Figure 4.
Figure 4. Polymerized MANI-FM shifts from cis-/medial to trans-Golgi cisternae. (a and b) HeLa cells were transfected with MANI-FM in the presence of AP (a), and then deprived of AP for 20 min (b) to induce polymerization. MANI-FM (15-nm gold) shifted from the cis-cisternae (labeled with the cis marker GM130; 10-nm gold; arrowheads) to the trans-cisternae after AP washout. (c–e) The distribution of MANI-FM across the stack at time 0 (c), 12 min (d), and 20 min (e) after AP washout, quantitated and expressed as LD normalized to that of the maximum. The normalized LD in the cis-cisternae is indicated in blue, in the medial in green, and in the trans in red. The schemes above the graph depict the shift of the MANI-FM peak from the cis/medial to the trans side of the stack. The intensities of the fill colors correspond to the normalized LD of the MANI-FM below (intensity scale on right). At least 20 stacks were analyzed for each time point. Data are mean ± SEM. (f–i) The shift of MANI-FM from the cis-/medial to the trans-Golgi was also visualized by confocal microscopy. HeLa cells were transfected with MANI-FM in the presence of AP (f), deprived of AP for 20 min (g), and then fixed and stained for MANI-FM (green), GM130 (blue), and TGN46 (red). The white arrow across the stacks was used for the line-scan analyses. The images are representative of >30 stacks analyzed for each condition. (h and i) Fluorescence intensity distribution of markers along the line scan (arrow in f and g). The fluorescent intensities were normalized to their respective peak values. The image and the corresponding quantitation are representative of at least 30 Golgi ministacks from three independent experiments. For this experiment, n = 30. (j) HeLa cells expressing MANI-FM or MANI-CFP cultured in the presence or absence of AP were treated with cycloheximide for the indicated times and the amount of protein was analyzed by lysing the cells followed by Western blotting. The quantification of the blots in panel j is shown in k. Whereas the polymeric MANI-FM (−AP) is degraded, the monomeric MANI-FM (+AP) is stable, like MANI-CFP and endogenous MANI. The results are typical of two independent experiments. Bars: (a and b) 220 nm; (f and g) 1 µm.

Polymerized MANI-FM shifts from cis-/medial to trans-Golgi cisternae. (a and b) HeLa cells were transfected with MANI-FM in the presence of AP (a), and then deprived of AP for 20 min (b) to induce polymerization. MANI-FM (15-nm gold) shifted from the cis-cisternae (labeled with the cis marker GM130; 10-nm gold; arrowheads) to the trans-cisternae after AP washout. (c–e) The distribution of MANI-FM across the stack at time 0 (c), 12 min (d), and 20 min (e) after AP washout, quantitated and expressed as LD normalized to that of the maximum. The normalized LD in the cis-cisternae is indicated in blue, in the medial in green, and in the trans in red. The schemes above the graph depict the shift of the MANI-FM peak from the cis/medial to the trans side of the stack. The intensities of the fill colors correspond to the normalized LD of the MANI-FM below (intensity scale on right). At least 20 stacks were analyzed for each time point. Data are mean ± SEM. (f–i) The shift of MANI-FM from the cis-/medial to the trans-Golgi was also visualized by confocal microscopy. HeLa cells were transfected with MANI-FM in the presence of AP (f), deprived of AP for 20 min (g), and then fixed and stained for MANI-FM (green), GM130 (blue), and TGN46 (red). The white arrow across the stacks was used for the line-scan analyses. The images are representative of >30 stacks analyzed for each condition. (h and i) Fluorescence intensity distribution of markers along the line scan (arrow in f and g). The fluorescent intensities were normalized to their respective peak values. The image and the corresponding quantitation are representative of at least 30 Golgi ministacks from three independent experiments. For this experiment, n = 30. (j) HeLa cells expressing MANI-FM or MANI-CFP cultured in the presence or absence of AP were treated with cycloheximide for the indicated times and the amount of protein was analyzed by lysing the cells followed by Western blotting. The quantification of the blots in panel j is shown in k. Whereas the polymeric MANI-FM (−AP) is degraded, the monomeric MANI-FM (+AP) is stable, like MANI-CFP and endogenous MANI. The results are typical of two independent experiments. Bars: (a and b) 220 nm; (f and g) 1 µm.

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