Studies on Plasmalemmal Caveola

1)质膜三磷酸肌醇受体样蛋白(PM-IP3R)定位于小窝。我们研究了它在小窝和肌动蛋白细丝连接中的作用。在正常的Pam 212细胞中，PM-IP3R密集分布在细胞间接触附近，肌动蛋白细丝也集中在那里。在细胞松弛素D处理的细胞中，PM-IP3R定位于无序的肌动蛋白聚集的部位；在电子显微镜下，PM-IP3R的标记出现在看起来像一串葡萄的小窝周围。PM-IP3R不能被Triton X-100或辛基葡萄糖苷增溶。这些结果表明PM-IP3R可能介导了小窝和肌动蛋白细丝的连接。2)用免疫细胞化学技术检测了GPI锚定蛋白、糖脂和鞘磷脂的分布。当培养的细胞被分子抗体处理，然后与标记的二次抗体孵育时，标记分布在整个表面。当细胞经一抗和二抗处理后升温至37℃时，10~30分钟后标记集中在小窝中。在这个过程中，GPI锚定的蛋白质和糖脂，或GPI锚定的蛋白质和鞘磷脂，形成共同的斑块，然后积累到共同的小窝。即使在细胞松弛素D和/或诺康唑存在的情况下，也能观察到再分布。结果表明，GPI锚定的蛋白质、糖脂和鞘磷脂只有在这些分子被抗体交联时才会发生隔离，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.