As part of a search for chemical agents able to promote the fusion of mouse A 9 fibroblasts, oleylamine, a positively charged compound, has been investigated for its fusogenic properties. In the pH range 5.5-7.5 and in the presence of dextran, fibroblast polykaryons were produced on treatment of monolayers of cultured cells with oleylamine dispersed directly in a modified Eagle's medium at concentrations of not less than 0.111 mg/ml. Electron microscopy demonstrated the absence of a dividing plasma membrane between the constituent nuclei of the polykaryons, and showed clustering of other subcellular organelles around their original parent nuclei. Fusion, which was preceded by rounding and swelling of the cells, occurred between cells in contact after 10-15 min. Oleylamine in lipid droplets containing glyceryl mono- and dioleate also caused swelling and fusion but to a lesser extent. Phosphatidylcholine appeared to have an inhibitory effect on oleylamine-induced fusion: lecithin liposomes containing oleylamine were only weakly fusogenic. The fusion process, but not the preceding swelling, was calcium-dependent; fusion was inhibited by low concentrations of lanthanum ions. While oleylamine inhibited cell division in monolayer cultures and prevented adhesion of fibroblasts in suspension to glass coverslips, oleylamine in lipid droplets was less toxic and is thus potentially more useful in this form for interspecific hybridization experiments.
Hen erythrocytes that were fixed after treatment with lysolecithin in aqueous solution for 30 s at 37 °C showed evidence of bridge formation between adjacent lysed cells. Generally, the homokaryons that were produced using lysolecithin in this way contained large numbers of nuclei. These giant syncytia had damaged nuclear membranes and unstable plasma membranes; complete disintegration of the syncytia occurred within 1 min of adding lysolecithin to the erythrocytes. In order to localize the action of lysolecithin, the fusing agent was incorporated into microdroplets of lipid. Cell fusion following the addition of lysolecithin in an aqueous glyceridelecithin emulsion was slower than with lysolecithin in aqueous solution, taking 10-30 min, and it was accompanied by considerably less damage to the plasma and nuclear membranes. The fused erythrocytes, which usually contained only two or three nuclei, lysed slowly during the 45 min following fusion, and lysis could be arrested by cooling the fused cells. The plasma membranes of lysed, multinucleated cells remained intact at 37°C for at least 90 h. Mouse fibroblast-hen erythrocyte heterokaryons formed with the aid of the emulsion were more stable than those produced with lysolecithin in solution, but the hybrid cells nevertheless had damaged subcellular organelles. Viable clones of hybrid mouse-hamster fibroblast cells were obtained using the emulsion although, possibly owing to reduced viability of the lysolecithin-treated cells, only at twice the frequency of spontaneously produced hybrids.