Zyxin-mediated Actin Assembly Is Required for Efficient Wound Closure

Abstract

Cytoskeletal regulation of cell adhesion is vital to the organization of multicellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly because zyxin recruits Enabled/vasodilator-stimulated phospho-proteins (Ena/VASP) to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin-deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin-coated surfaces and the formation of cell clusters were slower for zyxin-deficient cells than wild type cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin-deficient cells failed to recruit VASP to cell-cell junctions at the wound edge and had a slower wound closure rate than wild type cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion. Cytoskeletal regulation of cell adhesion is vital to the organization of multicellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly because zyxin recruits Enabled/vasodilator-stimulated phospho-proteins (Ena/VASP) to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin-deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin-coated surfaces and the formation of cell clusters were slower for zyxin-deficient cells than wild type cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin-deficient cells failed to recruit VASP to cell-cell junctions at the wound edge and had a slower wound closure rate than wild type cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion. IntroductionCell adhesion plays a central role during multicellular organization by interacting with surrounding extracellular matrix and neighboring cells. Adhesion proteins are abundant, but they alone do not sufficiently provide the necessary function and strength of cell adhesion. At cell-extracellular matrix and cell-cell junctions, the actin cytoskeleton promotes the formation and stabilization of adhesive contacts. The regulation of actin assembly is therefore crucial to the regulation of cell adhesion.Despite the presence of distinct adhesive receptors, some actin-binding proteins are shared by both cell-extracellular and cell-cell junctions. Zyxin is one such protein (1Crawford A.W. Beckerle M.C. J. Biol. Chem. 1991; 266: 5847-5853Abstract Full Text PDF PubMed Google Scholar, 2Beckerle M.C. Bioessays. 1997; 19: 949-957Crossref PubMed Scopus (184) Google Scholar). Interestingly, the localization of zyxin is mechano-sensitive. Zyxin is recruited to actin stress fibers upon uniaxial stretching of cells on a compliant substrate (3Yoshigi M. Hoffman L.M. Jensen C.C. Yost H.J. Beckerle M.C. J. Cell Biol. 2005; 171: 209-215Crossref PubMed Scopus (281) Google Scholar) or the application of external force on dorsal cell surface (4Colombelli J. Besser A. Kress H. Reynaud E.G. Girard P. Caussinus E. Haselmann U. Small J.V. Schwarz U.S. Stelzer E.H. J. Cell Sci. 2009; 122: 1665-1679Crossref PubMed Scopus (203) Google Scholar). In addition, stretch-induced actin polymerization at focal adhesion sites has been shown to depend on zyxin (5Hirata H. Tatsumi H. Sokabe M. J. Cell Sci. 2008; 121: 2795-2804Crossref PubMed Scopus (180) Google Scholar). Because of its unique ability to sense external mechanical cues and regulate actin polymerization, zyxin is an attractive candidate for regulating cell adhesion.Although zyxin does not interact directly with actin, zyxin contains binding sites for other actin-binding proteins. The N terminus of zyxin contains an ActA proline-rich domain that binds to the Enabled/vasodilator-stimulated phospho-protein (Ena 2The abbreviations used are: EnaEnabledVASPvasodilator-stimulated phospho-protein./VASP) family (6Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and an α-actinin binding site (7Crawford A.W. Michelsen J.W. Beckerle M.C. J. Cell Biol. 1992; 116: 1381-1393Crossref PubMed Scopus (198) Google Scholar, 8Reinhard M. Jouvenal K. Tripier D. Walter U. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 7956-7960Crossref PubMed Scopus (159) Google Scholar) whereas the C terminus contains three LIM domains. Internal deletion of the α-actinin binding domain disrupts zyxin localization to focal adhesions (8Reinhard M. Jouvenal K. Tripier D. Walter U. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 7956-7960Crossref PubMed Scopus (159) Google Scholar, 9Nix D.A. Fradelizi J. Bockholt S. Menichi B. Louvard D. Friederich E. Beckerle M.C. J. Biol. Chem. 2001; 276: 34759-34767Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). However, the expression of the LIM domains alone is sufficient for targeting zyxin to focal adhesions (9Nix D.A. Fradelizi J. Bockholt S. Menichi B. Louvard D. Friederich E. Beckerle M.C. J. Biol. Chem. 2001; 276: 34759-34767Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The proline-rich Ena/VASP binding domain is not required for the localization of zyxin to focal adhesions (9Nix D.A. Fradelizi J. Bockholt S. Menichi B. Louvard D. Friederich E. Beckerle M.C. J. Biol. Chem. 2001; 276: 34759-34767Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), but it is essential for the localization of Ena/VASP to focal adhesions (5Hirata H. Tatsumi H. Sokabe M. J. Cell Sci. 2008; 121: 2795-2804Crossref PubMed Scopus (180) Google Scholar, 6Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Hoffman L.M. Jensen C.C. Kloeker S. Wang C.L. Yoshigi M. Beckerle M.C. J. Cell Biol. 2006; 172: 771-782Crossref PubMed Scopus (121) Google Scholar). Furthermore, zyxin promotes VASP-dependent actin assembly when zyxin is enriched at the membrane (6Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) or mitochondria (11Fradelizi J. Noireaux V. Plastino J. Menichi B. Louvard D. Sykes C. Golsteyn R.M. Friederich E. Nat. Cell Biol. 2001; 3: 699-707Crossref PubMed Scopus (99) Google Scholar). VASP is thought to compete with actin-capping protein for binding to the barbed ends of actin filaments, thus promoting actin filament elongation (12Bear J.E. Gertler F.B. J. Cell Sci. 2009; 122: 1947-1953Crossref PubMed Scopus (203) Google Scholar). By recruiting Ena/VASP to focal adhesions, zyxin regulates focal adhesion assembly and actin organization.Previous studies focused on zyxin at focal adhesion sites, but little is known about the roles of zyxin at cell-cell adhesion. Both α-actinin binding domain and LIM domains of zyxin have the ability to localize to cell-cell contacts (13Hansen M.D. Beckerle M.C. J. Biol. Chem. 2006; 281: 16178-16188Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The overexpression of zyxin without LIM domains (zyxinΔLIM) enhances cell aggregation, but proline-to-alanine mutations in the proline-rich domain or the LIM domains of zyxin alone do not have such effects (13Hansen M.D. Beckerle M.C. J. Biol. Chem. 2006; 281: 16178-16188Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Yet, the overexpression of zyxinΔLIM with the proline-to-alanine mutations reduces the enhanced cell cluster formation observed in zyxinΔLIM-overexpressing cells, suggesting that the LIM domains negatively regulate the function of Ena/VASP binding sites (13Hansen M.D. Beckerle M.C. J. Biol. Chem. 2006; 281: 16178-16188Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).Ena/VASP has been shown to be important for the integrity of cell-cell adhesion and the underlying actin organization. For example, Ena/VASP-null mice suffer vascular defects, likely due to the disruption of endothelial barrier function (14Furman C. Sieminski A.L. Kwiatkowski A.V. Rubinson D.A. Vasile E. Bronson R.T. Fässler R. Gertler F.B. J. Cell Biol. 2007; 179: 761-775Crossref PubMed Scopus (89) Google Scholar). In primary keratinocytes, the expression of dominant negative VASP results in the disruption of adhesion zipper formation at cell-cell contacts (15Vasioukhin V. Bauer C. Yin M. Fuchs E. Cell. 2000; 100: 209-219Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar). Furthermore, sequestration of Ena/VASP proteins to the mitochondria results in reduced actin level at cell-cell contacts and cadherin-based adhesion plaques (16Scott J.A. Shewan A.M. den Elzen N.R. Loureiro J.J. Gertler F.B. Yap A.S. Mol. Biol. Cell. 2006; 17: 1085-1095Crossref PubMed Scopus (118) Google Scholar). Together, all previous results point to the intimate functional relationship between zyxin and Ena/VASP in facilitating actin assembly at the sites of focal adhesion and cell-cell adhesion.We sought to analyze the role of zyxin as a regulator of VASP localization and actin assembly at sites of cell-cell adhesion using epithelial cells stably expressing zyxin shRNA. In zyxin-deficient cells, actin assembly was reduced at both sites of focal adhesion and cell-cell adhesion. Zyxin-deficient cells had no migration defects, but exhibited slower cell spreading on an E-cadherin-coated surface and cell cluster formation. Furthermore, the monolayer of zyxin-deficient cells was unable to recruit VASP to cell-cell junctions at the wound edge and effectively close a small circular wound. Together, our results suggest that zyxin regulates E-cadherin-mediated cell-cell adhesion by facilitating actin assembly.DISCUSSIONIn addition to the roles of zyxin at focal adhesions (supplemental Fig. S1) (5Hirata H. Tatsumi H. Sokabe M. J. Cell Sci. 2008; 121: 2795-2804Crossref PubMed Scopus (180) Google Scholar, 6Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Hoffman L.M. Jensen C.C. Kloeker S. Wang C.L. Yoshigi M. Beckerle M.C. J. Cell Biol. 2006; 172: 771-782Crossref PubMed Scopus (121) Google Scholar, 11Fradelizi J. Noireaux V. Plastino J. Menichi B. Louvard D. Sykes C. Golsteyn R.M. Friederich E. Nat. Cell Biol. 2001; 3: 699-707Crossref PubMed Scopus (99) Google Scholar), our results show that zyxin also regulates actin assembly at sites of cell-cell adhesion. Actin incorporation at cell-cell contacts was significantly reduced in zyxin-knockdown cells, especially at multicellular junctions where zyxin is often enriched (Fig. 4B). Unlike at focal adhesions, VASP accumulated along cell-cell contacts in zyxin-deficient cells. Therefore, zyxin-mediated actin assembly along the sites of cell-cell adhesion is independent of VASP localization.On E-cadherin-coated substrates, actin assembly is attenuated at adhesion plaques and the lamellipodia in zyxin-knockdown cells (Fig. 3A). Interestingly, VASP is still localized to the tips of cadherin-based lamellipodia (Fig. 3C). Despite the absence of zyxin, VASP can interact with vinculin (20Brindle N.P. Holt M.R. Davies J.E. Price C.J. Critchley D.R. Biochem. J. 1996; 318: 753-757Crossref PubMed Scopus (161) Google Scholar, 21Reinhard M. Rüdiger M. Jockusch B.M. Walter U. FEBS Lett. 1996; 399: 103-107Crossref PubMed Scopus (127) Google Scholar), lipoma-preferred partner (22Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (119) Google Scholar), and Wiskott-Aldrich syndrome protein (23Castellano F. Le Clainche C. Patin D. Carlier M.F. Chavrier P. EMBO J. 2001; 20: 5603-5614Crossref PubMed Scopus (91) Google Scholar). In zyxin-deficient cells, these proteins may recruit VASP to sites of cell-cell contacts. However, the defect in actin assembly at leading edges is not dependent on VASP localization, consistent with our observations at cell-cell adhesion (Fig. 4). Because actin polymerization at the lamellipodia is necessary for the formation of nascent cell adhesion on extracellular matrix (24Galbraith C.G. Yamada K.M. Galbraith J.A. Science. 2007; 315: 992-995Crossref PubMed Scopus (218) Google Scholar, 25Choi C.K. Vicente-Manzanares M. Zareno J. Whitmore L.A. Mogilner A. Horwitz A.R. Nat. Cell Biol. 2008; 10: 1039-1050Crossref PubMed Scopus (573) Google Scholar), the reduced actin polymerization at the leading edge, rather than the reduction in E-cadherin level, is likely responsible for the slow cell spreading on E-cadherin substrate. This also suggests that actin assembly may be important for the early stage of cell-cell adhesion as observed in hanging drop assays. Our data show that zyxin plays a role in the formation of cadherin-mediated adhesion in a VASP-independent manner.Although VASP-positive junctions are often sites of actin assembly, it is not clear why the presence of VASP does not enhance actin assembly at cell-cell contacts of zyxin-knockdown cells. It is possible that the localization of VASP is not sufficient and VASP needs to be activated to promote actin assembly. Based on the analysis of overexpressed zyxin mutants and detergent insolubility as a measure of VASP activation, the LIM domains of zyxin have been suggested to negatively regulate the ability of the ActA domain to activate VASP at cell-cell adhesion sites (13Hansen M.D. Beckerle M.C. J. Biol. Chem. 2006; 281: 16178-16188Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This may explain how the VASP-zyxin interaction regulates zyxin-bound VASP, but it does not explain how the rest of VASP may be regulated at cell-cell contacts.Previous studies have shown that VASP has an intrinsic ability to regulate actin assembly. For example, the recruitment of VASP via the conserved proline-rich domain of ActA is sufficient to promote Listeria motility in a system with purified proteins (26Loisel T.P. Boujemaa R. Pantaloni D. Carlier M.F. Nature. 1999; 401: 613-616Crossref PubMed Scopus (797) Google Scholar). Furthermore, anticapping activity of VASP does not require any association with ActA (27Pasic L. Kotova T. Schafer D.A. J. Biol. Chem. 2008; 283: 9814-9819Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), suggesting that VASP is constitutively active. However, VASP is not an actin nucleator. Instead, VASP activity is essential for the organization of branched versus linear actin network (28Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar) and may not be essential for actin assembly at cell-cell adhesion sites. Actin assembly at sites of cell-cell adhesion may be regulated by other actin nucleators. For example, the Arp2/3 complex (29Yamada S. Nelson W.J. J. Cell Biol. 2007; 178: 517-527Crossref PubMed Scopus (355) Google Scholar, 30Verma S. Shewan A.M. Scott J.A. Helwani F.M. den Elzen N.R. Miki H. Takenawa T. Yap A.S. J. Biol. Chem. 2004; 279: 34062-34070Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 31Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), formin-1 (32Kobielak A. Pasolli H.A. Fuchs E. Nat. Cell Biol. 2004; 6: 21-30Crossref PubMed Scopus (301) Google Scholar), and Dia1 (33Carramusa L. Ballestrem C. Zilberman Y. Bershadsky A.D. J. Cell Sci. 2007; 120: 3870-3882Crossref PubMed Scopus (138) Google Scholar) have been shown to localize to cell-cell contacts. Interestingly, VASP localization at cell-cell adhesion depends on Dia1 (33Carramusa L. Ballestrem C. Zilberman Y. Bershadsky A.D. J. Cell Sci. 2007; 120: 3870-3882Crossref PubMed Scopus (138) Google Scholar). The relationship between zyxin and other actin nucleators remains VASP localization is independent of zyxin at cell-cell contacts (Fig. and at the tips of E-cadherin lamellipodia (Fig. our also that VASP depends on zyxin at the E-cadherin plaques (Fig. and at cell-cell junctions of cells (Fig. Interestingly, both junctions are at the ends of contractile actin suggesting that these junctions are mechanical and that zyxin, a may be in VASP wound the junctions of cells are an contractile that to the wound A. A. P. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). The circumferential actin are enriched with and they contractile A. A. P. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar, P. J. Cell Biol. 121: PubMed Scopus Google Scholar). The wound of a small depends on junctions the addition of wound closure Y. J. Cell Sci. Google Scholar). Furthermore, the interaction is shown to at cell-cell junctions of cells S. Y. T. A. M. Nat. Cell Biol. 12: PubMed Scopus Google Scholar). In addition, we that, VASP localization at cell-cell junctions (Fig. VASP at the edges of cell-cell junctions of cells depends on zyxin, and the absence of zyxin and VASP at these sites the rate of wound closure (Fig. Therefore, our data suggest that the of cells actin assembly by the these data suggest that the ability of zyxin is for VASP recruitment and the regulation of actin at force-bearing cell-extracellular matrix and cell-cell junctions. studies have shown that the actin network at sites of cell-cell adhesion is in M. M. T. Nature. 2008; PubMed Scopus Google Scholar, S. S. F. Nelson W.J. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), it remains how the mechanical the actin Our zyxin as a key regulator of actin assembly at the sites of force-bearing cell adhesion in epithelial cells. IntroductionCell adhesion plays a central role during multicellular organization by interacting with surrounding extracellular matrix and neighboring cells. Adhesion proteins are abundant, but they alone do not sufficiently provide the necessary function and strength of cell adhesion. At cell-extracellular matrix and cell-cell junctions, the actin cytoskeleton promotes the formation and stabilization of adhesive contacts. The regulation of actin assembly is therefore crucial to the regulation of cell adhesion.Despite the presence of distinct adhesive receptors, some actin-binding proteins are shared by both cell-extracellular and cell-cell junctions. Zyxin is one such protein (1Crawford A.W. Beckerle M.C. J. Biol. Chem. 1991; 266: 5847-5853Abstract Full Text PDF PubMed Google Scholar, 2Beckerle M.C. Bioessays. 1997; 19: 949-957Crossref PubMed Scopus (184) Google Scholar). Interestingly, the localization of zyxin is mechano-sensitive. Zyxin is recruited to actin stress fibers upon uniaxial stretching of cells on a compliant substrate (3Yoshigi M. Hoffman L.M. Jensen C.C. Yost H.J. Beckerle M.C. J. Cell Biol. 2005; 171: 209-215Crossref PubMed Scopus (281) Google Scholar) or the application of external force on dorsal cell surface (4Colombelli J. Besser A. Kress H. Reynaud E.G. Girard P. Caussinus E. Haselmann U. Small J.V. Schwarz U.S. Stelzer E.H. J. Cell Sci. 2009; 122: 1665-1679Crossref PubMed Scopus (203) Google Scholar). In addition, stretch-induced actin polymerization at focal adhesion sites has been shown to depend on zyxin (5Hirata H. Tatsumi H. Sokabe M. J. Cell Sci. 2008; 121: 2795-2804Crossref PubMed Scopus (180) Google Scholar). Because of its unique ability to sense external mechanical cues and regulate actin polymerization, zyxin is an attractive candidate for regulating cell adhesion.Although zyxin does not interact directly with actin, zyxin contains binding sites for other actin-binding proteins. The N terminus of zyxin contains an ActA proline-rich domain that binds to the Enabled/vasodilator-stimulated phospho-protein (Ena 2The abbreviations used are: EnaEnabledVASPvasodilator-stimulated phospho-protein./VASP) family (6Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and an α-actinin binding site (7Crawford A.W. Michelsen J.W. Beckerle M.C. J. Cell Biol. 1992; 116: 1381-1393Crossref PubMed Scopus (198) Google Scholar, 8Reinhard M. Jouvenal K. Tripier D. Walter U. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 7956-7960Crossref PubMed Scopus (159) Google Scholar) whereas the C terminus contains three LIM domains. Internal deletion of the α-actinin binding domain disrupts zyxin localization to focal adhesions (8Reinhard M. Jouvenal K. Tripier D. Walter U. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 7956-7960Crossref PubMed Scopus (159) Google Scholar, 9Nix D.A. Fradelizi J. Bockholt S. Menichi B. Louvard D. Friederich E. Beckerle M.C. J. Biol. Chem. 2001; 276: 34759-34767Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). However, the expression of the LIM domains alone is sufficient for targeting zyxin to focal adhesions (9Nix D.A. Fradelizi J. Bockholt S. Menichi B. Louvard D. Friederich E. Beckerle M.C. J. Biol. Chem. 2001; 276: 34759-34767Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The proline-rich Ena/VASP binding domain is not required for the localization of zyxin to focal adhesions (9Nix D.A. Fradelizi J. Bockholt S. Menichi B. Louvard D. Friederich E. Beckerle M.C. J. Biol. Chem. 2001; 276: 34759-34767Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), but it is essential for the localization of Ena/VASP to focal adhesions (5Hirata H. Tatsumi H. Sokabe M. J. Cell Sci. 2008; 121: 2795-2804Crossref PubMed Scopus (180) Google Scholar, 6Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Hoffman L.M. Jensen C.C. Kloeker S. Wang C.L. Yoshigi M. Beckerle M.C. J. Cell Biol. 2006; 172: 771-782Crossref PubMed Scopus (121) Google Scholar). Furthermore, zyxin promotes VASP-dependent actin assembly when zyxin is enriched at the membrane (6Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) or mitochondria (11Fradelizi J. Noireaux V. Plastino J. Menichi B. Louvard D. Sykes C. Golsteyn R.M. Friederich E. Nat. Cell Biol. 2001; 3: 699-707Crossref PubMed Scopus (99) Google Scholar). VASP is thought to compete with actin-capping protein for binding to the barbed ends of actin filaments, thus promoting actin filament elongation (12Bear J.E. Gertler F.B. J. Cell Sci. 2009; 122: 1947-1953Crossref PubMed Scopus (203) Google Scholar). By recruiting Ena/VASP to focal adhesions, zyxin regulates focal adhesion assembly and actin organization.Previous studies focused on zyxin at focal adhesion sites, but little is known about the roles of zyxin at cell-cell adhesion. Both α-actinin binding domain and LIM domains of zyxin have the ability to localize to cell-cell contacts (13Hansen M.D. Beckerle M.C. J. Biol. Chem. 2006; 281: 16178-16188Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The overexpression of zyxin without LIM domains (zyxinΔLIM) enhances cell aggregation, but proline-to-alanine mutations in the proline-rich domain or the LIM domains of zyxin alone do not have such effects (13Hansen M.D. Beckerle M.C. J. Biol. Chem. 2006; 281: 16178-16188Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Yet, the overexpression of zyxinΔLIM with the proline-to-alanine mutations reduces the enhanced cell cluster formation observed in zyxinΔLIM-overexpressing cells, suggesting that the LIM domains negatively regulate the function of Ena/VASP binding sites (13Hansen M.D. Beckerle M.C. J. Biol. Chem. 2006; 281: 16178-16188Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).Ena/VASP has been shown to be important for the integrity of cell-cell adhesion and the underlying actin organization. For example, Ena/VASP-null mice suffer vascular defects, likely due to the disruption of endothelial barrier function (14Furman C. Sieminski A.L. Kwiatkowski A.V. Rubinson D.A. Vasile E. Bronson R.T. Fässler R. Gertler F.B. J. Cell Biol. 2007; 179: 761-775Crossref PubMed Scopus (89) Google Scholar). In primary keratinocytes, the expression of dominant negative VASP results in the disruption of adhesion zipper formation at cell-cell contacts (15Vasioukhin V. Bauer C. Yin M. Fuchs E. Cell. 2000; 100: 209-219Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar). Furthermore, sequestration of Ena/VASP proteins to the mitochondria results in reduced actin level at cell-cell contacts and cadherin-based adhesion plaques (16Scott J.A. Shewan A.M. den Elzen N.R. Loureiro J.J. Gertler F.B. Yap A.S. Mol. Biol. Cell. 2006; 17: 1085-1095Crossref PubMed Scopus (118) Google Scholar). Together, all previous results point to the intimate functional relationship between zyxin and Ena/VASP in facilitating actin assembly at the sites of focal adhesion and cell-cell adhesion.We sought to analyze the role of zyxin as a regulator of VASP localization and actin assembly at sites of cell-cell adhesion using epithelial cells stably expressing zyxin shRNA. In zyxin-deficient cells, actin assembly was reduced at both sites of focal adhesion and cell-cell adhesion. Zyxin-deficient cells had no migration defects, but exhibited slower cell spreading on an E-cadherin-coated surface and cell cluster formation. Furthermore, the monolayer of zyxin-deficient cells was unable to recruit VASP to cell-cell junctions at the wound edge and effectively close a small circular wound. Together, our results suggest that zyxin regulates E-cadherin-mediated cell-cell adhesion by facilitating actin assembly.