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.

In epithelial cells, α-, β-, and γ-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. α-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell–matrix and cell–cell contacts, α-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell–cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and α-catenin. We show that α-catenin colocalizes at cell–cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to α-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The K d of the complex was determined to 2–4 × 10−7 M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of α-catenin is involved in this interaction.Complex formation of vinculin and α-catenin was challenged in transfected cells. In PtK2 cells, intact α-catenin and α-catenin1-670, harboring the β-catenin– binding site, were directed to cell–cell contacts. In contrast, α-catenin697–906 fragments were recruited to cell–cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo α-catenin, like vinculin, is tightly regulated in its ligand binding activity.

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...

Abstraet Using blot overlay techniques we have investigated the interaction of vinculm with u-a&tin. We show that an a-actinin binding site is located in the 90 kDa vinculin head and co&m a vinculin binding site in the C-terminal rod of a-actinin, as recently reported by McGregor et al. [(1994) B&hem. J. 310,2252331. The isolated vinculin head binds much more strongly to a-actinm than intact vinculin. Using a proteolytic 81 kDa head fragment, we show that vinculin residues l-107 are required for a-actinin binding. Antibodies directed against vinculin residues 808-850 inhibit the vmculin-u-actinin binding, suggesting that this sequence is directly involved in, or topographically related to, the a-actinin binding site.

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