Insulin-stimulated GLUT4 translocation is central to glucose homeostasis. Functional assays to distinguish individual steps in the GLUT4 translocation process are lacking, thus limiting progress toward elucidation of the underlying molecular mechanism.Here we have developed a robust method, which relies on dynamic tracking of single GLUT4 storage vesicles (GSVs) in real time, for dissecting and systematically analyzing the docking, priming, and fusion steps of GSVs with the cell surface in vivo. Using this method, we have shown that the preparation of GSVs for fusion competence after docking at the surface is a key step regulated by insulin, whereas the docking step is regulated by P13K and its downstream effector, the Rab GAP AS160. These data show that Akt-dependent phosphorylation of AS160 is not the major regulated step in GLUT4 trafficking, implicating alternative Akt substrates or alternative signaling pathways downstream of GSV docking at the cell surface as the major regulatory node.
However, monitoring fusion events with GLUT4一EGFP is extremely time-consuming, requires extensive training and tends to underestimate the fusion rate. Thus, it is desirable to develop a rather straightforward and easy-to-apply methodology to reliably detect fusion events at single vesicle level. Here we have eveloped a novel probe to monitor the translocation process of GLUT4 based on
dual-emission ratiometric fluorescence measurement. We have demonstrated that this probe is more than an order of magnitude sensitive than the current technology in detecting fusion event from single GLUT4 vesicles. The fusion rate constant of 3T3-L1 adipocytes was determined for the first time to be increased by 40 fold from basal condition by insulin stimulation. The probe can be also used to
monitor the pre-fusion behavior of GLUT4 vesicles. Further development of screening strategies employing this probe will facilitate the identification of key molecular players or novel drugs targeting at GLUT4 trafficking pathway.
Key words: Secretion Dockin Endocytosis Vesicle GLUT4 Priming Fusion Insulin
目录
摘要
Abstract
1绪论
1.1细胞分泌的循环及其调控的分子机制研究进展
1.2囊泡转运和膜蛋白的转运
1.3本研究的主要内容
2实验原理和研究方法
2.1全内反射荧光显微镜
2.2共聚焦激光扫描显微镜
2.3膜电容检测技术
2.4钙离子光解释放的原理
2.5电穿孔的实验原理
2.6 RNA干扰技术
3胰腺p细胞中网格蛋白依赖和非网格蛋白依赖的循环分泌颗粒中不
同dynamin亚型的交迭功能
3.1前言
3.2实验材料和实验方法
3.3实验结果
3.4讨论
4葡萄糖转运蛋白4转运步骤的解析及胰岛素的调控位点
4.1前言
4.2实验材料和实验方法
4.3实验结果
4.4讨论
5一种实时跟踪GLUT4储存囊泡转运的荧光比率探针
5.1前言
5.2实验材料和实验方法
5.3实验结果
5.4讨论
6总结
致谢
参考文献
附录1攻读学位期间发表的论文目录
附录2主要符号和缩略词表
