MECHANISMS OF MEMBRANE FUSION

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We propose to study the molecular factors which control exocytotic and endocytotic fusion events by means of simplified (sub-cellular) in vitro models for these processes. We will determine the role of individual lipid species and the effects of low pH and osmotic gradients on membrane fusion by means of liposome fusion assays. We also propose to identify the cytoplasmic factors and membrane proteins involved by following the fusion of isolated coated vesicles with lysosomes and the interaction of chromaffin granules with chromaffin cell plasma membranes. The individual projects can be categorized as follows: (1) Control of membrane fusion by individual lipid species: (i) the lipids involved in the stimulus-linked "phospholipid effect, "ie: the tri-, di-, and monophosphatidylinositols and their metabolic byproducts, phosphatidate, diglycerides and arachindonic acids; (ii) acyl chain composition (fluidity) within a particular phospholipid species; (iii) the gel-liquid crystalline and lamellar-hexagonal HII phase transitions; (iv) glycolipigs and lectins. (2) The role of low pH and osmotic grandients in membrane fusion: (i) molecular requirements of low pH (4.5-6.5)-induced membrane fusion, as a model for the fusion of lipid-enveloped viruses and the sorting-out and recycling of endocytotic vesicles; and (ii) the contribution of osmotic gradients to the kinetics and Ca++ requirements of the fusion of liposomes of varying composition and size. (3) Cytoplasmic and membrane proteins which mediate exocytosis: the role synexin and other cytoplasmic factors and granule membrane proteins in the Ca++ mediated interaction of isolated chromaffin granules with plasma membranes. (4) Molecular factors involved in the fusion of coated vesicles with lysosomes: fusion of coated vesicles with lysosomes with a fluorescence assay and the cytoplasmic factors and membrane proteins which mediate the uncoating of the coated vesicles and their fusion with the lysosomes.