In the present study we found that the polyethylene glycol (PEG)-induced membrane fusion involved primarily the disruption of lipid bilayers, and no active pore was found for membrane proteins. We propose to verify the importance of membrane lipids in PEG-induced fusion by studying vesicles and cells containing different lipids, and having different protein/lipid ratios. Vesicles reconstituted with erythrocyte membrane proteins, and exogenous lipids of different surface charge, headgroup hydration, bilayer stability, acyl chain length and saturation will be used. The lipids in human and avian erythrocyte and human lymphocyte membranes will be modified by lipid exchange techniques. The fusion efficiency of these samples will be analyzed in terms of known lipid properties. To access the unique characteristics of PEG, the interaction between PEG, phospholipids and the surface layer of water molecules will be studied by NMR, FTIR and radioactive binding assays. Peg-induced changes of membrane potential will be measured by fluorescence methods. Membrane fusion induced by electric pulses is said to involve the breakdown and the structural disruption of two tightly opposed bilayers. The postulated pore formation and reseal sequence will be investigated by time-resolved freeze fracture electron microscopy. The factors related to PEG-induced fusion, namely, surface charges, bilayer defects, susceptibility to form intramembranous particle-denuded areas and swelling will be tested for their effects on the dielectrophoresis and the reversible electrical breakdown processes. The results of this study are expected to lead to a more rational design of fusion protocols, and to contribute to the general understanding of membrane fusion mechanisms.
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