|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 2000 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1999 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1998 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1997 : ¥1,000,000 (Direct Cost : ¥1,000,000)
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.