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© The Rockefeller University Press, 0021-9525/2000//317 $5.00
The Journal of Cell Biology, Volume 148, Number 2, , 2000 317-324

Original Article

A Cell-Free System for Regulated Exocytosis in Pc12 Cells



Julia Averya, Darren J. Ellisb, Thorsten Langa, Phillip Holroyda, Dietmar Riedela, Robert M. Hendersonb, J. Michael Edwardsonb, and Reinhard Jahna

a Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, D-37077 Göttingen, Germany
b Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, United Kingdom
Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, D-37077 Göttingen, Germany.49-551-201-163949-551-201-1634

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

© 2000 The Rockefeller University Press

Abbreviations used in this paper: AFM, atomic force microscopy; SNARE, SNAP receptor.


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J. Cell Biol. 2000 148: 1-2. [Full Text] [PDF]





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