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Molecular and Cellular Biology, May 2007, p. 3456-3469, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.01719-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Insulin Stimulates Membrane Fusion and GLUT4 Accumulation in Clathrin Coats on Adipocyte Plasma Membranes{triangledown} ,{dagger}

Shaohui Huang,1 Larry M. Lifshitz,2 Christine Jones,1 Karl D. Bellve,2 Clive Standley,2 Sonya Fonseca,1 Silvia Corvera,1 Kevin E. Fogarty,2 and Michael P. Czech1*

Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, Massachusetts 01605,1 Biomedical Imaging Group, Department of Physiology, University of Massachusetts Medical School, 373 Plantation Street, Worcester, Massachusetts 016052

Received 12 September 2006/ Returned for modification 7 November 2006/ Accepted 15 February 2007

Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by ~4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.

* Corresponding author. Mailing address: Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, Massachusetts 01605. Phone: (508) 856-2254. Fax: (508) 856-1617. E-mail: michael.czech{at}umassmed.edu

{triangledown} Published ahead of print on 5 March 2007.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, May 2007, p. 3456-3469, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.01719-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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