Mechanism of insulin exocytosis in pancreatic β cells by analyzing single insulin granule motion with evanescent wave microscopy

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It is essential to use primary β cell for revealing the molecular mechanism of insulin exocytosis and the machinery behind impaired insulin release in type 2 diabetes, nevertheless, technical difficulties prevented us to analyze such a process. We have recently succeeded in monitoring the dynamics of insulin granules in primary β cell prepared from rat pancreas using TIRF imaging system with high time resolution. In the 1^<st>-phase of insulin release, the fusing granules occurred mostly from previously docked granules, but fusion granules in 2^<nd>-phase originated from newcomers. TIRF images could reveal that the docking time of newcomers on the plasma membrane was extremely short within 50 ms, in contrast to relatively long time in insulinoma MIN6 cells (37±11s), thus the exocytotic mechanism in 1^<st>-phase is quite different from that in the 2^<nd>-phase in primary β cells. We have then addressed the question if insulin exocytotic site is associated with SNAREs or related proteins. To study the interaction between t-SNAREs and the docking and fusion of insulin granules, we labeled endogenous syntaxin 1A and SNAP-25 with Cy3-labeled anti-syntaxin 1A and anti SNAP-25 antibodies by conjugating antibodies with HIV-1 TAT, that was rapidly transduced into the living β cells. TIRF image showed that t-SNAREs are distributed in numerous separated clusters (<400 nm) on the plasma membrane. Insulin granules were preferentially docked (about 80%) to sites on syntaxin 1A clusters, colocalizing with SNAP-25 clusters. The fusion was seen at the syntaxin 1A clusters. Now, we are investigating the relationship between site of insulin exocytosis and CAZ (cytomatrix at the active zone) proteins. Thus, our data revealed the molecular mechanism of insulin exocytosis in primary β cells.