Original Article

A Cell-free System for Regulated Exocytosis in PC12 Cells

Correspondence to: Reinhard Jahn, Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, D-37077 Göttingen, Germany. Tel:49-551-201-1634 Fax:49-551-201-1639 E-mail:rjahn{at}gwdg.de.

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes 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 that 42% of docked granules were released in a Ca²⁺-dependent manner dur- ing 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimula- tion. 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 biochemical and molecular tools.

Key Words: video microscopy, AFM, membrane fusion, in vitro, exocytosis

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