Fig. 4

BFA does not block endocytosis of FM 4–64 in BY-2 line upon inducible overexpression of Ntgnl1a ^M683L mutant allele. a-j In vivo confocal microscopy of FM 4–64 (2 μM, 20 min) uptake by 3-day-old tobacco BY-2 cells, Ntgnl1a ^M683L and NtGNL1a cells, non-induced (g, h, i, j) and induced with 3 μM β-estradiol (c, d, e, f), pre-treatment with mock DMSO (a, c, e, g, i) or 20 μM BFA (b, d, f, h, j) for 30 min. Confocal sections of perinuclear plane captured with confocal microscope (561 nm excitation). a Control (CTRL) cells with mock treatment (DMSO). FM 4–64-stained endosomes randomly distributed through the cytoplasm. Note the PM staining. b BFA (20 μM) inhibition of FM 4–64 uptake in CTRL cells. c, e, g, i FM 4–64-stained endosomes randomly distributed in the cytoplasm in induced and non-induced Ntgnl1a ^M683L cells (c, g) and NtGNL1a cells (e, i). f, h, j BFA (20 μM) inhibition of FM 4–64 uptake in non-induced Ntgnl1a ^M683L cells (h) and induced and non-induced NtGNL1a cells (f, j). Note the contrasting uptake of FM–64 in induced Ntgnl1a ^M683L cells (d) with BFA-induced FM 4–64 compartments observed in perinuclear area. k Relative area of internalized FM 4–64 (expressed in ‰ - per mil of the total cell area). Values represent means of the ratios between integral areas of the internalized FM 4–64 and the total area of the cell. Error bars represent SEM from three biological repetitions, n = 3