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 chromatin structureinmitoticchromosome functionsisun- demembranated spermnucleiintodiscretechromosomes, clarified extractsseverelycompromisedcondensationof Xenopus reduces chromosomelength chromosome 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 Chromosomes 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; mitosis;kinetochore; 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 Caenorhabditis elegans 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-