Ca 2+-Triggered Peptide Secretion in Single Cells Imaged with Green Fluorescent Protein and Evanescent-Wave Microscopy

• Published in: Neuron (Cambridge, Mass.) Vol. 18; no. 6; pp. 857 - 863
• Main Authors: Lang, Thorsten, Wacker, Irene, Steyer, Jürgen, Kaether, Christoph, Wunderlich, Ilse, Soldati, Thierry, Gerdes, Hans-Herman, Almers, Wolfhard
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 1997
• ISSN: 0896-6273; 1097-4199
• DOI: 10.1016/S0896-6273(00)80325-6

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 +], or of permeabilized PC12 cells with Ca 2+, led to Ca 2+-dependent loss of fluorescence from neurites. Ca 2+ 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 granules.