Microfilament-associated, cell:substrate and cell:cell junctions are the major load-bearing, force-transducing structures in non-muscle cells. The assembly and regulation of these adherens junctions is essential to many cell processes including wound healing, cell migration, and cellular adhesion. Loss of cell adhesion and the ability to form intercellular and cell:substrate junctions is tightly correlated with invasive and metastatic tumor cell behavior. Because vinculin is a diagnostic component of adherens junctions it is thought to play an important role in their assembly, structure, or function. The goal of this proposal is to define specific ways in which the properties of vinculin are important to adherens junction physiology. To learn how vinculin is recruited from cytoplasm to adherens junctions and how the interactions of vinculin with its ligands are regulated, the intramolecular association of head and tail (H/T) domains and the ability of vinculin to bind acidic phospholipids will be characterized. Antibody and enzyme probes that detect epitopes masked by the H/T interaction will be developed and used to identify the hypothesized open conformation of vinculin in which head is displaced from tail exposing the high affinity binding sites for talin and acidic phospholipid. These sites map in isolated head and tail domains, respectively. In vitro binding assays will be used to: assess whether the intramolecular interaction of vinculin head and tail is regulatory for the interaction of vinculin with alpha-actinin and actin; evaluate the hypothesis that the 46 kDa fragment of talin regulates the ability of the 190 kDa talin fragment to bind to vinculin; determine the effect of protein kinase C-mediated phosphorylation of talin on the Kd of vinculin/talin interaction define and characterize the smallest domain that retains the characteristics of the high-affinity, acidic phospholipid binding site found in the tail domain of vinculin; characterize the interaction between vinculin and phosphatidylinosito-4'5'-bisP (PIP2); and determine whether P1 and PIP2 modulate the binding of vinculin to talin, actin, or alpha-actinin. The hypothesis that vinculin plays a role in generation or maintenance of transmembrane force will be tested by downregulating the concentration of vinculin in RDA2 human rhabdomyosarcoma cells with antisense oligodeoxynucleotides. The effect of this perturbation on transmembrane force generation will be evaluated by measuring the ability of the cells to contract collagen gels. To gain insight on the requirement for E cadherin to trigger adherens junction assembly and function in L929 cells, the putative junction- dependent functions of cell compaction, development of cell surface polarity, restriction of motility, and invasiveness will be evaluated in E cadherin-transfected clones. The absence of cadherin-associated gamma- catenin in these cells will be explored with respect to its requirement for E cadherin-triggered junction assembly.
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