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Plasmalemmal caveolae and intracellular calcium regulation

质膜小窝和细胞内钙调节

基本信息

批准号：
08457001
负责人：
FUJIMOTO Toyoshi
金额：
$ 4.93万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457001/
关键词：
Caveola Plasma membrane Calcium Gi2a Cholesterol Freeze fracture Immunoelectron microscopy

项目摘要

1) A simple quick freezing method to observe the plasma membrane from monolayr cultured cells was invented. The metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation.2) rheta-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolysed and biotinylated (BCrheta) and used as a cytochemical probe. Because the plasma membrane keeps integrity even after the binding of BCrheta, the probe can be used not only for cytochemical labeling of fixed cells, but for pursuing the behavior of cross-linked cholesterol molecules in living cells. By using this new probe, it was revealed that cross-linked cholesterol in the plasma membrane are sequestered to caveolae.3) By immunoelectron microscopy, whether Gi2a is constitutively concentrated in caveolae was critically assessed. First, behavior of caveolin and Gi2a in density-equilibrium ultracentrifugation was reexamined. By collecting fractions efficiently, caveolin and Gi2a were found to distribute differently. Secondly, by novel immunocytochemical methods it was found that the labeling density of Gi2a was 2.29 times more in caveolae than in the non-caveolar plasma membrane. The results indicate that concentration of Gi2a in caveolae is less than deduced from most biochemical studies.

1)本文提出了一种观察单层培养细胞质膜的简便快速冷冻方法。金属夹心法简单，不需要任何昂贵的设备，并提供了一个大的质膜断裂平面，为随后的组织化学操作。2）rheta毒素（产气荚膜梭菌溶素O），胆固醇结合毒素，部分蛋白水解和生物素化（BCrheta），并用作细胞化学探针。由于即使在BCreta结合后质膜也保持完整性，因此该探针不仅可用于固定细胞的细胞化学标记，还可用于追踪活细胞中交联胆固醇分子的行为。通过使用这种新的探针，揭示了质膜中的交联胆固醇被隔离到小窝中。3）通过免疫电镜，严格评估Gi2a是否组成性地集中在小窝中。首先，重新检查了Caveolin和Gi2a在密度平衡超离心中的行为。通过有效收集组分，发现小窝蛋白和Gi2a分布不同。其次，通过新的免疫细胞化学方法，发现Gi2a的标记密度在小窝中是在非小窝质膜中的2.29倍。结果表明，Gi2a在小窝中的浓度小于从大多数生化研究推断的浓度。