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

1.重裂肌球蛋白修饰的F-肌动蛋白以两种方式连接到小鼠垂体前叶细胞的分泌颗粒。F-肌动蛋白的一端直接与颗粒膜结合。另一方面，F-肌动蛋白与颗粒膜以短的横桥以侧对侧的方式连接。免疫电镜显示，肌动蛋白和肌球蛋白在分泌颗粒附近呈胶团化。此外，肌球蛋白轻链激酶，肌动球蛋白系统的调节器，与分泌颗粒膜。这些结果表明，肌动球蛋白系统可能参与了分泌颗粒的细胞内运输. Annexin Ⅱ具有促进细胞膜融合的作用，定位于细胞膜与分泌颗粒膜的接触点和分泌颗粒膜之间。在那里，我通过冷冻复制技术发现了相邻膜之间的微小交叉桥（<10 nm）。当使用产气荚膜梭菌肠毒素引起Ca ^<2 +>内流时，这些桥消失，并诱导膜融合。因此，annexin II可能是跨桥的一个组成部分，在膜融合中起重要作用.用肌动蛋白聚合抑制剂mycalcantine B激活胰岛素颗粒在MIN6细胞（胰腺β细胞源性胰岛素分泌细胞系）中的运动，并使胰岛素颗粒向细胞周边移动，胰岛素分泌短暂增加。在各种促胰岛素分泌剂作用下，mycalctin B均能增加胰岛素分泌量。这些结果表明，F-肌动蛋白对胰岛素颗粒的胞内分布有重要影响，破坏F-肌动蛋白对胰岛素分泌有积极作用。

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.