Lysine-fixable dye tracing of exocytosis shows F-actin coating is a step that follows granule fusion in pancreatic acinar cells

Exocytosis, the fusion of a vesicle with the plasma membrane, involves a complex cascade of cellular events. We set out to develop a method to identify changes in the distribution of proteins associated with exocytotic events in secretory epithelial cells. Our model system, the mouse pancreatic acinar cell, contains many hundreds of secretory vesicles (zymogen granules). Cell stimulation with low, physiological concentrations of agonist leads to the exocytosis of only a few granules. Once the preparation is fixed it is impossible with light microscopy to determine which of the granules have fused. In our method we use a derivative of a fluorescent dye, lysine-fixable Texas Red dextran dye, where the dye has been conjugated to dextran containing lysine residues. This dye enters the zymogen granule on the opening of the fusion pore at the time of exocytosis. This dye can then be fixed with paraformaldehyde and the preparation used for conventional immunocytochemistry methods. In demonstrating the utility of the method we carried out experiments where we fixed the cells and counterstained F-actin with phalloidin. The results show that F-actin coating is associated specifically with dye-filled granules and therefore is a step that follows exocytosis