Figure 5

Consequences on ultrastructural parameters after CFTR inhibition. (A) Electron micrographs of a single beta-cell within an islet after incubation for one hour as indicated. The area within the dotted rectangle in the left image is highlighted to the right. The plasma membrane is indicated by a black solid line. The granules where defined as docked when the center of the granule was located within 150 nm from the plasma membrane (dashed line). N = nucleus. Scale bars: 2 μM (left) and 0.5 μM (right). (B) Bar graph of the total number of granules measured as volume density (N_v; granules*μm-3) after incubation in 1 mM glucose (1 G), 16.7 mM glucose (16.7 G), 10 μM forskolin (FSK) and 50 μM GlyH-101 (GlyH) as indicated in the color coding in D. (C) Bar graph of the docked granules as estimated by the surface density (N_s; granules*μm⁻²) under the different conditions as in D. (D) Relative distribution of granules at distance fractions from the PM. The distance at the x-axis is the upper border of each fraction. Color-coding for the different conditions is at the top right (n = 43 to 45 cells, N = 3 animals per condition). For B-D, data are presented as mean ± SEM. ^** P <0.01 16.7 G vs 1 G, ^*** P <0.001 16.7 G vs 1 G ^† P <0.05 FSK vs 16.7 G alone, ^†† P <0.01 FSK vs 16.7 G alone, ^‡ P <0.05 GlyH vs 16.7 G and FSK alone, ^‡‡‡ P <0.001 GlyH vs 16.7 G and FSK alone. (E) Localization of CFTR (yellow), syntaxin 1A (red) and insulin (green) in fixed single human islet cells detected using confocal immunocytochemistry (left). A bar graph describing the measured co-localization in the plasma membrane is shown to the right. The images are from a representative cell out of 39 from two human donors.