Beta cell insulin granule docking, priming and fusion

DESCRIPTION (provided by applicant): We have recently observed that depolymerization of the actin cytoskeleton in beta cell lines and primary isolated islets potentiates glucose-and KCI-stimulated insulin secretion. In parallel, the insulin granules localize to the cell periphery and become aligned juxtaposed to the plasma membrane. Furthermore in the basal state, the syntaxin1/SNAP25 complex co-immunoprecipitates with actin, however, following glucose-or KCI-stimulation there is a marked reduction in actin co-immunoprecipitated with the syntaxin1/SNAP25 t-SNARE complex. Based upon these data, we plan to investigate the molecular details of insulin granule docking/fusion in relationship to filamentous actin in the regulation of this process. To accomplish these goals, we propose three overall Specific Aims. Initially, we plan to examine the dynamic changes in actin structure. We will co-express YFP-actin with an insulin signal peptide-CFP fusion to monitor the time dependent changes in cortical actin structure with insulin granule trafficking. In parallel, we will identify the syntaxin 1 /SNAP25 domains responsible for interaction with F-actin, by expression of various syntaxin 1 and SNAP25 point and deletions mutants. Since F-actin most likely associates with the syntaxin 1/SNAP25 t-SNARE complex through intermediate proteins, we will determine the localization and interactions with known actin adaptor proteins. We will examine the mechanism of this potentiation of insulin secretion by determining calcium release properties, glucose and KCI-stimulated intracellular calcium levels will be determined by Fura2 measurments and the effect of calcium on insulin secretion in permeabilized cells assessed. In parallel, the relative extent of docked insulin granules will be determined by fluorescent microscopy, electron microscopy and by changes in the relative association state of the insulin granule v-SNARE protein synaptobrevin 2 (VAMP2) and Munc18a with syntaxin 1. Since Munc18a (n-sect) is a syntaxin 1 binding protein that appears to function as both a negative and positive regulator of membrane vesicle secretion, we will determine the effect of increased and decreased expression of Munc18a in the docking of insulin granules and in the interaction of syntaxin 1/SNAP25 with VAMP2 and F-actin. This will be directly compared to the effects of Munc18a blocking peptides and syntaxin 1 mutants that are locked in the open conformation state and are resistant to the inhibitory function of Munc18a.