MODELING DEGRANULATION WITH LIPOSOMES - EFFECT OF LIPID-COMPOSITION ON MEMBRANE-FUSION

Degranulation involves the regulated fusion of granule membrane with p lasma membrane. To study the role of lipid composition in degranulatio n, large unilamellar vesicles (LUVs) of increasing complexity in lipid compositions were constructed and tested for Ca2+-mediated lipid and contents mixing. Lipid-mixing rates of LUVs composed of phosphatidylet hanolamine (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 plas ma membranes) also showed very low rates of both lipid mixing and cont ents mixing. The addition of cholesterol significantly lowered the Ca2 + threshold for contents mixing and increased the maximum rates of bot h lipid and contents mixing in a dose-dependent manner. Membrane remod eling, which occurs in neutrophil plasma membranes upon stimulation, w as simulated by incorporating low levels of phosphatidic acid (PA) or a diacylglycerol (DAG) into complex LUVs containing 50% cholesterol. T he addition of PA both lowered the Ca2+ threshold and increased the ra te of contents mixing in a dose-dependent manner, while the DAG had no significant effect. The interaction of dissimilar LUVs was also exami ned. Contents-mixing rates of LUVs of two different cholesterol conten ts were intermediate between the rates observed for the LUVs of identi cal composition. Thus, cholesterol needed to be present in only one fu sing partner to enhance fusion. However, for PA to stimulate fusion, i t had to be present in both sets of LUVs. These results suggest that t he 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 fa ce 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.