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Actomyosin

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Actomyosin

    Actomyosin

    JournalofIntegrativePafBiology2006,48(1):53_1

    4

    wwwblackwellsynergy.corn;www.chineseplantscience.com

    Chun.LiLi',Zhi-Lingthen',andMingYuan'

    (1.StateKeyLaboratoryofPlantPhysiologyandBiochemistry;,DepartmentofanfSciences,CollegeofBiologicalSciences,China

    AgriculturalUniversity,Beijing100094,China;

    2.CollegeofLifeSciences,CapitalNormalUniversity,Beijing100037,China) Abstract

    Themicrotubulepreprophasebands(PPBs1participateinthesequenceofeventstopositioncellplatesin

    mostplants.However.themechanismofPPBformationremainstobeclarified.1nthepresentstudy,the

    organizationofPPBsinArabidops~suspensionculturedcellswasinvestigatedbyconfocallaserscanning

    microscopycombinedwithpharmacologicaltreatmentsofreagentsspecificforthecytoskeletonelements.

    DoublestainingofF-actinandmicrotubules(MTls)showedthatactinfilamentswerearrangedrandomlyand

    nocolocalizationwithcorticalMTswasobservedintheinterphasecells.However,corticalactinfilaments

    showedcolocaIizationwithMTsduringtheformationofPPBs.AbroadactinbandformedwiththebroadMT

    bandintheinitlationofPPBandnarroweddowntogetherwiththeMTbandtoformthePPB.Nevertheless.

    broadMTbandswereformedbutfailedtonarrowdownincellstreatedwiththeF-actindisrupt

orlatrunculin

    A.1ncontrast,inthepresenceoftheF-actinstabilizerphalloidin,PPBformationdidnotexhibitanyabnormality.

    Therefore,theintegrity,butnotthedynamics,oftheactincytoskeletonisnecessaryfortheformationof

    normalPPBs.Treatmentwith2,3-butanedinemonoxime,amyosininhibitor,alsoresultedintheformationof

    broadMTbands.indicatingthatactomyosinmaybeinvolvedintherearrangementofMTstoformthePPBs.

    DoublestainingofMTsandmyosinrevealedthatmyosinconcentratedonthePPBregionduringPPBformation.

    1tIssuggestedthattheactincytoskeletonatthePPBsitemayserveasaracktotransportcorticalMTsby

    usingmyosinwhenthebroadMTbandnarrowsdowntoformthePPB.

    Keywords:Arabidopsissuspensionculturedcell;2,3?butanedinemonoxime;microfitament;microtubulepreprophaseband

    myosin.

    Foragrowing.plantorgan,determinationofthedivision

    PIaneisofctiticaIimportance.ThemicrotubuIe

    preprophaseband(PPB)hasbeendemonstratedtopar.

    ticipateinthesequenceofeventstopositioncelIplatesin

    mostplants(Mineyuki1999;Smith2001).ThePPBnor.

    mallydevelopsduringpreprophase.Itconsistsofcompact.

    transverselyalignedmicrotubules(MTs).However,before

    cellsenterpreprophase,M"IsIocateinparallel,transverse

    arraysthroughoutthecortex.knownascorticaIMTs.The

    Received8Oct.2005Accepted22Oct.2005

    SuppoSedbytheStateKeyBasicResearchandDevelopment

    PlanofChina(2006CB100101)andtheNationalNaturalScience

    FoundationofChina(30421002,30370707and30100091).

    Authorforcorrespondence:T_eh+86(0)1062733436;Fax:+86 (O)1O62733491Email:<mingyuan@cau.edu.cn>. dynamicre0raanizati0nofcorticaIMTsoccursinashort periodoftimeatthetransitionfrominterphasetoMphase inthecellcycle.

    Microfilaments(MFs)arereportedtocolocalizewithPPB duringPPBformation(Palevitz1987;ClearyetaI.1992). TreatmentwithcytochalasinD(CD1doesnotinhibitthe formationofthePPB,butpreventsthenarrowingofthe PPBandresultsintheformationofbroadPPBs(Mineyuki andPaIevitz1990;EIeftheriouandPalevitz19921. Furthermore.broadPPBsarealsoobsewedwhenprotein synthesisisinhibitedbycycloheximidefNogamietaI. 19961.Treatmentwithkinaseinhibitorsdisruptsthede. velopmentofthePPBandalsoIoosensthePPB(Katsuta andShibaoka1992:NogamiandMineyuki1999). Therefore.complexmechanismsmaybeinvolvedinthe processofMTnarrowingtoformnormaIPPBs.

    Arecentstudyusingmammalianyellowfluorescent M

    54JournalofIntegrativeP|antBiologyVo1.48No.12006 protein-taggedCLlP-170tovisualizethedynamicplus endsofplantMTsintransfectedcowpeaprotoplastsand instablytransformedtobaccoBY-2cellsrevealedthat, duringPPBformation,thegrowthrateandcatastrophefre- quencyofplantMTsaredoubled,whereastheshrinkage rateandrescuefrequencyremainunchanged,makingMTs shorterandmoredynamic(DhonuksheandGadella2003). PreviousstudieshavealsosuggestedthatnewM1-sf0rm

    duringPPBfOrmation(Murataeta1.1991;Clearyeta1.1992; Panteriseta1.1995).Althoughitremainsuncertainwhether thosenewPPBM1-sfOrmatthePPBsiteoratthenuclear envelope(NE),previousreportshaveshownenhancement ofy-tubulinantibodystainingoftheNEduringthenarrow- ingofthePPB(Liueta1.1993)and,atthecondensedstate ofPPBformation,theNE-originatedMIsbecomemore dYnamicaccordingtotheirdYnamicParameters

    (DhonuksheandGadella2003).Therefore.itislikelythat atleastpartofthenewlyformedMTs.ifnotall.fOrmatthe NEandarethentransportedontothePPBsite.Hence. certainspecifictransportmaybeinvolvedtocontrolthese processes.Consideringthenecessityofactinfilamentsin PPBformation,myosin,themotorproteinbasedonthe actincytoskeleton.ispossiblyresponsibleforsuchtrans? portinvolvedinPPBorganization.

    Inthepresentstudyweexaminedtheeffectsofvarious reagents.knowntointerferewiththecytoskeletalelements, ontheformationofPPBs.OMrobse~ationssuggestthat actinfilaments,withtheirmotorproteinmyosins,doplaya roleintheorganizationofPPBMTs.

    Results

    IntegrityoftheactincytoskeletonisnecessaryforPPB formation

    Toinvestigatetheroleoftheactincytoskeleton,wedouble stainedMTsandF-actinandexaminedtheorganizationof theactincytoskeletonduringPPBformation.1ninterphase cellsbeforePPBfOrmation.corticalMIswerearrangedin paralle1.verticaltothelonggrowthaxis.whereasactinfila- mentswererandomlyarrangedbothatthecellcortexand

    inthecytoplasm(Figure1A).DuringtheinitiationofPPB formation.abroadMTbandemergedandthecorticalactin filamentsunderwentarearrangement;someparallelac- tinfilamentsappearedatthePPBsitetofOrmabroad actinbandcolocalizedwiththeformingMTPPB(Figure 1B1.WiththenarrowingofthebroadMTbandtofOrmthe PPB,thebroadactinbandalsonarrowedsothatanactin filamentbandlocalizedwiththePPBwasobse~ed(Figure 1C).ObservationofthecOlOcalizatiOnofactinbandswith MTPPBsisconsistentwithpreviousreportsfPalevitz1987; Clearyeta1.19921.Suchobse~ationssuggestthatthe actincytoskeletonmayhavecertainroleinPPBformation. ToexamiRewhetherMFSareinvolvedinPPB

    organization,wetreatedArabidopsissuspensioncultured cellswithlatrunculinA(UAtodisrupttheactinfilaments.

    AftertreatmentwithvariousconcentrationsofLATAfor1h. themorphologyofMTbandswasdifferentfrOmthatseen incontrolcellsfFigure2A).TheaveragewidthofMTbands wasincreasedinadose?dependentmannerbythepres- enceofLATA.Forexample.theaveragewidthofMTbands wasapproximately(4.89?0.32)Uminthepresenceof50

    nmol/LUA.whichasanapproximate50%increasecom- paredwithcontrolcells.inwhichtheaveragewidthofthe PPBwas(3.17?0.28)um(Figure2A).TheincreaseinMT

    bandwidthreachedamaximallevelwithcOncentratiOn> 50nmol/LLATAfFigure2A).

    Tomakesurethedisruptionofactinfilamentsaffects onlythemorphologyofPPBsandnottheirformation,the percentageofcellswithPPBsinbothtreatedandcontrol sampleswascalculated.Thiscalculationshowedthatthe

    percentageofcellswithbroadPPBswasapproximately 3.6%?0.2%insamplestreatedwithvariousconcentra- tionsofLATA.TherewasnosignificantdifferencefrOm controlsamples.inwhichapproximately3.8%?0.2%of

    cellsexhibitedPPBs(Table1).Therefore,theformationof broadPPBswasnothinderedbyLATAtreatment.

    Nevertheless.suchbroadPPBsfailedtonarrowdownto fOrmnormalPPBswhenactinfilamentsweredisrupted. Wefurtheraddressedthequestionastowhetheractin dynamicsortheintegrityoftheactincytoskeletonisnec- essaryforPPBorganization.WhenvariouscOncentratiOns ofphalloidinwereappliedtothesuspensionculturedcells tostabilizetheactincytoskeleton.therewasnosignificant differencebetweenthePPBsintreatedandcontroIcells. ThemorphologyofPPBswasnotaffectedbytheexposure ofcellstophalloidinatcOncentratiOnSrangingfrOm0.1tO 10limol/L.Measurementsshowedthatthemeanwidthof thePPBswasapproximately3LLminbothtreatedandcon. trolcells(Figure2B).BecausephalloidinbindstoF-actin andpreventsitsdepolymerization.theresultshereindi- carethatactindynamicsmaynotbeinvolvedinPPB formation.

    Basedonourexperimentalresultsinvestigatingthedis- ruptionandstabilizationoftheactinfilaments.wecon. cludethattheintegrityoftheactincytoskeletonisneces. saryforPPBorganization.

    NarrowingofPPBsishinderedwhenmyosinactivityis inhibited

    IftheactincytoskeletonfunctionsinPPBformation,oneof thepossibilitiesisthattheactincytoskeletonworkswith

itsmotor,myosin,totransportMTs.

    TorevealtheroleofmyosininPPBformation,the

    ActomyosinisInvolvedintheOrganizationofMicrotubulePreprephaseBand55 Figure1Organizationoftheaclincytoskeletonduringmicrgtubuieprepropnaseband{PPB)[ofm~l[onImagesJn(^)showMTsinredr

    imagesinlB1showactinfilamentsingreenandimagesin(c)aremergedimage~ofMTsandaeLinf1]amenl5toshowlhelrlocalLzation

    Bars2Oum

    cA1BeforePPBformatio~conlcamlcrotubu~es(MTs)wer8arrangedinparallelrecitaltothelonggrowlhawhe~Bsact~nfiiamBnts

    wererandomlyarrangedbothat1hecellc0nxandinthecytoplasm

    (B}FollowingtheInibat~onelPPBformationabroadMTband8mergeds.meparallelautinfilamentsappearedatthePPBsitetoforma

    broad~.clinbandcoJo~iizedWithIhef0m,ingMTPPB

    (C)Anarro1gdownelthebreadMTbandtotermeh

    PPBthebroadactinbandwasalsoR~rroweddowntoformanactinband coloCallzedwlthPPB

    I)caIizatronofmyosindiaPPBf0rmationwas

    invest'gated1mmunafiuorescenceofdoublestainingof

    myosinandMTsexhibitedadistinctspot'likedistribution

    ofmvoEinthreughoutthecYtoPIasm.13osPecific

    coloca?zationwithcorticalMTswasobsewedbeforePPB

    formationininterphasecells(Figure3A)However,

56JournalofIntegrativePfafBiologyVo1.48No.12006

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    ConcentrationofBDM(mmol/L)

    O1251O2O5O1OO

    ConcentrationofML7(p.mol/L)

    Figure2.Widthofthemicrotubulepreprophaseband(PPB)is affectedbydisruptingtheactincytoskeletonwithlatrunculinA (UA),butnotbystabilizingtheactincytoskeletonwithphalloidin. BDM.2.3butanediOnemonoxime.Dataarethemean?SEM.

    (A)ThewidthofthePPBofArabidopsissuspensioncultured cellsincreasedwithLATAinaconcentrationdependentmanner,

    (B)ThewidthofthePPBswasalmostunchangedwhencells weretreatedwithvariouscOncentratiOnsofphalloidin. myosinwasc0ncentratedatthePPBsiteandformedan obviousbandthatwascolocalizedwiththeMTPPBdurinq thefOrmationofPPBs(Figure3B).Whencellsweretreated with50mm0l/L2,3-butanedinemonoxime(BDM),themyo. sinbanddisappearedandmyosinwasdispersedintothe cytoplasm(Figure3C).Theconcentrationofmyosinatthe PPBsiteduringPPBformationsuggeststhatmyosinmay beinvolvedjntheformationofPPBs.

    BDMhasbeenreportedtobeamyosinjnhibitorinplant

    studies(Samaieta1.2000,Tashaeta1.2002)andwas thereforeusedinthepresentstudytoinvestigatetherole ofmyosininPPBformation.Whencellsweretreatedwith variousc0ncentrati0ns0fBDM.rangingfr0m2.0to50 mm0I/L.thewidthofthePPBsincreasedinaconcentra. tiondependantmanner(Figure4A).Themeanwidthof thebroadMTbandswasapproximately(4.46?0.41)umin

    thepresenceof50mm0l/LBDM.anapproximate50% increasecomparedwiththePPBsincontrolcells.thewidth ofwhichwasapproximately(3.01?0.27)Um(Figure4A).

    Theseresultsarequitesimilarwithobservationsinthe LATAexperiments.Similarly.thewidthofPPBsalso reachedamaximumlevelwhentheBDMc0nCentrati0n wasabove20mmol/L(Fgure4A).Calculationsshowed thatthepercentageofcellswithbroadPPBsinsamples treatedwithBDMwasapproximately3.8%?0.2%.which

    wasnotsignificantlydifferentfr0mcontrolsamples(Table 1).Therefore,theformationofbroadPPBsisnothindered bvBDMtreatment.

    However,applicationofML7,amyosinlightchain

    inhibitor.tothesuspensionculturedcellsgavedifferent results.ThewidthofPPBsincellstreatedwithvarious c0ncentrati0nsOfML7rangingfr0m1t0100pmol/L,

    showednosignificantdifferencefr0mcontrolcells(Figure 4B).Therefore,theactivityofmyosin(s)involvedinPPB formationmaynotbeaffectedbythemyosinlightchain inhibitorML7,suggestingthatspecialmyosin(s)isre

    sponsiblefortheformationofPPBs.

    Nevertheless.ourobsewationsofmyosinlocalization andtheresultsofBDMtreatmentsdemonstratethatmyosin

(s)mayplayacertainroleinMTPPBformation.

    Discussion

    OrganizationofthePPBiscomposedoftwostages Resultsofboththepresentstudyandthoseofprevious reportsjndicatethattheactincytoskeletonplaysanimpor. tantroleinPPBformation(MineyukiandPalevitz1990; EleftheriouandPalevitz1992;Mineyuki1999;Barlowand Balu~ka2000).However,antiactindrugsdonotinhibit

    theprocessofcorticalMTsreorganizingontoPPBsitesto f0rmabroadMTband.butrestraintheconsequentevent ofthenarrowingdownofthebandtof0rmthenormalPPB. Sofar,itjsnotknownwhatfunctiontheactincytoskeleton hasinPPBorganization.1naddition.broadPPBswere alsoobsewedwhencellsweretreatedwithaproteinsyn. thesislnhibitororkinaseinhibitors(Nogamieta1.1996: KatsutaandShibaoka1992;NogamiandMineyuki1999). Therefore,itseemsthattherearetwostagesforcorticaI MTstoreorganizeandconcentrateontothePPBsites. Table1.Percentageofthecellswithanormalandabnorma microtubulepreprophasebandindrugtreatedcells

    DataarethemeantSEM.

    CK,cellswithnodrugtreatments.Anormalmicrotubule preprophaseband(PPB)wasobsewedincellstreatedwithphal

    IoidinandML7.AbnormalPPBswereobsewedinceilstreated withlatrunculinA(LATA)and2,3-butanedionemonoxime(BDM).

ActomyosinIsInvolvedIntheOrganizationofMicrotubulePreprophaseBand57

    Figure3LocalizalionofmyosinnArab~dopmssuspensionculLuredcellsduringthe~ormatl

    onorthemiorotubulepreprophaseband

    IPPB)ImagesincA)8howMTsinredImagesin(B)ehowmyos~nsineenandimagesin

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