Studies on Plasmalemmal Caveola

1)质乳肌醇三磷酸受体样蛋白（PM-IP3R）定位于小泡中。我们研究了它在小泡和肌动蛋白丝连接中的作用。在正常的Pam 212细胞中，PM-IP3R密集分布在肌动蛋白丝集中的细胞间接触区附近。在细胞松弛素d处理的细胞中，PM-IP3R定位于无组织肌动蛋白聚集的部位；电镜下，PM-IP3R的标记出现在小泡周围，看起来像一串葡萄。PM-IP3R不能被Triton X-100或辛基葡萄糖苷溶解。这些结果表明PM-IP3R可能介导小泡和肌动蛋白丝的连接。2)免疫细胞化学技术检测gpi锚定蛋白、糖脂和鞘磷脂的分布。将培养的细胞用分子抗体处理后，再用标记的二抗孵育，标记分布在整个表面。但经一抗和二抗处理后，将细胞加热至37℃，10 ~ 30min后浓缩至小泡。在此过程中，gpi锚定蛋白与糖脂，或gpi锚定蛋白与鞘磷脂形成共同的斑块，然后积聚成共同的小泡。即使在细胞松弛素D和/或诺可唑存在的情况下，也观察到这种重新分布。结果表明，gpi锚定蛋白、糖脂和鞘磷脂的隔离仅在分子被抗体交联时发生，而不依赖于完整的细胞骨架。

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.