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DEVELOPMENT OF CELL REGULATION METHOD BY MANIPULATING CAVEOLAR FUNCTIONS

通过操纵细胞小泡功能的细胞调节方法的开发

基本信息

批准号：
12557001
负责人：
FUJIMOTO Toyoshi
金额：
$ 8.38万
依托单位：
NAGOYA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2003
项目状态：
已结题

项目摘要

Caveolae are plasmalemmal invaginations where receptors and signaling molecules are concentrated and may be important for signal transduction. We aimed to find out methods to regulate cellular functions by manipulating caveolae functions in living cells, and obtained the following results.1) A transmembrane segment plus a short extracellular portion of a membrane protein were fused to caveolin molecules, to make artificial transmembrane proteins. One of the fusion proteins showed colocalization with endogenous caveolin-1, and by crosslinking an extracellular epitope by antibodies, it showed local aggregation. This method may be used to manipulate caveolae distribution from the extracellular surface.2) By using interstitial cells of Cajal, we revealed that dihydropyridine-nonsensitive calcium oscillation is related to the pacemaker function, and that calcium influx through TRP4 in caveolae is involved in the oscillation. Caveolae also contains IP3 receptor and calcium pump, and thus TRP4 might be a target molecule for caveolae manipulation.3) Lipid droplets contain a large amount of cholesterol as ester, and could harbor caveolins under some circumstances. We found a Rab protein (LD-Rab) exists in lipid droplets. Overexpression of LD-Rab caused changes in the molecular composition of lipid droplets, and also induced structural changes. LD-Rab is likely to be involved in the intracellular cholesterol trafficking, and a change of its expression level may affect the membrane microdomain.

小窝是一种质膜内陷，受体和信号分子集中在这里，可能对信号转导起重要作用。本研究旨在通过对活细胞小窝功能的调控，寻找调节细胞功能的方法，获得了以下结果：1)将一段跨膜片段和一小段膜蛋白融合到小窝蛋白分子中，制成人工跨膜蛋白。其中一个融合蛋白与内源性小窝蛋白-1共定位，并通过抗体将细胞外表位交联，表现为局部聚集。2)利用Cajal间质细胞，我们揭示了二氢吡啶不敏感的钙振荡与起搏功能有关，钙内流通过TRP4参与了这种振荡。小窝还含有IP3受体和钙泵，因此TRP4可能是小窝操纵的靶分子。3)脂滴含有大量的胆固醇作为酯，在某些情况下可能含有小窝蛋白。我们发现在脂滴中存在一种Rab蛋白(LD-Rab)。LD-Rab的过表达引起了脂滴分子组成的变化，也引起了结构的变化。LD-Rab可能参与细胞内胆固醇的转运，其表达水平的改变可能会影响膜微区。