The Iong-term goal of this research is to understand how vinculin regulates cell adhesion and motility. inculin is essential for embryonic development in mice and for regulation of adhesion and motility on xtracellular matrix substrates. Current evidence supports a model in which vinculin's role in a focal adhesion is to provide a bifunctional interaction with talin-integrin complexes at vinculin's head domain and actin filaments at its tail domain. In purified vinculin the actin and talin binding sites are masked by an intramolecular interaction between the head (Vh) and tail (Vt) domains of vinculin. Regulation of the head/tail interaction to expose or hide the ligand binding sites is hypothesized to be the mechanism by which vinculin regulates the attachment of membrane proteins to cytoskeleton to control adhesion and motility in cells. A goal of the present proposal is to test this model in living cells. For this purpose, two mutants deficient in intramolecular head/tail interaction were developed and a fluorescence resonance energy transfer (FRET) probe that reports on the closed, open, and actin-bound conformations of vinculin have been constructed. When expressed in cells the mutants have a localization and dynamic phenotype consistent with the interpretation that they stabilize a subset of focal adhesion plaque proteins representing an intermediate in a process carried out by wild type vinculin. To elucidate this process and to test the requirement for regulation of the intramolecular head/tail interaction in the function of vinculin, we propose the following plan. ? Aim 1, Use the loss of function mutants to identify the requirement for regulated head/tail interaction in rescue of adhesion and motility phenotypes in vinculin null cells. ? Aim 2, test the hypothesis that the phenotype generated by the constitutive talin-binding conformation of vinculin represents a stalled intermediate in endocytosis of the a5bl integrin receptor for FN. ? Aim 3, use the conformation-sensitive vinculin FRET probe to determine when and where in a cell vinculin undergoes conformational regulation and to test the hypothesis that activation of vinculin responds to changes in mechanical forces and contractility. ? Aim 4, test the hypothesis that vinculin's effects on cell adhesion and motility are mediated through Rac, RhoA, or Cdc42 pathways. ? ?
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