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As described earlier, these different structures are initiated by the action of distinct nucleating proteins: thelong straight filaments produced by formins make bundles and the ARp complex makes webs. The actin filament cross-linking proteins that help to stabilizeand maintain these distinct structures are divided into tvvo classes:bundlingproteins and gel-forming proteins.
Bundling proteins cross-link actin filamentsinto a parallel array, while gel-forming proteins hold two actin filamentstogether at a large angle to each other, thereby creating a looser meshwork. BothEach type of bundling protein also determines which other molecules caninteract with an actin filament. Myosin II (discussedlater) is the motor proteinin stressfibers and other contractile arrays that enables them to contract. Thec o n t r a c t i l eb u n d l eg e l - l i k en e t w o r k1 0 0n mFigure 16-47 Actin arraysin a cell.A fibroblastcrawlingin a tissueculturedishis shownwith threeareasenlargedto showthe arrangementof actinfilaments.Theactin fifamentsare shown in red,withpointingtowardthe minusarrowheadsend.
Stressfibersare contractileand exerttension.Filopodiaarespike-likeprojectionsof the plasmamembranethat allowa cellto exploreits environment.The cortexunderliesthe plasmamembrane.1007THEIRCYTOSKELETALFILAMENTSHOWCELLSREGULATEspectrin(tetramer)fimbrin(monomer)0-actinin(dimer)5U nmvery closepacking of actin filaments causedby the small monomeric bundlingprotein fimbrin apparentlyexcludesmyosin, and thus the parallel actin filamentsheld togetherby fimbrin arenot contractile;on the otherhand,the looserpacking causedby the larger dimeric bundling protein a-actinin allows myosinmoleculesto enter,making stressfiberscontractile(Figuref6-49). Becauseofthe very different spacing between the actin filaments, bundling by fimbrinautomaticallydiscouragesbundling by o-actinin, and vice-versa,so that the twotypes of bundling protein are themselvesmutually exclusive.Villinis anotherbundling protein that, like fimbrin, has two actin-filamentbinding sites very closetogether in a single pollpeptide chain.Villin (togetherwith fimbrin) helps cross-linkthe 20 to 30 tightly bundled actin filaments foundin microvilli, the finger-like extensionsof the plasmamembraneon the surfaceof many epithelial cells (Figure l$-50).
A singleabsorptiveepithelial cell in thehuman small intestine,for example,has severalthousandmicrovilli on its apicalsurface.Eachis about 0.08pm wide and I pm long, making the cell'sabsorptivesurfacearea about 20 times greaterthan it would be without microvilli. \'Vhenvillin is introduced into cultured fibroblasts,which do not normally containvillin and haveonly a few small microvilli, the existingmicrovilli becomegreatlyelongatedand stabilized,and new onesareinduced.The actin filamentcoreofthe microvillus is attachedto the plasma membrane along its sidesby lateralsidearmsmade of myosinI (discussedlater),which has a binding site for filamentous actin on one end and a domain that binds lipids on the other end'one binding actin filamentsto eachother andThesetwo typesof cross-linkers,the other binding these filaments to the membrane, seem to be sufficient toform microvilli on cells.Interestingly,when the genefor villin is disrupted in amouse,the intestinal microvilli form with apparentlynormal morphology,indicating that other bundling proteins provide sufficient redundant function forthis purpose.However,the remodelingof intestinal microvilli in responseto certain kinds of stressor starvationis impaired.Figurel6-48 The modular structuresoffour actin-cross-linkingproteins.Eachofthe proteinsshown hastwo actinbinding sites(red)that are relatedinFimbrinhastwo directlysequence.sites,so that itadjacentactin-bindingholds its two actin filamentsvery closetogether(14nm apart),alignedwith thesamepolarity(seeFigure16-494).Thetwo actin-bindingsitesin cr-actininareseparatedby a spaceraround 30 nmlong,so that it forms more looselYpackedactinbundles(seeFigure16-49A).Filaminhastwo actin-bindingsiteswith a V-shapedlinkagebetweenactinfilamentsthem,so that it cross-linksinto a networkwith the filamentsorientedalmostat rightanglesto oneanother(seeFigure16-51).Spectrinis atetramerof two o and two 0 subunits,and the tetramerhastwo actin-bindingsitesspacedabout 200-nmapart (seeF i g u r e1 0 - 4 1 ) .Figure16-49 The formation of twotypes of actin filament bundles.(A)cr-actinin,whichis a homodimetactin filamentsinto loosecross-linksbundles,whichallowthe motor proteinmyosinll (not shown)to participateinactinFimbrincross-linksthe assembly.filamentsinto tight bundles,whichexcludemyosin.Fimbrinand s-actinintend to excludeone anotherbecauseofthe very differentspacingof the actinfilamentbundlesthat theYform.(B)Electronmicrographof purifiedc[-actininmolecules.(8,courtesyofJohnHeuser.)a c t i nf i l a m e n t sa n dfimbrina c t i nf i l a m e n t sa n dd-actinin50 nmcontractilebundlel o o s ep a c k i n ga l l o w sm y o s i n - l l(A)to enterbundlep a r a l l e lb u n d l et i g h t p a c k i n gp r e v e n t sm y o s i n - l lf r o m e n t e r i n gb u n d l e(B)1 0 0n m1008Chapter16:The Cytoskeletonamorpnous,denselys t a i n i n gr e g i o np l u se n d o fa c t i nf i l a m e n tmicrovillusacItnf ilamentbundlel a t e r a ls i d e a r m(myosin-1,calmodulin)(A)(B)(c)||1rtFigure16-50A microvillus.(A)A bundleof parallelactinfilamentscross-linkedby the actin-bundlingproteinsvillinandfimbrinformsthe coreof a microvillus.Lateralsidearms(composedof myosinI and the Ca2+-bindingproteincalmodulin)connectthe sidesof the actinfilamentbundleto the overlyingplasmamembrane.All the plusendsof the actinfilamentsareat the tip of the microvillus,wherethey areembeddedin an amorphous,denselystainingsubstanceof unKnown(B)Freeze-fracturecomposition.electronmicrographof the apicalsurfaceof an intestinalepithelialcell,showingmicrovilli.Actinbundlesfrom the microvilliextenddown into the celland arerootedin the terminalweb,wherethey are linkedtogetherby a complexsetof proteinsthat includesspectrinand myosinll.
Belowthe terminalweb is a layerofintermediate(C)Thinsectionelectronmicrographof microvilli.filaments.(8,courtesyof JohnHeuser;C,fromP.T.Matsudairaand D.R.Burgess,ColdSpringHarb.Symp.Quont.Biol.46:845-854,1985.With permissionfrom Cold SpringHarborLaboratoryPress.)Filaminand SpectrinFormActinFilamentWebsThe various bundling proteins that we have discussed so far have straight, stiffconnections between their two actin-filament-binding domains, and they tendto align filaments in parallel bundles. In contrast, those actin cross-linking proteins that have either a flexible or a stiff, bent connection between their twobinding domains form actin filament webs or gels,rather than actin bundles.projections called lamellipodiathathelp them to crawl acrosssolid surfaces.Fil-A very different well-studied web-forming protein i s spectrin,which was firstidentified in red blood cells. spectrin is a long, flexible protein made out of fourelongated pollpeptide chains (two cr subunits and two subunits), arranged soBthat the two actin-filament-binding sites are about 200 nm apart (comparedr.lr110 I(8)qlm '/66t 'Lsq-Svg:gtleql uorsstutad(ssar6{lrsranrunlolloJal)ou'Il!lllor.rl'f'to!B '1'pue{6o;oqdtoulle)uo;e1ar-ueq6uluunl tuorl)ilDaqlleqll)a;Jalo a)uasaJdeJoa)uasqea;6ursaneque)uralordle.rnl>nr1s/(lq6lLls!Lll')llelselauIueu jo ouosla;druexepuno;ordaql 6urlPllsuotuepuaaqI;1etlt;lltealPtuMousllafaql'palolsalarepueerpodr;;aue1leiuJouaulesaLll(8)'azlselselaulq)tqMutslle)euouPlaulsequorssaldxaurutplt,auelquaulo] ]ou pualpue{uood;nnetlr{aq}'}lnsore sv ,;sqalq,,pueerpodt;;euelqllMpora^o)olepealsulleulroualeulol alqelouuea^eqleq}slla)eurouelaurjo la^alMol{;;eutouqeareslla)asaql'ulJrelU'/ttltrousasne)ulurellr1ossol75-91ern6r3lla)lerulouqe;o dnol6V (V)'uollseJelul JulnlalorueJlul uP ^qaq uec uorleturoJuoc e^rlcE eq] o] SulqJlr1v\S'uollEluJoJuoc peploJ a^Il3eulJeqlaSol plaq eJe IuIIuJel-3 puB -N eql qllq^\ uIue pup 'utalord aueJqueusuBJl E puB uIIJP qloq ol spulq pue sazIJaIuoBIIo'suollgluJoJuoc 06l ul ls1xe ugl suleloldlpql uoqeuloJuoc pepuelxa e1llce uE'slEuBISrBlnlleJPJlxapuB mlnllacPrlul qtoq,(q pelup8ar erc sulelord ntrUgINUS,r(qpalerparu eupJqueu Eruseld eqt pue ullse uaamlaq slueuqcune eql'uEuoJnlEAq'v?cll su qJns 'sulal]uauoduos xrJlBur JelnllaJEJUa aql JoJ Joldacal eql-oJdoJ^18aueJqrueursueJ]aJotu Jo auo Jo af,EJJlluseldoldl aql ot sputq uletuop'sluaruBllJullJeJo sapls eq] o] fllJarlp spulq ulelord I trug ueIPurrurel-N eqJ.srsol{Jopue pue srsol-{3oxeur pe^Io^ur pue IlrJelodJo urEruop IpuruJa]-J aqJ'(ulseoulpupII3OJo eJupualureru aql JoJ pennbeJ eJe teql sJaqlueu Suleluos 'sulaloJd'urrnpeJ 'uuza 'srequtaruy'1gg aqlJelaaJqllsrlJ sll JoJ pelueu) tt11't;l-1-nlleJeJtul patelar.{1aso1cJo t{llrueJpeardsaprluV'poolsrapun dlprued lluo areeupJqueur eruseld aql ol uolela>1so$oullJe Ieoluoc eql Jo suollJeuuoo aql'auurqueu EursBIdaql;o sprdll Jo sulelord puE {Joo\-lau luauelrJ urlJg aq] ueegleq Slue111llJelleJIJIcedSSaJInbaJsaJnlsnJls esaqlJo sseue^IlcaJJaaql'sllaf, lullaqllda;o eare aceJJnse^IldJosqe eql eBJEIualeqlrllr^oJcrruuI selpunq uI]3E-uIIIF eql pue seueJqrueu eruseld aql sallJepun lEqlqe^a uIIJE-uIJlJads aqt :selduExa o^i\l passncsrp,{pearp e^eq eM'eueJqrxelueruseld aqt Jo adpqs eql a8ueqJ pue ueJJIlsqloq sernpnrls Ielale{sol,{c ullsvsauerqulewol sluauq)ellv {uery elew slueuall lelelalso}/()'euerquau aJeJrns aql ueJJIlspue adBr{so1 dlaq oqe -,(aqlalaq^r'sadd111acelPJqaua^raqlo lsoluJo xauoJ eq}'rfuepder e q8norql Sutzaanbsrage adeqsur punoJ aJeurrlceds Jo sellt€lal aso13'auuJqtualu eurseld 3utd1purSrro slr ol {JEq Suuds o} IIaJ poolq pal aql Surruolp-JeAoeq] ro; 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