Figure 6.
Figure 6. WA stimulates the formation of GM1-enriched domains and thickens actin bundles connecting secretory granules to the plasma membrane in nicotine-stimulated cells. (A) Confocal micrographs of cells treated for 1 h with 3 µM WA and stimulated with 10 µM nicotine in the presence of cholera toxin. Cells were then fixed and labeled with rhodamine-conjugated phalloidin to visualize F-actin. (B) Semiquantitative analysis of cholera toxin and F-actin labeling expressed in arbitrary units (±SEM; n = 15). Statistical significance for medians (black line) was determined using a Mann-Whitney test. The asterisks indicate statistical significance (**, P < 0.01; ***, P < 0.001) and the white lines represent the means. Similar results were obtained in two independent experiments performed on two culture preparations. (C) Electron micrographs of plasma membrane sheets prepared from nicotine-stimulated and WA-treated cells labeled with anti-actin antibodies and immunogold. (D) 3D model of the same secretory granule showing the bundling effect of WA on the actin network anchored to the granule surface. False colors were applied using a color-coded surface related to the topographic height on the right (Video 2). (E) Clipping plane of the side view of the isosurface representation of a tomogram showing an increase in the actin network anchored to the granule surface in a WA-treated cell. (F–I) Central tomographic section of a docked secretory granule and corresponding enlargement (inset, 2.5× zoom) showing anchored actin filaments and the granule surface marked with a dotted black line. Graphs (G and I) represent a plot profile along the red line perpendicular to the actin filament. Images were obtained from control (F) and WA-treated (H) cells stimulated with nicotine. Plot profiles presented are representative of several measurements (n = 15 for each plot profile) from different tomograms (n = 3 for each conditions). Individual 10-nm actin filaments anchored granules in control cells, whereas large actin bundles consisting of at least five actin filaments were observed in WA-treated cells. Bars: (A) 5 µm; (C) 200 nm; (E, F and H, main panels) 100 nm; (F and H, insets) 25 nm.

WA stimulates the formation of GM1-enriched domains and thickens actin bundles connecting secretory granules to the plasma membrane in nicotine-stimulated cells. (A) Confocal micrographs of cells treated for 1 h with 3 µM WA and stimulated with 10 µM nicotine in the presence of cholera toxin. Cells were then fixed and labeled with rhodamine-conjugated phalloidin to visualize F-actin. (B) Semiquantitative analysis of cholera toxin and F-actin labeling expressed in arbitrary units (±SEM; n = 15). Statistical significance for medians (black line) was determined using a Mann-Whitney test. The asterisks indicate statistical significance (**, P < 0.01; ***, P < 0.001) and the white lines represent the means. Similar results were obtained in two independent experiments performed on two culture preparations. (C) Electron micrographs of plasma membrane sheets prepared from nicotine-stimulated and WA-treated cells labeled with anti-actin antibodies and immunogold. (D) 3D model of the same secretory granule showing the bundling effect of WA on the actin network anchored to the granule surface. False colors were applied using a color-coded surface related to the topographic height on the right (Video 2). (E) Clipping plane of the side view of the isosurface representation of a tomogram showing an increase in the actin network anchored to the granule surface in a WA-treated cell. (F–I) Central tomographic section of a docked secretory granule and corresponding enlargement (inset, 2.5× zoom) showing anchored actin filaments and the granule surface marked with a dotted black line. Graphs (G and I) represent a plot profile along the red line perpendicular to the actin filament. Images were obtained from control (F) and WA-treated (H) cells stimulated with nicotine. Plot profiles presented are representative of several measurements (n = 15 for each plot profile) from different tomograms (n = 3 for each conditions). Individual 10-nm actin filaments anchored granules in control cells, whereas large actin bundles consisting of at least five actin filaments were observed in WA-treated cells. Bars: (A) 5 µm; (C) 200 nm; (E, F and H, main panels) 100 nm; (F and H, insets) 25 nm.

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