MANI-FM recycles from the trans- to the cis-/medial Golgi after depolymerization in the trans-Golgi. (a and b) HeLa cells were transfected with MANI-FM in the presence of AP and then deprived of AP for 20 min to induce polymerization and the transfer of MANI-FM to the trans-cisterna (a); finally, AP was added for 1.5 min (b). Arrowheads in b indicate the peri-Golgi carriers seen after AP addition. (c and d) The lateral distribution of MANI-FM along the cisternal length (c) and the distribution of MANI-FM in peri-Golgi carriers (d), expressed as LD. (e) The membrane length of vesicles and tubules as a measure of the surface area was quantitated and expressed as perimeter (in micrometers). (f) Colocalization of COPI and MANI-FM in vesicles and tubules (arrowheads) in cells fixed 1.5 min after addition of AP to depolymerize MANI-FM. (g) The change in distribution of MANI-FM from the trans- to the cis-/medial Golgi after addition of AP is expressed as LD normalized to that of the maximum (see Fig. 4 for description). Around 10–20 Golgi stack profiles were examined for each time point. Data are mean ± SEM. (h–m) The change in position of MANI-FM was also visualized under confocal microscopy. HeLa cells were fixed in the presence of AP (h and k), after 20 min of AP washout (i and l), and 5 min after readdition of AP (j and m). They were then stained for MANI-FM (green), GM130 (blue), and TGN46 (red). The white arrows across the stacks were used for line-scan analyses. Fluorescence intensity distribution of markers along the line scan (h–j, arrow), normalized to their respective peak values, is shown in k–m. The images are representative of >30 stacks analyzed for each condition from three independent experiments. For this experiment, n = 30. Bars: (a and b) 220 nm; (f) 120 nm; (h–j) 1 µm.