Polymerization prevents MANI-FM entry into vesicles and tubules. HeLa cells were transfected with MANI-FM in the presence of AP, and then deprived of AP for 15 min to induce polymerization. (a) Polymeric MANI-FM (−AP for 15 min) is nearly absent in Golgi vesicles (arrowhead) and tubules. The trans side of the stack was identified by clathrin-coated buds (arrow). (b) Quantification of MANI-FM in vesicles and tubules. Polymeric MANI-FM (−AP) is strongly depleted in vesicles and tubules compared with the monomer (+AP). (c) MANI-FM distribution along the cisternal length, expressed as the ratio between the LD in the rims versus the central part of the cisternae. MANI-FM shifts to a more central localization after polymerization. More than 20 stack profiles were analyzed for each condition. (d) The membrane length of vesicles and tubules, as a measure of the surface area, quantitated and expressed as perimeter (in micrometers) was not appreciably changed after polymerization of MANI-FM. The total surface area of tubules decreased by ∼25% after polymerization. (e) HeLa cells transfected with MANI-FM and KDEL receptor–GFP (KDELR-GFP) and kept in the presence or absence of AP (for 10 min) were treated with 6 µg/ml BFA for 8 min to induce tubules emanating from the Golgi, and then fixed and stained for MANI-FM. MANI-FM enters BFA-induced tubules in the presence of AP (monomers) but not in the absence of AP (polymers). (f) The number of BFA-induced tubules positive for MANI-FM was quantified. At least 10 cells per condition per experiment were analyzed, and the results are from three independent experiments. Data are means ± SEM. Bars: (a) 250 nm; (e, +AP) 15 µm; (e, −AP) 18 µm.