EVOLUTION OF LIPIDIC STRUCTURES DURING MODEL MEMBRANE-FUSION AND THE RELATION OF THIS PROCESS TO CELL-MEMBRANE FUSION

The sequence of events involved in poly(ethylene glycol)-mediated fusi on of small unilamellar vesicles (SUVs) has been studied, Fusion event s were monitored using light scattering for vesicle aggregation, the f luorescence lifetime of membrane probe lipids (DPHpPC and NBD-PS) for membrane mixing, the aqueous fluorescent marker (Tb3+/DPA and H+/HPTS) for contents mixing; and quasi-elastic light scattering for the chang e in the size of vesicles, Poly(ethylene glycol) is a highly hydrated polymer that can bring vesicle membranes to near molecular contact but is unable to induce vesicle fusion without manipulations that reduce packing density and encourage molecular motions in the backbone region s of both contacting membrane leaflets, Once this condition is achieve d, the sequence of events involved in vesicle fusion is shown here to be (1) outer leaflet mixing accompanied by (2) transient pore formatio n, both occurring on a time scale of similar to 10 s and leading to an initial, reversible intermediate; (3) a 1-3 min delay leading to form ation of a fusion-committed second intermediate; (4) inner leaflet mix ing on a time scale of ca. 150 s; and (5) contents mixing on a time sc ale of 150-300 s. Inner leaflet mixing, which has never before been sh own to be distinct from outer leaflet mixing, begins simultaneously wi th, but is completed before, contents mixing. Fusion products, which s eem to be large vesicles, are estimated to be formed from four to six SUVs. The fusion intermediates are shown to have merged outer leaflets and distinct inner leaflets prior to formation of fusion pores. Using quasi-elastic light scattering, the initial intermediate was shown to revert to SUVs upon removal of PEG, while the second intermediate irr eversibly continued to a fusion pore in the presence or absence of PEG , The sequence of events for this pure lipid bilayer fusion process sh ows remarkable homology to what is known about the sequence of protein -mediated cell membrane fusion events, suggesting a commonality betwee n these two processes.