Santiago Echeverry: Molecular determinants of insulin granule release probability

Abstract

Insulin release is crucial for glucose homeostasis, and its impairment leads to hyperglycemia and contributes to the development of type 2 diabetes. Pancreatic β-cells release insulin through Ca²+-triggered exocytosis, a process in which insulin-containing secretory granules dock at the plasma membrane, form molecular interactions that prepare the granule for release (priming), and fuse the plasma membrane following an elevation in intracellular Ca²+. Granule priming is regulated by secondary messengers, which confer cellular plasticity and allow β-cells to respond to their physiological context. However, the molecular mechanisms of insulin granule priming and its connection to secondary messengers are incompletely understood. This thesis investigates the molecular machinery governing insulin granule priming in dispersed β-cells through analysis of granule dynamics and exocytosis. We show that munc13 regulates granule priming through its dynamic recruitment to the plasma membrane in response to Ca²+ and diacylglycerol produced by cellular activity and signaling. Granules capable of release recruit, on average, six munc13 molecules. Once recruited to granules, Ca²+ binding to munc13 transiently increases granule release probability, thereby facilitating exocytosis. Furthermore, we demonstrate that elevated intracellular Ca²+ facilitates exocytosis in both diabetic and non-diabetic human β-cells. However, electrical activity alone failed to evoke Ca²+-dependent facilitation in human β-cells. Using quantitative microscopy and photoactivated localization microscopy, we estimated the copy number and localization of proteins at the granule docking site. We found that primed and unprimed granules differ in the abundance of munc13, RIMs, and liprinα-1. In addition, these proteins localize toward the center of the docking site, surrounding the core fusion machinery. Their lateral diffusion is slowed at the docking site center, suggesting binding to the fusion core machinery. Finally, we observed that syntaxin, a core fusion protein, binds to and diffuses freely together with its chaperone munc18 before arriving at the granule docking site. We conclude that insulin granules primed require recruitment of proteins to the granule docking site, as well as activation of these proteins by secondary messengers. Ultimately, priming is controlled by intracellular Ca²+ concentration through a mechanism dependent on munc13 translocation and activation, and dispersed human β-cells are capable of secretory Ca²+-dependent secretory facilitation.