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

1. 重肌球蛋白修饰的f -肌动蛋白以两种方式与小鼠垂体前叶细胞分泌颗粒相连。f -肌动蛋白的一端直接与颗粒膜结合。另一种方式，f -肌动蛋白与颗粒膜通过短交叉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共凝。此外，肌动球蛋白系统的调节因子肌球蛋白轻链激酶与分泌颗粒膜有关。从这些结果来看，肌动球蛋白系统可能参与了分泌颗粒的细胞内运输。膜联蛋白II具有加速膜融合的作用，定位于质膜与分泌颗粒膜之间以及颗粒膜之间的接触点。在那里，我通过冷冻复制技术在相邻膜之间发现了微小的交叉桥（<10nm）。当使用产气荚膜梭菌肠毒素引起Ca^<2+>-内流时，这些桥消失，引起膜融合。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可激活MIN6细胞（胰腺β细胞来源的胰岛素分泌细胞系）中胰岛素颗粒的运动，并使胰岛素颗粒向细胞周围移动，胰岛素分泌短暂增加。mycalolide b也增加了胰岛素在各种促分泌剂下的输出量。这些结果表明，F-actin对胰岛素颗粒的细胞内分布有很大的影响，F-actin的破坏对胰岛素的分泌有积极的影响。

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.