Modeling degranulation with liposomes: effect of lipid composition on membrane fusion

Degranulation involves the regulated fusion of granule membrane with plasma membrane. To study the role of lipid composition in degranulation, large unilamellar vesicles (LUVs) of increasing complexity in lipid compositions were constructed and tested for Ca(2+)-mediated lipid and contents mixing. Lipid-mixing rates of LUVs composed of phosphatidylethanolamine (PE) and phosphatidylserine (PS) were strongly decreased by the addition of either phosphatidylcholine (PC) or sphingomyelin (SM), while phosphatidylinositol (PI) had little effect. "Complex" LUVs of PC:PE:SM:PI:PS (24:27:20:16:13, designed to emulate neutrophil plasma membranes) also showed very low rates of both lipid mixing and contents mixing. The addition of cholesterol significantly lowered the Ca2+ threshold for contents mixing and increased the maximum rates of both lipid and contents mixing in a dose-dependent manner. Membrane remodeling, which occurs in neutrophil plasma membranes upon stimulation, was simulated by incorporating low levels of phosphatidic acid (PA) or a diacylglycerol (DAG) into complex LUVs containing 50% cholesterol. The addition of PA both lowered the Ca2+ threshold and increased the rate of contents mixing in a dose-dependent manner, while the DAG had no significant effect. The interaction of dissimilar LUVs was also examined. Contents-mixing rates of LUVs of two different cholesterol contents were intermediate between the rates observed for the LUVs of identical composition. Thus, cholesterol needed to be present in only one fusing partner to enhance fusion. However, for PA to stimulate fusion, it had to be present in both sets of LUVs. These results suggest that the rate of degranulation may be increased by a rise in the cholesterol level of either the inner face of the plasma membrane or the outer face of the granule membrane. Further, the production of PA can promote fusion, and hence degranulation, whereas the subsequent conversion of PA to DAG may reverse this promotional effect.