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 fusion of small unilamellar vesicles (SUVs) has been studied. Fusion events were monitored using light scattering for vesicle aggregation, the fluorescence 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 change 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 regions of both contacting membrane leaflets. Once this condition is achieved, the sequence of events involved in vesicle fusion is shown here to be (1) outer leaflet mixing accompanied by (2) transient pore formation, both occurring on a time scale of approximately 10 s and leading to an initial, reversible intermediate; (3) a 1-3 min delay leading to formation of a fusion-committed second intermediate; (4) inner leaflet mixing on a time scale of ca. 150 s; and (5) contents mixing on a time scale of 150-300 s. Inner leaflet mixing, which has never before been shown to be distinct from outer leaflet mixing, begins simultaneously with, but is completed before, contents mixing. Fusion products, which seem 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 irreversibly continued to a fusion pore in the presence or absence of PEG. The sequence of events for this pure lipid bilayer fusion process shows remarkable homology to what is known about the sequence of protein-mediated cell membrane fusion events, suggesting a commonality between these two processes.
Lee
J
J
Department of Biochemistry & Biophysics, University of North Carolina, Chapel Hill 27599-7260, USA.
Lentz
B R
BR
D001704:Biopolymers / Q000737:chemistry; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D008051:Lipid Bilayers / Q000737:chemistry*; D008561:Membrane Fusion / Q000187:drug effects; D008563:Membrane Lipids / Q000737:chemistry*; D008958:Models, Molecular; D010743:Phospholipids / Q000737:chemistry; D011092:Polyethylene Glycols / Q000494:pharmacology; D011522:Protons; D012542:Scattering, Radiation
D001704:Biopolymers; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010743:Phospholipids; D011522:Protons; D011092:Polyethylene Glycols