Red, yellow, green go! – a novel tool for microscopic segregation of secretory vesicle pools according to their age

Large dense-core vesicles (LDCVs) were labelled in cultured bovine adrenal chromaffin cells expressing fluorescent chimaeric ‘cargo’ proteins that were targeted to these secretory vesicles. When the cells were stimulated with nicotine 48 h after transduction, the fractional loss of fluorescent LDCVs was much greater than the fractional catecholamine secretion, implying selective release of newly assembled vesicles. This was confirmed using a fluorescent ‘timer’ construct that changes its fluorescence emission from green to red over several hours, and by measurement of the location and mobility of LDCVs in live cells by confocal fluorescence microscopy. Newly assembled (green) LDCVs were located mostly in peripheral regions of the cells, were virtually immobile and could be released by nicotine, but not by Ba²⁺; in contrast, older (red) LDCVs were centrally located and relatively mobile, and their exocytotic release was triggered by Ba²⁺, but not by nicotine. We describe the image restoration procedure that is necessary in order to analyse the behaviour of LDCVs labelled with this construct.