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Functions of myosin and myosin light chain kinase in the secretory mechanism of anterior pituitary cells

肌球蛋白和肌球蛋白轻链激酶在垂体前叶细胞分泌机制中的作用

基本信息

批准号：
09670007
负责人：
SENDA Takao
金额：
$ 1.92万
依托单位：
Fujita Health University (2000)Nagoya University (1997-1999)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

1. Heavy meromyosin-decorated F-actins were connected to the secretory granules of mouse anterior pituitary cells in two manners. One end of the F-actin directly bound to the granule membrane. In another way, F-actin was linked with the granule membrane by short cross-bridges in side-to-side manner. Immunoelectron microscopy showed that actin and myosin were colicalized near the secretory granules. Moreover, myosin light chain kinase, a regulator of the actomyosin system, was associated to the secretory granule membrane. From these results, the actomyosin system may be involved in the intracellular traffic of the secrtory granules.2. Annexin II, which has an effect to accelerate membrane fusion, was localized to the contact points between plasma membrane and secretory granule membrane, and between grarnnule membranes. There, I found by freeze-replica technique tiny cross-bridges (<10nm) between adjoining membranes. When Ca^<2+>-influx occurred with the use of Clostridium perfringens enterotoxin, these bridges disappeared, and the membrane fusion was induced. Thus, annexin II may be a component of the cross-bridges, and play important roles in membrane fusion.3. Movement of the insulin granules in MIN6 cells (pancreatic beta cell-derived insulin secreting cell line) was activated with the inhibitor of actin polymerization, mycalolide B.In addition, the insulin granules shifted to the cell periplnery, and the insulin secretion was transiently increased. The insulin-output under various secretogogues was also increased with mycalolide B.These results suggested that F-actin had a great effect on the intracellular distribution of the insulin granules, and that disruption of F-actin had a positive effect on the insulin secrction.

1.大分子肌球蛋白修饰的F-肌动蛋白以两种方式与小鼠垂体前叶细胞的分泌颗粒相连。F-肌动蛋白的一端直接与颗粒膜结合。在另一种方式中，F-肌动蛋白以侧对侧的方式通过短的跨桥与颗粒膜相连。免疫电子显微镜显示，肌动蛋白和肌球蛋白在分泌颗粒附近呈网状排列。此外，肌球蛋白轻链激酶是肌动球蛋白系统的调节者，与分泌颗粒膜有关。根据这些结果，肌动球蛋白系统可能参与分泌颗粒的细胞内交通。具有促进膜融合作用的膜联蛋白II定位于质膜与分泌颗粒膜、颗粒膜之间的接触点。在那里，我发现通过冷冻复制技术，相邻的膜之间有微小的交叉桥(&lt；10 nm)。当用产气荚膜梭菌肠毒素引起Ca~(2+)内流时，这些桥消失，膜融合被诱导。因此，膜联蛋白II可能是交叉桥的一个组成部分，并在膜融合中发挥重要作用。肌动蛋白聚合抑制剂mycalolide B激活了胰岛素颗粒在MIN6细胞(胰岛β细胞来源的胰岛素分泌细胞系)中的运动，并且胰岛素颗粒移位到细胞周围，胰岛素分泌一过性增加。Mycalolide B还能增加不同分泌期的胰岛素分泌。结果表明，F-肌动蛋白对胰岛素颗粒在细胞内的分布有很大影响，F-肌动蛋白的阻断对胰岛素的分泌有积极的作用。