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Fig. 7. Secretory granule trafficking is correlated with insulin secretion and refilling of the RRP. (A) Cells were transfected with proinsulin-EGFP and the overall fluorescence intensity of background-corrected images was plotted over time before and after glucose (blue) or KCl (red) was added. For nocodazole (Noc) treatment, cells were incubated with Noc prior to image acquisition and KCl was added (black). (B) FluoZin-3 fluorescence, shown in red, indicates insulin release before and after adding glucose and again after glucose washout. The blue bars show the percentage of granules with speeds >0.4 µm/second (fast-moving population), under identical conditions used to monitor insulin release with FluoZin-3. n=5 experiments. The error bars represent s.e.m. (C) pH dependency of proinsulin-EYFP-DsRed. See Materials and Methods for details. Left, normalized fluorescence ratio of EYFP to DsRed is plotted against pH. Right, raising the pH with a permeant base, NH₄Cl, caused an increase in the fluorescence ratio of EYFP/DsRed in live cells. (D) The fluorescence ratio of EYFP to DsRed (the lower the ratio, the more acidic) for each granule is plotted against the speed of movement from the tracking analysis. The fast-moving granules are indicated in red. a.u., arbitrary units.

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