PROJECT SUMMARY Human variants in the submembrane cytoskeleton-associated protein ankyrin-B (AnkB) have been identified as risk factors for diabetes, obesity and cardiometabolic diseases. Mice harboring these human variants have AnkB deficiency in multiple metabolic tissues, including skeletal muscle (SKM), and develop age-dependent obesity and systemic glucose mishandling. While SKM is a primary target of AnkB deficiency, how AnkB contributes to the regulation of SKM cellular metabolism and energetic capacity, and AnkB’s SKM-specific roles in promoting systemic metabolic homeostasis have not been elucidated. The goal of this study is to test the overarching hypothesis that AnkB forms a complex with mitochondria and endoplasmic reticulum (ER) resident proteins to promote the formation of mitochondria-ER contact sites and calcium transfer between the two organelles. I postulate that this function of AnkB is essential for maintaining mitochondria homeostasis and for proper skeletal muscle metabolism and bioenergetics. The first aim will define how AnkB interacts with mitochondria in skeletal muscle and the extent of its contribution to the formation of mitochondria-ER contact sites and to mitochondria calcium flux. The second aim will define the contribution of AnkB to SKM cellular respiration and the bioenergetic capacity of SKM. These studies will provide mechanistic and functional insights into a novel role of AnkB in skeletal muscle metabolism that may translate to other cell types with high energetic demand, and might be relevant to physiological processes, including adaptation to exercise and ageing. Moreover, through the proposed work I will uncover novel pathophysiological mechanisms of AnkB variants that will further explain their contribution to metabolic diseases.