Visualization of sequential exocytosis in rat pancreatic islet beta cells.
The examination of insulin exocytosis at the single cell level by conventional electrophysiologic and amperometric methods possesses inherent limitations, and may not accurately reflect the morphologic events of exocytosis of the insulin granule. To overcome some of these limitations, we show by epifluorescent microscopy of a fluorescent dye, FM1-43, its incorporation into the plasma membrane and oncoming insulin granules undergoing exocytosis, and their core proteins. Using this method, we tracked exocytosis in real-time in insulinoma INS-1 and single rat islet beta cells in response to KCl and glucose. We observed both single transient and multi-stepwise increases in membrane FM1-43 fluorescence, suggesting single granule exocytosis as well as sequential and compound exocytosis, respectively. Confocal microscopy of nonpermeabilized cells shows that some of the exocytosed insulin granules labeled by the FM1-43 dye could also be labeled with insulin antibodies, suggesting prolonged openings of the fusion pores and slow dissolution of the granule core proteins on the membrane surface.
Leung
Yuk Man
YM
Department of Medicine, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
Sheu
Laura
L
Kwan
Edwin
E
Wang
Guotang
G
Tsushima
Robert
R
Gaisano
Herbert
H
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007340:Insulinoma / Q000188:drug therapy / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000378:metabolism
D005456:Fluorescent Dyes; D007328:Insulin; D011189:Potassium Chloride; D005947:Glucose