Integrins and Actin Filaments: Reciprocal Regulation of Cell Adhesion and Signaling (2000)

extracellular matrix focal adhesions ezrin, radixin, and moesin vasodilator-stimulated phosphoprotein Wiskott-Aldrich syndrome protein integrin-linked kinase Integrin adhesion receptors link the extracellular matrix (ECM)1to the actin cytoskeleton and transmit biochemical signals and mechanical force across the plasma membrane. This enables cells to generate traction during migration and exert tension during matrix remodeling (1Choquet D. Felsenfeld D.P. Sheetz M.P. Cell. 1997; 88: 39-48Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar). Cytoskeletal linkages also enable integrins to mediate cell adhesion and regulate cell shape and gene expression (1Choquet D. Felsenfeld D.P. Sheetz M.P. Cell. 1997; 88: 39-48Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar). Here we will summarize the evidence for direct interactions between integrin cytoplasmic tails and specific actin-binding proteins and discuss how these interactions influence cell adhesion, cell spreading, and migration. Integrin α and β subunits are type I transmembrane proteins expressed in surface membranes as heterodimers. Each consists of a large extracellular domain, a single transmembrane segment, and a relatively short cytoplasmic tail. The latter contains anywhere from 20 to 70 amino acid residues, with the notable exception of the much larger β4 tail, which is linked primarily to intermediate filaments instead of actin filaments (2Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1674) Google Scholar). β-Cytoplasmic tails are necessary and sufficient to link integrins to the actin cytoskeleton (2Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1674) Google Scholar). In contrast, there is less evidence to date that α tails are directly linked to the cytoskeleton; indeed the removal of the α1, α4, or αIIb cytoplasmic tail appears to increase β tail-mediated interactions with the cytoskeleton (2Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1674) Google Scholar). Direct binding of the signaling adapter protein paxillin to α4 cytoplasmic tails has recently been demonstrated, and this binding regulates α4β1-mediated cell spreading, migration, and stress fiber formation (3Liu S. Thomas S.M. Woodside D.G. Rose D.M. Kiosses W.B. Pfaff M. Ginsberg M.H. Nature. 1999; 402: 676-681Crossref PubMed Scopus (294) Google Scholar). There is direct biochemical support for the interaction of α and β tails with each other (4Muir T.W. Williams M.J. Ginsberg M.H. Kent S.B.H. Biochemistry. 1994; 33: 7701-7708Crossref PubMed Scopus (84) Google Scholar, 5Haas T.A. Plow E.F. J. Biol. Chem. 1996; 271: 6017-6026Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and for the modulation of this interaction by the binding of ligands to the extracellular domain (6Leisner T.M. Wencel-Drake J.D. Wang W. Lam S.C. J. Biol. Chem. 1999; 274: 12945-12949Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Consequently, regulated changes in the interactions between the α and β tails may affect integrin-cytoskeleton linkages. In addition to mediating integrin linkages with the actin cytoskeleton, β cytoplasmic tails are important for adhesion, spreading, and migration of cells on ECM, processes dependent on an intact actin cytoskeleton. Integrins typically cluster within “matrix adhesions,” sites of close apposition of the cell membrane to the ECM. Matrix adhesions are extremely dynamic and heterogeneous structures with respect to size, composition, and orientation to actin filaments (7Zamir E. Katz B.Z. Aota S. Yamada K.M. Geiger B. Kam Z. J. Cell Sci. 1999; 112: 1655-1669Crossref PubMed Google Scholar, 9Hall A. Science. 1998; 23: 509-514Crossref Scopus (5278) Google Scholar). The largest ones are usually referred to as focal adhesions (FA), which are aligned at the ends of actin stress fibers (2Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1674) Google Scholar, 8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar, 9Hall A. Science. 1998; 23: 509-514Crossref Scopus (5278) Google Scholar). As such, FA represent a morphologically prominent association between integrins and the cytoskeleton, and investigation of integrin targeting to FA has shed light on the mechanisms of integrin-cytoskeleton association. Mutational analysis of the 47-amino acid β1 cytoplasmic tail has identified three clusters of amino acids important for integrin localization to FA, a membrane-proximal region and two conserved NPXY (single-letter amino acid code) motifs (10Reszka A.A. Hayashi Y. Horwitz A.F. J. Cell Biol. 1992; 117: 1321-1330Crossref PubMed Scopus (242) Google Scholar). Similar motifs in the β3 cytoplasmic tail are important for localization of β3 integrins to FA (11Ylanne J. Huuskonen J. O'Toole T.E. Ginsberg M.H. Virtanen I. Gahmberg C.G. J. Biol. Chem. 1995; 270: 9550-9557Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). An additional Thr-containing motif between the two NPXY sites has also been implicated in β2 integrin-cytoskeleton linkages (12Peter K. O'Toole T.E. J. Exp. Med. 1995; 181: 315-326Crossref PubMed Scopus (145) Google Scholar). Integrins are linked to actin filaments by specific actin-binding proteins, and there is now an emerging consensus concerning which proteins are involved (Fig. 1). Talin is composed of two ∼270-kDa subunits arranged as an anti-parallel homodimer (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar), and it co-localizes with integrins at certain sites of cell-substratum contact. Talin is a major structural component of FA along with actin and vinculin. It consists of an N-terminal ∼50-kDa globular head domain, which includes an ∼200-amino acid region with homology to the ezrin, radixin, and moesin (ERM) family of proteins, and an ∼220-kDa, C-terminal rod domain containing a conserved ILWEQ actin-binding domain (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar, 13Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (3058) Google Scholar). Talin contains binding sites for actin, vinculin, focal adhesion kinase, phospholipids, and the transmembrane protein laylin (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar, 14Borowsky M.L. Hynes R.O. J. Cell Biol. 1998; 143: 429-442Crossref PubMed Scopus (116) Google Scholar). Talin was the first actin-binding protein shown to directly bind integrins and was proposed to mediate the link to the actin cytoskeleton (2Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1674) Google Scholar). Talin binds to β1, β2, and β3 and more weakly to β7 integrin cytoplasmic tails (15Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 16Sampath R. Gallagher P.J. Pavalko F.M. J. Biol. Chem. 1998; 273: 33588-33594Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 17Calderwood D.A. Zent R. Grant R. Rees D.J.G. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 18Patil S. Jedsadayanmata A. Wencel-Drake J.J. Wang W. Knezevic I. Lam S.C.T. J. Biol. Chem. 1999; 274: 28575-28583Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Talin accumulation is an early step in FA formation and requires integrins but not vinculin (19Moulder G.L. Huang M.M. Waterston R.H. Barstead R.J. Mol. Biol. Cell. 1996; 7: 1181-1193Crossref PubMed Scopus (70) Google Scholar). Microinjection of antibodies to talin or talin antisense RNA disrupts stress fibers and inhibits adhesion, spreading, and migration of fibroblasts and HeLa cells (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar, 20Albiges-Rizo C. Frachet P. Block M.R. J. Cell Sci. 1995; 108: 3317-3329Crossref PubMed Google Scholar). The significance of talin for integrin function has been underscored by studies of talin-null ES cells, which exhibit extensive membrane blebbing, defects in cell adhesion and spreading, and a failure to assemble FA or stress fibers (21Priddle H. Hemmings L. Monkley S. Woods A. Patel B. Sutton D. Dunn G.A. Zicha D. Critchley D.R. J. Cell Biol. 1998; 142: 1121-1133Crossref PubMed Scopus (153) Google Scholar). These results suggest that talin is required for the integrin-cytoskeleton associations needed for FA and stress fiber formation. However, undifferentiated talin-null ES cells also express reduced levels of β1 integrin, vinculin, and α-actinin, which may contribute to the phenotype. Following differentiation of talin-null ES cells, only two morphologically distinct cell types emerged, and no organized tissues were formed (21Priddle H. Hemmings L. Monkley S. Woods A. Patel B. Sutton D. Dunn G.A. Zicha D. Critchley D.R. J. Cell Biol. 1998; 142: 1121-1133Crossref PubMed Scopus (153) Google Scholar). The differentiated cells expressed normal levels of β1integrin and vinculin and were capable of spreading and forming actin filaments and FA-like structures, indicating that in a subset of differentiated cell types, intact talin is dispensable for maintenance of β1 integrin expression and FA assembly. Integrin-binding sites have been localized to both the talin-head and rod domains (17Calderwood D.A. Zent R. Grant R. Rees D.J.G. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 18Patil S. Jedsadayanmata A. Wencel-Drake J.J. Wang W. Knezevic I. Lam S.C.T. J. Biol. Chem. 1999; 274: 28575-28583Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), suggesting that binding of two or more integrin β tails to the talin dimer may facilitate integrin clustering (Fig.1). Binding of both the head and rod domains are inhibited by Tyr to Ala mutations in the membrane-proximal NPXY motif of β1 and β3 integrins (15Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 17Calderwood D.A. Zent R. Grant R. Rees D.J.G. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 22Kaapa A. Peter K. Ylanne J. Exp. Cell Res. 1999; 250: 524-534Crossref PubMed Scopus (40) Google Scholar), consistent with the failure of integrins expressing this mutation to localize to FA (10Reszka A.A. Hayashi Y. Horwitz A.F. J. Cell Biol. 1992; 117: 1321-1330Crossref PubMed Scopus (242) Google Scholar, 11Ylanne J. Huuskonen J. O'Toole T.E. Ginsberg M.H. Virtanen I. Gahmberg C.G. J. Biol. Chem. 1995; 270: 9550-9557Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In v-Src-transformed cells, which exhibit reduced cell adhesion and a disorganized cytoskeleton, the NPXY motif in the β1 tail is phosphorylated on tyrosine, and talin binding is inhibited (23Tapley P. Horwitz A. Buck C.A. Duggan K. Rohrschneider L. Oncogene. 1989; 4: 325-333PubMed Google Scholar). Furthermore, synthetic peptides spanning the NPXY motif bind purified talin and inhibit talin binding to β1 (22Kaapa A. Peter K. Ylanne J. Exp. Cell Res. 1999; 250: 524-534Crossref PubMed Scopus (40) Google Scholar, 23Tapley P. Horwitz A. Buck C.A. Duggan K. Rohrschneider L. Oncogene. 1989; 4: 325-333PubMed Google Scholar). Thus, the talin-binding site in the β1 tail includes this sequence. However, other regions of the β tail are likely to contribute to the interaction with talin because the NPXY motif is highly conserved between integrin β subunits, but talin displays differential binding to various integrin β tails (15Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Indeed, deletion of the C-terminal 13 amino acids of the β1cytoplasmic tail, which leaves the membrane-proximal NPXY site intact, inhibits both talin binding in vitro and co-localization of talin and actin with clustered β1integrins in vivo (22Kaapa A. Peter K. Ylanne J. Exp. Cell Res. 1999; 250: 524-534Crossref PubMed Scopus (40) Google Scholar, 24Lewis J.M. Schwartz M.A. Mol. Biol. Cell. 1995; 6: 151-160Crossref PubMed Scopus (130) Google Scholar). In contrast, deletion of only the four most C-terminal amino acids from β1 has no effect on talin binding or recruitment of talin and actin to sites of clustered integrins (22Kaapa A. Peter K. Ylanne J. Exp. Cell Res. 1999; 250: 524-534Crossref PubMed Scopus (40) Google Scholar, 24Lewis J.M. Schwartz M.A. Mol. Biol. Cell. 1995; 6: 151-160Crossref PubMed Scopus (130) Google Scholar). Furthermore, a recent report concluded that talin could bind specifically to peptides corresponding to the membrane-proximal sequence of the β3 tail (18Patil S. Jedsadayanmata A. Wencel-Drake J.J. Wang W. Knezevic I. Lam S.C.T. J. Biol. Chem. 1999; 274: 28575-28583Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Thus, further work is required to determine the precise mode of interaction between integrin β tails and talin. Three distinct filamin genes have been reported, and alternative splicing allows for additional isoforms (25Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2122-2128Crossref PubMed Scopus (381) Google Scholar). Filamins are actin filament cross-linking proteins composed of two parallel 280-kDa subunits. Each subunit contains an N-terminal actin-binding domain composed of two calponin homology domains, followed by 23 repeating domains (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar, 13Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (3058) Google Scholar). Depending on the filamin:F-actin ratio, filamin reinforces loose microfilament nets such as those found in the cell cortex (Fig. 1) or tightly packed bundles as found in stress fibers (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar). Filamin also binds to the cytoplasmic domains of transmembrane proteins (e.g. GP Ibα) and to intracellular signaling molecules (e.g. RalA) (25Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2122-2128Crossref PubMed Scopus (381) Google Scholar, 26Fox J.W. Lamperti E.D. Eksioglu Y.Z. Hong S.E. Feng Y. Graham D.A. Scheffer I.E. Dobyns W.B. Hirsch B.A. Radtke R.A. Berkovic S.F. Huttenlocher P.R. Walsh C.A. Neuron. 1998; 21: 1315-1325Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar). Filamin localizes to the cortical actin cytoskeleton and along the length of stress fibers but is also found in some FA (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar). β1A, β2, β3, β7, and to a lesser extent β1D integrin tails can bind filamin, and Tyr to Ala point mutations in the membrane-proximal β1 NPXY motif inhibit binding (15Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 27Sharma C.P. Ezzell R.M. Arnaout M.A. J. Immunol. 1995; 154: 3461-3470PubMed Google Scholar). Both filamin and F-actin are recruited to β1-containing FA in response to mechanical stress, but F-actin recruitment does not take place in melanoma cells lacking filamin (28Glogauer M. Arora P. Chou D. Janmey P.A. Downey G.P. McCulloch C.A. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The gene encoding human filamin-1 is located on the X chromosome, and mutations of this gene are associated with periventricular heterotopia, indicating a requirement for filamin-1 in neuronal migration during brain development (26Fox J.W. Lamperti E.D. Eksioglu Y.Z. Hong S.E. Feng Y. Graham D.A. Scheffer I.E. Dobyns W.B. Hirsch B.A. Radtke R.A. Berkovic S.F. Huttenlocher P.R. Walsh C.A. Neuron. 1998; 21: 1315-1325Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar). Loss of filamin-1 expression in neuronal or melanocytic cells results in impaired migration and altered morphology (26Fox J.W. Lamperti E.D. Eksioglu Y.Z. Hong S.E. Feng Y. Graham D.A. Scheffer I.E. Dobyns W.B. Hirsch B.A. Radtke R.A. Berkovic S.F. Huttenlocher P.R. Walsh C.A. Neuron. 1998; 21: 1315-1325Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar, 29Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (503) Google Scholar). However, filamin-1 null melanocytic cells also have reduced levels of many cell surface receptors, including β integrins (30Meyer S.C. Sanan D.A. Fox J.E. J. Biol. Chem. 1998; 273: 3013-3020Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), which may account for some of these phenotypes. α-Actinin is another homodimeric actin-binding protein localized to FA (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar). Non-muscle α-actinin monomers are ∼100-kDa rodlike proteins containing three functional domains: an N-terminal actin-binding domain composed of two calponin homology domains, a central region of four spectrin-like repeats, and a C-terminal domain containing two EF hands. At least two α-actinin genes and alternative splicing allow for production of a number of α-actinin isoforms. In addition to binding F-actin, α-actinin binds the FA proteins vinculin, zyxin, and β1, β2, and β3 integrins (2Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1674) Google Scholar,8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar) (Figs. 1 and 2). α-Actinin targets to FA in microinjected cells and in a cell-free system, apparently by interaction with β cytoplasmic tails (31Pavalko F.M. Chen N.X. Turner C.H. Burr D.B. Atkinson S. Hsieh Y.F. Qiu J. Duncan R.L. Am. J. Physiol. 1998; 275: C1591-C1601Crossref PubMed Google Scholar, 32Cattelino A. Albertinazzi C. Bossi M. Critchley D.R. de Curtis I. Mol. Biol. Cell. 1999; 10: 373-391Crossref PubMed Scopus (30) Google Scholar). The binding sites for α-actinin have been localized to the membrane-proximal half of the β1 or β2 integrin tail, and binding to β2 is negatively regulated by sequences in the C-terminal region of the tail (16Sampath R. Gallagher P.J. Pavalko F.M. J. Biol. Chem. 1998; 273: 33588-33594Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The membrane-proximal location of the α-actinin-binding site within β tails is consistent with the observation that antibody-mediated clustering of β1integrins lacking the C-terminal 13 amino acids also induces clustering of α-actinin (24Lewis J.M. Schwartz M.A. Mol. Biol. Cell. 1995; 6: 151-160Crossref PubMed Scopus (130) Google Scholar). However, α-actinin binding to clustered integrins is not sufficient to recruit F-actin (24Lewis J.M. Schwartz M.A. Mol. Biol. Cell. 1995; 6: 151-160Crossref PubMed Scopus (130) Google Scholar, 32Cattelino A. Albertinazzi C. Bossi M. Critchley D.R. de Curtis I. Mol. Biol. Cell. 1999; 10: 373-391Crossref PubMed Scopus (30) Google Scholar). Overexpression of α-actinin in fibroblasts leads to more stable attachment sites whereas isolated integrin-binding fragments of α-actinin disrupt stress fibers, FA, and shear-induced mechanical signaling in fibroblasts and osteoblasts (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar, 31Pavalko F.M. Chen N.X. Turner C.H. Burr D.B. Atkinson S. Hsieh Y.F. Qiu J. Duncan R.L. Am. J. Physiol. 1998; 275: C1591-C1601Crossref PubMed Google Scholar). Recent reports suggest that additional proteins may serve as direct links between integrin tails and the cytoskeleton. In platelets, the two tyrosines in the β3 cytoplasmic tail become phosphorylated during agonist-induced cell aggregation (33Law D.A. DeGuzman F.R. Heiser P. Ministri-Madrid K. Killeen N. Phillips D.R. Nature. 1999; 401: 808-811Crossref PubMed Scopus (281) Google Scholar). Synthetic peptides corresponding to the tyrosine-phosphorylated β3 tail bind to the actin-binding protein, myosin (34Jenkins A.L. Nannizzi-Alaimo L. Silver D. Sellers J.R. Ginsberg M.H. Law D.A. Phillips D.R. J. Biol. Chem. 1998; 273: 13878-13885Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This interaction may be physiologically relevant because conversion of the two β3tail tyrosines to phenylalanine is associated with a mild bleeding phenotype in mice (33Law D.A. DeGuzman F.R. Heiser P. Ministri-Madrid K. Killeen N. Phillips D.R. Nature. 1999; 401: 808-811Crossref PubMed Scopus (281) Google Scholar). Skelemin, a cytoskeletal M-band protein, can bind β1 and β3 but not β2tails expressed in vitro (35Reddy K.B. Gascard P. Price M.G. Negrescu E.V. Fox J.E.B. J. Biol. Chem. 1998; 273: 35039-35047Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Skelemin co-localizes with stably expressed αIIbβ3 under some conditions, and microinjection of the integrin-binding domain of skelemin causes myoblasts to round up (35Reddy K.B. Gascard P. Price M.G. Negrescu E.V. Fox J.E.B. J. Biol. Chem. 1998; 273: 35039-35047Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Whether these interactions represent widespread or specialized cases of integrin-cytoskeleton linkages remains to be determined. Vinculin, an ∼120-kDa molecule, is one of the most abundant FA proteins and interacts with F-actin, talin, α-actinin, paxillin, and vasodilator-stimulated phosphoprotein (VASP) (8Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (432) Google Scholar, 36Bubeck P. Pistor S. Wehland J. Jockusch B.M. J. Cell Sci. 1997; 110: 1361-1371PubMed Google Scholar). Vinculin does not bind directly to integrins but may be recruited by integrin-bound talin or α-actinin (Fig. 1). However, although reduced levels of vinculin result in a reduction in the mechanical stiffness of the integrin-cytoskeleton linkage and increased cell motility, vinculin-null ES cells can differentiate in vitro into a variety of cell types (37Xu W. Baribault H. Adamson E.D. Development. 1998; 125: 327-337Crossref PubMed Google Scholar) and can spread and form talin-rich FA and stress fibers (21Priddle H. Hemmings L. Monkley S. Woods A. Patel B. Sutton D. Dunn G.A. Zicha D. Critchley D.R. J. Cell Biol. 1998; 142: 1121-1133Crossref PubMed Scopus (153) Google Scholar). Thus, despite ubiquitous expression, vinculin is not absolutely required for some integrin-F-actin linkages and is likely to function as a molecular to linkages. Integrins are to of binding by intracellular and this has been signaling or integrin P. Pfaff M. Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). signaling may by 1) changes in and the of integrin and clustering into These of modulation are not and may in a to both binding and binding N. J. Cell Biol. 1998; PubMed Scopus Google Scholar). signals to integrins from plasma membrane As with to other of the these receptors regulate integrins by such as that affect the localization of and in J.D. Science. 1997; PubMed Scopus Google Scholar). Thus, cytoskeletal proteins could signaling by the of molecules by to integrin cytoplasmic tails P. Pfaff M. Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). or in certain cytoskeletal proteins be to the integrin directly as the result of regulated changes in integrin-cytoskeleton linkages. Furthermore, in cases integrins bind to on other cells instead of ECM, integrin may be by of the membranes on the cells M.L. J.M. J. 1996; PubMed Scopus Google Scholar). As a of of integrin function by cytoskeletal β2 integrins from not β2 and with talin but not α-actinin (16Sampath R. Gallagher P.J. Pavalko F.M. J. Biol. Chem. 1998; 273: 33588-33594Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). However, cell by induces binding to the β2 integrins and talin and from β2 in a dependent on the a of cell β2 now with α-actinin and not talin, an association to result from a in of the β2 cytoplasmic tail (16Sampath R. Gallagher P.J. Pavalko F.M. J. Biol. Chem. 1998; 273: 33588-33594Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In this integrin be by of one integrin-cytoskeleton linkage and by This may also to of other but the precise may in is a relatively the of αIIbβ3 and it is for of cytoskeletal and signaling proteins, including the β3 cytoplasmic tail S. Williams M.J. Ginsberg M.H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). interactions between cytoskeletal proteins and integrin cytoplasmic tails also in a increase in a that with increased cell adhesion to M.L. J.M. J. 1996; PubMed Scopus Google Scholar). of these cells with of which actin filaments further has the whereas of inhibit cell of to induces clustering of and adhesion of the cells to M. Y. C.G. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar), and the is in cross-linking of the cell M.P. A. N. J. Cell Biol. 1998; PubMed Scopus Google Scholar). In it has been proposed that an protein, may the of in a dependent on by protein kinase Wang J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). Cytoskeletal linkages may also be involved in the of αIIbβ3 in in a of αIIbβ3 is associated with the membrane cytoskeleton, and relatively of or which