复旦细胞生物学课件09细胞骨架系统

CytoskeletonSystemA.ConceptionofCytoskeleton(Narrowsense)Acomplexnetworkofinterconnectedmicrofilaments,microtubulesandintermediatefilamentsthatextendsthroughoutthecytosol.Chapter9MicrobubulesMicrofilamemtsIntermediatefilaments1.IntroductionB.TechniquesforstudyingthecytoskeletonFluorescentmicroscopyandElectronmicroscopy:Immunofluorescence:fluorescently-labeledantibodyFluorescence:microinjectintolivingcellsVideomicroscopy:invitromotilityassaysElectron:TritonX-100,MetalreplicaQuickfreezing-deepetchingEMBiochemicalanalysis(invitro)Differencecentrifugation;SDS-PAGEDrugsandmutations(aboutfunctions)C.Theself-assemblyanddynamicstructureofcytoskeletalfilamentsEachtypeofcytoskeletalfilamentisconstructedfromsmallerproteinsubunits.Thecytoskeletonisanetworkofthreefilamentousstructures.Thecytoskeletonisadynamicstrucrurewithmanyroles.2.Microfilament,MFA.MFsaremadeofactinandinvolvedincellmotility.UsingATP,G-actinpolymerizestoformMF(F-actin)B.MFassemblyanddisassemblyCharacteristics:(1)WithinaMF,alltheactinmonomersareorientedinthesamedirection,soMFhasapolarityMyosinismolecularmotorforactins.(2)Invitro,(Polymerization)bothendsoftheMFgrow,buttheplusendfasterthantheminus.Becauseactinmonomerstendtoaddtoafilament’splusendandleavefromitsminusend----(3)DynamicequilibriumbetweentheG-actinandpolymericforms,whichisregulatedbyATPhydrolysisandG-actinconcentration.2.2Assembly◆Mechanismofactinpolymerization:3phasesofG-actinpolymerization.◆Criticalconcentration(Cc).Insteadystate,G-actinmonomersonlyexchangewithsubunitsatthefilamentendsbutthereisnonetchangeinthetotalmassoffilaments.◆Duringtheelongationstate,oneendofthefilament,the(+)end,elongatesfivetotentimesfasterthandoestheopposite(-)end.ThisisbecauseCcvalueismuchlowerforG-actinadditionatthe(+)endthanforadditionatthe(-)end.Figure6-17ThethreephasesofG-actinpolymerizationinvitro.(4)Dynamicequilibriumisrequiredforthecellfunctions.SomeMFsaretemporaryandotherspermanent.(5)ThenucleationofactinfilamentsatthePMisfrequentlyregulatedbyexternalsignals,allowingthecelltochangeitsshapeandstiffnessrapidlyinresponsetochangesinitsexternalenvironment.Thisnucleationiscatalyzedbyacomplexofproteinsthatincludestwoactin-relatedproteins,orARPs(Arp2andArp3).Actinarraysinacell.C.SpecificdrugsaffectpolymerdynamicsCytochalasins:PreventtheadditionofnewmonomerstoexistingMFs,whicheventuallydepolymerize.Phalloidin:Acyclicpeptidefromthedeathcapfungus,blocksthedepolymerizationofMFThosedrugsdisruptthemonomer-polymerequilibrium,soarepoisonoustocellsD.Actin-bindingproteinsThestructuresandfunctionsofcytoskeletonaremainlycontrolledbyitsbindingproteins2.4microfilament-bindingproteinsActinbindingproteinscontrolthestructureandbehaviorofactinfilament.◆actinbindingproteinse.g.proflin(promoteactingassembly),thymosinbeta4(inhibitsactinassembly).Somecytosolicproteinscontrolactinpolymerization.◆microfilament-bindingproteins◆3differenttypesofstalbeactinfilamentstructures：◆Parallelbundle:MFsisotacticparallelarrange，mainlyfoundinmicrovillusandfilopodium(丝状伪足).◆Contractilebundle:MFsanti-parallelarrange,mainlyfoundinstressfibers(应力纤维)andcontractiveringofmitosis(有丝分裂收缩环)。◆Gel-likenetwork:MFscross-linkedarrange,mostbefoundincellcortex(cytosol,细胞皮层).(2)MF-bindingproteins成核蛋白（nucleatingproteins）:actin-relatedproteins,ARPs单体–隔离蛋白（monomer-sequesteringprotein）:thymosin封端（加帽）蛋白（End-blocking(capping)proteins）:capZ单体–聚合蛋白（monomer-polymerizingproteins）:抑制蛋白（profilin）是一种与ATP-肌动蛋白单体结合的蛋白质肌动蛋白纤维解聚蛋白（actinfilament-depolymerizingproteins）:cofilin、ADF以及蚕食蛋白与肌动蛋白纤维的减端结合，大大促进肌动蛋白纤维解聚成单体。交联蛋白（cross-linkingproteins）:ABP280和细丝蛋白,促进形成近于正交相互联系的纤维松散网络纤维–切割蛋白（filament-severingproteins）:gelsolin膜结合蛋白（membrane-bindingproteins）:连接膜与肌动蛋白的蛋白质包括联结蛋白（vinculin），ERM家族的成员包括埃兹蛋白（ezrin）、根蛋白（radixin）和膜突蛋白（moesin）Modelofthecomplementaryrolesofprofilinandthymosinβ4inregulatingpolymerizationofG-actin.Actinfilamentsarelikewisestronglyaffectedbythebindingofaccessoryproteinsalongtheirsides.Actinfilamentsinmostcellsarestabilizedbythebindingoftropomyosin,anelongatedprotein.Whichcanpreventthefilamentfrominteractingwithotherproteins.Anotherimportantactinfilamentbindingprotein,cofilin,presentinalleucaryoticcells,whichdestabilizedactinfilaments(alsocalledactindepolymerizingfactor).Cofilinbindsalongthelengthoftheactinfilament,forcingthefilamenttotwistalittlemoretightly.Inaddition,cofilinbindingcausealargeincreaseintherateofactinfilamenttreadmilling.Themodularstructuresoffouractin-cross-linkingproteinsTheformationoftwotypesofactinfilamentbundles:Contractilebundlemediatedbyα-actininparallelbundlemediatedbyfimbrin.Gel-likenetworkActinfilamentsareoftennucleatedattheplasmamembrane.Thehighestdensityofactinfilamentsisatthecellperipheryformingcellcortex.Filamincross-linksactinfilamentsintoathree-dimensionalnetworkwiththephysicalpropertiesofagel.LossoffilamincausesabnormalcellmotilityE.FunctionsofMFs(1)Maintaincell’sshapeandenforcePM(2)Cellmigration(Fibroblastetal)Plateletactivationisacontrolledsequenceofactinfilamentsevering,uncapping,elongation,recapping,andcross-linking.(3)Microvi