CA2-TRIGGERED PEPTIDE SECRETION NEUROTECHNIQUE IN SINGLE CELLS IMAGEDWITH GREEN FLUORESCENT PROTEIN AND EVANESCENT-WAVE MICROSCOPY()

Green fluorescent protein fused to human chromogranin B or neuropeptid e Y was expressed in PC12 cells and caused bright, punctate fluorescen ce. The fluorescent points colocalized with the endogenous secretory g ranule marker dopamine beta-hydroxylase. Stimulation of live PC12 cell s with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated se cretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence micro scopy. Granules were seen to migrate; to stop, as if trapped by plasma lemmal docking sites; and then to disappear abruptly, as if through ex ocytosis. Evidently, GFP fused to secreted peptides is a fluorescent m arker for dense-core secretory granules and may be used for time-resol ved microscopy of single granules.