The goal of this project is to describe the events which occur, on a molecular level, during the fusion of vesicles and viruses to planar bilayer membranes. This will provide a model system for understanding the physiological process of fusion which includes exocytosis. Interactions between bound vesicles and planar membranes will be studied by incorporating acceptor/donor pairs of fluorescent phospholipids into vesicular and planar membranes. The vesicles will be bound to planar membranes with divalent cations. Fluorescence energy transfer measurements will be performed, employing video fluorescence microscopy, to characterize the bound state. Image processing and analysis will be used to determine the energy transfer in the contact region between vesicles and planar membranes. These experiments and analyses will yield: 1) the area of contact between vesicle and planar membrane, 2) the distances between the two membranes, and 3) whether lipids such as phosphatidylethanolamine accumulate in the region of contact. By reconstituting the ion-channel porin into the vesicular membranes, simultaneous voltage-clamp and fluorescence energy transfer measurements will be performed. The changes in energy transfer that precede and accompany fusion (noted as incorporation of porin into the planar membrane) will provide data on the rearrangements that lipids undergo during fusion. To study protein- mediated fusion, a model system of enveloped virus-mediated fusion of phospholipid vesicles to planar membranes will be developed. Viral envelopes will be fluorescently labelled and the binding of virus to the planar membrane will be studied as a function of membrane composition and pH. Large (several microns) phospholipid vesicles will be loaded with high concentrations of the fluorescent dye calcein. Virus-mediated binding of the vesicles to the planar membrane will be studied. Viral-mediated fusion will be monitored as transfer of dye to the side of the membrane free of viruses and vesicles (trans side). Release of dye to the vesicle- containing (cis) side will also be measured. By loading red blood cell ghosts with calcein, experiments similar to those described for the phospholipid vesicles can be performed with red blood cells. This will provide a model system for virally-mediated cell-cell fusion and hemolysis. Reconstituted viral envelopes (virosomes) will be loaded with calcein. Virosome binding to planar membranes will be studied and whether fusion occurs will be investigated. If fusion occurs, this would provide a direct model system for virus fusion to cell membranes and therefore viral entry into cells.
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