We have continued our combined biophysical and biochemical research on the molecular mechanisms of membrane fusion during exocytosis, the critical step in insulin, neurotransmitter, and immunoglobulin secretion, and in fertilization. Cortical vesicles (CV), the docked, release-ready, secretory organelles of sea urchin are isolated from concomitant processes such as vesicle mobilization from reserve pools and vesicle recycling, have all the factors essential to tight membrane-membrane contact, retain their capacity for Ca2+-triggered fusion in vitro, and are available in quantities sufficient for biochemical detection of membrane components. Our previously described model for multiple fusion complexes was used to analyze homotypic CV fusion, revealing that CV-CV fusion captures all the essential features of exocytosis, including the same number of operationally defined 'Fusion Complexes' per vesicle, and a similar Ca2+ sensitivity. CV-CV fusion is therefore a reduced model system for Ca2+-triggered exocytosis. Analysis of results with Sr2+ and Ba2+ also indicate that these divalent cations trigger fusion via the same common pathway as does Ca2+. Although SNARE complex disruption is Ca2+-triggered in this native membrane, disruption occurs at higher [Ca2+]free than is Ca2+-triggered fusion initiated. Furthermore, the Ca2+ sensitivity of the complex was unchanged in fusion-incompetent CV. Sr2+ and Ba2+ substituted for Ca2+ in triggering fusion but had no significant effect on complex disruption. Thus, contrary to current hypotheses in the literature, neither the formation, presence nor disruption of the SNARE complex is essential to fusion. We have also demonstrated this result for the fusion of endogenously docked CV to the plasma membrane. Thus, using both an experimental and a modeling approach, we find that the SNARE complex is not essential to the final steps of exocytosis. By exposing CV to Ca2+ while bringing them into contact (rather after the establishment of contact), results were obtained suggesting that inter-membrane SNARE complexes may enhance the Ca2+ sensitivity of the fusion reaction. Other membrane components or other SNARE protein functions, which must be essential for Ca2+-triggered membrane fusion, will be investigated next.
| Lizunov, Vladimir A; Lisinski, Ivonne; Stenkula, Karin et al. (2009) Insulin regulates fusion of GLUT4 vesicles independent of Exo70-mediated tethering. J Biol Chem 284:7914-9 |
| Biswas, Subrata; Yin, Shu-Rong; Blank, Paul S et al. (2008) Cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin. J Gen Physiol 131:503-13 |
| Shnyrova, Anna; Frolov, Vadim A; Zimmerberg, Joshua (2008) ER biogenesis: self-assembly of tubular topology by protein hairpins. Curr Biol 18:R474-6 |
| Plonsky, Ilya; Kingsley, David H; Rashtian, Afshin et al. (2008) Initial size and dynamics of viral fusion pores are a function of the fusion protein mediating membrane fusion. Biol Cell 100:377-86 |
| Akimov, Sergey A; Frolov, Vladimir A J; Kuzmin, Peter I et al. (2008) Domain formation in membranes caused by lipid wetting of protein. Phys Rev E Stat Nonlin Soft Matter Phys 77:051901 |
| Shnyrova, Anna V; Ayllon, Juan; Mikhalyov, Ilya I et al. (2007) Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain. J Cell Biol 179:627-33 |
| Hess, Samuel T; Gould, Travis J; Gudheti, Manasa V et al. (2007) Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc Natl Acad Sci U S A 104:17370-5 |
| Akimov, Sergey A; Kuzmin, Peter I; Zimmerberg, Joshua et al. (2007) Lateral tension increases the line tension between two domains in a lipid bilayer membrane. Phys Rev E Stat Nonlin Soft Matter Phys 75:011919 |
| Chernomordik, Leonid V; Zimmerberg, Joshua; Kozlov, Michael M (2006) Membranes of the world unite! J Cell Biol 175:201-7 |
| Chen, Xiaocheng; Arac, Demet; Wang, Tzu-Ming et al. (2006) SNARE-mediated lipid mixing depends on the physical state of the vesicles. Biophys J 90:2062-74 |
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