Our objective is to elucidate the molecular mechanisms by which cellular adhesion complexes form and remodel in response to mechanical load. Cell-cell and cell-matrix adhesions are a defining feature of metazoan life and are essential to the physiological function of virtually every tissue in the human body. Despite this central importance, only a few of the protein-protein interactions that make up adhesion complexes have been characterized biochemically, and even less is known about the underlying mechanisms by which these structures respond to mechanical load. This lack of quantitative data presents an unavoidable roadblock in the collective effort to understand how cells build and remodel multicellular tissues. We will use single-molecule biophysical approaches to develop a detailed understanding of how adhesion complexes templated by E-cadherin sense and transduce mechanical cues. Previously, we demonstrated that a complex of E-cadherin, β-catenin, and αE-catenin forms a minimal force-sensing unit at intercellular adhesions. Here, we build on this result to test the hypothesis that this complex lies at the heart of a mechanosensory assembly that converts small changes in input forces into dramatic alterations in adhesion architecture, size, and stability. In parallel work, we will use biophysical techniques unique to our laboratory to determine how directional interactions between proteins within adhesion complexes and filamentous (F)-actin may give rise to long-range organization in the cytoskeleton. Recently, we found that the protein vinculin, which is recruited to both cell- matrix and intercellular adhesions, forms a directionally asymmetric interaction with F-actin that is stabilized ~10- fold when load is oriented toward the pointed (-) vs. barbed (+) end of the actin filament. Preliminary data suggest that force-dependent, asymmetric binding interactions with F-actin are not unique to vinculin, and likely extend to other adhesion proteins. These observations suggest that asymmetric interactions between F-actin and proteins within adhesion complexes may play a central and previously unsuspected role in organizing cells and tissues, a hypothesis that we will test during the next funding period. Cell and developmental biological data indicate that αE-catenin plays a central role in organizing epithelial tissues through its interactions with zonula occludens-1 (ZO-1) and afadin, both of which bind F-actin and recruit other scaffolding and signaling proteins. We will perform the first detailed biochemical and biophysical characterization of the interaction of the cadherin-catenin complex with ZO-1 and afadin, and use cutting-edge imaging techniques to determine how these proteins interact in living cells. These studies will lay the foundation for a quantitative understanding of how intercellular adhesion complexes function as integrated, multifunctional force-sensing assemblies.