大三上課件-遺傳細(xì)胞運(yùn)動(dòng)遷徙

核基因編碼的線粒體蛋白質(zhì)需先在細(xì)胞質(zhì)中合成前體蛋白，前體蛋白由成熟形式的蛋白和N端的一段導(dǎo)肽序列共同組成，然后再轉(zhuǎn)移到線粒體內(nèi)，即先合成后轉(zhuǎn)移。rER合成的分泌蛋白質(zhì)是邊合成邊轉(zhuǎn)移，即共轉(zhuǎn)移。核基因編碼的蛋白質(zhì)向線粒體跨膜運(yùn)送與rER合成的分泌蛋白質(zhì)不同：約20-80個(gè)aa組成。富含帶正電荷的堿性氨基酸，特別是Arg。帶正電荷的氨基酸殘基有助于前導(dǎo)肽序列進(jìn)入帶負(fù)電荷的基質(zhì)中。含較多羥基氨基酸如Ser。幾乎不含帶負(fù)電荷的酸性氨基酸。導(dǎo)肽的結(jié)構(gòu)特征與作用導(dǎo)肽內(nèi)有識(shí)別線粒體的信息，而且導(dǎo)肽具有牽引蛋白質(zhì)通過(guò)線粒體膜進(jìn)行運(yùn)送的功能。導(dǎo)肽決定運(yùn)送方向，對(duì)被運(yùn)送的蛋白質(zhì)無(wú)特異性。Molecularchaperone細(xì)胞運(yùn)動(dòng)遷徙醫(yī)學(xué)遺傳系曹軒紡錘體微管微管組織中心應(yīng)力纖維微絲Cellshavetoorganizethemselvesinspaceandinteractmechanicallywiththeirenvironment.Cellshavetobeabletochangetheirshapeandmovefromplacetoplace.Cellshavetobeabletorearrangetheirinternalcomponentsastheygrow,divide,andadapttochangingcircumstances.Allthesespatialandmechanicalfunctionsaredevelopedtoaveryhighdegreeineucaryoticcells,wheretheydependonaremarkablesystemoffilamentscalledthecytoskeleton.TheCytoskeleton

細(xì)胞骨架Thecytoskeleton.AcellinculturehasbeenfixedandstainedwithCoomassieblue,ageneralstainforproteins.Threetypesofcytoskeletalfilamentsarecommontomanyeucaryoticcells:Intermediatefilaments（中間絲）providemechanicalstrengthandresistancetoshearstress.Microtubules（微管）determinethepositionsofmembrane-enclosedorganellesanddirectintracellulartransport.Actinfilaments（微絲）determinetheshapeofthecell'ssurfaceandarenecessaryforwhole-cellotion.MicrotubulesActinfilamentsIntermediatefilaments激光掃描共聚焦顯微鏡(laserscanningconfocalmicroscopy,LSCM)

ActinfilamentsMicrotubulesButthesecytoskeletalfilamentswouldbeineffectiveontheirown.Theirusefulnesstothecelldependsonalargenumberofaccessoryproteinsthatlinkthefilamentstoothercellcomponents,aswellastoeachother.Thissetofaccessoryproteinsisessentialforthecontrolledassemblyofthecytoskeletalfilamentsinparticularlocations,anditincludesthemotorproteinsthateithermoveorganellesalongthefilamentsormovethefilamentsthemselves.EachTypeofCytoskeletalFilamentIsConstructedfromSmallerProteinSubunitsCytoskeletalstructuresfrequentlyreachallthewayfromoneendofthecelltotheother,spanningtensorevenhundredsofmicrometers.Yettheindividualproteinmoleculesofthecytoskeletonaregenerallyonlyafewnanometersinsize.Thecellisabletobuildthelargestructuresbytherepetitiveassemblyoflargenumbersofthesmallsubunits,likebuildingaskyscraperoutofbricks.Becausethesesubunitsaresmall,theycandiffuserapidlywithincytoplasm,whereastheassembledfilamentscannot.Inthisway,cellscanundergorapidstructuralreorganizations,disassemblingfilamentsatonesiteandreassemblingthematanothersitefaraway.ManybiologicalpolymersincludingDNA,RNA,andproteinsareheldtogetherbycovalentlinkagesbetweentheirsubunits.Incontrast,thethreetypesofcytoskeletal"polymers"areheldtogetherbyweaknoncovalentinteractions,whichmeansthattheirassemblyanddisassemblycanoccur

rapidly,withoutcovalentbondsbeingformedorbroken.Thecytoskeletonandchangesincellshape.Theformationofproteinfilamentsfrommuchsmallerproteinsubunitsallowsregulatedfilamentassemblyanddisassemblytoreshapethecytoskeleton.Filamentformationfromasmallprotein.(B)Rapidreorganizationofthecytoskeletoninacellinresponsetoanexternalsignal.NucleationIstheRate-limitingStepintheFormationofaCytoskeletalPolymerForanewlargefilamenttoform,subunitsmustassembleintoaninitialaggregate,ornucleus,thatisstabilizedbymanysubunit-subunitcontactsandcanthenelongaterapidlybyadditionofmoresubunits.

Theinitialprocessofnucleusassemblyiscalledfilamentnucleation(成核作用),

anditcantakequitealongtime,dependingonhowmanysubunitsmustcometogethertoformthenucleus.Lagphase:nucleiareassemblingslowly.Elongation:subunitsaddquicklyontotheendsofthenucleatedfilaments.Steadystate:therateofadditionofnewsubunitstothefilamentendsisexactlybalancedbytherateofsubunitdissociationfromtheends.Theconcentrationoffreesubunitsleftinsolutionatthispointiscalledthecriticalconcentration,Cc(臨界濃度).Thelagphaseinfilamentgrowthiseliminatedifpreexistingnucleiareaddedtothesolutionatthebeginningofthepolymerizationreaction.FilamentsFormedfromMultipleProtofilamentsHaveAdvantageousPropertiesThestructureofamicrotubuleanditssubunit.(A)Thesubunitofeachprotofilamentisatubulinheterodimer,formedfromaverytightlylinkedpairofα-andβ-tubulinmonomers.TheGTPmoleculeintheα-tubulinmonomerissotightlyboundthatitcanbeconsideredanintegralpartoftheprotein.TheGTPmoleculeintheβ-tubulinmonomer,however,islesstightlyboundandhasanimportantroleinfilamentdynamics.Bothnucleotidesareshowninred.(B)Onetubulinsubunit(α-βheterodimer)andoneprotofilamentareshownschematically.Eachprotofilamentconsistsofmanyadjacentsubunitswiththesameorientation.(C)Themicrotubuleisastiffhollowtubeformedfrom13protofilamentsalignedinparallel.Thestructuresofanactinmonomerandactinfilament.TheactinmonomerhasaNucleotide(eitherATPorADP)boundinadeepcleftinthecenterofthemolecule.(B)Arrangementofmonomersinafilament.Althoughthefilamentisoftendescribedasasinglehelixofmonomers,itcanalsobethoughtofasconsistingoftwoprotofilaments,heldtogetherbylateralcontacts,whichwindaroundeachotherastwoparallelstrandsofahelix,withatwistrepeatingevery37nm.Allthesubunitswithinthefilamenthavethesameorientation.(C)Electronmicrographsofnegativelystainedactinfilaments.TheTwoEndsofaMicrotubuleandofanActinFilament

AreDistinctandGrowatDifferentRatesThemoredynamicofthetwoendsofafilament,wheregrowthisfast,iscalledtheplusend(正極),andtheotherendiscalledtheminusend（負(fù)極）.Onmicrotubules,αsubunitsareexposedattheminusend,andβsubunitsareexposedattheplusend.Onactinfilaments,theATP-bindingcleftonthemonomerpointstowardtheminusend.FilamentTreadmillingandDynamicInstabilityAreConsequencesofNucleotideHydrolysisbyTubulinandActinPlusendMinusendFilamentTreadmilling踏車行為Ataparticularintermediatesubunitconcentration,thefilamentgrowthattheplusendisexactlybalancedbythefilamentshrinkageattheminusend.TurnoverofFilamentInadditiontotheirabilitytoformnoncovalentpolymers,theactinandtubulinsubunitsarebothenzymesthatcancatalyzethehydrolysisofanucleosidetriphosphate,ATP

or

GTP,respectively.Forthefreesubunits,thishydrolysisproceedsveryslowly;however,itisacceleratedwhenthesubunitsareincorporatedintofilaments.Shortlyafterincorporationofanactinortubulinsubunitintoafilament,nucleotidehydrolysisoccurs;thefreephosphategroupisreleasedfromeachsubunit,butthenucleosidediphosphateremainstrappedinthefilamentstructure.TreadmillingandDynamicInstabilityRequireEnergybutAreUsefulAtfirstglance,thisdynamicbehavioroffilamentsseemslikeawasteofenergy.Cytoskeletalsystemsaredynamicandadaptable,organizedmorelikeanttrailsthaninterstatehighways.Itseemsthatthecelliscontinuallytestingawidevarietyofinternalstructuresandonlypreservingthosethatareuseful.Whenexternalconditionschange,orwheninternalsignalsarise,thecellispoisedtochangeitsstructurerapidly.ExceptionIncertainspecializedstructures,particularlyinvariousspecializedcellsinamulticellularorganism,partsofthecytoskeletonelessdynamic.Inaterminallydifferentiatedcellsuchasaneuron,forexample,itisdesirabletomaintainaconsistentstructureovertime,andmanyoftheactinfilamentsandmicrotubulesarestabilizedbyassociationwithotherproteins.TubulinandActinHaveBeenHighlyConservedDuringEucaryoticEvolutionTubulinmoleculesthemselvescomeinmultipleisoforms.Inmammals,thereareatleastsixformsofα-tubulinandasimilarnumberofformsofβ-tubulin,eachencodedbyadifferentgene.Thedifferentformsoftubulinareverysimilar.Yeastandhumantubulinsare75%identicalinaminoacidsequence.MostofthevariationamongdifferentisoformsoftubulinisfoundintheaminoacidsneartheC-terminus,whichformaridge脊onthesurfaceofthemicrotubule.Thus,variationsamongisoformsareexpectedtoaffectprimarilytheassociationofaccessoryproteinswiththesurfaceofthemicrotubule,ratherthanmicrotubulepolymerizationperse.Actinisfoundinalleucaryoticcells.Mostorganismshavemultiplegenesencodingactin;humanshavesix.Actinisextraordinarilywellconservedamongeucaryotes.Theaminoacidsequencesofactinsfromdifferentspeciesareusuallyabout90%identical.Invertebrates,therearethreesubtlydifferentisoformsofactin,termedα,β,andγ,thatdifferslightlyintheiraminoacidsequences.α-actin:inmusclecells,βandγ-actin:inalmostallnonmusclecells.YeastactinandDrosophilamuscleactinare89%

identical,yettheexpressionofyeastactininDrosophilaresultsinaflythatlooksnormalbutisunabletofly.Whatistheexplanationfortheunusuallystrictconservationofactinandtubulinineucaryoticevolution?Thestructureoftheentiresurfaceofanactinfilamentormicrotubuleisconstrainedbecausesomanyotherproteinsmustbeabletointeractwiththesetwoubiquitousandabundantcellcomponents.Amutationinactinthatcouldresultinadesirablechangeinitsinteractionwithoneotherproteinmightcauseundesirablechangesinitsinteractionswithanumberofotherproteins.Overtime,evolvingorganismshavefounditmoreprofitabletoleaveactinandtubulinalone,andaltertheirbindingpartnersinstead.Actinatthecrossroads.Actinbindstoaverylargevarietyofaccessoryproteinsinalleucaryoticcells.IntermediateFilamentAlleucaryoticcellscontainactinandtubulin.But,theintermediatefilament,isfoundinonlysomemetazoans（多細(xì)胞動(dòng)物）,includingvertebrates,nematodes（線蟲(chóng)）,andmolluscs（軟體動(dòng)物）.Evenintheseorganisms,intermediatefilamentsarenotrequiredineverycelltype.Intermediatefilamentsareparticularlyprominentinthecytoplasmofcellsthataresubjecttomechanicalstress,andtheirmajorfunctionseemstobetoimpartphysicalstrengthtocellsandtissues.雙股卷曲螺旋有極性反向交錯(cuò)并列

無(wú)極性-螺旋桿狀區(qū)：非螺旋區(qū)非螺旋區(qū)Thislargenumberofpolypeptidesalllineduptogether,withthestronglateralhydrophobicinteractions（側(cè)面疏水相互作用）typicalofcoiled-coilproteins,givesintermediatefilamentsarope-likecharacter.Theycanbeeasilybentbutareextremelydifficulttobreak.MajorTypesofIntermediateFilamentProteinsinVertebrateCells核纖層蛋白波形蛋白結(jié)蛋白膠質(zhì)絲酸性蛋白外周蛋白角蛋白神經(jīng)絲蛋白中胚層來(lái)源的細(xì)胞星形膠質(zhì)細(xì)胞Thediversityinkeratins（角蛋白）isclinicallyusefulinthediagnosisofepithelialcancers(carcinomas),astheparticularsetofkeratinsexpressedgivesanindicationoftheepithelialtissueinwhichthecanceroriginatedandthuscanhelptoguidethechoiceoftreatment.DrugsThatAffectActinFilamentsandMicrotubules鬼筆環(huán)肽細(xì)胞松弛素紫杉酚拉春庫(kù)林諾考達(dá)唑秋水仙堿長(zhǎng)春花堿Howmicrotubulesandactinfilamentsarenucleatedincells?MicrotubulesAreNucleatedbyaProteinComplex

Containingγ-tubulin.ActinFilamentsAreOftenNucleatedbytheARPComplexatthePlasmaMembrane.MicrotubulesAreNucleatedbyaProteinComplex

Containingγ-tubulinMicrotubulesaregenerallynucleatedfromaspecificintracellularlocationknownasamicrotubule-organizingcenter(MTOC).Microtubulesarenucleatedattheirminusend,withtheplusendgrowingoutwardfromeachMTOC.γ-tubulinisinvolvedinthenucleationofmicrotubulegrowthinorganismsrangingfromyeaststohumans.Aγ-tubulinringcomplex(γ-TuRC)hasbeenisolatedfrombothinsectandvertebratecellsandisanimpressivelyefficientnucleatorofmicrotubulegrowthinatesttube.Thecentrosome（中心體）.ThecentrosomeisthemajorMTOCofanimalcells.Locatedinthecytoplasmnexttothenucleus,itconsistsofanamorphousmatrix（不定形基質(zhì)）ofproteincontainingtheγ-tubulinringcomplexesthatnucleatemicrotubulegrowth.Thismatrixisorganizedbyapairofcentrioles（中心粒）.(B)Acentrosomewithattachedmicrotubules.Theminusendofeachmicrotubuleisembeddedinthecentrosome,havinggrownfromaγ-tubulinringcomplex,whereastheplusendofeachmicrotubuleisfreeinthecytoplasm.L-shapedconfigurationPolymerizationoftubulinnucleatedbyγ-tubulinringcomplexes.Modelforthenucleationofmicrotubulegrowthbytheγ-TuRC.Theredoutlineindicatesapairofproteinsboundtotwomoleculesofγ-tubulin.(B)Electronmicrographsofpurifiedγ-tubulinringcomplexes(top)andsinglemicrotubulesnucleatedfromthepurifiedγ-tubulinringcomplexes(middleandbottom.Thecenter-seekingbehaviorofacentrosome.Asinglecentrosomewasplacedintothewell,alongwithtubulinsubunitsinsolution.Asthemicrotubulespolymerize,nucleatedbythecentrosome,theypushagainstthewallsofthewell.黑色素細(xì)胞Amicrotubulearraycanfindthecenterofacell.Afterthearmofafishpigmentcelliscutoffwithaneedle,themicrotubulesinthedetachedcellfragmentreorganizesothattheirminusendsendupnearthecenterofthefragment,buriedinanewmicrotubule-organizingcenter.ActinFilamentsAreOftenNucleatedbytheARP

ComplexatthePlasmaMembraneActinfilamentnucleationmostfrequentlyoccursattheplasmamembrane.Consequently,thehighestdensityofactinfilamentsinmostcellsisatthecellperiphery.Theseactinfilamentsinthelayerunderlyingtheplasmamembrane,calledthecellcortex（細(xì)胞皮層）.Thisnucleationiscatalyzedbyacomplexofproteinsthatincludestwoactin-relatedproteins,orARPs,eachofwhichisabout45%identicaltoactin.The

ARPcomplex(alsoknownastheArp2/3complex)nucleatesactinfilamentgrowthfromtheminusend,allowingrapidelongationattheplusend.However,thecomplexcanalsoattachtothesideofanotheractinfilamentwhileremainingboundtotheminusendofthefilamentthatithasnucleated,therebybuildingindividualfilamentsintoatreelikeweb.NucleationandactinwebformationbytheARPcomplex.about45%identicaltoactinThetipoftheleadingedgeofacellnucleatesactinfilaments.Fibroblastsincultureweregentlypermeabilized（滲透化）usinganonionicdetergentandwerethenincubatedwithrhodamine-labeledactinmoleculestovisualizenewlyformedactinfilaments(red,543nm).After5minutes,thecellswerefixedandstainedwithfluorescein-labeledphalloidintostainallactinfilaments(green,488nm).(A)Alloftheactinfilaments,mostofwhichwereformedbeforethecellswerepermeabilized,areshowningreen.(B)Thelocationofthenewlyformedactinfilaments(red)showthattheleadingedgeisthepredominantsiteofactinfilamentassemblyinthecell.FilamentElongationIsModifiedbyProteinsThatBindtotheFreeSubunitsActinmonomersboundtothymosin（胸腺素）areinalockedstate,wheretheycannotassociatewitheithertheplusorminusendsofactinfilamentandcannothydrolyzeorexchangetheirboundnucleotide.ProfilinbindstothefaceoftheactinmonomeroppositetheATPbindingcleft,blockingthesideofthemonomerthatwouldnormallyassociatewiththefilamentminusend.Profilinboundtoanactinmonomer.Theprofilinproteinmoleculeisshowninblue,andtheactininred.ATPisshowningreen.ProfilinbindstothefaceofactinoppositetheATP-bindingcleft.Thisprofilinactinheterodimercanthereforebindtoandelongatetheplusendofanactinfilament,butitisstericallypreventedfrombindingtotheminusend.+--+Theprofilin-actincomplexcanreadilyaddontoafreeplusend.Assoonasthisadditionoccurs,aconformationalchangeisinducedintheactinthatreducesitsaffinityforprofilin,sotheprofilinfallsoff,leavingtheactinfilamentonesubunitlonger.Profilincompeteswiththymosininbindingtoindividualactinmonomers,andalocalactivationofprofilinmoleculesmovesactinsubunitsfromthesequesteredthymosin-boundpoolontofilamentplusends.Severaltypesofintracellularmechanismsregulatetheactivityofprofilin,includingprofilinphosphorylationandprofilinbindingtoinositol(肌醇)phospholipids.Thesemechanismscandefinethesiteswhereprofilinacts.Onemoleculeofthesmallproteinstathminbindstotwotubulinheterodimersandpreventstheiradditionontotheendsofmicrotubules.Stathminthusdecreasestheeffectiveconcentrationoftubulinsubunitsthatareavailableforpolymerization.Highlevelsofactivestathmininacelldecreasetheelongationrateofmicrotubules.Stathmin'sbindingtotubulinisinhibitedbythephosphorylationofstathmin,andsignalsthatresultinstathminphosphorylationcanincreasetherateofmicrotubuleelongationandsuppressdynamicinstability.Effectsofstathminonmicrotubulepolymerization.Stathminsequestersfreetubulinsubunits,bindingtwotubulinheterodimersperstathminmolecule.Asthepooloffreetubulinsubunitsshrinks,microtubuleelongationslows.ThismakesitmorelikelythattherateofGTPhydrolysiswillcatchupwiththerateofsubunitaddition,andtheGTPcapwillbelost,resultinginatransitionofthemicrotubulefromthegrowingstatetotheshrinkingstate.MotorProteinsMotorproteins:bindtoapolarizedcytoskeletalfilamentandusetheenergyderivedfromrepeatedcyclesofATPhydrolysistomovesteadilyalongit.Thecytoskeletalmotorproteinsassociatewiththeirfilamenttracksthrougha"head"region,ormotordomain,thatbindsandhydrolyzesATP.Theidentityofthetrackandthedirectionofmovementalongitaredeterminedbythemotordomain(head),whiletheidentityofthecargo(andthereforethebiologicalfunctionoftheindividualmotorprotein)isdeterminedbythetailofthemotorprotein.Actin-basedMotorProteinsAreMembersoftheMyosin（肌球蛋白）SuperfamilyAmyosinIImoleculeiscomposedoftwoheavychains(eachabout2000aminoacidslong(green)andfourlightchains(blue).Thecoiled-coilarrangementmakesanextendedrodinsolution,andthispartofthemoleculeiscalledthetail.twoglobularheadsthetailThemyosinIImoleculesaggregatebymeansoftheirtailregions,withtheirheadsprojectingtotheoutsideofthefilament.ThebarezoneinthecenterofthefilamentconsistsentirelyofmyosinIItails.Light-chainphosphorylationandtheregulationoftheassemblyofmyosinIIintothickfilaments.Achangeintheconformationofthemyosinhead,exposingitsactin-bindingsite.Releasethemyosintailfroma"stickypatch"onthemyosinhead,therebyallowingthemyosinmoleculestoassembleintoshort,bipolar,thickfilaments.MyosinLight-ChainKinaseregulatorylightchain,showninlightblueMyosinsuperfamilytreeTwoTypesofMicrotubuleMotorProteins:Kinesin（驅(qū)動(dòng)蛋白）andDynein（動(dòng)力蛋白）.Thereareatleasttenfamiliesofkinesin-relatedproteins,orKRPs,inthekinesinsuperfamily.MostofthemhavethemotordomainattheN-terminusoftheheavychainandwalktowardtheplusendofthemicrotubule.Thedyneinsareafamilyofminus-end-directedmicrotubulemotors.Structuresoffourkinesinsuperfamilymembers.Asinthemyosinsuperfamily,onlythemotordomainsareconserved.TheStructuralSimilarityofMyosinandKinesinIndicatesaCommonEvolutionaryOriginThemotordomainofmyosinsissubstantiallylargerthanthatofkinesins,about850aminoacidscomparedwithabout350.Thetwoclassesofmotorproteinstrackalongdifferentfilamentsandhavedifferentkineticproperties,andtheyhavenoidentifiableaminoacidsequencesimilarities.However,determinationofthethree-dimensionalstructureofthemotordomainsofmyosinandkinesinhasrevealedthatthesetwomotordomainsarebuiltaroundnearlyidenticalcores.X-raycrystalstructuresofmyosinandkinesinheads.Thecentralnucleotide-bindingdomainsofmyosinandkinesin(shadedinyellow)arestructurallyverysimilar.Theverydifferentsizesandfunctionsofthetwomotorsareduetomajordifferencesinthepolymer-bind