This project is centered on the mechanisms of endocytosis and exocytosis, the ubiquitous eukaryotic processes by which vesicles fuse to the plasma membrane and release their contents, and then are retrieved. We report two subprojects this year. 1. Formation of an endocytic vesicle is completed by scission of a thin membrane neck connecting the vesicle and the plasma membrane Though scission is intuitively associated with cutting and resealing, even transient permeabilization of cellular membranes could be damaging, especially for small vesicles whose content can quickly escape through even minute pores. Thus, fission of neck membranes is more likely to proceed via hemifission to avoid leakage. The neck fission involves extensive bending deformations conducted by a specialized protein machinery assembled on the membrane neck, where the GTPase dynamin is a key component). Crucial for many cell processes featuring fission, dynamin family members form dense collars around necks of budding vesicles and dividing organelles. Assembly of this collar triggers cooperative GTP hydrolysis, which is thought to force membrane remodeling. However, the pathway that links the GTPase activity of dynamin and membrane rearrangements during fission is obscure. The dynamics of membrane remodeling by short dynamin assemblies formed in the presence of GTP is difficult to characterize because the corresponding membrane transformations are expected to be fast and highly localized. In cellular systems, similar membrane transformations are studied by electrophysiology revealing a rich dynamic behavior of membrane necks of endocytic vesicles. Here, we applied these techniques to resolve dynamin's interaction with nanotubes pulled from lipid membranes. We measured the ionic permeability of the tube's interior to estimate average changes in the diameter of the tube and to resolve the kinetics of tube fission. We discovered that self-assembling dynamin scaffolds caused dramatic narrowing of the nanotubes, their final radius depending on the tube rigidity. However, fission required partial disassembly of long dynamin scaffolds triggered by GTP hydrolysis. GTPase cycles of dynamin were coupled to assembly and disassembly of short dynamin coats, producing membrane curvature and fission in a stochastic lipid-dependent manner. These results suggest that dynamin acts as a catalyst of membrane remodeling bringing membrane nanotubes to the point of spontaneous fission via creation of regulated curvature constraints. Membrane fission converges to a highly localized and fast restructuring of the lipid bilayer. Using sensitive time-resolved conductance measurements, we identified the key steps for fission of NT mediated by dynamin. Theoretical analysis of these data revealed that the fission is catalyzed in two critical steps: GTP-independent scaffolding of membrane curvature by dynamin followed by GTP-dependent disassembly of the scaffold, allowing lipid to complete membrane remodeling. Self-assembly of the dynamin scaffold induces NT narrowing until the scaffold reaches a length sufficient to trigger GTP hydrolysis. Depending on the curvature imposed on the NT, membrane detachment from the dynamin scaffold upon GTP hydrolysis can cause spontaneous hemifission followed by complete fission. This step is apparently stochastic: hemifission probability depends on the energy barrier for the hemifission transformation and on the timeframe during which the dynamin scaffold holds its rigidity upon GTP hydrolysis. On NT the scaffold softens 10 s after the hydrolysis. If fission does not happen within this time, NT expands with the softening of the scaffold and then a new squeezing cycle is initiated. Consistent with this scheme, cyclic assembly of fluorescently labeled dynamin in the presence of GTP has been visualized directly. Several sequential squeezing attempts might be needed to trigger hemifission of the NT. Our experiments reveal that dynamin activity is crucially modulated by lipid composition: final diameters of dynamin-coated tubes (without GTP) are not dictated by dynamin alone, but also depend on lipid composition. If dynamin scaffold had completely dominated the energetics of tube formation, then it would have satisfied its optimal protein packing constraints by forming the same diameter tube. If, however, the energy of scaffold polymerization is comparable to the energy of membrane bending, then the final tubule diameter will depend on lipid composition. Hence, dynamin-like proteins may function as GTP-dependent curvature agents. To summarize, our results reveal a tight coupling between dynamin and the lipid template. Dynamin is designed to selectively target highly curved membrane necks and probe their mechanical stability by repetitive squeezing. Fission critically depends on the geometry and mechanical parameters of the neck membrane. This dependence has a clear physiological significance: dynamin effectively cuts only those necks that are prone to fission, such as narrow necks formed at the final stages of vesicle detachment. On wider and/or more rigid necks, dynamin is expected to operate as a GTP-dependent curvature regulator. Hence, cooperation of dynamin and the lipid membrane provides a universal tool to control the behavior of the neck of a vesicle. 2. Insulin Regulates Fusion of GLUT4 Vesicles Independent of Exo70-mediated Tethering At the cellular level, glucose uptake is regulated by controlling the amount of the GLUT4 2 glucose transporter present in the plasma membrane. In insulin-responsive adipose and muscle cells, insulin regulates glucose uptake by promoting the exocytosis of specialized GLUT4 storage vesicles (GSV). Although many steps of the insulin signal transduction pathway have been elucidated, the mechanism bridging insulin signaling with the actual fusion of GSV with the plasma membrane, with concomitantly enhanced glucose uptake, is still missing. Increasing evidence suggests that insulin regulates GLUT4 exocytosis at many different levels such as intracellular sequestration of GLUT4 into GSV, traffic to the plasma membrane, and tethering and fusion. Recent reports using total internal reflection fluorescence (TIRF) microscopy and comprehensive kinetic analysis using rat adipose cells and 3T3-L1 adipocytes suggest that both tethering and fusion of GSV are primary steps regulated by insulin stimulation. Insulin regulates GLUT4 exocytosis at multiple steps involving trafficking, tethering, and fusion. The molecular events associated with stimulated GLUT4 exocytosis are likely to take place at both the vesicular surface and the plasma membrane. In the current study, we investigated the role of the Exocyst component Exo70 on the trafficking and tethering events of GSVs. We did not see any effect of overexpressing the wildtype Exo70 protein. Surprisingly, the Exo70-N mutant, reported to block the assembly of the Exocyst complex and GLUT4 exocytosis in differentiated 3T3-L1 cells, enhances the tethering in the primary adipose cells but does not affect the rate of fusion, neither in the basal nor in the insulin-stimulated state. The effect of the Exo70 mutant on the tethering in basal conditions was strikingly similar to insulin-induced tethering but is insufficient to induce fusion. This fact indicates that insulin regulates fusion steps of GLUT4 exocytosis independently of upstream tethering where Exo70 might be involved.
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