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Moreover, when anchored in amore complex way to the membrane, the same t)?e of force drives plasmamembrane invaginations, as it does during the endocytotic and phagocl'toticprocessesdiscussedin Chapter 13.It is interesting to compare the organization of the actin-rich lamellipodiumto the organization of the microtubule-rich mitotic spindle.
In both cases,thecell harnessesand amplifies the intrinsic dynamic behavior of the cltoskeletalfilament systems to generate large-scalestructures that determine the behaviorof the whole cell. Both structures feature rapid turnover of their constituentcytoskeletal filaments, even though the structures themselves may remain intactat steady state for long periods of time. The leading edge plasma membrane inthe lamellipodium fulfills an organizational role analogous to the condensedchromosomes in organizing and stimulating the dynamics of the mitotic spindle.
In both cases,molecular motor proteins help to enhance cltoskeletal filament flux and turnover in the large-scale arrays.Figure 16-91 Contribution of myosin ll to polarizedcell motility'(A)Myosinll bipolarfilamentsbind to actinfilamentsin the dendriticThe myosin-drivenmeshworkand causenetworkcontraction.lamellipodialreorientationof the actinfilamentsin the dendriticmeshworkformsanto generatingtheactinbundlethat recruitsmoremyosinll and contributesforcesrequiredfor retractionof the trailingedgeof the movingcontractilecan beof a keratocytecell.(B)A fragmentof the largelamellipodiumorfrom the maincellbody eitherby surgerywith a micropipetteseparatedby treatingthe cellwith certaindrugs.Manyof thesefragmentscontinueastheorganizationto moverapidly,with the sameoverallcytoskeletalintact keratocytes.Actin (btue)formsa protrusivemeshworkat the front ofthe fragment.Myosinll (pink)is gatheredinto a band at the rear'(FromA.
Verkovskyet al.,Curr.Biol.9:11-20,1999.With permissionfrom Elsevier,)Figure16-90 A model for protrusion ofthe actin meshworkat the leadingedge,Two time pointsduringadvanceof thewith newlyareillustrated,lamellipodiumat the latertimestructuresassembledpoint shownin a lightercolor.Nucleationis mediatedby the ARPcomplexat thefront.Newlynucleatedactinfilamentsareattachedto the sidesof preexistingfilaments,primarilyat a 70'angle.elongate,pushingthe plasmaFilamentsmembraneforward becauseof somesortof anchorageof the arraybehind.At asteadyrate,actinfilamentplusendsbecomecapped.After newly polymerizedactinsubunitshydrolyzetheir boundATPin the filamentlattice,the filamentsto depolymerizationbecomesusceptibleby cofilin.Thiscyclecausesa spatialseparationbetween net filamentat the front and net filamentassemblyat the reat so that the actindisassemblyfilamentnetworkas a wholecan moveforward,eventhoughthe individualwithfilamentswithin it remainstationaryresoectto the substratum.1040Chapter16:TheCytoskeletonHrc-cH'-cg,/\HrC-CHCHOHHzcCHilCHHCHOleading edge of cell(c)Hzc20pmauCellAdhesionandTractionAllowCellsto PullThemselvesForwardLamellipodia of all cells seem to share a basic, simple type of dynamic organization where actin filament assembly occurs preferentially at the leading edge andactin filament disassembly occurs preferentially at the rear.
However, the interactions between the cell and its normal physical environment usually make thesituation considerably more complex than for fish keratocFes crawling on a culture dish. Particularly important in locomotion is the intimate crosstalkbetweentightly to move forward. In these lamellipodia, the same cycle of localized nucleation of new actin filaments, depolymerization of old filaments, and myosindependent contraction continues to operate.
But because the leading edge isprevented physically from moving forward, the entire actin mesh moves backward toward the cell body instead, pulled by myosins (Figure 16-92). The adhesion of most cells lies somewhere between these two extremes,and most lamellipodia exhibit some combination of forward actin filament protrusion (like keratocJ,tes)and rearward actin flux (like Ihe Aplysia neurons).As a lamellipodium, filopodium, or pseudopodium extends forward over asubstratum, it can form new attachment sites at the cell front that remain stationary as the cell moves forward over them, persisting until the rear of the cellcatches up with them.
\.A/henan individual lamellipodium fails to adhere to the(D)cytochalasinBFigure 16-92 Rearwardmovement ofthe actin network in a growth-conelamellipodium.(A)A growthconefrom aneuronof the seaslugAplysiaisculturedon a highlyadhesivesubstratumandviewed by differential-interferencecontrastmicroscopy.Microtubulesandmembrane-enclosedorganellesareconfinedto the bright,rearareaofthegrowth cone (to the /eft),while ameshworkof actinfilamentsfillsthe(onthe right).(B)Afterlamellipodiumbrieftreatmentwith the drugcytochalasin,whichcapsthe plusendsofactinfilaments(seeTable16-2,p. 988),the actinmeshworkhasdetachedfromthe front edgeof the lamellipodiumand(C)At thehasbeenpulledbackward.time point shownin B,the cellwasfixedphalloidinand labeledwith fluorescentto showthe distributionof the actinfilaments.Someactinfilamentspersistatthe leadingedge,but the regionbehindthe leadingedgeis devoidof filaments.Notethe sharpboundaryof therearward-movingactinmeshwork.(D)ThecomplexcyclicstructureofcytochalasinB.(A-C,courtesyof PaulForscher.)Figure16-93 Lamellipodiaand rufflesatthe leadingedge of a human fibroblastmigratingin culture.Thearow in thisscanningelectronmicrographshowsthedirectionof cell movement.As the cellmovesforward,lamellipodiathat failtoattachto the substratumaresweotbackwardoverthe dorsalsurfaceof thecell,a movementknownas rufflinq.(Courtesyof JulianHeath.)1041THECYTOSKELETONAND CELLBEHAVIORsubstratum, it is usually lifted up onto the dorsal surface of the cell and rapidlycarried backward as a "ruffle" (Figure 16-93).The attachment sites established at the leading edge serve as anchoragepoints, which allow the cell to generate traction on the substratum and pull itsbody forward.
Myosin motor proteins, especially myosin II, seem to generatetraction forces.In many locomoting cells, myosin II is highly concentrated at theposterior of the cell where it may help to push the cell body forward like toothpaste being squeezed out of a tube from the rear (Figure 16-94; see also Figure16-91). Dictyostelium amoebae that are deficient in myosin II are able to protrude pseudopodia at normal speeds,but the translocationof their cell body ismuch slower than that of wild-type amoebae, indicating the importance ofmyosin II contraction in this part of the cell locomotion cycle. In addition tohelping to push the cell body forward, contraction of the actin-rich cortex at therear of the cell may selectivelyweaken the older adhesive interactions that tendto hold the cell back.
Myosin II may also transport cell body components forward over a polarized array of actin filaments.The traction forces generated by locomoting cells exert a significant pull onthe substratum (Figure 16-95). In a living animal, most crawling cells moveacross a semiflexible substratum made of extracellular matrix, which can bedeformed and rearranged by these cell forces. In culture, movement of fibroblasts through a gel of collagen fibrils aligns the collagen, generating an organizedextracellular matrix that in turn affectsthe shape and direction of locomotion ofthe fibroblasts within it (Figure 16-96).
Conversely, mechanical tension orstretching applied externally to a cell will cause it to assemble stressfibers andfocal adhesions, and become more contractile. Although poorly understood,this two-way mechanical interaction between cells and their physical environment is thought to be a primary way that vertebrate tissuesorganize themselves.Membersof the Rho ProteinFamilyCauseMajor Rearrangementsof the Actin Cytoskeleton5[mFigure16-94The localizationof myosinland myosinll in a normalcrawlingDictyosteliumamoeba.Thiscell wascrawlingtowardthe upperright at thetime that it wasfixedand labeledwithantibodiesspecificfor two myosinisoforms.Myosin| (green)is mainlyto the leadingedgeofrestrictedpseudopodiaat the front of the cell.Myosinll (red)is highestin the posterior,of theactin-richcortex.Contractioncortexat the posteriorof the cellbymyosinll may helpto pushthe cellbodyof YoshioFukui.)forward.(CourtesyCell migration is one example of a process that requires long-distance communication and coordination between one end of a cell and the other.
Duringdirected migration, it is important that the front end of the cell remain structurally and functionally distinct from the back end. In addition to driving localmechanical processessuch as protrusion at the front and retraction at the rear,the cltoskeleton is responsible for coordinating cell shape, organization, andmechanical properties from one end of the cell to the other, a distance which istypically several tens of micrometers for animal cells. In many cases,includingbut not limited to cell migration, large-scalecyoskeletal coordination takes theform of the establishment of cell polarity, where a cell builds different structureswith distinct molecular components at the front vs.
the back, or at the top vs. the1 0 0$ mFigure16-95Adhesivecellsexerttractionforceson the substratum.Thesehavebeenculturedon a veryfibroblaststhin sheetof siliconrubber.Attachmentofthe cells,followedby contractionof theirhascausedthe rubbercytoskeleton,to wrinkle.(FromA.K'Harris,substratumP.Wild and D. Stopak,Science208:177-179'from AAAS.)1980.With permission1042Chapter16:TheCytoskeletonFigure16-96Shapingof theextracellularmatrixby cellpulling.Thismicrographshowsa regionbetweentwo piecesof embryonicchickheart(tissueexplantsrichin fibroblastsandheartmusclecells)thatweregrownin cultureon a collagengelfor4 days.A densetractof alignedcollagenfibershasformedbetweenthetwoexplants,apparentlyasa resultoffibroblasts(FromD.Stopaktuggingon thecollagen.andA.K.Harris,Dey.press.)Blol.90:383-398,1982.WithpermissionfromAcademicbottom.
Cell locomotion requires an initial polarization of the cell to set it off ina particular direction. Carefully controlled cell polarization processesare alsorequired for oriented cell divisions in tissues and for formation of a coherent,organized multicellular structure. Genetic studies in yeast,flies, and worms haveprovided most of our current understanding of the molecular basis of cell polarity. The mechanisms that generate cell polarity in vertebrates are only beginningto be explored. In all known cases,however, the c],toskeletonhas a central role,and many of the molecular components have been evolutionarily conserved.For the actin cytoskeleton, diverse cell-surface receptors trigger globalstructural rearrangementsin responseto external signals.But all of these signalsseem to converge inside the cell on a group of closely related monomericGTPasesthat are members of the Rho protein family-cdc42, Rac, andRho.
Thesame Rho family proteins are also involved in the establishment of many kindsof cell polarity.Like other members of the Rassuperfamily, these Rho proteins act as molecular switches to control cell processesby cycling between an active, GTp-boundstate and an inactive, GDP-bound state (seeFigure 3-71). Activation of cdc42 onthe plasma membrane triggers actin polymerization and bundling to formeither filopodia or shorter cell protrusions called microspikes. Activation of Racpromotes actin polyrnerization at the cell periphery leading to the formation ofsheet-like lamellipodial extensions and membrane ruffles, which are actin-richprotrusions on the cell'sdorsal surface (seeFigure l6-93).
Activation of Rho promotes both the bundling of actin filaments with myosin II filaments into stressfibers and the clustering of integrins and associatedproteins to form focal contacts (Figure 16-97). These dramatic and complex structural changes occurbecause each of these three molecular switches has numerous downsueam rarget proteins that affect actin organization and dynamics.actinstaininga c t i ns t a i n i n g( A ) Q U I E 5 C E NCTE L L S(B) Rho ACTIVATION(C) RacACTIVATION(D) Cdc42ACTIVATION20 pmFigure 16-97 The dramatic effectsofRac,Rho,and Cdc42on actinorganizationin fibroblasts.In eachcase,the actinfilamentshavebeenlabeledwith fluorescentphalloidin.(A)Serum-starvedfibroblastshaveactinfilamentsprimarilyin the cortex,andrelativelyfew stressfibers.(B)Microinjectionof a constitutivelyactivatedform of Rhocausesthe raoidassemblyof manyprominentstressfibers.(C)Microinjectionof aconstitutivelyactivatedform of Rac,acloselyrelatedmonomericGTPase,causesthe formationof an enormouslamellipodiumthat extendsfrom theentirecircumferenceof the cell.(D)Microinjectionof a constitutivelyactivatedform of Cdc42,anotherRhofamilymember,causesthe protrusionofmanylong filopodiaat the cellperiphery.The distinctglobal effectsof thesethreeGTPaseson the organizationof the actincytoskeletonare mediatedby the actionsof dozensof other protein moleculesthatare regulatedby the GTPases.Thesetargetproteinsincludesomeof thevariousactin-associatedproteinsthat wehavediscussedin this chapter.(FromA.
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