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Microtubular

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MicrotubularMicrot

    Microtubular

Jan.2008,Volume2,No.1(SerialNo.2)JournalofLifeSciences,ISSN1934-7391,USA

    ;MicrotubularelementsofcortexinEuplotesWoodruffi ;LlYisong.LIULan-xia.GUFukang

    ;(SchoolofLifeScience,EastChinaNormalUniversity,Shanghai200062,China

    ;Abstract:Thecorticalciliaturemicrotubularorganellesofthe ;ciliatedprotozoanEuplotesWoodruffiwereanalyzedwith ;FLUTAXandanti0tubulinantibody.1tshowedthatthe ;corticalcytoskeletonwascomposedofnonciliaturestructure.

    ;ciliaturestructureincludingadoralzoneofmembranelles ;(AZM),undulatingmembranes(UM),frontalventraltransverse

    ;cirri(FVTC),caudalcirri(cc),dorsalkineties,aswellas ;baseassociatedmicrotubules.Thesilverlinesystemis ;composedoflongitudinalandtransversemicrotubules,concave ;structureappearedontheeelldorsalside.thebaseofDK ;containingrosettelikeskeletonstructure.Allthesesuggested ;thatthenonciliaturestructure.ciliaturestructureofEuplotesbe ;quitedifierentfromthatofotherspeciesofciliate,the ;silverlinesystembetruepareofcorticalmicrotubute ;cytoskeletoninEuplotes,rosettestructurebepartofthebase ;cytoskeleton.

    ;Kevwords:corticalmicrotubularcytoskeleton;FLUTAX: ;antidtubulin;EuplotesWDodruffi

    ;1.Introduction

    ;Theciliatecellshaveabundantformsof

    ;cytoskeletonincludingciliaturecytoskeleton,andother ;typesofmicrotubularcytoskeletonI1-5]

    ;,whichplaysan

    ;importantroleinshapepreservingandmorphogenesis.

    ;Recently,thefluorescentlabelingmethodhasbeenuse ;inthestudyonmicrotubularcytoskeletonofciliatecel1. ;itcouldfacilitatevisuaIizationoftheciliate ;microtubularsystem’sdistribution.Basedonthe

    ;resultsreceived.the0.microtubuleofcorticalIayerin

    ;EuplotesWoodruffiwaslabeledbybothFLUTAXand ;antic.microtubuleantibody.flseriesofnewstructures ;includingthebuccalcortexmicrotubules.dorsalkinety ;linesonthedorsalside.ventralanddorsalcortex ;Acknowledgments:ThisstudywassupportedbyNatural ;ScienceFoundationofChina(No.30270160).

    ;LIYisong(1972),Ph.D.,lecture;researchfield:celland ;molecularbiology.

    ;microtubulesetalthatneverreportedbeforehavebeen ;revealed,furthermore,theirfunctionhasalsobeen ;discussed.

    ;2.Materialandmethods

    ;2.1Material

    ;TheEuplotescellswerecollectedfromasmall ;lakeofChangfengpark,Shanghai.Clonalcultures ;wereestablishedandmaintainedinfiltratedwaterat ;roomtemperaturewithricegrainsasf0odsourceto ;enrichbacteria.

    ;2.2Methods

    ;2.2.1FIutax

    ;Thestainingmethodismodifiedwiththe

    ;referenceofthatreportedbyArregui[61.Theorganisms ;wereconcentratedwithsinteredgalssfilterofmedium ;porosityandplacedinadepressionslidecoveredwith ;polyLlysinesolution.permeabilizedwith0.5% ;saponininPHEMbuffer.after30s.thecellswerefixed ;ina2%paraformaldehydePHEMsolutionfor30s. ;extractedwith0,5%TritonX.1o0for1minandrinsed ;threetimeswithPBS.incubatedwithadropof ;FLUTAX2rMolecularProbes)for10rain.rinsed ;againwithPBS,andthencoveredwithaclean ;coverslip.Thepreparationwereobservedunderan ;OlympusBX5lfluorescencemicroscope.

    ;2.2_2Anti.0.microtubuleantibody

    ;Thismethodismodifiedwiththereferenceofthat ;reportedbyArreguil/1.Thecellswereconcentrated ;withsinteredgalssfilterofmediumporosityandplaced ;inadepressionslidecoveredwithpoly--L-?lysine ;solution.permeabilizedwith0.5%saponininPHEM ;buffer.after30s.thecellswereextractedwith0.5% ;MicrotubularelementsofcortexinEuplotesWoodruffi ;TritonX1oof0r5min.fixedina2%

    ;paraformaldehydePHEMsolutionforlh.andrinsed ;threetimeswithPBS+3%BSA.incubatedforlhinthe ;primaryantibody(abutulin,Sigma).Afterthree

    ;washesinthebuffertheywereincubatedinthe ;secondaryantibodyat1/1O0dilutionFlTC.1abelled ;goatantimouseIgGantibody,washedthreetimesin ;thebufferandmountedinCityfluor.Thepreparation ;wereobservedunderanOlympusBX51fluorescence

;microscope.

    ;3.Results

    ;Theresultsrevealedthatthecytoskeletonof ;EuplotesWoodruffiwerecomposedofciliature

    ciliature ;microtubularcytoskeletonandnon

    ;cicrotubularcytoskeleton.Theanchorshaped

    ;macronucleoluscouldeasilybeenobservedonthe ;greenbackground(Fig.1.

    ;3.1Ciliaturemicrotubularcytoskeleton

    ;Theciliatruemicrotubularcytoskeletonwere ;composedofadoralzoneofmembranelles(AZM), ;undulatingmembranes(UM),frontalventralcirri

    ;(FVC),caudalcirri,transversecirri(TC)and ;microtubularnetwork.

    ;3.1.1Ventralmicrotubularcytoskeleton

    ;TheJ-shapedAZMwascomposedof5565

    ;sheetlikestructureofmicrOtubulesinwhicheach

    like ;sheetissimilarinthickness(Fig.2),thesheet

    ;bracketoftheparaoralmembraneconsistsoftwo ;groupsofmicrotubule,onegroupparalleltoeachother ;transverselyandtheothergrouplongitudinally. ;ThenineFVCandfivetransversecirriTCwere ;arrangedintherightoftheventralsideofthecel1.Six ;ofFVClocatedonthetoprightoftheside,theleftin ;themiddle.Thekineticsassociatedmicrotubules,

    ;includingPosteriorLongitudinalMicrotubules(PLM), ;AnteriorLongitudinalMicrotubules(ALM),and ;RadiatingMicrotubules(RM),clearlyobservedand ;connectingwithothercorticalmicrotubules.form ;strongnetworktosupportthecell,theyarevariousin ;2

    ;lengthandorigination,forexample,ontheonehand, ;boththeALMandPLMarelongerthanRM.onthe ;otherhand,ALMoriginatingfromtopleftofthe ;kinetics,PLMfrombottomright.

    ;ThebaseofbothFVCandTClocatedinthe

    ;UshapedFibrillarCirrraiBasket(FCB).Thereexist ;somedifferencesinFBCexactstructure,forexample, ;thebottomisalittlethickerthanfringeinthatofFVC, ;whereasnotobservedinthatofTC.TheFVC

    ;microtubuleswereinterlacedwiththatofpellicle(Fig. ;21.Therewerefourcaudalcirrilocatedattheendofthe ;cell(Fig.2),whosebaseassociatedmicrotubules

    ;connectedwiththatoffrontalventralcirriformstrong

    ;networktostrengthenthemiddleandlatterparttocel1. ;3.1.2Dorsalkinetylinesonthedorsalsideofthe ;cell

    ;Thereareninelinesofkineticsondorsalsideofthe ;cell,thespacebetweeneachtwolines,locatingonthe ;formersideofthecell,isalittlelargerthanthatonthe ;latterofthecell,whichisconsistentwiththefigureof

    3O ;thecel1.Eachkinetylinewascomposedofabout25;kinetics(Fig.3),thelabeledkineticsappearpinshaped,

    ;therearerosetteshapedstructuresurroundingthe

    ;kinetieswereobserved(Fig.4).Concavestructures ;beneaththepelliclewerealsoobservedafterextraction, ;whichvariedinshape(Fig.1).

    ;3.2Non.ciliaturemicrotubulesinbuccaland ;ventralsideofthecell

    ;Buccalcortexmicrotubules,whichwerenearly ;parallellyarrangedandanchoredontwobundleof ;microtubules,wereobseserved,onebundleof ;microtubulesdisappearingwithhigherbiochemitry ;extraction(Fig.5).Therewerealsolatticeworkin ;ventralsideofthecell,butvariousinshapeand ;arrangement.someneartheAZ]Mwerearranged ;densely,otherswereloose(Fig.6).

    ;3.3Dorsalcorticalmicrotubules

    ;Twolinesoflattices,wovenbylongitudualand ;transversemicrobubulesandspreadinginthemiddleof ;twolinesofdorsalcirri.couldbeobserved.Theform ;oflatticesappearsdifferent,hexagonsformarather ;MicrotubularelementsofcortexinEuplotesWoodruffi ;prominentpartofthenetworkwhilerectangles,squares, ;etc.,arenotinfrequent.Thepattern,however,asseen ;inanyoftheindividualsisconstantandcharacteristic. ;Eachlatticeiscircledwithtwoparallelarrangedslick ;microtubules,whereas,twomorefiurenscentspots ;couldbeobservedonafewmicrotubules,andthespots ;weredifferentinsize,withhighermagnification,the ;largerspotswerecomposedofsomesmallspots.A ;bundleofmicrotubules,underneaththeconcave ;structureandspreadingalongthe|ongitudualanexof ;cell,couldbeobserved,granular-shapedmaterial ;covingtheotherpartofthecell(Fig.7).

    ;Thepellicleofciliatedisappearsafterbiochemical ;extraction,thecomplicatedmicrotubularnetwork ;appears,withhigherextractionintensity,transverse

    ;microtubulesdisappearingandonlylongitudualones ;left;beneaththepellicle,thereare,consistentwith ;previousobservationonEuplotessp.,twomorelayers ;offiberswithirregularlyarranged(Fig.8). ;4.Discussion

    ;4.1Thecolticaimicrotubulecharacterin

    ;EuplotesWoodruffi

    ;ThecorticalmicrotubuleofEuplotesWoodruffi ;wascomposedofciliatrueeytoskeletonand

    ;cytoskeletonassociatedfiberrevealedbytheboththe ;methodofFLUTAXandindirectflurescentlabeling. ;ThecorticalcytoskeletonincludeAdoralZoneof ;Membranelles(AZM),paraoralmembrane,

    ;fronto.ventralcirri.caudalcirrianddorsalkinetylines. ;OfwhichtheAZMcomposedofmembranellebracket. ;membranelleassociatedfiberetalparaora

    ;membranebracketcomposedofparalelledarranged

    associatedfibercomposedof ;microtubule,cirribase

    ;anteriorlongitudinalfibers,posteriorlongitudinal ;fibers,andradiatingfibers.Furthermore,kinetosome ;OfcirrilocatinginthefibrillarcirraJbasket.beneaththe ;pelliclethereexistsmoremicrotubulelayers. ;ThecorticalcytoskeletonofEuploteshavebeen ;reportedbefore…o1.Themethodsusua11vusedinclude

    ;bothsilverimpregnationandprotargoltechnique.But, ;theformercouldonlyrevealthekinetosomeandsilver ;silverlinesystem,anddifficulttoobservethese ;structureswithhighermagnification;thelatterisalso ;onekindofstainingmethodandnotparticularly ;labelledthemicrotubulesinciliate.Comparedwith ;thesetwomethods.FLUTAXandindirectflurescent ;labelingcanlabelthecorticalmicrotubular ;cytoskeleton,onthebaseofbiochemistryextraction, ;thefluorescencemicrographsofmicrotubular ;cytoskeletoninEuplotesWoodruffiarereported. ;4.2MicrotubularcytoskeletonofDKbasein

    ;EuplotesWoodruffi

    ;Regularlyarrangedconcavestructurewere

    ;observedspreadingonthedorsalsideofthecell,their ;distributioncharacterissimilartothatofthekinetics, ;andthishasnotbeenreportedbefore.rosetteshaped

    ;structurearoundthekineticscouldbeobservedwith ;highermagnification,thisindicatethatmore ;complicatedflameexistaroundthekinetics,whereas,

    ;moreexperimentsneedtobedonetorevealtheirexact ;functionandstructure.

    ;4.3Thecharacterofsilverlinesystem

    ;composedofmicrotubulesinEuplotes

    ;Thesilverlinesystemisakindofnetwork

    ;structure.whichcoversonboththedorsalandventral ;sideinEuplotes,andcouldonlybeobservedafter ;reactionwithsilvernitratewiththemethodofsilver ;impregnation,itsfigurecharacterisalsoakindof ;methodusedinciliatespeciesclassification112.The

    ;networkstructuretraitsrevealedbysilver ;impregnantionareinaccordancewiththatbyscanning ;electronmicroscopy,Moreover,thewholenetwork ;couldbeclearlyobservedaftertheEuplotescell ;demembranedwithbiochemistryandshorttime ;extractionmethod,alIofthesesupportedthefactthat ;thesilverlinesystemisarealstructureinciliate.The ;figureofnetworkinEuploteswoodruffilabeledby ;FLUTAXandantiamicrotubuleantibodyissimilarto ;thatrevealedbythemethodincludingprotargol,silver ;impregnation,andscanningelectronmicroscopy[11-12] ;,

    ;MicrotubularelementsofcortexinEuplotesWoodruffi ;so,wecouldgettheconclusionthatsilverlinesystemis ;onetruepartofcorticalmicrotubularcytoskeletonin ;Euplotes,whichhasneverbeenreportedbefore.The ;layermayfunctiononstrengtheningthecellpellicle, ;furthermore,itmayfunctiononintegratingthe ;structuresdistributingontheciliatecelJcortex. ;References:

    ;f1GaertigmJ.Molecularmechallismsofmicrotubular ;organelleassemblyinTetrahvmena.JEukMicrobiol, ;2000,47(3):l85.190.

    ;2HontsJE,WilliamsNE.Novelcytoskeletonproteinsin ;thecortexofTetrahymena.JEukaryotMicrobio1.20o3. ;50:914.

    ;3IflodeF’CleorotJC,LevilliersN,eta1.Tubulin

    ;polyglycylation:Amorphogenicmarkerinciliates.Bio ;C,,.20o0.92:615628.

    ;4LibusovaL,DraberP.Multipletubulinformsinciliated ;protozoan7,rahymenaandParameciumspecies. ;Protoplasma.2006.227:6576.

    ;51MclaughlinNB,HowardEB.Localizationbyindirect ;immunofluorescenceoftetrin,actin.andcentfintotheoral

    ;apparatusandbuccalcavityofthemacrostomalformof ;Tetrahymenavorax.JMicrobiol,2o04,J(2):

    ;253257.

    ;6ArreguiL,Mufioz-FontelaC,SerranoS,eta1.Direct ;visualizationofthemicrotubularcytoskeletonofciliate ;7

    ;8

    ;9

    ;10]

    ;12

    ;protozoawithafluorescenttaxoid.JMicrobio,.20o2. ;49:3l23l8.

    ;ArreguiL,MufiozFontelaC,GuineaA,eta1.FLUTAX

    ;faciliatesvisualizationoftheciliatureofoxytrichid ;hypotrichs.EuropJProtosto1.2003.39:169172.

    ;SchwarzMVJ,ZuendorfA,StoeckT.Morphology, ;ultrastructure,molecularphylogeny,andautecologyof ;EuploteselegansKahl,1932(Hypotrichida;Euplotidae) ;isolatedfromtheanoxicmariagerfjord.Denmark.J ;EukaryotMicrobio,,20o7,4(2):l25l36.

    ;SerranoS,SolaA,GuineaA,eta1.Cytoskeletonof ;Euplotes’focardii:Morphologyandmorphogenesis.Can.J.

    ;Zool,l992.70:20882O94.

    ;TIANQ,zHANGL,SUIS,eta1.Ciliaturecytoskeleton ;andmicrotubulinofrestingcystandvegetativecelIin ;Euplotesencysticus.ZoolRes,2O02,23(5):405408.

    ;RuffoloJ.Corticalmorphogenesisduringthecelldivision ;cycleinEuplotes:Anintegratedstudyusinglightoptical, ;scanningelectronandtransmissionelectronmicroscopy.J ;Morph..1976.148:489528.

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    ;(EditedbyAndrewJacksonChen)

    ;ExplanationofPlate:

    ;Fig.1Corticalmicrotubules(dorsalsideofthecelt)(Bigarrowshowedanchorshapedma

    cronucleus;smallalTOWSshowedround

    ;orellipticalconcave).

    ;Fig.2Cytoskeletonofadoralzoneofmembranelles(AZM),undulatingmembranes(UM),

    frontalventralcirri(FVC)and

    ;transversecirri(TC)andassociatedmicrotubules(PLM:Posteriorlongitudinalmicrotubu

    les;ALM:Anteriorlongitudinal

    ;microtubules;RLM:Radiatingmicrotubules;FCB:Fibrillarcirrralbasket);Arrowshowe

    dfourcaudalcirrilocatedatventralsideend

;ofthecel1.

    ;Fig.3Dorsalkinetylinesonthedorsalside(Ninekinetylinesparallelarranged). ;Fig.4Rosetteshapedstructurearoundthekineties.

    ;Fig.5Buccalcortex(BC)andbaseassociatedmicrotubules.

    ;Fig.6Ventralcortexmicrotubules(BigarrowshowedlatticeworkneartheAZMarrangeddensely;smallarrowshowed

    ;microtubulesharedbyadjacentlatticework).

    ;Fig.7Dorsalcortexmicrotubules.

    ;Fig.8Longitudinalmicrotubulelayerbeneaththepellicle. ;

    Fig.4thefluorescencelabelingofFLUTAX;Fig.1,2,3,5,6,7,8thefluorescencelabelingof

    antiatubulinantibody.Scalebar

    ;indicatesl0m.

    ;4

    ;MicrotubularelementsofcortexinEuplotesWoodruffi

    ;5

    ;

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