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Address correspondence to René-Marc Mège, INSERM U440, 17 rue du Fer à Moulin, 75005 Paris, France. Tel.: 33-1-45-87-61-36. Fax: 33-1-45-87-61-32. E-mail: mege{at}ifm.inserm.fr
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Abstract |
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Key Words: cell adhesion; cytoskeleton; signal transduction; migration; mechanotransduction
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Introduction |
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Cadherins are transmembrane glycoproteins that mediate cellcell adhesion through homophilic Ca2+-dependent interactions of their extracellular region and anchoring of their intracellular domain to the actin cytoskeleton (Yap et al., 1997). Cadherin ectodomains are thought to dimerize and to interact with dimers of the same cadherin species at the surface of adjacent cells (Shapiro et al., 1995; Pertz et al., 1999). On the other hand, the conserved cytoplasmic domain of cadherins is part of a multimolecular complex including the p120 phosphoprotein and catenins and ß, which link cadherins to the actin cytoskeleton and have a modulatory effect on cadherin adhesive function (Kemler, 1993; Anastasiadis and Reynolds, 2000). The integrity of this cadherincatenin complex and its correct association to the actin cytoskeleton are required for cell aggregation (Nagafuchi and Takeichi, 1988).
Although the developmental roles of cadherins are well documented, their mode of action is still a matter of intense investigation. In addition to the increasing understanding of the molecular mechanisms underlying specific adhesive interactions, other aspects (i.e., the dynamics and mechanisms of cell contact formation and the nature of the mechanochemical signals transduced in response to contact formation) remain largely unknown. Recently, we developed an approach allowing us to mimic and control cadherin function in the absence of actual cellcell contact (Lambert et al., 2000). We produced a fusion protein containing the N-cadherin ectodomain fused to the IgG Fc fragment (Ncad-Fc), which retains the homophilic Ca2+-dependent adhesive properties of native N-cadherin. Ncad-Fccoated beads specifically bind to N-cadherinexpressing cells, fully mimicking the formation of cellcell contacts. The bead binding induces the recruitment of preexisting cell membrane cadherincatenin complexes, and triggers the recruitment of tyrosine-phosphorylated proteins and the redistribution of actin filaments. These results support a model in which the homophilic adhesion of cadherin ectodomains induces the transduction of mechanochemical signals toward the intracellular signaling apparatus and the actin cytoskeleton. The signaling toward actin filaments might be of major importance, not only for the strengthening of cellcell contacts (Adams and Nelson, 1998; Vasioukhin et al., 2000), but also for the coupling of cadherin-based adhesion to the force-generating moving actin cytoskeleton. Although this question has been extensively studied for extracellular matrix adhesion receptors of the integrin family (Miyamoto et al., 1995a; Lauffenburger and Horwitz, 1996; Choquet et al., 1997), little is known in the case of cadherins.
Because of its importance in providing the driving force for cell sorting, cell migration, and growth cone navigation, we focused on the dynamics of functional anchoring of cadherins to the actin cytoskeleton, thus allowing the transduction of mechanical forces across the cell membrane. We combined our cellular model with a biophysical approach, enabling us to monitor the bidimensional movement of single or small clusters of proteins and to determine their level of anchoring to the cytoskeleton (Kusumi et al., 1993; Simson et al., 1995). Indeed, the movement of ligand-coated microparticles bound to membrane receptors can be monitored with nanometer precision by video microscopy and single-particle tracking (Sterba and Sheetz, 1998). So far it has been possible to follow (Kusumi et al., 1999) the anchoring of integrins or E-cadherin to the cytoskeleton (Choquet et al., 1997; Sako et al., 1998; Felsenfeld et al., 1999; Nishizaka et al., 2000). In the present work, N-cadherin molecules were triggered at the surface of C2 myogenic cells with Ncad-Fccoated beads. Optical tweezers were used to force the contact of the beads with different domains of the cell membrane. The dynamics of N-cadherin anchoring to the cytoskeleton were analyzed in the very first seconds after beadcell contact. We observed that N-cadherin receptors were initially free to undergo Brownian diffusion, and then became tightly anchored to the actin cytoskeleton. Ligand dose effect analysis, pharmacological perturbations, and cell transfection were used to approach the molecular mechanisms controlling this anchoring. Altogether, our results show for the first time the dynamics and molecular mechanisms leading to the anchoring of cadherins to the actin cytoskeleton, uncovering novel aspects of the mode of action of these adhesion receptors.
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Results |
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The movement of Ncad-Fc beads presents an initial freely diffusive phase
We postulated that the fast anchoring of Ncad-Fc or antiN-cadherin beads may result from a massive mobilization of N-cadherin at the beadcell contact. Hence, we hypothesized that the kinetics and/or extent of anchoring may directly depend on Ncad-Fc ligand density at the bead surface. Thus, beads were prepared with decreasing densities of Ncad-Fc corresponding to 30% (medium), 10% (low), and 1% of their maximal loading. Medium and low Ncad-Fc beads showed only a slight reduction in their cell binding capabilities (Table I). In contrast, Ncad-Fc 1% did not present binding capabilities statistically different from control Fc-coated beads (unpublished data). Despite their similar cell binding properties, medium and low Ncad-Fc beads behaved differently from high Ncad-Fc beads. Medium Ncad-Fc beads either remained diffusive or displayed a biphasic behavior characterized by an initial phase of diffusion followed by a phase of rearward transport similar to that observed with high Ncad-Fc beads. The diffusion coefficient of such beads was initially high and dropped rapidly by more than one order of magnitude when the bead adopted a directed movement (Fig. 2). The behavior of low Ncad-Fc beads was even more drastically shifted. Indeed, the majority of the low Ncad-Fc beads remained highly diffusive (diffusion coefficient, 4.6 ± 4.4 x 10-10 cm2/s, n = 25). Nevertheless, a few low Ncad-Fc beads displayed a biphasic behavior (Fig. 2).
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When the effect of Rac1 and Cdc42 mutants on Ncad-Fc bead anchoring was analyzed, we observed a drastic inhibition of the anchoring of Ncad-Fc beads bound on N17 Rac1-GFP expressing cells (Fig. 5 B). In these cells, Ncad-Fc beads remained diffusive over the time of the experiment, without directed movement (Fig. 6). By contrast, neither N17 Cdc42 (Fig. 5 B) nor the V12 forms of Rac1 or Cdc42 did alter Ncad-Fc bead anchoring (unpublished data). Thus, N17 Rac1 expression fully mimicked the lovastatin effect, inhibiting the anchoring to the cytoskeleton without affecting initial binding. These results indicate that the inhibition of Rac1, but not of Cdc42 activity, uncouples the initial homophilic binding of N-cadherin ectodomains from subsequent tight anchoring of N-cadherin to the actin cytoskeleton.
Dominant negative Rac1 or Cdc42 do not alter cell contact formation or stability
The inhibitory effect of N17 Rac1 on N-cadherin anchoring may parallel an effect on N-cadherinmediated cellcell contact formation or stability. Indeed, a strong inhibitory effect of N17 Rac1 and Cdc42 mutants on the formation and stability of E-cadherin mediated cellcell contacts has been described previously in keratinocytes (Braga et al., 1997). Thus, the morphology of cellcell contacts of C2 cells expressing transiently the N17 and V12 Rac1 mutants was analyzed after antiß-catenin immunofluorescent staining (Fig. 7, AB'). No major changes in the formation of cellcell contacts nor in the accumulations of ß-catenin at these sites were observed. To investigate whether perturbations of Rac1 activity may have a different effect on the recruitment of the other catenins, the cells were stained for -catenin and p120 (Fig. 7, CD'). Both
-catenin and p120 were accumulated similarly at cellcell contacts in N17 Rac1-GFPexpressing cells and untransfected cells. In addition, neither N17 nor V12 forms of Cdc42 were able to prevent cellcell contact formation and catenin accumulation in C2 cells (unpublished data). These results indicate that Rac1 and Cdc42 mutants had no major effect on cellcell contact formation and catenin accumulation at these sites.
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The inhibitory effect of N17 Rac1 may result from a direct perturbation on actin dynamics. This hypothesis prompted us to examine the recruitment of neoformed actin filaments at the beadcell contact. For this purpose, N17-Rac1 and V12-Rac1expressing cells were incubated in the presence of Ncad-Fc beads and permeabilized in the presence of rhodamine-labeled actin (Fig. 9). Strong accumulations of rhodamine-conjugated actin were observed at the beadcell contact in V12 Rac1 expressing cells and untransfected cells (unpublished data). In contrast, we were unable to detect these accumulations around beads in contact with N17 Rac1-transfected cells. Interestingly, rhodamine-labeled actin was strongly incorporated into focal adhesions in both conditions. These results indicate that Rac1 indeed plays an important role during actin incorporation/or recruitment at cadherin-mediated cell contacts.
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Discussion |
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The establishment of cellcell contacts is initiated by the adhesive interaction of cadherin ectodomains on adjacent cells. We showed that this initial binding is regulated differentially in various cell membrane subdomains, with a preferential binding of Ncad-Fc beads on lamellipodia. The molecular bases of these differences in homophilic binding of N-cadherin molecules on the lamellipodia versus the cell body are unknown. However, lamellipodia are characterized by a highly dynamic actin cytoskeleton. Moreover, a preferential binding of fibronectin beads to lamellipodia has been reported and attributed to a better avidity of the integrin receptors for their ligand in relation to changes in their association to the actin cytoskeleton (Nishizaka et al., 2000). Thus, the adhesive properties of cadherins may be different in lamellipodia and in the cell body, in relation to actin dynamics and/or the mode of association of the cadherin cytoplasmic tail to the actin cytoskeleton.
After bead binding, we used single-particle tracking to monitor the two-dimensional movement of bead-bound N-cadherin molecules, and to determine whether their diffusion within the membrane was restricted or not. An initial diffusive phase was clearly determined for all low Ncad-Fc beads, indicating that N-cadherin molecules were initially freely diffusive in the lamellipodia. The diffusion coefficient of N-cadherin during the initial diffusive phase (3.3 x 10-10 cm2/s) was very similar to that reported for another receptor, NCAM, known to remain unlinked to the cytoskeleton (Simson et al., 1998). It was also comparable to the diffusion coefficient determined for E-cadherin lacking the ß-catenin binding site (Sako et al., 1998). Thus, our data demonstrate that N-cadherin is not tightly linked or restrained by the cytoskeleton on the free surface of lamellipodia. However, high Ncad-Fc beads were immediately diffusion restricted and pulled away from the leading edge of the cells at the speed of actin treadmilling. This diffusion restriction, inhibited by actin depolymerization, can be attributed to an anchoring of N-cadherin to the actin cytoskeleton, as previously shown for ß1 integrin (Choquet et al., 1997). The behavior of these restricted N-cadherin molecules was similar to that observed by Sako et al. (1998) for an E-cadherin/-catenin fusion mutant constitutively linked to actin filaments. Our results indicate that the homophilic binding of Ncad-Fc beads to N-cadherin triggers the transition of this receptor from a freely diffusive to a cytoskeleton anchored state with a kinetics directly dependent on the density of ligand. This process may directly mimic an adhesion-triggered anchoring of cadherin taking place during normal cellcell contact formation.
Although this approach does not directly give insight into the molecular nature of the mechanisms involved, we propose that this anchoring may result from a ligand-induced cadherin recruitment. Indeed, Ncad-Fc beads have been shown independently to induce the recruitment of cadherincatenin complexes by lateral diffusion in the membrane (Lambert et al., 2000). Moreover, antiN-cadherin coated beads became spontaneously anchored, suggesting that antibody-induced clustering by itself may induce N-cadherin anchoring. Interestingly, Sako et al. (1998) reported the existence of both anchored and freely diffusive E-cadherin molecules at the surface of transfected L cells triggered for 30 min with antibody-coated particles. Altogether, these results support a model in which cadherin molecules are free to diffuse in the cell membrane before the initiation of the adhesion process and become anchored to the actin cytoskeleton as a result of their homophilic ligand triggered recruitment. Nevertheless, we cannot exclude that the occupancy of the ectodomain by the Ncad-Fc ligand may directly activate cadherins. Alternatively, both receptor clustering and ligand occupancy may be required for cadherin activation as reported for integrins (Miyamoto et al., 1995b).
The search for intracellular factors regulating the anchoring process prompted us to test the involvement of the small GTPases Rac1 and Cdc42. The dominant negative form of Rac1 inhibited the anchoring of Ncad-Fc beads, whereas the dominant negative form of Cdc42 had no effect, indicating that N-cadherin anchoring to the cytoskeleton specifically depends on Rac1 activity. However, N17 Rac1 did not inhibit the recruitment of catenins at the beadcell contact. Moreover, neither N17 Rac1 nor N17 Cdc42 prevented the formation of N-cadherinmediated cellcell contacts and the recruitment of catenins at these sites. They did not either induce the destruction of the preexistent cell contacts or the stability of the cadherincatenin complex. Furthermore, the Rac1 and Cdc42 effector IQGAP1 did not appear significantly associated with cellcell contacts in C2 cells, in contrast to what we and others observed in MDCK cells (Nakagawa et al., 2001; unpublished data). Braga et al. (1997) showed that N17 Rac1 or Cdc42 mutants microinjected in keratinocytes negatively regulate cadherins, although we and others did not note major effects of those mutants in transfected MDCK and C2 cells (Takaishi et al., 1997; unpublished data). These differences might be attributed to the expression level of mutant proteins achieved after microinjection or transfection. Alternatively, these differences might be related to molecular differences between cadherin species or to differences in the nature and regulation of cadherin-based cell contacts in different cell backgrounds. Indeed, very similar perturbations of GTPase activity had a differential effect on E-cadherin in keratinocytes and on VE-cadherin in endothelial cells (Braga et al., 1999). The absence of effect of N17 Rac1 on catenin recruitment observed here suggested that Rac1 may directly act on actin dynamics by regulating either actin polymerization or crosslinking. Indeed, we showed that N17 Rac1 inhibited the incorporation of rhodamine-labeled actin under the beads, arguing in favor of a role of Rac1 at the level of actin dynamics. Our results strongly specify the effect of Rac 1, as we report a specific effect of the perturbation of its activity on the functional anchoring of N-cadherin to actin, in conditions where its effect on overall cellcell contact morphology is not detectable. This regulation by Rac1 activity may have a strong physiological relevance for the control of cell migration or contact strengthening.
Upon ligand-induced anchoring, beads were transported on the lamellipodia surface by the rearward-moving actin cytoskeleton, indicating that cadherins can mediate ligand-dependent receptor cell migration in a way similar to what has been proposed for integrins (Choquet et al., 1997). However, cadherin anchoring to the cytoskeleton shows some remarkable differences compared to integrins. The ligand-dependent anchoring of integrins triggered by the binding of fibronectin-coated beads has been shown to be reinforced upon application of a restraining force to the bead (Choquet et al., 1997). In contrast, Ncad-Fc beads spontaneously escaped the laser trap and further application of the trap over the beads was not able to restrain their movement, suggesting that cadherin-cytoskeleton anchoring was spontaneously stiff. Thus, rigidity applied on cadherin mediated contacts does not appear as a pertinent factor regulating cadherin anchoring. Conversely, prevalence and activity of N-cadherin presented by adjacent cells may be an essential parameter, in agreement with the physiological role of cadherin adhesion receptors in migration of a cell over surrounding cells. We also observed that in contrast to fibronectin-coated beads (Nishizaka et al., 2000), Ncad-Fc beads were not released at the rear of the lamellipodia. Thus, once established, the cadherin-based contacts remain stable. These results are in agreement with the fact that in many cadherin-dependent morphogenetic processes, such as border cell migration in Drosophila (Niewiadomska et al., 1999), convergent extension during frog gastrulation (Zhong et al., 1999), cell sorting (Friedlander et al., 1989), or neurite outgrowth (Matsunaga et al., 1988), cells maintain contact and traction with other cells during migration and rearrangement.
This ligand-dependent linkage to the cytoskeleton, and the subsequent transduction of forces across the plasma membrane, appear as an essential aspect of the functional role of cadherins in these various biological processes requiring combined cellcell adhesion and migration. Based on the present findings, we propose a mechanistic model for cadherin action in cell rearrangement. In the case of cell sorting, the sorting between cells expressing different levels of cadherins is probably initiated by the extension of lamellipodia or filopodia contacting various distant cells. Those contacts established between higher expresser cells will be preferentially rendered efficient to transduce mechanical forces generated by the cell's motility system, via a faster anchoring of cadherins to the actin-cytoskeleton. This ligand density kinetics advantage will favor association of these cell bodies via their efficient actin-based traction on filopodia and lamellipodia.
In conclusion, the present data show for the first time that the adhesive interactions of cadherins induce their strong anchoring to the cytoskeleton, enabling the transduction across the cell membrane of mechanical forces generated by the actin treadmilling. These findings enlighten an essential aspect of the mode of action of cadherins in developmental processes such as cell sorting, cell migration, and growth cone navigation.
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Materials and methods |
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Cell culture
C2 mouse myogenic cells (Yaffe and Saxel, 1977) were cultured in DME containing 10% FCS at 37°C in 7.5% CO2. For single-particle tracking, cells were transfected 18 h before analysis, directly on coverslips by Fugen (Boehringer Mannheim) with expression vectors coding for the GFP-tagged N17 or V12 forms of Rac1 and Cdc42, a gift from Dr. Gauthier-Rouviére (CRBM/CNRS, Montpellier, France). For long-term bead cell binding assays, cells were transfected by electroporation (Easyject plus; Equibio) in OPTIMEM under 260 V, 1,500 µF. Cells were resuspended in DME containing 10% FCS and plated on 14-mm three-well glass slides at 5 103 cells/cm2. For protein analysis, cells were electroporated with N17 Rac1-GFP or V12 Rac1-GFP or a membrane anchored GFP (mGFP) expression vector together with a puromycin resistance plasmid pPUR (CLONTECH Laboratories, Inc.), and then subjected to puromycin selection for 24 h (5 µg/ml). Cells were then platted at high density and replaced in puromycin free medium for another 5 h.
Video microscopy and single-particle tracking
Experiments were carried out essentially as described in Choquet et al. (1997). Briefly, C2 cells were plated at sparse density on silane-treated 22-mm glass coverslips coated with laminin, and cultured for 18 h at 37°C in a phenol redfree DME plus 10% FCS and 20 mM Hepes, pH 7.2. Cells were either untreated or treated with 50 µM pervanadate for 10 min, 100 µM lovastatin a gift from Dr. Carnac (IGM/CNRS) for 18 h, or 1 µg/ml cytochalasin B for 10 min. Cells were mounted at 37°C in medium containing coated beads (0.10.2% vol/vol) and visualized under differential interference contrast through a 100x Planapo objective on a C2400 Camera (Hamamatsu). An optical trap was formed with the beam of a Ti:sapphire laser (Spectra-Physics) tuned at 800 nm, 200 mW. Beads were manipulated with the optical trap, and maintained in contact with the cell surface (515 s) to allow their attachment. Transfected cells were identified by the green fluorescence of GFP-tagged proteins. Video images were recorded at 25 Hz on a VCR over 50200 s for later analysis and bead positions followed using homemade software (Choquet et al., 1997) with an accuracy of 510 nm. For each recording, the MSD function and the apparent diffusion coefficient was calculated as previously. 2 or Student's t tests were performed on the different sets of data.
Protein extracts analysis
Cells were lysed in 50 mM Tris buffer, pH 8, 50 mM NaCl, 300 mM sucrose, 1% Triton X-100, plus protease inhibitors (Complete; Roche Diagnostics) for 20 min at 4°C. Cleared cell lysates (100 µg total proteins) were incubated first with protein A Sepharose beads (Amersham Pharmacia Biotech) loaded with rabbit nonimmune serum for 1 h at 4°C. Supernatants were then incubated for 4 h with protein A Sepharose beads loaded with a polyclonal antiß-catenin antibody (Sigma-Aldrich). Beads were washed four times with lysis buffer. Immunoprecipitated and total protein extracts were separated on 7.5% polyacrylamide-SDS gels and immunoblotted as previously described (Lambert et al., 2000) with either polyclonal antiß-catenin (1/5,000), anti-catenin (1/2,000; Sigma-Aldrich), or monoclonal anti-IQGAP1 (clone AF4, 1/2,000; Upstate Biotechnology) antibodies.
Long-term beadcell adhesion assay and immunofluorescent staining
Long-term beadcell binding assays were performed on cells grown for 24 h on 14-mm three-well slides as described previously (Lambert et al., 2000). Briefly, 6-µm Ncad-Fccoated beads were incubated at a concentration of 12% with N17 Rac1-GFP or V12 Rac1-GFP transfected C2 cells for 45 min at 37°C. Preparations were then washed extensively with DME, 10% FCS, and fixed. The percentage of transfected and untransfected cells bearing at least one bead was determined by manual counting in three independent experiments. Alternatively, fixed cells were permeabilized and immunofluorescently stained with either polyclonal anti-p120 (1/1,000), a gift of Dr. A. Reynolds (Vanderbilt University, Nashville, TN), anti-catenin (1/500) or antiß-catenin antibodies (1/500) and further analyzed with a TCS confocal microscope (Leica).
Rhodamine-conjugated actin incorporation
The experiments were performed according to Vasioukhin et al. (2000), with some modifications. Ncad-Fc beads were incubated for 35 min on C2 cells 36 h after electroporation with plasmids encoding N17 Rac1-GFP or V12 Rac1-GFP as described above. Cells were washed twice at room temperature with 20 mM Hepes, pH 7.5, 140 mM KCl, 3 mM MgCl2, 2.5 mM CaCl2, and then incubated for 10 min in the presence of 20 µg/ml of rhodamine-conjugated actin (Cytoskeleton), 0.02% saponin, and 1 mM ATP in the same buffer. The preparations were fixed 10 min in 0.5% glutaraldehyde and examined under a conventional fluorescence microscope (Olympus).
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Footnotes |
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Acknowledgments |
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This work was supported by institutional funding from INSERM and CNRS (Programme Physique et Chimie du Vivant, PCV 1998), as well as by grants from Association Française contre les Myopathies, Association Française de Recherche contre le Cancer, La Ligue contre le Cancer, and the Conseil Régional d'Aquitaine.
Submitted: 24 July 2001
Revised: 11 March 2002
Accepted: 14 March 2002
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