Spatial and Temporal Aspects of Exocytosis Studied on the Isolated Plasma Membranes.
Exocytosis of large-dense core vesicles in neuroendocrine cells is a highly regulated, calcium-dependent process, mediated by networks of interrelated proteins and lipids. Here, I describe experimental procedures for studies of selective spatial and temporal aspects of exocytosis at the plasma membrane, or in its proximity, using adrenal chromaffin cells. The assay utilizes primary cells subjected to a brief ultrasonic pulse, resulting in the formation of thin, flat inside-out plasma membranes with attached secretory vesicles and elements of cell cytoskeleton. In this model, secretion of plasma membrane-attached secretory vesicles was found to be dependent on calcium and sensitive to clostridial neurotoxins. Depending on the probe selected for secretory vesicle cargo, protein, and/or lipid detection, this simple assay is versatile, fast and inexpensive, and offers excellent spatial resolution.