Ca<SUP>2+</SUP>-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy

Green fluorescent protein fused to human chromogranin B or neuropeptide y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine β−hydroxylase. Stimulation of live PC12 cells with elevated [K<SUP>+</SUP>], or of permeabilized PC12 cells with Ca<SUP>2+</SUP>, led to Ca<SUP>2+</SUP>−dependent loss of fluorescence from neurites. Ca<SUP>2+</SUP> stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent−wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense−core secretory granules and may be used for time−resolved microscopy of single granule