Article |
Address correspondence to Alpha S. Yap, Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia. Tel.: 61-7-3346-2013. Fax: 61-7-3346-2101. email: a.yap{at}mailbox.uq.edu.au
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Abstract |
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Key Words: E-cadherin; cortactin; actin assembly; Arp2/3; epithelia
The online version of this article includes supplemental material.
Abbreviations used in this paper: hE-CHO, CHO cell stably transfected with human E-cadherin; hE/Fc, human E-cadherin fused to the Fc region of IgG; NTA, NH2-terminal acidic; RNAi, RNA interference; TIRF, total internal reflection fluorescence.
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Introduction |
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The molecular mechanisms that link cadherins and actin assembly are now beginning to be identified. One such mechanism is likely to involve interaction between E-cadherin and the Arp2/3 actin nucleator complex. Recently, we found that cadherin adhesive ligation can recruit the Arp2/3 complex to the cell surface (Kovacs et al., 2002b). Strikingly, Arp2/3 preferentially localizes to sites of actin assembly in newly forming cadherin contacts. Such cadherin-directed localization of Arp2/3 provides an attractive potential mechanism to optimally focus the polymerization of actin in newly forming contacts, thereby allowing its force-generating capacity to be concentrated to drive the extension of those contacts.
The molecules that regulate actin assembly at cadherin contacts remain to be elucidated. Cortactin has recently emerged as a potentially critical regulator of actin assembly in a variety of contexts that entail dynamic control of membrane movements (Olazabal and Machesky, 2001; Weed and Parsons, 2001; Higgs, 2002). First discovered as an src-kinase substrate in RSV-transformed fibroblasts (Wu et al., 1991), cortactin is a multidomain actin-binding protein (Wu and Parsons, 1993; Weed and Parsons, 2001). Cortactin can interact directly with both Arp2/3 (via an NH2-terminal acidic [NTA] domain) and F-actin (notably via the fourth of six tandem repeats located in the NH2-terminal half of the molecule; Weed et al., 2000). Direct binding of cortactin activates Arp2/3-driven actin nucleation (Uruno et al., 2001; Weaver et al., 2001, 2002), and this is enhanced when WASp-interacting protein associates with the cortactin SH3 domain (Kinley et al., 2003). Cortactin also inhibits disassembly of Arp2/3-generated actin filaments, an effect that can potentially stabilize the cortical actin network (Weaver et al., 2001). These properties of cortactin prompted us to examine its role in cadherin contact formation. We now report that cortactin is necessary for cadherin-directed actin assembly and contact zone extension.
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Results |
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Cortactin accumulates preferentially at the extending margins of cadherin adhesive contact zones
To better define the precise pattern of cortactin recruitment in cadherin contacts, we used total internal reflection fluorescence (TIRF) microscopy to visualize cortactin in cells adherent to hE/Fc-coated substrata (Fig. 3). In this assay, the cellsubstratum interface constitutes the zone of contact and, as cells adhere, they progressively extend their zones of contact by protruding cadherin-based lamellipodia (Fig. 3 and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200309034/DC1; Kovacs et al., 2002a, b). Although not designed to reproduce all events likely to arise when native cell surfaces are brought together, these planar spreading assays have the analytic advantage of allowing us to isolate dynamic cellular events that arise as specific responses to cadherin ligation, independent of any potential juxtacrine signals (Noren et al., 2001; Kovacs et al., 2002a, HREF="#BIB14">b; Yap and Kovacs, 2003). Moreover, many salient features displayed by cells in these assays, including the spatial confinement of Rac and PI3-kinase signaling to the outer, extending margins of the contact (Kovacs et al., 2002a), are also observed when migrating MDCK cells establish productive contacts with one another (Ehrlich et al., 2002). Combined with the ability of TIRF microscopy to image molecules located within 100 nm of the cell substrate boundary (Steyer and Almers, 2001), these assays thus provided the opportunity to examine dynamic events located at the cadherin adhesive interface itself.
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This suggested strongly that cortactin might preferentially associate with the outer margins where contact zones were undergoing extension. As a further test of this notion, we used TIRF to assess the patterns of fluorescence recovery in the adhesive interface after photobleaching of GFP-cortactin. As shown in Fig. 3 (DF) and in Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200309034/DC1), fluorescence first returned at the very outer margins of the cadherin-based lamellipodia, being readily detectable within 20 s after photobleaching. Measurement of fluorescence intensity showed clear recovery of fluorescence both at the very outer margins and within the lamellipodium proximal to the outer margin itself (Fig. 3 E). However, fluorescence began to increase earlier, and to a greater degree, at the outer margin than in more proximal regions of lamellipodia (Fig. 3 E), suggesting that this was a site for preferential recovery of GFP-cortactin. As a further test, we compared the distribution of fluorescence recovery in line scans through the photobleached area at various times after photobleaching (Fig. 3 F). This analysis showed that although fluorescence progressively increased throughout the photobleached area, recovery was most pronounced at the very outer margin. Together, these data indicate that cortactin accumulated preferentially at the outer margins of cadherin-based lamellipodia where contact zones were being actively extended.
Cortactin localizes with Arp2/3 and participates in actin remodeling at cadherin adhesive contacts
A major target of cortactin is the Arp2/3 actin nucleator complex (Uruno et al., 2001; Weaver et al., 2001). Consistent with this, we found that cortactin colocalized with Arp2/3 at sites of homophilic cadherin adhesion. In bead adhesion assays, cortactin accumulated with transiently expressed GFP-Arp3 at sites of adhesion to hE/Fc-coated beads (Fig. 2 B), but not after binding of Con Acoated beads (unpublished data). In planar spreading assays, cortactin also colocalized precisely with transiently expressed GFP-Arp3 at the outer margins of cadherin-based lamellipodia (Fig. 4). In both these assays, cortactin coaccumulated with F-actinat the sites of binding to cadherin-coated beads (Fig. 2 B) and in prominent bands at the outer margins of cadherin-based lamellipodia, which we have previously shown to correspond to sites of active actin assembly (Fig. 4; Kovacs et al., 2002b).
To test whether cortactin could participate in actin organization at cadherin adhesive contacts, we examined the effect of cortactin mutants on F-actin accumulation around hE/Fc-coated beads (Fig. 5). When transiently expressed, these cortactin mutants generated polypeptides of the predicted molecular weight and were readily detected by immunofluorescence (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200309034/DC1). F-actin accumulated prominently at the sites of adhesion between control cells and hE/Fc-coated beads (Fig. 5 B; hE/Fc, untransfected). Characteristically, intense F-actin staining was observed in a distinct circumferential pattern around the hE/Fc-coated beads (Fig. 2 and Fig. 5 B). In contrast, no circumferential actin staining was seen upon binding of Con A beads, which showed only occasional actin cables that appeared to originate and terminate in different focal planes than the beads (Fig. 5 B, Con A). Importantly, transient expression of the CA domain of N-WASp (Fig. 5 B, GFP-CA) largely abolished circumferential phalloidin staining around cadherin-coated beads. Because this fragment is a potent inhibitor of Arp2/3 activity (Rohatgi et al., 1999), it indicated that Arp2/3 activity was necessary for cadherin-based actin accumulation upon bead binding.
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Cortactin is functionally important for cadherin contact formation
Actin assembly by Arp2/3 is commonly implicated in generating the forces necessary for cell surface protrusion (Pollard et al., 2000). This led us to test whether cortactin might participate in cadherin-based contact zone extension. First, we examined whether cortactin mutants affected the ability of cells to form cadherin-based lamellipodia using an assay that measures the dimensions of outer margins as a proportion of the total cell periphery (Fig. 6; lamellipodial index, LI). We found that transient expression of mutants that disrupt either the ability of cortactin to bind F-actin (4) or its ability to interact with the Arp2/3 complex (
NTA) reduced the LI of hE-CHO cells by >50% compared with untransfected controls (Fig. 6). In contrast, neither the 3Y mutant nor a mutant that consisted only of the internal repeats region (Fig. 6, repeats) affected cadherin-based contact zone extension compared with untransfected control cells (Fig. 6).
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Discussion |
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Actin assembly has the capacity both to drive cell surfaces together to initiate contacts (Vasioukhin et al., 2000) and to promote extension of cadherin-based cell contacts upon one another after contacts are first made (Kovacs et al., 2002b). In the latter case, E-cadherin ligation itself is likely to be an active participant, by directing where actin assembly concentrates in contact zones. We found that cortactin mutants profoundly inhibit actin accumulation at adhesive contacts with cadherin-coated beads. We chose to use bead assays in these experiments because they provide spatially defined, cadherin-specific adhesive cues that are independent of cell spreading. Similar approaches demonstrated that G-actin incorporates at the adhesive contact with N-cadherincoated beads (Lambert et al., 2002), and we confirmed that F-actin accumulation at bead adhesions was inhibited by an N-WASp fragment that effectively inhibits Arp2/3-mediated actin assembly in vitro (Rohatgi et al., 1999). Therefore, our results suggest strongly that cortactin activity is necessary for the Arp2/3-dependent actin assembly that occurs in response to E-cadherin homophilic ligation.
In keeping with a role for actin assembly in extending adhesive contacts, we found that the ability of cells to extend cadherin contacts in planar spreading assays was dramatically reduced when cortactin activity was perturbed, either by expression of dominant-negative cortactin mutants or by RNAi-mediated knock-down of endogenous cortactin. Furthermore, depletion of cortactin in NMuMG cells significantly reduced the efficiency with which cells reestablished contacts with one another. Together, these findings indicate that cortactin plays a key role in cadherin-based contact zone extension. In separate analyses, we also recently established that Arp2/3 activity is necessary for extension of cadherin adhesive contacts in these assays (unpublished data). Thus, we interpret these findings to indicate that cortactin is necessary for Arp2/3-mediated actin assembly to effectively drive cell surfaces together during contact zone extension.
In vitro, cortactin can increase net actin assembly by directly or indirectly stimulating the catalytic activity of the Arp2/3 complex (Uruno et al., 2001; Weaver et al., 2001, 2002), as well as stabilizing actin filaments at a post-nucleation step (Weaver et al., 2001). Any of these actions, individually or in concert, may account for the contribution of cortactin to the cadherin-mediated actin assembly that we observed. Notably, our analysis of cortactin mutants suggests that both the Arp2/3-binding and F-actinbinding domains of the molecule are necessary for actin assembly at cadherin contacts. Hence, the ability of cortactin to act as a multidomain scaffold, simultaneously linking Arp2/3 and actin filaments, may be a key to its role in cadherin-directed actin assembly. The inhibitory effect of the cortactin NTA mutant might then be explained by its ability to displace endogenous cortactin from filaments (because this mutant retains the F-actinbinding site), whereas the
4 mutant would compete with endogenous cortactin for Arp2/3, but be incapable of binding F-actin.
Ultimately, for cortactin to potentiate Arp2/3 activity, this protein must also be strictly recruited to sites of actin assembly at the cell surface. Indeed, we found that cortactin colocalizes with the Arp2/3 complex exactly where cadherin adhesion induces actin assemblyaround nascent contacts with cadherin-coated beads and at the outer margins of contact zones in planar adhesion assays. Furthermore, after photobleaching, GFP-cortactin appeared to preferentially recruit to the outer margins of cadherin-based lamellipodia where contact zones were being actively extended (Kovacs et al., 2002b). Such selective accumulation might reflect either preferential delivery of cortactin to these regions and/or selective retention at these sites. Irrespective of the mechanism, this finding indicates that cortactin is recruited to specific subregions within individual cadherin contacts, suggesting that the adhesive contact zones are not biochemically or functionally uniform structures (Ehrlich et al., 2002; Kovacs et al., 2002b).
Our data further indicate that cortactin recruitment occurs as a consequence of cadherin homophilic ligation and, indeed, is associated with a biochemical interaction between cortactin and the cadherin adhesive complex. Most notably, presentation of a cadherin-specific adhesive signal (on beads or planar substrata) was sufficient to recruit cortactin to adhesive sites and induce cortactin to coimmunoprecipitate with E-cadherin. In contrast, nonspecific adhesive ligands did not recruit cortactin to the membrane, nor did they allow the cadherincortactin biochemical interaction to be sustained.
Although we have yet to elucidate the molecular mechanism that links cortactin to the cadherin adhesive complex, our data argue against it being a constitutive interaction. In planar spreading assays, much E-cadherin did not colocalize with cortactin, and in our current experiments only a small proportion of total cellular E-cadherin was found in the cortactin immunoprecipitates. Similarly, integrin-based cell migration also entails a functionally important interaction between small subpopulations of Arp2/3 and vinculin (DeMali et al., 2002). Although cortactin may interact with some members of the p120-catenin family (Martinez et al., 2003), this suggests that the contribution of cortactin to cadherin function involves a specific pool of cadherin molecules, presumably that is responsible for recruiting Arp2/3. Similarly, we observed earlier that only a subpopulation of the cellular pools of cadherin and Arp2/3 interacted biochemically during contact extension (Kovacs et al., 2002b).
Instead, it is likely that recruitment of cortactin to cadherin contacts is signal regulated, consistent with emerging evidence that multiple signals determine both Arp2/3 activity (Machesky and Insall, 1999) and cortactin localization (Head et al., 2003). A good candidate here is Rac, which clearly stimulates cortactin recruitment in motile cells (Weed et al., 1998). Moreover, Rac is directly activated by cadherin ligation (Nakagawa et al., 2001; Kovacs et al., 2002a), and is likely to be spatially confined to the outer margins in extending contact zones (Ehrlich et al., 2002; Kovacs et al., 2002a), exactly corresponding to the sites where cortactin recruits in our analyses. Spatio-temporal localization of signals may then be an important factor that directs cortactin to specific subregions within individual adhesive contact zones.
In summary, we have identified cortactin as a novel determinant of cadherinactin cooperativity during adhesive contact formation. We envisage that cortactin acts in newly forming contacts to promote Arp2/3-mediated cell surface protrusion, thereby driving the extension of nascent contacts to form stable zones of adhesion. However, the impact of cortactin is not confined to the early stages of contact formation. Cortactin mutants significantly inhibited the accumulation of E-cadherin in contacts between MDCK cells, a hallmark of adherens junctions assembly, accompanied by gross distortions of epithelial cell morphology (residual cortactin likely explains why morphologic changes were less apparent in RNAi-treated NMuMG cells). Interestingly, the cortactin mutants that most effectively perturbed cadherin-directed actin assembly and contact zone extension (4 and
NTA) were also the most potent in disrupting junctional assembly and epithelial morphology. These effects of cortactin on later stages of epithelial biogenesis may therefore reflect a requirement for efficient contact zone extension early in the process of contact formation. However, cortactin may also participate in other aspects of junctional biogenesis. For example, cortactin also binds directly to ZO-1, a cytoplasmic scaffolding protein of the tight junction in both invertebrate and vertebrate cells (unpublished data; Katsube et al., 1998), which can also associate with adherens junctions. Given its potential to support multiple intermolecular interactions (Weed and Parsons, 2001), cortactin may then act as a signal integrator in cadherin contacts, coupling adhesion to actin dynamics, cell signaling, and junctional assembly.
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Materials and methods |
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Antibodies
Primary antibodies were as follows: (1) a pAb directed against the ectodomain of human E-cadherin was raised in rabbits using hE/Fc as an immunogen. In Western blots, this pAb recognized a single 116-kD polypeptide band in lysates from MCF-7 and hE-CHO cells, but not in lysates from parental CHO cells that lack E-cadherin; (2) mouse mAb against the cytoplasmic tail of human E-cadherin (Transduction Laboratories); (3) mAb HECD1 against human E-cadherin (provided by Dr. M. Wheelock [University of Nebraska, Omaha, NE], with the permission of Dr. M. Takeichi [RIKEN CDB, Kobe, Japan]); (4) mouse mAb 3B8 directed against canine E-cadherin (a gift from Dr. Warren Gallin, University of Alberta, Edmonton, Alberta, Canada); (5) rat mAb DECMA-1 against mouse E-cadherin (Sigma-Aldrich); (6) rabbit pAb against the NH2 terminus of Xenopus ß-catenin (a gift of Dr. Barry Gumbiner, University of Virginia, Charlottesville, VA); (7) mouse mAb 4F11 against cortactin (Wu et al., 1991); (8) rabbit pAb C2 raised against chicken cortactin; (9) anti-FLAG mAb M5 (Sigma-Aldrich); (10) rabbit anti-GFP pAb (Molecular Probes, Inc.); and (11) anti-GFP mAb (Roche). F-actin was identified using Alexa® 488 or Alexa® 568labeled phalloidin (Molecular Probes, Inc.). Secondary antibodies were species-specific antibodies conjugated with Alexa® 350, Alexa® 488, or Texas red (Molecular Probes, Inc.).
Plasmids
The pEGFP-CA construct bearing the CA fragment of N-WASp was a gift of Drs. H. Miki and T. Takenawa (University of Tokyo, Tokyo, Japan). GFP-Arp3 (constructed by Dr. Matt Welch) was a gift from Dr. D. Schaefer (Washington University, St. Louis, MO). GFP-cortactin and the cortactin mutants 4, Repeats, and 3Y were described earlier (Weed et al., 2000; Kinley et al., 2003). The cortactin
NTA expression construct was created by PCR amplification of mp85.L7 (Miglarese et al., 1994), with a 5' primer flanking codon 85 containing a KpnI restriction site and a 3' primer flanking the stop codon (546) containing an EcoRI restriction site. The amplified PCR product was digested with KpnI and EcoRI and subcloned in frame into KpnIEcoRI-digested pcDNA3FLAG2AB. The final construct was tested by DNA sequencing.
To generate the pSUPER-Cort construct, the pSUPER vector was digested with BglII and HindIII and then ligated with the annealed oligonucleotides 5'-gatccccCACATCAACATTCACAAGCttcaagagaGCTTGTGAATGTTGATGTGtttttggaaa-3' and 5'-agcttttccaaaaaCACATCAACATTCACAA-GCtctcttgaaGCTTGTGAATGTTGATGTGggg-3' (cortactin sequences are in uppercase, corresponding to bases 284302 in the murine cortactin cDNA; Miglarese et al., 1994). Oligonucleotides were HPLC purified and 5'-phosphorylated before annealing. The pSUPER-Cort sequence was verified by DNA sequencing.
Synthetic RNA duplexes based on the sequences 5'-AAAGCTTCGAGAGAATGTCTT-3' and 5'-AAGACTGAGAAGCATGCCTCC-3' were prepared using the SilencerTM kit (Ambion). Cells were transfected with a cocktail of both duplexes using siPORTTM Amine (Ambion) as per the manufacturer's instructions.
Immunofluorescence microscopy and quantitation
Samples were prepared for immunofluorescence analysis as described previously (Kovacs et al., 2002a,b). Fixed material was mounted in 1% N-propyl-gallate in 50% glycerol:PBS for epi-illumination and laser-scanning confocal microscopy; glycerol was omitted from the mounting medium for TIRF. Fixed material was examined by epi-illumination using microscopes (model AX70 or IX81; Olympus) with 60x, 1.4 NA or 100x, 1.4 NA objectives (Olympus), and was imaged with cameras (Orca 1 or Orca-ER; Hamamatsu). TIRF imaging was performed using a microscope (model IX81; Olympus) and an objective (60x, 1.45 NA; Olympus) illuminated with a 10-mW argon laser. For live-cell imaging, cells were incubated in phenol redfree HBSS at 37°C. All acquisition and movies were controlled using MetaMorph® software (Universal Imaging Corp.). Photobleaching was performed by closing the iris field diaphragm in the epi-illumination pathway and illuminating with a 100-W Hg lamp for 10 s. Laser-scanning confocal microscopy was performed using a scanning system (MRC 600; Bio-Rad Laboratories) mounted on a microscope (Axioskop; Carl Zeiss MicroImaging, Inc.) equipped with a Plan-Apochromat 63x objective (Carl Zeiss MicroImaging, Inc.) running under Bio-Rad Laboratories software. Some epi-illumination images were deconvolved from z-stacks using three-dimensional blind deconvolution (Autoquant). Movie images were compiled and converted to QuickTime stacks using ImageJ. Figures were assembled for presentation in Adobe Photoshop®.
To quantitate cadherin-based contact formation in planar spreading assays, phalloidin-stained images were analyzed using MetaMorph® software. For each cell, the lamellipodial index was calculated as follows: lamellipodial outer margins were identified by their broad phalloidin staining, were measured, and the total length of outer margins in each cell was expressed as a percentage of the cell perimeter for that cell. Contact formation in calcium jump experiments was quantitated by measuring the lengths of individual cadherin contacts (defined as lines of continuous cadherin staining between cell vertices). As a semiquantitative estimate of actin accumulation around hE/Fc-coated beads, the degree of phalloidin staining around beads was assessed using a scoring system based on the proportion of the circumference of the bead showing intense F-actin accumulation. 4+, complete circumferential F-actin accumulation; 3+, 75% of the perimeter; 2+,
50% perimeter; 1+,
25%; 0, no actin accumulation.
Immunoprecipitations
Cells were lysed in 1 ml lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 50 mM NaF, 2 mM sodium vanadate, 0.1% BSA, and complete protease inhibitors [Roche]). Protein complexes were immunoprecipitated with either a polyclonal E-cadherin antibody or a C2 pAb directed against cortactin bound to protein Aagarose beads and were separated by SDS-PAGE. Immune complexes were blotted for E-cadherin and cortactin. Blots were scanned and compiled in Adobe Photoshop® (v. 6).
Online supplemental material
Online supplemental material includes expression characterization of the cortactin mutants used in this work (Fig. S1), graphical illustration of the regions chosen to analyze FRAP (Fig. S2), and the videos of GFP-cortactin localization imaged by TIRF (Videos 1 and 2) that are illustrated in Fig. 3. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200309034/DC1.
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Acknowledgments |
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The work in Australia was supported by grants to A.S. Yap from the National Health and Medical Research Council (Australia), Wellcome Trust, and Human Frontiers Science Program. S.A. Weed is supported by grant K22 DE 14364 from the National Institute of Dental and Craniofacial Research. Support is also provided by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (RO1 DK061397) to A.S. Fanning and James M. Anderson; and A.S. Yap is a Wellcome Trust Senior International Medical Research Fellow.
Submitted: 5 September 2003
Accepted: 15 January 2004
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