Functional and spatial segregation of secretory vesicle pools according to vesicle age.

Synaptic terminals and neuroendocrine cells are packed with secretory vesicles, only a few of which are docked at the plasma membrane and readily releasable. The remainder are thought to constitute a large cytoplasmic reserve pool awaiting recruitment into the readily releasable pool (RRP) for exocytosis. How vesicles are prioritized in recruitment is still unknown: the choice could be random, or else the oldest or the newest ones might be favoured. Here we show, using a fluorescent cargo protein that changes colour with time, that vesicles in bovine adrenal chromaffin cells segregate into distinct populations, based on age. Newly assembled vesicles are immobile (morphologically docked) at the plasma membrane shortly after biogenesis, whereas older vesicles are mobile and located deeper in the cell. Different secretagogues selectively release vesicles from the RRP or, surprisingly, selectively from the deeper cytoplasmic pool. Thus, far from being equal, vesicles are segregated functionally and spatially according to age.

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.

New technologies in exocytosis and ion movement.

Evolving new technologies for the study of exocytosis have been successfully exploited for the analysis of secretory events in the well-characterized chromaffin cell system. These technologies include amperometry, fluorescence resonance energy transfer (FRET), confocal and total internal reflection fluorescence (TIRF), and organelle-targeted aequorins.

Efficacy of Semliki Forest virus transduction of bovine adrenal chromaffin cells: an analysis of heterologous protein targeting and distribution.

In using chromaffin cells as a model for studying the mechanism of regulated exocytosis, there is a requirement for an efficient, safe, and robust system for the transduction and expression of heterologous cDNA in these cells. We have used Semliki Forest virus to transduce cDNAs encoding various proteins fused to enhanced green fluorescent protein (EGFP) into cultured bovine adrenal cells. Transduction is highly efficient but has no significant effect on the steady state levels of several endogenous proteins or of catecholamines in the transfected cells. Furthermore, the transfected cells show depolarization-induced calcium currents and nicotine-induced catecholamine release. We present data to show that virally transduced proteins are targeted to their intracellular locations correctly in chromaffin cells. The fusion protein pro-ANF-EGFP is specifically targeted to large dense-core vesicles as shown by its colocalization with acidophilic dyes and chromogranin A, making this a useful system for the study of secretory vesicle dynamics.