Ca2+-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 beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ 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.
Lang
T
T
Max-Planck-Institut für Medizinische Forschung, Heidelberg, Federal Republic of Germany.
Wacker
I
I
Steyer
J
J
Kaether
C
C
Wunderlich
I
I
Soldati
T
T
Gerdes
H H
HH
Almers
W
W
D000818:Animals; D002118:Calcium / Q000502:physiology*; D015550:Cell Degranulation; D002864:Chromogranins / Q000378:metabolism; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence / Q000379:methods; D016501:Neurites / Q000378:metabolism; D009478:Neuropeptide Y / Q000378:metabolism; D016716:PC12 Cells; D010455:Peptides / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins; D014741:Video Recording
D002864:Chromogranins; D008164:Luminescent Proteins; D009478:Neuropeptide Y; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004299:Dopamine beta-Hydroxylase; D002118:Calcium