RESONANCE ENERGY-TRANSFER IMAGING OF PHOSPHOLIPID VESICLE INTERACTIONWITH A PLANAR PHOSPHOLIPID MEMBRANE - UNDULATIONS AND ATTACHMENT SITES IN THE REGION OF CALCIUM-MEDIATED MEMBRANE MEMBRANE ADHESION

Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the ves icle membrane adheres to the planar membrane. A resonance energy trans fer (RET) imaging microscope, with measured spectral transfer function s and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances betwee n tile membranes within this zone. Large vesicles (5-20 mu m diam) wer e labeled with the donor fluorophore coumarin-phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodami ne-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 fr om the RET rate constant (k(t)) computed from the acceptor:donor emiss ion intensity ratio. In the absence of an osmotic gradient, the vesicl es stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of p lanar membrane, coincident with the vesicle image, over which rhodamin e fluorescence was sensitized by RET. The total area of the contact re gion depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. T he changes in area and separation were probably related to divalent ca tion effects on electrostatic screening and binding to charged membran es. 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 less than or equal to 2 nm were localized to discrete sites that formed in an ordered arrang ement throughout the contact region. The area of the contact region oc cupied by these punctate attachment sites was increased at high [Ca2+] . Membrane fusion may be initiated at these sites of closest membrane apposition.