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A Rho signaling network links microtubules to PKD controlled carrier transport to focal adhesions

Research Article
Cite this article as: eLife 2018;7:e35907 doi: 10.7554/eLife.35907
9 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
RhoA activates PKD at the TGN.

HeLa cells were transfected with G-PKDrep plus control vector, or a plasmid encoding HA-tagged RhoA wt or RhoA Q63L. One day after transfection cells were fixed and stained for G-PKDrep phosphorylation (pSer294) and F-actin (Alexa633-labelled phalloidin). Images were quantified by ratiometric calculation of GFP and Alexa546-labelled pSer294 signal. (A) Shown are representative confocal images, scale bar 10 µm. (B) The box plot shows the results of three independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 72 sample points each. The significance of differences was analyzed by a one-way ANOVA (Kruskal-Wallis test) followed by a Dunn’s multiple comparison test. ****p<0.0001 (control vs. RhoA Q63L). (C) HeLa cells were transfected with a plasmid encoding GFP-tagged RhoA Q63L, fixed 24 hr later, and stained for the trans Golgi protein p230 (red). Shown is a confocal image, scale bar 10 µm. Yellow regions indicate co-localization. * indicates cells with a compact Golgi complex, # indicates cells with a fragmented Golgi complex.

https://doi.org/10.7554/eLife.35907.002
Figure 1—figure supplement 1
RhoB activates PKD at the TGN.

HeLa cells were transfected with G-PKDrep plus control vector or a plasmid encoding CFP-tagged RhoB Q63L. One day after transfection cells were fixed and stained for G-PKDrep phosphorylation (pSer294). Images were quantified by ratiometric calculation of GFP and Alexa546-labelled pSer294 signal. (A) Shown are representative confocal images, scale bar 10 µm. (B) The box plot shows the results of a representative experiment. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 31, 28, 28 sample points. Statistical analysis was done using a one-way ANOVA followed by a Dunn’s multiple comparison test. ****p<0.0001 for control vs. RhoB Q63L. (C) HeLa cells were transfected with a plasmid encoding CFP-tagged RhoB Q63L plus GFP-tagged G-PKDrep and fixed 24 hr later (red). Shown is a confocal image, scale bar 10 µm. Yellow regions indicate co-localization. (D) Co-localization of GFP-tagged RhoA and RhoB wt and Q63L with Rab6. HeLa cells were transfected with the indicated plasmids, fixed 24 hr later, and stained for Rab6 (Alexa 633). Left panel: The box plot shows the co-localization results of two independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 52, 61, 56, 51 sample points. Statistical analysis was done using a one-way ANOVA (Kruskal Wallis) followed by a Dunns multiple comparison test. *p<0.05. **p<0.01. Right panel: Shown are representative confocal images, scale bar 10 µm.

https://doi.org/10.7554/eLife.35907.003
Figure 2 with 3 supplements
Nocodazole-mediated PKD activation at the TGN requires GEF-H1.

(A) HEK293T cells were transfected with spGEF-H1, spNT was used as a control. Three days after transfection cells were stimulated with nocodazole for 60 min, lysed and analyzed for PKD activity (pPKD (Ser744/748) and expression of PKD1, 2, and 3. Detection of alpha tubulin served as a loading control. Successful depletion of GEF-H1 was verified by detection with a GEF-H1 specific antibody. Shown is a representative Western blot. The integrated density of the pPKD and PKD1-3 signal was measured, corrected for background signals and the ratio (pPKD/PKD) calculated. Data were normalized to the control (spNT -nocodazole), which was set to 100. The graphs show the mean ± SEM of three independent experiments. The significance of differences was analyzed by a Ratio paired t-test (two-tailed). ***p=0.0007 (ratio pPKD Ser744/748/PKD1) and p=0.0002 (ratio pPKD Ser744/748/PKD2), **p=0.0014 (ratio pPKD Ser744/748/PKD3). (B) FlpIn T-Rex 293 EGFP-GEF-H1 wt or FlpIn T-REx 293 EGFP-GEF-H1 C53R cells were left untreated (-) or treated with doxycyline for 18 hr (+). Cells were lysed and analysed for PKD activity, PKD1-3 expression and endogenous as well as ectopic GEF-H1 expression. Detection of alpha tubulin served as loading control. Shown is a representative Western blot. Quantification of Western blot data was done as described in Figure 2A. Data were normalized to the control (GEF-H1 wt untreated), which was set to 100. The graphs show the mean ± SEM of three independent experiments. The significance of differences was analyzed by a Ratio paired t-test (two-tailed). *p=0.0365 (ratio pPKD Ser744/748/PKD1) and **p=0.0023 (ratio pPKD Ser744/748/PKD2). (C) FlpIn T-Rex HeLa EGFP-GEF-H1 wt or EGFP-GEF-H1 C53R cells were transfected with a Cherry-tagged G-PKDrep. One day after transfection cells expression of GEF-H1-GFP was induced by doxycyline. One day later, cells were fixed and stained for G-PKDrep phosphorylation (pSer294). Images were quantified by ratiometric calculation of Cherry and Alexa633-labelled pSer294 signal. Cells without doxycycline treatment were used as control. The box plot shows the results of three independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 89, 56, 23 sample points. The significance of differences was analyzed by a one-way ANOVA (Kruskal-Wallis test) followed by a Dunns multiple comparison test. ****p<0.0001 (control vs GEF-H1 wt or GEF-H1 C53R). (D) HeLa cells were transfected with the indicated siRNAs. Two days later, cells were transfected with the RhoA Biosensor along with mRuby-Golgi-7 (ratio 6:1) and, after 24 hr, stimulated with nocodazole and imaged. Left panel, representative images taken at 0 and 30 min of stimulation are shown; scale bar 10 µm. Right panel, the graph shows the mean ± SEM of at least 40 cells imaged in two independent experiments. The arrow indicates the time point of nocodazole stimulation. Successful depletion of GEF-H1 was verified by Western Blot analysis. Detection of alpha tubulin served as a loading control.

https://doi.org/10.7554/eLife.35907.004
Figure 2—figure supplement 1
Validation of the requirement of PLCε and GEF-H1 for nocodazole-induced PKD activation.

HEK293T cells were transfected with single siRNAs specific for GEF-H1 or PLCε, spNT was used as a control. Three days after transfection cells were stimulated with nocodazole for 60 min, lysed and analyzed for PKD activity (pPKD Ser744/748) and expression of PKD2 and PKD3. Detection of alpha tubulin served as a loading control. GEF-H1 depletion was verified by immunoblotting using a specific antibody. Successful depletion of PLCε was verified by RT-qPCR. Relative expression was calculated by normalization to actin using the ΔCq method.

https://doi.org/10.7554/eLife.35907.005
Figure 2—figure supplement 2
GEF-H1 mediated PKD activation is independent of ERK

(A) Nocodazole-mediated PKD activation is independent of the MAPK pathway. HEK293 cells were left untreated (-) or stimulated with nocodazole for 30 min. Prior to nocodazole stimulation cells were treated with DMSO or UO0126 (10 µM) for one hour. Cells were lysed and analyzed for phosphorylation and expression of PKD1-3, MEK1/2, and ERK1/2. Detection of tubulin served as a loading control. (B) Localization of GEF-H1 wt and GEF-H1 C53R. FlpIN T-REx Hela EGFP-GEF-H1 wt or C53R cells were treated with doxycycline to induce expression of GFP-GEF-H1. 24 hr later cells were fixed and stained for p230. Shown are representative confocal images, scale bar 10 µm.

https://doi.org/10.7554/eLife.35907.006
Figure 2—figure supplement 3
The RhoA biosensor localizes to the Golgi complex.

HeLa cells were transfected with plasmids encoding the RhoA Biosensor and mRuby-Golgi-7 (ratio 6:1). Cells were imaged one day later at the Spinning disc microscope. Scale bar 10 µm.

https://doi.org/10.7554/eLife.35907.007
Figure 3 with 2 supplements
The Rho effector PLCε is required for GEF-H1 mediated PKD activation at the TGN.

(A) HeLa cells were transfected with spRNAs as indicated, spNT was used as a control. Two days after transfection cells were transfected with G-PKDrep and RhoA Q63L and, after 24 hr, fixed, stained and analyzed as described in Figure 1A. Shown are representative confocal images, scale bar 10 µm. (B) The box plot shows the results of three independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 90 sample points each. The significance of differences was analyzed by a one-way ANOVA followed by a Dunns multiple comparison test. ***p<0.0001. (C) Left panel, silencing efficiency of mDia1 was analyzed in lysates by immunoblotting using a mDia1-specific antibody. Equal loading was verified by detection of alpha tubulin. Right panel, successful depletion of PLCε was verified by RT-qPCR. Relative expression was calculated by normalization to GAPDH using the ΔCq method. Shown is the mean ± SEM of two independent experiments. (D) Left panel, HEK293T cells were transfected with the spPLCε, spNT was used as a control. Three days post transfection cells were left untreated or stimulated with nocodazole for 60 min. Detection of active PKD and expression of PKD1-3 was performed in cell lysates using specific antibodies. Equal loading was verified by detection of alpha tubulin. Shown is a representative Western blot. Right panel, quantification of Western blot data was done as described in Figure 2A. Data were normalized to the control (untreated spNT cells), which was set to 100. The graphs show the mean ± SEM of three independent experiments. The significance of differences was analyzed by a Ratio paired t-test (two-tailed). ***p=0.0007 (ratio pPKD Ser744/748/PKD1), *p=0.0334 (ratio pPKD Ser744/748/PKD2), and **p=0.0026 (ratio pPKD Ser744/748/PKD3). Successful depletion of PLCε was verified by RT-qPCR. Relative expression was calculated by normalization to Actin using the ΔCq method. Shown is the mean ± SEM of three independent experiments. The significance of differences was analyzed by a Ratio paired t-test (two-tailed). *p=0.0157 (E) Left panel, FlpIn T-REx 293 EGFP-GEF-H1 C53R cells were transfected with spPLCε, spNT was used as a control. Two days later, GFP-GEF-H1 C53R expression was induced by doxycycline treatment. After one day, cells were lysed and analysed for PKD activity, PKD1-3 expression and endogenous as well as ectopic GEF-H1 expression. Detection of alpha tubulin served as a loading control. Shown is a representative Western blot. Right panel, quantification of Western blot data was done as described in Figure 2A. Data were normalized to the control (spNT cells without doxycycline), which was set to 100. The graphs show the mean ± SEM of three independent experiments. The significance of differences was analyzed by a Ratio paired t-test (two-tailed). *p=0.0266 (ratio pPKD Ser744/748/PKD1), p=0.0106 (ratio pPKD Ser744/748/PKD2), and p=0.0152 (ratio pPKD SerS744/748/PKD3). Successful depletion of PLCε was verified by RT-qPCR. Relative expression was calculated by normalization to Actin using the ΔCq method. Shown is the mean ± SEM of three independent experiments.

https://doi.org/10.7554/eLife.35907.008
Figure 3—figure supplement 1
ROCK1 and ROCK2 are not involved in PKD activation at the TGN.

(A) HeLa cells were transfected with the indicated spRNAs, spNT was used as a control. Two days after transfection cells were transfected with G-PKDrep and RhoA Q63L and, after 24 hr, fixed, stained and analyzed as described in Figure 1A. The box plot shows the results of three independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 90 sample points each. The significance of differences was analyzed by a one-way ANOVA followed by a Dunns multiple comparison test. ****p<0.0001. Right panel, successful depletion of ROCK1 and ROCK2 were verified by immunoblotting using specific antibodies. Alpha tubulin detection served as a loading control. (B) HeLa cells were transfected with G-PKDrep and RhoA Q63L. After 24 hr, cells were treated with DMSO or with 10 µM H1152 for 6 hr. Subsequently, cells were fixed, stained and analysed as described in Figure 1A. The box plot shows the results of three independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 90, 60, 61 sample points. The significance of differences was analyzed by a one-way ANOVA followed by a Dunn’s multiple comparison test. ****p<0.0001.

https://doi.org/10.7554/eLife.35907.009
Figure 3—figure supplement 2
Validation of PLCε as Rho-effector protein in PKD activation at the TGN using an independent siRNA.

Left panel, HEK293T cells were transfected with a PLCε-specific single siRNA, spNT was used as a control. Two days after transfection cells were transfected with G-PKDrep plus an empty vector or HA-tagged RhoA Q63L. 24 hr later, cells were lysed and phosphorylation plus expression of G-PKDrep was analyzed by Western blotting using the pSer294-specific and GFP-specific antibody, respectively. Expression of RhoA Q63L was verified using a HA-specific antibody. Activity of endogenous PKD was detected using the pSer910 antibody. Detection of alpha tubulin served as a loading control. Right panel, successful depletion of PLCε was controlled by quantitative RT-PCR. The graph shows a representative experiment.

https://doi.org/10.7554/eLife.35907.010
Figure 4 with 1 supplement
PLCε is required for basal and trypsin-induced PKD activity at the TGN.

(A) Top panel, HeLa cells were transfected with spPLCε, spNT was used as a control. Two days later, cells were transfected with a plasmid encoding PKD1kd-GFP. After 24 hr, cells were fixed and stained for p230. Representative confocal images are shown; scale bar 10 µm. CellProfiler images show the masks defining the Golgi complex (blue) and the cytosol (yellow) merged with the PKD1kd-GFP image (shown in greyscale). Bottom panel, the fluorescence intensities of the GFP signal were measured under both masks and the Golgi/Cytosol ratio was calculated for each cell. The graph shows the mean ± SEM of at least 150 cells analysed. (B) HeLa cells were transfected with siRNAs as described in E. Two days later, cells were transfected with G-PKDrep. Visualization and analysis of G-PKDrep phosphorylation was performed as described in Figure 1A. Shown are representative confocal images, scale bar 10 µm. (C) Left panel, the box plot shows the results of three independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 114, 104 sample points. The significance of differences was analyzed by a two-tailed t-test (Mann-Whitney test), *p<0.05. Right panel, successful depletion of PLCε was verified by RT-qPCR. Relative expression was calculated by normalization to GAPDH using the ΔCq method. Shown is the mean ± SEM of two independent experiments. (D) HEK293T cells were left untreated or stimulated with trypsin. Cells were lysed and detection of active PKD (pPKD Ser744/748) and expression of PKD1-3 was performed using specific antibodies. Equal loading was verified by detection of alpha tubulin. (E, F) HeLa cells were transfected with spRNAs as indicated, spNT was used as a control. Two days after transfection cells were transfected with G-PKDrep. 24 hr later, cells were left untreated or stimulated with trypsin, fixed, stained and analyzed as described in Figure 1A. (E) Shown are representative confocal images of the spNT control, scale bar 10 µm. (F) The scatter dot blot shows the result of three independent experiments, line indicates the mean. Each dot represents one experiment with at least 30 cells analysed. The significance of differences was analyzed by a one-way ANOVA (Friedman test) followed by a Dunn’s multiple comparison test. *p=0.0306 (spNT – trypsin vs. spNT + trypsin). All other comparisons were not significant. Successful depletion of the proteins was verified by Western blot or RT-qPCR (data not shown).

https://doi.org/10.7554/eLife.35907.011
Figure 4—figure supplement 1
PAR stimulation through thrombin and trypsin activates PKD at the Golgi.

HeLa cells were transfected with G-PKDrep. 24 hr later, cells were starved in serum free medium and treated with CRT0066101 at 5 µM for one hour, DMSO was used as a control. Subsequently, cells were treated with thrombin (10 nM) or trypsin (10 nM) for 5 min. Cells were fixed and stained for G-PKDrep phosphorylation (pSer294). Images were quantified by ratiometric calculation of GFP and Alexa546-labelled pSer294 signal. Shown are representative confocal images, scale bar 10 µm. The box plot shows the results of a representative experiment. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 39, 38, 58, 50, 38, 48 sample points each.

https://doi.org/10.7554/eLife.35907.012
Loss of the RhoGAP DLC3 promotes PKD activation at the TGN.

(A) HeLa cells were transfected with spRNAs as indicated. Two days after transfection cells were transfected with G-PKDrep and, after 24 hr, fixed, stained and analyzed by ratiometric measurement as described in Figure 1A. Representative confocal images are shown, scale bar 10 µm. (B) Top panel, the box plot shows the results of three independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 90 sample points each. The significance of differences was analyzed by a two-tailed t-test (Mann-Whitney test). ****p<0.0001. Bottom panel, silencing efficiency of DLC3 was analyzed in lysates by immunoblotting using a DLC3-specific antibody. Equal loading was verified by detection of alpha tubulin. (C) Cells were transfected with spRNAs as indicated. Two days after transfection cells were transfected with G-PKDrep and, after 24 hr, fixed, stained and analyzed by ratiometric imaging as described in 1A. Representative confocal images are shown, scale bar 10 µm. (D) The box plot shows the results of three independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 83, 73, 73, 75, 73, 75 sample points. The significance of differences was calculated by a one-way ANOVA (Kruskal-Wallis test) followed by a Dunn’s multiple comparison test. ****p<0.0001, *p=0.0393. ns, not significant. Right panel, silencing efficiency of GEF-H1 and DLC3 were verified in lysates by immunoblotting using specific antibodies. Detection of alpha tubulin served as a loading control.

https://doi.org/10.7554/eLife.35907.013
Figure 6 with 1 supplement
PKD recruits Rab8 to the TGN and co-localizes with Rab6.

(A) HeLa cells were transfected with a plasmid encoding wt PKD1-GFP. One day after transfection, cells were fixed and stained for Rab8 (top panel) or Rab6 (bottom panel) plus p230 and analyzed by confocal microscopy. Scale bar 10 µm. Right panel, in the histogram (profile scan) the fluorescence intensities of the three signals along the white line are depicted. (B) HeLa cells were transfected with an empty GFP vector, PKD1-GFP or PKD2-GFP constructs and stained for Rab6 or Rab8 and the TGN marker protein p230. Co-localization analysis of Rab8 or Rab6 with the TGN in GFP positive and GFP negative cells was performed using Cell Profiler software as described in the material and methods section. The graph shows the mean ± SEM of three independent experiments. Significance of differences was analyzed by a two-way RM ANOVA followed by a Bonferroni’s multiple comparison test. ***p=0.0004, **p=0.001.

https://doi.org/10.7554/eLife.35907.014
Figure 6—figure supplement 1
PKD co-localizes with Rab6 and Rab8 at the Golgi compartment.

HeLa cells were transfected with a GFP control vector or GFP-tagged versions of PKD1 or PKD2. 24 hr later cells were fixed and stained for p230 and Rab6 or Rab8 according to the protocol for indirect immunofluorescence. Images were acquired as described in the methods section. Shown are the different channels overlaid with the segmentation mask of the Golgi complex generated in CellProfiler.

https://doi.org/10.7554/eLife.35907.015
Figure 7 with 2 supplements
GEF-H1, PLCε and PKD are required for localized delivery of Rab6 to FAs.

(A, B, C, D, E) Cells were transfected with spRNAs as indicated. Two days later cells were transfected with plasmids encoding GFP-tagged Rab6 and DsRed2-tagged paxillin. After 24 hr cells were analyzed at a TIRF-equipped Spinning disc microscope. The amount of Rab6-positive vesicles arriving at FAs was assessed within a two minute time interval. (A) The box plot shows the results of three independent experiments. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 26, 26, 26, 20 sample points. The significance of differences was assessed by a one-way ANOVA (Kruskal Wallis test) followed by a Dunn’s multiple comparison test. ****p<0.0001, ***p=0.002. (B) Top panel, number of FAs per cell. Bottom panel, number of vesicles arriving at FAs within two minutes. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 26, 26, 26, 20 sample points. (C) Representative TIRF images. Red dots indicate Rab6-positive vesicles arriving at FAs within the two minute time interval. Scale bar 10 µm. (D) Left panel, silencing efficiency of GEF-H1, PKD2 and PKD3 was analyzed in lysates by immunoblotting using specific antibodies. Equal loading was verified by detection of alpha tubulin. Right panel, successful depletion of PLCε was verified by RT-qPCR. Relative expression was calculated by normalization to actin using the ΔCq method. (E) Cells were imaged 5 min prior to stimulation with thrombin or PBS (as a control). After addition of the reagent, imaging was continued for 30 min. Left panel: The box plot shows the result of three independent experiments. The vesicles arriving at FAs per minute before and after stimulation were calculated and presented as ratio ‘number of vesicles at FA after/before stimulation’. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism 7 software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 7, 12, 10 sample points (cells analyzed). The significance of differences was assessed by a one-way ANOVA (Kruskal Wallis test) followed by a Dunn’s multiple comparison test. **p<0.01, ns not significant. Right panel: Representative TIRF kymographs of spNT and spPKD2/3 cells. Arrows indicate vesicles arriving at FAs.

https://doi.org/10.7554/eLife.35907.016
Figure 7—figure supplement 1
PLCε knockdown does not impair microtubule-FA interaction.

HeLa cells were transfected with spRNAs as indicated. Two days later cells were transfected with plasmids encoding GFP-tagged Clip170 and DsRed2-tagged paxillin. After 24 hr cells were analyzed at a Spinning disc microscope in phenolred-free RPMI supplemented with 10% FCS as described in the material and methods section. Shown are merged channel images at time point zero. The kymographs represent the intensity change in the selected lines over a 2 min time interval. Kymograph lines are illustrated by a white rectangular frame. Scale bar 10 µm. Right panel, successful depletion of PLCε was verified by RT-qPCR. Relative expression was calculated by normalization to actin using the ΔCq method.

https://doi.org/10.7554/eLife.35907.017
Figure 7–video 1
Loss of GEF-H1, PLCε, or PKD impairs the localized delivery of Rab6-positive vesicles to FAs.

HeLa cells were transfected with spRNAs as indicated. Two days later cells were transfected with plasmids encoding GFP-tagged Rab6A (green) and DsRed2-tagged paxillin (red). After 24 hr, cells were imaged in phenolred-free RPMI supplemented with 10% FCS at the Spinning disk microscope as described in the material and methods section. Stacks were acquired simultaneously in green and red channels each second for a time interval of 2 min. The movies show the 3D rendering of the merged channel image with 10 frames per second speed. Scale bar 10 µm.

https://doi.org/10.7554/eLife.35907.018
Figure 8 with 1 supplement
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.

https://doi.org/10.7554/eLife.35907.019
Figure 8–video 1
Loss of GEF-H1, PLCε, or PKD blocks transport of TNFα at the Golgi complex.

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.

https://doi.org/10.7554/eLife.35907.020
Schematic illustration of the Rho signaling pathway at the TGN.

GPCR signaling activates GEF-H1 by releasing it from microtubules. GEF-H1 in turn promotes an increase in cellular RhoA activity. At the TGN, active RhoA binds to its effector PLCε, which hydrolyzes PtdIns(4)P to generate DAG and IP2. DAG recruits and activates nPKCs and PKD, which gets further activated by nPKC-mediated phosphorylation. The activation of PKD by RhoA is counterbalanced by the RhoGAP DLC3. Active PKD recruits Rab8 to TGN membranes and induces the fission of Rab6-positive vesicles containing cargo such as TNFα destined for FA-targeted transport along microtubules.

https://doi.org/10.7554/eLife.35907.021

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line (human)HEK293TATCCATCC Cat# CRL-3216, RRID:CVCL_0063
Cell line
(human)
Flp-In T-Rex-293Thermo Fisher ScientificRRID:CVCL_U427
Cell line
(human)
Flp-In T-Rex-HeLaothergenerated by Elena Dobrikova
and Matthias Gromeier, Duke
University Medical Center,
Durham, NC, USA
Cell line
(human)
HeLaATCCATCC Cat# CRM-CCL-2, RRID:CVCL_0030
Recombinant
DNA reagent
pEGFP-N1-PKD1
wt/K612W
DOI: 10.1038/ncb1289
Recombinant
DNA reagent
pEGFP-N1-PKD2DOI: 10.1038/ncb1289
Recombinant
DNA reagent
pCMV5-EGFP-GEF
-H1 wt
DOI: 10.1038/ncb773
Recombinant
DNA reagent
pCMV5-EGFP-GEF
-H1 C53R
DOI: 10.1038/ncb773
Recombinant
DNA reagent
pTriEx-RhoA
FLARE.sc
DOI: 10.1038/nature04665Addgene #12150
Recombinant
DNA reagent
mRuby-Golgi-7Addgene #55865
Recombinant
DNA reagent
Rab6a-GFPotherprovided by Francis Barr,
Oxford University
Recombinant
DNA reagent
DsRed2-PaxillinDOI: 10.1038/ncb1094
Recombinant
DNA reagent
G-PKDrepdoi: 10.1111/j.1600–
0854.2009.00918.x
Recombinant
DNA reagent
Clip170-GFPotherprovided by Niels Galjart,
Erasmus Medical Center,
Rotterdam
Recombinant
DNA reagent
pcDNA5/FRT/TO-EGFP
-GEF-H1 wt/C53R
this paper
Recombinant
DNA reagent
pECFP-Endo/RhoBClontechClontech #6934–1
Recombinant
DNA reagent
pECFP-Endo/Rhob
Q63L
this papersite-directed mutagenesis
using pECFP-RhoB as a
template
Recombinant
DNA reagent
pEGFP-RhoBthis papersubcloned from
pECFP-Endo/RhoB
Recombinant
DNA reagent
pEGFP-RhoB Q63Lthis papersubcloned from
pECFP-Endo/RhoB Q63L
Recombinant
DNA reagent
pEGFP-RhoADOI: 10.1242/jcs.163857
Recombinant
DNA reagent
pEGFP-RhoA Q63LDOI: 10.1242/jcs.163857
Recombinant
DNA reagent
pcDNA3.1-HA-RhoA
Q63L
DOI: 10.1242/jcs.163857
Recombinant
DNA reagent
pcDNA3.1-HA-RhoADOI: 10.1242/jcs.163857
Recombinant
DNA reagent
Str-KDEL-TNFa-SBP-
mCherry
Addgene #65279
Recombinant
DNA reagent
pOG44Thermo Fisher ScientificThermo Fisher Scientific V600520
Antibodyp230BD BiosciencesBD Biosciences Cat# 611280, RRID:AB_398808
AntibodyGFPRocheSigma-Aldrich
Cat# 11814460001,
RRID:AB_390913
AntibodyTGN46Bio-Rad/AbD SerotecBio-Rad/AbD Serotec
Cat# AHP500,
RRID:AB_324049
Antibodyalpha-tubulinMilliporeMillipore Cat# 05–829,
RRID:AB_310035
AntibodyDLC3Santa Cruz BiotechnologySanta Cruz Biotechnology
Cat# sc-166725,
RRID:AB_2197829
AntibodyPKD1Cell SignalingCell Signaling Technology
Cat# 2052, RRID:AB_2268946
AntibodyPKD2Cell SignalingCell Signaling Technology
Cat# 8188S,
RRID:AB_10829368
AntibodyPKD3Cell SignalingCell Signaling Technology
Cat# 5655S,
RRID:AB_10695917
AntibodyGEF-H1Cell SignalingCell Signaling Technology
Cat# 4076, RRID:AB_2060032
Antibodyphospho-PKD
(Ser744/748)
Cell SignalingCell Signaling Technology
Cat# 2054S,
RRID:AB_2172539
AntibodyRab8Cell SignalingCell Signaling Technology
Cat# 6975S,
RRID:AB_10827742
AntibodyRab6Cell SignalingCell Signaling Technology
Cat# 9625S,
RRID:AB_10971791
AntibodypS910DOI: 10.1083/jcb.200110047
AntibodyERK1/2Cell SignalingCell Signaling Technology
Cat# 9107S,
RRID:AB_10695739
AntibodyMEK1/2Cell SignalingCell Signaling Technology
Cat# 8727,
RRID:AB_10829473
AntibodypERK1/2
(Thr202/Tyr204)
Cell SignalingCell Signaling Technology
Cat# 4094S,
RRID:AB_10694057
AntibodypMEK1/2
(Ser2017/221)
Cell SignalingCell Signaling Technology
Cat# 9154,
RRID:AB_2138017
AntibodyROCK1MilliporeMillipore Cat# 04–1121,
RRID:AB_1977472
AntibodyROCK2BD BiosciencesBD Biosciences Cat# 610623,
RRID:AB_397955
AntibodypS294DOI: 10.1038/ncb1289IF 1:750
AntibodyHRP goat anti-rabbitDianovaJackson ImmunoResearch
Labs Cat# 111-035-144,
RRID:AB_2307391
AntibodyHRP goat anti-mouseDianovaJackson ImmunoResearch
Labs Cat# 115-035-062,
RRID:AB_2338504
AntibodyAlexa Fluor labelled
secondary antibodies
Thermo Fisher ScientificThermo Fisher Scientific
Sequence-based
reagent
ON-Targetplus
smartpools
DharmaconDharmacon
Sequence-based
reagent
Silencer select
siPLCe
Thermo Fisher ScientificThermo Fisher Scientific
s27660
Sequence-based
reagent
Silencer select
siGEF-H1
Thermo Fisher ScientificThermo Fisher Scientific
s17546
Sequence-based
reagent
Quantitect primers
for RT-PCR
QiagenQiagen
Peptide, recombinant
protein
TrypsinThermo Fisher ScientificThermo Fisher Scientific
15090046
Peptide, recombinant
protein
ThrombinMilliporeMillipore 605195
Commercial assay
or kit
QuantiTect SYBR
Green RT-PCR Kit
QiagenQiagen 204243
Commercial assay
or kit
Rneasy plus KitQiagenQiagen 74104
Chemical compound,
drug
NocodazoleSigma-AldrichSigma-Aldrich M1404
Chemical compound,
drug
CRT0066101Tocris BioscienceTocris Bioscience 4975
Chemical compound,
drug
H1152Enzo Life ScienceEnzo Life Science
ALX-270–423 M001
Chemical compound,
drug
UO126Cell SignalingCell Signaling #9903
Chemical compound,
drug
BlasticidinThermo Fisher ScientificThermo Fisher Scientific
R21001
Chemical compound,
drug
Hygromycin BThermo Fisher ScientificThermo Fisher Scientific
10687010
Chemical compound,
drug
DoxycyclinSigma-AldrichSigma-Aldrich D9891
Chemical compound,
drug
ZeocinThermo Fisher ScientificThermo Fisher Scientific
R25001
Chemical compound,
drug
Collagen RServaServa 47254
Chemical compound,
drug
BiotinSigma-AldrichSigma-Aldrich B4501
Chemical compound,
drug
Blocking reagentRocheRoche 11096176001
Software, algorithmCell Profilerhttp://cellprofiler.orgCellProfiler Image
Analysis Software,
RRID:SCR_007358
Software, algorithmGraphPad PrismGraphPad Prism (https://graphpad.com)Graphpad Prism,
RRID:SCR_002798
Software, algorithmImage Studio
Lite 4.0
https://www.licor.com/bio/products/software/image_studio_lite/?utm_source=BIO+Blog&utm_medium=28Aug13post&utm_content=ISLite1&utm_campaign=ISLiteImage Studio Lite,
RRID:SCR_014211

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