The aim of this application is the elucidation of the roles of key elements in the mechanism of membrane fusion as it occurs in neurosecretory processes. The complexity and limited accessibility of synaptic membranes virtually preclude molecular dissection in vivo and model systems appear to constitute the only feasible approach to unraveling the molecular mechanism of neurosecretion. Because of the difficulty of assaying for fusion, however, such systems have not utilized the natural membrane constituents that may be critical to the efficient and controlled operation of the process. This application describes a novel assay for membrane fusion and its proposed use in a model neurosecretory system that utilizes components of the natural membranes that participate in neurotransmitter release. Synaptic vesicles, loaded with high, self-quenched concentrations of the fluorescent dye, calcein, will be assayed for fusion to a planar model membrane reconstituted from membrane components spread as a monolayer. Fusion will be assayed by monitoring and recording the """"""""flashes"""""""" of fluorescence resulting from the sudden dequenching of fluorescence as calcein is released on the trans side of the membrane. Differentiation between fusion and rupture of vesicles attached to the planar bilayer will be accomplished by taking advantage of the unusual sensitivity of calcein fluorescence to quenching by cobalt ion, which can be introduced on either cis or trans sides of the membrane and by selective focussing of the optical detection system. The capacity of synaptic vesicles to fuse with a purely lipid planar bilayer, after adhesion, will be investigated. Since calcium ion is known to participate in neuroscretion in vivo, the specificity for calcium ion of both adhesion and fusion will be determined. The possibility that stress in secretory vesicle membranes drives the integration of membranes will be tested by assessing the effect of osmotic stress on fusion. Using planar membranes reconstituted from presynaptic membranes, the participation of presynaptic membrane components will be examined to determine whether they influence; a) the adhesion process with respect to either specificity for or concentration dependence on calcium ion, b) the efficiency of fusion, or c) both.
| Perin, M S; MacDonald, R C (1989) Fusion of synaptic vesicle membranes with planar bilayer membranes. Biophys J 55:973-86 |
| Perin, M S; MacDonald, R C (1989) Interactions of liposomes with planar bilayer membranes. J Membr Biol 109:221-32 |
| MacDonald, R C; MacDonald, R I (1988) Membrane surface pressure can account for differential activities of membrane-penetrating molecules. J Biol Chem 263:10052-5 |
| MacDonald, R C; Simon, S A (1987) Lipid monolayer states and their relationships to bilayers. Proc Natl Acad Sci U S A 84:4089-93 |
