A Cell-Free System for Ca2+-Regulated Exocytosis

• Published in: Methods (San Diego, Calif.) Calif.), 1998-10, Vol.16 (2), p.209-214
• Main Author: Edwardson, J.Michael
• Format: Article
• Language: English
• Subjects: Amylases - secretion; Animals; Calcium - metabolism; Cell Fractionation; Cell Membrane - metabolism; Cell-Free System; Cytoplasmic Granules - metabolism; Enzyme Precursors; Exocytosis; Membrane Fusion; Pancreas - cytology; Pancreas - metabolism; Rats; Spectrometry, Fluorescence
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1006/meth.1998.0669

The reconstitution of a membrane fusion event in a cell-free system makes possible a biochemical investigation of the molecular mechanisms underlying it. We have developed anin vitroassay for the fusion of pancreatic zymogen granules with the plasma membrane. The lipid-soluble fluorescent probe octadecylrhodamine is loaded into the granule membrane, and the granules are then incubated with unlabeled plasma membranes. Membrane fusion results in a dilution of the probe, which is detected through the dequenching of its fluorescence. The properties of thein vitrofusion event are impressively similar to those of exocytosis from permeabilized pancreatic acini, indicating that dequenching is detecting a physiologically relevant process. In particular, exocytotic membrane fusion bothin vitroand in permeabilized acini is stimulated by Ca2+with an EC50of 1 μM, and enhanced by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) with an EC50of 10–20 μM. Another parallel between the two systems is the incomplete inhibition of fusion/exocytosis by tetanus toxin, despite complete cleavage of synaptobrevin 2 on the zymogen granule membrane. Recently, thein vitroassay for membrane fusion has been used to indicate a role in the control of exocytosis for syncollin, a granule membrane protein that binds to syntaxin in a Ca2+-sensitive manner. The assay should continue to provide information about this exocytotic membrane fusion event in the future.