Jiang Li, Fan Junmei, Bai Li, Wang Yan, Chen Yu, Yang Lu, Chen Liangyi, Xu Tao
Joint Laboratory of Institute of Biophysics & Huazhong University of Science and Technology, National Laboratory of Biomacromolecules, Chinese Academy of Sciences, Beijing, China.
J Biol Chem. 2008 Mar 28;283(13):8508-16. doi: 10.1074/jbc.M708688200. Epub 2007 Dec 6.
Insulin-stimulated GLUT4 translocation to the plasma membrane constitutes a key process for blood glucose control. However, convenient and robust assays to monitor this dynamic process in real time are lacking, which hinders current progress toward elucidation of the underlying molecular events as well as screens for drugs targeting this particular pathway. Here, we have developed a novel dual colored probe to monitor the translocation process of GLUT4 based on dual color fluorescence measurement. We demonstrate that this probe is more than an order of magnitude more sensitive than the current technology for detecting fusion events from single GLUT4 storage vesicles (GSVs). A small fraction of fusion events were found to be of the "kiss-and-run" type. For the first time, we show that insulin stimulation evokes a approximately 40-fold increase in the fusion of GSVs in 3T3-L1 adipocytes, compared with basal conditions. The probe can also be used to monitor the prefusion behavior of GSVs. By quantifying both the docking and fusion rates simultaneously, we demonstrate a proportional inhibition in both docking and fusion of GSVs by a dominant negative mutant of AS160, indicating a role for AS160 in the docking of GSVs but not in the regulation of GSV fusion after docking.
胰岛素刺激下葡萄糖转运蛋白4（GLUT4）向质膜的转位是血糖控制的关键过程。然而，目前缺乏方便且可靠的实时监测这一动态过程的检测方法，这阻碍了当前在阐明潜在分子事件以及筛选靶向该特定途径的药物方面的进展。在此，我们开发了一种新型双色探针，基于双色荧光测量来监测GLUT4的转位过程。我们证明，该探针在检测单个GLUT4储存囊泡（GSV）的融合事件方面比现有技术敏感一个数量级以上。发现一小部分融合事件属于“亲吻-逃离”类型。首次，我们表明与基础条件相比，胰岛素刺激可使3T3-L1脂肪细胞中GSV的融合增加约40倍。该探针还可用于监测GSV的预融合行为。通过同时定量对接和融合速率，我们证明AS160的显性负性突变体对GSV的对接和融合均有比例抑制作用，表明AS160在GSV的对接中起作用，但在对接后GSV融合的调节中不起作用。
