A cell-free system for regulated exocytosis in PC12 cells

We have developed a cell-free system for regulated exocytosis in the PC12 n euroendocrine cell line. Secretory vesicles were preloaded with acridine or ange in intact cells, and the cells were sonicated to produce flat, carrier -supported plasma membrane patches with attached vesicles. Exocytosis resul ted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light Bashes by fluorescence dequenching, Exocytosis in vitro requires cytosol and Ca2+ at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction limited resolution revealed tha t 42% of docked granules were released in a Ca2+-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the p resence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the mem brane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for bi ochemical and molecular tools.