Keeping kinetochores on track

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Keeping kinetochores on track

MERALDI, Patrick

Abstract

The multiple functions of kinetochores are reflected in their complex composition, with over a hundred different proteins, which self-associate in several functional subcomplexes. Most of these kinetochore proteins were identified over the last 10-12 years using a combination of genetic, cell biological, biochemical, and bioinformatic approaches in various model organisms. The key challenge since then has been to determine the structural architecture of kinetochores, define the functions of its different subcomponents, and understand its regulation, both in response to the rapid changes in microtubule dynamics or to sense erroneous attachments for spindle checkpoint signalling. Here, we present some of the key advances obtained in the last six years on the biology of kinetochores, both through our work and through the work of many other groups studying this exciting structure.

MERALDI, Patrick. Keeping kinetochores on track. European Journal of Cell Biology , 2012, vol. 91, no. 2, p. 103-6

DOI : 10.1016/j.ejcb.2011.10.002 PMID : 22104445

Available at:

http://archive-ouverte.unige.ch/unige:28852

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European Journal of Cell Biology

jo u r n al h o m e p a g e :w w w . e l s e v i e r . d e / e j c b

Mini-Review

Keeping kinetochores on track ^夽

Patrick Meraldi

InstituteofBiochemistry,ETHZurich,Schafmattstrasse18,CH-8093Zurich,Switzerland

a r t i c l e i n f o

Articlehistory:

Received22September2011 Accepted6October2011

Keywords:

Kinetochore

Chromosomemovement Functionalsubcomplexes

a b s t r a c t

Themultiplefunctionsofkinetochoresarereflectedintheircomplexcomposition,withoverahundred differentproteins,whichself-associateinseveralfunctionalsubcomplexes.Mostofthesekinetochore proteinswereidentifiedoverthelast10–12yearsusingacombinationofgenetic,cellbiological,biochem- ical,andbioinformaticapproachesinvariousmodelorganisms.Thekeychallengesincethenhasbeen todeterminethestructuralarchitectureofkinetochores,definethefunctionsofitsdifferentsubcom- ponents,andunderstanditsregulation,bothinresponsetotherapidchangesinmicrotubuledynamics ortosenseerroneousattachmentsforspindlecheckpointsignalling.Here,wepresentsomeofthekey advancesobtainedinthelastsixyearsonthebiologyofkinetochores,boththroughourworkandthrough theworkofmanyothergroupsstudyingthisexcitingstructure.

© 2011 Elsevier GmbH. All rights reserved.

Introduction

Ineverycellcyclethegenomemustbeduplicatedandequally distributedintotwofuturedaughtercells.Eachchromatidisrepli- cated during theS-phase and the duplicatedsister chromatids aresegregated duringmitosis.Chromosomesegregationis con- trolledineukaryotesbythemicrotubule-basedmitoticspindleand kinetochores,whichare microtubulebindingsitesbuiltonspe- cialisedchromosomalregionscalledcentromeres(Santaguidaand Musacchio,2009;WalczakandHeald,2008).Themitoticspindle hasabipolarstructure,asmicrotubulesareanchoredwiththeir minusendsatbothspindlepoles,andbindthekinetochoresvia theirplus ends.Faithful chromosome segregationrequires that eachsisterkinetochorepairbindsmicrotubulesemanatingfrom oppositespindlepoles(amphitelicorbipolarattachment).Incase oferrors,orabsenceofattachments,kinetochoresengagethespin- dlecheckpoint,leadingtoadelayinanaphaseonset(Khodjakovand Pines,2010).Thissafeguardmechanismguaranteesthatanaphase onlyoccursonceallkinetochorepairsareattachedinabipolarman- ner.Thissetofeventsisessentialtopreventgeneticinstability atthechromosomallevelandthedevelopmentofchromosomal imbalances(aneuploidy),aphenomenonthatispresentinabout 85%ofsolidhumantumours(WeaverandCleveland,2006).

Kinetochoresarenotonlythecentromericmicrotubuleattach- mentsitesandkeysensorsforthespindlecheckpoint,theyalso formthemachinerythatisresponsibleformitoticchromosome

夽ThisarticleisbasedontheWalther-Flemming-Medallectureandhasbeenpub- lishedinparallelin“CellNews”,theNewsletteroftheDeutscheGesellschaftfür Zellbiologie.

E-mailaddress:patrick.meraldi@bc.biol.ethz.ch

movements, in particular congression to the metaphase plate andseparationofthesisterchromatidsatanaphaseonset(Kops etal.,2010).Kinetochoresregulatechromosomemovementsboth throughmicrotubulemotorproteins(kinesinsanddynein)andby regulatingthe dynamicsof themicrotubule plus endstheyare boundto.Kinetochores alsostabilisethemicrotubulestheyare boundto,ensuringthatchromosomesremainattachedtothespin- dleduringallphasesofchromosomesegregation.Theimportance ofkinetochoresisillustratedbythefactthatinunicellularorgan- isms,suchasthebuddingyeastSaccharomycescerevisiaeandthe fissionyeastSchizosaccharomycespombe,orinmulti-cellularorgan- ismssuchasthenematodeCaenorhabditiselegansorthefruitfly Drosophilamelanogaster,mostkinetochoreproteinsareessential.

Thisisalsotrueinvertebrates,wheremanykinetochoresareessen- tial,evenatthecellularlevel.

Kinetochores,ahighlyconservedstructureonlyatsecond look

Thestudyofkinetochoreproteinshasbeendifficultforalong timefortwomainreasons:firstlytheproteinsareonlypresentin lowabundance,anduntilveryrecentlythepurificationofentire kinetochoreswaselusive(Akiyoshietal.,2010).Thisdelayedthe functionalstudyof kinetochoreproteins, asonlyafew ofthem wereknown.Theseconddifficultyisthattheproteinsequences ofmostkinetochoresubunitsareevolutionarilyhighlydivergent.

For a long time, the sequences of fungal kinetochore proteins werenotsufficienttoidentifymetazoankinetochoreproteinsand vice versa in a BLAST search, a fact that considerably delayed comparativefunctionalstudies.Asanexample,in2005onlytwo structuralproteinswereknowninD.melanogaster,comparedto over60inS.cerevisiae(Heegeretal.,2005).Tomakethingsmore 0171-9335/$–seefrontmatter© 2011 Elsevier GmbH. All rights reserved.

doi:10.1016/j.ejcb.2011.10.002

104 P.Meraldi/EuropeanJournalofCellBiology91 (2012) 103–106

complicated,itwasalsoknownthatthestructureandsizeofkine- tochoresvarystronglyfromorganismtoorganism(Przewlokaand Glover,2009).Thekinetochoresofmosteukaryotesarebuiltonso- calledregionalcentromeres,longstretchesofarepetitiveAT-rich sequencethatlackanysequence-specificity.Suchkinetochorescan bebuiltoncentromeresthatareassmallasafewkilobasesand onlybindthreemicrotubules(S.pombe),orsitoncentromeresof severalmegabasesandbindupto25microtubules(mammalian cells).Therangeisevenwiderifoneconsidersthekinetochoresof certainbuddingyeasts(includingS.cerevisiae),whicharebuilton short(125bp)andsequence-specificpointcentromeresandonly bindonemicrotubule,ortotheotherextreme,certainnematodes (includingC.elegans),whichpossessholocentriccentromeresthat spantheentirelengthofthechromosomes andareboundbya multitudeofmicrotubules.Giventhehighdiversityin structure andsequence,itwasuncleartowhatextentthecompositionof kinetochoresineukaryoteswouldbeconserved.

However,aparallelsteadyincreaseinfullysequencedgenomes andinthenumber ofannotatedkinetochoreproteinsover time alloweda more precise bioinformatic analysis, which revealed thatmost kinetochoreproteinsare conserved amongst eukary- otes.Inparticular,ourgroupdevelopedanalgorithmtoidentify novelkinetochoreproteinsbothin fungi,plants andmetazoans usingknownS.cerevisiaeor S.pombekinetochoreproteinsas a startingpoint(Meraldietal.,2006).Sincethesequencesofkine- tochoreproteinsareoftenonlyconservedinveryshortstretches, wegeneratedmultiple-sequencealignmentsoffungalkinetochore proteinstoidentifytheseshortstretchesandusethemassearch patternsagainstmetazoanorplantdatabases.Thisapproachiden- tifiedsevennovelorthologouskinetochoreproteinsinmammals, threenovelproteinsin D. melanogasterand twonovelproteins inplants. These resultsimpliedthat mostkinetochoreproteins are conserved in eukaryotes, a finding that was confirmed by biochemicalandgeneticstudiesidentifyingthosesameproteins asbonafidekinetochoreproteins(Cheesemanetal.,2004;Foltz etal.,2006;Obuseetal.,2004;Okadaetal.,2006;Schittenhelm etal.,2007).Theoverallconclusionofthesestudieswasthatthe coreof eukaryotickinetochores is composedof two largecon- servedproteinnetworks:ononesidetheKMNnetwork, which consistsofKnl-1,thehetero-tetramericMIND/Mis12subcomplex, andthehetero-tetrameric-NDC80subcomplex,andontheother sidetheCCANnetwork(ConstitutiveCentromereAssociatedNet- work,whichisalsocalledCENP-ANAC/CADorCENP-H/Icomplex), which consists of 15 subunits. Soon after the NDC80 subcom- plexwasrecognisedas thekey microtubule attachmentsite at kinetochores,whichisessentialforthebindingtospindlemicro- tubulesinalleukaryotes(Cheesemanetal.,2006;DeLucaetal., 2006).

Kinetochores,keydriversofchromosomemovements

Once kinetochores have established a stable microtubule- attachmentthrough theNDC80 complex,one of theiressential functionsisto drivethechromosomes tothemetaphaseplate, aprocessthat isless wellunderstood.Inmammaliancells, one can distinguish two phases in this process: first kinetochores establishlateralattachmentstomicrotubules(inanNDC80inde- pendent manner), allowing them to congress to the centre of thespindle toform stablebipolar, end-onattachments viathe NDC80complex (Cai et al., 2009). Microtubule motors, in par- ticularthe kinesinCENP-E and dynein, control themovements inthisinitialphase (Kapooretal.,2006;Vorozhkoetal.,2008).

Inasecondstep,oncekinetochorepairsareattachedinabipo- lar manner,chromosomemovements aredriven by changes in thedynamicsofkinetochore-microtubules(Jaqamanetal.,2010;

Tirnaueretal.,2002).However,themechanismsbywhichkineto- chorescontrolplus-endmicrotubuledynamicshaveonlystarted to emerge recently. One difficultyis that these dynamics can- not be easilymeasured since kinetochores are not bound to a singlemicrotubule, butrather are attachedto25microtubules, whicharebundledintoakinetochore-fibre,allofwhichhavedif- ferent inherent dynamics(VandenBeldtetal., 2006).Therefore, one key step in the study of kinetochore movements wasthe jointdevelopment(incollaborationwiththeDanuser, McAinsh andSwedlowgroups)ofakinetochore-trackingassayinhuman cells(Jaqamanetal.,2010).Thisassay,whichwasbasedonthe computationalanalysisofrapidandhigh-resolutionrecordingsof GFP-labelledkinetochoresindividinghumancells,allowedforthe firsttime toquantify and characterisetheexact movementsof humansister-kinetochoresin4-dimensions,andtoscreenforper- turbationsthataffectthesemovements.Itrevealedthatmetaphase sister-kinetochorepairsundergosemiregularoscillationsalongthe spindleaxis,aphenomenon thathadbeenpreviouslydescribed in a qualitative manner(Skibbens etal., 1993).Our automated assayfurthershowedthatthespeedofkinetochoremovements iscontrolledantagonisticallybytwomicrotubule-depolymerases locatedatkinetochores,thekinesinsMCAKandKif18a,consistent withpreviousstudiesonKif18a(Jaqamanetal.,2010;Stumpffetal., 2008).However,thedepletionofeitherdepolymerasedidnotaffect thesemi-regularity of sister-kinetochoreoscillations, indicating thatothercomponentsmustbecontrollingtheregularityofchro- mosomemovements.Soonafter,usingthesameassaywecould showthat theCCAN networkis essential forsister-kinetochore oscillationsand that it playsan essentialrole in thecontrol of microtubuledynamics(Amaroetal.,2010).Indeed,CENP-Hdeple- tion,which disruptstheentireCCANcomplex,abolishesregular sister-kinetochoreoscillations,leadingtorapidanderratickine- tochoremovementsandadisorganisedmetaphaseplate(Fig.1).

ThisphenotypeisduetotheabilityoftheCCANnetworktocon- troltheturnoverofkinetochore-microtubules.Whilefreemitotic microtubuleshaveahalf-lifeof10–15s,kinetochore-microtubules haveamuchslowerturnoverof4–6min(Zhaietal.,1995).How- ever,intheabsenceofCENP-H,kinetochore-microtubuleplus-ends showaturnoverof10–15s,indicatingthatkinetochoreshavelost theabilitytosuppresstherapidturnoverofmitoticmicrotubules (Amaroetal.,2010).Importantly,theCCANnetworkismostlikely directlycontrollingmicrotubuledynamics:atleastoneCCANsub- unit,CENP-Q,canefficiently bindmicrotubulesinvitroand our livecellimagingofGFP-CENP-I,anotherCCANsubunit,indicates thatitpreferentiallyaccumulatesonthesister-kinetochorebound togrowingmicrotubules,a behaviour thatis only knownfor a fewmicrotubule-bindingproteins(Amaroetal.,2010).Interest- ingly,theCCANnetworkdirectlybindstothecentromericCENP-A nucleosomesandcontributestotheassemblyofthecentromeric nucleosomes,suggestingthatitactsasalinkbetweencentromeric DNAandthemicrotubuleplus-ends(Carrolletal.,2009;Foltzetal., 2006;Okadaetal.,2006).Onecriticalchallengeforthefuturewill betodeterminethemolecularmechanismsbywhichtheCCAN networkcontrolstheturnoverrateofkinetochore-microtubules, andtheprecisefunctionoftheindividualcomponentsofthislarge proteinnetworkinthisprocess.

Kinetochorefunctionhasaglobaleffectonthedynamicsof themitoticspindle

OurinvestigationofCCANkinetochoreproteinssuggestedearly onthatkinetochoresarenotjustcontrollingchromosomemove- ments,butthatinadditiontheygloballyaffectspindlemorphology anddynamics.Indeed,whenwedepletedtheCCANproteinsCENP- OorCENP-L,wefoundthatthosedepletionsledtoanaccumulation

Fig.1. DisruptionoftheCCANnetworkimpairscorrectchromosomemovements.ShownarerepresentativeimmunofluorescencepicturesofCENP-H-orcontrol-depleted HeLametaphasecellsstainedwithantibodiesagainstCREST(red;kinetochores),antibodiesagainstalpha-tubulin(green;microtubules)and4,6-diamidino-2-phenylindole (DAPIforDNA;blue;courtesyofA.C.Amaro).Notethetightmetaphaseplateincontrol-depletedcells,andthemultipleunalignedchromosomesincellslackingtheCCAN network(CENP-Hdepletion;whitearrows).Scalebar=10␮m.

of transient monopolar spindles in about 30–40% of the cells (McAinshetal.,2006;McClellandetal.,2007;Mchedlishvilietal., inpress;Tosoetal.,2009).Thissuggestedthatkinetochoresarenot onlydrivingchromosomemovementsatthelocallevel,buthave amoreglobaleffectonspindlearchitecture.Ourlive-cellimag- inganalysisofCENP-OorCENP-L-depletedcellsrevealedthatthis effectwaslinkedtotherespectivehistoryofcentrosomeseparation atthesinglecelllevel.Ifcellshadbeenabletoseparatetheircen- trosomesbeforenuclearenvelopebreakdown,CENP-OorCENP-L depletiondidnotaffectspindleformation,chromosomealignment orchromosomesegregation(Mchedlishvili etal.,in press;Toso etal.,2009).Incontrast,ifcellshadnotyetseparatedtheircentro- somesatnuclearenvelopebreakdown–aphenomenonthathas

beenobservedinmanycancerousorprimarycelllines–CENP- OorCENP-Ldepletiondelaycentrosomeseparationby6–9min, leadingtoaprolongedmonopolarspindleconformation(Fig.2;

Mchedlishviliet al.,inpress;Tosoetal.,2009).Ourrecentdata showthatevensuchasmalldelaywillresultinaveryseveredelay ofchromosomealignmentanda2.5-foldincreaseintheerrorrate ofchromosomesegregation,indicatingthattherapidformation ofabipolarspindlewiththehelpofakinetochore-basedpushing forceplaysacrucialroleinfaithfultransmissionofchromosomes (Mchedlishvili et al., in press). At the more mechanistic level, ourinvestigationsfurtherrevealedthatthiskinetochore-pushing force in earlyprometaphase depends on the ability of kineto- chorestoincorporatetubulinsubunitsatthemicrotubuleplusend,

Fig.2. DepletionoftheCCANkinetochoreproteinCENP-Lleadstotheformationoftransientmonopolarspindlesin30%ofthecells.Shownarerepresentativeimmunoflu- orescencepicturesofCENP-LdepletedHeLacellsinprometaphasestainedwithantibodiesagainstgamma-tubulin(red;spindlepoles),antibodiesagainstalpha-tubulin (green;microtubules)and4,6-diamidino-2-phenylindole(DAPIforDNA;blue;courtesyofN.Mchedlishvili).WhilethemajorityofCENP-Ldepletedcellsdisplayanormal bipolarspindle(leftpanel),about30%ofcellslackingCENP-Lshowamonopolarconfiguration.Thispopulationofcellswithmonopolarspindlesreflectstheinabilityof CENP-Ldepletedcellstorapidlyseparatethetwospindlepolesduringprometaphase.Thisshowshowalocaldysfunctionatkinetochorescanaffecttheglobalarchitecture ofthemitoticspindle.Scalebar=10␮m.

106 P.Meraldi/EuropeanJournalofCellBiology91 (2012) 103–106

causingbipolarlyattachedkinetochorestoacceleratethesepara- tionofthetwospindlepoles(Tosoetal.,2009).BothCENP-Oand CENP-Ldepletionsreducethisforce,byweakeningkinetochore- microtubules,reflecting theinfluence of theCCAN complex on kinetochore-fibredynamics.Thesestudiesthereforeindicatethat kinetochoresarenotjustlocalplayersthatcontrolthemovements ofindividualchromosomes,butthattheyplayacrucialglobalrole inthedynamicsoftheentiremitoticspindle.Thesestudiesalso openupa newexciting field for thefuture where wewill not onlyhavetounderstandthefunctionand compositionofasin- glekinetochore,butwewillincreasinglyhavetodissecthowlocal microtubuledynamicsatkinetochoresatoneend,andspindlepoles attheotherendarecoordinatedduringcelldivision,toensurethe correctsystemicfunctionofthemitoticspindle.

Acknowledgments

IthankJasonSwedlow(UniversityofDundee),GaudenzDanuser (HarvardUniversity)andinparticularAndrewMcAinsh(University ofWarwick)forourfruitfulcollaborations,allmyco-workersfor theirexcitingresearch,andtheSwissNationalFund,theETHZurich, EURYI,andtheSwissCancerLeagueforfinancialsupport.

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