|Budget Amount *help
¥7,000,000 (Direct Cost : ¥7,000,000)
Fiscal Year 1995 : ¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1994 : ¥3,800,000 (Direct Cost : ¥3,800,000)
1) Plasmalemmal inositol trisphosphate receptor-like protein (PM-IP3R) is localized in caveolae. We investigated its role in the linkage of caveolae and actin filaments. In normal Pam 212 cells, PM-IP3R was distributed densely in the vicinity of intercellular contacts where actin filaments were also cencentrated. In cytochalasin D-treated cells, PM-IP3R was localized in spots where disorganized actin was aggregated ; by electron microscopy, the labeling for PM-IP3R was seen around caveolae appearing like a bunch of grapes. PM-IP3R was not solubilized by Triton X-100 or octylglucoside. These results indicate that PM-IP3R may mediate the linkage of caveolae and actin filaments.
2)Distribution of GPI-anchored proteins, glycolipids and sphingomyelin was examined by immunocytochemical techniques. When cultured cells were treated with antibodies to the molecules, and then incubated with labeled secondary antibodies, the labeling was distributed in the entire surface. But when the cells were warmed to 37C after treated with the primary and secondary antibodies, the labeling was concentrated to caveolae after 10 to 30 min. During the process, GPI-anchored proteins and glycolipids, or GPI-anchored proteins and sphingomyelin, formed common patches and then accumulated to common caveolae. The redistribution was observed even in the presence of cytochalasin D and/or nocodazole. The results indicate that sequestration of GPI-anchored proteins, glycolipids and sphingomyelin occurs only when the molecules are cross-linked by antibodies, and does not depend on intact cytoskeleton.