CELL SCIENCE AT A GLANCE 3577 Components of cell- microfilaments and apparently play an subunits, each of which contains a large important role in cell spreading and extracellular domain responsible for matrix adhesions migration. ligand binding, a single transmembrane Eli Zamir and Benjamin Geiger* domain and a cytoplasmic domain that FAs are transmembrane anchorage sites, can interact with the cytoskeleton. There Department of Molecular Cell Biology, The α β Weizmann Institute of Science, Rehovot 76100, in which the ECM is indirectly linked to are several - and -subunits, the Israel the actin cytoskeleton via a web of combination of which defines the *Author for correspondence (e-mail: submembrane ‘anchor proteins’. Thus, binding specificity to the ECM. [email protected]) characterization of the molecular components of these adhesions and their Across the membrane, FAs contain a Journal of Cell Science 114, 3577-3579 (2001) © The Company of Biologists Ltd precise organization appears to be meshwork of ‘anchor proteins’ that link important if we are to understand the the membrane to the actin cytoskeleton. Focal adhesions (FAs), also known as function of FAs in both the mechanical As can be seen in the poster, the ‘focal contacts’, are specialized, interaction with the matrix and the molecular complexity of this ‘sub- extracellular matrix (ECM) attachment generation and transduction of adhesion- membrane plaque’ is enormous, and >50 and signaling organelles, usually mediated signals. Immunofluorescence different proteins have been reported to measuring a few square microns, located and immunoelecron microscopy studies localize to it. Given the rather sporadic along the ventral plasma membrane of have revealed that FAs contain a manner at which FA components were adherent cultured cells. Interference surprisingly large number of proteins. discovered over more than two decades, reflection microscopy, electron The major transmembrane ECM it appears likely that the actual microscopy and micro-mechanical receptors in these sites belong to the number of molecular residents of measurements indicate that the cell integrin family, but additional trans- FA (constitutive and transient) is membrane in these sites is tightly membrane molecules, such as heparan considerably larger. Given modern tools connected with the substrate surface and sulfate proteoglycans, the hyaluronan that allow cell-based functional genomic that the gap remaining is only ~10-15 binding molecule layilin and urokinase screening, it is likely that the full nm. At their cytoplasmic aspects, FAs receptor, appear also to be present. repertoire of FA molecules will be soon are associated with bundles of actin Integrins are heterodimers of α and β unraveled. Adhesion site F-Actin G-Actin Filamin ERM Actopaxin/ EAST Abl Nexilin Fimbrin Parvin Dbl PSTPIP Profilin AND-34 Vinexin Vimentin PTP1B CAS Calpain II VASP/ PTP-PEST SrcFK ENA PSGAP Ponsin Trio PTEN DOCK180 Zyxin Csk AKT C3G Crk Paxillin PKL Crp1 Vinculin PAK PKC PYK2 Hic-5 PIX Cbl Palladin Grb7 α SOS Graf -Actinin FAK Tensin Nck-2 Grb2 ASAP1 Talin Syntenin Shc PI 3-K CASK PINCH IRS-1 ILK PLC-γ CH-ILKBP SHP-2 DRAL Syndesmos Synectin LIP.1 PtdIns (4,5)P2 Caveolin-1 Plasma membrane IR LAR PTP SHPS-1 Integrins Syndecan-4 uPAR Polycystin-1 Layilin ECM Heparan-sulfate- Hyaluronan Extracellular matrix proteins binding proteins (See poster insert) 3578 JOURNAL OF CELL SCIENCE 114 (20) The molecular complexity of the FA is interactions between the various molecular complexity of matrix probably considerably greater than that components, as well as interactions adhesions appears to provide important shown, not just because many of the between these components and insights into the structural and functional components are still unknown but because molecules not stably localized in FA. As diversity of these sites in living cells. As many of these molecules can appear in the poster shows, some of the indicated here, the known repertoire of more than one form - owing to post- interactions within FAs are mediated by FA components is probably incomplete, translational modification, proteolytic known binding motifs, such as SH2 and and the information on their assembly processing, alternative splicing and SH3 domains, but the majority of following interaction with the ECM is conformational reorganization. For binding motifs are still poorly still very limited. The greatest challenge example, tyrosine-specific protein phos- characterized. Note that some of the in future characterization of these sites phorylation, one of the hallmarks of FAs, interactions (e.g. those between SH2 will be to understand the mechanisms of can affect binding to SH2 domains in domains and tyrosine phosphorylated adhesion-mediated signaling in these different proteins, and PtdIns(4,5)P2- proteins) can be regulated by specific sites and how this affects cell behavior induced conformational changes in enzymatic activities (tyrosine phos- and fate. vinculin can alter its interactions with phorylation or dephosphorylation, in this some of its partners (see below). case) and thus may be switched on and Abbreviations, synonyms and approximate molecular weights: α-actinin, 103 kDa; Abl, 145 off. kDa; Actopaxin/Parvin, 42 kDa; AKT (PKB), 55 Let us take a closer look at the known kDa; AND-34, 93 kDa; ASAP1, 130 kDa; C3G, components of FA. Sorting these Evidently, many of the components of 145-155 kDa; Calpain II, 80 kDa and 30 kDa proteins according to their biochemical FA can interact with more than one subunits; CAS, Crk-associated substrate, 130 kDa; CASK, calcium/calmodulin-dependent serine activity reveals among them (often 5-10 or more) molecular partner; protein kinase, 104 kDa; Caveolin-1, 21-24 kDa; cytoskeletal proteins (defined by their this suggests that they can - at least Cbl, 120 kDa; CH-ILKBP, calponin homology direct or indirect association with actin in theory - assemble in numerous domain-containing ILK-binding protein, 53 kDa; and the absence of enzymatic activity), alternative combinations, giving rise to Crk, 33 kDa; Crp1, cysteine-rich protein-1, 23 α kDa; Csk, C-terminal Src kinase, 50 kDa; Dbl such as tensin, vinculin, paxillin, - highly diverse supramolecular com- (MCF-2), 115 kDa (proto-Dbl) or 66 kDa (Dbl); actinin, parvin/actopaxin and talin, plexes. However, only a fraction of the DOCK180, 180 kDa protein downstream of tyrosine kinases, such as members of interactions identified by biochemical Crk, 180 kDa; DRAL, downregulated in the Src family, FAK, PYK2, Csk and studies can actually take place rhabdomyosarcoma LIM-protein (FHL2 and SLIM3), 32 kDa; EAST, EGFR-associated protein Abl, serine/threonine kinases, such as simultaneously, since many of the with SH3 and TAM domains, 68 kDa; ECM ILK, PKC and PAK, modulators of partners either compete at the same site proteins, extracellular matrix proteins (a wide small GTPases, such as ASAP1, Graf or sterically interfere with the binding of variety of proteins such as fibronectin, vitronectin and PSGAP, phosphatases, such as other molecules to nearby locations (see, and collagen); ERM family, ezrin radixin moesin, 68-69 kDa; F-Actin, filamentous (polymerized) SHP-2 and LAR PTP, and other for example, the interactions of many actin; FAK, focal adhesion kinase, 125 kDa; enzymes, such as PI 3-kinase and the anchor proteins with the short Filamin, 280 kDa; Fimbrin, 68-70 kDa; G-Actin, protease calpain II. Are all these cytoplasmic tail of integrin, the multiple globular (monomeric) actin, 42 kDa; Graf, components constitutively located in all SH2- and SH3-mediated interactions of GTPase regulator associated with FAK, 95 kDa; ECM adhesions in all cells? Probably FAK etc.). This notion does not really Grb2, growth-factor-receptor-bound protein 2, 25 kDa; Grb7, growth-factor-receptor-bound protein not, because many of the components ‘simplify’ the picture but instead 7, 59 kDa; Heparan-sulfate binding proteins, may be expressed in a cell-type-specific suggests that FAs are a molecularly many ECM molecules, growth factors, protease manner, and others may be differentially diverse family of structures or consist of inhibitors, lipoproteins and others; Hic-5, enriched in specific subtypes of FA or a heterogeneous array of supramolecular hydrogen-peroxide-inducible clone 5, 55 kDa; Hyaluronan, hyaluronic acid ([-β(1,4)-GlcUA- FA-related adhesions, such as fibrillar complexes. Such heterogeneity could, β(1,3)-GlcNAc-]n), 1000-10000 kDa; ILK, adhesions, podosomes or focal com- of course, affect the function of the integrin-linked kinase, 59 kDa; Integrins, variable plexes. Thus, the scheme shown should specific adhesion. Indeed, quantitative αβ heterodimer receptors of ECM proteins; IR, not be regarded as a representative microscopy indicates that adhesion sites insulin receptor, oligotetramer composed of two heterodimers of 130 kDa and 90 kDa subunits; snapshot of FA architecture. can be molecularly heterogeneous IRS-1, insulin receptor substrate 1, 185 kDa; LAR structures that have diverse PTP, leukocyte-common-antigen-related protein What are the molecular interactions that morphologies, molecular compositions tyrosine phosphatase, 200 kDa (proteolytically take place between FA components? and phosphotyrosine levels (e.g. fibrillar processed into 150 kDa and 85 kDa subunits); Layilin, 55 kDa; LIP.1, LAR-interacting protein This question can be slightly rephrased adhesions, focal complexes and 1, 160 kDa; Nck-2, 43 kDa; Nexilin, 72-78 kDa; according to the experimental approach podosomes). Given the relatively