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 Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ 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 Ca2+. 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 Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-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 Ca2(+)-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 La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-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 Ca2+.

Human neutrophil annexin I promotes granule aggregation and modulates Ca(2+)-dependent membrane fusion.

“…We showed in a previous report that plasma membranes can fuse with liposomes in a Ca2 -dependent manner (3). In this report, we show that the 36-kD protein both enhanced and inhibited this reaction.…”

“…Annexin I did not promote lipid mixing between these membranes; however, it did affect lipid mixing between the plasma membrane fraction and liposomes. We and others have previously shown that plasma membranes fuse with liposomes in a Ca2l -dependent manner (3,21 ). The 36-kD protein enhanced Ca2 -dependent lipid mixing between plasma membranes and liposomes at 30 ,gM Ca2+ (Fig.…”

“…One likely candidate is Ca2+, since it promotes fusion of artificial membranes and its concentration is elevated near forming phagolysosomes in activated neutrophils ( 1,2). However, we have recently shown that Ca2+ by itselfdoes not promote fusion between neutrophil granule and plasma membrane fractions using a cell-free fusion assay (3). Therefore other components must be required to bring about phagolysosomal formation.…”

“…Neutrophil granule and plasma membrane fractions were isolated using nitrogen cavitation and Percoll gradient centrifugation according to a previously published study (3). Fractions were kept on ice before use.…”

The Formation of Ceramide-1-phosphate during Neutrophil Phagocytosis and Its Role in Liposome Fusion

“…Although the conditions required for PMN activation have been extensively studied, much remains to be learned about the cofactors and mechanisms that promote fusion between granules and the plasma membrane. Recently it has been shown that Ca 2ϩ alone does not promote fusion between neutrophil granules and plasma membrane fractions using a cell-free fusion assay (3). Therefore, other components must be required to bring about phagolysosomal formation.…”