Sequential exocytosis of insulin granules is associated with redistribution of SNAP25

Abstract

We have investigated sequential exocytosis in beta cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in beta cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-beta-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.

Figure 1.

Insulin exocytic events revealed by two-photon excitation imaging in mouse pancreatic islets. Islets were superfused with a solution containing 0.7 mM SRB and were stimulated by exposure to 20 mM glucose. (A and B) En face views of insulin exocytic events. The numbers below each panel represent time after the onset of exocytosis. The Ω-shaped profile of the primary exocytic granule (arrow) in B becomes the target for a secondary exocytic event (arrowhead); the two granules finally flattened out within the plasma membrane. (C and D) Changes in fluorescence intensity (AU, arbitrary unit) within the ROIs (white outlines in the insets) containing the exocytic granules shown in A and B, respectively. The fluorescence value before exocytosis was set to zero. (E) Three additional examples of sequential exocytic events. (F) Latency histogram for the delay between the onsets of the primary and secondary events during sequential exocytosis.

Figure 2.

Distributions of the fluorescence intensity of individual exocytic events and of the diameter of exocytosed granules. (A) Distributions of the increase in fluorescence intensity associated with individual exocytic events in islets stimulated with 20 mM glucose (black bars) or with 20 mM glucose and 2 μM forskolin (white bars). The mean intensities were 4,671 ± 1,950 AU (n = 97) and 4,680 ± 1,530 AU (± SD, n = 82), respectively. The arrow indicates an apparent excess of large components. (B) Distributions of granule diameter estimated from the fluorescence intensity distributions shown in A. The smooth lines represented the Gaussian distribution with a mean and SD of 0.42 and 0.05 μm, respectively.

Figure 3.

Exocytic events triggered by photolysis of a caged-Ca^{2 +} compound. (A) SRB fluorescence image of an islet that had been preloaded with NP-EGTA and fura-2. (B) Fura-2 (Ca²⁺) images obtained 1.2 s before and after UV irradiation of the islet shown in A. (C) Representative time courses of normalized fura-2 fluorescence (black) and of [Ca²⁺]_i estimated with fura-2FF (blue). Fura-2 fluorescence (F) was normalized by the resting fluorescence (F₀), and the trace shown is for the islet in B. (D) Exocytic event (arrow) triggered by UV photolysis of NP-EGTA. Ω Profiles often persisted for tens of seconds. (E) Two examples of time courses of sequential exocytosis (blue and red) and an example of that of solitary exocytosis (black) triggered by photolysis of NP-EGTA. The traces were aligned at the onset of exocytosis. (F) Time courses of the decay of SRB fluorescence associated with exocytic events induced by 20 mM glucose (black and red) or by photolysis of NP-EGTA (green). The traces are averages of >10 exocytic events, which were aligned at the peak of SRB fluorescence and normalized by the peak intensity. The data correspond to solitary events (black and green) or to the primary event of sequential exocytosis (red).

Figure 4.

Redistribution of SNAP25 during sequential exocytosis. SRB (A) and ECFP-SNAP25 (B) fluorescence images of an islet. The islet was transfected with an adenoviral vector encoding ECFP-SNAP25 and immersed in a solution containing polar tracers, SRB. Simultaneous measurement of SRB (C) and ECFP-SNAP25 (D) fluorescence during a sequential exocytic event. The number below each image in C represents the time after the onset of exocytosis. The blue dashed circle represents the ROI. Each image in D was obtained by averaging 5–10 images in the three time periods shown in E. (E) Time courses of fluorescence of SRB (black) and ECFP-SNAP25 (red) in the ROI shown in C. Open horizontal bars represent time periods between −14.4 and 0 s after the onset of exocytosis (pre), between 6.4 and 12.8 s (a), and between 14.4 and 20.8 s (b). Dashed horizontal lines show baseline fluorescence levels. (F) Difference images obtained by subtracting image pre from three images in D. (G–I) Time courses of fluorescence for another example of sequential exocytosis (H), and examples of solitary exocytic events in a control cell (G) and in a cell treated with 15 mM methyl-β-cyclodextrin for 30 min (I).

Figure 5.

Amplitudes and latencies of the redistribution of ECFP-SNAP25. (A) Histograms for the maximal increase in fluorescence intensity of ECFP-SNAP25 between 5 and 20 s after primary exocytosis for solitary events (blue), the primary events of sequential exocytosis (red), and arbitrary regions of the plasma membrane (black). The vertical dashed line represents the noise level. (inset) Cumulative intensity plots for the three histograms. (B and C) Latency histograms for the delay between the onset of exocytosis (the primary events of sequential exocytosis) and that of the ECFP-SNAP25 signal (B) and between the onset of the ECFP-SNAP25 signal and the secondary event of sequential exocytosis (C). Values shown above the plots are means ± SD.