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

利用原代β细胞来揭示2型糖尿病胰岛素胞吐的分子机制和胰岛素释放受损的机制是必不可少的，但技术上的困难阻碍了我们对这一过程的分析。我们最近利用高时间分辨率的TIRF成像系统成功地监测了大鼠胰腺原代β细胞中胰岛素颗粒的动态变化。在胰岛素释放的第1阶段，融合颗粒主要来自以前对接的颗粒，而在第2阶段的融合颗粒则来自于新人。TIRF图像显示，新生物在质膜上的对接时间在50ms内极短，而胰岛素瘤MIN6细胞的对接时间相对较长(37±11s)，因此原代β细胞的胞吐机制与原代Min-6细胞的胞吐机制有很大不同。然后，我们解决了胰岛素胞吐部位是否与SNARES或相关蛋白质相关的问题。为了研究T-SNARs与胰岛素颗粒的对接和融合的相互作用，我们用Cy3标记的抗Synaxin 1A和抗SNAP-25的抗体与HIV-1 Tat偶联，将其快速导入活的β细胞，标记内源性Synaxin 1A和SNAP-25。TIRF图像显示，T型陷阱分布在细胞膜上的大量分散的团簇中(&lt；400 nm)。胰岛素颗粒(约80%)优先对接于Synaxin 1A簇上的位点，与SNAP-25簇共定位。融合出现在Synaxin 1A簇处。现在，我们正在研究胰岛素胞吐作用的部位与CAZ蛋白之间的关系。因此，我们的数据揭示了原代β细胞胰岛素胞吐的分子机制。

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.