Title

Insulin stimulates membrane fusion and GLUT4 accumulation in clathrin coats on adipocyte plasma membranes

UMMS Affiliation

Graduate School of Biomedical Sciences; Program in Molecular Medicine; Department of Physiology; Program in Gene Function and Expression

Date

3-7-2007

Document Type

Article

Medical Subject Headings

3T3-L1 Cells; Adipocytes; Animals; Cell Membrane; Clathrin; Exocytosis; Glucose Transporter Type 4; Insulin; Kinetics; Membrane Fusion; Mice; Microscopy, Fluorescence; Time Factors

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

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 approximately 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.

Rights and Permissions

Citation: Mol Cell Biol. 2007 May;27(9):3456-69. Epub 2007 Mar 5. Link to article on publisher's site

Related Resources

Link to Article in PubMed

PubMed ID

17339344