Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Abstract

Confocal imaging of GFP-tagged secretory granules combined with the use of impermeant extracellular dyes permits direct observation of insulin packaged in secretory granules, trafficking of these granules to the plasma membrane, exocytotic fusion of granules with the plasma membrane, and eventually the retrieval of membranes by endocytosis. Most such studies have been done in tumor cell lines, using either confocal methods or total internal reflectance microscopy. Here we compared these methods by using GFP-syncollin or PC3-GFP plus rhodamine dextrans to study insulin granule dynamics in insulinoma cells, normal mouse islets, and primary pancreatic beta cells. We found that most apparently docked granules did not fuse with the plasma membrane after stimulation. Granules that did fuse typically fused completely, but a few dextran-filled granules lingered at the membrane. Direct recycling of granules occurred only rarely. Similar results were obtained with both confocal and total internal reflection microscopy, although each technique had advantages for particular aspects of the granule life cycle. We conclude that insulin exocytosis involves a prolonged interaction of secretory granules with the plasma membrane, and that the majority of exocytotic events occur by full, not partial, fusion.

Fig. 1.

Time course of syncollin–GFP expression in MIN6 (A) and mouse (B) beta cells and PC3–GFP in MIN6 cells (C). Images represent typical appearance of expression in cells at times indicated. Arrows indicate regions with peripheral punctate fluorescence. Asterisks indicate regions of diffuse fluorescence. (Scale bars = 10 μm.)

Fig. 2.

Colocalization of syncollin–GFP and insulin in beta (A and B) and MIN6 (C) cells. Colocalization of fluorescence is greater near cell periphery than in the perinuclear region. (A) Orthogonal views (XY, XZ, YZ) through one beta cell demonstrating localization of syncollin–GFP (green) and insulin staining (Cy3, red); colocalization produces yellow color. (B) Single optical section showing insulin staining (Left, red) in cell interior (asterisk) and syncollin–GFP (Center, green). (Right) Overlay image. (C) Three-dimensional maximum intensity reconstruction of a MIN6 cell showing colocalization (arrow) of insulin (red) and syncollin–GFP (green). Animated reconstructions (see Movie 1) show cellular distribution of both probes as well as the degree of colocalization per vesicle. (Scale bars, 5 μm.)

Fig. 3.

Three-dimensional intercellular distribution of 3-kDa dextran–TMR in a mouse islet (XYZ). (A) Three-dimensional reconstruction of confocal images of dextran–TMR fluorescence obtained ≈5 min after application to live mouse islets. White arrows indicate bright “creases” at intersections between multiple beta cells. Asterisks indicate areas of uniform, less intense staining of narrow spaces between cells. Similar patters are obtained with FM-464 after staining (data not shown). (B) Maximum intensity projections of bright dextran–TMR-bathed unstimulated a mouse islet (Left) and same cells after depolarization with 40 mM KCl (Right). Omega-like figures (arrows) appear after depolarization (see Movie 2). Here, bright spots (arrows) indicate sites of rapidly changing dextran–TMR-filled profiles that mainly occur at cell edges. (Scale bars, 10 μmin A and 5 μmin B.)

Fig. 4.

Exocytosis of GFP–syncollin-labeled granules in MIN6 cells. See Movie 3 for full experiment (panels show 66.3–120.9 s, upper left to lower right, after 40 K⁺ addition). Two fusions (arrows) occurring at the right edge of a MIN6 cell are indicated in frames with asterisks. Filling of the green (GFP) insulin granule with extracellular dextran–TMR results in a yellow structure. Note that some dextran filled structures slightly expand beyond the diameter of the associated GFP vesicles and that most dextran-filled granules fully disappear (see Movie 3, 147-s total duration, and see Movie 4). Image sample rate was 0.9 s per image. (Scale bar, 3 μm.)

Fig. 5.

Exocytosis of syncollin–GFP granules in isolated mouse beta cells and PC3–GFP granules in MIN6. (A) Exocytosis in isolated mouse beta cells. Interface between two cells (faint horizontal line) is filled with dextran–TMR (red) and shows development and collapse of omega-like figure in bottom cell. Syncollin–GFP is expressed only in lower cell. Colocalization of syncollin–GFP and dextran–TMR (Lower) results in yellow granule (arrows). Images were acquired 11.8, 13.0, and 14.4 s after depolarization. (B) Fusion (arrow) of a PC3–GFP granule in MIN6 cells; images were collected at 700 ms per scan (left to right). (Scale bars, 1 μm.)

Fig. 6.

TIRF images of syncollin–GFP-labeled granule exocytosis after depolarization with 40 mM KCl. (A) MIN6 cell (see Movie 5). (B) Primary bovine chromaffin cell (see Movie 6). Asterisks indicate the onset of granule fusions. (Right) Quantitation of intensity at the site of granule fusion (blue lines) and a nearby background (pink lines). Note the persistence of the granule after full disappearance in A and the partial discharge in B.