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. Sequencesimilaritytothe In vitroexperimentsshowthat from thespindletophragmoplastsandnuclearenvelopeintelophaseI,backplastidsurfacesprophase II. land plants.TheG9antibodytofissionyeast ole pairembeddedinanamorphous matrixwithastralmicrotu- centrosome, acompoundstructurecomposedofcentralcentri- Oakley, 2000).AtypicalMTOCinanimalandalgalcellsisthe to playanessentialroleinmicrotubulenucleation(reviewedby ganizing centers(MTOCs)ineukaryoticcells,whereitisthought stricted totheminusendsofmicrotubulesatmicrotubuleor- not affectedbythedestructionofmicrotubulesoryzalin.Weconcludethat 466-8550, Japan www.plantcell.org/cgi/doi/10.1105/tpc.016501. Article, publicationdate,andcitation informationcanbefoundat hiroshima-u.ac.jp; fax81-824-24-0734. 1 Although seedplantshave g f e d c b a Tetsuya Horio, Naohisa Nishihara, Masaki Shimamura, The PlantCell,Vol.16,45–59,January2004,www.plantcell.org©2003AmericanSocietyofBiologists in theliverwort trosomal plantspindle.Wehaveisolatedandcharacterizedthe plants hasrevealedasurprisingvarietyofMTOCsthatmayrepresentmilestonesintheevolutiontypicaldiffuse acen- trosomes, andtheorganizationofmicrotubulearraysinplantshasremainedenigmatic.Spindledevelopmentbasal land gal andanimalcells,theydonothavediscretemicrotubuleorganizingcenters(MTOCs)comparableto cen- dimers aremajorcomponentsofmicrotubules, tubules canberepolymerizedfromtheisolatedplastids. INTRODUCTION orderly successionofmicrotubulearrays. ciated withmicrotubulenucleationinbasallandplantsandthatithasacellcycle–dependentdistributionessential forthe (Oak of bryophytes).Changesinthedistribution basal landplants(e.g.,discretecentrosomeswithandwithoutcentriolestheplastidsurfaceinmonoplastidic Immunohistochemistry withtheG9antibodyclearlydocumentedassociationof Organizing Centers and ImplicationintheEvolutionofAcentriolarMicrotubule

Otsuka DepartmentofMolecularNutrition,SchoolMedicine,UniversityTokushima,Tokushima770-8503,Japan

To whomcorrespondenceshould beaddressed.E-mailmineyuk@ - isthethirdmemberoftubulinsuperfamily,with Department ofBiology,GraduateSchoolScience,OsakaUniversity,Toyonaka,560-0043,Japan Department ofBiology,UniversityLouisiana,Lafayette,Louisiana70504 Department ofBiologicalScience,GraduateSchoolHiroshimaUniversity,Higashi-Hiroshima739-8526,Japan Department ofNutrition,SchoolMedicine,UniversityTokushima,Tokushima770-8503,Japan Division ofMolecularMycologyandMedicine,NagoyaUniversityGraduateSchool65Tsurumai, Plant MolecularPhysiologyLaboratory,ResearchInstituteofInnovativeTechnologyfortheEarth,Kizu,Kyoto619-0292,Japan

30% aminoacidsequenceidentitywith

-Tubulin inBasalLandPlants:Characterization,Localization,

ley andOakley,1989).Although

Dumortiera hirsuta

g

andYoshinobuMineyuki

a

Ken-IchiTomizawa,

a,b

RoyC.Brown,

-tubulin, aubiquitouscomponentofcentrosomesassociatedwithmicrotubulenucleationinal-

-tubulin isdetectableonthesurfaceofisolatedplastidsandnuclei

.

-tubulin geneofhigherplantssuggeststhatthe

-Tubulin changesitslocalizationfromtheplastidsurfaceinprophaseItospindle, - and

c BettyE.Lemmon, - and b -tubulin hetero- a,1 KatsuhikoYoshimoto, -tubulin isre- -tubulin recognizedasinglebandof55kDinimmunoblotsfrombryophytes. -tubulin occurinacellcycle–specificmannerduringmonoplastidicmeiosis - -Tubulin localizationpatternsonplastidandnuclearsurfacesare c TomohiroAkashi, and McIntosh,1980). the centrosomeandtheirplusendsfacingoutward(Heidemann . Microtubulesalwayshavetheirstableminusendstoward the nucleationofmicrotubulesandinspindlepoleformation in bules radiatingfromit.Thepericentriolarmaterialisimportant in (Joshi etal.,1992; JoshiandZhou,2001). the centrosomeandmorphogenesis ofthemitoticspindle antibody disruptsboththenucleation ofnewmicrotubulesfrom provided evidenceforanimportant roleof Zheng etal.,1991,1995).Numerous experimentalstudieshave fungal spindlepolebodies(Horioetal.,1991;Stearns imal polarityofmicrotubulesrelativetoanimalcentrosomesand hance thenucleationofmicrotubulesandsetupminus-endprox- Shu andJoshi,1995).Forexample, microinjectionof trosome function(Felixetal.,1994; StearnsandKirschner,1994; form largerring-shapedcomplexes.These it appearstoassociatespecificallywithseveralotherproteins to -tubulin genefromaliverwort,oneoftheextantbasalland f HironoriDeguchi, -tubulin isahighlyconservedproteinasso- d KoichiMizuno, -Tubulin isenrichedinMTOCsites,where -tubulin geneishighlyconservedin -tubulin withvariousMTOCsitesin a HiroshiHosoya, D. hirsuta e -tubulin ringsen- -tubulin incen- a , andmicro- -tubulin The Plant Cell oughly documentthedistribution andlocalizationof mation regarding 1984; VaughnandRenzaglia,1998).However,thereisnoinfor- trosomes arisedenovoandserveasspindleMTOCs(Robbins, lope. Inspermatogenouscellsofbryophytes,centriolarcen- tubule polymerizationoccurredotherthanatthenuclearenve- and Gunning,1989),oneoffewcasesinplantswhichmicro- demonstrated microtubuleregrowthfromtheplastids(Busby experimental studiesusingmicrotubule-depolymerizingdrugs microscopy (BrownandLemmon,1982,1987),importantly, the plastidenvelopehavebeenshownbytransmissionelectron tus (BrownandLemmon,1997).Microtubulesemanatingfrom comitantly serveasMTOCsofthemitoticandmeioticappara- surfacefunctioninpartitioning,andplastidscon- grates beforenucleardivision.Microtubulesradiatingfromthe al., 2003).Likethecentrosome,plastiddividesandmi- well occurincellsthatcontainasingleplastid(Shimamuraet 1993). Inmanybryophytes,meiosisandsometimesmitosisas between greenalgaeandseedplants(BrownLemmon, this mitoticapparatusisconsideredtobeatransitionalstate organizers orcentrosomes(BrownandLemmon,1990), arise fromacentriolarsphericalstructuresdescribedaspolar have well-definedMTOCs.Inliverworts,mitoticspindlepoles phytes, thisgroupisexceptionalinthattheircellsalsomay systemsmentionedabovearefoundinbryo- for consideringtheevolutionoflandplants.Althoughfive (mosses, liverworts,andhornworts),representapivotalgroup (Palevitz, 1993). MTOC iscloselyassociatedwitheasilyidentifiedcentrosome withmodelsbasedonanimalcells,inwhichthe diffuse andmigratory.Thisfactmadeitdifficulttoidentify bule dynamicsbyShawetal.(2003),MTOCsofseedplantsare evident fromtherecentdirectobservationofcorticalmicrotu- phase, preprophasebands,spindles,andphragmoplasts.Asis dial arraysfromthenuclearsurface,corticalofinter- Five distinctmicrotubulesystemsgenerallyarerecognized:ra- characterized byanorderlysuccessionofmicrotubulearrays. manner. Dividingvegetativecellsofvascularlandplantsare and plastidenvelopes,relocateinacellcycle–specific endomembranes, includingendoplasmicreticulumandnuclear appear tobedistributedonthecytoplasmand/oravarietyof ble tocentrosomes.Instead,nucleationsitesformicrotubules ens encoding present study,weisolatedand characterizedagenomicclone function postulatedtohaveoccurredduringplantevolution. standing oftheevolutionarychangesinMTOClocalizationand could beexpectedtoprovideevidenceincreaseofunder- sequenced inonlytwospecies,themoss ThePlantCell 46 japonicum in diverseMTOCs inbryophytes. However, beyondtheseedplants, been clonedandsequenced(OvenchkinaOakley,2001). bule systems.Analysisof -tubulin. WethenusedtheG9 anti- The oldestlivinglineageoflandplants,thebryophytes Typical landplantcells,however,havenoMTOCscompara- A largenumberofplant andthefern , andcomparedthesequences amonglandplant -tubulin fromaliverwort(Bryophyta), Anemia phyllitidis -tubulin distributionintheseuniquemicrotu- -tubulin distributioninbryophytes -tubulin cDNAsandgeneshave (Fuchsetal.,1993).Inthe -tubulin antibodytothor- -tubulin cDNAhasbeen

Physcomitrella pat-

Conocephalum

-tubulin

The First, itwasimmediatelyapparentthat cum cephalum japonicum japonicum moss with restrictionenzymesandprobedaDNAprobeofthe cum in thehaploidgenomeof with These dataindicatethattherearenoothergeneshomologous quences ofisolatedgenesweredetected(datanotshown). of Bryophytes those ofotherlandplants( are mostconsistentifthereisonlyone lin was74.7%tothatof lin–like proteinTub4p,oneoftheunconventional belonged totheconventionalgroupof First, weidentifiedthe Analysis oftheGeneEncoding RESULTS We usedthemonoclonalanti- G9 Anti- P. patens and aComparisonof maize, 91.0%).Theaminoacididentityof 96.4%; Arabidopsis,90.1%;rice,89.9%;tobacco,89.2%; raised against idues. Throughouttheprocessof frame of1428bpthatencodedaprotein475aminoacidres- Connecting 11exonsrevealedthattherewasanopenreading gene encompassing3582bpandwasintervenedby10introns. to thatofthefissionyeast blot hybridization. possibility ofthepresencehomologousgenesbyDNAgel conventional shared atleast67.5%aminoacididentitywithotherknown vealed thatthe method. SequencingofbothstrandsthePCRfragmentsre- cum of the used todesignPCRprimersisolatetheentiregenomicDNA amplification wasperformedusingtheseprimersand lin was39%identicaltothe such asthefern conserved sequencesofknownconventional designed oligonucleotideprimersthatcorrespondedtothe among plant plants (Figure1).Theresultsofouraminoacidcomparison amplification, ofthepresencemorethanone phism amongtheisolatedclonesaftervector-annealingPCR fragment ofmorethanonesizeorobvioussequencepolymor- we encounterednoevidence,suchasamplificationofgenomic

We comparedthededucedaminoacidsequenceof

genomicDNA.Thedeterminednucleotidesequencewas

-tubulin in -tubulin geneproductishighlyconservedamongland -tubulin withthesequencesofotherknown

-tubulin showed89.2to97.7%aminoacididentity

-tubulin. Onlythebandsofexpectedsizesfromse-

-tubulin genebymeansofthevector-annealingPCR

.

-Tubulin Antibodyanda55-kD Peptide

,asisthecasewithotherlowerlandplants,

-tubulins. Ontheotherhand,

S. pombe

-tubulins isshowninFigure1B.The

C. japonicum

A. phyllitidis

-tubulin of

C. japonicum

. Toaccessthegenomicinformation,we

Chlamydomonas reinhardtii -Tubulin AminoAcidSequences -tubulin genefromtheliverwort

C. japonicum

-tubulin (Horioet al.,1999),todetect

Schizosaccharomyces pombe

Saccharomyces cerevisiae (Fuchsetal.,1993)andthemoss

C. japonicum

P. patens . Thus,weconcludethatourdata

-tubulin antibodyG9,whichwas

genomicDNAwasdigested

-Tubulin fromaLiverwort

-tubulin geneidentification,

. Wefurtherexploredthe

, 97.7%;

C. japonicum

-tubulin geneinthe

C. japonicum -tubulins, becauseit

C. japonicum wasencodedbya

-tubulins. PCR

-tubulin gene A. phyllitidis

and69.3% C. japoni- C. japoni-

C. japoni- -tubulins. -tubulins.

-tubulin . Cono- -tubu- -tubu- -tubu- C. , The Plant Cell method witharithmeticmeanalgorithm. The (A) Figure 1. lins werealigned, andaminoacidsidenticaltothose ofthe plant (B) ate thetreearelistedin -tubulin isshowninthetoprow.Amino acidsequencesofplant Deducedphylogenytreegenerated bytheunweightedpairgroup Aminoacidsequencecomparison ofthe -tubulins. Thededucedaminoacid sequence ofthe Characterization ofthe (B) . -Tubulin GenesofBasalLandPlants. -tubulins usedtogener- C. japonicum C. japonicum andother C. japoni- -tubu- was determinedthattheG9antibodydidnotrecognize unusual quadripolarspindle(BrownandLemmon,1997). In tetrad nuclear division.Thepositioningofthefourplastidsat I,thesingleplastiddividesintofourplastidsbefore The sporocytesofbryophytestypicallyhaveasingleplastid. In QMS inMossMeiosis cross-reactive materialsinputativeMTOCsbryophytes MTOC sites,weexaminedthelocalizationofG9anti- The cence stainingofseedplants(OvenchkinaandOakley,2001). ure 2B).G9hasbeenusedsuccessfullyforimmunofluores- ety ofbryophytesmostlikelyis These factsindicatedthattheepitopedetectedbyG9inavari- munofluorescence stainingusingG9(ourunpublisheddata). with asinglebandat cum S. pombe bule systems.Toascertaintheoccurrenceof cortical microtu- Reproductive cellsgenerallylackdistinctive the RMS,theseMTOCshaveneverbeenseenin for Except (Brown andLemmon,1988;Shimamuraetal.,1998). radial microtubulesystems(RMSs)duringmeiosisin marchantialean liverworts(BrownandLemmon,1990,1992), Renzaglia, 1998),polarorganizers(POs)ofmitoticspindles spindle polesincellsundergoingspermatogenesis(Vaughnand (QMSs) ofmeioticcells(BrownandLemmon,1997),centriolar such astheplastidsurfaceinquadripolarmicrotubulesystems Bryophytes areknowntohaveavarietyofuniqueMTOCs, Subcellular LocalizationofG9Anti- narrowed downtoaminoacidresidues97111of epitope detectedbytheG9antibodyhasbeenstudiedand molecular massofthepredicted in Bryophytes Cross-Reactive MaterialsinVariousMTOCSites immunoblots ofextractsfromsporophytes pombe sporophytes fromanotherbryophyte, region isalmostcompletelyconservedamongknownplant pom phy (Cj), cer thaliana H. sap underlined. for thealignment.TheinferredrecognitionepitopeofG9antibody is cum -tubulin homologsinvariousspeciesofbryophytes.The -tubulins (Figure1B,underlined) andisfairlywellconservedin -tubulins (GGGAGNNWANGYSHA; ourunpublisheddata).This -tubulin -tubulins (Figure2A,lanes3and4)butclearlycross-reacted , (Ap),

Saccharomyces cerevisiae (Sp), is53,359D.Similarresultswereobtainedinextractsof Conocephalum japonicum -tubulin areindicatedbydashes.Dotsindicategapsintroduced -tubulin ofArabidopsisexpressedinthefissionyeast , poles isassociatedwithaQMSandtheformationofan ; hasbeendetectedbyimmunoblotanalysisandim- Homo sapiens N. tab Anemia phyllitidis Schizosaccharomyces pombe

-tubulin (11of15aminoresiduesareidentical).In (Nt), Nicotiana tabacum ; X. lae 55 kD(Figure2A,lane5).Theestimated ; C. rei , . ; Xenopus laevis TblnadMO vlto 47 -Tubulin andMTOCEvolution P. pat (Cr), -tubulin. -tubulin proteinof (Pp), ; ; Chlamydomonas reinhardtii Z. may A. nid -Tubulin Dumortiera hirsuta Physcomitrella patens ; , (Zm), A. tha Aspergillus nidulans C. japonicum -tubulin inthese (At), Zea mays seed plants. C. japonicum

Arabidopsis

S. pombe C. japoni-

-tubulin

;

(Fig- C. jap

- or

; ; ; , it

S.

A. S. S.

The Plant Cell sis in spindle (BrownandLemmon,1992).Duringarchesporialmito- novo outsideofthenuclearenvelope,arefociaprophase donotserveasMTOCs.Instead,POs,whicharisede During mitosisinpolyplastidiccellsofmarchantialeanliverworts, Marchantia polymorpha POs andMetaphaseSpindlesinArchesporialMitosisof in Figure3Earethepositionofcentriolarcentrosomes. pata final spermatogenouscelldivisionintheliverwort Renzaglia, 1998).Figures3Dto3Fshowthecelljustbefore the finalmitosisofspermatogenousdivision(Vaughnand for bicentrioles andassociatedpericentriolarmaterialseparate in MTOCsjustoutsidethenuclearenvelope.Onceformed, In spermatogenouscellsofbryophytes,bicentriolesarisedenovo Centriolar MTOCsinMitosisofSpermatogenousCells ous punctae(Figure3B,arrowheads). distributed aroundthesurfacesoffourplastidsasnumer- QMS (Figure3A).G9anti- bules emanatefromindividualplastidsandinteracttoforma prophase Isporocytesofthemoss ThePlantCell 48 (Figure 3J).TheG9stainingpatterninthemetaphasespindleis POs of (cf. Figures3Gand3H).Asthemetaphasespindledevelops, was nocolocalizationofmicrotubulesand cross-reactive materials(Figure3H)correspondtoPOs.There crete POs(Figure3G).DistinctivedotsofG9anti- against without primaryantibodies;lanes 3and4,monoclonalantibodies Lane 1,Coomassiebrilliantbluestaining; lane2,controlimmunoblot (B) (A) indicated atleft. sitions ofmolecularmassmarkers and theirmolecularmasses(kD)are Sporogenous TissueoftheBryophytes Figure 2. C.japonicum D. hirsuta . Twodistinctivefluorescentdotsofthe Marchantia polymorpha M. polymorpha - and Immunoblot AnalysisofG9AntibodyinProteinExtractsfrom . -tubulin, respectively;lane5,G9anti- . disappearandspindlepolesdisperse -tubulin cross-reactivematerialsare , prophasespindlesfocusondis- C. japonicum Entodon seductrix -tubulin homolog -tubulin homolog and -tubulin. Thepo- Makinoa cris- D. hirsuta , microtu- -tubulin . Members ofthegenus in Spindles, RMSs,andPhragmoplastsduringMeiosis the poleregions(Figure3K). more diffusethanthatinprophase,butitisclearlyrestrictedto netic apparatusduringmeiosisin from telophasenucleiareanimportantfeatureofthecytoki- the elongatesporocyteintoalineartetrad.RMSsemanating second roundofmeiosis,simultaneouscytokinesispartitioned clearly biasedtowardthespindlepoles(Figure3N).After crotubules ofthematurespindle,butwithdistribution G9 anti- other anddeliverthechromosomestobroadtruncatedpoles. foci. Thus,thespindlemicrotubulesareorientedparalleltoeach are barrelshapedwithbroadpolesthatneverconvergeinto Lemm some uniquemicrotubulesystemsduringmeiosis(Brownand antibody (G9)was omitted,plastidswereunstained (Figures4I surface (Figures4Gand4H).In controlsinwhichtheprimary bules, themajorityof portion of calization of confocal laserscanningmicroscopy demonstrateddifferentlo- the dividingplastids(Figures4D and4F).Opticalsectionsby anti- emanated fromdividingplastids(Figures4Cand4E).G9 ure 4B).Inplastiddivisionduringprophase, tive materialsweredistributedrandomlyinthecytoplasm(Fig- spanned thecytoplasm.DotsofG9anti- In premeioticinterphase,areticulatearrayofmicrotubules Premeiotic PlastidDivisionandPartitioning 2001). sis in material. Themicrotubuleandplastiddistributionduringmeio- anti- ognized MTOCsites,furtherstudiesofthedistributionG9 cells of Becausemeiotic Figure10). moplasts (telophaseIandII)(see region (metaphaseIandII)tothenuclearsurfacephrag- from theplastidsurface(prophaseIandII)tospindlepole and positionduringthecellcycle(BrownLemmon,1997) In monoplastidicbryophytes,meioticMTOCschangeinform MTOC Cyclein Cross-Reactive MaterialsduringtheMonoplastidicMeiotic Change intheLocalizationofG9Anti- 3O). arrowheads) thanformicrotubules(Figure conspicuous whenstainedfor stained midzonewherethecellplateisdepositedwasmore MTOCs oftheRMSandinphragmoplasts(Figure3P).Theun- spicuous bothatnuclearsurfacesthesitesofputative moplasts. G9anti- equal distributionofcytoplasmandthepositioningphrag- Conocephalum -tubulin cross-reactivematerialswereperformedwiththis D. hirsuta -tubulin cross-reactivematerialswerelocalized around -tubulin on, 1988;Shimamuraetal.,1998).Metaphasespindles D. hirsuta -tubulin cross-reactivematerialslocalizedalongmi- -tubulin homologseemedtolocalize alongmicrotu- -tubulin homologsandmicrotubules. Althougha havebeendescribed(Shimamuraetal.,2000, D. hirsuta arerelativelylarge( -tubulin cross-reactivematerialswerecon- -tubulin homologoccurredatthe plastid Conocephalum -tubulin homologs(Figure3P, Conocephalum 30 (Marchantiidae)have -Tubulin -tubulin cross-reac- m) witheasilyrec- , ensuringthe The Plant Cell anti-plant-tubulin, G9anti- Figure 3. C. japonicum ( (A) Sites inBryophytes. Microtubules ( poles tion of Marchantia polymorpha dots (D) plastids (arrowheadsin MTOCs, thenuclearsurface,andphragmoplasts (G) M) to to to to (E) (N) (F) (P) (C) (L) -tubulin extendingalongproximalportions isseeninametaphasespindlethathasdispersedMTOCs attwodistinctiveMTOCs . RMSsemanatingfromtelophasenuclei (asterisksin Aprophasespindlewithcentriolesinthefinalspermatogenousmitotic divisionoftheliverwort Abarrel-shapedmetaphasespindle inmeiosisI( AprophasespindlewithPOs( AQMSduringmeioticprophaseofthemoss Triple Labelingfor . Notethat [A] , [D] , [G] . Notethatalthoughaprophasespindle arisesfromthediscretePOswhere [B] -tubulin homologslocalizedonthe mature spindlemicrotubules,butdistributionclearlywasbiasedtoward thebroadspindle , [J] -tubulin, and4 andasterisksin -Tubulin, , [M] (D) , and areconsideredtobeequivalentcentriolarcentrosomes. and [G] [O] ,6-diamidino-2-phenylindole (DAPI),respectively.Bars [C] to ), -tubulin homologs( ) fromwhichmicrotubulesemanate [I] -Tubulin, andDNAShowstheSubcellularLocalizationof (P) ) andametaphasespindlewithbroadpoles( . Arrowheadsin Entodon seductrix [M] and [O] ) andphragmoplastsdevelopbetween nuclei. [N] [B] (P) ) andRMSsphragmoplastsinmeiotic telophaseII( showthreenewlyformingcellplates thatappearasdark,unstainedlines. , [E] , [H] . Notethat , [K] (A) , [N] . -tubulin homologs , and [P] [J] ), andnuclei( to 10 -tubulin homologslocate [L] m. Makinoa crispate ) duringarchesporialmitosisoftheliverwort -Tubulin HomologsinSomeTypicalMTOC (B) TblnadMO vlto 49 -Tubulin andMTOCEvolution arelocalizedaroundthefourdaughter [C] (K) -Tubulin homologslocalizedatputative , [F] . , [I] , and . Twodistinctivefluorescent [O] and [L] (H) ) werestainedusing , thepolardistribu- [P] ) oftheliverwort The Plant Cell 0ThePlantCell 50 in (E) (C) the [B] Figure 4. (A) of mologs duringPlastidDivisionandPartitioninginMonoplastidicMeiosis [D] D. hirsuta and to and ). Notethatdotsof ). Notethe (H) (B) (D) Amid-prophase cell withfourdaughterplastids(asterisks in Apremeioticinterphasecellwitha singleplastid(asteriskin Changes intheSubcellularLocalizationof Anearlyprophasecellwithtwodaughter plastids(asterisks (PremeioticInterphasetoProphase I). (B) -tubulin homologslocalizedaround thetwoplastids. withreticulararraysofmicrotubules -tubulin homologsaredistributed randomly in (A) -Tubulin Ho- . nations. Bar and G9 anti- [D] [F] (I) optical sectionsof daughter plastidsbecomesmoreprominent. the nuclearsurfaces.Opticalsectionsclearlyshowlocaliza- bled betweenallsisternuclei(Figures6Eand6F).Atthisstage, the surfacesoffournuclei,andphragmoplastsreassem- ing telophaseII,radialmicrotubulesonceagainemanatedfrom lar regionsofmetaphaseIIspindles(Figures6Cand6D).Dur- anti- spindles wereidenticaltothoseofthemetaphaseIspindle.G9 surfaces (Figure6B).DevelopmentalfeaturesofmetaphaseII tion tothespindlemicrotubules.Translocationof set ofseveralmicrotubulesemanatedfromtheplastidsinaddi- dots. Contrarytothelocalizationof poles onthetipsofdistortednucleusasdistinctfluorescent disappeared fromtheplastidsurfaceandappearedatspindle In lateprophaseI,G9anti- Meiosis I through theprocessofdetergentextraction(seeMethods). and 4J).Autofluorescenceofplastidswasremovedcompletely in Microtubules (greensignalin observed. [E] [I] prophase nuclei(Figure6A).PunctatestainingofG9anti- prophase II,microtubulesemanatingfromplastidssurrounded completing newcellwalls,abinucleatedwasformed.In Because phragmoplastsintelophaseIdisappearedwithout Meiosis II clearly absentfromtheplastidsurfaces(Figures5Gand5H). bothinphragmoplastsandonthenuclearsurfacebutwere tected cell plate.G9anti- wasverywelldevelopedbutincompetenttodeposit a moplast Lemmon, 1988;Shimamuraetal.,1998),thefirstdivisionphrag- As showninpreviousstudiesof emanated fromthenuclearsurfaces,andphragmoplasts. crotubule systemsappeared:theradialmicrotubules,which and (Figure5F,arrowheads).IntelophaseI,twomi- regions ofthespindleduringmetaphase(Figures5Cand5D) cross-reactive materialswerelocalizedconspicuouslyatpolar gions oftheformingspindle.InmeiosisI,G9anti- spindle (Figures5Aand5B).Distinctspotsmarkedthepolarre- seemed toprecedethereorganizationofmicrotubulesinto -tubulin homologsweredetectedinphragmoplastsandon -tub and ). Theprimaryantibody(G9)isomitted(asimilarstageshownin [A] ). Notethatthelocalizationof ). Crosstalkandnonspecificsignalofsecondaryantibodywerenot , [F] (H) , [C] -tubulin cross-reactivematerialswerelocalizedinthepo- ulin cross-reactivematerialsreappearedontheplastid (J) , , [H] -tubulin, respectively. and Amid-prophasecellwithfourdaughterplastids(asterisksin , [E] , and (I) , [G] 10 and m. [J] (E) , and (J) ) werestainedusinganti-plant-tubulin, DAPI,and and showthesamecellswithdifferent colorcombi- -tubulin cross-reactivematerialswerede- [I] (F) ), and , respectively. [A] (A) , [C] -tubulin homologs(redsignalin -tubulin cross-reactivematerials and -tubulin homologsaroundthefour , [E] Conocephalum (B) , [G] -tubulin homologs,asub- , (C) , and (G) and and [I] (D) ), DNA(bluesignal -tubulin homolog , (E) (H) (Brownand showsingle and -tubulin (F) , (G) [B] , The Plant Cell 10 and [F] in Figure 5. (E) chore side. calized alongspindlemicrotubulearraysbutareabsentfromthekineto- (C) [A] (A) hirsuta mologs duringtheProgressionofMeiosisIinMonoplastidicCells polar surfaceofeachdaughternucleus(arrowheadsin Microtubules (greensignalin plate isshownbyarrowheads. in phragmoplastsandonthenuclearsurface.Thepositionofcell (G) -tubulin, respectively. [A] and and(B) , and and ). Notethatthe and m. (H) , [C] (PrometaphasetoAnaphaseI). showthesamecellswithdifferent colorcombinations.Bar (F) (H) (D) [H] , [E] AcellinanaphaseI. Aprometaphasecellwithfourdaughterplastids(asterisksin Changes intheSubcellularLocalizationof AcellintelophaseI. AcellinmetaphaseI.Notethat ) werestainedusinganti-plant-tubulin, DAPI,andG9anti- , and -tubulin localizesdistinctlynearthespindlepoles. [G] ), and (A) and -tubulin homologs(redsignalin [A] -Tubulin homologsarelocalizedatthe -Tubulin homologsarelocalizedboth (B) , [C] , (C) , [E] and , and -tubulin homologsarelo- (D) [G] , (E) ), DNA(bluesignal and [F] -Tubulin Ho- ). (F) ,

[B] and , [D] (G) D. , RMSs (Figure8A).Cellstreatedwith20 more sensitivetothetreatmentthanwerespindlefibersand disorganization. Microtubulesemanatingfromplastidswere cum The presentstudyshowsthat the The DISCUSSION (Figures 8Gand8H). G9 anti- Longer oryzalintreatments(2h)didnotaffectthelocalization of and microtubuleremnantsofRMSs(Figure8E,arrowheads). and 8F).Therewasnocolocalizationof surface wasnotaffectedbytreatmentwithoryzalin(Figures8E The localizationof remnant microtubulesofspindlefibers(Figures8Cand8D). anti- to disorganization.Inmetaphasecells,thedistributionofG9 by oryzalin.Spindlemicrotubulesseemedtobemostresistant face (Figure8B,asterisks)whenmicrotubulesweredisrupted materials weredetectableonorneartheprophaseplastidsur- prominent asincontrolcells,G9anti- and RMSs(Figures8A,8C,8E).Althoughitwasnotas no microtubulesotherthansomeremnantsofspindlefibers try. Solutionsof ing anti-microtubuleandG9anti- were treatedwithoryzalinsolutionfor20minandexaminedus- crotubule system,sporocytesof in MTOCsiteswasaffectedbythedisorganizationofmi- To testwhetherthespecificlocalizationof after TreatmentwiththeAnti-MicrotubuleDrugOryzalin Distribution ofG9Anti- surfaces. Suchmicrotubulesmayobtainalengthof10to20 (Figures 7Gand7H)wereabletonucleatemicrotubulesattheir bulin purifiedfromanimalsources,unfixedisolatedplastids was investigated.Whenincubatedwithrhodamine-labeledtu- tubule-initiating activityfromthesurfacesofisolatedorganelles indicated byimmunofluorescence(Figures7Cand7F).Micro- plastids andnucleihad terns (Figures7A,7B,7D,and7E).Lessthan50%ofisolated from otherorganellesbysize,shape,andDNAstainingpat- D. hirsuta tive materialsonisolatedorganellesfrommeioticsporocytesof attempt wasmadetoconfirmthelocalizationofG9cross-reac- that Because immunofluorescenceobservationsstronglysuggested on IsolatedPlastidsandNuclei Distribution ofG9Anti- cleus (Figures6Gand6H). nuclear surface,andtherewasnosignalfromwithinthenu- tion of m (Figures7Iand7J). mostlikelyis present asasingle-copygene inthege- -tubulin cross-reactivematerialswasrestrictedalongthe -tubulin isassociatedwithorganellesduringmeiosis,an -Tubulin GeneIsConservedin LandPlants -tubulin homologsatpresumptiveMTOCsitesonthe . Isolatedintactplastidsandnucleiweredistinguished -tubulin cross-reactivematerialsaroundtheplastids 5 -tubulin homologsonthetelophasenuclear M oryzalincauseddrasticmicrotubule -tubulin homologsattheirsurfaces,as -Tubulin Cross-ReactiveMaterials -Tubulin Cross-ReactiveMaterials TblnadMO vlto 51 -Tubulin andMTOCEvolution -tubulin immunocytochemis- D. hirsuta -tubulin geneof -tubulin cross-reactive M oryzalincontained -tubulin homologs inmeioticstages -tubulin homologs C. japoni- The Plant Cell show thesamecellswithdifferentcolor combinations.Bar and [A] bulin, respectively. Figure 6. ThePlantCell 52 Microtubules (greensignalin and (C) tion of (A) hirsuta mologs duringtheProgressionofMeiosisIIinMonoplastidicCells tween daughternuclei. ized atthenuclearsurfaceandearlyphragmoplastsarelocalizedbe- (E) mologs inthepolarregionofspindlesisidenticaltothatprophaseI. to and and , [C] (F) [H] (H) (ProphaseIItoTelophaseII). -tubulin homologsaroundtheplastids(asterisksin , respectively. , ) werestainedusinganti-plant-tubulin, DAPI,andG9anti- (D) (B) [E] AcellintelophaseII.Notethat AcellinmetaphaseII.Thebroadlocalizationof AcellinprophaseII.Notethereappearanceoflocaliza- Changes intheSubcellularLocalizationof , and [G] (A) ), and and (G) -tubulin homologs(redsignalin and (B) [A] , (C) (H) , [C] and showsingleopticalsectionsof , [E] (D) , and -tubulin homologsarelocal- , (E) and [G] ), DNA(bluesignalin (F) , and -Tubulin Ho- -tubulin ho- [B] [B] (G) 10 ). , [D] and m. -tu- , [F] (H) (E) D. , found asasingle-copygeneinthefern nome, asitisinotherlowerlandplants.The plastids in (G) lin. Rhodamine-labeledtubulinnucleates fromtheplastid. (I) (G DNA ( nuclear surface. (D) plastid surface. (A) of and Microtubule-InitiatingActivityofIsolatedPlastidsfromSporocytes Figure 7. also isseeninthese images.Bar red filtertodetecttherhodamine signal. Chlorophyllautofluorescence ence contrastviews. ing DAPIandG9anti- 2001). Aphylogeneticanalysisofpredictedproteinsequences dopsis (Liuetal.,1994)andmaize(OvenchkinaOakley, occurred throughtheevolutionoflandplants;atleasttwo have (Silflow etal.,1999).Duplicationofthe al., 1993),themoss -tubulin genesarerecognizedinseedplantssuchasArabi- and ) and D. hirsuta to to and [B] (F) (C) (J) (H) and (I) Anisolatednucleus. Anisolatedplastid. Isolated plastidafterincubationwith rhodamine-labeledtubu- Localization of showdifferentialinterferencecontrast viewsofthesame (H) Unfixed isolatedplastidjustafterisolation. . [E] and ) and (J) (A) , respectively. -tubulin homologs( P. patens -tubulin. to -Tubulin HomologsinIsolatedOrganelles, (C) -Tubulin homologsarelocalizedonthe -Tubulin homologsarelocalizedonthe and (A) , andthegreenalga 10 and (D) (H) m. to and (D) [C] (F) showdifferentialinter showthesamecells. (J) and A. phyllitidis showviewsthrougha -tubulin genemay [F] -tubulin genewas ) werestainedus- C. reinhardtii (Fuchset fer- The Plant Cell -tubulin, respectively. and (E) [A] (H) Note thatno (C) Plastid-based microtubulesareabsent (A) Localization of Figure 8. (G) localization onthenuclearsurface. tubules ofRMS.Oryzalintreatmentdidnotaffectthe Microtubules (greensignalin that mologs aredetectedaroundtheplastids -Tubulin homologsarelocalizedalongtheremnantmicrotubulebundles. and and and showthesamecells withdifferentcolorcombinations. Bar , [C] and [H] -tubulin homologsstillremainaround theplastids(asterisksin , [E] (H) ) werestainedusinganti-plant-tubulin, DAPI,andG9anti- (D) (F) (B) Effects oftheAnti-MicrotubuleDrugOryzalinonSubcellular Anearlymeioticcelltreatedfor2h with20 AtelophaseIIcelltreatedfor20minwith AmetaphaseIcelltreatedfor20minwith AprophaseIcelltreatedfor20minwith , and -tubulin homologsarelocalizedalongtheremnantmicro- -Tubulin HomologsinMonoplastidicCellsof [G] ), and (A) and -tubulin homologs(redsignalin [A] (B) , [C] , (C) , [E] and (B) , and (A) (D) . . Notethat , (E) [G] ), DNA(bluesignalin and -tubulin homolog M oryzalin.Note (F) -tubulin ho- , M oryzalin. M oryzalin. [B] and D. hirsuta M oryzalin. , [D] (G) 10 [H] , [F] and m. . ). , the 10intronsof trioles denovo.Neitherhasbeenstudiedmolecularly.Nineof and cycadshaveflagellatedspermatozoidsproducecen- Among theextantseedplants(Spermatophyta),onlyginkgo the presenceorabsenceofcentrosomesinlifecycle. plants (Figure1A).Theoccurrenceoftwogroupswithregardto revealed monophylywithtwodistinctivebranchesofland Boergesenia forbesii been reportedinalgaeandanimalcells.Inthegreenalga 2001). Themechanismofcellcycle–dependenttranslocation ing meiosis(BrownandLemmon,1997;Shimamuraetal., of bryophytesbasedonchangingpatternsmicrotubulesdur- were commonintheMTOCcycleduringmonoplastidicmeiosis cycle–dependent distributionpatternsof sporocytes oftheliverwort We wereabletoprovidedetailsonthechangesinlocationof MTOC Cycle Correlation ofthe 4F, 4H,5H,6B,6F,6H,9C,10B,10C,10F,10G,and10H). 10H), andmembrane-associatedMTOCs(Figures3B,3P,4D, (Figures 3K,3N,3P,5D,5F,5H,6D,6F,6H,9D,10E,10F,and ures 3Hand9B),diffuseMTOCssimilartothoseinseedplants (Figures 3Eand9A),discreteMTOCswithoutcentrioles(Fig- tative MTOCsitesofbryophytes,suchascentriolarMTOCs antibody, wedemonstratedthat andOakley,2001;Horioetal.,2002).Usingthis (Ovenchkina bryophytes aswellinawidevarietyofotherorganisms nobl The G9antibodyspecificallyrecognizes highly conservedinlandplants. structure andthepredictedproteinof genes. Thepredictedaminoacidsequenceofthe the samesitesasnineintronsofArabidopsis microtubules emanate fromtheseplastidenvelopes directlyor croscopy andimmunofluorescence microscopysuggestedthat tid migrationanddivision(Shimamura etal.,2003).Electronmi- the spindleapparatustobeorganized inassociationwithplas- gle plastidtodivideinanticipation ofnucleardivisionandfor In monoplastidicmeiosisofbryophytes, itistypicalforthesin- Plastid-Based MTOCs prophase (KhodjakovandRieder,1999). trosome-associated trosomes throughoutthemitoticcycle,amountofcen- cells,inwhich centrosome fromprophaseonward(Motomuraetal.,2001). In trosome duringinterphasebutbegantoconcentrateatthe -tubulin (angiospermsandnonfloweringplants)mightreflect -tubulin isnotclear.Thislittle-understoodphenomenonhas -tubulin duringthemeioticcycleinlargemonoplastidic -tubulin oflandplants.Theseresultssuggeststhatboththe -tubulins -tubulin proteinis89.2to97.7%identicalthoseofother -Tubulin CharacterizesDiverseMTOCsinBryophytes ots ofproteinextractsandinimmunohistochemistry -Tubulin LocalizationPatternwiththe , C. japonicum -tubulin neverthelessincreasesduring -tubulin wasnotdetectedatthecen- -tubulin alwaysassociateswithcen- D. hirsuta TblnadMO vlto 53 -Tubulin andMTOCEvolution -tubulin characterizesthepu-

. Weexpectedthatthecell -tubulin geneoccurredat -tubulin (Figure10) -tubulin inimmu- -tubulin geneare C. japonicum -tubulin The Plant Cell MTOC with thenuclearenvelope.Shouldpresenceofsuchnon- tion ofspindlesthatinsteadresideswith ary relic,andthis ciation of with proplastidsinwheatroottipcells.Perhapssuchanasso- 2001) reported lin ontheplastidsurface.Arecentstudy(Dibbayawanetal., stages ofthemeioticcycle,therewasnolocalization both prophaseI(Figure4)andII6B).Atother peripheral localizationof face seemtooccurconcurrently.Duringmeiosisin surface andthenucleationofmicrotubulesfromplastidsur- tid envelopeinmonoplastidiccells. ports thecontentionthatMTOCsareassociatedwithplas- position toserveastheMTOCbothinvivoandvitrosup- from sporocytesinmeiosis.Althoughthevitroincubation tivity wasrestrictedtoasubpopulationoftheplastidsisolated ation withthesurfaceofisolatedplastidsinvitro.MTOCac- 2001). Here,weshowmicrotubule-nucleatingactivityinassoci- plant cells(Mizuno,1993;Stoppinetal.,1994;JoshiandZhou, microtubules hasbeendemonstratedinvariousanimaland plants. ies willbeneededtosearchfortheirpossiblepresencein (C) (B) (A) Figure 9. in vivo(BusbyandGunning,1989).Thedemonstrationof covery ofmicrotubulesfromanti-microtubuledrugtreatments ability oftheplastid-basedMTOChasbeenshownbyre- tids (BrownandLemmon,1997).Themicrotubule-nucleating from discretebodiesofelectron-densematerialsneartheplas- ThePlantCell 54 Microtubules, plastids, nuclei,and (D) -tub The cellcycle–dependentlocationof The abilityofisolatedcentrosomesand/ornucleitonucleate Acellwithcentrioles.Ingreenalgae andinspermatogenouscellsofbryophytes, AcellwithPOs. Thespindleduringmonoplastidicmeiosis insomebryophytesarisesfromdaughterplastids. Inhigherplants, ulin attheperipheryofdividingplastidsinproper -tubulin provetobewidespread,thenadditionalstud- Schemes oftheDiversity -tubulin andplastidsinseedplantsisanevolution- -tubulin, butnotmicrotubules,inassociation -Tubulin localizesdistinctlyatacentriolar POsofsomeliverworts. -tubulin localizesindistinctlyatpolar regionsofdiffusespindles. -tubulin haslostitsfunctionintheorganiza- -tubulin onplastidswasobservedin -tubulin areshown asdarkgreen,limeblue,and red,respectively. -Tubulin LocalizationinProphaseSpindles. -tubulin attheplastid -tubulin associated D. hirsuta -tubu- , evolutionarily conservedcomponentofthespindleMTOCin poles inbryophytessupportsthepremisethat Plant MTOCs Localization inProphaseSpindlesReflectstheEvolutionof Spindles andPhragmoplasts:theDiversityof the caseofplastid-basedMTOCs. relate withacellcycle–dependentmigrationoftheMTOC,asin to thebroadpolarregionofmaturespindleappearscor- in 1984) inwhichsubsetsofmicrotubulesconverge.Thechange poles oftenconstitutenumerousdistinct“minipoles”(Schnepf, pointed polestobarrelshapedwithbroadpoles.The dle. Theshapeoftheplantspindlerangesfromfusiformwith spindlesresembledthetypicalanastralplantspin- spindles developed,andthedistributionof liverworts. Interestingly, clusively atthediscretePOsthatorganizemitoticspindlein Our observationsclearlyshowthat without POs: investigation. mental stageslackMTOCactivity.Theelucidationoftherole bule fromplastidsorifisolatedcertaindevelop- whether G9antibodyblockstheinvitronucleationofmicrotu- studying thefunctionof with labeledtubulinand -Tubulin localizationinvarioustypesofprophasespindle -tubulin inmicrotubulenucleationfromplastidsneedsfurther -tubulin positionfromacloseassociationwithdiscretePOs -tubulin localizespreciselyatcentriolar centrosomes. -Tubulin LocalizesinDiscreteMTOCs -Tubulin localizesontheplastidsurface. -tubulin staininggraduallydiffusedas -tubulin, itwasdifficulttodetermine -tubulin antibodyisimportantfor -tubulin isassociatedex- -tubulin inmature -tubulin isan -Tubulin The Plant Cell (F) (E) (D) the twomeioticdivisions. (C) (B) (A) in Bryophytes. Figure 10. TelophaseI. MetaphaseI. Premeioticinterphase. Earlyprophase. Lateprophase. Midprophase.QMSandcytoplasmic lobingpredictthepolarityof Schemes oftheMTOCCycleduringMonoplastidicMeiosis ing throughoutthephragmoplast.Furthermore,association both asdistinctdotsatthenuclearsurfaceanddiffusestain- clearly showtheopposite;incytokinesis, nuclei (Liuetal.,1993).Ourobservationsofliverwortmeiosis been shown(Lambert,1980).Theperinuclearlocalizationof the associationofmicrotubuleswithnuclearenvelopehas et al.,1994),andanelectronmicroscopicimagethatsuggests envelope istheprincipalsiteofmicrotubuleassembly(Stoppin In seedplants,thereisageneralconsensusthatthenuclear with theNuclearMembrane MTOCs ontheNuclearSurface: plants. tribution patternalsoareindistinguishablefromthoseofseed of bryophytephragmoplastsandtheassociated 1993, 1995;JoshiandPalevitz,1996;thisstudy).Thestructure of the crotubules, theunstainedzoneatcellplateiswiderwhen persed inthephragmoplastandappearstocolocalizewithmi- rected bythephragmoplast.Although tablishment ofthematuremetaphasespindleinlandplants. microtubules inthepolarregionseemstocorrelatewithes- Joshi andPalevitz,1996). situation commonlyobservedinhigherplants(Liuetal.,1993; nearly allmetaphasespindlesbutabsentfromkinetochores,a spindle poles.Inbryophytes, change of through theevolutionoflandplantsseemtoberelated 1991). Thelossofmotilecellsandthemultiplicationplastids of mostlandplants(Pickett-Heaps,1975;GrahamandKaneko, olar centrosomes.Thesespindlesarebarrelshaped,likethose algae, whichdonotproduceflagellatezoospores,lackcentri- diffuse spindles(Figure9D)(Liuetal.,1993,1994).Somegreen area. the diffuseprophasespindleisorganizedfromperinuclear lutionary stagesofplantspindledevelopment.Inhigherplants, ious typesofprophasespindlepolesalsomayreflecttheevo- the diffusespindlesofseedplants. tional stagesbetweencentriolarspindlesofgreenalgaeand character, POsandplastid-basedspindlesseemtobetransi- 9C). Althoughitisdifficulttodeterminethemoreancestral lin wasdistributedassmalldotsontheplastidsurface(Figure worts (Figure9B).Inmonoplastidiccellsofbryophytes, cells ofbryophytes(Figure9A)andtheacentriolarPOsliver- and specificallyatcentriolarcentrosomesofspermatogenous formation inplants. . Figure9showsthediversityofprophasespindle lime green,blue,and red,respectively. Microtubules, plastids,nuclei,and (H) (G) -tubulin isevidentinprophasenucleibutfainttelophase -tubulin Cytokinesis inlandplantsinvolvescellplateformationdi- EarlytelophaseII. EarlyprophaseII. -tubulin atdistalregionsofthephragmoplast(Liuetal., -tubulin isviewedexclusively,indicatingaconcentration -Tubulin localizesindistinctlyatthepoleregionof -tubulin localizationandtheacquisitionofdiffuse -Tubulin wasdistributedconspicuously TblnadMO vlto 55 -Tubulin andMTOCEvolution -Tubulin localizationalongspindle -tubulin areshownasdarkgreen, -tubulin isdistributedalong -Tubulin CanAssociate -Tubulin localizationinvar- -tubulin iswidelydis- -tubulin occurred -tubulin dis- -tubu- The Plant Cell site proteinandmayhaveadditionalrole(s)inplantcells. that plant Joshi andPalevitz,1996).Suchfindingsraisethepossibility minus ends(Liuetal.,1993,1994,1995;Hoffman1994; lin ispresentalongorganizedmicrotubulearraysaswell. sites.However,manystudieshaveshownthat initiating strated thepresenceof All studiesof MTOCs andDispersedalongMicrotubuleArrays Two DifferentLocalizationsof ciation of lin-treated cells(Figures8Eand8F),suggestingthetightasso- they werenolongerlocatedintheremnantsofRMSoryza- another (BrownandLemmon,1988;Shimamuraetal.,1998). phragmoplasts developwhereopposingRMSscontactone cytokinetic apparatusduringmeiosisinbryophytesbecause ated withtelophasenucleiareimportantcomponentsofthe crotubule-nucleating capacityisstagespecific.RMSsassoci- mi- that nuclei, buttheseobservationssupportthecontention are neededtodetectmicrotubulenucleationfromisolated crotubule nucleationfromisolatednuclei.Clearly,morestudies surfaces ofsomeisolatednucleiinvitro,wedetectednomi- ures 4and6B).Although ing (Figures5H,6F,and6H)thaninprophase(Fig- of ThePlantCell 56 presence of evolutionary taxa.TheplantMTOC canberecognizedbythe specific organellesandendomembrane systemsinthemajor specific manner.Furthermore, it iscapableofassociatingwith plants andthatitisamotileentity thatmigratesinacellcycle– present ubiquitouslyatsitesof microtubulenucleationinland . other functions,suchasmicrotubulestabilization,inadditionto post-translational molecularformsandcombinationsaswell ization inbryophytesalsomightreflecttheexistenceofmultiple (Dryková etal.,2003).Twodifferentpatternsof for nucleation activity,butactivitywasnotobserved associated largecomplexinArabidopsishasmicrotubule flowering plants(Stoppin-Melletetal.,2000).Themembrane- cleus) MTOCs. on able disorganizationofmicrotubulesbuthadlittleornoeffect localization patterns.Oryzalintreatmentsinducedconsider- vides additionalinformationaboutdifferencesinthesetwo might gounnoticed.Theanti-microtubuledrugstudypro- have distinctMTOCs,thetwodifferentlocalizationpatterns plast arraysofmicrotubules.Infloweringplantsthatdonot and theotheralonglengthofspindlephragmo- (plastid surface,nuclearcentrosomes,andPOs), nificant differentlocalizationpatterns:oneatMTOCsites -Tubulin homologsremainedonthenuclearsurface,although -Tubulin followsmicrotubulespreciselybutisbiasedtoward -Tubulin The presentstudyclearlyshowsthat In conclusion,datafromthisstudy indicatethat -Tubulin ispresentinproteincomplexesofvarioussizes -tubulin withthenuclearenvelopewasmoredis the smallercomplexthatbindslaterallytomicrotubules -tubulin localizationatorganelle-based(plastidandnu- -tubulin oritscomplexonthenuclearenvelope. -tubulin isnotexclusivelyamicrotubule-initiating -tubulin, andthere aretransitionalstagesinex- -tubulin distributioninplantcellshavedemon- -tubulin atsuspectedmicrotubule- -tubulin occurredasdotsonthe -Tubulin: Concentratedat -tubulin hastwosig- -tubulin local- -tubulin is tinct dur- -tubu- pBluescript KS were isolatedbyvector-annealing PCRamplification.First,thevector quence. GenomicDNAfragments containingtherestof primers weredesignedaccording tothedeterminednucleotidese- was isolated,purified,andsubjected todirectsequencing.Additional fied DNAwasrunonanagarosegel, andthespecificallyamplifiedband served aminoacidsequencesofknown C orT.Alloftheseprimersweredesignedtocorrespondthecon- S denotesCorG;DA,G,T;RAandY 291 (5 Total DNAof Molecular Cloning Hills). tions growingasweedsinthenurserydistrictofcentralLouisiana(Forest trix the Higashi-HiroshimacampusofHiroshimaUniversity. noa crispata Conocephalum japonicum Plant Materials METHODS plified furtherusingprimersPS3A(5 GGNAA-3 using redundantoligonucleotideprimers294(5 DNA wasstoredat mated byethidiumbromidestainingafter1%gelelectrophoresis.The was dissolvedin20to50 was discardedandthepelletdriedundervacuum.Then,DNA the tubeseveraltimes.Aftercentrifugationfor2min,supernatant 20,000 The tubeswereplacedat cold absoluteethanolwasadded,andthesolutionmixedgently. 20,000 ume ofchloroformwasadded,mixedgently,andcentrifugedfor5minat 20,000 ume ofbufferedphenol.Thesolutionwascentrifugedfor20minat ferred toanewmicrotubeandmixedgentlyfor10minwithanequalvol- centrifuged for20minat20,000 the digestionmixture.Thesuspensionwasmixedgentlyfor10minand at 37 and 0.2mg/LproteinaseK(TEbuffer).Extractedsolutionwasincubated HCl, pH8.0,20mMEDTA,0.5MNaCl,1%(v/v) powder wassuspendedinextractionbuffercomposedof50mMTris- samples weregroundtoapowderinliquidnitrogenusingpestle.This and excesswaterwasremovedfromsampleswithapapertowel.Frozen mg dryweight).Sampleswerewashedseveraltimeswithdistilledwater, ity of identifying thepathway(s)thatcontrolsmigrationandactiv- meiosis ofbryophytes.Futureeffortsneedbedirectedtoward lin withtheplastidenvelopeinmonoplastidicmitosisand lar organizersofhepaticsandthediffuseassociation clude theconcentratedpresenceof with thenuclearenvelopeofhigherplants.Thesestagesin- centrosomes (asinalgalandanimalcells)todiffuseassociation tant plantsindicatinganevolutionfromcorpuscularcentriolar Isolated genomicDNAwasusedasthetemplateforPCRamplification and C for2to3h.Anequalvolumeofbufferedphenolwasadded -NCCNGANCCNGTNCCNCC-3 g. TheDNApelletwasrinsedwithcold70%ethanolbyinverting g. Aftertransferoftheupperlayertoanewtube,2.5volumes g. Aftertransferofthesupernatanttoanewtube,anequalvol- -tubulin inplantcells. Marchantia polymorpha ) andPA4(5 werecollectedfromnaturalpopulationsinshadycornersof C. japonicum (Stratagene,San Diego, CA)wasdigestedwithtwodif- 20 -NCCNGANCCNGTNCCNCC-3 C. , L ofTEbuffer.DNAconcentrationwasesti- wasextractedfromdriedspecimens( Dumortiera hirsuta 20 C for2handcentrifuged5minat werecollectedfromlong-livedpopula- g. Then,theupperphasewastrans- -AARGAYGTNTTYTTYTA-3 ), whereNdenotesA,C,GorT; -tubulins. Theresultingampli- -tubulin atacentriolarpo- subsp -GCSGGDCCRTGY- N-lauroylsarcosinate, tatunoi ). DNAwasam- Entodon seduc- , and -tubulin -tubu- ) and Maki- 200 The Plant Cell probe overnightina5 Hybridization wasperformedbyincubatingthemembraneand presence ofalkali-labileDIG-11-dUTP:unlabeleddTTPataratio1:10. ment, Tokyo,Japan). sequences wereanalyzedusingGENETYX-MAC(SoftwareDevelop- using thePCRdirectsequencingmethod.Nucleotideandaminoacid nucleotide sequenceofeachfragmentwasdeterminedonbothstrands gene wereamplifiedbyPCRusinggenomicDNAasthetemplate.The overlapping fragmentscoveringtheentirecodingregionof quence weredesignedaccordingtothedeterminedsequence.Four sequence wasisolated.Specificprimerscoveringtheentirecodingse- repeated usingthevector-annealingPCRmethoduntilentirecoding and thepossiblepresenceofmorethanone than onecloneweredeterminedandcomparedvisuallyforPCRerrors boring fragments.Ateachcloningstep,nucleotidesequencesofmore were determinedandusedtodesigntheprimersisolateneigh- son, WI).Nucleotidesequencesoftheinsertfragmentsinisolatedclones T EasyvectorusingthepGEM-TVectorSystem(Promega,Madi- by theseprimers.AmplifiedDNAfragmentswereligatedintothepGEM- ments ofthe end forPCRamplification.GenomicDNAfragmentscontainingseg- side ofpBluescriptKS the genomicDNAwereusedatoneend.Generalprimerslocatedout- fication. Primersspecifictothe cation. nomic DNAwasusedasthetemplatefortwo-stepnestedPCRamplifi- to thedouble-digestedvector.Then,resultingvector-conjugatedge- with oneoftheenzymesusedfordigestionvectorandligated with noncohesiveends.ThegenomicDNAof ferent restrictionenzymesinthemultiplecloningsites,yieldingavector 1-tubulin, gelblotanalysis.The the cross-reactivityofantibodiesto the activity oftheantibodiestopartialfragments ofthe nized epitopesoftheseantibodies have beenmappedbytestingthere- 0.1 containing 0.1%SDSsolutionatroomtemperatureandthentwicewith Diagnostics) at42 roylsarcosine, 0.02%(w/v)SDS,and2%blockingreagent(Roche sodium citrate)solutioncontaining50%formamide,0.1%(w/v) TGGGCCAGTGGGTA-3 ponicum Japan). A2.0-kbregionofaclonedgenomic probes bymeansofPCRwithKOD-DNApolymerase(Toyobo,Osaka, with aminormodification:theDIG-11-dUTPwasincorporatedinto tics, Mannheim,Germany)accordingtothemanufacturer’sinstructions with DNAprobelabeledalkali-labileDIG-11-dUTP(RocheDiagnos- away, NJ).ThemembranetowhichDNAwasblottedhybridized blotted ontoaHybond-N with BamHI,HindIII,orPstI,fractionatedona0.7%agarosegel,and lans saccharomyces pombe monoclonal antibodyclonesraisedagainstbacteriallyexpressed A monoclonalanti- Anti- Star (NewEnglandBiolabs,Beverly,MA). labeled anti-digoxigeninantibody(RocheDiagnostics)andwithCDP- beled probeonthemembranewasdetectedwithalkalinephosphatase– pressed ineitherbacteria orthefissionyeast For DNAgelblotanalysis,genomicof LA Taqpolymerase(TakaraSyuzo,Kyoto,Japan)wasusedforampli- , andthefissionyeast SSCcontaining0.1%SDSsolutionat65 -Tubulin Antibody wasamplifiedbyPCRwiththeprimersmossF2(5 Arabidopsis thaliana -tubulin genewereisolatedbyamplifyingtheDNAflanked C. Themembranewaswashedtwicewith2 -tubulin antibody(G9)wasused.G9isoneofthe

-tubulin (Horioetal.,1999,2002).Therecog- ) and78(5 multiplecloningsiteswereusedattheother SSC(1 membrane(AmershamBiosciences,Piscat- Schizosaccharomyces japonicus -tubulins usedforthetest—

-tubulin genesequencethatannealto 2-tubulin, thefungus -CTTTTGTGAACCTGTAAC-3 SSCis0.15MNaCland0.015 -tubulins ofotherspeciesby -tubulin gene.Cloningwas C. japonicum C. japonicum S. pombe C for15mineach.Thela- -tubulin geneof -tubulin. Wetested Aspergillus nidu- . Homo sapiens wasdigested wasdigested -AACAAC- —were ex- -tubulin Schizo- ) inthe N-lau- C. ja- SSC 6.9) at4 IgG (H rabbit IgG(H for 60minat37 for 90minat37 in polyclonalanti- PBS inamoistchamberfor90minat37 bated withmonoclonalanti- cells wereair-driedonaglassslideandthenextractedwithmethanolat ton X-100and1%gelatinfor1h.AfterseveralwasheswithPMEbuffer, ter abufferwash,cellswereextractedinPMEcontaining1%Tri- cocktail (BoehringerMannheim,Tokyo,Japan),and0.3Mmannitol.Af- tolyase (Kikkoman,Tokyo,Japan),0.5%TritonX-100,proteaseinhibitor containing 1% had beenadded.Cellwallsweredigestedfor20to30minwithasolution EGTA, and0.5mMMgSO Tubulin frombovinebrainwasisolatedwithbufferA(20mMPipes,1 Isolated Plastids Preparation ofTubulinandAssemblyMicrotubulesfrom shown). ure 4I.Similarexperimentsalsowereperformedinothercases(datanot crosstalk oftubulinsignals.WepresentanegativecontrolimageinFig- of G9andDAPItoconfirmthatsignalsdonotderivefromthe staining oftubulin,G9,andDAPI,wealsoexaminedthedouble though theimmunocytochemicalimagespresentedhereshowtriple excitation sequentially(Ar,488nm;He/Ne,543andUV,365nm).Al- Carl Zeiss,Tokyo,Japan).Imageswerecollectedusingmonochromatic were observedusingaconfocallaserscanningmicroscope(LSM410; 2-phenylindole (DAPI)wasaddedinthemountingmedium.Preparations EGTA, 33%glycerol,0.1mMGTP, 0.5 mMMgSO eluate wasdialyzedagainstbufferB (100mMPipes,0.5DTT,1 was elutedwithbufferAcontaining 0.8MNaClfromthiscolumn.The Sephacel. Afterwashingwithbuffer Acontaining0.3MNaCl,tubulin Tubulin thenwaspurifiedbycolumn chromatographyonDEAE- polymerizationanddepolymerization(Shelanskietal.,1973). dependent Probes). TostainDNAinnucleiandorganelles,1 Slides weremountedusingtheSlowFadeAntifadekit(Molecular and 1mMMgSO 4.5% formaldehydein50mMPMEbuffer(50Pipes,5EGTA, Sporogenous andspermatogenoustissueswerefixedfor60minwith Immunofluorescence Microscopy method and3,3 munoperoxidase stainingwasperformedwithanavidin-biotincomplex game, CA)wasappliedinadilutionof1:200.AfterrinsingTPBS,im- IgG (H (0.05% Tween20inPBS)secondaryantibody,biotinylatedanti-mouse tibody (N357;Amersham)diluted1:2500inPBS,ormonoclonalanti- (N356; Amersham)diluted1:2500inPBS,monoclonalanti- perature for1handtreatedwithmonoclonalanti- et al.(1998).Themembraneswereblockedin1%gelatinatroomtem- ferred topolyvinylidenedifluoridemembranesaccordingMurata-Hori and separatedon10%polyacrylamidegels,proteinsweretrans- extracts werespuninamicrocentifuge.Thesupernatantsboiled and pestlein5volumesofLaemmlisamplebuffer(Laemmli,1970).The Sporophytes of Protein GelElectrophoresisandImmunoblotAnalysis -tubulin antibody(G9)diluted1:500inPBS.AfterrinsingTPBS -tubulin 20 C for10min.AfterwashingwithPBSmin,cellswereincu- C overnight.GTP was addedto1mM,andthecycle of temper- L) antibody(BA-2000Vectastain; VectorLaboratories,Burlin- L) (MolecularProbes,Eugene,OR)diluted1:200withPBS. L) diluted1:100withPBSandAlexa594goatanti-mouse -glucuronidase (Sigma-Aldrich,St.Louis,MO),1%Pec- C withamixtureofOregonGreen488–labeledgoatanti- -diaminobenzidine substrate(VectastainABCkit). C. WashedagainwithPBS,thesampleswereincubated C. japonicum 4 ·7H Vigna 2 O, pH6.8)towhich0.1Msucroseand1%DMSO tubulindiluted1:500withPBSwasperformed 4 , pH6.9)bythreecyclesoftemperature- -tubulin (G9)antibodydiluted1:200with and TblnadMO vlto 57 -Tubulin andMTOCEvolution D. hirsuta C. AfteraPBSwash,incubation weregroundwithamortar 4 , and80mMNaCl,pH g/L 4 -tubulin antibody ,6-diamidino- -tubulin an- The Plant Cell Arabidopsis thaliana TUBG2 follows: The accessionnumbersforthesequences showninFigure1Bareas The GenBankaccessionnumberfor Accession Numbers please contactY.Mineyuki,[email protected]. similar termsfornoncommercialresearchpurposes.Toobtainmaterials, lication willbemadeavailableinatimelymannertoallinvestigatorson 0.3 Msucrosealone(datanotshown). lution using0.2%DMSOin0.3Msucroseshowednodifferencefrom tions atconcentrationsfrom1to20 and immunostainingwasperformedasdescribedabove.Oryzalinsolu- the oryzalinsolutionwasremovedandreplacedwithfixative tration of20 was addedtoanequalvolumeofcellsuspensiongiveafinalconcen- and thepooledcellsweredividedbetweentreatments.Oryzalinsolution Meiotic-stage sporangiaof Oryzalin Treatment formed asdescribedabove. 3.6% formaldehydein50mMPMEbuffer,andimmunostainingwasper- frommembrane-lysedprotoplastswerefixedfor60minwith the observationof pus, Tokyo,Japan)todetecttheclusterofmicrotubuleformation.For action mixturewasobservedbyfluorescencemicroscopy(BX-50;Olym- DMSO, and1mMGTP,pH6.9)at27 merization bufferC(50mMPipes,1EGTA,MgSO added tothepreparationofisolatedplastids.Afterincubationinpoly- tion of0.4mg/mLthatneverinducesspontaneouspolymerization,was of rhodamine-labeledtubulinandunlabeledtubulin,atafinalconcentra- at 4 lysed protoplastswereprecipitatedbycentrifugationat1000 nizer using3to11strokesbyhand.Plastidsreleasedfrommembrane- ride-HCl, and10 2-mercaptoethanol, 0.1mM NaCl, 2mMEDTA,0.15spermine,0.5spermidine,20 shearing inchilledmedium[10mMMesbuffer,0.25Msucrose,10 twice with0.85Mmannitol.Theprotoplaststhenwererupturedby plasts weresedimentedbycentrifugationfor5minat500 RS and0.043%PectolyaseY23.Afterincubationat30 solution ofmannitol,pH5.5,thatcontained0.43%CellulaseOnozuka and depolymerizationthenstoredinliquidnitrogen. purified furtherbytwocyclesoftemperature-dependentpolymerization lymerized at0 30 minat154,000 ture waslaidonthecushionbufferwith60%glycerolandcentrifugedfor preparation wasaddedundervortexing.After10min,thereactionmix- was dissolvedinDMSOat100mM,and1/20thvolumeofmicrotubule 8.0. Carboxytetramethylrhodaminesuccinimidylester(MolecularProbes) centration of50mg/mLinaPipesbuffersimilartoBexceptatpH min at36 glycerol insteadof33%asacushionandcentrifugedat154,000 bulin inbufferB,thepreparationwaslaidonBcontaining60% (1991). Afterpolymerizationofmicrotubule-associatedprotein–freetu- preparation ofmicrotubule-associatedprotein–freetubulin. twice. Theresultingtubulinpreparationwasstoredinliquidnitrogenasa ature-dependent polymerizationanddepolymerizationwasrepeated ThePlantCell 58 Upon request,materialsintegraltothefindingspresentedinthispub- The lysismediumforsporocytesof Rhodamine-labeled tubulinwaspreparedaccordingtoHymanetal. C. Forthedetectionofactivitytoorganizemicrotubules,mixture Homo sapiensTUG1 C. Theprecipitatedmicrotubulesweresuspendedtoacon- M in0.2%DMSOand0.3sucrose.After20minor2h, C aftersuspension.Therhodamine-labeledtubulinwas g/mL leupeptin,pH6.0]withaglassTeflonhomoge- g. Theresultingprecipitateofmicrotubuleswasdepo- -tubulin localizationinisolatedplastidsandnuclei, D. hirsuta , M61764; p , U03990; -4-(2-aminoethyl) benzenesulfonylfluo- M hadsimilareffects.Acontrolso- werebrokenupin0.3Msucrose, C foranappropriatetime,there- C. japonicumgtb1 D. hirsuta Xenopus laevisXgam Nicotiana tabacum nttug1 consistedofa0.85M C for1h,proto- isAF511487. g andwashed g for5min , M63446; g for30 4 , 10% , AB051679; Brown, R.C.,andLemmon,B.E. Brown, R.C.,andLemmon,B.E. Brown, R.C.,andLemmon,B.E. Brown, R.C.,andLemmon,B.E. Brown, R.C.,andLemmon,B.E. Busby, C.H.,andGunning,B.E.S. Brown, R.C.,andLemmon,B.E. Brown, R.C.,andLemmon,B.E. REFERENCES Received August19,2003;acceptedOctober21,2003. Y.M. fromtheJapanSocietyforPromotionofScience. Grants-in-Aid forScientificResearchB(09044225) andC(12640651)to for JapanSocietythePromotionofSciencefellowstoM.S.andby for DNAsequences.ThisworkwassupportedbyGrant-in-Aid 200001095 (University ofTokushima),andMiyukiShimizuTokushima) antibody andHiromiTsubota(HiroshimaUniversity),NatsuTakemura We thankMasayukiYamato(UniversityofTokushima)forpreparingG9 ACKNOWLEDGMENTS TUB4 Aspergillus nidulansmipA coding gtb1 Dibbayawan, T.P.,Harper,J.D.I.,andMarc,J. litidis tubg Dryková, D.,Cenklová,V.,Sulimenko,Voic,J.,Dráber,P.,and Felix, G.W.,Antony,C.,Wright,M.,andMaro,B. Fuchs, U.,Moepps,B.,Maucher, H.P.,andSchraudolf,H. 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