Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

Abstract

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca 2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca 2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca 2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca 2+, with threshold and 50% maximal rate of fusion occurring at 2 μM and 50 μM, respectively. Furthermore, the fusion was highly specific for Ca 2+; other divalent cations such as Ba 2+, Mg 2+ and Sr 2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca 2+-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca 2+-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La 3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca 2+. Furthermore, GTPγS was found to enhance Ca 2+-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca 2+.