Imaging secretory vesicles by fluorescent protein insertion in propeptide rather than mature secreted peptide.
We combined confocal and live-cell imaging with a novel molecular strategy aimed at revealing mechanisms underlying glucose-regulated insulin vesicle secretion. The 'Ins-C-GFP' reporter monitors secretory peptide targeting, trafficking, and exocytosis without directly tagging the mature secreted peptide. We trapped a green fluorescent protein (GFP) reporter in equimolar quantity within the secretory vesicle by fusing it within the C peptide of proinsulin which only after nascent vesicle sealing and acidification is cleaved from the mature secreted A and B chains of insulin. Ins-C-GFP expression in mouse islets without fail exhibited punctate distribution of green fluorescence by confocal microscopy. Ins-C-GFP colocalized GFP with insulin at vesicle dense cores by immuno-electron microscopy. Glucose stimulation decreased vesicle fluorescence coordinately with enhanced secretion from islets of C-GFP detected by anti-GFP Western blots, and of insulin detected by anti-insulin radioimmunoassay. An insulin secretagogue with a red fluorescent label, glibenclamide BODIPY TR, was applied to islets expressing Ins-C-GFP. The stimulus response was imaged as a rise in red secretagogue leading to marked loss in green granules. Since neuropeptides as well as peptide hormones are processed from propeptides after sealing of secretory granules, vesicle trapping likely is widely applicable for studies on targeting, trafficking, and regulated release of secretory peptides.
Watkins
Simon
S
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Geng
Xuehui
X
Li
Lehong
L
Papworth
Glenn
G
Robbins
Paul D
PD
Drain
Peter
P
D000818:Animals; D002096:C-Peptide / Q000378:metabolism; D005089:Exocytosis; D005947:Glucose / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D013453:Sulfonylurea Compounds / Q000494:pharmacology
D002096:C-Peptide; D007328:Insulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D013453:Sulfonylurea Compounds; D049452:Green Fluorescent Proteins; D005947:Glucose