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J. Biol. Chem., Vol. 283, Issue 13, 8508-8516, March 28, 2008

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Direct Quantification of Fusion Rate Reveals a Distal Role for AS160 in Insulin-stimulated Fusion of GLUT4 Storage Vesicles^*

Li Jiang¹, Junmei Fan¹, Li Bai¹, Yan Wang, Yu Chen, Lu Yang, Liangyi Chen, and Tao Xu²

From the Joint Laboratory of Institute of Biophysics & Huazhong University of Science and Technology, National Laboratory of Biomacromolecules, Chinese Academy of Sciences, Beijing 100101, China and the College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China

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 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.

Received for publication, October 19, 2007 , and in revised form, November 26, 2007.

^* This work was supported by National Science Foundation of China Grant 30630020, Major State Basic Research Program of China Grant 2006CB70570, and Chinese Academy of Sciences Project Grant KSCX1-YW-02-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Movies S1 and S2.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Tel.: 86-10-64888469; Fax: 86-10-64888513; E-mail: xutao{at}ibp.ac.cn.

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