Interactions between metal ions and poly(ethylene glycol) in the fusion of human erythrocytes

Summary

Diffusion of the fluorescent membrane probe, Dil-C₁₆ (3), from labelled to unlabelled human erythrocytes has been employed to monitor hemi-fusion (membrane fusion) in monolayers of cells exposed to poly(ethylene glycol) (PEG). Diffusion of the cytoplasmic probe, 6-carboxyfluorescein, was used similarly to monitor cell fusion (cytoplasmic mixing). Hemi-fusion, which is normally seen when erythrocytes are exposed to dehydrating concentrations of commercial PEG 6000, did not occur when the PEG was pretreated with Chelex 100 resin to remove metal ions. Cytoplasmic mixing, which is normally observed when the dehydrated erythrocytes are substantially rehydrated, also failed to occur when both PEG 6000 and the rehydrating buffer had been treated with Chelex 100. The re-addition to Chelex-treated PEG of components removed by the resin, and the addition of 10 μ mM concentrations of La³⁺ or Al³⁺, restored its ability to induce hemi-fusion and cell fusion. Higher concentrations of several other metals, including Ca²⁺, were also effective. These observations show that metal ions are required for hemi-fusion with erythrocytes in the presence of PEG, and that dehydration alone is insufficient to induce hemi-fusion. Phosphatldylserine was apparently not accessible in erythrocytes treated with PEG 6000 until the cells were rehydrated. This indicates that metal ions do not assist the hemi-fusion of erythrocytes by forming trans complexes with surface phosphatldylserine when the cells are dehydrated by PEG. It is suggested that tri—and divalent metal ions facilitate the PEG-induced fusion of human erythrocytes by perturbing the polar groups of phosphatidylcholine in the outer leaflet of closely apposed cells, thus acting synergistically with dehydrating concentrations of PEG to increase membrane hydrophobicity.

Key Words:

• Cell fusion
• poly(ethylene glycol)
• erythrocyte
• phospholipid vesicle
• lipid mixing