BRIEF COMMUNICATION VESICLE-MEMBRANE FUSION Observation of Simultaneous Membrane Incorporation and Content Release DIXON J. WOODBURY AND JAMES E. HALL Department of Physiology and Biophysics, University of California, Irvine, California 92717 ABSTRACT Vesicle fusion, the central process of neurotransmitter release and hormonal secretion, is a complex process culminating in simultaneous incorporation of vesicle membrane into the plasma membrane and release of the vesicular contents extracellularly. This report describes simulteous observation of membrane incorporation and content release using a model system composed of a planar bilayer and dye-filled vesicles. Exocytosis, the fusion of vesicles with the plasma mem- brane, is central to many physiological processes, most notably quantal transmitter release at nerve terminals and secretion of prepackaged materials such as adrenalin, insulin, and other hormones. Identifying the steps involved in exocytosis and the factors that control them is a major problem of cell biology. Artificial vesicles and planar membranes provide a simple system with which to model exocytosis. Such model systems make it possible to ask how defined sets of components facilitate fusion and the path by which fusion occurs. Previous workers have demonstrated channel inser- tion or vesicle content release from synthetic vesicles added to planar bilayers (1, 2). However, these experiments could not distinguish between fusion and other possible vesicle- bilayer interactions such as channel incorporation without trans ("extracellular") content release or content release without channel incorporation (bursting). For exocytosis to be efficient, the probability of nonfusion events must be low. Thus, it is important to ask if true fusion (channel insertion and trans content release) occurs in model sys- tems and if so, with what probability. This report describes the simultaneous observation of channel incorporation and trans content release in a model system. In our system fusion has a probability of 10%; most of the time vesicles burst. With different experimental conditions higher probabilities have been observed (3). The observation of true fusion in a model system demonstrates a beginning of understanding for the steps involved in exocytosis. MODEL SYSTEM The model system is composed of a planar bilayer and dye-loaded vesicles containing porin channels. Fusion is induced by forming a transmembrane osmotic gradient (2-6). Content release is detected through a microscope as release of the fluorescent dye, calcein, from large unilamel- lar vesicles. Vesicle-membrane incorporation is detected by the appearance of vesicle-borne porin channels in the planar membrane. Finally, trans content release is deter- mined by selectively masking fluorescence due to cis release. (The cis side is the side to which the vesicles are added.) This is done by adding a divalent metal ion such as manganese (Mn2") to the cis side of the planar membrane. Thus it is possible to measure simultaneously vesicle- membrane fusion and release of vesicle contents to the trans side. Release of calcein from vesicles appears as a sudden change in vesicle fluorescence. For vesicles that contain submillimolar concentrations of calcein release appears as a sudden disappearance of vesicle fluorescence due to release and diffusion of dye away from the original location of the vesicle. For vesicles that contain self-quenched concentrations of calcein (>6 mM, reference 6 and manu- script in preparation), release appears as a flash of fluores- cent light. The flash arises because initially dye is quenched inside the confined interior of a vesicle, but when a vesicle releases its contents (either by fusion or bursting) the dye expands into the surrounding space, quenching diminishes, and the total fluorescence intensity increases. The increase is brief (tens of milliseconds) and is followed by a slow decrease in fluorescence as the dye dilutes by diffusion to submillimolar levels. In a typical experiment the membrane was formed with neutral lipids in a chamber filled with standard (low Ca2") buffer. A pipette filled with vesicles was positioned close to the cis side of the membrane. Pressure was applied and vesicles were squirted at the membrane. To induce fusion, the cis side of the membrane was perfused with standard buffer made hyperosmotic by the addition of urea. Usually several serial perfusions with increasing concentrations of urea were performed. Perfusion of urea-containing solutions caused a fraction of the adherent vesicles to release contents. This was BIOPHYS. J.o Biophysical Society * 0006-3495/88/08/345/05 $2.00 Volume 54 August 1988 345-349 345