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Imaging Secretory Vesicles by Fluorescent Protein Insertion in Propeptide Rather Than Mature Secreted Peptide by Simon Watkins; Xuehui Geng; Lehong Li; Glenn Papworth; Paul D. Robbins; Peter Drain is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.
What is Imaging Secretory Vesicles by Fluorescent Protein Insertion in Propeptide Rather Than Mature Secreted Peptide about?
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
Who reads Imaging Secretory Vesicles by Fluorescent Protein Insertion in Propeptide Rather Than Mature Secreted Peptide?
It is typically read by researchers, students, and practitioners in Biochemistry, Genetics and Molecular Biology.
- Author
- Simon Watkins; Xuehui Geng; Lehong Li; Glenn Papworth; Paul D. Robbins; Peter Drain
- Publisher
- John Wiley and Sons; Wiley (Blackwell Publishing); Blackwell Publishing Inc.; Wiley (ISSN 1398-9219)
- Published
- 2002
- Language
- EN
- Field
- Biochemistry, Genetics and Molecular Biology (Life Sciences)