This article outlines the present knowledge of the architecture, molecular composition, and dynamics of focal contacts of adhesive animal cells. These structures, developed at the plasma membrane at sites where cells touch their substratum, are essential for cellular attachment in tissue formation during embryogenesis and wound healing. In tissue culture, they are particularly prominent and thus amenable to detailed investigation. Focal contacts consist of a cytoplasmic face, comprising cytoskeletal elements, a transmembrane connecting region, and a extracellular face composed of proteins of the extracellular matrix. The molecular anatomy of the numerous proteins involved, the basis for classifying them as structural or regulatory components, and their in vitro interactions are described. Based on this information, current models on the dynamics of their assembly and of possible regulatory mechanisms involving a variety of signal transduction pathways are discussed.

Abstract. Vinculin, a major structural component of vertebrate cell-cell and cell-matrix adherens junctions, has been found to interact with several other junetional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli.In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin illaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893--1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction.In fixed and permeabilized cells (cell models), as well as after microinjeetion, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments.Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures. "~ 7"INCULIN is an important component of junctional complexes. Its ll6-kD polypeptide chain, folded I' into a large NH2-terminal head and an extended rodlike tail (7,8,24,25) was localized at the cytoplasmic face of both, cell-matrix and cell-cell junctions in vertebrates and invertebrates. It is indispensible for correct cell attachment and mobility, as shown by several independent lines of evidence. Focal adhesions of cultured fibroblasts are effectively disrupted upon microinjection of monoclonal antibodies directed against several epitopes in the vinculin head (34), and interference with the correct level of vinculin expression leads to drastic alterations in cellular morphology, adhesiveness and motility (10-12). Mutations in the single vinculin gene are incompatible with normal development in the nematode (2). In vitro, the protein, as isolated from chicken smooth muscle, interacts with several other junctional components, i.e., talin, ot-actinin, paxillin, tensin, and with itself, and also with acidic phospholipids (see refer- ences 14, 16, 23, 26, and 27). While talin, ~actinin and tensin are all believed to bind to act.in as well, for vinculin, the corresponding experimental data (17-19, 29, 34) have been controversely discussed (26,35,36). In particular, the purity of vinculin preparations used to show binding to filamentous actin was questioned, and contamin...

Vinculin, a structural protein of animal cells, is critically involved in the assembly of microfilament/ plasma membrane junctions at cell contacts. To understand its role in organizing the distal portions of microfilaments into specific, morphologically distinct structures at these sites in more detail, we characterized its interaction with filamentous actin and with itself by means of in vitro assays. Using recombinant proteins comprising different parts of the vinculin tail fused to the maltose-binding protein of Escherichia coli, we show in sedimentation assays that this part of vinculin harbors two discrete sites that can bind to actin independently. They reside within amino acid residues 893-985 and 1016-1066 of the 1066-residue polypeptide chain. However, both sites are necessary to cross-link or bundle actin filaments, as demonstrated by low shear viscometry. Crosslinking and bundling are alternatives determined by the molar ratio of fusion protein to F-actin. Both actin-binding sequences are capable of oligomer formation, as shown in chemical-cross-linking and dot-overlay assays. These data allow us to propose a possible role for vinculin in organizing the distal ends of microfilaments at the plasma membrane into the pointlike structure characteristic for cell-matrix contacts.

Vinculin, a prominent protein component of microfilament-membrane attachment sites, consists of three major domains: an N-terminal, compact head and a C-terminal rod-like tail that are connected by a flexible, proline-rich hinge. In vitro, the protein has been shown to interact with numerous ligands, including other components of the microfilament system. To characterize the ligand recruitment ability of the different vinculin domains in a cellular environment, we used a novel approach of comprising chimeric proteins of either the vinculin head, hinge or tail regions, fused to the membrane anchor sequence of ActA, a surface protein of the intracellular bacterial pathogen Listeria monocytogenes. When PtK2 cells were transfected with the corresponding constructs, the ActA membrane anchor directed the chimeric polypeptides to mitochondrial membranes. In this position, they accumulated microfilament proteins, as seen by immunofluorescence analysis. A chimera comprising the full length vinculin clone recruited a substantial amount of the cellular F-actin, the vasodilator stimulated phosphoprotein (VASP) and paxillin, but little alpha-actinin and talin. The presence of only the vinculin head directed some of the fusion protein to focal contacts, and alpha-actinin recruitment was still ineffective. Prominent recruitment of F-actin and of VASP required the presence of the tail and proline-rich hinge, respectively. Reducing the vinculin tail to short pieces harboring only one of the two F-actin binding sequences, which were defined by in vitro experiments, resulted in loss of activity, possibly by incorrect polypeptide folding. The proline-rich hinge domain could be exchanged for the analogous region of the ActA protein, and the number of such proline-clusters, containing an FPPPP motif, correlated with the extent of VASP recruitment. The results show that this system can be used to analyze in vivo the activity of vinculin domains responsible for the assembly of various cytoskeletal ligands.