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Address correspondence to Kris DeMali, Lineberger Comprehensive Cancer Center, CB #7295, University of North Carolina, Chapel Hill, NC 27599. Tel.: (919) 966-1904. Fax: (919) 966-1856. E-mail: kdemali{at}med.unc.edu
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Abstract |
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Key Words: vinculin; Arp2/3 complex; integrins; adhesion; migration
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Introduction |
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The ability to extend protrusions is driven by actin polymerization. Proteins that regulate the assembly and disassembly of actin have been localized to the leading edge of migrating cells (for review see Borisy and Svitkina, 2000; Cooper and Schafer, 2000; Pollard et al., 2000; Condeelis, 2001). A key component regulating nucleation of polymerization is the Arp2/3 complex, which is comprised of seven subunits, actin-related protein (Arp)*2, Arp3, p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc in mammalian cells (Welch et al., 1997). The Arp2/3 complex becomes activated downstream from Cdc42 and Rac1. GTPases that induce filopodial and lamellipodial extension, respectively. Rac1 activates the Arp2/3 complex via the Scar/WAVE proteins (Machesky and Insall, 1998; Miki et al., 1998), whereas Cdc42 binds and activates Wiskott-Aldrich Syndrome protein (WASP) or N-WASP via a conserved acidic domain (Rohatgi et al., 1999). Activated WASP binds and induces a conformational change in the Arp2/3 complex that permits the Arp2 and Arp3 subunits of the complex to form the template for the daughter filament (Robinson et al., 2001). Nucleation of actin polymerization in this fashion triggers the addition of actin monomers close to the membrane. In addition, the Arp2/3 complex also binds to the sides of existing filaments (Svitkina and Borisy, 1999). This binding to F-actin activates the Arp2/3 complex in a manner cooperative with the activation by the WASP/Scar family of proteins (Higgs and Pollard, 2001). Nucleation of existing filaments in this manner forms a highly branched, dendritic network (Mullins et al., 1998; Svitkina and Borisy, 1999; Blanchoin et al., 2000; Pantaloni et al., 2000; Bailly et al., 2001), and this branching has been shown to be essential for lamellipodial extension (Bailly et al., 2001). The continued addition of actin monomers in this branched network is thought to provide the driving force for the extension of membrane that occurs during lamellipodial protrusion.
Another group of proteins localized and activated in extending lamellipodia are the integrins, cell surface receptors that serve as transmembrane links between the extracellular matrix on the outside and cytoskeleton on the inside of the cell (Nishizaka et al., 2000; Kiosses et al., 2001). The integrins bind a number of cytoskeletal proteins, including talin, -actinin, and filamin, all of which bind F-actin (Hemler, 1998; for review see Critchley, 2000; Liu et al., 2000; Zamir and Geiger, 2001a,b). Additionally, both talin and
-actinin bind vinculin, another actin-binding protein (Jockusch and Isenberg, 1981; Johnson and Craig, 1995a). Together, these observations have led to the idea that multiple protein complexes mediate attachment of integrins to actin filaments.
In order for cells to migrate effectively, it is important that protrusive activity is linked to adhesion. Potential mechanisms by which this may occur are via integrin engagement activating Rac1 (Clark et al., 1998; Price et al., 1998; Del Pozo et al., 2000) or recruiting activated Rac1 to these sites (Del Pozo et al., 2002). The local activation of Rac1 at the leading edge of cells (Kraynov et al., 2000) is thought to stimulate the Arp2/3 complex in this region, thereby triggering actin polymerization and growth of the dendritic network of actin filaments in the lamellipodium. It seemed to us that besides this signaling pathway linking adhesion to actin polymerization, there may also exist a structural link between integrin engagement and actin polymerization, so that newly engaged integrins might couple to the Arp2/3 complex. Here, we have identified an interaction between vinculin and the Arp2/3 complex that occurs transiently in response to signals that trigger membrane protrusion. This interaction is regulated by phosphatidylinositol-3-kinase (PI3K) and Rac1 activity, and is sufficient to recruit the Arp2/3 complex to new sites of integrin clustering.
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Results |
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To explore the interaction of vinculin with Arp2/3, we examined their coimmunoprecipitation from cultured fibroblasts. Only low levels of association were detected (unpublished data). To examine whether the interaction might be stimulated when cells are induced to extend membrane ruffles and lamellipodia, we looked for coimmunoprecipitation from A431 cells, which form large membrane ruffles when stimulated with EGF (Brunk et al., 1976; Diakonova et al., 1995). EGF induced a transient association of the Arp2/3 complex with vinculin. This peaked within 30 s and was diminished by 1520 min, depending on the experiment (Fig. 1 A). The time at which the Arp2/3 complex was bound to vinculin is similar to the time frame for induction of ruffles by EGF in A431 cells (unpublished data). In addition, recruitment of the Arp2/3 complex to vinculin did not correlate with actin binding, which was constant with it being independent of actin binding. This interaction was specific for vinculin, because talin, another actin binding protein, was unable to recruit the Arp2/3 complex in response to EGF stimulation (Fig. 1 B). Membrane protrusion is also stimulated by cell spreading on extracellular matrix (ECM). Using Hs68 cells, we investigated the association of the Arp2/3 complex with vinculin that was immunoprecipitated from stationary cells or from those spreading on a fibronectin (FN) matrix. For this experiment, adhesion and spreading were accelerated by centrifuging the cells onto FN-coated coverslips. A low amount of the Arp2/3 complex was detected associating with vinculin in stationary cells, and this was greatly increased as cells spread on FN (Fig. 1 C). Similar to the EGF-treated cells, the interaction was transient and over by 1520 min, at which time spreading was largely complete.
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The Arp2/3 complex binds directly to vinculin amino acids 850881
To explore the role of this interaction between the Arp2/3 complex and vinculin, we considered generating a mutant form of vinculin that could not bind the Arp2/3 complex. This would allow us to examine the phenotype of this vinculin when expressed in vinculin-null cells. We mapped the Arp2/3 binding site on vinculin using vinculin fragments expressed as GST fusion proteins (Fig. 6 A). Several overlapping fragments bound to the Arp2/3 complex and identified amino acids 850881 as the critical domain (Fig. 6 B). Because these experiments were carried out using crude platelet extracts, we determined whether the association of Arp2/3 complex depended on the ability of vinculin to recruit actin. We examined whether the Arp2/3 complex was retrieved from platelet extracts using a vinculin fusion protein containing an intact actin binding site (Vin 8811066), but lacking an Arp2/3 binding site. The converse experiment was also performed with a vinculin fusion protein containing an intact Arp2/3 binding site (Vin 811881), but lacking intact actin binding site. Fusion proteins that bound actin were not able to recruit the Arp2/3 complex and vice versa (Fig. 6 C). We conclude that actin binding to vinculin is not required for binding of the Arp2/3 complex.
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To ensure that the mutation was capable of interferring with binding of the Arp2/3 complex in the context of full length vinculin, we subcloned the mutation into full length vinculin and assessed its ability to recruit the Arp2/3 complex in vinculin immunoprecipitates. In response to FBS stimulation, the mutant was unable to recruit the Arp2/3 complex to wildtype levels (Fig. 6 E). This impaired binding was not due to differences in the levels of expression as both the WT and mutant vinculin were expressed at similar levels (unpublished data).
We next examined whether the mutant could carry out functions normally ascribed to vinculin. We examined whether the mutant vinculin was able to localize to and concentrate in focal adhesions when expressed in the vinculin-null cells. We found a normal distribution of vinculin in focal adhesions in cells reexpressing the WT or mutant vinculin (Fig. 8). No vinculin staining was detected in the vinculin null cells transfected with the vector alone.
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Because vinculin has also been implicated in cell adhesion and migration, we examined if the mutant vinculin was impaired in its ability to regulate these events. We assessed the ability of the vinculin-null, WT, or mutant reexpressing cells to adhere to FN. We observed that cells reexpressing both the WTand mutant vinculin adhered to similar extents, which were 1.9- and 1.7-fold more than the vinculin-null cells, respectively (Fig. 9 A). Migration assays were performed using Transwell filters in which the lower surface of the filter was coated with FN. Migration was allowed to proceed for 6.0 h. Consistent with previous findings, we found that the vinculin null cells migrated faster than the cells reexpressing WT vinculin (Xu et al., 1998b). Furthermore, the mutant vinculin lacking an intact Arp2/3 complex binding site was indistinguishable from WT cells. Thus, ablation of the binding site for the Arp2/3 complex on vinculin had no effect on cell adhesion or migration through a Transwell filter, although it did affect development of lamellipodia and cell spreading.
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Discussion |
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At the outset of this work, we were motivated to look for proteins that might directly couple matrix adhesion to nucleation of actin polymerization. We have found here a transient interaction of the Arp2/3 complex with vinculin that is stimulated by matrix adhesion and by growth factor treatment. The interaction of the Arp2/3 complex with vinculin only occurs at sites of newly engaged integrins and is not detected in mature adhesive structures, such as focal adhesions. Together these observations lead us to suggest that this interaction is one way that the actin nucleating machinery that drives membrane extension can be coupled to new sites of cellECM adhesion. Further evidence supporting this idea comes from our studies of vinculin-null cells reexpressing either WT vinculin or a mutant form of vinculin that does not bind Arp2/3. Whereas the WT vinculin restores normal formation of lamellipodia, the mutant unable to bind Arp2/3 does not (see below).
Vinculin is a prominent component of focal adhesions and it is notable that the Arp2/3 complex has not been detected in these structures either in previous localization studies (Welch et al., 1997) or in this study using a p34GFP chimera. Many vinculin-binding proteins have been identified, and so far the Arp2/3 complex is unusual among them in not colocalizing with vinculin in focal adhesions. The Arp2/3 complex is prominent in the leading edge of cells, where vinculin is also found, but much less prominently than its localization in focal adhesions. This implies that most of the vinculin in the cell is not associated with the Arp2/3 complex, which is what we have found biochemically. In quiescent, stationary cells, the amount of Arp2/3 coprecipitating with vinculin is very small and often difficult to detect. Indeed, we would probably have missed this association if we had not first observed it occurring in crude platelet fractions. With hindsight, we suspect that some of the platelets used to prepare these fractions had become activated thereby stimulating this interaction between vinculin and the Arp2/3 complex.
The transient nature of the interaction of the Arp2/3 complex with vinculin is striking. We observed that it is induced by both adhesion to ECM and by stimulating with EGF. Pursuing the pathways leading to this association, we determined that it was blocked by agents that inhibit PI3K or Rac1 activity. The interaction of the Arp2/3 complex with vinculin could be triggered by activated Rac1. In many systems, PI3K is upstream of Rac1 activation and Rac1 has been shown to stimulate the Arp2/3 complex via the WASP/Scar family of proteins (Aspenstrom et al., 1996; Symons et al., 1996; Miki et al., 1998). Upon activation, the WASP/Scar proteins undergo a conformational change exposing the COOH-terminal VCA domain that binds and activates the Arp2/3 complex. Thus, one way that Rac1 might promote binding of the Arp2/3 complex to vinculin would be through activation of the Arp2/3 complex. We tested this possibility by adding the recombinant VCA domain to cell lysates before immunoprecipitating vinculin. We found that this significantly increased the amount of Arp2/3 binding to vinculin, confirming that the interaction required activated Arp2/3 complex. However, we also found that the binding could be stimulated even further by addition of PIP2 to the lysates. PIP2 binds to vinculin (Johnson and Craig, 1995b), inducing a conformational change that dissociates the head domain from the tail domain (Johnson and Craig, 1995b; Gilmore and Burridge, 1996; Weekes et al., 1996; Steimle et al., 1999). This exposes binding sites on vinculin for several of its binding partners. Together, these results suggest that the interaction of the Arp2/3 complex with vinculin requires both activation of Arp2/3 and the open vinculin conformation.
We were curious whether the interaction of vinculin with Arp2/3 stimulated its actin nucleating activity. However, using actin polymerization assays, we found no evidence that vinculin is able to induce nucleation via the Arp2/3 complex. Furthermore, vinculin does not bind the Arp2/3 complex via an acidic domain like other Arp2/3 activators. We conclude that the interaction serves to localize the activated Arp2/3 complex to sites of new integrin engagement. The transient nature of the interaction also raises the question of how this association is turned off. One possibility is that the Arp2/3 complex is displaced by another protein that binds to vinculin in the same region. Several proteins (VASP, vinexin, and ponsin) have been identified that, like the Arp2/3 complex bind to the hinge region of vinculin (Huttelmaier et al., 1998; Kioka et al., 1999; Mandai et al., 1999). Currently, we know little about the function of ponsin and vinexin and even less about how their interaction with vinculin may be regulated. In future work, we hope to explore whether any of these proteins displace the Arp2/3 complex from vinculin and thus confine the interaction to the newest sites of adhesion. Presumably, whatever mechanism dissociates the Arp2/3 complex from vinculin also prevents recruitment of the Arp2/3 complex to focal adhesions.
Vinculin-null cells have provided clues to the function of vinculin in cells. Although the vinculin knockout mice are embryonic lethal (Xu et al., 1998a), the fibroblasts from these mice are surprisingly normal. Interestingly, the vinculin-null cells are less adherent to ECM and show reduced rates of spreading when plated on ECM-coated surfaces (Coll et al., 1995; Xu et al., 1998a, b). Contrary to many expectations, these cells can develop focal adhesions and are able to migrate. Indeed, migration is enhanced relative to cells expressing vinculin (Coll et al., 1995; Xu et al., 1998a,b). Presumably, this reflects that the migration rate of these cells is limited by the strength of their adhesions. To explore specifically the role of the interaction of the Arp2/3 complex with vinculin, we generated a point mutation in the binding site on vinculin for Arp2/3. This mutant vinculin was still able to bind to the other proteins that also bind to the hinge region of vinculin, indicating that the mutation was not disrupting vinculin's conformation and affecting multiple interactions. The mutant vinculin targeted to focal adhesions and restored the adhesive properties of the cells to the same extent as the WT vinculin. Whereas reexpression of WT vinculin restored cell spreading and lamellipodia formation to normal levels, the mutant vinculin did not. In these characteristics, the cells resembled the null cells. As noted above, these observations support the idea that the binding of the Arp2/3 complex to vinculin functions to promote the extension of lamellipodia and cell spreading.
We were interested in comparing the mutant and WT vinculin-expressing cells with respect to migration. Our initial expectation was that because the mutant vinculin-expressing cells showed diminished extension of lamellipodia, that these cells would migrate more slowly than the cells expressing WT vinculin. However, we found little difference in the rate of migration of the two cell types and that both migrated more slowly than the null cells. The cells reexpressing WT or mutant vinculin have increased adhesion to the substratum and we conclude that this retards their rate of migration. This result suggests that the rate limiting step in these cells is the release of adhesions rather than protrusion at the leading edge.
It is clear that the vinculin-null MEFs can generate lamellipodial protrusions, albeit less well than cells in which WT vinculin has been expressed. However, there are vinculin-deficient F9 embryonic carcinoma cells that have also been studied. Like the MEFs devoid of vinculin, these cells also reveal reduced adhesion and spreading on FN. In contrast to the vinculin-null MEFs, these F9 cells lacking vinculin were unable to develop lamellipodia (Coll et al., 1995), but continued to develop filopodia. Recent work from Goldmann and Ingber has found that these F9 cells deficient in vinculin fail to develop lamellipodia in response to Rac activation (Goldmann and Ingber, 2002). This is restored by reexpression of WT vinculin, leading to the conclusion that vinculin is critical in lamellipodial extension. However, because MEFs lacking vinculin can generate lamellipodia, we conclude that multiple mechanisms are involved. For some cells (e.g., the F9 cells) vinculin appears to be critical for this activity, whereas for others, such as the fibroblasts used here, the coupling of Arp2/3 to vinculin provides just one of possibly several mechanisms that contribute to lamellipodial extension.
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Materials and methods |
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Constructs
The GSTvinculin fusion proteins Vin 1399, 399881, 8811066 were provided by D. Critchley (University of Leicester, Leicester, UK). All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GSTP876A and GSTP878A, site-specific mutagenesis was carried out using the GSTVIN 811881 template according to the Quickchange Manual (Stratagene). These mutants were subcloned into full-length vinculin in pCMV-myc (CLONTECH Laboratories, Inc.) with a modified multiple cloning site. pGEX-VCA domain was a gift from H-Y. H. Ho and R. Rohatgi (Harvard Medical School, Boston, MA). The GFP fusion protein, p34GFP, was constructed by RT-PCR amplification a Sal-BamH1 p34-Arc subunit of the Arp2/3 complex cDNA from mRNA obtained from HeLa cells and subcloning the resulting DNA into pEGFP-N3 (CLONTECH Laboratories, Inc.).
Reagents and antibodies
Wortmannin (Sigma-Aldrich) and LY294002 (Alexis) were resuspended according to the manufacturer's specifications. Human FN was prepared (Ruoslahti et al., 1982) or purchased from GIBCO-BRL. PIP2 was a gift from Andrew Morris (University of North Carolina, Chapel Hill, NC). The GSTVCA protein was prepared as described (Rohatgi et al., 2000). The Arp2/3 complex for the actin polymerization studies was purified as described (Welch and Mitchison, 1998). The Arp2/3 complex used in the binding studies was provided by D. Yaarar (University of California, Berkeley, CA). The Arp2/3 complex was blotted using a rabbit antibody raised against a peptide that included amino acids 179204 of the p34-Arc subunit of the Arp2/3 complex. Vinculin was immunoprecipitated and blotted using a rabbit antibody raised against purified chicken gizzard vinculin. Antibodies against VASP and Rac1 were obtained from Transduction Labs, against HLA from Pharmingen, and against integrins (TS2/16) from American Type Culture Collection. Affinity-purified, anti-vinexin antibody was a gift from N. Kioka (Kyoto University, Kyoto, Japan). The ponsin antibody (Mandai et al., 1999) was a gift from Y. Takai (Osaka University School of Medicine, Osaka, Japan).
Bead experiments were carried out as previously described with minor modifications (Miyamoto et al., 1995). Latex beads (3.0 µm) were coated with 50 µg/mL human plasma FN or 100 µg/mL of poly-lysine for 1.0 h at 37°C and then blocked using 10 mg/mL BSA. HeLa or Vin-/- cells transiently expressing p34GFP or GFP were plated on collagen-coated coverslips for 1.0 h at 37°C, washed in PBS, and 1.0 x 106 beads were incubated with the coverslips for the times indicated. The coverslips were fixed, permeabilized, and stained with antibodies against vinculin, ß1-integrins or HLA, followed by a Texas redconjugated secondary antibody. For each experiment, 150 beads were scored for the presence or absence of fluorescence around the beads. Values are number of positive beads ± SEM from at least three independent experiments.
Rac1 activity
To measure Rac1 activity, A431 cells were left resting or stimulated with EGF at specified time points, cells were washed, lysed, and active GTP-bound Rac1 was precipitated using GST-PBD beads, as described (Bagrodia et al., 1998). Densitometric analysis of films was performed as described (Liu and Burridge, 2000).
Immunoprecipitation and Western blot analysis
For the EGF stimulation experiments, subconfluent A431 cells were starved for 24 h in DME + 0.1% FBS and then stimulated with human EGF in 2 mM acetic acid, 10 mg/mL BSA at a concentration of 100 ng/mL. Control cells were treated with an equivalent amount of 2 mM acetic acid, 10 mg/mL BSA. For the cell spreading on FN experiments, cells were trypsinized, washed three times in DME, and plated onto FN-coated coverslips submerged in a 6-well plate at a density of 1.33 x 105 cells. The plates were centrifuged at 300 g for 5 min and allowed to spread for the times indicated at 37°C. Subsequent to stimulation of cells by EGF or spreading on FN, the cells were washed in HBS (20 mM HEPES, pH 7.4, 150 mM NaCl + 2 mM Na3VO4) and lysed in ice-cold EB (1% Triton X-100, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 0.1% BSA, 20 µg/mL aprotinin, 1 mM PMSF, 2 mM Na3VO4). For the experiments addressing whether PIP2 and/or GSTVCA can induce binding of the Arp2/3 complex to vinculin, the Triton X-100 cleared lysates were incubated with 10 mM PIP2 and/ or 30 µg GSTVCA. Vinculin was immunoprecipitated with a polyclonal antibody raised against chicken gizzard vinculin, the immunoprecipitates were washed four times in ice-cold EB, fractionated by SDS-PAGE, transferred to PVDF membranes, and subjected to Western blot analysis. 20% of the immunoprecipitate was analyzed for vinculin recovered, 10% was used for the actin blots, and between 70 and 80% was analyzed for p34-Arc coprecipitation. The blots were developed with Western blot detection reagents (ECL; Amersham Biosciences), and the signal was detected on x-ray film.
Migration assays
Migration assays were performed using Transwell cell culture chambers containing polycarbonate membrane inserts with 8.0 µm pores (Becton-Dickinson) as previously described (Xu et al., 1998a). The filters were processed for immunofluorescence as described above and stained with DAPI (Sigma-Aldrich) to stain nuclei.
Adhesion assays
Cells were plated on coverslips coated with 50 µg/mL of human FN, allowed to adhere for 45 min at 37 °C, rinsed three times with PBS, and then processed for immunofluorescence and stained with DAPI (Sigma-Aldrich).
Immunofluorescence
Cells were fixed, permeabilized, and washed as described (Liu and Burridge, 2000). Cells were incubated with primary antibodies for 1.0 h at room temperature, washed, and incubated with secondary antibody for 1.0 h at room temperature. Vinculin and myc monoclonal antibodies (Sigma-Aldrich) were used at 1:50 and 1:1,000, respectively. Alexa fluoro 594-conjugated phalloidin (Molecular Probes) was used to visualize actin at 1:200. Images were obtained as described (Liu and Burridge, 2000).
Spreading assays
Cells were seeded onto coverslips coated with 50 µg/mL of human FN and incubated for 45 or 120 min at 37°C. The cells were then processed for immunofluorescence.
Microinjection
Cells were cultured and microinjected as previously described (Nobes and Hall, 1995).
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Footnotes |
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Acknowledgments |
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This work is supported by National Institutes of Health grants #GM29860 and HL45100, and postdoctoral fellowship GM20610.
Submitted: 10 June 2002
Revised: 24 October 2002
Accepted: 25 October 2002
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References |
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