Figure 8. The Rho signaling pathway controls vesicle fission and cargo transport at the level of the TGN.

(A) HeLa cells were transfected with a construct encoding Str-KDEL-TNFα-SBP-mCherry together with Rab6-GFP. 24 hr later, biotin was added for 35 min followed by fixation. Cells were stained for p230 and analyzed by confocal microscopy. Shown is a maximum intensity projection, scale bar 10 µm. (B, C, D) HeLa cells were transfected with spRNAs as indicated, spNT was used as a control. (B, C) Two days later cells were transfected with plasmids encoding Str-KDEL-TNFα-SBP-mCherry and Rab6-GFP and, after 24 hr, biotin was added and cells were subjected to the RUSH assay as described in the material and methods section. After fixation, cells were stained for p230. Shown are representative confocal images, scale bar 10 µm. (C) Left panel, the scatter dot blot shows the result of three independent experiments, line indicates the mean. Each dot represents one experiment with at least 190 cells analysed. The significance of differences was analyzed by a one-way ANOVA followed by a Holm-Sidak’s multiple comparison test, **p=0.0065. All other comparisons were not significant. Right panel, successful depletion of the proteins was verified by RT-qPCR. Relative expression was calculated by normalization to actin using the ΔCq method. The graphs represent the mean ± SEM of three independent experiments. (D) HeLa cells were transfected with a plasmid encoding Str-KDEL-TNFα-SBP-mCherry and release from the ER was induced by biotin addition 24 hr later. The graph shows integrated fluorescence intensity in the Golgi region at each time point, corrected for background and normalized to the maximum value. Curves depict the measurement of at least 22 cells of a representative experiment. Error bars, SEM.

Figure 8–video 1. Loss of GEF-H1, PLCε, or PKD blocks transport of TNFα at the Golgi complex.

Download video file ^{(2.3MB, mp4)}

DOI: 10.7554/eLife.35907.020

HeLa cells were transfected with spRNAs as indicated. Two days later cells were transfected with a plasmid encoding Str-KDEL-TNFα-SBP-mCherry. After 24 hr, cells were imaged in biotin-free FluoroBrite DMEM medium supplemented with 10% FCS at the Spinning disk microscope as described in the material and methods section. Stacks were acquired every minute. The movie replays at a speed of 10 frames per second. Scale bar 10 µm.