Title: Mechanisms of Compartmentalized cAMP Signaling Project summary: The overall goal of the proposed research is to provide a mechanistic understanding about how cyclic AMP (cAMP) regulates a variety of different cellular functions with exquisite specificity. As a ubiquitous second messenger, cAMP regulates a variety of processes that are critical to cell physiology, such as cell growth, proliferation, metabolism, survival and mobility. In pancreatic β cells, cAMP is produced in response to glucose or hormones such as glucagon-like peptide-1 (GLP-1), and in turn modulates the Ca2+ signal, directly influencing exocytotic insulin release as well as regulating gene expression, acting through its effector molecules cAMP-dependent protein kinase (PKA) and exchange proteins activated by cAMP (Epac). The concept of “cAMP compartmentation”, wherein unique changes in second messenger levels and effector activities occur in both space and time, was put forward to help understand the exquisite signaling specificity of cAMP, yet the mechanisms underlying cAMP compartmentation remain elusive. In the previous funding period, we made a breakthrough discovery. We discovered that the PKA regulatory subunit RIα undergoes liquid-liquid phase separation (LLPS) and that these condensates are critical for cAMP compartmentation by serving as a dynamic buffering system, and allowing PDEs to create cAMP microdomains. This discovery was enabled by the development of a new class of fluorescent biosensors that can be targeted to proteins at the endogenous level, allowing probing of signaling dynamics at the native stoichiometry. We have also created a novel class of fluorescent biosensors that allow direct visualization of PKA activities in superresolution and elucidated how our previously discovered oscillatory circuit is regulated spatially by A-Kinase Anchoring Protein 79/150. Altogether we published 29 peer-reviewed journal articles (with 4 additional manuscripts in revision). In the current proposal, building on these discoveries, we will test our hypothesis that PKA RIα phase separation, by sequestering cAMP and the catalytic subunit of PKA, enables compartmentalized cAMP signaling and allows cAMP to achieve high signaling specificity and functional diversity in β cells. Specifically, we will develop novel molecular tools to interrogate the spatiotemporal regulation of cAMP/PKA signaling in living cells, examine the impact of RIα phase separation on cAMP/PKA signaling and determine the functional roles of RIα phase separation in β cells. Our proposed studies should lead to a better understanding of the molecular mechanisms and functional roles of the spatiotemporal regulation of cAMP/PKA signaling, particularly in the context of the regulation of cell functions. As aberrations in the cAMP signaling pathway are implicated in clinical conditions such as obesity and type 2 diabetes mellitus, such an understanding should help identify sites of dysfunction in type ...