Studies on the Mechanism of Cell Fusion Using Fluorescent Probes Polyethylene Glycol-mediated Membrane and Cytoplasmic

The mechanism by which polyethylene glycol (PEG) mediates cell fusion has been studied by examining the movements of membrane lipids and proteins, as well as cytoplasmic markers, from erythrocytes to monolayers of cultured cells to which they have been fused. Fluorescence and freeze-fracture electron microscopy and fluorescence recovery after photobleaching have yielded the following results: (a) In the presence of both fusogenic and nonfusogenic PEG 1 membranes are brought together at closely apposed contact regions. (b) Fluorescent lipid probes quickly spread from the membranes of erythrocytes to cultured cells in the presence of both fusogenic and nonfusogenic PEG. (c) Proteins of the erythrocyte membranes were never observed to diffuse into the cultured cell membrane. (d) Water-soluble proteins did not diffuse from the erythrocyte interior into the target cell cytoplasm until the PEG was removed. These data suggest that the coordinate action of two distinct components is necessary for fusion as mediated by PEG. Presumably, the polymer itself promotes close apposition of the adjacent cell membranes but the fusion stimulus is provided by the additives contained in commercial PEG. Polyethylene glycol (PEG) of high molecular weight is widely used to mediate ceU-ceU fusion in the production of somatic cell hybrids, including hybridomas, and more recently in the fusion injection of macromolecules from erythrocytes (1, 2) or liposomes into cultured cells (3). PEG offers advantages over other fusogens in that it permits fusion of a variety of cell types which may differ in species or even in kingdom and under the proper conditions produces high fusion efficiencies with minimal toxicity. Yet little is known of the mechanism by which PEG operates. PEG causes the redistribution of intramembrane particles (IMPs) of cellular membranes, this ability being attributed to the ordering of water by high concentrations of the polymer (4). When aqueous solutions of PEG exceed 35%, cell aggreDr. J. Lucy has pointed out that the term "nonfusogenic PEG" does not apply to all cell fusion protocols. He and his co-workers (29) have shown that extracted PEG (6) will fuse hen erythrocytes in a protocol requiring a 15-rain incubation in PEG. gation and fusion are observed, although maximum fusion efficiency occurs at concentrations between 40 and 50%. Since all water is bound to PEG in solutions having concentrations of 35% by weight or greater, dehydration appears to play a role in PEG-mediated fusion (5, 25). However, pure PEG does not appear to be a complete fusogen. Recently, Honda et al. (6) have demonstrated that antioxidants and/or polymerization agents added to commercial PEG are responsible for the fusion ~ctivity since removal of these agents through organic solvent extraction renders the PEG nonfusogenic) Earlier work had shown that membrane active chemicals such as glyceryl monooleate (GMO) are only capable of inducing cell-cell fusion when administered in conjunction with high molecular weight polymers such as dextrans (5). Our interest in the mechanisms of PEG-induced fusion was prompted by our use of the erythrocyte-mediated microinjection method to introduce fluorescent macromolecules into the cytoplasm of cultured cells (7) and subsequently to measure their diffusion rates by the fluorescence recovery after photoTHE JOURNAL OF CELL BIOLOGY • VOLUME 96 JANUARY 1983 151-159 © The Rockefeller University Press 0021-9525/83/01/0151/09 $1.00 1 51 on O cber 9, 2017 jcb.rress.org D ow nladed fom bleaching (FRAP) technique. Experimentally, this method offers both relative biological simplicity and a high fusion efficiency. To approach the question of the molecular mechanisms occurring during fusion, we planned to examine both the movement of membrane lipids and proteins from one cell to another and the cytoplasmic communication between cells which follows the fusion event. MATERIALS AND METHODS