Molecular and cellular morphological analysis of sugar transporter dynamics between cell membrane and organelles

细胞膜和细胞器之间糖转运蛋白动力学的分子和细胞形态学分析

基本信息

批准号：
15390053
负责人：
TAKATA Kuniaki
金额：
$ 9.86万
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：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Sugar transporter GLUT4 is recruited from the intracellular compartments and is translocated to the cell membrane upon insulin stimulation, which increases the sugar transport activity at the cell surface. GLUT4, therefore, plays a pivotal role in the regulation of blood glucose level. I first examined the localization of GLUT4 in 3T3-L1 cultured adipocytes by immunofluorescence microscopy. Insulin-stimulation mobilized only a small portion of intracellular GLUT4. The paucity of translocation of GLUT4 was a serious drawback in analyzing the mechanism of transporter trafficking by morphological methods. Aquaporin 2 water channel is also translocated from the intracellular pool to the cell membrane upon anti-diuretic hormone-stimulation in principal cells of the kidney collecting ducts, which increases the water permeability at the apical cell membrane. This system provides a unique opportunity to study the translocation mechanism between intracellular compartments and cell membrane. I examined the immunolocalization and trafficking of aquaporin 2 with special reference to intracellular organelles and microdomains of the embrane such as rafts. Aquaporin 2 was recovered in Triton X100-insoluble fractions, suggesting the contribution of membrane rafts. Stimulation with forskolin induces the translocation of aquaporin 2 to the cell membrane, where it was colocalized with caveolin 1. Wash-out of forskolin initiated the retrieval of aquaporin 2 to the EEA1-positive early endosomes by endocytosis. Such trafficking was affected by drugs that perturb membrane microdomains, suggesting an important role of membrane microdomains in the trafficking of aquaporin 2.

糖转运蛋白 GLUT4 从细胞内区室募集，并在胰岛素刺激下转运至细胞膜，从而增加细胞表面的糖转运活性。因此，GLUT4在血糖水平的调节中起着关键作用。我首先通过免疫荧光显微镜检查了 3T3-L1 培养脂肪细胞中 GLUT4 的定位。胰岛素刺激仅动员细胞内 GLUT4 的一小部分。 GLUT4易位的缺乏是通过形态学方法分析转运蛋白运输机制的一个严重缺陷。在肾脏集合管主细胞中受到抗利尿激素刺激后，水通道蛋白 2 水通道也会从细胞内池转移到细胞膜，从而增加顶端细胞膜的水渗透性。该系统为研究细胞内区室和细胞膜之间的易位机制提供了独特的机会。我检查了水通道蛋白 2 的免疫定位和运输，特别关注细胞内细胞器和膜的微结构域（例如筏）。在 Triton X100 不溶组分中回收到水通道蛋白 2，表明膜筏的贡献。毛喉素的刺激诱导水通道蛋白 2 易位至细胞膜，并与小窝蛋白 1 共定位。毛喉素的洗出启动了水通道蛋白 2 通过胞吞作用恢复至 EEA1 阳性早期内体。这种运输受到扰乱膜微结构域的药物的影响，这表明膜微结构域在水通道蛋白 2 的运输中发挥着重要作用。