Abstract This proposal seeks to establish the role of kindlin-3 in physiological and pathophysiological responses. Kindlin-3 (FERMT3) is one of the three member family of FERM domain intracellular adapter proteins, which has >20 binding partners and thereby control numerous responses in numerous cells types. Our contributions to the kindlin field have included the demonstration of their essential role in integrin activation; delineation of the molecular basis for their interaction with integrin beta subunits; identification of a disease, LADIII, associated with severe bleeding, compromised immunity, increased susceptibility to infections and osteopetrosis arising from a deficiency of kindlin-3, demonstration of not only integrin dependent, but also integrin independent functions of kindlins (e.g. perturbed hemostasis and vascular permeability); and the implication of a central role of the kindlins in cancer biology. Adding to the importance of kindlin-3 is evidence that it is not restricted to hematopoietic cells, but is present in and contributes to the biological responses of endothelial cells and cancer cells, particularly to the progression and metastasis of breast cancer. Despite the clear significance of kindlin-3 in vascular biology and pathology, it is this kindlin that has been particularly resistant to analysis arising from the lack of cellular and in vivo models to understand the breadth of its biological functions. We have resolved some of these barriers and are now poised to resolve key questions regarding kindlin-3. We have developed assays and mouse strains in which the molecular basis of kindlin-3’s function can be dissected. Specifically, we have in hand mouse strains in which the integrin-dependent and independent responses can be distinguished (integrin binding site disabled) and tissue specific knockouts of kindlin-3 to delete it from monocyte/macrophages and erythroid cells. We have also developed CRISPR/cas9 technology which has allowed us to knockout or replace kindlin-3 in cancer cells. Importantly, we have identified a unique phosphorylation site in kindlin-3 that is not conserved in the other two kindlins, and have shown that this post-translational event is of functional significance in hematopoietic and breast cancer cells and distinguishes between kindlin-3 binding partners. Our specific aims are: 1) identify the role of integrin- dependent and -independent functions in breast cancer progression and metastasis; 2) determine the importance of phosphorylation of kindlin-3 in vascular and cancer cells; and 3) determine how kindlin-3 influences erythropoiesis and erythroid cell shape. Overall, these studies will establish basic mechanisms by which kindlin-3 exerts its known functions, may identify previously unappreciated functions of this molecule and may establish if kindlin-3 can serve as a biomarker and as a therapeutic target.