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Hall,Science279:509-514,1998.Withpermissionfrom AAAS.)1043THECYTOSKELETONAND CELLBEHAVIORSome key targets of activated Cdc42 are members of the WASp protein family. Human patients deficient in WASp suffer fromWiskott-Aldrich Syndrome, asevere form of immunodeficiency where immune system cells have abnormalactin-based motility and platelets do not form normally.
AlthoughWASp itself isexpressedonly in blood cells and immune system cells, other family membersare expressed ubiquitously that enable activated Cdc42 to enhance actin polymerization. WASp proteins can exist in an inactive folded conformation and anactivated open conformation. Association with Cdc42-GTP stabilizes the openform of WASp, enabling it to bind to the ARP complex and strongly enhancingthis complex's actin-nucleating activity (seeFigure 16-34).
In this way, activationof Cdc42 increasesactin nucleation.Rac-GTP also activates WASp family members, as well as activating thecrosslinking activity of the gel-forming protein filamin, and inhibiting the contractile activity of the motor protein myosin II, stabilizing the lamellipodia andinhibiting the formation of contractile stressfibers (Figure l6-98A).Rho-GTP has a very different set of targets. Instead of activating the ARPcomplex to build actin networks, Rho-GTP turns on formin proteins to constructparallel actin bundles. At the same time, Rho-GTP activatesa protein kinase thatindirectly inhibits the activity of cofilin, leading to actin filament stabilization.The same protein kinase inhibits a phosphatase acting on myosin light chains(seeFigure 16-72). The consequent increase in the net amount of myosin lightchain phosphorylation increasesthe amount of contractile myosin motor protein activity in the cell, enhancing the formation of tension-dependent structures such as stressfibers (Figure 16-988).In some cell types, Rac-GTPactivatesRho, usuallywith kinetics that are slowcompared to Rac'sactivation of the ARP complex.
This enables cells to use theRac pathway to build a new actin structure while subsequently activating theRho pathway to induce a contractility that builds up tension in this structure.This occurs, for example, during the formation and maturation of cell-cell contacts. As we will explore in more detail below the communication between theRac and Rho pathways also facilitates maintenance of the large-scaledifferencesbetween the cell front and the cell rear during migration.ExtracellularSignalsCanActivatethe ThreeRhoProteinFamilyMembersThe activation of the monomeric GTPasesRho, Rac, and Cdc42 occurs throughan exchange of GTP for a tightly bound GDP molecule, catalyzed by guaninenucleotide exchangefactors (GEFs).Of the 85 GEFsthat have been identified in<_.:J-Krnase/t'/t\wASpfamilyPl(4)P5-kinase,/\/\pAK_llltl++ARPFilaminPl(4,5)P2(web(branching|Jnucleator) crosslinker)'"".??:?: I---i::forminsR h ok i n a s eMLCKIbranchedactin webin lamellipodia(decreased)myosinactivityIlessstressfiberformationcofilin (increased)myosinactivity(A)actinbundlegrowth-vmore stressfibers?i n t e g r i nc l u s t e r i n ga n dfocal adhesionformation(B)Figure 16-98 The contrastingeffectsofRacand Rho activation on actinorganization.(A)Activationof the smallRacleadsto actinnucleationbyGTPaseinthe ARPcomplexand otheralterationsactin accessoryproteinsthat tend tofavorthe formationof actin networks,asdifferentSeveralin lamellipodia.pathwayscontributeindependently.activatesmembersof the WASpRac-GTPproteinfamily,which in turn activateactinnucleationand branchedwebformationby the ARPcomPlex.In aactivatesaparallelpathway,Rac-GTPprotein kinase,PAK,which hasseveraltargetsincludingthe web-formingfilamin,which is activatedbycrosslinkerphosphorylation,and the myosinlightwhich is inhibitedchainkinase(MLCK),The resultingby phosphorylation.ofthein phosphorylationdecreasemyosinregulatorylightchainleadstoand amyosinll filamentdisassemblydecreasein contractileactivity.In somecells,PAKalsodirectlyinhibitsmyosinllof the myosinactivityby phosphorylationheavychain(MHC).Anothersetofpathwaysdownstreamof Racactivationlipidis mediatedby phosphoinositideLocalcreationof PlPz[Pl(4,5)Pz]signals.may help to reducethe activitYofcappingprotein,to furtheraid actinpolymerization.Activationof Pl 3-kinase,leadstoPlP3from PlP2,whichgeneratesfurther activationof Racitselfvia apositivefeedbackloop.(B)Activationof the relatedGTPaseRholeadsto nucleationof actinfilamentsbycontractionbyforminsand increasesmyosinll, promotingthe formationofactinbundlessuchasstresscontractilefibers.Activationof myosinll by Rhoproteinkinaserequiresa Rho-dependentcalledRock.Thiskinaseinhibitsthephosphatasethat removesthe activatingphosphategroupsfrom myosinll lightit mayalsodirectlYchains(MLC);phosphorylatethe myosinlight chainsinsomecelltypes.Rockalsoactivatesotherwhichsuchas LIMkinase,oroteinkinases,to the formationofin turn contributesactinfilamentbundlesstablecontractileby inhibitingthe actindepolymerizingfactorcofilin.A similarsignalingpathwayis importantfor formingthe contractilefor cytokinesis(seering necessaryFigure'17-52).1044Chapter 16:The Cytoskeletonthe human genome, some are specific for an individual Rho family GTpase,whereas others seem to act on all three family members.
The number of GEFsexceeds the number of Rho GTPasesthat they regulate because different GEFsare restricted to specific tissues and even specific subcellular locations, and theyare sensitive to distinct kinds of regulatory inputs. Various cell-surface receptorsactivate GEFs.An example is the Eph receptor tyrosine kinase involved in neurite growth cone guidance, which is discussed in detail in chapter 15.
Interestingly, several of the Rho family GEFs associatewith the growing ends of microtubules by binding to one of the +TIPs.This provides a connection between thedlnamics of the microtubule cytoskeleton and the large-scale organization ofthe actin cltoskeleton, which is important for the overall integration of cellshape and movement.The Rho family GTPases are also primary determinants of cell polarity inbudding yeast, where extensive genetic analyses have increased our understanding of the general mechanisms involved. on starvation, yeasts,like manyother unicellular organisms, sporulate.
But sporulation can occur only in diploidbudding yeast cells, whereas budding yeasts mainly proliferate as haploid cells.A starving haploid individual must therefore locate a partner of the oppositemating type, woo it, and mate with it before sporulating. yeast cells are unable toswim and, instead, reach their mates by polarized growth. The haploid form ofsignal molecule, which under normal circumstances would direct it toward anamorous a cell located nearby.This polarized cell growth requires alignment of the actin cytoskeleton inresponse to the mating factor signal. \.A/henthe signal binds to its receptol thereceptor activates cdc42, which in turn induces assembly of actin fllaments atthe location closestto the source of the signal.Local activation of Cdc42 is furtherenhanced by a positive feedback loop, requiring actin-dependent transport ofcdc42 itself as well as its GEF and other signaling components along the newlyassembled actin structures toward the site of the signal.
subsequently, actincables are assembled pointing toward the site of cdc42 accumulation due to theupstream and dor,rmstream pathways have been identified through geneticFigure 16-99 Morphologicalpolarizationof yeast cellsin responseto matingfactor. (A) Cellsof Soccharomycescerevisiaeare usuallyspherical.(B)Theybecomepolarizedwhen treatedwithmatingfactor from cellsof the oppositematingtype.The polarizedcellsarecalled"shmoos."(C)Al Capp'sfamouscartooncharacter,the originalShmoo.(A and B,courtesyof MichaelSnyder;C, @ 1948CappEnterprises,Inc.Usedby permission.)'(umoqslou) 6ulrpruloj lla) proldeqaqt 6uuedard'(Straldeq) ur pessn)stp)ape)spraseurldV4 e q6norqlsesuodsarraqlo sra66r.l1uorlenrl:eroldata.r'uolrlppeu1'edeqsooLuqse uorlrslnbte]opue'qunor6pezuelod'uolleulloJalqe) url)e o1{;;eur;pue'uotlent})euruiloj puPoqu o^tsualxaol spPalsil.ll'po)uequaraqun, sr,tlnlr>e zt>p){qalaqm dool lreqpaa; anrlrsode aleal)qv"ror6luaue;U pue uorleal)nuurl)e;ero;eql'6urpurqrol)e16ulteu Jo allsaql le uolleal)nuurl)e le)olot 6urpeal'xelduordUVaql sale^tl)e1eq1uralotd,(1;ue1d5yg e sete^tl)eZt)p)'slla)uerleuureuJur sV'Zt)pl'utalold 6u;pulq-619,(1;ueJoqu aql sate^ll)eurnl ulslr.ll'(Stroldeqf ur passnf,stp)uralo.rd-g)rJauJuloleleqe uoi] ]tunqnsDgpunoq-dfg aql Jo uotle!tossrps.re66u1roloa)er aq] jo uolle^lt)v'auerquiauleurse;deqt ur loldatel pa;dnor-urelord-ge ol spulq rolre; 6ug1eurelnlla)eltxaaq1'asuodsarto1rel Eulleu lsea{ aqlu; Iernqled 6ur;eu6rsaq1 96 1-9 1 arn6r3'lg-sLiLf prl slorlsllqnduellrur)en urorJuorssrurad'lllM'666 ['tDN'(t-9 ernbrl osleaas)]! 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