Coordinating cell polarity with cell division in space and time

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Reference

Coordinating cell polarity with cell division in space and time

PANBIANCO, Costanza, GOTTA, Monica

Abstract

Decisions of when and where to divide are crucial for cell survival and fate, and for tissue organization and homeostasis. The temporal coordination of mitotic events during cell division is essential to ensure that each daughter cell receives one copy of the genome. The spatial coordination of these events is also crucial because the cytokinetic furrow must be aligned with the axis of chromosome segregation and, in asymmetrically dividing cells, the polarity axis. Several recent papers describe how cell shape and polarity are coordinated with cell division in single cells and tissues and begin to unravel the underlying molecular mechanisms, revealing common principles and molecular players. Here, we discuss how cells regulate the spatial and temporal coordination of cell polarity with cell division.

PANBIANCO, Costanza, GOTTA, Monica. Coordinating cell polarity with cell division in space and time. Trends in Cell Biology , 2011, vol. 21, no. 11, p. 672-80

DOI : 10.1016/j.tcb.2011.08.006 PMID : 21930382

Available at:

http://archive-ouverte.unige.ch/unige:25587

Disclaimer: layout of this document may differ from the published version.

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Coordinating cell polarity with cell division in space and time

Costanza Panbianco and Monica Gotta

DepartmentofGeneticMedicineandDevelopment,FacultyofMedicine,UniversityofGeneva,1rueMichelServet, 1211Geneva4,Switzerland

Decisionsofwhenandwheretodividearecrucialforcell survival and fate, and for tissue organization and homeostasis.Thetemporalcoordinationofmitoticevents during cell division is essential to ensure that each daughter cell receives one copy of the genome. The spatial coordination of these events isalso crucial be- causethe cytokineticfurrowmustbealignedwiththe axisofchromosomesegregationand,inasymmetrically dividing cells, the polarity axis. Several recent papers describe how cell shape and polarity are coordinated withcelldivisioninsinglecellsandtissuesandbeginto unravel the underlyingmolecular mechanisms, reveal- ingcommonprinciplesandmolecularplayers.Here,we discuss how cells regulate the spatial and temporal coordinationof cellpolaritywithcelldivision.

Celldivision:gettingspaceandtimeright

Toachievesuccessfulcelldivision,cellulareventsmustbe tightly regulated both spatially andtemporally. The ge- nomemustbefullyreplicatedbeforethecellentersmitosis andchromosomesproperlyattachedtothemitoticspindle beforesegregationbegins.Cytokinesiscannotstartunless the chromosomeshaveseparated, anditmust occurper- pendiculartotheaxis ofchromosomesegregation.These eventsarecoordinatedwithcellgrowth,cellpolarityand, inmulticellularorganisms,tissuearchitecture.Disruption canresultinaneuploidy,celldeath,abnormalproliferation and developmental disorders [1–3]. Toensure successful coordination,cellshavemultiple,oftenredundant,strate- gies:(i)couplingofeventssuchthatalatereventdoesnot occur until thepreviousoneiscompleted, (ii)checkpoint surveillancemechanismsthatsensewhetheraneventhas beencompletedproperly,and(iii)feedbackmechanismsin which the output of an event can either be reinforced (positivefeedback)orinhibited(negativefeedback).

Although our knowledge on cell cycle regulation has progressedenormously,westillknowlittleabouthowcell shapeandpolarityinfluencecelldivision.Inrecentyears, workinvariousmodelsystemshasstartedtouncoverthe molecularmechanismsbehindthecrosstalkbetweenthese processes.

Here,wedescribehowcellshapeandpolarityinfluence the positionofthespindle and, therefore,the site ofcell division in unicellular and multicellular organisms. We thendiscusshowshapeandpolarityexerttemporalcontrol

over cell division and, conversely, how mitotic kinases controlcellpolarity.Theemergingdatapointtoregulation viafeedback mechanismstoensuretight coordination of theseprocesses.

Spatialregulationofcelldivision

In non-polarized cells, proper cleavage-plane orientation ensuresthatthecytoplasmisaccurately partitionedand thatcellsdevelopinthecorrectenvironment(Figure1a).

In polarized cells, cleavage-plane orientation also deter- mineswhetherthedivisionwillbesymmetric, producing identicaldaughters,orasymmetric,producing daughters withdifferentfates(Figure1b).Belowwediscusshowcells ensurespatialcoordinationoftheirdivisionaxis.

Symmetricallydividingcells

Typically, the positionof the mitoticspindle determines thesiteofcytokinesis,andphysicallymovingaspindlecan changethe cytokinesis plane [4,5]. Both astral andmid- zonemicrotubulesarerequiredtodeterminethecleavage- planesite[6].Intheabsenceofpolaritycues,cellstendto divideperpendicularlytotheirlongestaxis,however,this isnot auniversal rule.In a recentstudy,individual sea urchineggs,whicharenon-adherentanddividesymmet- rically,wereplacedintomicrofabricatedwellswithdefined shapestostudyhowgeometryinfluencesthepositionofthe cleavageplane[7].Thenucleuspositionedinthecentreof thecellinamicrotubule-dependentmannerandstretched alongapreciseaxis;afternuclearenvelopebreakdownthe spindleelongatedalongthesameaxis,dictatingtheposi- tionofthecleavagefurrow. Formostshapes, theaxis of spindleelongationcorrespondedtothelongaxisofthecell, butforparticularshapesthisruledidnotapply,suggesting that shapealone candeterminethe planeof division,at leastinembryoniccells(Figure2a).

Molecularinsightintohowcellsmightsensetheirshape and find their middle comes from studies in the fission yeast Schizosaccharomyces pombe, rod-shaped cells that growbytipextensionanddivideinthemiddle.Aftercell division, daughter cellselongate onlyat the oldend but startgrowing atthenewendafterDNAreplication.The position ofthe cleavage plane isregulated by a positive signalemanatingfromthenucleusandaninhibitorysignal emanatingfromthecelltips[8–11].Bothsignalsregulate thelocalizationoftheanillin-likeproteinMid1(Figure2b) [12,13].Ininterphase,Mid1localizesinthenucleusandto medial cortical patches (termed cortical nodes) [13,14].

ThislatterlocalizationdependsontheSADkinaseCdr2

Correspondingauthor:Gotta,M. (monica.gotta@unige.ch).

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[15,16]. The DYRK family kinase Pom1 prevents Mid1 from spreading towards the non-growing end. This depends on the ability of Pom1 to phosphorylate Cdr2 (belowandFigure2b).Anadditionalunknownsignalacts fromthegrowingend[10,11,17,18] (Figure2b).In G2/M, phosphorylationofnuclearMid1bythemitotickinasePolo- like kinase (Plk1) triggers Mid1 nuclear export and its abilitytorecruitcontractileringcomponentstothemiddle ofthecell,temporallycoordinatingmitosiswithcytokinesis [19]. Therefore, microtubule-dependentpositioning ofthe nucleus[20]andinhibitorysignalsatthecelltipsdictatethe positionofthedivisionplane(reviewedin[21]).

Both sea urchin and fission yeast regulate cleavage- planepositioning basedonly oncellshape,independently ofanyexternalsignal.Bycontrast,symmetricallydividing animal cells establish cell matrix adhesions or contacts withneighboringcellsand,invivo,coordinatetheirdivision plane with tissue architecture. In vitro, adhesion to the extracellularmatrix(ECM)playsamajorroleinorienting thedivisionplane[22].HeLacellsgrownoncoverslipswith patterned shapes offibronectin (e.g. rectangular,discoid, triangular)assumedthe correspondingshapes duringin- terphase. In mitosis, the spindle orientedparallel tothe substrate and according to the interphase shape despite

cellrounding:alongthelongaxisinthemajorityofrectan- gularcells,randominthediscoidcells,andparalleltothe hypotenuse in triangular cells. Additional experiments suggested that shape is not the only factor influencing spindleorientation.CellsgrownondifferentECMpatterns thatallledtoasquaredshapehadmitoticspindleorienta- tionsbasedonECMdistribution,notshape(Figure2c).The ECM orientsthemitoticspindlebyinfluencingthedistri- butionofcorticalcues.These cuesoriginateininterphase cellsandaremaintainedduringmitosiswheretheyassoci- atewithretractionfibers,membranetubesthatmaintain cellconnectionswiththeECMatcellrounding.Theforces applied by retraction fibers provide a cortical polarizing signalthatisessentialtoorientthespindle[23].

IsECM-dependentspindleorientationrelevantinvivo, whereorientationofthedivisionplaneiscrucialtodevelop and maintain tissue architecture? Drosophila cells vary theircleavage-plane positionin responsetothe shapeof neighboringcellstomaintainthegeometricregularityof epithelia [24].This mightresult from the distributionof cell–cellcontactsduringinterphase,similartothebehav- iorofculturedcells. Integrins–celladhesion receptors– are required for spindle orientation in several systems [25,26]. In the mammalian epidermis, integrins control (a)

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TRENDS in Cell Biology

Figure1.Spindlepositioninginnonpolarizedandpolarizeddividingcells.(a)Anon-polarized,symmetricallydividingcelldeterminesthefateofitsdaughtersbyorienting themitoticspindle.Dependingontheorientation,daughtercellswillassumedifferentpositionsinthetissueafterdivision.(b)Inpolarizedcells(reddots:cytoplasmic determinant;blueline:corticaldeterminant),theorientationofcelldivisioncaneitherleadtoanasymmetricdivisionwherebythetwodaughtercellsaredifferentinfate (left),ortothebirthoftwoidentical,butpolarizedcells(right).

Review TrendsinCellBiology November2011,Vol.21,No.11

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asymmetric apical localization of polarity proteins and LGN (Box 1). This correctly aligns the spindle and pro- motes skin stratification andproper differentiation [27].

Although these are asymmetrically dividing cells, it is likelythatadhesiontotheECMregulatesthedistribution andpossibly activityofLGNandNUMA, majorspindle- positioningregulators,inallcells.

Therefore, shape alone has the ability to control the positionofthecleavageplane.Cellsintissuescanoverride this,however, becausetheymustcoordinate theirsiteof divisionwithneighboringcellsorwiththeaxisofthetissue tomaintainproperarchitecture.

Asymmetricallydividingcells

Inasymmetricallydividingcells,polarityorientsthedivi- sionaxisandovercomesshapeorcelladhesioninformation [28].Here,wesummarizetwopolarizedsystemsinwhich spindle positioning has been extensively characterized, revealing common principles: the Drosophila neuroblast andtheCaenorhabditiselegansembryo (Box1,Figure3;

[29,30]forreview).

In Drosophila neuroblasts, apico-basal polarity regu- lates spindle orientation via a set ofconserved proteins (Box1, Figure 3a). Neuroblastshave a secondpathway, however: the telophase rescue pathway. In Inscuteable Axis of division

Key: Long axis

(a) Sea urchin

Pom1 Mid1

Non-growing cell end

Growing cell end contractile ring assembly

(b) S. pombe

Pom1

(c) HeLa cell Key:

Key: Axis of division

Pom1 and ?

Figure2.Cellgeometryregulatesthepositionofthecytokinesisfurrow.(a)Seaurchinone-cellembryos(blacklines)areplacedinchambersofdifferentshapes(grey lines).Microtubule-generatedforcespositionthenucleuscentrallyandpromotecleavage-planepositioning.The‘long-axisrule’predictsthatcellsdivideperpendicularto thelongestaxisinthecell.Theleft-handcellfollowsthisrule,however,theright-handcelldoesnot(longestcellaxis,blackdottedline;axisofcelldivision,reddottedline).

(b)InS.pombe,tworegulatorysignals,bothactingontheanillin-likeproteinMid1(yellowcircles),positionthecytokinesisfurrow.Apositivesignalcomesfromthe nucleus(largeyellowcircle),whichiscentrallylocatedinamicrotubule-dependentfashion(greylines).AsecondPom1-dependentnegativesignalfromthepoles(pink gradient)inhibitslateralspreadingofMid1.Fromthegrowingendanadditionalunknownsignalparticipatesinthisinhibitionbecauseinpom1mutantsMid1onlyspreads tothenongrowingend.(c)HeLacells(blacklines)constrainedontwinbars,cross,orL-dotfibronectinmicropatterns(greyshapes)allassumeasquareshapebuthave differentspatialdistributionofattachmentsitestotheECMandelongatethespindle(blackrhomboid)accordingtotheseattachmentsites(axisofdivision,reddottedline).

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mutants(Box1)themitoticspindlecaninduceapolarity axis. This rescue occurs in late mitosis, is mediated by interaction of the mitotic spindle with the cortex and requires PINS, the tumor suppressor Discs Large (a PSD95family member)andKh63 (a kinesin). PINSand Gabecomelocalized inacrescent closetoaspindle pole resultinginpolarization ofthecell cortexrelativetothe mitoticspindle, andthis allowsasymmetric cell division [31]. Whether both poles can polarize the cortex is unknown.

AsimilarrescuemechanismwasproposedinC.elegans [32]andwas recentlydescribed. Similarly toDrosophila neuroblasts,spindle positioningisregulatedbyanterior–

posteriorpolarityintheone-cellembryo(Box1,Figure3b).

Conserved anterior and posterior PAR proteins occupy about half of the embryonic cortex. PAR domain length is altered before cytokinesis in particular mutants (too shortortoolong)butitiscorrectedduringfurrowingres- sion [33,34]. Abnormally small PAR domains, whether anterior or posterior, expand to reach the site of cell division.PARdomaincorrectiondependsonGa,GPR-1/2 and LIN-5, and occurs possibly via Ga control of microtubule–cortex interactions. Theseinteractions drive acto- myosin-based cortical reorganization, which repositions the PAR boundary. Thus, in C. elegans early embryos,

polaritycontrolsthepositionofthemitoticspindle,which determines the site of furrow ingression. In turn, the cytokinesis furrow contributesto positioning thepolarity boundary, regulatingproper segregationof PARproteins (Figure3b).

These rescue mechanisms provide a robust feedback pathwaythathelpsensurethesuccessofasymmetriccell divisions. Aninteresting questionis whether thesecells delay mitosis to correct polarity defects, suggesting a polaritycheckpoint(seebelow).

The plane ofcytokinesis can also be determined in a spindle-independentbutpolarity-dependentmanner.My- osin forms a furrow on the basal cortex of Drosophila neuroblastsevenintheabsenceofamitoticspindle[35].

Inmudmutants(Box1),wherethespindleisorthogonalto the normal polarity axis, myosin is still enriched at the basal cortex. These cells initiate two furrows: onebasal constrictionthatoftenresultsinananucleatedbasal‘polar body’,andaspindle-drivenfurrow.Basalasymmetricen- richment of myosin is dependent on PINS revealing a polarity-dependent and spindle-independent mechanism for furrow ingression (Figure 3c). A similar mechanism exists in C. elegans neuroblasts [36]. In these cells the mitoticspindleisinitiallycentredbutelongatesasymmet- ricallytogenerateasmalleranteriorcell.Myosinisasym- metrically enriched at the anterior, and inactivation of myosinleadstoamoresymmetricdivision.Myosinasym- metrydependsonpolarity,asinDrosophila neuroblasts.

The authors propose that asymmetric cortical tension drivenbymyosin,ratherthanspindlepositioning,defines thecleavagefurrow.

Commonconceptsarisefromtheexamplesinthissection:

shapeandpolaritycontrolspindlepositioning,whichinturn regulatescleavage-planepositioninmanycells.Inaddition, reciprocalinteractionbetweencellpolarityandfurrowpo- sitioningincreasestherobustnessofthesystembyensuring thecoordinationofthesetwoprocesses(Figure3c).

Temporalregulationofcelldivision:linkingcellpolarity tocellcycleprogression

Thedecisionofwhentodivideisasimportantaswhereto divide.The timing ofdivision determinesthe positionof the cell in adeveloping organism.In addition, polarized cellsonlydivideafterpolarityisfullyestablished,inorder thatpolarizedfactorsarecorrectlysegregatedtodaughter cells(Figure1b).Here,wediscussfindingsinyeastdem- onstratingthatthetimingofcelldivisioniscoupledtocell sizeandmorphology.Wethenreviewametazoanpolarized systemwherecell polarityregulatescell divisiontiming.

Finally,wereviewhowmitotickinasesareimplicatedin cellpolarityregulation.

Regulationofcelldivisiontimingbycellgeometry inyeast

Yeastcellsentermitosisonlyafterreachingthepropercell size. What is the correct size and how is it measured?

Importantly, howiscell growth timed with celldivision?

InS.Pombe,Cdk1/Cdc2ismaintainedinaninactivestate during G2 by Wee1-dependent phosphorylation. Dephos- phorylationofCdk1/Cdc2bytheCdc25phosphatasetrig- gers entryinto mitosis[37].In wee1mutants, cells enter Box1.C.elegansembryosandDrosophilaneuroblasts

divideasymmetrically

Cellpolarityandasymmetriccelldivisionareessentialfeaturesof many, if not all, animal cells. Together they ensure the proper inheritance of localized molecules essential for cell diversity. A commonsetofmoleculesregulatepolarityandspindlepositioning inC.elegansembryosandDrosophilaneuroblasts.

Sperm entry defines the first axis of polarity, the anterior–

posterior(A–P)axis,inC.elegansembryos.Subsequenttosperm entry,sixPARproteins(for‘partitioning-defective’),aswellasthe atypicalproteinkinaseC(PKC-3),localizeatthecortexalongtheA–P axis.ThePAR-3–PAR-6–PKC-3complexlocalizesattheanteriorand PAR-2andPAR-1attheposterior.Asmitosisprogresses,themitotic spindle initially forms in the center of the embryo and is then displaced towards the posterior pole by cortical pulling forces exertedonastralmicrotubulesundertheregulationofPARpolarity.

Two Ga subunits of heterotrimeric G proteins, their receptor- independent activatorsGPR-1/2 andthecoiled-coil protein LIN-5, controlcorticalpullingforces.GPR-1/2andLIN-5becomeenriched at the posterior cortex after nuclear envelope breakdown (Figure3b).This enrichment directs dynein-mediated forces that actontheposteriorpole,resultinginasymmetricspindleposition- ing. The first embryonic division segregates the anterior PAR complextotheanteriorlargercell,whereasPAR-2andPAR-1are restrictedtothesmallerposteriorcell.

Drosophilaneuroblastsbecomepolarizedduringearlyprophase.

Bazooka(PAR-3),Par6andtheatypicalkinaseaPKC(PKC-3)together with the adaptor protein Inscuteable (Insc) localize at the apical plasmamembrane.Subsequently,theNotch-DeltainhibitorNumb localizes to the basal cortex. After apico-basal polarity is established, themitotic spindleissetup alongthe polarityaxis. Insc recruitsandlinkstheapicalPAR complextoGaandits activator PINS(PartnerofInscuteable,GPR-1/2inC.elegans),whichinturn ensures correct orientationof the mitotic spindlevia themicro- tubule-associateddynein-bindingproteinMUD(functionalhomolog ofLIN-5).Thespindle isasymmetricandcytokinesisgenerates a larger apical cell that remains a neuroblast and a smaller basal ganglionmothercell(GMC).Inmammaliancells,heterotrimericG proteinstogetherwithLGN(PINS/GPR-1/2)andNuma(Mud/LIN-5) alsoregulatespindleorientation.

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mitosisprecociously,indicatingaroleforWee1inthecoor- dinationbetweensizeanddivision[38].Cdk1andWee1use aPom1gradientasaspatialcueforsensingcellsize[16,18].

Wee1localizes at medialcortical nodeswith its inhibitor Cdr2.Bycontrast,Pom1formsagradientemanatingfrom thetips.InshortcellsthePom1gradientoverlapswiththe medialcorticalregionsallowingPom1toinhibitCdr2.Asa result,Wee1isactiveandentryintomitosisisdelayed.As the cell elongates, Cdr2 is released from the inhibitory influenceofPom1andinactivatesWee1,allowingentryinto mitosis (Figure4). Pom1isalsoimportantforcontrolling division-plane positioning, as described above. Using the samemoleculestoregulatethetimingandpositionofdivi- sionprovidesarobustandefficientmeansofcoordinating theseevents.Additionalmechanismsareinvolved,however,becauseS.pombe cellsdepletedofPom1areonly10%

shorterthanwild-typecells.

Pom1gradientformationoccursthroughdynamicmem- brane binding of Pom1. Pom1 autophosphorylates and phosphorylatedPom1isunabletobindtothemembrane.

ThelandmarkproteinTea4,whichlocalizestothetips[39], recruits Pom1 and the protein phosphatase Dis2 [40], which locally dephosphorylates Pom1 [41]. This allows binding of Pom1 to membranes at the cell tips. Lateral

diffusionofPom1awayfrom theTea4 domainresultsin autophosphorylationandmembranedetachment[41].In- tracellulargradientssuchasthisarecrucialfortheregu- lationofmitoticeventsinmanycells[42].

S.cerevisiaehasalsoevolvedamechanismtocoordinate thetimingofcelldivisionwithcellgeometry.Afteraperiod of isotropic growth, mother cells initiate budding at a definedcorticalsite.Topreventformationofabinucleated cell,S.cerevisiaedelaysentryintomitosisuntilabudhas formedina mechanism calledthe morphogenesis checkpoint[43],which reliesontheSAD-relatedkinaseHsl1.

Hsl1localizesatthebudneckwhereitbindsseptins,small GTP-bindingproteinsessentialforcytokinesis.Oncethe bud forms, Hsl1 recruits the Wee1-homolog Swe1 and regulatesitsdegradation,allowingmitoticentry.Incells whereabudfailstoform,orwheretheactincytoskeletonis perturbed,Hsl1localizestothebudneck,asinwild-type cells,butremainsinactive.AsaresultSwe1isnotdegrad- ed,leadingtoadelayinentryintomitosis(Figure4)[44].

WhattriggersHsl1activationisnotknownbutithasbeen proposed thatthe localgeometryof septinsplaysarole [43,45]. Although the morphogenesis checkpoint does not appear to use a gradient-based signal for geometry sensing,bothfissionandbuddingyeastuseaSADkinase

Polarity

Spindle Positioning Cytokinesis

Furrow Polarity

Spindle Positioning Cytokinesis

Furrow

PINS, Galpha, MUD Inscutable

Baz, PAR6, aPKC

Myosin Numb GPR-1/2, LIN-5

PAR-3, PAR-6 PKC-3 PAR-2 (a)

(c) Key:

(b)

DNA Key:

Centrosome Microtubule

Figure3.Crosstalkbetweencellpolarity,spindlepositioningandcytokinesisfurrowingression.(a)Drosophilaneuroblastsarepolarized,withBaz,PAR6andaPKC complex(blue),PINS,GaandMUD(yellow),andInscuteable(pink)attheapicalcortexandNumb(green)atthebasalcortex.Corticalpolaritycontrolsthepositionofthe mitoticspindleviaPINS(arrowfromtheapicalcrescenttothespindle),whichinturndictatesthepositionoffurrowingression.Intheabsenceofamitoticspindle,PINScan asymmetricallypositionmyosin(red)forasymmetriccelldivision(arrowfromtheapicalcrescenttomyosinatthefurrowingression).Ifanaxisofpolarityisnotproperly established,interactionofthemitoticspindlewiththecortexallowsthecorrectlocalizationofPINSandGainamechanismreferredtoastelophaserescue(arrowfromthe spindletotheapicalcrescent).(b)Theone-cellC.elegansembryoispolarized,withPAR-3,PAR-6andPKC-3attheanterior(blue)andPAR-2(red)attheposterior.PAR polaritycontrolstheasymmetricenrichmentofGPR-1/2andLIN-5(yellow)attheposterior.Ga,GPR-1/2andLIN-5positionthespindle,whichdeterminesthesiteof cytokinesisfurrowingression(arrowsfromthespindletofurrowingression).IncellswherethePARdomainlengthisaltered,thecytokinesisfurrow,viaGa,canreposition thePARdomaintomatchthesiteofcelldivision(arrowfromfurrowtopolarity).InC.elegans,neuroblastpolarityasymmetricallypositionsmyosinforpolarizeddivision.

Whetherthisistrueintheembryoisunknown(dottedline).(c)Schematicrepresentationofthecrosstalkbetweenpolarity,spindlepositioningandfurrowpositioning.

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(Cdr2andHsl1)inresponsetoaspatialcuetoregulatecell cycleprogression.

By contrast, cell cycle progression can also regulate shape/morphogenesis. In S. cerevisiae, DNA replication checkpoint mutants have aberrant bud morphogenesis [46].Consistentwiththis,thereplicationstresscheckpoint proteinRad53physicallyinteractswithseptinsandloca- lizesatthebudneck[47],suggestingaroleincoordinating cellmorphologywithDNAreplication.Similarly,comple- tionofDNAreplicationandpolarizedgrowthinS.pombe cellsarealso linkedvia arecentlydescribedmechanism that requires the checkpoint proteins Chk2/Rad53 and Rad3 (ATR)[48]. Why polarized growthis linked tocell cycleprogressionattheDNAreplicationlevelisunknown.

Biased segregation of DNA strands has been shown in many cells including S. pombe [49]. We speculate that coordination of cell polarity and DNA replication might be essential for such asymmetric segregation of DNA strands.

Regulationofcelldivisiontimingbycellpolarity inmetazoancells

Itislikelythat,asinyeastcells,polarityalsoregulatescell cycleprogressioninmetazoancells.Themechanismscou- pling polarity and cell cycle progression remain elusive, however.Suchcoordinationcouldbeachievedbypolarity proteins regulatingthe activity or localizationof mitotic kinases (e.g. Cdk1 regulation by Pom1) or by polarity checkpoints,similartomorphogeneticcheckpoints.

C. elegans one-cell embryos divide asymmetrically to formalargeranteriorcell(AB)andasmallerposteriorcell (P1). Cortical PAR polarity controls enrichment of the

cytoplasmic mitotic kinase Polo-like kinase (PLK-1) at the anterior of the one-cell embryo [50–52]. As a result, ABinheritshigherlevelsofPLK-1atdivision.Thehigher PLK-1levelsdriveABintomitosisearlierthanitsposteri- orsisterP1[51,52].par-3mutants,whichhavelowPLK-1 levelsinbothABandP1,haveasynchronouscellcycleat thetwo-cellstage,resemblingtheslowertimingoftheP1 cell. Conversely, par-2mutants, whichhave high PLK-1 levelsinbothABandP1,haveasynchronousbutfastcell cycle,asdoABcells[51,52].InC.elegansembryos,therefore, polarity controlsPLK-1 distribution,which in turn regulatesasymmetricentryintothecellcycleofABandP1.

ThesefindingsdemonstratethatPARpolaritycanregulate differentialcellcycleentryatthetwo-cellstage.However, whetherpolaritycontrolsmitoticentryviaPLK-1inone- cellembryosisnotknown.

Regulationofcellpolaritybymitotickinases inmetazoancells

Several studies highlight a role for mitotic kinases in polarity control. The mitotic kinase Aurora A is highly conservedandimplicatedincentrosomematuration,spin- dleassemblyandmitoticentry[53].InAuroraAmutant Drosophila neuroblasts,the cell fate determinant Numb failstolocalize asymmetricallytothebasalcortexandis segregatedintobothdaughtercells[54–56].Numblocali- zation depends on Aurora A phosphorylation of Par-6, which is found in a complex with the polarity protein Lethal giant larvae (Lgl) and aPKC. Phosphorylation of Par-6triggersthereleaseofLglallowingBazookatobind to the Par-6-aPKC complex. aPKC then phosphorylates Numbtoregulateitsbasallocalization(Figure5a)[57].

Cdr2

Cdr2 S.pombe S.cerevisiae

Septin

Septin ring

Septins organised Septins

perturbed Long cell

Pom1 Cdr2 / Hsl1

Cdk1 / Cdk1

G2 delay Entry into M G2 delay Entry into M

Wee1 / Swe1 Short cell

+

+ + - -

-

- Hsl1

Hsl1 Cdk1

Wee1 Cdk1

Swe1 Pom1

Key: Pom1

Figure4.Coordinationofcellgeometryandcelldivisioninyeast.InS.pombePom1(pink)formsagradientemanatingfrombothpoles,whereastheWee1inhibitor,the SADkinaseCdr2(yellowcircles),islocalizedtoamedialcorticalregion.Inshortcells,Pom1inactivatesCdr2.Wee1phosphorylatesandinhibitsCdk1,leadingtoaG2delay.

Inlongcells,Cdr2inhibitionbyPom1isrelieved,allowinginhibitionofWee1,activationofCdk1andmitoticentry.InS.cerevisiaetheSADkinaseHsl1(yellowcircles)is recruitedtothebudneckbyseptins.Uponbudformation,Hsl1recruitsandregulatesthedegradationofSwe1,therebypromotingentryintomitosis.Incellswhereseptin organizationisperturbed,Hsl1isrecruitedtothebudneck,whereit‘senses’thisdisorganization.Hsl1remainsinactiveleadingtoaG2delay.

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Similarly to Aurora A mutants, aPKC and Numb are mislocalizedinmutantsofthemitotickinasePolo(plk1).

PolophosphorylatesPon(partnerofNumb),whichlocalizes Numb asymmetrically tothe basal cortex of neuroblasts [58]. Therefore, Numb localization is controlled by two mechanisms: Aurora A- and aPKC-dependent exclusion

fromtheapicalcortex,andPolo-andPon-dependentlocali- zationtothebasalcortex(Figure5a).

In Drosophila, AuroraA isalsoimportant foranother aspect of asymmetric cell division: spindle positioning.

Using an elegant S2 cell culture system designed to studyasymmetriccelldivisions,itwasshownthatspindle (a) Aurora A and Polo in Drosophila neuroblasts polarity

Aurora A in spindle positioning in S2 culture cells Aurora A

Aurora A PAR6

Basal

Numb

TPR

L L

L Mud

Dynein

Disc large Khc73

Microtubule capture Microtubule pulling

Aurora A

Aurora A Apical

PAR6 PAR6 PAR6

aPKC aPKC

aPKC LGL

LGL Baz

Pon Pon

Polo

Polo Polo

P

P P

P

P P

P P P

P

P P

Exchange

Exclusion of Numb from the

apical cortex

Asymmetric localisation of Numb

at the basal cortex

Aurora A- dependent exclusion of

Numb Polo- dependent

Numb localization

(b)

Key:

P

P P P P P P P

Figure5.Polarityandspindle-positionregulationbyAuroraAandPolo.(a)InDrosophilaneuroblastspolarityiscontrolledbyAuroraAandPolo.AuroraA(yellow) phosphorylatesPAR6(blue)inthePAR6/aPKC/LGLcomplexattheapicalcortextriggeringtheexchangeofLGLforBaz.aPKCcanthenphosphorylateandexcludeNumb (pink)fromtheapicalcortex.Concomitantly,Polo(green)phosphorylatesPon,whichinturnlocalizesNumbtothebasalcortex.(b)AuroraAregulatesspindlepositioning inS2culturecells.AuroraAphosphorylatesPINS(blue)onthelinkerdomain(L).Oncephosphorylated,PINSmediatesmicrotubule(green)captureviaDiscsLargeandthe kinesinKhc73.CompletePINSfunctionrequiresitsTPRdomain,whichbindsMudfordynein-dependentmicrotubulepulling.

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positioningdependsupontwodomainsofPINS[59].One, the PINS linker, is phosphorylated by Aurora A. After phosphorylationitpromotespartialspindlepositioningby bindingtothekinesinKhc73viaDiscs large.The partial functionisprobablyduetothefactthatKhc73isaplusend kinesinand thereforeunabletoproducepullingforceson centrosomes. Full function requires the TPR domains of PINS,which bindMud (Box1).Thissuggestsamodelin whichthePINSlinkercapturesastralmicrotubules,while the TPR domains, by binding to Mud, promote dynein- dependentpulling(Figure 5b). The linkerdomain,which isphosphorylatedbyAuroraA,wouldalsoprovidecoordi- nationbetweencellcycleprogressionandspindleposition- ing[59].

InC.elegansembryos,downregulationoftheAuroraA homolog,AIR-1,causescellpolaritydefects[60,61].PAR-2, normally found at the posterior of one-cell embryos, is found at both poles in air-1(RNAi) embryos, whereas PAR-6 is found in a central band [60]. It is not known whether AIR-1phosphorylates PAR-6 or any other PAR proteininC.elegans.

PLK-1is requiredfor asynchronouscell cycleentry of theanteriorandposteriorcellsintwo-cellembryos.How- ever,depletionofplk-1alsoresultsindefectsinthelocali- zationofPARproteinsinone-cellembryos.Furthermore, depletion of plk-1 canpartially rescue the lethality and polarity defects of par-2 temperature-sensitive mutants, suggestingthatPLK-1iseitherapositiveregulatorofthe anterior PAR proteins or a negative regulator of PAR-2 [60].WhetherPARproteinsthemselvesaresubstratesof PLK-1isunknown.

TheCdc2homologCDK-1controlsthetimingofspindle positioninginC.elegans.Thisensuresthatthespindleis fullyassembledandfunctionalwhen itisdisplaced [62].

Thetarget(s)ofCDK-1inthisprocesshasnotbeenidenti- fiedbutproteinsrequiredforspindlepositioningarelikely substrates.

Therefore,mitotickinasescanregulatepolarity,provid- ingameanstolinkcellcycleprogressiontocellpolarity.In somecases,however,theroleofthesekinasesinpolarity regulationmightbeindependentfromtheircellcyclerole [63],butthisrequiresfurtherstudy.

Concludingremarks

Celldivisionoffersabeautifulexampleofspatiotemporal coordination.Inasymmetricallydividingcells,orincellsin tissues,celldivisionmustbecoordinatedwiththepolarity axisorwiththearchitectureofthetissue.Achallengefor thefutureistoidentifythemolecularmechanismsbehind thecoordinationbetweencellshape/polarityandcelldivi- sion.Mitotickinasesregulatepolarity;however,whether andhowsuch regulation constitutes a couplingbetween cell division and cell polarity, or instead represents a mitotic-independentroleforthesekinases, isnotalways clear.Furthermore,howpolarityregulatescelldivisionin metazoancellsremainselusive.Are theseprocesses cou- pledsuchthatcompletionofcellpolaritytriggersentryinto mitosis? Or do surveillancemechanisms (polaritycheck- points)arrestcellcycleprogression ifpolarityisnotnor- mal?Studies in C.elegans andDrosophila will pavethe wayforstudiesinotherorganisms.Indeed,theseprocesses

are highly conserved at the molecular level, conceptual level,orboth.

Spatiotemporalcoordinationduringcelldivisionises- sentialtoensuretheproperbalancebetweensymmetric and asymmetric divisions, to properly position cells in tissuesand tocontrol thebalancebetween proliferation and differentiation. Disruptions in this coordination mightresultintumorigenesisordevelopmentaldisorders, underscoringtheimportanceofstudyingthecrosstalkand coordination between these fundamental cell biological processes.

Acknowledgments

We thank Patrick Meraldi and Sophie Martin for comments on the manuscript.Wealsothankthereviewersforthoroughandconstructive comments. Workin the authors laboratoryis supportedby the Swiss NationalScienceFoundation,theUniversityofGenevaandaEuropean MolecularBiologyOrganisation(EMBO)YoungInvestigatorProgramme award.

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