Abstract Clathrin-mediated endocytosis is the main port of entry into our cells for medically relevant substances including cholesterol-laden particles and viruses such as influenza and hepatitis. By engulfing signaling receptors, this fundamental cellular process also tunes our sensitivity to the potentially pathological actions of growth factors and neuromodulators. As such, understanding how the underlying endocytic machinery is regulated promises to reveal novel mechanisms that could be harnessed to control neoplastic, neurodegenerative, cardiovascular, and viral diseases. At the heart of the endocytic process lies the AP2 clathrin adaptor complex which appears to undergo a conformational change during vesicle formation to actively couple membrane and cargo to the clathrin coat. Despite the central role of AP2, we lack critical details about how this molecular machine is regulated in vivo and how this regulation influences multicellular systems. To address this need, we have developed innovative tools in C. elegans that allow us to quantify AP2 activity at multiple levels and have employed deep genetic screens to identify three conserved protein families that appear to govern AP2 conformation and activity. Our goal is to illuminate how these allosteric regulators of the endocytic machinery function mechanistically. In Aim 1 we will validate our hypothesis that adaptiN-Ear-Binding Coat-Associated Proteins (NECAP)s counteract the active (open) conformation of AP2 to ensure proper recycling of adaptor complexes. We have discovered that AP2 accumulates in an active state in NECAP mutants, and that NECAPs specifically bind open, phosphorylated forms of AP2. Using cryo- EM we have determined that the phosphorylated AP2 core bound to NECAP is conformationally inactive. We will validate this structure in vivo and whether it reflects the end product of NECAP activity. Previously it was thought that membrane phospholipids, cytosolic cargo domains, and phosphorylation by the AP2-associated kinase (AAK1) activate AP2. Our preliminary data indicate that a conserved region of the membrane- associated Fer/Cip4 Homology Domain-only (FCHo) proteins is required to promote endocytosis by converting AP2 to an active complex. We have named this functionally important domain the AP2 Activator, or APA. In Aim 2 we will determine where the APA binds AP2 using cryo-EM and test whether the APA is sufficient to induce a structural rearrangement of AP2, as well as defining the roles of membrane, cargo, and phosphorylation in that process. We will evaluate the physiological significance of AP2 phosphorylation by characterizing kinase mutants. In our new Aim 3 we will examine how membrane trafficking influences tissue physiology using our suite of assays ...