PROJECT SUMMARY Insulin secretion deficiency is a hallmark of diabetes. In this process, the cortical actin of β cells has recently been recognized as a critical player contributing to diabetes pathogenesis. β cells from type-2 diabetes (T2D) patients show significant signaling alteration associated with cortical actin remodeling, and restoring such abnormality rescues insulin secretion to normal levels. Cortical actin is thought initially as a simple barrier to prevent insulin secretion, but recent studies challenge this view and imply an active role. Mounting evidence further indicates that cortical actin is a converging point and effector of multiple metabolic signals in β cells. However, the molecule and nanostructure basis of its remodeling remains unresolved. Addressing this question becomes imperative to understanding the regulation of insulin secretion and developing new therapeutics to restore β cell function in T2D. This proposal will investigate ArpC3, a gene encoding a subunit of the Arp2/3 complex that dictates actin filament branching and cortical remodeling. Although multiple signaling pathways converge on Arp2/3, the mechanism downstream Arp2/3 activity to regulate insulin secretion remains unknown. Based on recent progress in the field and our new data on β cell imaging and functional assays, we hypothesize that ArpC3-dependent actin polymerization regulates insulin exocytosis by driving the local cortical actin disassembly-reassembly cycles. Through this remodeling, cortical actin filaments may actively coordinate the critical steps of granule trafficking and insulin secretion. We have assembled a team of investigators with expertise in diabetes, β cell biology, vesicle trafficking, and super-resolution microscopy to test this hypothesis. We will focus on three specific aims. First, determine the in vivo role of ArpC3-mediated cortical actin remodeling in insulin secretion and glucose homeostasis. We have generated a new mouse model where ArpC3 is deleted selectively in adult β cells to ablate Arp2/3 activity and cortical actin branching. Second, define ArpC3 function in cortical actin nano-remodeling and its role in insulin granule recruitment, docking, and exocytosis. Third, evaluate the role of cortical actin branching in human β cells and its dysfunction in T2D patients. The results from this work will fill a long-standing knowledge gap between cortical actin remodeling and insulin secretion, providing mechanistic insights into T2D pathogenesis induced by cortical actin dysfunction.