Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis

Abstract

We compared secretion kinetics for four different fluorescent cargo proteins, each targeted to the lumen of insulin secretory vesicles. Upon stimulation, individual vesicles displayed one of four distinct patterns of fluorescence change: i), disappearance, ii), dimming, iii), transient brightening, or iv), persistent brightening. For each fusion protein, a different pattern of fluorescence change dominated. Furthermore, we demonstrated that the dominant pattern depends upon both i), the specific choice of fluorescent protein, and ii), the sequence of amino acids linking the cargo protein to the fluorescent protein. Thus, in beta-cells, experiments involving fluorescent cargo proteins for the study of exocytosis must be interpreted carefully, as design of a fluorescent cargo protein determines secretion kinetics at exocytosis.

FIGURE 1

Depolarization stimulated heterogeneous patterns of fluorescence change for fluorescent cargo proteins expressed in pancreatic β-cells. (A) High speed imaging of C-emGFP revealed transient brightening and rapid release, as fluorescence collapsed to background within 200 ms. (B–E) Single vesicles (montages and top traces) responded with four patterns of fluorescence change and, as indicated, each fluorescent cargo protein had a preferred pattern. Whole-cell fluorescence changes (middle traces) paralleled changes at the single-vesicle level, whereas fluorescence 3 μm from the edge of the cell (bottom traces) increased regardless of fluorescence changes for single vesicles, suggesting that protein was released and diffused away. Arrows indicate application of elevated potassium. For detailed descriptions of figures, see Supplementary Material.

FIGURE 2

Persistent brightening of rIAPP-EGFP does not require an insulin/zinc electron-opaque core (A versus B) as demonstrated by the similar behaviors of vesicles labeled with rIAPP-EGFP in β-cells from CPE +/+ and CPE −/− mice (C versus D).

FIGURE 3

Design of a fluorescent cargo protein determines secretion kinetics at exocytosis. In pancreatic β-cells, the amino acid sequence (shown in italics) linking IAPP to EGFP influenced the behavior of labeled vesicles at exocytosis. Arrows indicate processing sites.