Insulin Stimulates Membrane Fusion and GLUT4 Accumulation in Clathrin Coats on Adipocyte Plasma Membranes

摘要: 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 computer program (Fusion Assistant) that enables direct analysis docking/fusion kinetics hundreds exocytic fusion events. Insulin stimulation increases frequency GLUT4 vesicles by approximately 4-fold, increasing content in membrane. Remarkably, insulin signaling modulates process, decreasing vesicle tethering/docking duration prior to fusion. In contrast, molecules spreading out from sites is unchanged insulin. As accumulates membrane, it also immobilized punctate on cell surface. A previous report suggested these are (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF using proteins fused clathrin light chain or clathrin-coated patches. Taken together, data show accelerates transition docking GLUT4-containing their with and promotes accumulation clathrin-based endocytic