Dissecting Multiple Steps Of Vesicle Trafficking And Elucidating Their Regulatory Mechanisms

The process of vesicle translocation and fusion involve multiple steps and complex protein interactions. Extensive efforts have been made to study the whole process in recent years, but it is still not clear when and where do these proteins function? The obstacle lead to failure to uncover the molecular mechanisms is lacking an effective method, which can dissect the multiple steps at high spacial and temporal resolution during vesicle trafficking and fusion process. Taking advantage of total internal reflection fluorescence microscope (TIRFM), we marked the vesicles with fluorescence protein, studied the multiple steps and molecular mechanisms during vesicle translocation in living cells. In this paper, we compared the different molecular mechanisms of vesicle translocation and membrane fusion between GTP triggered and Ca²⁺ induced exocytosis in PC12 cells. In addition, we developed a new way to dissect the multiple steps in vesicle translocation and elucidated the mechanisms of insulin induced membrane protein GLUT4 trafficking in 3T3-L1 adipocytes.Many cells utilize a GTP-dependent pathway to trigger exocytosis in addition to Ca²⁺-triggered exocytosis. However, little is known about the mechanism by which GTP triggers exocytosis independent of Ca²⁺. We used dual-color evanescent field microscopy to compare the motion and fusion of large dense core vesicles stimulated by either mastoparan in Ca²⁺-free conditions or high K+ in the presence of Ca²⁺ in PC12 cells. We demonstrate that mastoparan is hardly effective in triggering the fusion of the pre-docked vesicles, but predominantly mobilizes cytosolic vesicles. In contrast, Ca²⁺-dependent exocytosis is largely due to pre-docked vesicles. Fusion kinetics analysis and carbon-fibre amperometry reveal that mastoparan induces a brief'kiss-and-run'fusion and releases only a small amount of the cargo, whereas Ca²⁺ stimulates a more persistent opening of the fusion pore and larger release of the contents. Furthermore, we show that mastoparan-released vesicles require a much shorter time to reach fusion competence once they approach the plasma membrane. Our data suggest the involvement of different mechanisms not only in triggering and fusion, but also in the docking and priming process for Ca²⁺- and GTP-dependent exocytosis.GLUT4 is the only one of GLUTs family can be translocated to the plasma membrane in response to insulin. Though the translocation constitutes a key process for blood glucose control, research on the mechanisms of insulin-induced GLUT4 translocation has thus progressed, functional assays to distinguish multiple steps along the GLUT4 vesicle trafficking cascade are still lacking, which hinders current progress towards the elucidation of the underlying molecular events and the identification of the key step(s) that is/are regulated by insulin. Here we developed an unequivocal method for dissecting and systematic analyzing the docking, priming and fusion steps of GLUT4 vesicle in 3T3-L1 adipocytes. Using this method, we show that insulin can increase the docking rate of GSVs. Furthermore, we identify a key step regulated by insulin, which is the preparing of GLUT4 vesicle for fusion competence after docking at the plasma membrane. Also, we demonstrate that docking involves activation of PI3K and its downstream effector AS160. This method, in combination with various molecular interventions, will greatly facilitate our understanding of the molecular events of GLUT4 trafficking cascade and how these events are precisely regulated by insulin signaling.