MEMBRANE-PROTEINS AT THE INTERFACE OF ERYTHROCYTES FUSED BY TREATMENTWITH POLYETHYLENE-GLYCOL

Fusion of human red cells through the action of polyethylene glycol gi ves rise to pairs or higher clusters with a common membrane envelope, in which a barrier at the position of the original interface can be se en in phase contrast. At early times this septum contains lipids, as j udged by labelling with a fluorescent lipophile, and transmembrane pro tein; this was shown by the presence of the preponderant component, ba nd 3, detected by a fluorescent label, covalently attached before fusi on at an extracellular site, or by immunofluorescence with anti-band 3 antibody. Ankyrin, which is bound to band 3, is also observed in the septum, The lipid thereafter disappears from the interface, carrying m ost of the band 3 with it. A continuous membrane skeletal network, def ined by the presence of spectrin (detected by immunofluorescent staini ng in epifluorescence and confocal microscopy) appears to persist for long periods, but in many cells interruptions develop in the septum. I n other fused pairs, particularly at longer times, the interface is se en to have vanished completely. Protease inhibitors have no discernibl e effect on any of these observations. The results suggest a model for the events that follow fusion. Covalent cross-linking of membrane pro teins beyond a critical level causes inhibition of fusion, suggesting that proteins, probably the membrane skeletal network, regulate the fu sion process, The efficiency of fusion is strikingly dependent on the composition of the isotonic medium, being relatively high at an orthop hosphate concentration of 5 mM and minimal at 20 mM.