Divalent Cation Induced Fusion and Lipid Lateral Segregation in Phosphatidylcholine-Phosphatidic Acid Vesicles

Abstract

The interactions of unilamellar vesicles containing phosphatidylcholine (PC) and phosphatidic acid (PA) in the presence of calcium and magnesium were examined by fluorometric assays of vesicle lipid mixing, contents mixing, and contents leakage and by spray-freezing freeze-fracture electron microscopy. These results were correlated with calorimetric and fluorometric measurements of divalent cation induced lateral segregation of lipids in these vesicles under comparable conditions. PA-PC vesicles in the presence of calcium show a rapid but limited intermixing of vesicle lipids and contents, the extent of which increases as the vesicle size decreases or the PA content increases. Calcium produces massive aggregation and efficient mixing of the contents of vesicles containing high proportions of dioleoyl-PA or egg PA, but vesicle coalescence in the latter case is followed rapidly by vesicle collapse and massive leakage of contents. The effects of magnesium are similar for vesicles of very high PA content. However, in the presence of magnesium, vesicles containing lower amounts of PA exhibit "hemifusion", a mode of interaction in which vesicles aggregate and mix approximately 50% of their lipids, apparently representing the lipids of the outer monolayer of each vesicle, without significant mixing of vesicle contents or collapse of the vesicles. Fluorometric measurements of lipid lateral segregation demonstrate that lateral redistribution of lipids in PA-PC vesicles begins at submillimolar concentrations of divalent cations and shows no abrupt change at the "threshold" divalent cation concentration, above which coalescence of vesicles is observed. By correlating calorimetric and fluorometric measurements of lipid lateral segregation and mixing of vesicle components, we can demonstrate that lipid segregation is at least strongly correlated with calcium-promoted coalescence of PA-PC vesicles and is essential to the magnesium-promoted interactions of vesicles of low PA contents.

Ion Regulation of Homotypic Vacuole Fusion in Saccharomyces cerevisiae

“…Divalent ions can also drive fusion of protein-free liposomes (35)(36)(37). It has been unclear how each of these two ion effects relate to physiological membrane fusion.…”

“…The addition of low concentrations (0.1-1 mM) of divalent ion chelators in various combinations promotes fusion, with maximal fusion occurring in the presence of all three chelators (TPEN, EDTA, and BAPTA), a condition where any free divalent ions would be at subnanomolar concentrations. Although divalent ions can directly associate with lipid bilayers to promote fusion (35)(36)(37), these data suggest that free divalent cations are not required for association with signaling proteins during vacuole membrane fusion.…”

GTPγS and phorbol ester act synergistically to stimulate both Ca2+‐independent secretion and phospholipase D activity in permeabilized human platelets

“…There are multiple mechanisms through which PA generated by PLD might enhance secretion. These include activation of PKC, as suggested by the GTPyS-induced phosphorylation of pleckstrin [l], stimulation of other protein kinases [25], effects on factors that regulate low-M, GTP-binding proteins [26] and a direct membrane-fusogenic action [27].…”

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

“…In the presence of 300 pM POPG vesicles, the onset of aggregation appears to begin at approximately 0.9 pM HNP-2, where, at most, 1070 of the lipid bilayer charge has been neutralized. This suggests that bound defensins are probably recruited by diffusion and concentrated in regions of close bilayer contact because aggregation can only occur when the surface charge in the contact area is neutralized (Leventis et al, 1986).…”

“…The apparent lack of contents mixing and the limited mixing of bilayer lipids detected with the NBDhhodamine assay indicates that the defensin-induced fusion of the inherently stable POPG LUV is not a significant issue. If fusion occurs, it is limited to hemi-fusion resulting from the mixing of outer-monolayer lipids at bilayer contact sites (Leventis et al, 1986).…”