We have continued our research on membrane fusion, the fundamental step in secretion, viral infection, fertilization and neuro-transmission. Sea urchin cortical granule exocytosis, an example of calcium triggered membrane fusion, has served as a model system. Fusion kinetics were modeled using a new principle for interpreting the calcium dependence of calcium-triggered exocytosis: calcium controls the number of committed fusion complexes at docking sites and exocytotic granules have spare fusion complexes that are Poisson distributed among the population of granules. The random model for multiple fusion complexes that we presented last year was extended by explicitly including a committed state following calcium-dependent activation. The probability of a committed fusion complex to fuse and the average number of committed fusion complexes were linearly dependent on pCa such that the fusion probability per average number of fusion complexes was constant over a range of calcium concentrations. This suggests that the overall probability to fuse depends on both the number of committed fusion complexes and an intrinsic fusion probability. We have also use isolated cortical granules (CG) for combined biochemical and physiological studies to identify essential proteins and protein complexes. Based on the SNARE hypothesis of membrane targeting/docking/fusion, a heterotrimeric core complex of the proteins VAMP, Syntaxin and SNAP-25 plays a central role in docking and fusion; according to the hypothesis, bilayer fusion is driven by the dissociation of this complex. We have identified these proteins as both monomers and as constituents of a heterotrimeric complex found on isolated sea urchin CG in suspension and between contacting CG. Free Ca2+ concentrations triggering maximal CG-CG fusion result in complete dissociation of the core complex, and this dissociation is inhibited by N-ethylmalaimide at concentrations that also inhibit fusion. In contrast, lysophosphatidylcholine, which blocks a late step in membrane fusion, does not inhibit Ca2+-induced complex dissociation; thus, dissociation is not a result of membrane fusion, but must be a prior or concomitant process. Using a variety of tools and treatments including alternative divalent cations to trigger fusion, tetanus and botulinum toxins to specifically remove SNARE proteins, we have more extensively analyzed the relationship between membrane fusion and the presence of SNARE protein complexes and monomers. The results dispute a direct fusogenic role for the SNARE protein monomers or complex and suggest that these proteins likely function during docking.
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| 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 |
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| Chernomordik, Leonid V; Zimmerberg, Joshua; Kozlov, Michael M (2006) Membranes of the world unite! J Cell Biol 175:201-7 |
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