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First published online 29 November 2005
doi: 10.1242/jcs.02684

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Regulation of two insulin granule populations within the reserve pool by distinct calcium sources

¹ Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA
² Department of Electrical Engineering and Computer Science, Vanderbilt University, Nashville, TN 37232, USA

View larger version (59K): [in a new window]   Fig. 1. Phogrin-EGFP is effectively targeted to the insulin granules in ßTC3 cells. (A-C) Cells stably transfected with phogrin-EGFP were plated for 48 hours and then immunostained with an insulin antibody and an Alexa Fluor 546 secondary antibody. (D-F) Cells were co-transfected with phogrin-EGFP and proinsulin-ECFP (pseudocolored red). Measures were taken to minimize and correct for crossover fluorescence. Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 2. Tracking of insulin granules in ßTC3 cells reveals two granule populations. (A-C) Three representative frames, at different time points, from a time-lapse movie of secretory granules labeled with phogrin-EGFP before and after KCl stimulation. The first 60 frames (120 seconds) were taken under unstimulated condition before 30 mM KCl was added, and the movie continued for another 120 frames to record KCl-stimulated granule movement. Several granules are manually tracked through 30 frames to show their paths in D-F. The movie can be viewed in the supplementary material. Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 3. Histograms of average velocity reveal segregation of granule populations upon stimulation. The histograms show the number of granules at different velocities traveled. The inset bar graphs are derived from Table 1 and show the percentage of granules with speeds >0.4 µm/second, with the error bars representing s.e.m. For all panels, a time series of granule movement was first taken under unstimulated condition and then after each treatment. The treatments were sequential, in the order shown in the symbol legend. Images were taken immediately after KCl was added and 2 minutes after glucose was added. *P<0.001 in a paired t-test. (E) The average velocity values were divided into 24 intervals and an average of net displacement (distance between the positions of a granule at the first and the last time points) of all the granules within each interval was plotted against the average velocity. Because various numbers of granules fall into different velocity intervals, each data point represents an average of net displacement from different numbers of granules. (F) Three granules were selected to represent the three types of motion. Data points were fitted to a moving average, linear regression and second degree polynomial curve using Microsoft Excel and labeled as type i, ii, iii, respectively. The inset shows an enlarged view of curves i and ii.

View larger version (27K): [in a new window]   Fig. 4. Insulin granules are differentially regulated by localized Ca2+ upon stimulation. The histograms in A-D were generated similarly to the ones described in Fig. 3. TG, thapsigargin. Measurements were taken 2 minutes after the addition of Bay K8644, nimodipine and glucose, and after 20 minutes of TG treatment. (E) ßTC3 cells were co-transfected with phogrin-EGFP and ECFP-ER, and stimulated with glucose. An image of ECFP-ER was taken prior to acquiring the time-lapse movie of phogrin-EGFP labeled granules. The distance of each granule to the ER, measured by the fluorescence intensity of ECFP-ER at that position, is plotted against the speed of that granule from the tracking analysis. The fast-moving granules, as defined in this paper, are indicated in red. (F) A similar experiment as in E was performed using ECFP-Golgi instead of ECFP-ER. *P<0.001, #P<0.01, &P<0.05, and ^P>0.1, as assessed by paired t-tests. a.u., arbitrary units.

View larger version (46K): [in a new window]   Fig. 5. Effects of cytochalasin D and jasplakinolide on granule mobility. (A-F) The effects of cytochalasin D (cyto D) and jasplakinolide (Jas) treatment on the actin network in ßTC3 cells. Cells were incubated with 10 µg/ml Cyto D or 1 µM Jas for 20 minutes at 37°C before being stained with Alexa Fluor 488-phalloidin. (G,H) The histogram of average velocity under basal condition (green), after Cyto D (G) or Jas (H) treatment (black), and after adding KCl (red). The inset bar graphs show the percentage of granules with speeds >0.4 µm/second, with the error bars representing s.e.m. *P<0.001, #P<0.01, &P<0.05 and ^P>0.1, as assessed by paired t-tests. Bar, 10 µm.

View larger version (34K): [in a new window]   Fig. 6. Effect of nocodazole on granule mobility. (A-D) The effect of nocodazole (Noc) treatment on the microtubule network in ßTC3 cells. Cells were incubated with 10 mM Noc for 20 minutes at 37°C. They were then stained with -tubulin antibody and Alexa Fluor 488 secondary antibody. (E) The histograms of average velocity under basal condition (green), after Noc treatment (black), and after adding KCl (red). The inset bar graphs show the percentage of granules with speeds >0.4 µm/second, with the error bars representing s.e.m. &P<0.05 and ^P>0.1, as assessed by paired t-tests. Bar, 10 µm.

View larger version (22K): [in a new window]   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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