The primary goal of this project is to understand the processes which occur during the fusion of vesicles to planar bilayer membranes and how they relate to the physiological process of exocytosis. The fusion of phospholipid vesicles to planar membranes has been shown to be enhanced by divalent cations which promote the close association of vesicles to the planar membrane. Osmotic swelling of these vesicles is the driving force for fusion. In cell membranes, phospholipids are arranged in a bilayer structure; we will now establish which aspects of biological exocytosis can be accounted for solely on the basis of phospholipid interactions. Utilizing large (10 um) vesicles in which fusion to planar membranes can be electrically assayed by measuring step-wise increases in the capacitance of the planar membrane, the vesicle-planar membrane interactions will be directly viewed under a fluorescence microscope. By loading the vesicles with fluorescent dye we will determine whether soluble vesicular contents are exclusively transferred to the other (trans) side of the planar membrane as occurs in biological exocytosis, or if there is leakage of contents back into the vesicle containing (cis) side. We will determine the pathway by which vesicles come in contact with the planar membrane and establish to what area of the membrane the vesicles fuse. We will observe whether only isolated vesicles fuse to the membrane or compound exocytosis can occur. The efficacy of fusogenic agents will be tested using a previously established conductance assay and their mechanisms of action investigated. To more closely model biological exocytosis, the fusion of biological intracellular secretory granules to planar membranes will be studied. Using catecholamine-containing chromaffin granules we will determine if conductance increases in the planar membrane accompany fusion; and measure release of granule contents to the trans side. The long range effort will be to determine the conditions necessary to induce fusion, the parameters which control its rate, and the forces and energetics - in other words the underlying mechanisms - of the fusion process in this semi-natural system.
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