Components controlling vesicle trafficking and regulated exocytosis in pancreatic beta-cells

Regulated exocytosis is a sophisticated, well-organized and conserved multistage process underlying release of both neurotransmitters and hormones. Exocytosis is mediated by SNARE proteins that act at the center stage by forming a SNARE complex with the intrinsic capability to execute membrane fusion. In addition, supplementary protein families and also mechanisms such as protein phosphorylation are emerging as critical fine-tuning and regulatory components. SNARE proteins are also crucial for regulated insulin secretion from pancreatic β-cells. In response to elevated glucose levels β-cells secrete insulin, the fundamental metabolic and glucose homeostasis regulating hormone. To resolve molecular mechanisms and components operating in the regulation of insulin exocytosis from β-cells, we have in this work investigated the expression, subcellular distribution and functional significance of Cdk5 activators p39 and p35. We demonstrate that both p35 and p39 are present in primary β-cells, and that Cdk5, activated by the p39 subunit, augments Ca-induced insulin exocytosis. Moreover, we show that Cdk5/p39 acts, at least partly, by phosphorylation of Munc18-1, a member of the SM protein family. To explore the potential presence of additional Cdk5 substrates mediating enhancement of insulin release, we investigated the role of a supplementary Munc18 variant, Munc18-2. By transient overexpression studies using WT and kinase phosphorylation mutants of Munc18-1 and Munc18-2 proteins we established a difference in the subcellular compartmentalization of the two Munc18 isoforms and a stimulation-induced redistribution of Munc18-1. These results also illustrate the importance of the phosphorylation events in cycling and localization of SM proteins, in particular of Munc18-1. With further investigations of the two homologous Munc18 isoforms, employing slow uncaging of Ca followed by high-time resolution membrane capacitance recordings, we prove that phosphorylated Munc18-1 and Munc18-2 as well as both Cdk5 activators, p35 and p39, modulate the heterogeneity of Ca-sensitivity and kinetic dynamics of insulin exocytosis. Detailed evaluation of the significance of the p39 activator was performed using a combination of in vitro techniques, on the level of individual β-cells and pancreatic islets, and in vivo investigations in a mouse mutant lacking the p39 protein. The loss of the p39 activator result in abnormalities related to β-cell function and insulin exocytosis. In addition to deficiencies in release kinetics, Ca-sensitivity of exocytosis and impaired VGCC activity recorded in single β-cells, p39-deficient mice exhibit insufficient glucose tolerance and lower serum insulin levels. These observations suggest that the p39 activator is an essential component for an adequate secretory response and glucose homeostasis in β-cells. By investigating other components that may also be involved in the spatial and temporal regulation of insulin exocytosis, we here report the presence and inhibitory effect of tomosyn, a syntaxin 1 associating protein. To be able to describe and comprehend different steps of the insulin secretory pathway we have studied novel molecular mechanisms and components of regulated membrane fusion. Taking advantage of in vitro and in vivo techniques, insulin exocytosis was investigated at different levels of biological complexity, from single β-cells to the level of the whole organism. In this thesis work we provide evidence that different Munc18 isoforms, p39 and p35 activators and the tomosyn protein, in concert with dynamic fine-tuning mechanisms such as phosphorylation, are involved in the regulation of the complex insulin secretory cascade. LIST OF PUBLICATIONS This thesis is based on the following publications and manuscripts, referred to by their Roman numbers.