第9章细胞周期及调控

第九章细胞周期与调控TheculturedratkidneycellinmetaphaseBlue:condensedchromosomes;red:microtubulesofthespindleapparatus;1green:theinnernuclearenvelopeproteinPOM121ThecellcycleThecell-cyclecontrolsystemThemechanicsofcelldivision21、ThecellcycleThecellcycleentailsanorderedseriesofmacromoleculareventsthatleadtocelldivisionandtheproductionoftwodaughtercellseachcontainingchromosomesidenticaltothoseoftheparentalcell.Theeukaryoticcellcycleisdividedintofourphases:Interphase(间期)•Gap1phase(G1期):theperiodbetweenmitosisandtheinitiationofnuclearDNAreplication•DNAsyntheticphase(S期,DNA合成期):theperiodofnuclearDNAreplication•Gap2phase(G2期):theperiodbetweenthecompletionofnuclearDNAreplicationandmitosisMitosisphase(M期,有丝分裂期)•Mitosis•CytokinesisCyclingcell（周期中细胞）3Quiescentcell（静止期细胞，G0phase）StagesofmitosisinananimalcellProphase(前期),Prometaphase(前中期),Metaphase(中期),Anaphase(后期),Telophase(末期);Cytokinisis(细胞质分裂期)4ThephasesofMitosisinaplantcell5Meiosis6Variationsingenerationtimeforcellsofdifferenttissues•Cells,suchasnervecells,musclecells,orredbloodcells,thatarehighlyspecializedandlacktheabilitytodivide•CellsthatnormallydonotdividedbutcanbeinducedtobeginDNAsynthesisanddividewhengivenanappropriatestimulus(eg,thelivercells,thelymphocytes)•Cellsthatnormallypossessarelativelyhighlevelofmitoticactivity(thestemcells)7Cellcycletimes(hr)8Embryoniccellcycles2、ThecellcyclecontrolsystemThediscoveryofthecellcyclecontrolsystem—Thecellcyclecontrolsystemissimilarinalleukaryotes•Yeasts:buddingyeastandfissionyeast•Animaloocytesandembryos•MammaliancellsSeaurchin9Thecellfusionexperiment—Prematurechromosomecondensation,PCC(早熟染色体凝集)—TheseresultssuggestedthatthetransitionsfromG1toSandfromG2toMwerebothinducedbythepresenceofsomestimulatoryagent10FusionofaG1cellwithaMcellFusionofaScellwithaMcellFusionofaG2cellwithaMcellThediscoveryandcharacterizationofMPFMaturationpromotingfactor,MPF(成熟促进因子)orM-phasepromotingfactor,MPF(M-期促进因子)—Tolearnmoreaboutthenatureofthecytoplasmicchangethatresponsiblefortriggeringmaturation11UniversityofTorontoYaleUniversityThematurationandearlydevelopmentofXenopus—Theprocessesofgerminalvesiclebreakdown(胚泡破裂)andfirstmeioticdivisionarereferredtoasmaturation•Oocytesinthefrogovary,theyreplicatetheirDNAandbecomearrestedinG2for8months•Stimulatedbyamale,secretingthesteroidhormoneprogesterone,inducingtheG2-arrestedoocytestoentermeiosisIandarrestedinthesecondmeioticmetaphase.Atthisstagethecellsarecalledeggs.•Fertilizationandmitoticdivision12Xenopus:非洲爪蟾oocyte:卵母细胞;germinalvesicle,GV:生发泡•SolatedfrogoocytesatvariousIdentificationofMPFstages,treatedwithprogesterone•Injected40-60nlofthedonorcytoplasmintofullygrown,immatureoocytesthathadnotbeentreatedwiththehormone•Referredtothecytoplasmicsubstancsthatinducematurationinrecipientoocytesas“maturationpromotingfactor，MPF”Successivetransfersofcytoplasmfrommaturingoocytestoimmatureoocytes.13AdiffusiblefactorinarrestedXenopuseggspromotesmeioticmaturation.ThebehaviorofMPFduringearlyamphibiandevelopment•In1978,WilliamWassermanandDennisSmithofPurdueUniversity•DiscoveredthatMPFactivityundergoesdramaticfluctuationincleavingeggsthatcorrelatewithchangesinthecellcycle•MPFdoesmorethatsimplycontrolthetimeofoocytemuturationand,mayplayakeyroleinregulatingthecellcycleofdividingcellsCyclingofMPFactivityinfertilizedR.pipienseggs1st:firstcytokinesisat180min2nd:thesecondcleavagecycle(Ordinate:percentrecipientoocytesundergoinggerminalvesiclebreakdown(胚泡破裂)inresponseto80nlofcytoplasmfromfertilizedeggs;Bascissa:cytoplasmtakenfrom14cleavingfrogeggsatthetimeafterfertilization)MaturationpromotingactivityofHelacellsextractsduringdifferentstagesofthecellcycle•ExtractsfromculturedHelacellspreparedfromearlyG1,lateG1,S-phasecellslackMPFactivity•MPFactivityappearsinearlyG2,risesdramaticallyinlateG2,andreachesapeakinmitosis15GVBD:germinalvesiclebreakdownSummaryMPFactivityinXenopusoocytes,eggs,andearlyembryospeaksascellsentermeiosisandmitosis16CSF:cytostaticfactorCelldivisioncyclegene(cdc)LelandH.Hartwell(1960s)—CellCycleMutants—Cellcyclecheckpointinbuddingyeast—start17LelandH.HartwellCellcycleofSaccharomycescerevisiaeThebehaviorofatemperature-sensitivecdcmutantRestrictive(high)temperaturePermissive(low)temperatureLelandH.Hartwell,etal.Genetics1973;74:267-286.18Buddingyeastcellsarrestedbyacdcmutationcdc28yeastcellsgrownatrestrictiveNormalproliferatingyeastcells19temperaturearearrestedatG1Wild-typecell-divisioncycle(CDC)genescanbeisolatedfromaS.cerevisiaegenomiclibrarybyfunctionalcomplementationofcdcmutants•Mutantcellswithatemperature-sensitivemutationinaCDCgenearetransformedwithagenomiclibrarypreparedfromwild-typecellsandplatedonnutrientagaratthenonpermissivetemperature.•Therarecellthattakesupaplasmidcontainingthewild-typeversionofthemutantgene(inthiscaseCDC28)iscomplemented,allowingthecelltoreplicateandformacolonyatthenonpermissivetemperature.•PlasmidDNAisolatedfromthiscolonycarriesthewild-typeCDCgenecorrespondingtothegenethatisdefectiveinthemutantcells.•Thesameprocedureisusedtoisolatewild-typeCDCgenesinS.pombe.20CDC28TheCDC28productIsaphosphoprotein(p36)Proteinkinaseactivity(P40)AntibodiespreparedagainstaCDC28-IacZfusionproteinImmunoprecipitate,preparedfromyeastcelllysates,immunoprecipitatetheinvivoCDC28product(1-4,35S-incubatedwith[r-32P]ATP;lanes1,2,and3,wildtype,labeledsamples;lanes5-8,32p-labeledsamples)cdc28-4,andamixtureofthetwo,respectively,alllanes1and5,immuneserum,wild-typecells;lanes2and6,assayedat23°C.Lanes4,5,and6,wildtype,cdc28-13,preimmuneserum,wild-typecells;lanes3and7,immuneandcdc28-4,respectively,againassayedat23°C.Lanesserum,CDC28plasmid-containingcells;lanes4and8,7-9,duplicateimmunoprecipitates(equivalenttolanes214-preimmuneserum,plasmid-containingcells.6)preincubatedat38°Candassayedat38°CPaulNurse(1970s)—cdc2orp34cdc2PaulM.Nurse22Cellcycleoffissionyeast(Schizosaccharomyces.pombe)cdc2突变wee1突变23WeeMutantsinFissionYeast(weeistheScottishwordforsmall)IsolationofCdc2geneCdc2-3329°C36°CPermissivetemperatureRestrictivetemperatureNormalgrowthCellcyclearrestTransformationwithDNAlibraryNoyeastcolonyFromwild-typeyeastorhumancells36°CCdc2-33Cdc2+NormalgrowthColonyformationBeachetal.(1982)Nature300:70624Leeetal.(1987)Nature327:31StudiesshowedthatS.cerevisiaecdc28andS.pombecdc2arefunctionallyhomologousgenes(P34cdc2/cdc28)Molecularcloningandnucleotidesequencingrevealedthatcdc2encodedaproteinkinase—thefirstindicationoftheprominentroleofproteinphosphorylationinregulatingthecellcycle25ThediscoveryofCyclin•In1983,TimHuntandhiscolleaguesreportedonseveralproteinsthataresynthesizedinfertilizedseaurchinbutnotunfertilizedeggs•Namedtheprotein“cyclin”CorrelationofthelevelofcyclinwiththecelldivisioncycleTimHunt•ProteinA:varyingaccordingtothecellcycle,namedcyclin•ProteinB:nocellcyclefluctuation•Opensquares:thepercentageofcellsundergoingdivisionatanygiventimeperiodisgivenasthecleavageindexAsuspensionofeggswasfertilized,andafter6minutes,[35S]methioninewasadded.Samplesweretakenforanalysisbygelelectrophoresisat10-minute26intervals,startingat16minutesafterfertilization.Oscillationexpressionofcyclinsduringearlyembryoniccellcycle•CyclinAisdegradedduringa5-6minuteperiodbeginningjustbeforethemetaphase-anaphasetransition•CyclinBisdegradedafewminutesafterthistransition27ThefirstclearlinkbetweencyclinandMPF(DemonstratedbyJoanRudermanandhercolleaguesattheWoodsHoleMarineBiologicalLaboratory)WhensyntheticcyclinAmRNAwasinjectedintoXenopusoocytes,thecellunderwentgerminalvesiclebreakdown(胚胞破裂,GVBD)andchromosomecompactionoveratimecoursenotunlikethatinducedbyprogesteronetreatmentTheriseincyclinA,whichoccursnormallyduringmeiosisandmitosis,hasadirectroleinpromotingentryintoMphase.28KineticsofXenopusoocyteactivitybyprogesteroneandcyclinAmRNAMPFpurification(byJamesMallerandhiscolleaguesatUniversityofColoradoSchoolofMedicinein1988)29(C)ChromatographyofMonoQfractionsonMonoSMonoQfractions10-14werepooledandchromatographedonaMonoScolumn.Thecross-hatchedregionindicatesfractionsthathadactivityinthecell-freeassay.Fractions11-13inducedGVBDwheninjectedintooocytes.30PolyacrylamidegelanalysisoffractionselutingfromtheMonoScolumn•Twopolypeptides:onehavingamolecularmassof32KDaandtheotherof45KDa•ThepurifiedMPFpreparationpossessedahighlevelofproteinkinaseactivity(A)Silver-stainedpolyacrylamidegelofpurifiedMPF.Theactivityofthefractionsinthecell-freeassay(NEBD,核膜破裂)andintheoocytemicroinjectionassay(GVBD)isshownbelowthegel.(B)AutoradiographofthesilverstainedgelshowninA.(C)H1kinaseactivityofpurifiedMPF.MonoSfractions6-15wereassayedforH1kinaseactivity.Theautoradiographoftheregionofthegelwithhistone1isshown.Fraction12hadaspecificactivityof～270nmol/min/mg.A45-plaliquotoffractions5-16wasincubatedwith[g-*32P]ATPand31electrophoresedthrougha10%NaDodSO4/poly-acrylamidegelWhatisMPF?Antibodiesformedagainstcdc2fromfissionyeastwereshowntoreactspecificallywiththe32-kDacomponentofMPFisolatedfromXenopuseggs—thiscomponentofMPFisahomologueofthe34-kDayeastkinaseSequenceanalysisshowedthat45KDcomponentofMPFwasthecyclininseaurchin32MPF=Cdc2+CyclinBTheseinitiallyindependentapproachesconvergeddramaticallyin1988,whenMPFwaspurifiedfromfrogeggsinthelaboratoryofJamesMaller(P32andP45)CoveragewithyeastgeneticsandstudiesofseaurchinembroyesAdimerofCdkandcyclin•Cdks(cyclin-dependentproteinkinases,周期蛋白依赖性蛋白激酶):acatalyticsubunit•Cyclin(周期蛋白):aregulatorysubunit33LelandHHartwell，PaulNurseandTimHunt(NobelPrizein2001)“Fortheirdiscoveriesofkeyregulatorsofthecellcycle”34ThefunctionofMPFMPFinducesmultiplenuclearandcytoplasmicchangesattheonsetofMphase,bothbyactivatingotherproteinkinasesandbyphosphorylatingproteinssuchascondensinsandthenuclearlamins.TargetsofMPF35(凝集蛋白)RegulationoftheCellCycleThecell-cyclecontrolsystem(1).Moleculescontrollingcellcycle36Asimplifiedviewofthecoreofthecell-cyclecontrolsystem37(2).Regulationofcyclin-Cdkcomplex5.Proteolysis(SCF/APC)•Cyclin-Cdkcomplex•Cdkphosphorylationstate•Cdkinhibitors•ControlledproteolysisCAKWee1/cdc2538MechanismsofCdkregulationCAK:Cdk-activatingkinaseCdkphosphorylationstateCAK(Cdk-activatingkinase)•Phosphorylatethreonineresidue(Thr161)Wee1/CDC25—mutation?•Wee1:phosphorylatetyrosineresidue(Tyr15),inactive•Cdc25:aphosphatase,active(Wee1)39CdkinhibitorsCKI(cyclin-dependentkinaseinhibitor)•INK4family•CIP/KIPfamily40ProteolysisInG1andSphaseInmetaphase/anaphaseM-cyclinsE1—ubiquitin-activatingenzyme,E2—ubiquitin-conjugatingenzyme,E3—ubiquitinligase:SCF(skp1-cullin-F-boxprotein),APC(anaphase-promotingcomplex)41(3).CellcyclecheckpointsCheckpointsaremechanismsthathalttheprogressofthecellcycleif(1)anyofthechromosomalDNAisdamaged,or(2)certaincriticalprocesses,suchasDNAreplicationduringSphaseorchromosomealignmentduringMphase,havenotbeenproperlycompleted.G1checkpointSphase•Startpointinyeastcellscheckpoint•Therestrictionpointorcommitment42pointinanimalcellsCell-cycleprogressionisblockedbyDNAdamage:DNAdamagecheckpointATRCellcyclearrestCellcyclearrestModelsforthemechanismofactionoftwoDNA-damagecheckpointsATM:ataxiatalangiectasia-mutatedgene(共济失调-毛细血管扩张症突变基因)43ATR:ataxia-telangiectasiaandRad3-relatedRoleofP53inG1arrest—PrevententryintoSphaseHowDNAdamagearreststhecellcycleinG144ControloftheS,G2checkpointPrevententryintomitosis453、ThemechanicsofcelldivisionHowdoesthecellescapefromthisstableG1statetoinitiateSphase?GrowthfactorsandcyclinD46E2F:E2factorCyclinDandRb47CurrentmodelforregulationoftheeukaryoticcellcycleGrowthfactors(CycD/cdk4,6)(CycB/cdc2)(CycA/cdc2)(CycE/cdk2)(CycA/cdk2)48ExitfrommitosisrequiretheinactivationofM-Cdk•InactivationofM-CdkbyproteolysisofM-cyclins(Cdh1-APCcomplex)cyclinBExit49SummaryCellcycle,cyclinandcdkInterphase:G1，S，G2Mphase•Mitosis:prophase;prometaphase;metaphase;anaphase;telophase•Cytokinesis5051Cell-cyclecontrolsystemandcheckpointsGrowthfactor↙Rb↙52思考题细胞周期及各时相的主要事件。细胞周期的调控机制。53TheEndThankYou