ARTICLES Cell Research (2004); 14(6):480-486 Dynamic tracking and mobility analysis of single GLUT4 storage vesicle in live 3T3-L1 cellsChen hong Li1,*, Li Bai1,*, Dong dong Li1, Sheng Xia1, Tao Xu1,2,** 1Institute of Biophysics and Biochemistry,
Huazhong University of Science and Technology, Wuhan 430074, China Received, Aug 6, 2004 Revised, Oct 22, 2004 Accepted, Nov 8, 2004 *These authors contributed equally to this work.
Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is unclear how GSVs are arranged based on their mobility. We examined this issue in 3T3-L1 cells via investigating the three-dimensional mobility of single GSV labeled with EGFP-fused GLUT4. A thin layer of cytosol right adjacent to the plasma membrane was illuminated and successively imaged at 5 Hz under a total internal reflection fluorescence microscope with a penetration depth of 136 nm. Employing single particle tracking, the three-dimensional subpixel displacement of single GSV was tracked at a spatial precision of 22 nm. Both the mean square displacement and the diffusion coefficient were calculated for each vesicle. Tracking results revealed that vesicles moved as if restricted within a cage that has a mean radius of 160 nm, suggesting the presence of some intracellular tethering matrix. By constructing the histogram of the diffusion coefficients of GSVs, we observed a smooth distribution instead of the existence of distinct groups. The result indicates that GSVs are dynamically retained in a continuous and wide range of mobility rather than into separate classes. Keywords: Insulin, GLUT4, GLUT4 storage vesicle (GSV), 3T3-L1, total internal reflection, fluorescence microscopy, single particle tracking.
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