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

通过倏逝波显微镜分析单个胰岛素颗粒运动来研究胰腺 β 细胞中胰岛素胞吐作用的机制

基本信息

批准号：
14570130
负责人：
OHARA Mica
金额：
$ 1.92万
依托单位：
Kyorin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

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.

利用原代胰岛β细胞研究2型糖尿病胰岛素分泌受损的分子机制是必要的，但技术上的困难阻碍了我们对这一过程的分析。我们最近成功地用高时间分辨率的TIRF成像系统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1^<st>-相，融合颗粒主要来自先前停靠的颗粒，而2^ -相的融合颗粒<nd>则来自新加入的颗粒。TIRF图像显示，新生细胞在质膜上的对接时间非常短（50 ms），而胰岛素瘤MIN 6细胞的对接时间相对较长（37± 11 s），因此原代β细胞的1^ -期<st>和2^ -期的胞吐机制有很大不同<nd>。然后我们讨论了胰岛素胞吐位点是否与SNARE或相关蛋白相关的问题。为了研究t-SNARE与胰岛素颗粒的对接和融合之间的相互作用，我们用Cy 3标记的抗syntaxin 1A和抗SNAP-25抗体通过将抗体与HIV-1达特缀合来标记内源性syntaxin 1A和SNAP-25，将其快速转导到活β细胞中。TIRF图像显示，t-SNARE在质膜上分布成许多分离的簇（<400 nm）。胰岛素颗粒优先对接（约80%）的网站syntaxin 1A集群，共定位与SNAP-25集群。在syntaxin 1A簇处观察到融合。目前，我们正在研究胰岛素胞吐位点与CAZ蛋白的关系。因此，我们的数据揭示了原代β细胞中胰岛素胞吐的分子机制。