Movements of fluorescent probes in the mechanism of cell fusion induced by poly(ethylene glycol)

Abstract

It has been claimed that purified poly(ethylene glycol) (PEG) is able only to aggregate cells and not to fuse them. In our hands, purified PEG 6000 (recrystallized/dialysed) induces both aggregation and fusion of human erythrocytes, and the mechanism of fusion by the purified polymer has been investigated with fluorescent probes. No movement of a carbocyanine probe or of octadecyl rhodamine B chloride from labelled to unlabelled cells occurred in the absence of PEG or with cells treated with concanavalin A, protamine or spermine. With 40% PEG, however, both probes immediately started to diffuse into the membranes of unlabelled cells. This indicates that continuity between the phospholipid bilayer membranes of adjacent erythrocytes (i.e. membrane fusion) is established within seconds in concentrated solutions of the polymer, and precedes the cell fusion event that is induced by purified PEG. These observations are consistent with the idea that micro-regions of shared phospholipid bilayer may be formed in the membranes of cells when they are forced together as a consequence of the dehydrating action of PEG. Intact erythrocytes were cytoplasmically labelled with 6-carboxyfluorescein to avoid the possibility that loading the cells with a cytoplasmic marker by hypotonic haemolysis might modify their response to PEG. Unlike the lipid probes, carboxyfluorescein did not diffuse from labelled to unlabelled cells in the presence of 40% PEG, and there was little diffusion on subsequent dilution of the polymer solution to 13%. However, after the PEG solution had been replaced by an isotonic buffer, a rapid transfer of the cytoplasmic fluorophore to unlabelled cells often occurred. This is considered to be more consistent with the osmotic rupture of a membranous barrier, such as a shared bilayer, between the labelled and unlabelled cells than with the return of cytoplasmic viscosity to normal when the PEG is removed. Possible reasons are discussed for the reported inability of purified PEG to fuse fibroblasts with hypotonically loaded human erythrocytes.