The objectives of this research are threefold. First, to develop the analysis of phospholipid vesicle fusion at two levels: (i) Kinetic - to ascertain which step of the overall process is rate limiting and (ii) Structural - identifying the intermembrane intermediates involved either during or subsequent to fusion. The effects of lipid composition,cation binding temperature and liposome size on the destabilization and fusion of these liposomes is being developed into a rigorous biophysical theory of membrane interactions. This theory is centered upon relating equilibrium thermodynamic properties of the lipids, e.g., phase transitions and morphological changes, to the initial interaction and destabilization of apposed bilayers. Our second objective is to enlarge our study of cytoplasmic fusion agents using liposomes as target membranes. The protein synexin and the polyamine spermine have both been implicated as mediators of intracellular fusion events involved in exocytosis. Our preliminary data show that both these agents act on liposomal membranes solely by enhancing aggregation rates, i.e., the rate of close approach. The destabilization of the apposed bilayers requires another factor, e.g., bound Ca2+. We will establish the generality of this result with other membranes. Of particular interest here is elucidating the effect of triphosphoinositides on membrane fusion in the presence of the polyamines. Our third objective is to begin studies on biological fusion proteins, specifically hemagglutinin from the influenza virus and the (putative) myoblast fusion protein. For hemagglutinin, we will focus on the role of cell surface receptors on the fusion mechanism using intact virus and reconstituted systems, virosomes, which we will develop into a reliable method for delivery of macromolecules into cells. For the myoblasts, we will focus on the ability of muscle cell vesicles, secreted during active myoblast fusion, to induce the fusion of a cloned line of nonfusing myoblasts.
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