Abstract
To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.
Highlights
To elucidate the role of phospholipid asymmetry in calcium-phosphate-inducedfusion of human erythrocytes,we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributionsof phospholipids
We have shown recently that vesicular stomatitis virus fuses rapid with lipid-symmetric erythrocyte membranes at acidic pH, but shows little or no fusion with lipid-asymmetric erythrocyte membranes (Herrmann et al, 1990b).The role of phospholipid asymmetry in fusion of membranes is sustained by recent investigations on artificially induced cell-cell fusion
Fusion was monitored either by light microscopy or fluorescence dequenching assay which is based on the insertion of the fluorescent amphiphile octadecylrhodamine B chloride (R18)into one population of ghosts at a self-quenching concentration andthe dequenching of fluorescence following fusion with an unlabeled membrane (Hoekstra et al, 1984; Herrmann and Hillebrecht, 1991).We found that the redistribution of aminophospholipids, in particular PS to the outer leaflet, facilitated membrane fusion
Summary
After removal of buffy coat and plasma, RBC were washed three times in buffer A (150 mM NaC1, 5.8 mM phosphate buffer, pH 7.4). The procedure for resealed lipid-symmetric ghosts is based on that of Williamson et al (1985) (Clagueet al., 1990; Herrmann et al, 199Ob).RBC were lysedby adding 15 volumes of 10 mM Tris, 0.1 mM EGTA (pH 7.2) containing 1mM CaCI2.Resealing of ghosts was performed by adding concentrated buffer solution consisting of 150 mM Na2HP04,50 mM KH2P04,1.22 M NaCl, 30 mM KC1,l mM CaC12,incubating the suspension at 37 "C for 40 min, and subsequently washing 3 times in buffer A. The molar ratio of fluorophore to endogenous lipids was kept at 6 mol %, assuming a weight ratio of protein to lipid of 1:lfor erythrocyte membranes or, alternatively, 4 X molof phospholipid/cell (Shiga et al, 1979).Ghosts or intact RBC were labeled with R18 in the dark (room temperature) for 15 min as described (Herrmann et al, 1988; Herrmann and Hillebrecht, 1991) and thereafter washed twice in buffer D (150mM NaC1,O.l mM EDTA, 10 mM phosphate buffer, pH 7.4). The fusion index, FI (Perez et al, 1974; Herrmann et al, 1988),was calculated
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