This project is centered on the mechanisms of exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. We report two subprojects this year, both related to the fact that the major exocytotic proteins are clustered. [unreadable] [unreadable] 1. Cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin. [unreadable] [unreadable] Successful infection by influenza virus requires that the envelope spike protein, hemagglutinin (HA), catalyzes fusion between the viral envelope and the intracellular endosomal membrane of the target cell and creates a pore large enough to release the viral genome. There is a growing appreciation that membrane lipids play a role in this critical event, coming mostly from experiments and theory on lipid composition in relationship to membrane monolayer curvature stress. Recently there has been consideration given to the role of membrane phase behavior and membrane micro-domains on the lateral distribution, sorting and interactions of lipids with membrane proteins in general, and viral envelope glycoproteins in particular. Cholesterol is a major and vital constituent of eukaryotic cell membranes. Its unique structure, a small hydrophilic head group and rigid, hydrophobic, fused-rings, favors preferential association with saturated acyl-chain lipids and sphingolipids to form liquid-ordered micro-domains (termed lipid rafts) in phospholipid bilayer membranes of the right composition. Lipid rafts are hypothesized to exist in the cell plasma membrane at specialized sites where proteins, having favorable associations with the ordered, cholesterol-rich, environment, are concentrated.[unreadable] [unreadable] Specific intermediates of membrane fusion, catalyzed by the influenza virus protein hemagglutinin, are regulated by cholesterol. The extent of early lipid transfer in Sf9 cells expressing HA (HAS cells) is similar to that previously observed in mammalian cell systems, but the initial fusion pore is small and pore expansion is stunted. A three-fold increase in cellular cholesterol (see Methods) leads to 1) faster lipid dye transfer kinetics, 2) increased amount of aqueous dye transferred, 3) increased extent of aqueous dye transferred, and 4) an increase in the rate of pore conductance. The cholesterol dependent increase in fusion efficiency required an intact HA TMD and optimal pH. Overall, these results support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: 1) an early, lipidic stage prior to fusion pore opening and, 2) a later stage during fusion pore expansion. How can the physical properties of cholesterol influence fusion?[unreadable] [unreadable] We have shown that cholesterol promotes both lipid transfer (hemi-fusion) and fusion pore expansion in the cell-cell membrane fusion mediated by influenza HA. The cholesterol effect requires a complete TMD and optimal pH. We hypothesize that cholesterol promotes fusion pore expansion 1) by virtue of its negative intrinsic curvature and 2) the same specific cholesterol/lipid/HA interactions that mediate the 1-10 nm scale clustering of HA in the plane of the membrane, the mobility of HA in fibroblasts, and the phase behavior of the influenza envelope. These structural forces act at the fusion stage of viral invasion to facilitate fusion pore widening.[unreadable] [unreadable] 2. Domain formation in membranes caused by lipid wetting of protein[unreadable] [unreadable] The classic fluid mosaic model views the lipid environment of a plasma membrane as essentially homogenous. But over the past decade it has been increasingly realized that non-homogeneities in membranes are central to biological functions that depend on protein-protein interactions within membranes. Membrane non-uniformities must exist because lipid and protein interactions mediated by hydrophobic, Van der Waals, electrostatic and chemical forces will cause some lipids and proteins to cluster into domains and others to repel. Over thirty years ago it was demonstrated by electron spin resonance that small spatial inhomogeneities existed. It was found that boundary lipids surrounding a protein exhibit about a 10-fold reduction in hop time compared to lipids that are not associated with proteins. In our project, formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. [unreadable] [unreadable] We calculated thermodynamic functions of wetting lipid films by using a mean-field theory of liquid crystals as adapted to biomembranes. We show that either molecular or macroscopic films can form, depending on the values of parameters such as membrane thickness, hydrophobic height mismatches, spontaneous curvature of lipids, and protein radius. We show that a single protein of r 1 nm is not large enough to induce a local phase transition to form a protein-lipid raft. But a macroscopic wetting film can form around a lipid/protein aggregate of more than tens of nanometers in diameter. We have assumed that the lipids in such aggregates are in a liquid-ordered state, analogous to wetting of solid surface. The validity of this assumption is supported by calculations and simulations made in the context of the capillary wave model of Mouritsen. Wetting films that coat an aggregate could be quite important because they facilitate merger of domains. Also, a wetting film prevents a protein from leaving an aggregate and thereby promotes accumulation and clustering of proteins.