Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: Observation of mixing of vesicular inner monolayers

Large unilamellar vesicles containing phosphatidylinositol (PI), neutral ph ospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by P I-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing: of vesicular aqueous contents. An average of 2-3 vesicles merge int o a large one in the fusion process. Vesicle fusion is accompanied by leaka ge of vesicular contents. A novel method has been developed to monitor mixi ng of lipids located in the inner monolayers of the vesicles involved in fu sion, Using this method, the mixing of inner monolayer lipids and that of v esicular aqueous contents are seen to occur simultaneously, thus giving ris e to the fusion pore. Kinetic studies show, for fusing vesicles, second-ord er dependence of lipid mixing on diacylglycerol concentration in the bilaye r. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle f usion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing o f outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC ac tivity leads to complete fusion.