Direct quantification of fusion rate reveals a distal role for AS160 in insulin-stimulated fusion of GLUT4 storage vesicles.
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 approximately 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.
Joint Laboratory of Institute of Biophysics & Huazhong University of Science and Technology, National Laboratory of Biomacromolecules, Chinese Academy of Sciences, Beijing, China.
Li
L
Jiang
Junmei
J
Fan
Li
L
Bai
Yan
Y
Wang
Yu
Y
Chen
Lu
L
Yang
Liangyi
L
Chen
Tao
T
Xu
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D010122:Cystinyl Aminopeptidase / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000187:drug effects* / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D008561:Membrane Fusion / Q000187:drug effects*; D051379:Mice; D011485:Protein Binding; D021381:Protein Transport; D013997:Time Factors
D015415:Biomarkers; D020690:GTPase-Activating Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; C485187:PHluorin; C505647:Tbc1d4 protein, mouse; D049452:Green Fluorescent Proteins; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase