The formation of cell-matrix (focal adhesions) and cell-cell (adherens junctions) adhesion complexes links signals at the cell surface to the actin cytoskeleton, and these direct cell migration, cell growth and survival, and the morphological changes that are needed for proper development. Signaling from focal adhesions or adherens junctions is directed by integrin or cadherin transmembrane receptors, respectively, and their links to the actin cytoskeleton require activation of the cytoskeletal protein vinculin, which binds to proteins that directly interact with these receptors, such as talin, a-actinin, and a-catenin, as well as to components of the machinery that controls cell migration. During the past funding cycle of GM071596 we defined the structure of the closed, inactive conformation of vinculin, which is compromised of five, loosely-packed helical bundle domains that are held in a closed-clamp conformation via extensive hydrophobic interactions of its N-terminal seven-helical bundle (Vh1) domain with its five-helical bundle tail (Vt) domain. Our studies also defined the atomic changes that accompany vinculin activation, where the Vh1 domain undergoes remarkable structural changes that displace Vt domain from a distance, and which release the domains of vinculin to allow binding to its partners. Finally, we demonstrated that talin and a-actinin are physiological triggers that can activate vinculin, and that they must first undergo structural alterations to bind to and activate vinculin, establishing that adhesion signaling involves a chain reaction of structural alterations. While these surprising and exciting advances defined the mechanism and structural alterations that control vinculin activation, very little is known regarding how activated vinculin binds to its numerous partners, or how it directs such diverse processes throughout the cell. Here we propose to address these important questions in a head-on fashion, together with functional studies by solving the crystal structures of activated vinculin in complex with three partners that control cell adhesion and cell migration, and the localized production of adhesion components at nascent junctions. Further, we will solve the crystal structure of metavinculin, an isoform of vinculin that is exclusively expressed in muscle tissue, and we will also define the interactions that are required for metavinculin function. Collectively, the proposed studies will resolve how activated vinculin and metavinculin direct their diverse functions, and they will lay the foundation for targeting their interactions for the treatment of diseases having vinculin or metavinculin involvement, in particular metastatic cancer, ischemia, and myopathies. PUBLIC HEALTH RELEVANCE Cells require distinct adhesion complexes at their cell surface to form contacts with their neighbors or with the extracellular environment, and the protein vinculin plays essential roles in linking these adhesion complexes to the actin cytoskeleton, and in directing the cell migration machinery. The formation of these links requires that vinculin transition from its closed, inactive conformation to its activated state, and the studies supported by R01 GM071596 defined the structure of inactive and activated vinculin, and revealed its mechanism of activation. However, essentially nothing is known regarding the interactions of activated vinculin with its binding partners in the cell, and our new studies in this revised competitive renewal application of R01 GM071596 will define the structure and function of vinculin in complex with three of its partners that play essential roles in adhesion complexes, in cell migration, and in the localized production of components of adhesion junctions. Finally, we will also define the structure and function of metavinculin, an isoform of vinculin that plays essential roles in the formation and function of muscle tissue.