Sequential exocytosis of insulin granules is associated with redistribution of SNAP25.

Isolation of mouse pancreatic islets

Pancreatic islets were isolated from 8- to 12-week-old ICR mice by collagenase digestion and were maintained for 1–12 h under a humidified atmosphere of 5% CO 2 at 37°C in DME containing glucose (1.0 mg/ml) and supplemented with 10% FBS, 100 μU/ml penicillin, and 100 mg/ml streptomycin. The islets were transferred with a Pipetman (Gilson) to thin (0.1-mm) glass coverslips (Matsunami-glass) in the recording chamber. The standard external bathing solution (Sol A) contained 150 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM Hepes-NaOH, pH 7.4, and 2.8 mM glucose. Imaging experiments were performed within 20 min after placing an islet in Sol A containing 0.7 mM SRB (Molecular Probes). Forskolin (Sigma-Aldrich) or PMA (Sigma-Aldrich) was initially dissolved in DMSO at 10–50 mM and was subsequently diluted in Sol A. Methyl-β-cyclodextrin (Sigma-Aldrich) was directly dissolved in Sol A.

Two-photon excitation imaging

Two-photon excitation imaging of islets was performed with an inverted microscope (model IX70; Olympus) and a laser-scanning microscope (model FluoView; Olympus) equipped with a water-immersion objective lens (UplanApo60×W/IR; numerical aperture, 1.2). The laser power at the specimen was 3–10 mW, and two-photon excitation was effected at 830 nm, with images acquired every 0.3–2 s. The fluorescence of SRB, of fura-2 and fura-2FF, and of ECFP was measured at 550–650 nm, 400–530 nm, and 400–490 nm, respectively. In ECFP measurement, laser power and photomultiplier voltage were set to 8 W and 500 V, respectively. 12-bit images were color-coded with autumn color codes of FluoView. All experimental procedures were performed under yellow light illumination (FL40S-Y-F; National) to prevent unintended photolysis of the caged-Ca 2+ compound (see the following section). Most experiments were performed at RT (24–25°C), although the temperature of the bathing solution was increased to 33–35°C with a heater (Daia Medical System) in some experiments. Exocytic events were counted in a region of interest (ROI) with an area of 3,000–5,000 μm 2 and were normalized to an area of 2,000 μm 2 . Given that the thickness of the optical section ( T ) in our setup is ∼0.8 μm, the volume of the normalized ROI is 1,600 μm 3 . We expressed the number of events in the normalized ROI as events per cell, given that the volume of individual β cells is ∼1,600 (β cells have a mean diameter of 14.0 μm and constitute 90% of cells in an islet, or one β cell/1,596 μm 3 of an islet). For example, a frequency of 11.2 (or 45) events per cell per minute can also be expressed as 0.018 (or 0.073) events per square micrometer of plasma membrane per minute ( a ). In this example, the mean frequency of events (λ) within 1 min and a distance of