Project Summary Insulin secretion from pancreatic β cells is crucial to maintaining blood glucose homeostasis, allowing cells throughout the body to take up glucose from the blood and obtain nutrients for normal cellular processes. In type 2 diabetes, however, peripheral tissues become insulin resistant, β cells over-secrete insulin to compensate, and eventually β cells become dysfunctional. It is therefore necessary to thoroughly understand the mechanisms of insulin secretion in order to identify potential therapeutic targets for type 2 diabetes treatment. Insulin secretion is known to predominantly occur at the vascular face of the β cell, at regions termed “hot spots.” Hot spots are characterized by proteins common to the active zone of neurons as well as proteins of cortical microtubule-stabilizing complexes and focal adhesions. Our laboratory has also found that microtubule destabilization promotes insulin secretion specifically at hot spots. Others have shown that the actin cytoskeleton also plays a critical regulatory role in insulin secretion. Given the known connections of microtubules, focal adhesions, and actin with known hot spot proteins, it is likely that the interplay of these cytoskeletal elements is essential for the hot spot organization and function. I therefore hypothesize that insulin secretion hot spots are “secreting adhesions”: mechanosensitive subcellular domains which use cytoskeletal regulation to accomplish directed and clustered secretion. My specific aims are to 1) characterize the cytoskeleton networks, their interplay, and other hot spot components in β cells; and to 2) test if MT- regulated RhoA-dependent contractility promotes clustered secretion via secreting adhesion assembly. To address these aims, I will use high- and super-resolution fixed and live-cell imaging techniques combined with computational analyses. Improving the understanding of hot spot identity and establishment via the cytoskeleton will contribute to overall knowledge of insulin secretion, and potentially uncover novel targets for diabetes therapeutics.