Rab5 Isoforms Orchestrate a “Division of Labor” in the Endocytic Network; Rab5C Modulates Rac-Mediated Cell Motility

<div><p>Rab5, the prototypical Rab GTPase and master regulator of the endocytic pathway, is encoded as three differentially expressed isoforms, Rab5A, Rab5B and Rab5C. Here, we examined the differential effects of Rab5 isoform silencing on cell motility and report that Rab5C, but neither Rab5A nor Rab5B, is selectively associated with the growth factor-activation of Rac1 and with enhanced cell motility. Initial observations revealed that silencing of Rab5C expression, but neither Rab5A nor Rab5C, led to spindle-shaped cells that displayed reduced formation of membrane ruffles. When subjected to a scratch wound assay, cells depleted of Rab5C, but not Rab5A or Rab5B, demonstrated reduced cell migration. U937 cells depleted of Rab5C also displayed reduced cell motility in a Transwell plate migration assay. To examine activation of Rac, HeLa cells stably expressing GFP-Rac1 were independently depleted of Rab5A, Rab5B or Rab5C and seeded onto coverslips imprinted with a crossbow pattern. 3-D GFP-Rac1 images of micro-patterned cells show that GFP-Rac1 was less localized to the cell periphery in the absence of Rab5C. To confirm the connection between Rab5C and Rac activation, HeLa cells depleted of Rab5 isoforms were starved and then stimulated with EGF. Rac1 pull-down assays revealed that EGF-stimulated Rac1 activity was significantly suppressed in Rab5C-suppressed cells. To determine whether events upstream of Rac activation were affected by Rab5C, we observed that EGF-stimulated Akt phosphorylation was suppressed in cells depleted of Rab5C. Finally, since spatio-temporal assembly/disassembly of adhesion complexes are essential components of cell migration, we examined the effect of Rab5 isoform depletion on the formation of focal adhesion complexes. Rab5C-depleted HeLa cells have significantly fewer focal adhesion foci, in accordance with the lack of persistent lamellipodial protrusions and reduced directional migration. We conclude that Rab5 isoforms selectively oversee the multiple signaling and trafficking events associated with the endocytic network.</p></div

Rab5C depletion significantly inhibits cell migration.

<p>A) DIC images of stable Rab5 isoform knock-down (KD) HeLa cells taken with light microscope at 40X magnification (left panel). Arrows indicate membrane ruffles. KD of Rab5 isoforms (right panel) in these stable cell lines is shown in the immunoblots following SDS-PAGE as described in Experimental Procedures. B) 0.5–1 mm width wounds were made on a monolayer of HeLa stable control or Rab5 isoform KD cells. 5–7 wounded spots in each dish were imaged with time-lapse microscope every 5 minutes for 20 hours. C) The percentage of wound closure (left panel) was calculated from images acquired at time 0 and 16 hours with ImageJ. For each sample, at least 5 images were used to calculate the percentage of wound closure in each experiment. The graph represents the Mean± S.E. from four independent experiments. U937 cells (right panel) transiently transfected with siRNA against Rab5 isoforms were seeded in the upper chamber of the Transwell plates and allowed to migrate towards 10% FBS in the bottom chamber for 24 hours. Migrated cells were measured as indicated in Material and Methods. Data are normalized to initial seeding cell numbers. The graph represents the Mean± S.E. from four independent experiments. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test.(*P<0.05, ***P<0.001)</p

Depletion of Rab5C reduces cell adhesion.

<p>A) HeLa cells were seeded on coverslips O/N and then transfected with GFP or Rab5 isoform siRNAs. The focal adhesion complex was visualized by immunostaining with vinculin antibody. The numbers of focal adhesion complexes were determined with ImageJ. The graph represents Mean± S.E. from 30 cells. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test. P<0.0001. B) HeLa cells transfected with GFP or Rab5 isoform siRNAs were re-suspended and re-plated on fibronectin-coated plates for indicated times. At the end of each time point, cell lysates were extracted and prepared for SDS-PAGE and Western bloting. The activation of focal adhesion kinase was determined with phospho-FAK antibody. Total levels of FAK were not determined. The data represents Mean± S.E. from three independent experiments. Analysis was carried out with a two-way ANOVA, Bonferroni’s post-test. P<0.05.</p

PI3K signaling in response to Rab5 isoform depletion.

<p>A) HeLa cells were transfected with GFP (as negative control) or Rab5 isoform-specific siRNA. 48 hours post-transfection, cells were starved and then stimulated with EGF for indicated times. Cell lysates were subjected to SDS-PAGE and probed with antibodies as indicated. Band intensity was quantified with AlphaEaseFc 4.0 software. Bars represent the mean value ± S.E. from four independent experiments. Analysis was carried out with a two-way ANOVA, Bonferroni’s post-test. P<0.05. B) HeLa cells were transfected with indicated siRNA. 48 hours post-transfection, cells were seeded onto micropatterned coverslips coated with fibronectin, and then allowed to spread out for 2 hours in starvation medium. Starved cells were stimulated with 10 % FCS for 3 minutes and then fixed for PIP₃-FITC antibody immuno-staining. Images shown here are average projections of PIP₃ staining from 30–35 cells.</p

Loss of Rab5C reduced translocation of Rac1 to cell periphery and Rac1 activation in response to EGF stimulation.

<p>A) HeLa cells stably expressing GFP-Rac1 were transfected with scrambled or Rab5 isoform siRNAs. 48 hours post-transfection, cells were seeded onto coverslips imprinted with crossbow micro-patterns. 3-D GFP-Rac1 images of at least 40 micro-patterned cells were acquired for each sample. Each GFP-Rac1 3-D image stack was subjected to Maximum intensity projection and then grey scale normalization. Next, the max projections of 88 GFP-Rac1 images (from two independent experiments) were aligned and averaged (upper panel, in pseudo-color). Image subtraction was carried out between averaged image of scramble control and that of individual Rab5 isoform siRNA-treated sample. The resulting image after subtraction (siScr minus siRab5) is shown in pseudo-color (bottom panel). B) HeLa cells stably expressing GFP-Rac1 were transfected with scrambled or Rab5 isoform siRNAs. 48 hours post-transfection, cells were separated into membrane (Mem) and cytosolic (Cyt) fractions as described in Material and Method. Relative amounts of GFP-Rac1 in each fraction were analyzed by SDS-PAGE and Western blot. Densitometry of the bands was quantified using AlphaEaseFC 4.0 software. The numbers represent the ratio of GFP-Rac1 in cytosol or membrane/total. C) HeLa cells were transfected with scrambled or Rab5 isoform-specific siRNA. 48 hours post-transfection, cells were starved and then stimulated with EGF for two minutes. Cell lysates were prepared and subjected to Rac1 pull-down assays. Proteins were eluted, separated by SDS-PAGE and blotted for Rac. Total lysates were also probed for Rac1 to determine the total Rac1 level is equal in all samples. The intensity of the bands from western blots was quantified with AlphaEaseFc 4.0 software. The relative amount of Rac-GTP from pull-downs was normalized to that of total Rac1 from total cell lysates. The adjacent graph represents the mean value ± S.E. from four independent experiments. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test. (*P<0.05, **P<0.01)</p

Interactions between expressed Fbw8, CUL7 and TBC1D3.

<p>HeLa cells were transfected with HA-CUL7, Flag-TBC1D3 and Myc-Fbw8 constructs. At 18 h after transfection, cell lysates (200 µg) were immunoprecipitated with anti-TBC1D3 (2C7) (left panel) or anti-Myc (right panel) antibodies, separated by SDS-PAGE and immunoblotted as indicated.</p

CUL7-mediated TBC1D3 degradation requires a CUL7-associated Fbw8.

<p>HeLa cells were transiently transfected with Myc-TBC1D3 along with mock, full-length CUL7 (1–1698) or truncated CUL7-mutant (268–1698) that is unable to recruit Fbw8. The cells were starved and stimulated with 10% FCS for different times. Lysates were resolved by SDS-PAGE at the indicated times. Immunoblotting was carried out with anti-Myc, -tubulin and -HA antibodies (left panel). Right panel shows mean ± SE of 3 independent experiments. TBC1D3 expression is normalized by tubulin. The initial level of TBC1D3 expression in each group is set to 100%.</p

CUL7 siRNA silencing delays TBC1D3 degradation.

<p>(A) HeLa cells were transfected with scramble siRNA or CUL7 siRNA. At 24 h post-transfection, Myc-TBC1D3 was expressed in the cells for another 18 h. The cells were then starved for 3 h and stimulated with 10% FCS for different time periods. Cell lysates were prepared, separated by SDS-PAGE and immunoblotted with anti- TBC1D3, CUL7 and tubulin antibodies. (B) Summary of results. Mean ± SE of 3 independent experiments. TBC1D3 expression is normalized by tubulin. The initial level of TBC1D3 expression in each group is set to 100%.</p

CUL7 mediates the degradation and ubiquitination of TBC1D3.

<p>(A) CUL7 increased TBC1D3 ubiquitination. HeLa cells were transfected with Myc-TBC1D3 and with or without HA-CUL7. TBC1D3 was immunoprecipitated with anti-Myc antibody. Polyubiquitination of TBC1D3 was analyzed by immunoblotting, using anti-ubiquitin antibody. (B) CUL7 expression enhances TBC1D3 degradation. HeLa cells were transfected with Myc-TBC1D3 and with or without HA-CUL7. Cells were starved for 3 h and stimulated with 10% FCS. Lysates were separated by SDS-PAGE and immunoblotted with anti- TBC1D3, CUL7 and tubulin antibodies. (C) Summary of results. Mean ± SE of 3 independent experiments. TBC1D3 expression is normalized by tubulin. The initial level of TBC1D3 expression in each group is set to 100%.</p

TBC1D3 interacts with Fbw8 in a phosphorylation-dependent manner.

<p>(A) <i>In vitro</i> interaction between TBC1D3 and Fbw8. Separate sets of HeLa cells were transfected with GFP-TBC1D3 or Myc-Fbw8. At 18 h after transfection, the GFP-TBC1D3-expressing cells were starved and stimulated with 10% FCS. To prepare TBC1D3-beads, GFP-TBC1D3 was immunoprecipitated with mouse anti-GFP antibody on Protein G beads. The TBC1D3-beads were treated with or without alkaline phosphatase (AP) for 1 h at 37°C. TBC1D3-beads were incubated with Fbw8-cytosol prepared from HeLa cells expressing Myc-Fbw8. Following incubation at 4°C, the beads were washed and the bound proteins were separated and analyzed by immunoblot using anti-Myc antibody. TBC1D3-beads pulled down Fbw8 and alkaline phosphatase treatment blocked TBC1D3-Fbw8 interaction. (B) <i>In vivo</i> interactions between TBC1D3 and Fbw8. HeLa cells were co-transfected with GFP-TBC1D3 and Myc-Fbw8. After 18 h, the cells were starved and stimulated with 10% FCS. Cell lysates were treated with and without AP and immunoprecipitated with anti-GFP antibody. The precipitates were separated by SDS-PAGE followed by immunoblot analysis. (C) GST-Fbw8-Skp1 pull-down of TBC1D3 is phosphorylation dependent. Glutathione beads with bound GST-Fbw8-Skp1 complex or GST alone were incubated with GFP-TBC1D3 expressing lysates, treated with or without AP for 1 hour at 37°C. The beads were washed and eluted proteins were separated by SDS-PAGE. Immunoblotting with monoclonal anti-TBC1D3 antibody (2C7) showed that GST-Fbw8-Skp1 pull-down of TBC1D3 was nearly abolished by prior alkaline phosphatase treatment (right panel). The right panel shows the Ponceau S staining of the transferred membrane. The experiments were repeated three times.</p