Fig. 1

Uptake of FM 4–64 is differently inhibited in the presence of trafficking inhibitors, auxins and cytoskeletal drugs. a-n In vivo confocal microscopy of FM 4–64 (2 μM, 20 min) uptake by 3-day-old tobacco BY-2 cells pre-treated for 30 min with inhibitors. Confocal sections of perinuclear plane captured with confocal microscope (561 nm excitation). a Control (CTRL) cells with mock treatment (DMSO). FM 4–64 endosomes are randomly distributed through the cytoplasm. Note the PM staining. b TYR A23 (50 μM) inhibition of the endocytosis in contrast to (c) inactive TYR A51 (50 μM), note the absence of the PM staining. d WM (33 μM) inhibition of endocytosis. e FIL (15 μM), (f) DNS (80 μM) and all auxins (g) 1- NAA, (h) IAA, (i) 2,4-D (5 μM) tested, partially inhibiting FM 4–64 uptake. j BFA (20 μM) inhibition of endocytosis. k Cyt D (40 μM), (l) Lat B (500 nm), (m) ORY (15 μM) and (n) TX (10 μM) partial inhibition of FM 4–64 uptake. Scale bar =10 μm. o Relative area of internalized FM 4–64 (expressed in ‰ - per mil of the total cell area) for all inhibitors. Values are means of the ratios between integral areas of the internalized dye (without PM attached endosomes) and total area of the cell. Error bars represent standard error of the means (SEM) from four biological repetitions, n = 4. p Number of PM attached internalized FM 4–64 endosomes. Values represent the proportion of FM 4–64 endosomes attached to PM to the total internalized FM 4–64. Note the correlation between the amount of cortical (PM attached) endosomes and the rate/inhibition of endocytosis. Error bars represent SEM from four biological repetitions, n = 4