2996 Research Article

IntroductionIntegrins are the major family of receptors that mediate cell-matrixadhesion (Hynes, 2002). In interphase cells, which are generallywell spread, integrins assemble into adhesive structures linked toactin fibres (Geiger et al., 2001). As cells enter into mitosis, focaladhesions and actin stress fibres disassemble, leading to cellretraction along actin retraction cables and cell rounding (Cramerand Mitchison, 1997; Maddox and Burridge, 2003). As cell divisionnears completion at the beginning of telophase, daughter cells beginto spread on the substratum, as focal adhesions and stress fibresare reformed. To date, the signals and pathways regulating thedynamics of actin fibres and adhesion structures as cells enter andexit mitosis remain unknown. Importantly, in human mammaryepithelial cells (HMEs), the level of β1 integrin expressed at theplasma membrane remains unchanged during mitosis, arguing fora regulation of integrin activity (Suzuki and Takahashi, 2003).Nevertheless, the nature of such a regulator of integrin-dependentadhesion, which would itself be regulated during mitosis, remainsto be elucidated.

Experimental evidence obtained during the last decade has shownthat the small GTPase Rap1 acts as a potent activator of manyintegrins (Bos, 2005; Caron, 2003), such as α3β1, α4β1, α5β1,αLβ2, αMβ2 and αIIbβ3 (Arai et al., 2001; Bertoni et al., 2002;Caron et al., 2000; de Bruyn et al., 2003; Enserink et al., 2004;Katagiri et al., 2000; Reedquist et al., 2000). To summarise, Rap1seems to activate all of the integrins connected to the actincytoskeleton (e.g. from the β1, β2 and β3 families), but not integrinsconnected to intermediate filaments, such as α6β4 (Enserink et al.,2004). To date, two Rap1 effectors have been clearly linked to

integrin activation by Rap1. RAPL (regulator for cell adhesion andpolarization enriched in lymphoid tissues), which is mainly expressedin lymphoid organs and tissues, interacts specifically with GTP-bound Rap1. Expression of RAPL is sufficient to stimulate theactivation of αLβ2 integrins and polarise their distribution inlymphocytes, in a manner that is dependent on the ability of RAPLto bind both Rap1 and the αL-integrin subunit (Katagiri et al., 2003).Accordingly, lymphocytes from RAPL-knockout mice are severelyimpaired in their abilities to adhere in vitro and to reach their homingorgans in vivo (Katagiri et al., 2004). Nevertheless, the restrictedexpression of RAPL might indicate that other Rap1 effectors areinvolved in controlling adhesion in non-lymphoid cells. In particular,RIAM (Rap1-interacting adhesion molecule) is another Rap1 effectorwhose expression is sufficient to promote αLβ2-integrin- and α4β1-integrin-dependent adhesion of Jurkat cells; moreover, RIAMsilencing suppresses the ability of a constitutively active Rap1 mutantto stimulate the adhesion of Jurkat cells to fibronectin and ICAM,or to activate αIIbβ3 integrin when ectopically expressed in Chinesehamster ovary (CHO) cells (Han et al., 2006; Lafuente et al., 2004).Interestingly, talin, a well-described component of focal adhesions,contains in its N-terminal head domain a B4.1 motif whoseassociation with a NPX[Y/F] motif, which is present in many β-subunits, is sufficient to promote integrin activation (Tadokoro etal., 2003). Moreover, talin colocalises and is co-immunoprecipitatedwith RIAM when both molecules are overexpressed, suggesting thatRap1 activates integrins by promoting the recruitment of talin tointegrins through RIAM (Han et al., 2006).

As a consequence of the ability of Rap1 to stimulate integrinactivation and cell adhesion, expression of the constitutively active

At the onset of mitosis, most adherent cells undergo cell retractioncharacterised by the disassembly of focal adhesions and actinstress fibres. Mitosis takes place in rounded cells, and the twodaughter cells spread again after cytokinesis. Because of the well-documented ability of the small GTPase Rap1 to stimulateintegrin-dependent adhesion and spreading, we assessed its roleduring mitosis. We show that Rap1 activity is regulated duringthis process. Changes in Rap1 activity play an essential role inregulating cell retraction and spreading, respectively, before andafter mitosis of HeLa cells. Indeed, endogenous Rap1 is inhibitedat the onset of mitosis; conversely, constitutive activation of Rap1inhibits the disassembly of premitotic focal adhesions and of the

actin cytoskeleton, leading to delayed mitosis and to cytokinesisdefects. Rap1 activity slowly increases after mitosis ends;inhibition of Rap1 activation by the ectopic expression of thedominant-negative Rap1[S17A] mutant prevents the roundedcells from spreading after mitosis. For the first time, we provideevidence for the direct regulation of adhesion processes duringmitosis via the activity of the Rap1 GTPase.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/16/2996/DC1

Key words: Rap1, Mitosis, Integrin, Spreading, Focal adhesion

Summary

Dynamic changes in Rap1 activity are required for cellretraction and spreading during mitosisVi Thuy Dao1,*,‡, Aurélien Guy Dupuy1,*,§,¶, Olivier Gavet2, Emmanuelle Caron3,** and Jean de Gunzburg1

1Institut Curie, Centre de Recherche, Inserm U528, 26 rue d’Ulm, 75248 Paris, France2Wellcome Trust/Cancer Research UK, Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, UK3Imperial College, Molecular and Cellular Biology Department, CMMI2, Flowers Building, London SW7 2AZ, UK*These authors contributed equally to this work‡Present address: Pérouse Médical, 52 rue des Meuniers, 92220 Bagneux, France§Present address: Imperial College, Molecular and Cellular Biology Department, CMMI2, Flowers Building, London SW7 2AZ, UK¶Author for correspondence (aurelien.dupuy@yahoo.fr)**Deceased July 2009

Accepted 6 May 2009Journal of Cell Science 122, 2996-3004 Published by The Company of Biologists 2009doi:10.1242/jcs.041301

JCS ePress online publication date 28 July 2009

2997Rap1 controls adhesion in mitosis

Rap1[Q63E] mutant or negative regulators of Rap1 (such asRap1GAP or Spa-1) respectively stimulates or inhibits the spreadingof HeLa cells on fibronectin (Arthur et al., 2004; Tsukamoto et al.,1999). Similarly, knockout of the Rap1 activator C3G in mouseembryo fibroblasts impairs their adhesion and spreading, andaccelerates migration (Ohba et al., 2001). Interestingly,overexpression of the Rap1 orthologue similarly stimulatesspreading of Dictyostelium discoideum cells (Rebstein et al., 1997;Rebstein et al., 1993). Recently, a genetic screen has led to theidentification of the Phg2 kinase (phagocytosis 2) as a Rap1 effectornecessary to mediate the stimulation of Dictyostelium cell spreadingby Rap1 (Jeon et al., 2007; Kortholt et al., 2006); no mammalianorthologue of Phg2 has been isolated so far.

Because of its apparent ability to regulate all of the differentintegrins engaged in adhesion, Rap1 constitutes a good candidatefor regulation of the turnover of adhesive structures and for controlof the retraction as well as spreading that occur during mitosis.

In this work, we show that Rap1 is transiently inactivated duringmitosis and that constitutively active or dominant-negative Rap1mutants respectively inhibit the cell-retraction and cell-spreadingphenomena that normally occur during the mitosis of HeLa cells.Our data suggest that Rap1 is a key element in the control ofadhesion and spreading dynamics during mitosis.

ResultsModulation of Rap1 activity affects the morphology of HeLacellsRap1 activity controls the activation of many integrins, including theα5β1 integrin that mediates cell adhesion and spreading ontofibronectin (Arai et al., 2001). Because integrin activation is aprerequisite for integrin outside-in signalling, Rap1 activity ultimatelyinfluences integrin outside-in signalling and thereby regulates cellmorphology (Arthur et al., 2004; Tsukamoto et al., 1999). Todetermine whether Rap1 activity controls HeLa-cell morphology, weexpressed a constitutively active mutant (Rap1[Q63E]) or a powerfuldominant-negative mutant (Rap1[S17A]) (Dupuy et al., 2005) of Rap1in cells seeded on fibronectin-coated plates. Transfected cells wereeasily identifiable by their increased immunofluorescence whenstained with an anti-Rap1 antibody (data not shown). As previouslydescribed (Arthur et al., 2004), expression of the constitutively activeRap1[Q63E] mutant stimulated the spreading of HeLa cells (Fig. 1A).In comparison with untransfected cells, Rap1[Q63E]-expressingcells exhibited few peripheral actin stress fibres but a very densenetwork of thin actin stress fibres throughout the cell body. Eachfibre was connected to a vinculin-labelled structure that was generallysmaller than those found in untransfected cells; the vinculin-richstructures were scattered throughout the cell body. By contrast, cellsexpressing the dominant-negative Rap1[S17A] mutant were roundedand not spread (Fig. 1A). Actin cables, essentially lining the cellperiphery, were connected at their ends to peripheral vinculin-stainedstructures comparable in size to those displayed by untransfected cells.Hence, despite their rounded morphology, Rap1[S17A]-expressingcells exhibit focal-adhesion structures apparently similar to those ofuntransfected cells. This explains why these cells are still adherentand do not detach even after performing a shake-off, a techniqueknown to detach poorly adherent cells such as those in mitosis (datanot shown).

To determine whether these phenotypes resulted from a long-lasting modulation by Rap1 mutants of HeLa-cell adhesion andspreading capacities, cells expressing Rap1[S17A] or Rap1[Q63E]were trypsinised and replated onto a fibronectin-coated surface. As

shown in Fig. 1C, cells expressing Rap1[S17A] indeed had adrastically impaired ability to re-adhere, as only 4% of transfectedcells adhered to fibronectin 6 hours after replating. By contrast,Rap1[Q63E]-expressing cells re-adhered more rapidly thanuntransfected cells. Accordingly, measurements of cell area showedthat cells expressing the constitutively active mutant of Rap1 spreadmore rapidly and to a larger extent than untransfected cells uponreplating, whereas cells expressing dominant-negative Rap1 wereunable to spread after replating (Fig. 1B,D). No vinculin-richadhesion structures were observed in cells expressing theRap1[S17A] mutant, hence explaining their inability to adhere andspread (Fig. 1B). By contrast, cells expressing the constitutivelyactive Rap1[Q63E] mutant displayed the same morphologicalcharacteristics upon replating as in Fig. 1A; at all time pointsobserved, replated cells also exhibited numerous thin actin stressfibres and small vinculin-rich structures throughout the cell body(Fig. 1B). Moreover, these cells adopted a much roundermorphology than control cells, which exhibited the polygonal shapecharacteristic of HeLa cells. In conclusion, Rap1 activity modulatesthe distribution and size of adhesion structures and actin fibres, andconsequently contributes to the maintenance and dynamics of cellmorphology in HeLa cells.

Recently, Rac1 was suggested to mediate Rap1-dependentstimulation of cell spreading (Arthur et al., 2004). To investigatewhether Rac1 could play a role in our system, we assessed thelevel of active Rac1 in cells expressing either a dominant-negativeor constitutively active mutant of Rap1 (Fig. 1E). Surprisingly, wefound that Rac1 was activated in rounded cells expressingdominant-negative Rap1, and that Rac1 was inactivated inoverspread cells expressing active Rap1. Because Rac1 and Rap1activities did not appear to vary in sync, we conclude that Rac1was not involved in the Rap1-induced changes of cell morphologythat we observed.

Rap1 activity is regulated during mitosisHaving observed that modulations of Rap1 activity stimulate orinhibit cell spreading, we hypothesised that Rap1 activity shouldbe tightly regulated during the cell cycle and particularly in mitosis.Indeed, most adherent cells undergo a phase of retraction at theonset of mitosis and, following cytokinesis, the two daughter cellsspread to assume their interphasic shape (Cramer and Mitchison,1993). To test this hypothesis, HeLa cells were synchronised,blocked at the beginning of the S phase by a double thymidine block,and progressively released into the cell cycle. Rap1 activation levelswere assessed by pull-down assays whilst progression along thecell cycle was simultaneously controlled by FACS analysis. Asshown in Fig. 2A, at 2 hours after release from the thymidine block,cells were in S phase, well spread and exhibited a high Rap1 activity.After a further 7 hours (i.e. 9 hours after release), most of the cellswere approaching mitosis: 50% were in G2 or M phase, close toperforming mitosis, whereas 39% were ending their replication and12% of the cells had already undergone mitosis and were back toG1. At this time point, global Rap1 activity was reduced by 50%.After a further 2 hours (i.e. 11 hours after release), while most ofthe cells (71%) had performed their mitosis and 17% were eitherin mitosis or late G2 phase, the amount of active Rap1 was at itslower level (reduced by 80% as compared with pre-mitotic cells).After another 2 hours (i.e. 13 hours after release) cells had allreturned to the G1 phase and were spread; surprisingly, despite adoubling in Rap1 activity from its minimum, it remained low (40%of the level measured before mitosis) until 15 hours after release

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(approximately 4 hours after mitosis), after which GTP-Rap1 levelsremained high until cells entered another S phase.

To confirm these results obtained from a pool of synchronisedcells at a single level, we monitored the changes in Rap1 activityusing a Rap1 FRET (fluorescence resonance energy transfer) probedeveloped in the Matsuda laboratory (Mochizuki et al., 2001). Theoriginal Raichu-Rap probe comprises the Ras-binding domain(RBD) of Raf fused in frame to the C-terminal part of Rap1A. Inthis probe, the YFP and CFP fluorescent domains are fused in frameto the N-terminus of Rap1A and to the C-terminus of the RBD,respectively; the membrane-targeting signal (comprising the last 18amino acids, including the CAAX sequence) of Rap1A is transferredfrom the C-terminus of Rap1 to the C-terminus of the FRET probe.Upon activation (GTP loading) of Rap1A, binding of the RBD to

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Rap1-GTP leads to a conformational change that brings togetherthe two fluorophores and causes an increase in the FRET signal.Because a weak decrease in the intrinsic FRET signal is usuallyobserved during mitosis, as a result of cell morphological changes,irrespective of the probe used (O.G., unpublished observations andFig. 2B), we constructed a non-responsive Rap1 probe to serve asan internal negative control. The resulting Raichu-Rap[R452L]probe presents a mutation in the RBD that was previously shownto abrogate the interaction of Raf with active Ras (Fabian et al.,1994). As shown in Fig. 2B, entry into mitosis, detected as thebreakdown of the nuclear envelope (as assessed by phase contrastand set to t=0 in the graph), was followed by a weak decrease inthe FRET signal of the control probe (CTR). By contrast, FRETfrom the wild-type Raichu-Rap probe dropped abruptly upon entry

Fig. 1. Rap1 activity affects HeLa-cell adhesion and spreading abilities. (A) HeLa-cell morphology is modulated by Rap1 activity. At 24 hours after transfectionwith empty vector (control; CTR) or mutant alleles of Rap1, HeLa cells were fixed and stained for Rap1 (not shown, cells expressing Rap1 mutants are markedwith an asterisk), F-actin (green), vinculin (red) and DNA (blue). Insets are enlarged in the lower panel. Scale bars: 20μm. (B) Rap1 activity regulates thedistribution of adhesion sites and actin fibres during HeLa-cell spreading. At 24 hours after transfection, mock-transfected (CTR) or Rap1-expressing HeLa cellswere trypsinised and tested for re-adhesion on a fibronectin matrix. After 1, 3 or 6 hours, cells were fixed and stained for F-actin (green), vinculin (red), DNA(blue) and Rap1 (not shown). Typical examples are shown. Scale bars: 20 μm. (C) Cell adhesion requires Rap1 activity. Cells were treated as described in B andcells remaining bound 1, 2, 3 or 6 hours after plating were counted. Results are expressed relative to the number of control cells adhering after 6 hours (arbitrarilyset to 100). (D) Rap1 activity is required for cell spreading. Cells were treated as described in B. Surface areas of 200 cells per condition were measured asdescribed in the Materials and Methods. (E) Rac1 activity correlates inversely from Rap1 activity. Levels of active Rac1 were assessed by pull down in cellstransformed by an empty plasmid (CTR) or expressing either Rap1[Q63E] or Rap1[S17A]. The quantification graph displays levels of active Rac1 normalised tototal Rac1. The result is representative of three independent experiments.

2999Rap1 controls adhesion in mitosis

into mitosis, indicating that Rap1 activity is negatively regulatedduring the process. Interestingly, whereas the downregulation ofRap1 activity preceded nuclear-envelope breakdown and took placewithin a few minutes, the post-mitotic recovery of Rap1 wasconsiderably slower, confirming our biochemical results (see Fig.2A). Taken together, these results show that Rap1 activity istransiently downregulated during mitosis.

Rap1 inactivation is required for the disassembly of adhesioncomplexes during mitosisBecause endogenous Rap1 is transiently inactivated during mitosis,we investigated the impact of the constitutive activation of Rap1on the mitotic process by expressing the constitutively activeRap1[Q63E] mutant. Control mitotic cells, as revealed by the typicalHoechst staining of metaphasic chromosomes, exhibited theircharacteristic rounded morphology (Fig. 3), with F-actin no longer

organised as stress fibres, but rather as retraction fibres (Cramerand Mitchison, 1993), and a loss of mature adhesion complexes asattested by the absence of vinculin labelling. By contrast,Rap1[Q63E]-expressing mitotic cells maintained the tight networkof thin actin fibres they exhibited in interphase, with a noticeabledeformation of the network as it circumvented the area surroundingthe chromosomes. Unlike control cells, Rap1[Q63E]-expressingcells also retained vinculin-labelled structures throughout the cellbody, and these structures appeared larger at the cell periphery (Fig.3). We also checked that the phenotype observed was specific toRap1 by showing that the expression of the constitutively activeform of either H-Ras, R-Ras or Rac1 did not phenocopy theinhibition of focal-adhesion disassembly and cell retraction duringmitosis induced by active Rap1 (data not shown). Hence, persistenceof Rap1 signalling does not prevent cells from undergoing mitosis,but specifically opposes the loss of actin stress fibres and focal

Fig. 2. Rap1 signalling is transiently downregulated during mitosis. (A) Rap1activity was assessed by pull-down of the GTP-bound Rap1 fraction from cellssequentially released from a double thymidine block (time indicated). Top,representative kinetics showing the levels of GTP-bound and total Rap1. Thegraph is a quantification of the GTP-bound Rap1 fraction relative to the totalRap1 level. Representative pictures of synchronised cells are shown (bottom),together with their cell-cycle status as monitored by FACS. Results arerepresentative of two independent experiments. (B) Quantification of Raichu-Rap FRET during mitosis. YFP and CFP signals from the non-responsive(Raichu-Rap[R452L]; CTR) or wild-type (Raichu-Rap) probes were quantifiedin single cells. The graph displays the variation of the FRET signal duringmitosis as expressed as the mean of YFP:CFP ratio (±s.d.), calculated fromfive independent cells per probe. Nuclear-envelope breakdown (observed byphase contrast) was set to t=0. The two curves become statistically different 3minutes before nuclear-envelope breakdown (P<0.05).

Fig. 3. Rap1[Q63E] expression prevents remodelling of the actin cytoskeletonand inhibits disruption of adhesion sites during mitosis. HeLa cells weretransfected with either empty vector or a Rap1[Q63E]-encoding vector, thenstained for Rap1, vinculin (red), F-actin (green) and DNA (Hoechst, blue).(1) and (2) indicate representative examples of two Rap1[Q63E]-expressingcells. Scale bars: 20 μm.

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adhesions that leads to the characteristic cell rounding observedduring the mitosis of HeLa cells.

Misregulation of Rap1 affects the regulation of cell morphologyduring mitosisTo analyse how the misregulation of Rap1 activity alters thedynamics of the actin cytoskeleton and adhesion sites during mitosis,we performed live video-microscopy experiments on cellsexpressing either the dominant-negative Rap1[S17A] or theconstitutively active Rap1[Q63E] mutants. Because a previous studyhad shown that Rap1 mutants are not fully active when fused to aN-terminal tag (Dupuy et al., 2005), we co-transfected cells withuntagged mutant alleles of Rap1 and a vector encoding redfluorescent protein (RFP) in the ratio 3:1. Under these conditions,more than 95% of RFP-expressing cells showed markedly increasedRap1 levels compared with control untransfected cells, as assessedby immunofluorescence using anti-Rap1 antibodies (not shown).In further experiments, RFP expression was therefore taken as anindicator of the overexpression of the mutant.

Time-lapse video-microscopy was performed between 4 and48 hours after transfection, a time period sufficient for HeLa cellsto accomplish two full division cycles (Fig. 4A; supplementarymaterial Movies 1-3). Cells ectopically expressing Rap1 mutantsundergo a first mitosis that is undistinguishable from that of mock-transfected cells (control; CTR): cells round up and go throughall of the steps of mitosis with normal morphology and kinetics(Fig. 4A, top panel). However, following mitosis, cells expressingthe constitutively active Rap1[Q63E] mutant displayed a morerapid and pronounced post-mitotic spreading than untransfectedcells, exhibiting a bigger cell area as well as a large lamellipodiumon the opposing side from the sister cell. By contrast, cellsexpressing the dominant-negative Rap1[S17A] mutant started tospread, but eventually retracted and remained rounded thereafter.These results are reminiscent of those obtained in adhesion andspreading experiments (see Fig. 1). In order to ascertain thetemporal correlation of this phenotype with the expression of thedominant-negative Rap1 mutant, the same experiments wereperformed using RFP-tagged forms of the Rap1 mutants ([S17A]or [Q63E]) (Fig. 4B). Strikingly, expression of the fusion proteinsonly started to be detectable after cells had accomplished the firstmitosis and initiated spreading. In particular, increased RFP-Rap1[S17A] expression correlated with retraction of transfectedcells, suggesting that interfering with Rap1 activity resulted ininhibition of post-mitotic spreading and eventually in cellretraction. Despite their rounded morphology, Rap1[S17A]-expressing cells were able to proceed through a further mitosis,again with normal kinetics and morphology except that the cellsretained a rounded shape throughout, and the two sister cellsremained round and close to one another after cytokinesis (Fig.4A, lower panel; supplementary material Movie 3). Thisphenotype was highly reproducible because it similarly affectedall cells expressing Rap1[S17A].

The effects of Rap1[Q63E] expression were even more striking.These cells, which were very widely spread following the firstmitosis, were unable to round up prior to the second mitosis, despiteclear morphological signs of attempted retraction (seesupplementary material Movie 2). They proceeded as spread cellsthrough the chromosomal steps of mitosis until cytokinesis, inagreement with the fact that these cells retained actin stress fibresand adhesion sites during mitosis as shown in Fig. 3. As aconsequence, 50% of cells expressing active Rap1 were unable to

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complete cytokinesis, and exhibited a total regression of thecleavage furrow, resulting in large and spread binucleated cells(supplementary material Movie 2 and data not shown). This failureof cytokinesis was also observed in a different batch of HeLa cellsas well as with NIH3T3 cells. We then measured the duration oftwo successive cell cycles, and analysed the results, taking intoaccount the results of Fig. 4B that the mutant Rap1 proteins areonly efficiently expressed after the end of the first mitosis. As shownin Fig. 4C, cells expressing the constitutively activated mutant ofRap1 were delayed in their cell cycle, especially during mitosis,which lasted twice as long as in control cells. Despite their roundedmorphology, the cell-cycle kinetics of cells expressing dominant-negative Rap1[S17A] were undistinguishable from those of controlcells. In conclusion, the regulation of Rap1 activity plays animportant role in the cell-shape changes that occur during mitosis.Too much Rap1 activity prevents the cells from de-adhering at theonset of mitosis, eventually resulting in a delayed cell cycle andmitosis as well as a high proportion of binucleated cells, whereasthe inability to re-activate Rap1 following mitosis prevents the cellsfrom spreading on the substratum to assume their normal interphasicmorphology.

DiscussionRap1 activity has been shown to stimulate the adhesive propertiesof many integrins, as assessed by adhesion assays, ligand-bindingassays and using activation-specific reporter antibodies. Less studiedis the impact of modulating Rap1 activity on cell morphology, orhow Rap1 activity is regulated during the morphological changesthat accompany physiological processes. In this article, we havecharacterised both the effect of modulating Rap1 activity on HeLa-cell spreading and its regulation during mitosis.

Consistent with its role in inside-out integrin activation (Bos,2005), we show that Rap1 overactivation stimulates cell adhesionand spreading onto a fibronectin matrix (Fig. 1). Cells expressingconstitutively active Rap1 displayed a more homogeneousdistribution of their adhesion sites connected to a denser networkof actin stress fibres. These results show that integrin activationby Rap1 is sufficient for the establishment and maturation ofadhesion sites and their connection to the actin cytoskeleton (Fig.1B,D). Comparatively, cells expressing a dominant-negative Rap1mutant did not spread and remained rounded (Fig. 1A). Thesecells are also completely inhibited in their abilities to re-adhereonce trypsinised, because no adhesion-related structures could beobserved (Fig. 1B). These results clearly show that Rap1 activityis required for cell adhesion onto fibronectin and that it mediatesa non-redundant pathway that leads to integrin activation. Becausetime-lapse microscopy showed that cells retract concomitantly tothe expression of dominant-negative Rap1, one hypothesis couldbe that Rap1 function is also required for the maintenance of pre-formed adhesion sites (Fig. 4B). However, these cells do not detachbut retain some adhesion- and actin-related structures. Rather thanRap1 playing a role in maintaining already formed adhesion sites,it is likely that adhesion sites are subject to constant recyclingand that Rap1 activity is required to initiate the formation of newsites. According to this hypothesis, the adhesion sites that remainin cells expressing dominant-negative Rap1 could be explainedby the fact that these sites are less prone to recycling, maybebecause the tension exerted by the actin cytoskeleton at these sitesis somehow different. Further FRAP experiments to study thedynamics of vinculin-rich structures in these cells could beinformative.

3001Rap1 controls adhesion in mitosis

Correlated with the fact that expression of either constitutivelyactivated or dominant-negative mutants respectively stimulate orinhibit cell adhesion and spreading (Fig. 1), we clearly demonstratedthat cell retraction at the beginning of mitosis requires Rap1inactivation. Indeed, both pull-down and FRET experiments showedthat endogenous Rap1 is inactivated during mitosis (Fig. 2); moreover,ectopic expression of constitutively active Rap1 is not compatiblewith the disruption of adhesion sites and actin fibres that normallyoccur during this process (Fig. 3). Downregulation of Rap1 activityat the onset of mitosis could be ensured by the inhibition of a guaninenucleotide exchange factor (GEF) activity, stimulation of GTPase-

activating protein (GAP) activity or a combination of bothmechanisms. In Fig. 4B, the effects of expressing dominant-negativeRap1, which blocks the activation of endogenous Rap1 by titratingGEFs (Dupuy et al., 2005; Feig, 1999), takes place within 2 hours.Comparatively, Rap1 is inactivated within 10 minutes of the onsetof mitosis (Fig. 2B). Such a rapid inhibition might indicate thatincreased GAP activity is required. Interestingly, Rap1GAP has beenshown to be directly phosphorylated by Cdc2, the protein that initiatesthe onset of mitosis (Janoueix-Lerosey et al., 1994). WhetherRap1GAP activity is modulated by phosphorylation is unknown, asis the physiological relevance of its phosphorylation by Cdc2.

Fig. 4. Modulations of Rap1 activity affect cellmorphology during the cell cycle.(A) Dominant-negative and constitutivelyactive Rap1 mutants inhibit post-mitotic cellspreading and pre-mitotic retraction,respectively. HeLa cells were co-transfectedwith pEmRFP-C2 and either empty vector(CTR) or vectors expressing Rap1[S17A] orRap1[Q63E] mutants as indicated. Immediatelyafter transfection, cells were filmedsimultaneously by multi-positioning time-lapsevideo-microscopy for 48 hours. The firstmitosis is shown in the top panels and begins attime t1, whereas the second mitosis begins attime t2 and is shown in the bottom panels.Relative time intervals are indicated at the topof each column; total time elapsed since thebeginning of the first mitosis is indicated in theupper left corner of each frame. Arrowsindicate transfected cells undergoing mitosis (asassessed by RFP fluorescence visualised at theend of the live recording; see supplementarymaterial Movies 1-3). (B) Cell retraction afterthe first mitosis is correlated to the expressionof Rap1[S17A]. HeLa cells were transfectedwith Rap1[S17A] fused in frame to themonomeric RFP protein. Pictures arerepresentative of what was observed in 100% ofRFP-Rap1[S17A]-expressing cells.(C) Expression of Rap1[Q63E] delays cell-cycle progression. The duration of the firstmitosis, interphase and second mitosis weremeasured on movies from 20 cells percondition. Mitosis lengths were evaluated asstarting with cell rounding and finishing withthe first signs of separation of the two daughtercells (beginning of cytokinesis). As indicated,only the interphase and second mitosis of cellsexpressing constitutively active Rap1 aresignificantly different from the control, asassessed by Student’s t-test (P<0.05).

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To date, cell rounding has been shown to require a RhoA-Rockpathway, probably by acting on actin-myosin contractility throughphosphorylation of myosin light chain (MLC) (Maddox andBurridge, 2003). More recently, two different studies havesuggested a role for moesin during mitosis of Drosophila S2 cells.Moesin belongs to the ERM (ezrin, radixin, moesin) family ofproteins that link the plasma membrane to the actin cytoskeleton.Moesin is the only member of this family expressed in Drosophila,in which it is required for complete cell retraction and correctorientation of the mitotic spindle (Carreno et al., 2008; Kunda etal., 2008). How Rap1 and moesin pathways are coordinated toimplement cell retraction during mitosis presently remains unclear.However, it is unlikely that a structural protein such as moesincould directly regulate the activity of the signalling molecule Rap1;rather, it is conceivable that two pathways, one directly regulatingadhesion and another pathway promoting cell contractility, couldcooperate to induce cell rounding during mitosis. To support thisidea, signs of contractility remain visible in cells inhibited in theirability to retract upon expression of the constitutively active Rap1mutant as well as in cells expressing dominant-negative Rap1 (Fig.4A; supplementary material Movies 1-3). These observationssuggest that several pathways regulate cell rounding duringmitosis.

Surprisingly, once mitosis has been completed, Rap1 activity onlyincreased slowly, with maximal activation only reached after cellshad fully spread (4 hours after mitosis, Fig. 2A). However, Rap1activity is required to allow cell spreading, because the inhibitionof Rap1 by expression of the dominant-negative Rap1[S17A] mutantcompletely blocks cell spreading (Fig. 4A). This delayed activationraises questions about the role of Rap1 in cell spreading after mitosis.It seems that a weak Rap1 activation overall is sufficient to initiatespreading, maybe because a subset of Rap1 molecules is activatedat specific locations or because active Rap1 is targeted to specificregions of the cell. We did not observe any specific local increasein FRET signal with the Raichu-Rap1 probe, possibly owing to itsoverexpression. Nevertheless, because levels of active Rap1 arehigher in cells plated on fibronectin compared with cells maintainedin suspension, it is likely that Rap1 in turn is activated by integrinengagement (Ohba et al., 2001). Such an auto-stimulatory loop couldhelp reinforce and secure newly formed adhesion sites, and mightalso allow cells to spread directionally.

Finally, we show that expression of the dominant-negativeRap1[S17A] mutant has no effect on cell-cycle progression. Despiteremaining rounded, cells progress along the cell cycle and inparticular into mitosis within the same time course as control cells(Fig. 4C). These results suggest that neither mitosis nor cytokinesisrequire fully developed adhesion sites in order to proceed. Comparedwith control cells, interphase and mitosis last longer in cellsexpressing the constitutively active Rap1[Q63E] mutant. Inparticular, mitosis lasts 100% longer than in control cells. This couldbe related to the finding that cell rounding helps to stabilise spindleassembly (Kunda et al., 2008). Interestingly, Kunda et al. showedthat the unstable metaphase spindles observed in non-fully retractedcells lacking moesin could be rescued by artificially rounding thecells. A possible explanation is that, in non-rounded cells, astralmicrotubules can no longer contact the cortex to separate the twoasters of the spindle (Rosenblatt, 2008). This might also occur incell expressing constitutively active Rap1, in which the mitoticspindle is not as stable as in control cells, turning from side to sidebefore chromosome segregation (data not shown). Nevertheless,another simple explanation could be that the lack of actin-fibre

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disruption disturbs the organisation of the mitotic spindle. Becausecytokinesis is a process mainly dependent of acto-myosincontractility (Matsumura, 2005), maintenance of the actin-fibrenetwork might also explain the cytokinesis defects and the fusionof the cells expressing constitutively active Rap1 (Fig. 4A).

To conclude, we provide strong evidence that cell-adhesiondynamics play an important role in regulating the progression ofmitosis, and that activity of the integrin activator Rap1, which isfinely tuned during this process, is involved. Further challenges willbe to unveil the signals from the cell cycle that trigger cellrounding, and decipher the pathways upstream and downstream ofRap1 that act in this process.

Materials and MethodsConstructspRK5 empty vector, pRK5-Rap1[S17A] and pRK5-Rap1[Q63E] were previouslydescribed (Dupuy et al., 2005). pEmRFP-C2 was generated by replacing the GFP-encoding BamHI-NotI fragment from the pEGFP-C2 vector (Clontech) with thesequence encoding the monomeric RFP (a kind gift of Roger Y. Tsien, University ofCalifornia at San Diego, La Jolla, CA), amplified by PCR. pRaichu-Rap1[R452L]was built by targeted mutagenesis of pRaichu-Rap1 (a precious gift of MichiyukiMatsuda, Kyoto University, Kyoto, Japan) with the QuikChange kit (Stratagene).

Cell culture and transfectionHeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing10% foetal bovine serum (FBS) on plastic dishes or glass coverslips that had beenprecoated with fibronectin (10 μg/ml). Plasmids were transfected with LipofectaminePlus (Invitrogen). For video microscopy, a 35-mm culture dish was first pre-coatedwith fibronectin (10 μg/ml), and then filled with polydimethylsiloxane polymer (DowCorning) in which three wells were subsequently cut out to enable the simultaneousrecording of three distinct experiments (see below).

ImmunofluorescenceCells were prepared for indirect immunofluorescence using a method that preservesthe actin retraction fibres observed during mitosis (Mitchison, 1992). Briefly, cellsseeded on glass coverslips were incubated for 1 minute with cytoskeleton buffer [10mM 2-(N-morpholino) ethanesulfonic acid buffer, pH 6.1, 138 mM KCl, 3 mM MgCl2,2 mM EGTA] containing 0.32 M saccharose and 0.1% Triton X-100, then fixed for20 minutes in a 4% solution of paraformaldehyde in cytoskeleton buffer containing0.32 M saccharose. Coverslips were then washed three times in Tris-buffered saline(TBS: 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl) and paraformaldehyde was quenchedfor 20 minutes with 0.1 M NH4Cl. After three washes in TBS, cells were permeabilisedfor 10 minutes with TBS containing 2% BSA and 0.2% Triton X-100 (TBS-BT).After three washes in TBS containing 0.02% Triton X-100 (TBST), samples wereincubated for 1 hour with primary antibodies: affinity-purified rabbit polyclonal anti-Rap1 antibodies (Beranger et al., 1991), mouse monoclonal anti-vinculin VII F9 (akind gift of Marina Glukhova, Institut Curie, Paris, France), diluted in TBST. Afterfour washes in TBST, cells were incubated for 45 minutes with Alexa-Fluor-488-coupled phalloidin (1/250 in TBST; Molecular Probes) and the fluorescent-coupledsecondary antibodies Cy3-coupled goat anti-mouse IgG (1.3 μg/ml in TBS-BT;Jackson) and Alexa-Fluor-647-conjugated goat anti-rabbit IgG (2 μg/ml in TBS-BT;Molecular Probes). Cells were washed four times in TBS and incubated for 2 minutesin 1 μg/ml Hoechst 33258 in TBS-T. Finally, cells were washed in TBS and coverslipswere mounted with Gold Prolong reagent (Molecular Probes).

Adhesion and spreading assaysAt 1 day after transfection with vectors encoding mutants of Rap1 (dominant negativeor constitutively active) or RFP as a control, HeLa cells were trypsinised for 5 minutesat 37°C, washed once with PBS, incubated for 20 minutes in a rotating tube at 37°Cin DMEM containing 10% FBS, and finally seeded on coverslips coated withfibronectin (10 μg/ml). At the indicated times, cells were gently washed with PBSand processed for immunofluorescence to label Rap1 and F-actin according to theimmunofluorescence protocol mentioned above. At least five fields for eachcondition were photographed with a 40� lens. Adhesion was measured as the numberof adherent cells expressing either Rap1 mutant (as assessed by immunofluorescencewith an anti-Rap1 antibody) or RFP (control), expressed as a percentage of the totalnumber of cells adhering after 6 hours. From pictures acquired, the cell peripheryof more than 200 cells per condition was drawn by hand according to the F-actinstaining and cell surface was measured with the ImageJ freeware(http://rsb.info.nih.gov/ij).

Pull-down of GTP-bound Rac1Assays were performed with the Rac1 activation assay Biochem Kit (Cytoskeleton)according to the manufacturer’s instructions.

3003Rap1 controls adhesion in mitosis

Pull-down of GTP-bound Rap1 in cells synchronised by a doublethymidine blockTo synchronise cells, a double thymidine block was performed by incubating cells for19 hours at 37°C in normal medium (DMEM containing 10% FBS) supplemented with2 mM thymidine; cells were washed three times with normal medium, then incubatedfor 10 hours in pre-warmed normal medium containing 30 μM deoxycitidine. Finally,cells were blocked again by incubating them for at least 19 hours at 37°C in normalmedium supplemented with 2 mM thymidine. Cells were sequentially released into thecell cycle by replacing the medium with normal medium containing 30 μMdeoxycitidine. The levels of Rap1-GTP were assessed using a pull-down assay aspreviously described (Franke et al., 1997). Briefly, cells were washed with cold PBSand lysed in a buffer containing 50 mM Tris-HCl, pH 7.5, 15 mM NaCl, 20 mM MgCl2,5 mM EGTA, 1% Triton X-100, 1% N-octylglucoside, 100 μM PMSF, 10 μM leupeptinand 10 μM aprotinin. Lysates were cleared by centrifugation, and Rap-GTP complexeswere recovered on glutathione-Sepharose beads precoupled to GST fused with theRas/Rap-binding domain of RalGDS. Precipitates were washed three times with lysisbuffer and solubilised in SDS sample buffer. Aliquots of cleared cell lysates were keptfor detection of total Rap1 contents. Following western blotting, Rap1 was detectedwith affinity-purified polyclonal antibodies (Béranger et al., 1991). Actin was detectedusing the monoclonal AC-74 anti-β-actin antibody (Sigma). Proteins were visualisedon western blots by chemiluminescence (SuperSignal West Femto, Pierce); images werecaptured with a FUJI LAS-1000 CCD camera and quantified using FUJI Image Gaugesoftware. In parallel to the pull down, the cell-cycle stage was assessed by flowcytometry. Briefly 2�106 synchronised cells were trypsinised, washed with PBS, fixedwith ice-cold 70% ethanol, washed again twice with PBS, incubated for 5 minutes witha 1 mg/ml RNAse-A solution in PBS, centrifuged and then resuspended in 400 μl ofPBS, 50 μg/ml propidium iodide. Fluorescence was measured at 620 nm with excitationat 488 nm.

Live video-microscopy and immunofluorescence microscopyLive video recordings were acquired on a Leica DMIRBE microscope equipped witha 37°C thermostated, 5% CO2-equilibrated motorised stage. Immunofluorescencepictures were acquired on a Leica DM6000B Epifluorescence microscope with a63� Apochromat lens (Leica Microsystems). All acquisitions were made with aPhotometrics CoolSnap HQ camera and analysed with the Metamorph software(Universal Imaging).

FRETHeLa cells (3�106) were electroporated (250 V, 1500 μF, infinite resistance) with 25μg of FRET probe DNA using an Easyjet plus machine (Equibio). Then, cells wereseeded in glass-bottom dishes (Intracel, Royston) pre-coated with fibronectin (10μg/ml). At 7 hours after transfection, cells were synchronised in S phase with 2.5 mMthymidine for 24 hours. Cells were then released in fresh DMEM. For time-lapseexperiments, the culture medium was replaced by Leibovitz’s L-15 medium(Invitrogen), containing 10% FBS. DIC and fluorescence images were acquired witha Deltavision microscope equipped with a cascade II EMCCD camera (Photometrics)using a binning of 1�1 and a gain of 140. A 40�/1.35 UApo oil objective was usedfor all images. Filter configuration was 436/10 for the CFP excitation filter, and 465/30and 535/30 for the CFP and YFP emission filters, respectively. The exposure time was200 milliseconds for CFP and YFP images using a 32% ND filter. ImageJ softwarewas used to quantify fluorescence signals. Briefly, after background subtraction, theYFP:CFP ratio of the whole cell was calculated and used to represent the FRETefficiency.

The authors are indebted to M. Bornens and M. Théry for stimulatingdiscussions and help with video-microscopy, and to M. Gluckhova andM. Matsuda for providing anti-vinculin antibodies and Raichu-RapFRET probe, respectively. This work was funded in part by a grantfrom the Association pour la Recherche contre le Cancer. A.G.D. wassuccessively supported by post-doctoral fellowships from the Cancerand Solidarity Foundation (Geneva) and the ‘Fondation pour laRecherche Médicale’. V.T.D. was the recipient of a post-doctoralfellowship from the Institut Curie Genhomme project.

The authors are extremely saddened by the loss of their collaboratorEmmanuelle Caron. She was a young talented scientist, senior lecturerat Imperial College London, recognised by her peers and loved by herstudents. Emmanuelle was also a great person, full of energy and joyof life, always available to others. She supervised Aurélien Dupuy fortwo years and helped him considerably while he was finishing the workfor this manuscript. Emmanuelle was more than a supervisor; she wasa mentor. She will be terribly missed. This work is dedicated to hermemory.

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