-233f of SINEs,234 l-50

o

|NDEX

somatic-cell, 226 Transposons, 227-229, 227f-229f , 265-26 6, 350. Seea/soRetrotransposons bacterial,227-228, 227f definitron of,227 eukaryotic,228-229, 229f in exon shuffling,235 multiplicationof, 229, 229f Transthyretin,282-283, 309-310, 310{ Transverse tubules,7 40, 7 41.f Treadmilling in actinfilamentassemblg7Zl-722,721.f , 722f in microtubules, 753, 763f in mitosis,785, 786f, 787f, 788 Triacylglycerides, 48 Triacylglycerol, 491 Tricarboxylicacid cycle,487489, 489f Triglycerides,48,491,.Seealso Lipid(s) TrimericC proreins.355.637-639,638f in signaling,634 in, 644 subunitdissociatron Trimolecularcargocomplex,573 Triplet code.SeeGeneticcode Triplermicrotubules, 7o0, 760f Triskelions, 598,598f TrisomS 887 Trithoraxproteins,302, 303, 306,982-983 Triton X-100, 428,428f trk oncogene,1.128,1L28f Trks, in neuronsurvival,938 I R N A ( t r a n s f eRr N A ) , 1 1 2 , 2 1 7 , 2 2 2 t in amino acid activation,131 amino acid linkageto, 129, 1.30f,L3L in,127,129 anticodons codon recognitionby, 727-1.31' cognate,131 diversityof, 129 folding of, 129,1.30f functionsof, 127, 1.27{,1.29 13 1 isonine-containing, 133 methionine-containing, nonstandard basepairingand, 130, 131f processing o\ 363, post-transcriptional 363f,364f processing of, 363-364, 365f p r o m o t e ri sn . J l 7 , 3 t 7 f rRNA and, 137 structureof, 118, 1.29,1.30f synthesisof. SeealsoTranscription III in, 278,279t RNA polymerase in translation,1.27-131. Trophectoderm, 960 Trophicfactors,in apoptosis,243f,936, 942-943 Trophoblast, 962,962f Tropomodulin,723, 740, 740f Tropomyosin,740-7 41., 7 41f Troponin,740-747, 741.f trp operon,1.24f ,21.7 True-breedingstrains, L67-1-69 Trypsin activationof, 91 activesite of, 80f, 82-83, 82f Tryptophan,42, 42f. Seealso Amino acid(s) TSCl/TSC2,354-355 Tsix, tn genomicimprinting,958-959 TTR, 282-283, 283f of, 309-310,310f transcription Tuberoussclerosis, 354, 355 Tubulin, 7 58, 760f ct form of, 758,760f B formof,758,760f in neurogenesis , 929, 930f of, 763 critical concentratton 1 formof,761,,762f

homologyin, 244, 2451 sequence Tumor(s),1108 benign,1109 hypoxic,1.L09, 1.1.L2-1113 Seealso Cancer malignant,1.1.09-7110. Tumor cells,identificationof, 7716-1J.13,1.11.8f 1111 Tumor microenvironment, Tumor necrosisfactor alpha (TNFo), 703 Tumorstemcells,1111 l1.l8f identificationof, 1.1.16-1119, Tumor-suppressorgenes,882, 1107, 1122t inherited mutations in, 1-123 mutationsin, 148,'l'1'23 loss-of-function in, 11'24,1'1'25f lossof heterozygosity Rb protein,882-883, 882f Tumor-suppressor in cell-cycleregulation,891 Tumor suppressors, Tumorviruses,158-159 1108 Tumorigenesis, Turgor,377-378 Turgor pressure,444, 830 Turnovernumber,8L 20S proteasome,87-88, 87f gel electrophoresis, 95-96, Two-dimensional 95f, 106 symporteg466457, Two Na*/one-glucose 465f, 471. t/one-leucine symporteg467468, 467f Two Na zJz, zSzr Iy elements, Tyrosine,42, 421.Seealso Amino acid(s) U1 snRNA,330, 331f U1 snRNP,332,332f U2 snRNA,330, 331f U2 snRNR332,3321 U4 snRNP,332,332f U5 snRNl 332,332f,333 U6 snRNl 332,332f Ubiquinone,in electrontransport,495t,496, 496f Ubiquitin, 88, 858 610 in endocytosis, pathway,protein Ubiquitin/proteasome degradationin, 555 Ubiquitin-proteinligases.Seealso APC/C complex in cell-cycleregulation,850, 850f, 851' 8 5 8 ,8 5 9 f ,8 7 6 - 8 7 7 , 8 8 38, 8 6 t Ubiquitination in cell-cycleregulation,850f, 851 in anaphaseinitiation, 867-869, 868f of B-typecyclins,858, 858f in mammals,850, 850f,851, 858' 859f,876-877 in Xenopouslaeuis,858,859f in yeast,876-877 867-859, 868f in chromosomesegregation, of histones,250, 250f, 251'-252 Ultravioletradiation leukemiadue to, 1139-1140 mutationsdte to,'1.46-1.47 Umami tasteperception,1035f, 1036 Unc proteins,1045 spacerDNA, 220t, 225-226 Unclassified Uncouplers,5l0 Unfolded-proteinresponse,555, 555f Uniporters,440, 441'443, 441446, 441'f, 442f. Seea/soTransporters transport,471',47If in transepithelial Unipotentstemcells,907 Unsaturatedfatty acids,47-48, 47t Untranslatedregions(UTRs),124 285, 3L6 Upstreamactivatingsequences, Upstreamtranscription,1.20,121.f , 277, 285, 316 IJracll,44, 45t, 113-11'4.Seea/so Base(s) in RNA, 118 structureo1,44,44f Ushersyndrome,1033-1034

V - c l a s sp r o t o n p u m p s , 4 4 7 f , 4 4 8 , 4 5 3 4 5 4 , 453f. See a/so Pumps in synaptic vesicle transport, 1019-1020 V segments in heavy chains, 1069, 1069f,1071,-1,073, L072f in light chains, 1069-7071, 1069f, 1070f i n T - c e l l r e c e p t o r s ,1 0 8 8 , 1 0 8 9 - 1 0 9 1 , , 1 , 0 9 0 f v-SNAREs, 585, 586f, 591., 1022 v-s/c proto-onc ogene, | 121-1 122 Vaccines, 1 1,07-1,1,02 V a c u o l a r m e m b r a n e ,3 7 7 - \ - 8 , 3 - - f Vacuoles,373f acidification of, H* MPases in, 453-454 contractile, 444 plant, 377-37 8, 469-47 0, 469 f Y a l i n e , 4 2 , 4 2 f . S e ea / s o A m i n o a c i d ( s ) Valinomycin, 502 VAMB 591, 1022 V a n d e r W a a l s i n t e r a c t i o n s ,3 7 - 3 8 , 3 8 f i n p h o s p h o l i p i d b i l a y e r ,4 1 1 , 4 1 8 4 1 9 Variable region of heavy chains,1066f, 1067 of lighr chains,1,066-1067,10661 Variation, genetic, 7. See also Mutations in evolution, 28-29 V a s c u l a rC A M - 1 , 8 3 5 Vascular endothelial growrh factor, in cancerr e l a t e d a n g i o g e n e s i s1, 1 1 2 - 1 1 1 3 Vascular muscle, protein kinase G and, 656-557, 657f V a s c u l a rt i s s u e ,i n p l a n t s , 8 3 9 , 8 3 9 f VASP,726 Vault RNA,222t Vector(s) BAC,179 bait,310,311f definition of, 176 DNA insertion rnto, 777-178 D N A p r o c e s s i n gf o r , 1 7 6 - 1 7 7 , 1 , 7 6 f expression, 1,94-197. See also Gene expression studies bacterial, 194-1.95, 794-196, 195f e u k a r y o t i c , 1 , 9 6 - L 9 8 ,l 9 6 f - 7 9 8 f in gene/protein tagging, 197-198, 1,98f plasmid, 195-196, 795f retroviral, 197, l97l fish,310,311f plasmid, 178-179, 178f, 179f e x p r e s s i o n ,1 9 4 - 1 9 6 , 1 95 f in prorein production, 195-196, 795f shuttle, 183, 183f viral, 6 Vector DNA, 176-178. See also Clones/cloning, DNA Vegetal pole, 963 VEGF proteins, in cancer-relaredangiogenesis, 1112-1713 VEGF receptor antagonists, for cancer, 1113 VelocitS maximal (V",",), 80, 80f Ventricles, cerebral, 916 Vesicles aggregatedproteins in, 602 a u t o p h a g i c ,5 1 , 4 - 6 1 6 , 6 l 5 f budding of, 580, 581f, 586-587,586f, 587f c o a t a s s e m b l ya n d , 5 8 6 - 5 8 8 , 5 9 2 f donor/destination organelles in, 585 dynamin in, 599-600, 599f,600f i n e n d o c y t o s i s ,4 1 4 f , 5 8 1 , 6 1 0 ,

612-614, 6r3f, 1023 pinching off and,599-500,5991, G00f in retroviruses, 614 from trans-Golgi,599-600, 599f in vesicularrransporr,599-600,599f, 600f, 6L3. Seealso Vesicular

rransporr clathrin/AP-coated, 393, 586-589,587t, 589t,598-600,598f-500f i n o r g a n e l lpeu r i f i c a r i o n 3 .9 3 - 3 9 4 3. 9 3 f pinchingoff of. 59e-o00.5q9f, 600f, 1023 dynaminin, 1023 in receptor-mediated endocytosis, 606-607,607f uncoatingof, 600 COPI,s86-589, 587,587r,589t, 595 C O P I I ,5 8 6 - 5 8 9 5, 8 7 f , 5 8 7 t 5 , 8 8 f ,5 8 9 t , 592-593, 592f-594f, 595 fusionof, 535, 585, 585f,589-591,590f in exocytosis,414f, 580, 591 synaptic,1022 GoIgt,376-377, 377f secretory,373f, 376-377, 376f, 580, 602-603 constitutive,602 regulated,602-603 synaptic, 1019-1022, 1020f, 1021f. See a/soSynapticvesicles rransporr,376-377, 376f, 579-606. See a/soVesiculartransport uncoatingof, 500 Vesicularglutamatetransporters(VGLUTs),1020 Vesicular transport,376-)77, 549-606.See a/soCell movement/migration anterograde, 580, 58lf, 592-593,592f, 595-597,596f cargoproteinsin, 580, 588-589,593 in cls-Golgi,592-597, 592f-596f cisternalmaturationin, 580, 596, 597, 597f clathrin/AP-coated vesiclesin, 393, 586-589,587t,598-600,598f-600f pinchingoff of, 599-600,599f,600f uncoatingof, 500 coat assembly in, 587-588,588f endocytosisin, 414f, 581 endoplasmicreticulumto Golgi, 592-593, 592f-594f enerBysources for. 587-588,5ql exocytosisrn, 414f, 580, 591 experimentalstudiesof, 580-586 future researchdirectionsfor, 61.7 intercompaftmental k i n e s i nisn , t a l - 7 - 2 , 7 7 l f lysosomal,600-602, 600f, 601f, 608-610, 609f,612-616 mannose6-phosphatein, 600-602,500f, 601.f mechanismsof, 586-592 membrane fusionin, 474f, 535,585, 586f, 5 8 9 - 5 9 1 5, 9 0 f microtubules in, 593, 769-770,770f mitosis-promoting factor in, 866 overviewof, 580, 581f proteintargetingin, 588-591,600-602. SeealsoProteintargeringl Sorring signals to apicalor basolateral membrane, 504-60s,605f transcytosis in, 605 proteolyticprocessingin, 503-604, 603f, 604f Rab proteinsin, 589-591,590f retrograde, 580, 581f, 592, 594-595,596, 596f in secretorypathway,533-535, 534f, 579-606. Seealso Secretorypathway in early stages,592-597 in later stages,597-606 of secretoryproteins, 602-605 signalingin, 589, 589t,594-595,594f, 600-602. Seealso Sortingsignals SNARE proteins/complexes in, 586, 586f,

591 in trans-Golgi,597-606, 598f-601.f, 603f, 604f vesiclebuddingin, 585-587, 599-600, 599f, 600f, 613. Seea/soVesicles, buddingof vesicledockingin, 589-590, 590f vesicletargetingin, 589-591 i n y e a s t5, 8 4 ,5 8 4 f , 5 9 1 . , 5 9 6 , 5 9 7 f Vibrio cholerae,639-640, 816 Villi, intestinal, 471,916, 976f Vimentin,793t,794 Viral capsids,154-155,155f Viral DNA. replication of. 142-143,142(.l43f Viral envelope,154, 155f Viral infections time courseof, 1101f vaccinesfor, 1,1.01.-71.02 Viral oncoproteins,1121.-1723,11,22f, 1.1.28-L1.29, L1.29f Virions, 154 b u d d i n go f , 1 5 8 , 1 5 8 f progeny,1.56,1.56f,158, 158f,159f Virus(es),6, 154-1.59.Seealso Retrovirus(es) animal,154 buddingof,5l4, 675f cancer-causing, 1.1.21-11.23, 11.22f, L128-1.L29,1.L29f cloningof, 155 definitionof, 154 DNA, 154 oncogenic,1122-1123 DNA replicationin, 142-143, 1,42f,l43f enveloped, 154-155,155f retroviruses as, 158, 159f as experimentalorganisms,25 genomeof, 154 helical,154, 155f host rangeof, 154 icosahedral, 154, 155f infectivemechanisms of, 1057 integrinsand, 838 membraneinvasionby, 409, 409f mRNA transportin, 346-347, 346f oncogenic,1121-11,23 phage,154, 158 plant,154 plaqueassayfor, 155, 156f quantificationof, 155 replication of,1.057 lytic, 156-158,155f,1.57f nonlytic,158, 159f in plaqueassay,155 reproduction of,5,5f RNA, 154 structureof,6,6f tumor,158-159 veclor, b Virus-associatedRNA, in translation, 356 Viscometrg in actin polymerizatron, 7 19 Vision, 1027-1031. See also Eye acuity of, 1028 alternative splicing in, 340 bipolar cells in, 1.027f, 1029-7030 color, 7027f, 1028 evolution of, 1028-L029 image interpretation in, 1029-1031, 70301 integrative processesin, 1029-7037, 1.029f-1.03If neuronal organization in, 1,029-1031,, 1,029f p a t t e r n r e c o g n i t i o ni n , 1 0 2 9 - 1 0 3 1 , 1 0 3 1 f primary cilia in, 780 receptive fields in, 1028-7030, L029f signal transduction tn, 641-645, 642f-645f s p a t i a l p r o c e s s r n gi n , 1 0 3 1 , 1 0 3 1 f Visual adaptation, 644-645 INDEX

t-51

Visualcortex, 1028 Visualpathways,1002f Vitamin C deficiency,825 V-,* (maximalvelocity),80, 80f Voltage,electrochemical gradientand, 439, 464,465f Voltage-gated ion channels.SeeIon channels Von Behring,Emil, 1062-1063 Von \Willebrandfactor,834 VSV G protein transport,582-583, 582f Wall-associated kinases(\7AKs),84L-842 WASpprotein,725-726, 725f Water.Seea/so Solutions membranetransportof, 444445, 444f446f Water-channel proteins(aquaporins), 423424, 444445, 444f446f, 445 in diabetesinsipidus,445 structureof, 424, 424f, 446f Watermolecule,dipole natureof, 34, 35f Watson-Crickbasepairs, 114-715, 11.5f Watson,James,114 Weeprotein,in mitosis,860,862,862f Weel kinase,886t Weel protein-tyrosine kinase,852 Westernblotting, 98, 99f White blood cells.SeeLeukocyte(s) Whole genomeshotgunsequencing, 187 Whoopingcough,640 Wiechaus,Eric, 17l, 999-1000 Wiesel,Torsten,1031 \fild type organisms,166, 167l Wilkins, Maurice, 114 Wilms' tumoq 290,290f Wilson,H.V., 805 wingless, 699,702 Wnt pathway,667f, 697-698, 699-700 in axon guidance,1046-1047 in body segmentation, 909,914-915 in cancer,1125 in limb development,992-994,993f in stemcell differentiation,909,91,4-91,5 Wnt receptors,666f Wobbteposition,nonstandardbasepairing at, 130-131,131f Wound healing,dendriticepidermalT cellsin, 91,s 'Wounded-cell monolayerassay,748, 7 50f

l-52

.

INDEX

X chromosome, 1.3,1.3f,1,9,1.9f,955 dosagecompensation and, 253, 9 58-959, 9 59f inactivationof, 253, 958-959, 959f in heterochromatin formation,253-254 X-inactivationcenter,958, 959f X-linked recessive inheritance,200, 200f X-ray crystallography,22, 37, 38f Xenopuslaeuis mitosisregulationin, 856-858,856f,857f, 867-869, 868f neurogenesis in, 929, 989-990 oocytematurationin, 854-856, 854f, 855f n4ttPtnrno

rn

anterior-posteri or, 965-966, 966f 963-965 dorsal-ventral, 42, 1'1,42t Xerodermapigmentosum,148-1,49, 1.'1. Xist protein,222t,253 in genomicimprinting,958-959 XMAP2157 , 8 5 ,7 8 5 f Xolloid,965,966f

Y c h r o m o s o m e , 1 , 3 , 7139f ,, 1 , 9 f , 9 5 5S. e ea l s o Sexchromosomes Yeast,5. Seealso Ftngi in. autonomously replicating sequences 261,262f buddingin,872,873f c d cm u t a t i o nisn , 1 7 0 - 1 -l , l - 0 f cell-cycleregulationin, 851-852, 852f, 853t, 859-853, 860f-863f cell-typespecificationtn, 921-924, 922f-924f in, 261-263, 262f, 263f centromeres cytoplasmicinheritancein, 237-238, 2 3 7 - 2 3 82. 3 8 f . 2 3 8 1 as diploid vs. haploid organism,166, 169f 1.69-1.70, DNA replication, 877-879, 878f as experimentalorganisms,26 functionsof, 5 geneinactivationin, 205, 206f meiosisin, 895 mitochondrialDNA in, 237, 238f myosinV motors in, 743-744,743f nucleartransportin, 343-344, 344f

protein translocationin, 540-541, 542f pyruvatesynthesisin, 485 reproductionin, 7,71 in, 350-363 rRNA processing pathwayin, 584,584f secretory telomeresin, 261.,2621,263 transcription repressionin, 299-301.,300f, 301f transfectionexperimentsrn, 261-263, 262f vesiculartransportin, 584, 584f, 591, 596, 597f Yeastgenomiclibraries,180-181, 182-184, 18 3 f Yeastmatingsystems,MAP kinasesignaling in, 691.-692,692{ Yeastmating type(sl, 7, 7f alu, 169,170f in asymmetriccell division,930-93L, 9311 cell-typespecificationin, 922-923, 9221,923f 299-300, Yeastmating type silencersequences, 300f YeastSR proteins,343-344,344f Yeasttwo-hybrid systems,310, 311f YeastTy elements,232,232f YXXO sortingsignal,589, 589t,609-610

Z d\sk, 7 39, 739f, 7 40, 741.f desminand, 794 z DNA, 115, 115f Zebrafish as experimentalorganisms,26 transportproteinsin, 469, 469f Zellweggersyndrome,568 Zig-zagribbon, in chromatin,249,249f Zinc-I\nger motif, 69-7 0, 70I, 29 1',29 1'f. See a/soNuclearreceptor(s) 95 5-957,9 56f Zona pellucida, Zone of polarizingactivity,991, 99lf in fertilization,956f, 957 ZP glycoproteins, ZwtllelPinhead,920 Zwitterions, 52 Zygotes,S, 854, 950 of, 950, 960-96L,960f cleavage as stemcells,907 Zyklon B, 498499 Zymogens,9L