The long term goals of this proposal are to elucidate the molecular mechanism of membrane fusion involved in exocytotic processes, in order to have a better understanding and control of biological secretion phenomena under normal and pathological conditions. For this purpose, parallel studies of membrane fusion for both model membranes and biological membranes in secretory systems will be performed. Membrane fusion in model membrane systems (lipid vesicles, monolayer and bilayer membranes) induced by divalent (or polyvalent) cations, temperature and osmotic pressure gradients will be examined for the cases with and without proteins or cholesterol incorporated in the membranes. Effects of various fusogenic substances, biomolecules (i.e., arachidonic acid and polyamines) and calcium-phosphate on membrane fusion will also be studied. Membrane fusion will be correlated with the degree of free-energy increase in the membrane surface due to ion binding to and dehydration of bound water from the membrane surface for the case of divalent cation induced fusion, due to membrane expansion for the case of temperature or osmotic pressure gradient induced membrane fusion, and due to membrane surface conformational change possibly mediated by calcium binding proteins. In order to apply our knowledge about membrane fusion in model lipid membrane systems to biological membrane systems, the interaction and fusion between lipid membranes and biological membranes (synaptic vesicles, chromaffin granules, erythrocyte vesicles, etc.) will be examined along the same lines as summarized above. Methods to be used for these experiments will include fluorescence and luminescence fusion assays, light scattering measurements, dynamical light scattering techniques, weight measurements with a microbalance, NMR, electron microscopy, phase contrast microscopic observation, and other physico-chemical (surface chemistry, etc.) techniques to determine properties of the membranes and their constituents.
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