Exocytosis studies in a chromaffin cell-free system: imaging of single-vesicle exocytosis in a chromaffin cell-free system using total internal reflection fluorescence microscopy.

We have developed a system for the real-time study of regulated exocytosis in living, cultured bovine adrenal chromaffin cells (BCCs). Exocytosis was monitored by the use of total internal reflection fluorescence (TIRF) microscopy to image single large dense-core secretory vesicles (LDCVs). Fluorescent labeling of LDCVs was achieved either with the membrane-permeant weak base, acridine orange (AO), or by transduction of BCCs so as to express a fluorescent chimeric "cargo" protein that is targeted to LDCVs. In either case, exocytosis is visible by the disappearance of a vesicle accompanied by a bright flash as the fluorescent contents leave the acidic LDCV lumen, move towards the source of the evanescent wave, and disperse into the extracellular medium. Furthermore, for the first time, we have developed a broken-cell system for real-time imaging in BCCs, in which individual plated cells are mechanically "unroofed" with a jet of intracellular medium, leaving a membrane patch with docked vesicles on the coverslip. In this cell-free system, a subpopulation of docked granules undergoes exocytosis in response to calcium. This approach allows us direct experimental access to membrane-docked LDCVs in order to investigate the dependence of exocytosis on defined protein components and intracellular conditions at the single-vesicle level. In addition, this system can be used for a reconstitution analysis of the exocytosis machinery. Finally, we demonstrate the use of 2D+1 image analysis for visualizing single-vesicle exocytosis. We use this approach for a rapid analysis of larger numbers of imaged vesicles.