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Calmodulin

By Larry Hunt,2014-07-06 10:03
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Calmodulin

    Calmodulin

    Calmodulinregulatesthepost?anaphaserepositionofcentriolesduringcy? tokinesis

    YueYueYU,GuDAI,FeiYanPAN,JieCHEN,ChaoJunLI

    JiangsuKeyLaboratoryforMolecular&MedicalBiotechnology.SchoolofLifeSciences.NanjingNormalUniversity

    Nanjing210097,China

    ABSTRACT

    Atransientpostanaphaserepositioningofthecentrioleisfoundtocontrolthecompletionofcytokinesis.Usinga

    greenfluorescentprotein

    calmodulinfusionproteinasallyingcellprobe.wehavepreviouslyfoundthatcalmodulinis associatedwiththeinitiationandprogressionofcytokinesis.Inthisstudy.wefurtherstudiedtheeffectofcalmodulin

    ontherepositioningofthecentrioleandsubsequentcellcycleprogression.Whenactivityofcalmodulinisinhibited.the

    regressionofthecentriolefromtheintercellularbridgetothecellcenterisblocked.andthusthecompletionofcell

    divisionisrepressedandtwodaughtercellsarelinkedbylongercellbridgeinperturbedcells.W7treatmentduring

    cytokinesisalsoresultsinunfinishedcytokinesisandstoppedG.phase.Theseresultssuggestthatcalmodulinactivityis

    requiredforcentriolerepositioningandcanaffectthecompletionofcytokinesisandcellcycleprogression.

    Keywords:CaM,centriolereposition,cytokinesis,W7

    INTRoDUCTIoN

    Cytokinesisisaverycomplicatedandcarefullyorches

    tratedprocess.Duringthelaststepofthisprocess,an intercellularbridgeisformedbetweenthetwodaughter cells.Anumberofstudiessuggestthatthisintercellular bridgeisnotmerelyaremnantofthemitoticspindleand cellmembrane.Infact.itplaysasignificantroleincy

    tokinesiscompletionandthefollowingprogressionofcell cyclefromGltoSphasel1.Inadditiontomicrotubules,

    numerousproteinshaverecentlybeenidentifiedatthe intercellularbridgeandtheyplayaroleincytokinesis completion.Theseproteinsincludecytoskeletonproteins (actin,intermediatefilamentproteins),chromosomalpas

    sengerproteins(INCENP,TD60).motorproteins

    (CENPE,KLP3A),proteinkinases(PoloandAurora families),andy-tubulin[2,3.Someoftheproteinslo

    calizeatthecentrioleregionduringmetaphase,redistrib

    utetocentralspindle.andfinallylocalizetotheintercellu

    larbridge.

    TheCa2+/CaMsignalhaslongbeenthoughttoplayan importantroleincytokinesisduetotheobservationthat Correspondence:ChaoJunLI

    Tel/Fax:+862583598812:

    Email:licj@njnuedu.ca

    therewasacloseassociationbetweenthelocalizedeleva

    tionofCaandtheonsetofcytokinesis4,51.Calmodulin

    (CaM)alsorelocalizesfromthespindlepoleduring metaphasetothecentralspindleandintercellularbridge duringcytokinesis[6,7.CaMplaysasignificantrolein

    cytokinesisincludingfurrowformationandregression.The associationofCaMwithcentralspindleregulatestheinitia-

tionofcytokinesis[8.Theinjectionofeitherantisense

    mRNAofCaMorCvIinhibitingpeptidealwaysresultsin failedcytokinesis9,101.HerewereportthatCaMisas

    sociatedwithregulationofcentriolereposition,aremark

    ablemorphologicalchangethatcontrolsthecompletionof cytokinesisandsubsequenttransitionofthecellcyclefrom G,t0Sphase.ThedatagivesfurtherevidencethatCaM couldregulatethecompletionofcytokinesis. MATERIALSANDMETHoDS

    Cellcultureanddrugtreatment

    BothHeLacellsandPtK,cellswereusedinthisstudy. PtK,cellsaremorefiatthanHeLacellsduringmitosisand thecellbridgecanbemoreeasilyobserved.Cellswere culturedasamonolayerat37.CinMEMsupplemented with10%fetalcalfserumand100i.u./mlpenicillinand streptomycin.

    Thecellpermeableantagonistofcalmodulin.W7IN

    (6Aminohexy1)5cholro1-naphthalenesulfonamide.HC1]. CellResearch,15(7):548552.July20051www.cell-research.corn

    whchi_1llibjtCa/CaMdependentph~~sphc)diesteraseand liIVOS1I1ligiltchItkiliase(CalbiochemLaJoIla.CA),was addedtoreedILInl()flhesvtichronizedce1sThenonce11

    tratjonwacaIibratedinotllelexpertnlentsbyulngdif- fel'elitconcentrath)nsThelllaX1mum1nhlhtorN'efrectW:.IS

    litI5IllM.andwasreversibeAIheexpertmentswere

    IepeatedlndependentvLit1east3tirues

    CelIsynchrnni#ation

    SvnchrolliZUliOil'fPtKccI1swiI^aceon1DIishedbv

    usiligtIledoub1ethvn1idinebIOCknle11.od.Ce1Iswere b1ockedatSphaseandt1lelli11cubatedii1J"reshreediLIIll r12l1untiIcvtokiliesibet'oieW7V.%ISapp1ied Toobtansynchrt)11izednlelaphaseHeLaceIls.cell cvcIedtomJt(sjsweI'cbckcdbvn()c()dazole (CaIbjochenlLaJol_a.CAatafinaIconcentraljonof0.2 la?/I111lor101Sv11ohmnized111itolicHeLaceIIsweI.e thencollectedandrecoveredl11freshreed1unlfor0II1n

    bemreW71Ieatmellc.

    BrdUinnnrpnration

    ToC0111paletheBrdUjncorp()ratIonrateIi1W7treated anduntreatedHeLacelsBIdU(ZymedLabSouthSan

    FranciSCOCAwasaddeddirectvtotilecuturemed1aI11

    ofthesynchrlinIzedHeI.acc】】swit1afIi1aJO0ncentraticln c)fl(uM.AI"letilinilbationrI5~!(Jl1.ce1_soncover

    slipswcI?(ixedandthenimnlersedlI1formalilideat1tar'C rnr5mhltodenatureDNAbe^)restallliligwithantiBtdU

    monocIon_antlhtdv

    lmmunoffnoresceneestaininll

    Tl1cjmI11uI1oajnjnprocedureessenlia[Ivt'ollowedthat 0fHarlowandLanelJ01Formicrotubulesstaining,Tri. tollX1I)0wasusedtopermeah[ecel_[1getaclearImage

    102,added1nnxa"onhuIfer

    TheIll{]useillonoclo11alantiBtubnlinantihodvwas

    purchasedl-romSaraLabISussex,England1.rabhit

    polyclonalantT-tubaliI1antibodvonlSantaCJuzlab

    ISantaCruz.CA1andlilt)uselnonoc]onalantiBrdLanti

    bodvfrolnZyreedLabfSouthSanFrancisco.CAITexas

    RedorFITcconjugatedgoalantiIllOUSesecondaryanti

bodiesLindTexas..Redconjugatedgoatantirabbitsecond

    arYantjbodvwereobtajnedfr'1nlCabiochelll

    Nos,abiochemCorp(LaJolla,CA)

    Microscopy

    Inal1cases.imnlunostainedcellwerecostainedfor

    DNAwithHoechsl33342(MolecularPr(1hesEugene.OR

    MicroscopicobservationswerenladeOllaLeicaDMR microscopeequippedwithal()0xfNA1.4Iobiective

     ImageswerecapturedwjIJ]acooledchareCOUpedde

    Ycecamera(Dignosticlllt,irtunenM1)andprocessed

    usingSPOTsoftwarecDignosticlnstruliletits.MI

    WWWcellresearch.cornJCellResearchI71:548552.July2005

    A

    B

    :

    广十]ControIceltsW7treatedcells

    Fig.1W7treatnlenltilterstilelengthtIthelnlerceIlu1arbrldgefAl r,4ItrotubuI12inlnlunosntiningshox~esthattheinlerceI1ularbridReof

    theW7n'eatndlK2eel1inmchl)llgel{blthanthaioftheDornlaJ ce11sIaiBar=ItlFiillB1Thenvcr?IgcIeilgthofIhelntercelluIat biidgelnot'eased]rolll}lillea[1t1j7I4~0q7bun{n=30)lntheC011Lro1

    CCIkCOnnlca[il'11I256?l4Iuinfn=3t))l11theW7treatedcells(: <001)

    RESUTS

    CaMafieetedintercellularbridgeabscission

    Successfulcvtokinesisrequirestheabscissionandeven

    tuallyresovingofthelntercellularbridgecotlnectingtlne F~S.Odaughtercells}l0havefoulidthatCaMconcentrates tothecellbridgeaftercellsentertelophase.especiallynear the"FlenlmingBi)dy..wherecellbridgeabscissionhap.

    pens111.nordertoexaminewhetherthei11tercellular bridgeassociatedCaMisais{)involvedjnCVtOIdnests co1_pletion,wetreatedsynchronlzedPtK2cel1swjthW7 1l5IAMlfor4hatalatesta~einc},lokinesiswhenmost cellshavebeguntoft1rmlntelcellularbridgesWhenthe c【】ITIPIetl_OfCytokInesiswaspro1ongedbyanv perlurbations.therw']daughtercellswouldbe1_likedbya longerintercellularbridgeI2AHsho~d,,ninFiglA,w7

    ntercelIular treateddaughter'cellswereljnkedbvIon.per

    hrdeh_d【】【h('e(1ftheunueaedcells.Theaveraebde

    len星【hinc1?sedlgnricantlyIH)m7l4mfn=3O)inthe c(1l1cells(jI256um(n=3()InfheW7treatedce

    (Fig.Bd)nTh1suggeW7trementc0udpef

    LLlrbheabscisi('l10ftheintcl'ce]LlIarbrId?eandthenthe

    exitrr0mcv0knesjs.

    642O8642O

    nj?亏u.u10口罢口

    L]=?uJc一?L|~0L|一口c00L|J_

A[

    muchhigberpercelltageoleelIsintheno~ashotflgroup

    hadbothpaill1IcenLnllEcsposilinnedthevicinityofthe bridgefFig2BP<00I)TheseresuhsindicatethatW7 mayaffectthemotilit)0fccntriolcsatthefinal~tageof cytokinesi~

    W7inhihitedc~tokinesiscompletion

    J"klrthcrInv__gatewhelheilhedetanedctroIes

    J11theviciniwoftheinrJILIIrbJidlereflectedaRignal fdeI1vcdexifr0l11cyl__ki1iweheckedthepro

    res1oI1f)IceIcvc【】fFIL".IfterW7treatilletitIthHv hcnillLllcIIcl1halIhece1IIldidnlni11cvtokmeslH werrctedatG".Igeand~OtlIdi1'cnterSphase

    A

    l+1Fi3IAIBrd/Lncorp~1rlIiOllLic0IC~CL]hyW7IrcaImcafter

    l5hIre~tlnet~l(anh'1ulrollPhwi~]ll1_IIo[ipccI1[rol

    glOtlP}{BI1chIstOgli]1showsIhepercentag~olcI1ing~ll~o raUngBL.mthre~dIn'umdII5.iand2(IhlllcrBrd?addldon

    TheW71aldct'l~howcd.IdcinBrdIm1T1i1andthe

    L]~CIinlo1.~l_bvll?h1nhcl?'waIr'1unc-P10?

    

    

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    151.WefoundthatW7treatedcells(boththewashout andno.washoutgroups,showedadecreaseinBrdUstain- ing(Fig.3A,B1.Thisdecreasewasparticularlynotablein theno.washoutgroup(P<0.011.Thisresultconfirmed thatinhibitionoftheactivityofCaMcouldblockthe completionofcytokinesis.

DISCUSSION

    Cytokinesis,theconceptuallysimpleeventreferringto theseparationofthetwodaughtercellsaftermitosis.isin factapreciselyregulatedprocess.Severalimportantstruc- turesareformedduringthisprocess.Ourpreviousstudy appearedthatCaMmightregulatetheinitiationofcytoki- nesisbyassociatingwiththecentra1spindle81.Thein-

    tercellularbridgeisthekeystructureincontrollingcy- tokinesiscompletion.Consistentwiththisview.1otsof proteinshavebeenshowntodistributeattheintercellular bridgeduringcytokinesis2,3.Someoftheseproteins

    mayserveasstructura1componentsoftheintercellular bridge.whileothersmayfunctionassigna1proteinsthat mediatetheprogressionofcytokinesis.Inourprevious study,wehavefoundthatCaMmightfunctionduringthe completionofcytokinesisbecauseitdistributedinthe middleofthecel1bridgenearthe"Hemmingbody"where thefina1abscissionhappens111.

    Althoughtheintrinsicmechanismisunknown.ithas beensuggestedthatthecentriole(especiallythemother centriole1can'examine'thespatia1organizationofthecel1. andthendeterminewhetherthefina1cutshouldoccur 13,141.Furtherstudieshavedemonstratedthatthemo. tilityofthecentriolesisdependentonthemicrotubule. networkandregulatedbymotorproteins,suchasdynein andHsEg5[16,17].Intriguingly,bothdyneinandHsEg5 containCaMbindingsites,andCaMcanregulatedynein's activityinflagellarmotility181.Thedatapresentedin

    thisreportindicatethatCaMisrequiredforthesuccess. fu1completionofcytokinesis.Whencellsweretreated

    withaCaMinhibitor.centriolesretainedinthevicinityof theintercellularbridgeandtheabscissionwasalways deficient.Theseresultsareconsistentwithsevera1other reportsthatthepost..anaphaserepositioningofthecentri.. olescontrolstheexitfromcytokinesis13,141.

    Itisapparentthattherepositioningofcentriolecon. trolsthereleaseofcentra1microtubulesfromthemid. bodyandthusregulatesthecompletionofcel1division. CaMmayalsodirectlyaffectthedynamicstabilityofthe microtubulesoftheintercellularbridge.sinceitiswel1 knownthatCaMcaninteractwithsevera1MAPs

    (microtubuleassociatedproteins1suchastauandMAP2 19.21],andthusindirectlyaffectthecentriolesmotility. WehopethatthefurtherinvestigationofCaM'starget proteinincytokinesiscompletionwil1provideuswithmore WWW.cellresearch.comlCellResearch,15(7):548552,July2005

    YueYueYU

    informationaboutthiscrucialproteinandleadustoabet- terunderstandingofthiscomplicatedprOcess——

    cytokinesis.

    ACKNoWLEDGEMENTS

    ThisworkwassupportedbyNationa1Natura1Science FoundationofChina(GrantNo.301704531andFounda. tionofMinistryofEducationofChinaforDistinguished YoungTeachers(GrantNo.200139)awardedtoChao.

    JunII.

    Received,Apr4,2005

    Revised,June28,2005

    Accepted,June29,2005

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