3225Short Report

IntroductionDuring cell motility on the extracellular matrix (ECM), actin-drivenprotrusion is stabilized by new adhesive sites forming at theextending edge (Geiger et al., 2009; Le Clainche and Carlier, 2008).Lamellipodia are stabilized by clustering and activation of integrinsthat steady the link between their extracellular ligands and actin(Galbraith et al., 2007; Vicente-Manzanares et al., 2009). This linkrequires the dynamic regulation of focal adhesions (FAs) (Broussardet al., 2008). Although many of the molecular events involved inthe formation of FAs have been identified, understanding of themolecular mechanisms underlying their dynamics during motilityis still limited.

The liprin family includes α and β dimeric multidomain adaptorproteins (Serra-Pagès et al., 1998). Liprin-α1 interacts withleukocyte common antigen-related (LAR) tyrosine phosphatasereceptors at FAs (Serra-Pagès et al., 1995) and is implicated in theregulation of cell motility (Shen et al., 2007), whereas, in neurons,liprin-α is required for the assembly of pre-synaptic active zones(Stryker and Johnson, 2007). The role of this protein in cell motilityremains to be defined. Here, by using COS7 cells plated onfibronectin (FN), we show that liprin-α1 is localized at the edge ofspreading cells, where it affects active integrin distribution and isessential for efficient protrusive activity.

Results and DiscussionLiprin-α1 is stably associated to the cytoplasmic side of theplasma membraneIn transfected COS7 cells, liprin-α1 localized at the periphery (Fig.1A). In ventral plasma membranes (VPMs) prepared from ECM-attached transfected cells (Cattelino et al., 1997; Cattelino et al.,1999), liprin-α1 remained associated with the substrate-bound

plasma membrane (Fig. 1B). Endogenous liprin was enrichedtogether with talin in VPMs prepared from adherent fibroblasts (Fig.1C). Staining with purified anti-liprin-α1 antibodies (supplementarymaterial Fig. S1) showed that endogenous liprin partially colocalizedwith larger, more-central FAs (Fig. 1D). At up to 3 hours ofspreading on FN, endogenous liprin was enriched at the cellperiphery (supplementary material Fig. S2). Interestingly, co-staining with antibodies for liprin and either paxillin or talin clearlyshowed little colocalization of endogenous liprin with peripheralFAs. In fact, during spreading, liprin was localized just behind thesmall nascent FAs present at the cell edge (Fig. 1E; supplementarymaterial Fig. S2). The overlap with older FAs and poor localizationwith newly formed FAs at the cell edge was observed also forexogenous liprin expressed at low levels (Fig. 1F). Exogenous liprinwas excluded also from FAs concentrated at the edge of transfectedcells with higher levels of exogenous liprin-α1 (supplementarymaterial Fig. S3).

Liprin-α1 potentiates cell spreading by enhancing theformation of lamellipodia and FAs at the cell edgeTo study the function of liprin-α1 in the response of cells to theECM, we altered its expression by either overexpression ordownregulation by short interfering RNA (siRNA) and analyzedthe effects on spreading. Overexpression of liprin-α1 affected cellmorphology and evidently increased spreading (Fig. 2A,B) bystrongly enhancing the extent of actin-positive lamellipodia aroundthe cells (Fig. 2C,D), and by inducing the redistribution of newlyformed, nascent FAs that became densely packed at the cell edge(Fig. 2E; supplementary material Fig. S4). Reduced spreading ofcells that were co-transfected with liprin-α1 and dominant-negativeN17-Rac1 indicated the dependence of liprin-enhanced spreading

Integrin activation is needed to link the extracellular matrixwith the actin cytoskeleton during cell motility. Protrusionrequires coordination of actin dynamics with focal-adhesionturnover. We report that the adaptor protein liprin-α1 is stablyassociated with the cell membrane. Lipin-α1 shows a localizationthat is distinct from that of activated β1 integrins at theedge of spreading cells. Depletion of liprin-α1 inhibits thespreading of COS7 cells on fibronectin by affecting lamellipodiaformation, whereas its overexpression enhances spreading, andlamellipodia and focal-adhesion formation at the cell edge.Cooperation between liprin-α1 and talin is needed, becauseeither talin or liprin depletion prevents spreading in the presence

of the other protein. The effects of liprin on spreading, but notits effects in the reorganization of the cell edge, are dependenton its interaction with leukocyte common antigen-relatedtyrosine phosphatase receptors. Therefore, liprin is an essentialregulator of cell motility that contributes to the effectiveness ofcell-edge protrusion.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/18/3225/DC1

Key words: Cell motility, Focal adhesions, Integrins, Talin

Summary

Liprin-α1 promotes cell spreading on the extracellularmatrix by affecting the distribution of activatedintegrinsClaudia Asperti, Veronica Astro, Antonio Totaro, Simona Paris and Ivan de Curtis*Cell Adhesion Unit, Department of Neuroscience, San Raffaele University and San Raffaele Scientific Institute, 20132 Milano, Italy*Author for correspondence (decurtis.ivan@hsr.it)

Accepted 2 July 2009Journal of Cell Science 122, 3225-3232 Published by The Company of Biologists 2009doi:10.1242/jcs.054155

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Fig. 1. Liprin-α1 is stably associated with the plasma membrane. (A) Reconstruction (z-axis) from confocal sections of COS7 cells transfected with FLAG–liprin-α1 and fixed after 1 hour on FN. Integrins (TS2/16 mAb) and liprin-α1 were associated to both ventral and dorsal plasma membrane. (B) Liprin (green) remainedassociated with VPMs from COS7 cells. Blue, DAPI. (C) Enrichment of endogenous liprin and FA proteins in VPMs from chicken embryo fibroblasts (CEFs).15 μg of lysate (Lys), VPM Triton-insoluble fraction (TX-insol) or VPM Triton-soluble fraction (TX-sol) were analyzed for immunoblotting. (D) Endogenousliprin and paxillin in cells cultured on FN. The arrow shows the part magnified in E. (E) Plot profile: poor overlap of endogenous liprin with paxillin-positive FAsat the cell edge after 1 hour spreading. Vertical broken line indicates the segment analyzed in the graph. (F) The distribution of GFP-liprin (low levels) was similarto that of endogenous liprin. Scale bars: 20 μm (A,B,D); 5 μm (E,F).

Fig. 2. Liprin-α1 is required for cell spreading. (A) Spreading of COS7 cells on FN was enhanced by liprin-α1 overexpression, compared with control cells (β-galactosidase). (B) Bars are average values of projected cell areas ± s.e.m. (n=50 per condition). (C) Liprin-α1 overexpression enhances the formation oflamellipodia (phalloidin, red) during spreading on FN. (D) Bars are percentages of cell perimeter (± s.e.m., n=25 per experimental condition) positive for F-actin-rich lamellipodia. (E) Increase in the density of FAs (paxillin) at the edge of liprin-α1-transfected cells spreading on FN compared with controls. (F) Reducedspreading in liprin-α1-depleted cells: siRNA-transfected cells were stained for the co-transfected β-galactosidase (red, arrows) and for β1 integrins (TS2/16, green).(G) Quantification of spreading (right) in cells transfected with two different siRNAs for liprin-1α (lip1a and lip1b). Bars are average values ± s.e.m. (n=50 cellsper condition). The blot shows liprin-α1 depletion upon treatment. luc, luciferase. (H) Liprin depletion inhibits the formation of FAs and lamellipodia duringspreading on ECM. COS7 cells co-transfected with β-galactosidase (not shown) and siRNA for either liprin-α1 or luciferase were trypsinized and plated for 1 houron FN before immunostaining. (I) Depletion of liprin-α1 in COS7 cells by siRNA did not affect adhesion at different times and concentrations of FN. Control cells= 100% adhesion. Upper graph: time points are average values ± s.e.m. from four wells/condition from a representative experiment. Lower left: bars are meanvalues of adhesion ± s.e.m. (n=eight experiments, four wells/condition/experiment). Lower right: bars are mean values of adhesion ± s.e.m. (n=two experiments,eight wells/condition/experiment). (J) Overexpression of liprin-α1 in COS7 cells did not affect adhesion to FN for the indicated times (control cells = 100%adhesion). Bars are mean values ± s.e.m. (three experiments for t=7.5 minutes; four experiments for t=30 and t=60 minutes; four wells/condition/experiment).(K) Quantification of the persistence of GFP-paxillin in adhesion complexes. n=75 FAs from five different cells. (L) Number of FAs formed during spreading onFN. Bars are average values from ten different fields from cells co-transfected with GFP-paxillin together with either liprin-α1 or β-galactosidase. All P-values(shown above the bars) were determined by Student’s t-test. Scale bars: 20 μm.

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Fig. 2. See previous page for legend

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on Rac GTPase (supplementary material Fig. S5A). Moreover, wefound that activation of endogenous Rac1 was increased after 30minutes of spreading on FN upon liprin-α1 overexpression(supplementary material Fig. S5B).

Depletion of liprin-α1 by two specific siRNAs (88±6% s.d., n=4)inhibited spreading (Fig. 2F,G) by affecting lamellipodia formationand the concentration of newly formed FAs at the cell edge (Fig.2H), as confirmed by time-lapse analysis during spreading(supplementary material Fig. S5C). Conversely, cell adhesion wasnot affected by alteration of the cellular levels of liprin-α1 (Fig.2I,J). Time-lapse analysis of cells spreading on FN (supplementarymaterial Movies 1 and 2) showed that GFP-paxillin persisted forshorter times in newly formed FAs at the periphery of cellsoverexpressing liprin-α1 compared with controls (Fig. 2K). Bycontrast, liprin-α1 overexpression induced an increase in thenumber of FAs formed during spreading (Fig. 2L). Overall, thesedata strongly support a function of liprin-α1 in the turnover of FAsat the periphery of spreading cells.

Liprin and talin cooperate for efficient cell spreadingIntegrins can exist in an inactive or activated state, with low or highaffinity for their ligands, respectively (Calderwood, 2004). Talin-1and talin-2 are important regulators of integrin activation (Critchleyand Gingras, 2008). The interaction of the talin head with thecytoplasmic region of integrin β-subunits causes conformationalchanges that increase the affinity of integrins for their ligands(Calderhood et al., 1999; Tadokoro et al., 2003), whereas the roddomain of talin links integrins to the actin cytoskeleton (Jiang etal., 2003; Tanentzapf and Brown, 2006) and is necessary forpersistent cell spreading (Zhang et al., 2008).

Here, we analyzed the effects of liprin and/or talin overexpressionon cell spreading and on the activation of endogenous β1 integrins.To detect integrin activation, we used surface labeling of thetransfected cells with the 9EG7 antibody specific for activated β1integrins (Lenter et al., 1993). Interestingly, enhanced cell spreadingthat was induced by liprin overexpression (Fig. 3A,B) wasaccompanied by the accumulation of activated β1 integrin at theedge (Fig. 3A,D), with a reduction of the fraction of projected cellarea occupied by active 9EG7-positive integrins compared withcontrol cells (Fig. 3A,C). We found that transfection with eithertalin-1 or talin-1H did not affect cell spreading (Fig. 3A,B), butinduced an increase in the activation of β1 integrins (Cluzel et al.,2005) (Fig. 3A,C). Moreover, the co-transfection of talin-1 or ofits isolated head domain (talin-1H) with liprin prevented both liprin-enhanced spreading (Fig. 3A,B) and talin-induced integrin activation(Fig. 3A,C). These results suggest that the levels of liprin and talininfluence the functions of each other during cell spreading. Inparticular, our findings show that an excess of talin prevents theability of liprin to enhance cell spreading, possibly by enhancingintegrin activation to form more central, mature FAs. By contrast,the coexpression of liprin with talin reduced the hyperactivation ofintegrins at the cell surface compared with cells transfected withtalin-1 or talin-1H alone (Fig. 3A,C), in agreement with thehypothesis that the cellular levels of liprin and talin reciprocallymodulate the function of each other on cell spreading and integrinactivation.

COS7 cells mainly express talin-1 (Fig. 3E). Three independentsiRNAs for talin-1 were used to deplete talin (65±9% depletion± s.d., n=9) (Fig. 3F; supplementary material Fig. S6). Talin-1depletion confirmed its requirement for spreading on FN (Zhanget al., 2008) and showed that talin was needed for liprin-enhanced

spreading (Fig. 3G,H). Conversely, liprin-α1 depletion inhibitedtalin-mediated spreading (supplementary material Fig. S7). Intalin-depleted cells, there was more activated β1 integrins whenliprin was overexpressed (Fig. 3G). One possible explanation isthat some residual talin-1 that was left after knockdown of talinby siRNA might be more efficiently used for integrin activationwhen the cellular levels of liprin are increased by overexpression.This would be consistent with the hypothesis that liprin-α1 isrequired for efficient talin-mediated integrin activation during cellmotility.

Our results point to the fact that, although talin is necessary forintegrin activation, it is not sufficient to induce cell spreading inthe absence of liprin-α1. By contrast, liprin overexpression cannotrescue cell spreading in the absence of talin. Therefore, liprin andtalin are important for the function of each other in spreading, duringwhich they play cooperative and complementary roles in theregulation of cell-edge motility.

LAR is implicated in liprin-dependent spreadingWe tested whether the interaction between liprin-α1 and LAR(Serra-Pages et al., 1998) was required for the effects of liprin onspreading. LAR depletion (82±18% s.d., n=3) (Fig. 4A) reducedspreading to the same extent of depletion of both liprin and LAR(Fig. 4B,D), suggesting that these proteins participate in the samepathway. LAR depletion in cells overexpressing liprin-α1 preventedalso liprin-enhanced spreading (Fig. 4C), but did not prevent theredistribution of FAs at the edge of spreading cells (Fig. 4E), nordid it preclude the localization of endogenous liprin at the edge ofspreading cells (data not shown).

LAR interacts with liprin by binding to the sterile alpha motif 2(SAM2) domain (Olsen at al., 2006). The liprin-ΔSAM2 mutant,which cannot bind LAR (Fig. 4F) (Serra-Pagès at al., 1994), couldnot induce increased spreading (Fig. 4G) but, similar to liprin, wasstill able to induce the formation of large lamellipodia (data notshown) and the increase in concentration of FAs at the cell edge(Fig. 4H,I). The cell perimeter positive for activated β1 integrinswas increased both in liprin-ΔSAM2- (79.3±3.1%, n=20 perimetralfields) and liprin- (91.5±1.1%, n=20 perimetral fields) expressingcells compared with control cells (67.9±2.5%, n=20 perimetralfields). Moreover, the projected cell area occupied by FAs in liprin-ΔSAM2-transfected cells was similar to control cells but higher thanin liprin-transfected cells (Fig. 4J), reflecting the higher density ofcentral FAs in liprin-ΔSAM2-transfected cells (Fig. 4H). These dataindicate the uncoupling of the liprin-mediated reorganization of the

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Fig. 3. Talin is required for liprin-enhanced spreading. (A) Coexpression ofliprin with talin-1 or talin-1H prevents liprin-enhanced spreading. Staining fortransfected proteins (green/blue), active β1 integrins (9EG7, red), total β1integrins and F-actin. Two upper rows show corresponding fields with mergedimages (upper) or active β1-integrin staining only. Asterisks in lower rowsindicate single- or double-transfected cells. (B) Quantification of spreading(n=35 cells per experimental condition). (C) Fraction of projected cell areaoccupied by active β1-integrin-positive FAs and clusters (n=12 cells perexperimental condition). (D) Percentage of FA area at the cell edge (n=12 cellsper experimental condition). Bars are mean values ± s.e.m. P-values (shownabove the bars) were calculated using Student’s t-test. (E) COS7 cells expressmainly talin-1. Lysate (50 μg) from P7 mouse brain or COS7 cells wasimmunoblotted for talin or talin-2. (F) Immunoblots on lysates of control cells(Luc) and cells depleted of talin-1 (talin-1a): talin-1 depletion was 69±2.7%s.d., n=3. (G) Downregulation of talin expression by siRNA affected spreadingon FN (1 hour) in control (GFP + siRNA Talin) and liprin-transfected (liprin-α1 + siRNA Talin) cells when compared with the respective controls (GFP +siRNA Luc, and Liprin-α1+siRNA Luc). (H) Quantification of spreading. Barsare means ± s.e.m. (n=30 cells per condition). Scale bars: 20 μm.

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Fig. 3. See previous page for legend

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cell edge from the LAR-mediated effects. Liprin-α1 and LARcooperate in cell spreading, and two distinct activities can beidentified in this process: on one hand the binding of LAR to liprinis needed for cell spreading, but not for the ability of liprin toenhance either the formation of FAs or integrin activation at theperiphery of spreading cells. In this way, liprin can enhance theactivation of peripheral integrins independently of its binding to

LAR. On the other hand, liprin requires the interaction with LARto induce the actin-mediated protrusive events necessary to movethe lamellipodia forwards.

ConclusionsCell motility requires the dynamic regulation of actin and FAturnover at the protruding edge of the cell (Vicente-Manzanares et

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Fig. 4. Binding of liprin to LAR is needed for liprin-induced spreading but not for the increased number of FAs at the cell edge. (A) LAR depletion by RNAinterference. (B) Inhibition of spreading on FN by depletion of liprin and LAR. Bars are means ± s.e.m. (50 cells/condition). (C) Inhibition of liprin-inducedspreading by LAR siRNA in control cells and in cells overexpressing liprin. Bars are means ± s.e.m. (50 cells/condition). (D) LAR depletion (GFP-positive cells)affects spreading. (E) LAR depletion does not prevent liprin-induced potentiation of FAs at the cell edge. (F) Lack of interaction between liprin-ΔSAM2 and LAR.Immunoprecipitates of FLAG-liprin or FLAG–liprin-ΔSAM2 (200μg lysate) blotted for LAR and liprin. (G) Binding of liprin to LAR was required for liprin-induced spreading. Transfection of liprin-ΔSAM2 prevented enhanced spreading on FN. Bars are means ± s.e.m. (50 cells/condition). (H) Increased FAs at the edgeof liprin- and liprin-ΔSAM2-transfected cells (green). (I) Percentage of spreading cells with evident increase in FA density at the edge (n=48 fields, 40-50μm perfield, from 24 cells/condition). P<0.01 (ΔSAM2 vs βGal), and P<0.005 (liprin vs βGal) by the χ2 test. (J) Percentage of the total projected cell area occupied by FAs(average ± s.e.m.; n=24 cells/condition). (K) Hypothetical model for liprin-α function at the cell edge of a moving cell. See text for explanation. Scale bars: 20μm.

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al., 2009). We have shown here that changes in the cellular levelsof liprin-α1 have profound effects on the morphology andorganization of cells spreading on ECM. Our data indicate reciprocaleffects of talin and liprin on each other in the control of cellspreading, and suggest that a balance between the cellular levelsof these proteins is required for proper dynamics at the cell edgeduring cell motility. The positive role of liprin-α1 in protrusion wasreflected by the increase of lamellipodia and FAs at the periphery(Fig. 2). The concentration of liprin near the cell edge and its poorcolocalization with activated peripheral β1 integrins suggest a roleof liprin in the dynamic turnover of FAs during motility, which issupported by the dynamic analysis presented in this study of theeffects of liprin expression on FAs.

We propose a model in which ubiquitous liprin-α1 acts as adynamic regulator of protrusion by impinging on the mechanismof talin-mediated integrin activation (Fig. 4K). The effects onprotrusion observed by changing the cellular levels of liprin-αwould require the specific recruitment of liprin near the cell edgethrough unknown molecular interactions. This mechanism, byrecruiting liprin at the centripetal side of newly formed FAs, wouldsomehow enhance their turnover, and result in a more efficientprotrusive activity at the cell edge (supplementary material Movies1 and 2). The action of liprin-α on cell-edge dynamics cannot besimply explained by an effect of liprin on integrin inactivationand/or FA formation, because this would result in the inhibition ofprotrusion rather than in its potentiation in cells overexpressingliprin. By contrast, a simple potentiation of FA formation by liprinwould result in FAs with a longer life, which is opposite to whatwe found in cells overexpressing liprin. In fact, liprinoverexpression actually enhanced both the formation of newlyformed FAs accumulating at the cell periphery and increased theirturnover, whereas liprin-α1 depletion inhibited both FA formationand cell spreading. We therefore propose that liprin-α plays acomplex role in the dynamic events occurring at the edge of amoving cell. We expect that liprin functions at the cell edge aremediated by a molecular network that is assembled by a multi-domain adaptor protein such as liprin-α. We have shown that LARis probably part of this network, but it is not sufficient for the fullaction of liprin in cell motility. Future work will be aimed atidentifying the other players linking liprin-α to talin-mediatedintegrin activation and lamellipodia formation.

The steady association of liprin to the plasma membrane suggeststhat liprin interacts with as-yet-unidentified components of theplasma membrane. Because liprin localization at the cell edge isindependent from interaction with LAR, we propose that liprin, bybinding to both the plasma membrane and LAR, affects protrusiveactivity by recruiting the molecular machinery needed to regulateefficient FA turnover and actin dynamics. Therefore, liprin is animportant regulator of cell-edge dynamics, where balanced levelsof expression of liprin and talin are important for productiveprotrusion during motility.

Materials and MethodsAntibodiesMonoclonal antibodies (mAbs) for FLAG, α-actinin, talin, tubulin (Sigma-Aldrich);vinculin (Upstate); paxillin, phosphotyrosine, LAR 150- and 200-kDa forms (BDBiosciences); TS2/16 (Hemler et al., 1984) (American Type Culture Collection) and9EG7 (Lenter et al., 1993) (BD Biosciences) recognizing total and activated humanβ1 integrin, respectively, were used. Polyclonal antibodies against the cytoplasmicdomain of β1 integrin (Tomaselli et al., 1988); FLAG and actin (Sigma-Aldrich);FAK and LAR 85-kDa P subunit (Santa Cruz Biotechnology); GFP (Molecular Probes)were used. The anti-liprin-α1 rabbit polyclonal antibody raised to a GST-fusion proteinincluding the C-terminal human liprin-α1 (amino acids 818-1202) was affinity purified

on a maltose-liprin(818-1202) fusion protein adsorbed to amylose resin affinity matrix(New England Biolabs).

Constructs and transfectionsGFP–liprin-α1 and FLAG–liprin-ΔSAM2 plasmids were obtained from theFLAG–liprin-α1 plasmid. Human GFP–talin-1 and mouse GFP–talin-1H (amino acids1-433) were from Anna Huttenlocher (University of Wisconsin-Madison, WI). ThepSP65-SRα2-HPTPδ and pSP65-SRα2-LAR plasmids (Pulido et al., 1995) were fromRobert Kypta (Imperial College, London). The GFP-paxillin plasmid was from VictorSmall (Austrian Academy of Sciences, Vienna, Austria).

siRNAs used for silencing (from Sigma or Qiagen) were: siRNA liprin-1a(targeting 5�-TTCCAAGGTACAAACTCTTAA-3�) and liprin-1b (targeting 5�-CACGAGGTTGGTCATGAAAGA-3�); LAR siRNA (HS-PTRF_6 HP validated,from Qiagen); and talin-1a (targeting 5�-AATCGTGAGGGTACTGAAACT-3�)(Manevich et al., 2007), talin-1b (targeting 5�-CAGCTCGAGATGGCAAGCTTA-3�) and talin-1c siRNAs (targeting 5�-CCGCATTGGCATCACCAATCA-3�). ControlsiRNA targeting the luciferase sequence 5�-CATCACGTACGCGGAATAC-3� wasused. Cells transfected with Lipofectamine 2000 (Invitrogen) and 2-3 μg of plasmids,or siRNAs (50-100 nM) were used after 1-2 days as specified.

Immunoprecipitation, western blotting, pulldowns and proteindeterminationCells lysed with 0.5-1% Triton X-100, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1mM sodium orthovanade, 10 mM sodium fluoride, and protease inhibitors wereimmunoprecipitated from equal amounts of lysate using antibodies coupled to proteinA Sepharose (Amersham). For immunoblotting, primary antibodies were visualizedby ECL or 125I-anti-mouse Ig or protein A (Amersham). For evaluation of Rac-GTP,transfected COS7 cells were lysed (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 10%glycerol, 2 mM MgCl2, 1% NP-40) 30 minutes after plating on FN, and cleared lysateswere incubated with GST-PAK1-CRIB-adsorbed glutathione Agarose beads for 30minutes at 4°C. Washed beads were used for immunoblotting with anti-Rac1 mAb(Upstate).

Preparation of VPMsVPMs were prepared from chicken embryo fibroblasts (CEFs) or COS7 cells on 10μg ml–1 FN, as described (Cattelino et al., 1999). Briefly, VPMs were prepared witha jet of ice-cold buffer with anti-proteases and anti-phosphatases (20 mM HEPES-NaOH pH 7.6, 0.3 mM PMSF, 10 mM NaF, 1 mM NaV), and used forimmunofluorescence. Triton-soluble and -insoluble fractions from VPMs were usedfor immunoblotting.

Spreading and adhesion assaysCOS7 cells were trypsinized 1-2 days after transfection. A total of 25,000-30,000cells were plated on each 13-mm-diameter coverslip coated with 10 μg ml–1 FN.Cells were fixed after 1 hour and processed for immunofluorescence. Images wereanalyzed with ImageJ. Cell-adhesion assays were as described (Cattelino et al.,1995).

Morphological analysisCells and VPMs were incubated with the indicated antibodies after fixation. For9EG7 and TS2/16 antibodies recognizing extracellular epitopes of the β1 integrin,cells were incubated 15 minutes on ice with 5 μg ml–1 of purified IgG beforefixation. F-actin was revealed by FITC- or TRITC-conjugated phalloidin. Cellswere observed with Axiophot or Axiovert microscopes (Zeiss), or confocalmicroscopes (PerkinElmer and Leica Microsystems SpA). Images were processedusing Photoshop (Adobe) and analyzed with ImageJ. The analysis of the fractionof the projected cell area occupied by active β1 integrins was performed onthresholded images, by measuring the total area occupied by FAs and/or integrinclusters larger than 0.5 μm2. The values obtained were represented as percentagesof the total projected cell area on the substrate. The percentage of cell perimeterpositive for FAs was calculated from different perimetral fields by measuring thefluorescence intensity of 9EG7-positive integrin clusters. Data in the bar graphsare expressed as mean ± s.e.m. of one of at least two or three repetitions in which70-150 cells per experimental condition were analyzed. P-values were calculatedby Student’s t-test (two-tailed distribution, two-sample unequal variance). Livecells were observed with an UltraVIEWERS microscope (PerkinElmer).Measurements of FA lifespan during cell spreading on FN were made by countingthe amount of time lapsed between the first and last frame in which an individualadhesion could be observed. Adhesion formation was evaluated by counting thenumber of new GFP-paxillin-positive FAs appearing during 30 minutes in cellsspreading on FN.

We thank John Collard for the pGEX-PAK1-CRIB construct, PietroDe Camilli for the anti-talin-2 antibody, Anna Huttenlocher for the talinplasmids, Robert Kypta for the LAR plasmids and Victor Small forpEGFP-paxillin. We also thank Jacopo Meldolesi and Flavia Valtortafor critical reading of the manuscript, and Marzia De Marni, Mario

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Faretta (IFOM) and Cesare Covino (Alembic) for technical support.This work was supported by Telethon-Italy (grant GGP05051), AIRC(Italian Asssociation for Cancer Research, grant no. 5060) andFondazione Cariplo.

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