Rather, the plectin antibodies in A generate a predominantly filamentous staining pattern. integrin-positive focal contacts. We have investigated the function of 4-laminin and v3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and Rabbit polyclonal to VWF whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis. == INTRODUCTION PD-159020 == In cultured cells, the focal contact or focal adhesion is a region of close interaction between cells and the matrix on their substrate (Dogicet al., 1999). Focal contacts are dynamic structures that show motility even in stationary cells and are the conduits of signals from the matrix to the cytoplasm of the cell and vice versa (Howeet al., 1998;Smilenovet al., 1999;Zamiret al., 2000). Microfilament bundles associate indirectly with the cytoplasmic domains of matrix receptors of the integrin family that are enriched in focal contacts (Simon and Burridge, 1994;Dogicet al., 1999). The extracellular domains of integrins bind matrix elements such as fibronectin, vitronectin, and laminin in the connective tissue, thereby providing a structural link between the extracellular matrix and the actin-based cytoskeleton system of a cell (Hynes, 1992). In fibroblasts at least two types of matrix adhesions have been identified (Zamiret al., 1999,2000;Katzet al., 2000). These can be distinguished by their molecular composition and distribution. The classic focal contact tends to be located at the periphery of the cell and is enriched in such proteins as vinculin and paxillin. On the other hand, so-called fibrillar adhesions tend to be concentrated toward the center of the cells and contain little if any paxillin and vinculin, while being rich in PD-159020 tensin. Both the focal contact and fibrillar adhesion are associated with the microfilament cytoskeleton. In addition to focal contacts and, possibly, fibrillar adhesions, certain epithelial cells utilize a structurally distinct matrix adhesive device called a hemidesmosome to interact with the connective tissue (Joneset al., 1998). Like a focal contact, each hemidesmosome contains an integrin, namely, the 64 heterodimer, and is involved in both adhesion and signal transduction (Mainieroet al., 1995;Joneset al., 1998). However, the hemidesmosome is quite different from the focal contact in that it interacts with the keratin cytoskeleton network and fails to show any obvious association with microfilament bundles (Simon and Burridge, 1994;Joneset al., 1998). The hemidesmosome links epithelial cells to laminin in the basement membrane. Specifically, the 64 integrin of the hemidesmosome binds to the G-domain of the 3-subunit of the laminin-5 heterotrimer (Spinardiet al., 1993;Bakeret al., 1996;Joneset al., 1998). Of all the laminin -chains so far identified, the 3-subunit is structurally closest to the 4 laminin subunit in that they both contain a truncated N-terminal short arm domain, containing a single rod-like segment consisting of epidermal growth factor-like repeats. By comparison, the short arms of the 1, 2, and 5 laminin subunits contain three globular subdomains that are separated by rod-like segments (Ryanet al., 1994;Iivanainenet al., 1995;Frieseret al., 1997;Mineret al., 1997;Niimiet al., 1997;Aumailley and Smyth, 1998). Whereas the 3 laminin subunit is synthesized primarily by epithelial cells, the 4 laminin subunit is expressed in endothelium, bone marrow, adipocytes, lung fibroblasts, heart, lung, and skeletal muscle, smooth muscle, and dermis (Iivanainenet al., 1995;Liu and Mayne, 1996;Richardset al., 1996;Frieseret al., 1997;Mineret al., 1997;Niimiet al., 1997;Pierceet al., 1998;Guet al., 1999). Furthermore, the 4 laminin subunit is a component of laminin-8 and 9, heterotrimeric matrix molecules composed of 4-, 1-, and 1-chains and 4-, 2-, and 1-chains, respectively (Aumailley and Smyth, 1998;Kortesmaaet al., 2000). The goal of this study was PD-159020 to analyze the organization of the 4 laminin subunit in endothelial extracellular matrix using a new monoclonal antibody we developed. During the course of our studies, we have identified a matrix adhesion site that, like the hemidesmosome, associates with the intermediate filament cytoskeleton. However, this site of adhesion also possesses many of the characteristics of a focal contact in that it is enriched in vinculin, associates with the microfilament.