Imaging Secretory Vesicles by Fluorescent Protein Insertion in Propeptide Rather Than Mature Secreted Peptide

Abstract

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 antiinsulin radioimmunoassay. An insulin secretagogue with a red fluorescent label, glibenclamide BODIPY A 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. Glucose-stimulated insulin secretion is regulated over a wide range of time scales. It exhibits day to hour regulation of nuclear transcription (5), hour to minute regulation of cytoplasmic translation (6-8), hour to minute regulation of vesicular trafficking and recycling (9-13), and minute to millisecond regulation of exocytosis at the plasma membrane (14-23). Live-cell monitoring of the spatial and temporal features of the underlying molecules and mechanisms would be facilitated by a method that avoids modification of the mature bioactive secretory peptide for not only glucose-regulated insulin secretion but also regulated peptide secretion in general.Regulated secretory peptide granule trafficking and exocytosis have been typically measured by using insulin antibodies labeled by radioactivity or enzymes (24), by using capacitance changes (16), or by using amperometry (25). By far the most popular measure of insulin vesicle trafficking and secretion in the basic research lab or clinic is by immunoassays. The assays depend on the use of a relatively largesized biopsy or perfusate tissue and costly I 125 radioisotopes or enzymes conjugated to a secondary antibody. Though in widespread use, these methods reveal little about the underlying molecular and cellular mechanisms at work within the cell, in a live-cell, real-time format with dynamic spatial (subcellular, one-cell, multi-cell, islet) and temporal (millisecond, second, min, to hour) monitoring.The fluorescent labeling approach developed here provides the following advantages for studying complex molecular...

Glucose Acutely Decreases pH of Secretory Granules in Mouse Pancreatic Islets

“…Localization of Lysosensor DND-160 in ␤-Cells-The localization of Lysosensor DND-160 in ␤-cells and MIN6 cells was determined by comparison with Ins-C-GFP, a reporter of insulin secretory granules (38). The two signals exhibited a similar localization in both ␤-cells (Fig.…”

“…In intact cells, the probe is expected to accumulate in acidic compartments (26). To assess the intracellular localization of Lysosensor DND-160, secretory vesicles were labeled by expressing a green fluorescent protein fused within the C peptide of proinsulin (Ins-C-GFP) (38). The Ins-C-GFP virus was prepared and kindly provided by P. Drain (Pittsburgh, PA).…”

Insulin granule dynamics in pancreatic beta cells

“…Using this approach, both insulin itself or other granule proteins have been tagged with various variants of GFP [41,79,86,87,88,89]. Although the mo-lecular weight of GFP is 4 to 5 times larger than that of insulin, the physical dimensions of the proteins are not vastly different (5.1 nm×3.7 nm×3.5 nm for EGFP vs 3.2 nm×3 nm×2.5 nm for the insulin monomer) [88].…”

Human Insulin Vesicle Dynamics During Pulsatile Secretion

“…The fourth was made by fusing the rodent isoform of islet amyloid polypeptide to enhanced green fluorescent protein (rIAPP-EGFP). Construction of adenovirus containing the gene for either C-emGFP or rIAPP-EGFP has been described previously (12,14). To make adenovirus containing C-EGFP and C-Bright, we used standard molecular biological techniques to modify the C-emGFP construct by replacing emerald green fluorescent protein separately with either enhanced green fluorescent protein or the bright mutant of dsRed fluorescent protein.…”

“…Because it is known that the function of pancreatic ␤-cells is disturbed by dissociation into individual cells (11), we limited our study to cells that resided within small clusters (5-10 cells). To label insulin vesicles, we transduced the cells with a viral vector encoding a vesicle-targeted fluorescent cargo protein (12). By using a low titer of virus, we were able to find clusters containing only a single cell with fluorescent vesicles, allowing us to study the dynamics of individual insulin vesicles in single pancreatic ␤-cells.…”