Abstract
Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin-phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of closest membrane apposition.
Highlights
Membrane fusion is a key mechanism underlying many cellular processes, including exocytosis, intracellular membrane trafficking, and fertilization, as well as pathological events such as the infection of cells by bacteria and viruses (White, 1992)
The fluorescence and resonance energy transfer (RET) properties of cou- and rhoPE in membranes viewed with the video RET microscope were the same as those determined by spectrofluorimetry of dye-containing liposomes
The sensitization ratios of the liposomes were greater than those of the planar membranes at the same proportion of labeled to unlabeled probe. This was because /1) valuesmeasured in themicroscopewere emission intensities integrated over the passband of the donor emission pathway of the microsope, which were proportionally larger than the fluorimetric cou-PE intensities measured at a single wavelength for the liposomes
Summary
Membrane fusion is a key mechanism underlying many cellular processes, including exocytosis, intracellular membrane trafficking, and fertilization, as well as pathological events such as the infection of cells by bacteria and viruses (White, 1992). Membranes containing anionic lipids require Ca 2+ or other divalent cations to form the adherent, prefusion state (Akabas et al, 1984). This stage is long lived, because bilayers are stable (Evans and Waugh, 1977; Needham and Haydon, 1983) and externally applied energy, such as osmotic swelling of the vesicles (Cohen et al, 1980), is necessary to drive the membranes into the second stage, in which the two membranes mix into a single lipid bilayer and the vesicle contents are released to the opposite side of the planar membrane
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