The Journal of Cell Biology

JCB

is thecondensincomplex,whichwasfirstpurifiedfrom state tomitoticchromosomefunctionsispoorlyunderstood. (Heck, 1997).However,thecontributionofcondensed separation oftheduplicatedgenomeintotwodiscretesets and iscrucialtophysicallyresolveentanglementsallow in spindle assembly andchromosome segregation The condensincomplex isrequired forproper function in machinery, thecondensincomplex,inspindleassemblyand clear. Here,weaddresstheroleofmajorcondensation structureinmitoticchromosome functionsisun- demembranated spermnucleiintodiscretechromosomes, clarified extractsseverelycompromisedcondensationof Xenopus reduces chromosomelength architectureiscondensation,whichinvertebrates 1999; KarsentiandVernos,2001).Fundamentaltomitotic microtubule stabilization,andspindleorganization(Andersen, chromosome armsalsocontributetomovements, Salmon, 1998;Maneyetal.,2000).Proteinsassociatedwith dictate chromosomealignmentandsegregation(Rieder of eachsisterchromatidattachtospindlemicrotubulesand during celldivision.Kinetochoresassembledatthecentromere playanactiveroleintheirownsegregation C ofMolecularDepartment andCellBiology, University ofCalifornia,Berkeley, Berkeley, CA94720 Sarah M. Wignall, Renée Deehan, Thomas J.Maresca, andRebecca Heald defects inanaphasechromosome segregation. Although nuclei capableofformingspindles,andcausingdramatic around chromosomes, reducingthepercentage of sperm of condensininhibitedmicrotubulegrowth andorganization Introduction  condensation;anaphase Key words: microtubule; ;; E-mail: [email protected] Berkeley, CA94720-3200.Tel.:(510) 643-5493.Fax:(510)643-6791. Biology, 311LifeSciencesAddition, UniversityofCalifornia,Berkeley, Address correspondencetoRebecca Heald,Dept.ofMolecularandCell The onlineversionofthisarticlecontains supplementalmaterial. http://www.jcb.org/cgi/doi/10.1083/jcb.200303185 The Journal ofCellBiology

TheRockefeller University Press, 0021-9525 Article Strongly implicatedinmitoticchromosomecondensation

Xenopus

cell division, butthepreciseroleofhigherorder resolution andsegregationofsisterchromatids during hromosome condensationisrequiredforthephysical

laevis

Xenopus laevis

eggextracts.Depletionofthiscomplexfrom

, Volume 161, Number 6,June 23, 20031041–1051 eggextracts egg extracts. Immunodeple 100-fold relativetointerphase, /2003/06/1041/11 $8.00 tion presence ofatypeItopoisomerase,purified targeting orregulatoryrolesincondensinfunction.Inthe 1997; Uhlmann,2001),whichhavebeenproposedtoplay SMC proteins(XCAP-H,-G,and-D2;Hiranoetal., and Jessberger,1999).Condensinalsocontainsthreenon- compensation, andrecombination-mediatedrepair(Strunnikov organization andfunction,includingsistercohesion,dosage 1997). SMCproteinsplaymultiplerolesinchromosome XCAP-E; HiranoandMitchison,1994;etal., ( ATPase superfamilythatformacoiled-coilheterodimer conserved structuralmaintenanceofchromosomes(SMC)* consists of state (Hiranoetal.,1997).Theactivecondensincomplex both theestablishmentandmaintenanceofcondensed also causeddefects,pointingtoaroleforthecomplexin and inhibitionofcondensinaftercondensationhadoccurred XCAP, assembly andchromosome segregation. chromosomal architecture necessaryforproperspindle support animportantroleforcondensininestablishing was alsoimpaireduponcondensininhibition. These results around DNA-coated beadsintheabsenceofkinetochores indicating itscontinuousrequirement.Spindleassembly during anaphasealsoinhibitedchromosome segregation, mass was notresolved. Inhibitionofcondensinfunction poleward duringanaphase,thedisorganizedchromosome the motorCENP-Ewas recruitedtokinetochores pulled structural maintenanceofchromosomes; topoII,topoisomeraseII resonance energytransfer;GEF,guanine nucleotideexchange *Abbreviations usedinthispaper:CSF, cytostaticfactor;FRET,fluorescence coiling ofDNAbyasinglecondensincomplex,suggesting 1999). Electronspectroscopicimaginghasrevealedsuper- dependent manner(KimuraandHirano,1997;Kimuraetal., densin canreconfigureDNAstructureinanATPhydrolysis– Xenopus Xenopus chromosome–associatedprotein[XCAP]-Cand five proteinsincludingtwomembersofthehighly chromosome–associatedprotein. Xenopus factor; SMC, con- 1041 ; The Journal of Cell Biology 1042 some condensationinclarified quired fortheestablishmentandmaintenanceofchromo- chore organizationinmonocentricorganisms. whether thecondensincomplexalsocontributestokineto- condensation (Hagstrometal.,2002).However,itisunclear netochore defectsarisefromafailureinglobalchromosome persed sequencescoalesce,ithasbeenproposedthattheseki- cause holocentrickinetochoresarethoughttoformasdis- merotelic spindleattachments(StearandRoth,2002).Be- condensin subunitsresultedinchromosometwistingand poles (Hagstrometal.,2002),andmutationofonethe stricted orientationofkinetochorestowardoppositespindle RNAi analysisrevealedthatcondensinisrequiredforthere- part toafailureinholocentrickinetochoreorganization. peared moresubtleuponlossofcondensinfunctionin al., 2000).However,chromosomecondensationdefectsap- (Strunnikov etal.,1995;Lavoie2000;Ouspenski beled locionamitoticchromosome,supportingthismodel yeast increasedtheaveragedistancebetweenfluorescentlyla- ing anaphase.Mutationofcondensinsubunitsinbudding tion preventsthechromosomesfrombeingdisentangleddur- 1994; Hiranoetal.,1997),isthatagrossfailureincondensa- 2002). In matically affected(Steffensenetal.,2001;Hagstrom and longitudinalshorteningofchromosomeswerenotdra- morphology, whereasmetaphasechromosomecompaction sophila sis comesfromseveralorganisms.In matin loops(Bazett-Jonesetal.,2002). that itfunctionsbygeneratingpositivelysupercoiledchro- servations in simplest explanationforthesedefects,consistentwiththeob- 2002; Hagstrometal.,StearandRoth,2002).The Ouspenski etal.,2000;Steffensen2001;Bhalla Bhat etal.,1996;Sutani1999;Lavoie2000; sults inchromosomesegregationdefects(Sakaetal.,1994; saccharomyces pombe port factorimportin RCC1, causing localizedreleaseofcargoes from thetrans- matin-bound guaninenucleotide exchangefactor(GEF) ated primarilybyRanGTP,which isgeneratedbythechro- stabilizationof microtubulesisthoughttobemedi- dependent spindle assembly(Healdet al.,1996).Thechromatin- demonstrating asubstantial role formitoticchromatinin assembly intheabsenceofcentrosomes andkinetochores, addition, plasmidDNA-coatedbeadsdrivebipolarspindle chore kinesin-likeproteinCENP-E(Woodetal.,1997).In sai etal.,1999),dependentonfactorsincludingthekineto- and anaphasesegregationinvitro(Murrayetal.,1996;De- tional kinetochoresthatmediatechromosomealignment in the presenceandabsenceofkinetochores.Uponincubation ties duringmitosis,becausespindlescanbeformedbothin dent examinationofbothkinetochoreandchromatinactivi- 1997). Thissystemhastheadvantageofallowingindepen- can supportspindleassemblyandfunction(Hiranoetal., mitosis hasnotbeenstudiedusingconcentratedextractsthat Although thecondensincomplexhasbeenshowntobere- Evidence supportingaroleforcondensinfunctioninmito- Xenopus The JournalofCellBiology or C. elegans C. elegans eggextracts,spermchromosomesformfunc-

Xenopus

, anaphasedefectsmaybedueatleastin , lossofcondensinsubunitfunctionre-

, resultinginamorediffuseprophase

eggextracts(HiranoandMitchison, ,

thatpromotespindle assemblyspecif-

Saccharomyces cerevisiae,

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Volume 161,Number6,2003

Xenopus extracts,itsrolein and Schizo- Dro- , mitosis. Bar,10 The reactionswere fixedinmetaphase,60minafter initiationof which werethencycledthrough andbackintomitosis. bule organizationduringspindleassemblyandisrequired chromosomal architecturethatpromotespropermicrotu- egg extracts.Wefindthatcondensinactivityestablishesa depleted crudeextracts. more diffuse.(D)Chromosomesassembled incontrolandcondensin- of the complexarepresent,thoughXCAP-Happearslighterand coupled beadsandelutedusingXCAP-G peptide.Allfivesubunits complex purifiedfrom control extract.(C)Silver-stained 8%gelof XCAP-E antibody,whichrecognizedasinglebandof140kDin XCAP-G antibodies( Xenopus with Hoechst33258.Bar,10 were incubatedinCSFextractfor90min,andfixedstained XCAP-E antibody( assembled inclarifiedextracttowhichbuffer(Control)or0.26mg/ml causes defectsinchromosomecondensation. Figure 1. sembly andanaphasechromosomesegregationin the localizationandfunctionofthesefactorsisnotknown. lationship betweenmitoticchromosomearchitectureand Funabiki andMurray,2000;Buddeetal.,2001),butthere- and segregation(Vernosetal.,1995;Antonio2000; Polo kinases,playessentialrolesinchromosomealignment tors, suchaschromosomalkinesinmotorsandAurora 2002). InadditiontoRCC1,otherchromatin-boundfac- and Zhang,2001;Dasso,Hetzeretal.,2002;Macara, ically inthevicinityofchromosomes(forreviewsseeClarke Here, weaddresstheroleofcondensinduringspindleas- eggextractsdepletedusingIgG(Control)orXCAP-Eand Condensin depletionfromcrude m.

-XCAP-E) wasadded. Xenopus XCAP), andprobedwithaffinity-purified Xenopus m. (B)Westernblotanalysisofcrude eggextractusingXCAP-Gantibody– sperm nuclei were added to extracts, spermnucleiwereaddedtoextracts, Xenopus Xenopus Xenopus (A)Chromosomes spermnuclei eggextracts condensin Xenopus The Journal of Cell Biology of anti–XCAP-Etodiluted,highspeed nodepletion ofcondensinusingtheseantibodiesoraddition and Hirano,2000).Consistentwithpreviousstudies,immu- to thosedescribedpreviously(Hiranoetal.,1997;Kimura complex, XCAP-EandXCAP-G,usingsequencesidentical plex from we firstestablishedconditionstodepletethecondensincom- To addresstheroleofchromosomearchitectureinmitosis, extracts causeschromosomecondensationdefects Depletion ofthecondensincomplexfromcrude tion duringanaphase. continuously toallowchromosomeresolutionandsegrega- densin complexfromcrude(10,000 we testedtheabilityofourantibodiestodepletecon- system doesnotfullymimicinvivocondensation.Therefore, and,therefore,chromosomecondensationinthis al., 1997).However,theseclarifiedextractsarearrestedin A andnotdepicted;HiranoMitchison,1994;et resulted indefectivespermchromosomecondensation(Fig.1 Results tion usingamixtureofbothantibodies, transiting throughthecellcycle.Aftertworoundsofdeple- bodies againsttwocomponentsofthe Xenopus eggextracts.Wegeneratedpeptideanti- g)extractscapableof Xenopus Xenopus 98% ofXCAP-E eggextracts

condensin

Condensin functioninspindleassemblyandanaphase| morphologically similartothosedescribedinclarifiedex- crude extractsandresultsinmitoticchromosomedefects Therefore, condensinimmunodepletioncanbeachievedin fied condensincomplextothedepletedextract(Fig.1D). tion andresolutioncouldberescueduponadditionofpuri- just asforthehighspeedreactions,chromosomecondensa- hibition inthehighspeedextract(Fig.1A).Importantly, chromosomes weresimilartothoseseenuponcondensinin- tract atthestartofmitosis(notdepicted),andaberrant were observedifXCAP-Eantibodiesaddedtotheex- condensation andresolutionwereimpaired.Similardefects somes werenotapparent,suggestingthatbothchromosome chro absence ofthecondensincomplexwereaberrant.The ble (Fig.1D).However,chromosomesassembledinthe tracts, individualcondensedchromosomeswereclearlyvisi- DNA replication,andthenbackintomitosis.Incontrolex- crude extracts,whichwerecycledthroughinterphasetoallow sperm nucleiwereaddedtocontrolandcondensin-depleted (Fig. 1C).Totesttheeffectsonchromosomemorphology, body beadswithpeptideyieldedhighlypurifiedcondensin complex wasdepleted.Elutionofthefromanti- published data),indicatingthattheentire13Scondensin (Fig. 1B).AsimilarreductioninXCAP-Gwasobserved(un- was removedfromtheextract,asjudgedbyWesternblot matin formedadiffusemassandindividualchromo- appear redandchromosomesblue.Bar,10 microtubules. Inallimages,microtubules reduced normal spindles,abnormalstructures,and of thethreecategoriesquantifiedinBandC: 60 minaftertheinductionofmitosis.Thethree condensin antibodies( formed inextractsdepletedusingIgG(Control)or of spermspindleassembly. Figure 2. or 0.26mg/ml To eachspindlereaction,eitherbuffer(Control) assembly. Twoseparateexperimentsareshown. effect ofXCAP-Eantibodyadditiononspindle structures werecounted.(C)Quantificationofthe category depictedinA.Foreachcondition, bars) showingthepercentspermnucleiineach start ofmitosis.Twoexperiments(redandblue depletion onspindleassembly60minafterthe (B) Quantificationoftheeffectcondensin XCAP antibodies( reactions inextractsdepletedwithIgG(Control)or thecategoriesdepictedinA.(D)Spindleassemblyto 60 minandthemicrotubulestructureswereassigned added atthestartofmitosis.Sampleswerefixed normal spindlesisshown. microtubule structureswereexamined.Percent fixed at60minafterthestartofmitosisand complex wasadded(Add-back).Sampleswere extract towhichpurified XCAP imagesshowstructuresrepresentative Condensin inhibitionreducesthefidelity -XCAP-E antibody( XCAP), andinXCAP-depleted XCAP) thatwerefixed Xenopus (A) Mitoticstructures Wignalletal. condensin -XCAP) was m. 100 1043 The Journal of Cell Biology 1044 30, 35,and40min.Bar,10 anaphase Once metaphasespindleswereassembled(t (Control) orcondensinantibodies( some segregationinextractsdepletedwithIgG anaphase chromosomesegregation. from Figure 3. observed (Fig.2B).However,theextentofinhibitionvaried reduction inthepercentageofnormalspindleswasalways tures areshowninFig.2A.Aftercondensindepletion,a ing areducednumberofmicrotubules.Representativestruc- mal spindle,anabnormalstructure,orastructurecontain- the microtubulearraysurroundingitwasclassifiedasanor- 60 min.Eachspermnucleusontheslidewasexamined,and tify theseverityofspindledefects,reactionswerefixedat tubule polymerizationwaslessrobust(Fig.2A).Toquan- spindles, thoughmanyweresmaller,suggestingthatmicro- spindlesweremorphologicallysimilartocontrol depleted percentage ofnormalstructuresformed.Thecondensin- tion ofthecondensincomplexsignificantlyreduced formation ofbipolarspindlesby60min.However,deple- mitosis. Incontrolextracts,mostspermnucleidirectedthe which weremonitoredat15-minintervalsafterentryinto Rhodamine-labeled tubulinwasaddedtothereactions, rant spermchromosomeslackingthecondensincomplex. Next, wetestedwhetherspindlescouldformaroundaber- spindle assembly Condensin depletionreducesthefidelityof condensation machineryunderbothconditions. tracts, suggestingthatthecondensincomplexismajor Bar, 10 were fixed30minafteranaphaseinitiation. complex wasadded(Add-back).Thestructures extract towhichpurified antibodies ( in extractsdepletedwith IgG(Control)orXCAP extract. Bar,10 areshowninthecondensin-depleted spindles are markedwithasterisks(*). side-by-side Two after anaphaseinduction.Thepolesofeachspindle ( during anaphaseincontrolandcondensin-depleted (C) Deconvolutionmicroscopyofchromosomes sperm nucleiineachcategoryisdepicted. depletion ( were countedforthecontrolandcondensin separated, orunseparated.188and162structures were classifiedascompletelyseparated,partially initiation, andthechromosomesineachspindle shown. Sampleswerefixed40minafteranaphase segregation. Onerepresentativeexperimentis of theeffectcondensindepletiononchromosome XCAP) extracts.Thesampleswerefixed35min Xenopus The JournalofCellBiology m. was inducedandsampleswerefixedat Depletion ofthecondensincomplex XCAP), respectively.Percent XCAP), andintheXCAP-depleted eggextractscausesdefectsin m. (D)Chromosomesegregation Xenopus m. (B)Quantification

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Volume 161,Number6,2003 condensin (A) Chromo- XCAP). 0’), sperm chromosomesin quired forformationofbipolarmicrotubulearraysaround we concludethatalthoughcondensinfunctionisnotre- condensin tothedepletedreactions(Fig.2D).Therefore, could besubstantiallyrescueduponadditionofpurified with thecondensationdefects,spindleassemblyphenotypes cubation withpeptide(unpublisheddata).Furthermore,as C). Effectsofantibodyadditionwereneutralizedbyprein- condensin functionbyaddingXCAP-Eantibodies(Fig.2 served similarvariabilityamongextractswhenweinhibited tract preparations.Insupportofthishypothesis,weob- depletion, butratherresultedfromdifferencesamongex- variability didnotarisefromalteredefficiencyofcondensin were indistinguishablebyWesternblot,suggestingthatthe an averageof64%.Thelevelsdepletioninallexperiments ments, thepercentreductionvariedfrom32to93%,with depending ontheexperiment.Insevenindependentexperi- phase andevaluatedtheeffectsonchromosomesegregation. the condensincomplexwerefunctional,weinducedana- of To determinewhetherthespindlesformedinabsence segregate duringanaphase Chromosomes lackingcondensinfailtoresolveand enhances thefidelityofspindleassembly.

Xenopus eggextracts,itsignificantly The Journal of Cell Biology on spindlesinanaphase.Individualchromosomesresolved higher resolution,weperformeddeconvolutionmicroscopy pared with5%intheabsenceofcondensin(Fig.3B). 40 min,revealing82%segregationincontrolextracts,com- phologically normalspindleswithsegregatedchromosomesat representative experimentbycountingthenumberofmor- unsegregated massofDNA.Thiseffectwasquantifiedina condensin-depleted extractsbegantodisassemble,leavingan thelss, spindlepolesseparated,andby40minthespindlesin center ofthespindleandwerenotmovedtopoles.Never- densin-depleted extractsremainedadisorganizedmassinthe 40 min(Fig.3A).Incontrast,thechromosomesincon- gan movingtothepolesandwerecompletelysegregatedby ples at5-minintervals.Incontrolextracts,chromosomesbe- chromosome segregationwasmonitoredbyfixationofsam- or itsdownstreamtarget,CamKII(Morinetal.,1994),and Anaphase wastriggeredinextractsbytheadditionofcalcium To examinethechromosomesegregationdefectata

Condensin functioninspindleassemblyandanaphase| to ensureproperchromosomesegregationduringanaphase. quired tocondenseandresolvechromosomearmsinorder D). Therefore,thecondensincomplexisspecificallyre- condensin wasaddedbacktothedepletedextracts(Fig.3 some segregationdefectscouldlargelyberescuedifpurified somes werenotapparent.Importantly,anaphasechromo- anaphase, itremainedasonemassandindividualchromo- extractsappearedstretchedtowardthepolesduring depleted (Fig. 3C).Incontrast,althoughtheDNAincondensin- from oneanotherandmovedpolewardincontrolspindles strom etal.,2002;StearandRoth,2002).However,these al., 2000;Steffensenet2001;Bhalla2002;Hag- 1996; Sutanietal.,1999;Lavoie2000;Ouspenski densin subunitinhibition(Sakaetal.,1994;Bhat other organismsshowingsegregationproblemsaftercon- The anaphasedefectsobservedareconsistentwithstudiesin Condensin functionisrequiredthroughoutanaphase http://www.jcb.org/cgi/content/full/jcb.200303185/DC1. induction. Bar,10 either astheextractwasenteringmitosis( presence ofbufferor0.26mg/mlXCAP-Eantibody,added sperm chromosomesin chromosome segregationdefects. before mitosisorafteranaphaseinitiationcauses Figure 4. ( ( IgG (Control),XCAP-Eantibodypreincubatedwithbuffer counted. (C)Chromosomesegregationinthepresenceof or unseparated.Foreachreaction, were classifiedascompletelyseparated,partially 40 minafteranaphaseinduction,andthechromosomes addition onchromosomesegregation.Spindleswerefixed (B) Quantificationoftheeffectcondensinantibody and lateanaphase(EarlyAnaLateAna).Bar,10 Samples werefixedatmetaphase(Metaphase),andearly immediately afteranaphaseinduction( -XCAP -XCAP), orXCAP-Eantibodypreincubatedwithantigen pep). Samples were fixed 35 min after anaphase pep).Sampleswerefixed35minafteranaphase Inhibition ofthecondensincomplexeither

m. Videos1–6areavailableat

Xenopus eggextractsinthe (A) Segregationof 100 structureswere Wignalletal. -XCAP ana). -XCAP) or 1045 m. The Journal of Cell Biology 1046 RCC1 activity. WiththeexceptionofRCC1, however,we chromokinesins, chromatin-associated kinases,orlossof densin inhibitiondonotarise frommislocalizationofthe cate thatthedefectsinspindle assemblycausedbycon- sperm nuclei(Fig.5Aandnot depicted).Theseresultsindi- still observedasignificantRanGTP gradientsurrounding speed extractstowhichcondensin antibodywasadded,we of mitoticchromatin(Kalabetal.,2002).Inhighorlow but notRanGDP,exhibitingalossofFRETinthevicinity energy transfer(FRET)probe.TheprobebindsRanGTP, local generationofRan-GTPusingafluorescenceresonance despite recruitmentoftheRanGEFRCC1,weexamined whether thechromosomalRan-GTPgradientwasdisrupted nases AuroraBandPlx1(unpublisheddata).Todetermine Xklp1 andXkid,theRanGEFRCC1,ormitoticki- sins significant changesinthelocalizationofchromokine- rescence microscopyofmetaphasespindlesdidnotreveal spindle assemblyandanaphasewasaffected.Immunofluo- whether thelocalizationofknownproteinsrequiredfor both chromosomearmsandkinetochorestodetermine fects observedintheabsenceofcondensin,weexamined To begininvestigatingtheoriginofspindleassemblyde- morphology isdisrupted condensin-depleted extracts,butkinetochore Many proteinstargetproperlytochromosomesin maintaining thatstructurethroughoutanaphase. tecture capableofbeingsegregated,andalsoinactively densin isinvolvedbothinestablishingachromosomearchi- chromosome condensation(Hiranoetal.,1997).Thus,con- densin complexisnecessarytoestablishandmaintainproper lished data).Theseresultssupportobservationsthatthecon- www.jcb.org/cgi/content/full/jcb.200303185/DC1; unpub- the antibody wasaddedeitheratthestartofmetaphaseor observed chromosomesegregationdefectswhencondensin been added.Consistentwithanalysisoffixedsamples,we trol extractsandintowhichXCAP-Eantibodyhad tion usingtime-lapsefluorescencevideomicroscopyincon- (Fig. 4,AandB).Wealsomonitoredchromosomesegrega- chromosomes wasobservedcomparedwithcontrolspindles conditions, ahigherfrequencyoflaggingandmis-segregated though chromosomesmovedtowardthepolesunderthese body wasaddedimmediatelyafteranaphaseinduction.Al- metaphase plate,spindleswereassembledandXCAP-Eanti- chromosomes haveproperlycondensedandalignedonthe completely neutralizeditseffects(Fig.4C). tion ofthecondensinantibodywithpeptideantigen seen incondensin-depletedextracts(Fig.3A).Preincuba- chromosome segregationdefects(Fig.4A),similartothose XCAP-E antibodyatthetransitiontomitosiscausedsevere fects onanaphasechromosomesegregation.Additionof tions atdifferenttimepointsandmonitoringsubsequentef- vitro extractsystem,byaddingantibodiestospindlereac- anaphase. Thisquestioncanbeeasilyaddressedinthe and resolution,orcontinuouslythroughoutmetaphase requiredearlyinmitosisforchromosomecondensation is studies couldnotdistinguishwhethercondensinfunction To testwhethercondensinfunctionisalsorequiredonce start ofanaphase(Videos4–6availableathttp:// The JournalofCellBiology

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Volume 161,Number6,2003 were oriented along theaxisofspindle, suggestingthat CENP-E waslocalizedinirregular lines.Manyoftheselines al., 1997;Fig.5A).Incontrast, aftercondensindepletion, punctate patterncorresponding tokinetochores(Woodet 1997). Incontrolmetaphaseextracts, CENP-Elocalizedina associated kinesin-likeprotein CENP-E(Woodetal., microscopy usingantibodies against theouterkinetochore- by condensindepletion,weperformedimmunofluorescence tors areaffected. do notknowwhethertheactivitiesofthesechromatinfac- min aftertheinductionofmitosis.Bar,10 morphology intheabsenceofcondensin.Sampleswerefixed60 or condensinantibodies( (green) inspindlesformedextractsdepletedusingIgG(Control) surrounding chromosomesinclarifiedextractstreatedwithbufferor FRET (blue)tohigh(red).Gradientsappearedindistinguishable the FRETratiosignal(I when boundtoRanGTP.Fluorescenceimagesshowspermnuclei, was visualizedbyadditionofaprobethatshowslossFRET Ran-GTP gradient,butdisruptskinetochoremorphology. Figure 5. and partiallyalleviateditsdistortionin nocodazole tometaphasereactionsincreasedtheCENP-Esignal -XCAP-E. Bar,10 To evaluatewhetherkinetochoreformationwasaffected Depletion ofthecondensincomplexdoesnotaffect m. (B)ImmunofluorescenceimagesofCENP-E FRET /I CFP XCAP), revealingaberrantkinetochore ), andanoverlay.Ratiosrangefromlow

XCAP extracts.Bar,10

m. (C)Additionof1

(A) RanGTP M m. The Journal of Cell Biology tin proteinswhenincubatedin recruiting thecondensincomplexandothermitoticchroma- Plasmid DNAbeadsfunctionas“artificialchromosomes,” lacking kinetochoresbyusingDNA-coatedbeads(Fig.6). effects ofcondensininhibitionontheassemblyspindles fects inkinetochoreformation.Therefore,weanalyzedthe assembly aftercondensindepletionwascausedsolelybyde- sought todetermineifthereductioninfidelityofspindle functions, butdidaffectkinetochoremorphology,wenext several chromatin-associatedproteinswithknownmitotic As condensininhibitiondidnotleadtomislocalizationof fidelity ofDNAbeadspindleassembly Depletion ofthecondensincomplexreduces stretched andtheirmorphologyisdisrupted. microtubules movechromosomes,kinetochoresbecome tolosestructuralintegritysothatwhenspindle we proposethatdepletionofthecondensincomplexcauses more normal,punctatedistribution(Fig.5C).Therefore, extractscausedtheCENP-Estainingtoresumea depleted crotubule depolymerizingdrugnocodazoletocondensin- chromatin. Insupportofthishypothesis,additionthemi- force onthekinetochoresandstretchingunderlying the alteredmorphologywasduetomicrotubulesexerting As withtheeffectsonspindleassemblyaroundspermnuclei, abnormal orreducedmicrotubulepolymerization(Fig.6A). assembly, resultinginahigherpercentageofstructureswith densin complexreducedthefidelityofDNAbeadspindle based motors(Walczaketal.,1998).Depletionofthecon- tin beadsandaresortedintoabipolararraybymicrotubule- In thissystem,microtubulespolymerizearoundthechroma- et al.,1996;KimuraandHirano,2000;unpublisheddata). Xenopus eggextracts(Heald

Condensin functioninspindleassemblyandanaphase| tivity ofotherfactors. sembly isindirect,bypromotingthelocalizationand/orac- propose thatthecontributionofcondensintospindleas- tion inducedbyisolatedcentrosomes(unpublisheddata),we condensin inhibitiondidnotaltermicrotubulepolymeriza- densation contributestomitoticchromatinfunction.As the presenceofcondensin,indicatingthatchromosomecon- mitotic chromatintoinducespindleassemblyisenhancedin in chromatincondensation.Inconclusion,theactivityof DNA-coated beadsmayrenderthemlesssensitivetodefects ture andshortDNAsequencescharacteristicoftheplasmid spindle assemblyinhibition.Alternatively,thecompactna- that kinetochoredefectsmayincreasetheseverityofsperm 2, BandC,Fig.6,C).Thisobservationsuggests paring eitherimmunodepletionorantibodyaddition(Fig. sembly werelessseverethanwithspermnuclei,whencom- ochores. However,thedefectsobservedinbeadspindleas- of spindleassemblyinextracts,eventheabsencekinet- condensation issufficienttocauseareductioninthefidelity in Fig.6B.Thesedataindicatethatdisruptingchromatin Quantification oftworepresentativeexperimentsisshown hibition averaging33%infiveindependentexperiments. the severitywasvariableamongextracts,withpercentin- tion afteranaphaseinductiontodeterminewhetherkineto- this issue,wefirstexaminedCENP-Ekinetochorelocaliza- ure inchromosomecompactionandresolution.Toaddress gation inanaphaseappearedtobethedirectresultofafail- absence ofcondensin,theinhibitionchromosomesegre- In contrasttothespindleassemblydefectsobservedin effects onchromosomesegregation Depletion ofthecondensincomplexlikelyhasdirect in A. structures weregroupedintothecategories depicted Samples werefixedat90min,and themicrotubule antibody ( structures, andreducedmicrotubules.Bar,10 quantified inBandC:normalspindles,abnormal show structuresrepresentativeofthethreecategories initiation ofmitosis.Thethree either buffer(Control)or0.26mg/ml experiments. Toeachspindleassemblyreaction, addition onbeadspindleassemblyintwoseparate (C) QuantificationoftheeffectXCAP-Eantibody bead clustersineachcategoryaredepicted. to thecategoriesdepictedinA.Foreachreaction, the microtubulearraysurroundingitwasassigned of 10ormoreDNAbeadswasexaminedand fixed 90minafterthestartofmitosis.Eachcluster experiments (redandbluebars).Sampleswere depletion onbeadspindleassemblyintwoseparate (B) Quantification oftheeffectcondensin ( depleted usingIgG(Control)orcondensinantibodies structures formedaroundDNAbeadsinextracts of DNAbeadspindleassembly. Figure 6. 100 structureswerecounted.Percentof XCAP) thatwerefixedinmetaphase90minafter Condensin inhibitionreducesthefidelity -XCAP) wasaddedatthestartofmitosis. (A) Microtubule Wignalletal. XCAP images -XCAP-E

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m.

The Journal of Cell Biology 1048 movement ofchromosome arms(Antonioet al.,2000;Funa- chromokinesin Xkidmustbe degradedtoallowpoleward (Losada etal.,1998;Uhlmann etal.,1999).Inaddition,the proteolysis ofacohesinsubunit, termedXRad21in tween sisterchromatidsmust occur,whichisduetospecific defects inchromosomearchitecture. Lossofcohesionbe- metaphase toanaphasetransition thatcouldbeaffectedby ipate intheregulationofchromosomesegregationat defects intheabsenceofcondensin,otherfactorsalsopartic- matid resolutionmaybesufficienttoexplainthesegregation that mediatemicrotubuleattachmentandforcegeneration. CENP-E isabletotargetdiscretesitesonchromosomes tion suggeststhatintheabsenceofcondensinfunction, stretched segmentsofDNA(Fig.7A,arrow).Thislocaliza- these structures,itwasclearlyvisibleattheendsof and Fig.7A).AlthoughCENP-Estainingwasaberrantin though thechromosomeswereunabletoseparate(Fig.3 C observed trailsofchromatinextendingtowardthepoles,al- mosomes poleward(Fig.7A).Aftercondensindepletion,we lustrating kinetochoremovementsthatnormallydragchro- the endsofsisterchromatidsmigratingtowardpoles,il- control extracts,punctateCENP-Estainingwasobservedon poleward forcesintheabsenceofcondensinfunction.In chore–microtubule attachmentswerecapableofgenerating antibodies ( spindles formedinextractsdepletedusingIgG(Control)orcondensin in anaphaseorXRad21degradation. Figure 7. initiation. Bar,10 as aloadingcontrol(notdepicted). condensin. TheblotwasalsoprobedwithantibodiesagainsttopoII a similarreductionofXRad21inthepresenceandabsence and blottedusingaffinity-purifiedXRad21antibodies,revealing metaphase oranaphase.Proteinswereseparatedonan8%gel with IgG(control)orcondensinantibodies( in anaphase.Chromosomeswerepurifiedfromextractsdepleted kinetochores arestillfunctional.(B)AnalysisofXRad21degradation is stretchedtowardthespindlepoles(arrow),suggestingthat Although structuraldisorganizationandlossofsisterchro- The JournalofCellBiology Condensin depletiondoesnotaffectCENP-Elocalization XCAP). Sampleswerefixed15minafteranaphase m. CENP-EislocalizedtoregionsofDNAthat

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Volume 161,Number6,2003 (A)CENP-Estaining(green)in XCAP) ineither Xenopus biki andMurray,2000).Finally,topoisomeraseII resolution andsegregationduringanaphase. requirement forcondensinfunctiontoallowchromosome tion (Fig.4),theseresultsindicateadirectandcontinuous tion thatcondensinisrequiredevenafteranaphaseinduc- metaphase–anaphase transition.Togetherwithourobserva- does notappeartoimpairproteinsfunctioningatthe tracts uponanaphaseinduction.Thus,condensininhibition chromosomes inbothmock-andcondensin-depletedex- Xkid (unpublisheddata)weresimilarlydiminishedfrom ously (FunabikiandMurray,2000).XRad21(Fig.7B) metaphase orinducedtoenteranaphaseasdescribedprevi- phase, spermnucleiwereisolatedfromextractsarrestedin inhibition. ToexaminethelossofXRad21andXkidinana- ing thatbulktopoIIactivitywasnotaffectedbycondensin or XCAP-Eantibodyaddition(unpublisheddata),suggest- plast decatenationwasnotalteredaftercondensindepletion tracts (ShamuandMurray,1992).Theefficiencyofkineto- plast DNAaddedtocontrolandcondensin-depletedex- nevertheless affected,weassayedthedecatenationofkineto- unpublished data).TotestwhethertheactivityoftopoIIwas depletion onthelocalizationoftopoII(Hiranoetal.,1997; ported inclarifiedextracts,wefoundnoeffectofcondensin proteins incontrolandcondensin-depletedextracts.Asre- ray, 1992).Therefore,wecomparedthebehaviorofthese activity isrequiredforsistersegregation(ShamuandMur- Discussion sembly in Mitotic chromatinisamajordrivingforceforspindleas- Condensin andthefunctionofmitoticchromatin some functionsincrude ined theroleofcondensincomplexinmitoticchromo- tion andorientationofkinetochores.Here,wehaveexam- chromosome architecturemayaidinthestructuralorganiza- strength towithstandspindleforces.Third,thecondensed sister chromatids,andisthoughttoprovidemechanical ond, condensationisrequiredfortheresolutionoftangled spindle toseparatethegenomeintotwodiscretesets.Sec- exceed thelengthofcell,compactionisessentialfor gation forseveralreasons.First,ifinterphasechromosomes Condensation isrequiredforsuccessfulchromosomesegre- phase isinduced. and dramaticallyinhibitchromosomesegregationwhenana- nificant impactontheabilityofmitoticspindletoform, mised whencondensinisdepleted.Thesedefectshaveasig- that chromosomecondensationandresolutionarecompro- extracts examinedpreviously(Hiranoetal.,1997),wefind reconstituting spindleassemblyinvitro.Astheclarified tubule polymerization andspindleassembly intheabsence kinetochores (Healdetal.,1996). Theinhibitionofmicro- induce spindleassemblyinthe absenceofcentrosomesand cient, asillustratedbytheability ofDNA-coatedbeadsto Murray, 2000;Buddeetal.,2001). Chromatinitselfissuffi- 1995; Ohbaetal.,1999;Antonio etal.,2000;Funabikiand are essentialforproperspindle assembly(Vernosetal., tors, suchasRCC1,chromokinesins,andthekinasePlx1, Xenopus eggextracts.Chromatin-associatedfac- Xenopus eggextractscapableof (topoII)

The Journal of Cell Biology ers willbeextremely informativetoelucidate howchromo- fluorescence videomicroscopy analysisofkinetochoremark- microtubules andactedonby anaphaseforces.Inthefuture, their distortedmorphology,the kinetochoresareattachedto the spindlepoles.Thisobservation suggeststhatdespite of condensinfunction,CENP-E fociwerestretchedtoward eral failureofspermchromosome segregationintheabsence chromosome movement.Duringanaphase,despitethegen- cannot resistdeformationuponmicrotubuleattachmentand condensation, thekinetochoreandunderlyingchromatin spindle assembly,suggestingthatintheabsenceofproper CENP-E distributionbecomesstretchedanddistortedupon CENP-E todiscretefoci(Woodetal.,1997).However, cated bytherecruitmentofouterkinetochorekinesin Kinetochores formupondepletionofcondensin,asindi- Condensin andkinetochores might contributetothereducedfidelityofspindleassembly. ochore morphologyseenincondensin-depletedextracts around spermnuclei,and,therefore,thedisruptionofkinet- tively, kinetochoresmaycontributetospindleformation to disorganizationbylackofcondensinfunction.Alterna- densed spermchromosomes,makingthemlesssusceptible local concentrationofchromatinfactorsthanthedecon- physical nature,theplasmidDNAbeadsgenerateahigher 2001). Onepossibleexplanationisthatduetotheircompact matin proteins(KimuraandHirano,2000;Buddeetal., beads, althoughbothrecruitasimilarcomplementofchro- was moredramaticallyaffectedthanthatinducedbyDNA found thatspindleassemblyaroundspermchromosomes plex inspindleformation. portant todistinguishthegeneralityofrolecom- nation ofcondensinfunctioninothercelltypeswillbeim- et al.,2000;Ouspenski2000).Moreextensiveexami- ported tohavedefectsinmicrotubuleorganization(Lavoie ganisms, buddingyeastcondensinmutantshavebeenre- densin functionhavenotbeenwellelucidatedinotheror- eggs. Althoughspindleassemblydefectsduetolossofcon- served amongextractpreparationsfromdifferentbatchesof sembly invitro,andthattheseactivitiesarenotequallypre- ple andpartlyredundantmechanismspromotespindleas- phenotype variabilitylikelyresultsfromthefactthatmulti- tion efficiencieswereidenticalbyWesternblot.Spindle which webelieveisduetoextractvariabilitybecausedeple- tion onspindleassemblyfromexperimenttoexperiment, altered association,phosphorylationstate,oractivity. presence orabsenceofcondensinmayrevealproteinswith proteins recruitedtospermnucleiorDNAbeadsinthe assay. Inthefuture,biochemicalcomparisonofchromatin subtle defectsintheRanGTPgradientundetectableour (unpublished data;Fig.5A),thoughwecannotruleout not revealsignificantchangesuponcondensindepletion tion ofaRanGTPgradientinthevicinitychromatindid chromatin proteinsbyimmunofluorescence,andthegenera- ganization. However,ourexaminationofseveralknown of chromatinfunctionduetodefectsinitshigher-orderor- of condensinfunctionis,therefore,likelyduetoimpairment Despite thespindlephenotypevariability,weconsistently We observedvariationintheeffectsofcondensininhibi-

Condensin functioninspindleassemblyandanaphase| ment withinthespindle. some structureimpactskinetochoreattachmentandmove- complex inanaphasechromosomeresolution. Altogether, thesedatasupportadirectroleforthecondensin chromosomes inacondensinmutant(Bhallaetal.,2002). budding yeastthatshowedScc1isproperlyremovedfrom normally intheabsenceofcondensin,supportingwork subunit XRad21.Bothofthesefactorsappeartobedegraded separation includethechromokinesinXkidandcohesin somal factorswhoseproperregulationisrequiredforsister is capableofbeingefficientlydecatenated.Otherchromo- may berequiredtogenerateachromosomearchitecturethat complex modulatesthefunctionoftopoIIdirectly,thoughit published data).Therefore,itisunlikelythatthecondensin 1997) orinhibitthedecatenationofkinetoplastDNA(un- localization tochromosomesincrudeextracts(Hiranoetal., clarified extracts,condensindepletiondidnotaltertopoII tion (ShamuandMurray,1992).Consistentwithresultsin topoII, whichisrequiredforanaphasechromosomesegrega- was thatcondensininhibitioninterferedwiththefunctionof condensation andsisterentanglements.Anotherpossibility actively maintainchromosomeorganization,preventingde- enzymatic roleindecatenation,itismorelikelyrequiredto the cellcycle.Althoughitispossiblethatcondensinplaysan system allowedustoinhibitcondensinatanytimeduring tion tothesameextentasimmunodepletion,andinvitro because additionofcondensinantibodiesinhibiteditsfunc- chromosome segregationdefects.Thisanalysiswaspossible anaphase, asitsinhibitionafteranaphaseinductioncaused We foundthatcondensinfunctionisrequiredthroughout An activeroleforcondensininanaphase typic difference betweendepletionoftheentire condensin variable phenotypes.Forexample, theremaybeapheno- level ofcondensininactivation indifferentsystemscauses fied condensin.Itispossible thatvariabilitybetweenthe fects incondensation,whichare rescuedbyadditionofpuri- densin depletionfromlowspeed extractscausesseverede- the highspeedextractsystem. Wehaveshownthatcon- nopus ences betweenthephenotypesofcondensininhibitionin (Stear andRoth,2002).Ourstudiesshowthatthediffer- twisting phenotypethatdisruptskinetochoreorientation fects inmutantscanalsobeattributedtoachromosome anaphase segregation(Hagstrometal.,2002).Anaphasede- chromosome compactionduringmetaphase,butinhibited condensin subunitsdidnotcauseadramaticdecreasein effects wereobservedin disrupted duringanaphase(Steffensenetal.,2001).Similar appeared normal,butchromatidresolutionwasstrikingly densin component,longitudinalshorteningofchromosomes neuroblasts ofa compaction uponlossofcondensinfunction.Inmitotic systems reportedmoremoderatedefectsinchromosome ogy seenin In contrasttothedramaticeffectsonchromosomemorphol- of condensin? Does chromosomecondensationoccurintheabsence andothersystemsarenotattributabletoanartifactof Xenopus Drosophila (Hiranoetal.,1997),studiesinother C. elegans mutantlackinganSMCcon- embryos,asRNAiagainst Wignalletal. 1049 Xe- The Journal of Cell Biology 1050 some stretchinganddistortioninresponsetospindleforces. resolution, kinetochoreorientation,andpreventschromo- generate achromosomestructurethatallowssisterchromatid during mitosis,butthatcondensinfunctionisrequiredto ism, otherfactorsmaycontributetochromatincompaction extracts. Theemergingmodelisthatdependingontheorgan- KCl, 1mMMgSO (48% glycerol,11%formaldehydeinMMR[5mMHepes,pH7.8,2 CaCl 2001). Toinduceanaphase,0.1volumeofCaCl MgCl For antibodyaddition, Antibody addition,immunodepletion,andrescue driven intointerphasebyadding0.1extractvolumeofCaCl clei/ CSF extractsweresupplementedwithdemembranatedsperm(final500nu- In vitrospindleassemblyandanaphase were agiftfromP.Budde(UniversityofCalifornia,Berkeley). Gadde (UniversityofCalifornia,Berkeley).Plx1andAuroraBantibodies (University ofCalifornia,Berkeley).CENP-EantibodywasagiftfromS. et al.,1992).XCAP-GserumwasagiftfromN.CozzarelliandV.Holmes oratories. Antibodieswereaffinity-purifiedasdescribedpreviously(Sawin Peptide synthesisandantibodyproductionwereperformedbyZymedLab- ously (Hiranoetal.,1997;Losada1998;KimuraandHirano,2000). XCAP-E, XCAP-G,andXRad21wereidenticaltothosedescribedprevi- nuclei wereagiftfromG.O.Nads(UniversityofCalifornia,Berkeley). pared asdescribedpreviously(Murray,1991;Healdetal.,1996).Sperm (model TLS-55;BeckmanCoulter).SpermnucleiandDNAbeadswerepre- tracts, CSFextractwasspunanadditional90minat55,000rpminarotor rpm inarotor(modelHB-6,Sorvall)for15min.Toprepareclarifiedex- Desai etal.,1999),exceptthatthecrushingspinwasperformedat10,200 rested inmetaphaseofmeiosisIIasdescribedpreviously(Murray,1991; Crude cytostaticfactor(CSF)extractswerepreparedfrom Preparation of Materials andmethods and ensureproperchromosomesegregationin is requiredthroughoutmitosistopromotespindleformation chromosome condensationpathwaysindifferentorganisms. ternatively, itispossiblethattherearedifferencesinthe complex, comparedwithmutationofoneitssubunits.Al- condensation reactionsweresetupasinHiranoetal.(1997). was agiftfromM.Doree(CNRS,Montpellier,France).Clarifiedextract was addedasdescribedpreviously(Morinetal.,1994).CamKIIplasmid natively, anactivatedversionofCamKIIexpressedinreticulocytelysate fix, 1- with X-rhodamine–labeledtubulin(50 extract waskeptoniceandmixedfrequently. to depletea70- peptin, pepstatin,andchymostatin. (Desai etal.,1999).Beadswereused night. Aftercoupling,thebeadswere washedwithCSF-XB extract to0.2mg/ml. dialyzed intoXB(Desaietal.,1999)toremovethepeptideandadded to fold molarexcessofXCAP-Epeptideorbufferovernight.Reactionswere added. Toblockwithantigen,20 duction. Asacontrol,equivalentvolumesofCSF-XBorrabbitIgGwere nal) atthestartofinterphase,uponentryintomitosis,orafteranaphasein- described previously(Hiranoetal., 1997; KimuraandHirano,2000).100 brought to200 were addedto55 into acolumn,and washedwith80columnvolXBE2-gly (containing20 BRL). Beadswere incubated witha2-mlhighspeedextract for4h,poured g/ml Hoechst33258)andsquashedwithacoverslip(WignallHeald, g g In summary,ourstudiesshowthatthecondensincomplex Synthetic peptidescorrespondingtotheCOOH-terminalregionsof Xenopus Extracts weredepletedofcondensinintworounds.Foreachround,15 2 -XCAP-E and7 -XCAP-G wascoupledto200 2 , 10mMHepes,pH7.7,150sucrose,100KCl,and1 l) orwithDNAbeadsonice,andtransferredto20 ). Tocycleintomitosis,anequalvolumeofCSFextractwasadded The JournalofCellBiology l sampleswerespottedontoslides,overlaidwith5 13Scondensinwaspurifiedfrommitotic clarifiedextractsas l withTBS Xenopus 4 l CSFextractintwo1-hrounds.During eachround,the , 100mMNaCl,2CaCl l ofproteinA–Dynabeads(Dynal),thevolumewas g -XCAP-E wasaddedtoextract0.26mg/ml(fi- -XCAP-G ( eggextracts,DNA,andantibodies 0.1%TritonX-100,androtatedat4

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g Volume 161,Number6,2003 XCAP) or23 l proteinA–agarosebeads(GIBCO -XCAP-E wasincubatedwithafive- g/ml final;Hymanetal.,1991).To 2 , and0.1mMEGTA],5 2 bufferwasadded.Alter- g rabbitIgG(control) X. laevis C. Extractswere Xenopus 2 l ofspindlefix buffer(4mM 10 eggsar- C over- g leu- egg paragraphs. Additionof60 tion: aninvertedmicroscope(modelIX70;Olympus)with40 was performedusingaDeltaVision(AppliedPrecision,Inc.)imagingsta- independent experimentswereperformed.Deconvolutionmicroscopy At least75structureswereevaluatedforeachcondition,andatfive of spindlephenotypes,microtubulestructureswerevisuallycategorized. spun ontocoverslipsthrougha4-mlcushion(BRB80 otin dUTP(25 changes. Inbrief,120 Chromosomes werepurifiedasinFunabikiandMurray(2000)withminor Chromosome purification 1 mlofdilutionbuffer BRB80, 30%glycerol,and0.5%TritonX-100)fixedbytheadditionof cations. Inbrief,10- coverslips asdescribedpreviously(Desaietal.,1999)withminormodifi- To performimmunofluorescenceonspindles,reactionswerespunonto Fixation andstainingofspindleschromosomes Accepted: 30April 2003 Revised: 27March 2003 Submitted: 27March2003 doctoral NationalScienceFoundation fellowship(toS.M.Wignall). Heald; grantGM57839),thePewCharitable Trust(toR.Heald),andapre- script; andtheHealdlabforhelpful discussions. P. Budde,K.Hagstrom,andKaufman forcriticalreadingofthemanu- convolution microscopy;P.Kalabfor helpwithRanGTPgradientanalysis; We thankM.Johnson(UniversityofCalifornia,Berkeley)forhelpwithde- full/jcb.200303185/DC1. (microtubules). Videos1–6areavailableathttp://www.jcb.org/cgi/content/ shown togetherinVideo4,andseparately5(DNA) 6 tibody wasaddedatthestartofmitosis.ThemicrotubulesandDNAare 1. Video2showstheDNAalone,and3microtubules. somes ingreen.ThemicrotubulesandchromatinareshowntogetherVideo added atthestartofmitosis.Microtubulesareshowninredandchromo- rate of10frames/s. vious section.Frameswerecollectedevery30s,andaredisplayedat a All videoswereacquiredusingthemicroscopeset-updescribedinpre- Online supplementalmaterial ported intoAdobePhotoshop Co.), andMetamorphsoftware(UniversalImagingCorp.).Imageswereim- Hamamatsu), ashuttercontroller(modelLambda10-2;SutterInstrument scope (modelE600;Nikon)equippedwithaCCDcameraOrcaII; Images andtime-lapsemovieswerecollectedusingafluorescencemicro- Microscopy anddataanalysis performed asdescribedpreviously(FunabikiandMurray,2000). form metaphaseoranaphasechromosomes,respectively.Purificationwas 100 tive deconvolution(Chenetal.,1996). metrics; RoperScientific).AllimagesweretakenwithaZ-stepsizeof0.2 30% glycerol).Coverslipswerefixedafterin aldehyde (Losadaetal.,2000)andspunthrougha5-mlcushion(XBE2 ture, 10- 5,500 rpmfor20minusinganHB-4rotor.Topreservechromosomestruc- Kalab (UniversityofCalifornia,Berkeley). fore startingthecondensationorspindlereactions.ProbewasagiftfromP. the probe(final4 probe YFP-RBD-CFPasdescribedpreviously(Kalabetal.,2002),except Hoechst 33258inPBS. ing FITC-conjugatedsecondaryantibodies.DNAwasstainedwith1 blocked with3%BSAinPBS,andprocessedforimmunofluorescenceus- concentrators (Amicon),andaddedtodepletedextractsat1:10. mg/ml XCAP-Gpeptide,concentratedto1–2usingMicrocon-30 column volXBE2-gly.CondensinwaselutedwithXBE2-glycontaining0.8 mM l) cycledtheextractintomitosis.ControlorCaCl m, savedasthree-dimensionalstacks,andsubjectedtoconstraineditera- This workwassupportedbytheNational InstitutesofHealth(toR. Videos 4–6showanaphaseinextracttowhich0.26mg/mlXCAP-Ean- Videos 1–3showchromosomesegregationinextracttowhichbufferwas The RanGTPgradientwasvisualizedusingaRanGTP-bindingFRET , 1.35NAoilimmersionlenses,andacooledCCD(modelPhoto- -glycerophosphate), 10-columnvolXBE2-gly l reactionswerefixedfor10minwith100 M) wascycledintointerphaseasdescribedinprevious M) waspreincubatedwithextractonicefor10minbe- l reactionswereaddedto1mlofdilutionbuffer(1 l extractcontainingspermnuclei(2500/ 5%formaldehyde.After10min,sampleswere l freshextractandcyclinB ® softwareforprocessing.Forquantification 20 2 methanolfor5min, bufferwasaddedto 0.4MKCl,and10- l XBE2 40%glycerol)at 90 (final24 , 1.3NAor 2%form- l) andbi- g/ml g/ The Journal of Cell Biology Dasso, M.2001.RunningonRan:nucleartransportandthemitoticspindle. Desai, A.,A.Murray,T.J.Mitchison,andC.E.Walczak.1999.Theuseof Lavoie, B.D.,K.M.Tuffo,S.Oh,D.Koshland, andC.Holm.2000.Mitoticchro- Kimura, K.,V.V.Rybenkov,N.J.Crisona,T.Hirano,andN.R.Cozzarelli.1999. Karsenti, E.,andI.Vernos.2001.Themitoticspindle:aself-mademachine. Kalab, P.,K.Weis,andR.Heald.2002.VisualizationofaRan-GTPgradient in Hyman, A.A.,D.Drechsel,Kellogg,S.Salser,K.Sawin,P.Steffen,L.Worde- Hirano, T.,R.Kobayashi,andM.Hirano.1997.Condensins,chromosomecon- Hirano, T.,andT.J.Mitchison.1994.Aheterodimericcoiled-coilproteinrequired Hetzer, M.,O.J.Gruss,andI.W.Mattaj.2002.TheRanGTPaseasamarkerof Heck, M.M.1997.Condensins,,andchromosomearchitecture:howto Heald, R.,R.Tournebize,T.Blank,Sandaltzopoulos,P.Becker,A.Hyman,and Hagstrom, K.A.,V.F.Holmes,N.R.Cozzarelli,andB.J.Meyer.2002. Clarke, P.R.,andC.Zhang.2001.RanGTPase:amasterregulatorofnuclear Funabiki, H.,andA.W.Murray.2000.ThechromokinesinXkidisessentialfor Kimura, K.,andT.Hirano.2000.Dualrolesofthe11Sregulatorysubcomplex in Kimura, K.,andT.Hirano.1997.ATP-dependentpositivesupercoilingofDNA Chen, H.,D.D.Hughes,T.A.Chan,J.W.Sedat,andD.A.Agard.1996.IVE(Im- Budde, P.P.,A.Kumagai,W.G.Dunphy,andR.Heald.2001.RegulationofOp18 Bhat, M.A.,A.V.Philp,D.M.Glover,andH.J.Bellen.1996.Chromatidsegrega- Bhalla, N.,S.Biggins,andA.Murray.2002.MutationofYCS4,abuddingyeast Bazett-Jones, D.P.,K.Kimura,andT.Hirano.2002.Efficientsupercoilingof Antonio, C.,I.Ferby,H.Wilhelm,M.Jones,E.Karsenti,A.R.Nebreda,and Andersen, S.S.L.1999.Balancedregulationofmicrotubuledynamicsduringthe References 104:321–324. mosome condensationrequiresBrn1p,the yeasthomologueofBarren. writhe: implicationsforchromosomecondensation. 13S condensinactivelyreconfiguresDNAbyintroducingglobalpositive interphase andmitotic Enzymol. man, andT.J.Mitchison.1991.Preparationofmodifiedtubulins. homolog ofthe densation proteincomplexescontainingXCAP-C,XCAP-Eanda for mitoticchromosomecondensationinvitro. 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