Arfaptin2 binds preferentially to membrane tubes connected to GUVs when Arf1 is in its active GTP-bound state.

<p>Membrane tube networks were pulled from Golgi-mix GUVs containing 1% red fluorescent lipid BodTRCer and 1% biotinylated lipid Biot-CAP-PE by a truncated biotinylated version of kinesin1. Tube networks (red panel) were incubated with (A) 1 µM Arfaptin2-Alexa⁴⁸⁸, (B) 1 µM Arfaptin2-Alexa⁴⁸⁸ and 1 µM Arf1:GDP, or (C) 1 µM Arfaptin2-Alexa⁴⁸⁸ and 1 µM Arf1:GTPγS. Arrows ending with a triangle point to highly curved membrane regions (tubes) where Arfaptin2 is bound. Arrows ending with a circle point to weakly curved membrane regions (vesicles) where Arfaptin2 binding is not detected. Scale bar: 10 µm.</p

The binding of Arfaptin2 to liposomes requires Arf1 activation and increases with membrane curvature.

<p>A. Flotation assay. Arfaptin2 and Arf1:GDP (left) or Arf1:GTP (right) (0.75 µM) were co-incubated with Golgi-mix liposomes (0.75 mM lipids) extruded through filters with pores of decreasing sizes (200 nm, 100 nm, 50 nm and 30 nm). The suspension was adjusted to 30% w/v sucrose and overlaid with two cushions of decreasing sucrose density. After centrifugation, the top (liposomes) and bottom (unbound proteins) fractions were collected and analysed by SDS-PAGE. Direct Alexa⁴⁸⁸ fluorescence (top panels) was observed before Sypro-orange staining (bottom panels). B. Protein quantification from the Sypro-orange stained gels shown in A. The black bars correspond to Arfaptin2 and the white/black striped bars to Arf1. C. FRET assay. Arfaptin2-Alexa⁴⁸⁸ (0.5 µM) and myr-Arf1-H6 were mixed with Golgi-mix liposomes of various sizes containing 1 mol% TRITC-DHPE. At the indicated times, GTP (0.1 mM), EDTA (2 mM), MgCl2 (2 mM) and ArfGAP1 (10 nM) were added. Alexa⁴⁸⁸ fluorescence was followed in real time. Arfaptin2 was recruited to the membranes after Arf1 activation (GTP). Arfaptin2 recruitment increased as a function of membrane curvature.</p

Arfaptin2 induces membrane tubulation at high concentration.

<p>Golgi-mix GUVs containing 1% of the red fluorescent lipid BodTRCer were incubated with (A) 3 µM Arfaptin2-Alexa⁴⁸⁸ and 3 µM Arf1:GDP or (B) 3 µM Arfaptin2-Alexa⁴⁸⁸ and 3 µM Arf1:GTPγS. Scale bar: 10 µm.</p

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

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

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

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

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

Several deregulated genes have their expression correlated with the expression of <i>MKI67</i>, a proliferation marker gene.

<p>The Pearson correlation coefficient (r) between the expression of the deregulated genes and the expression of proliferation marker gene, <i>MKI67</i>, was calculated for <i>FGFR3</i>-mutated tumors in the TaG1G2 group (n = 28) and for <i>FGFR3</i>-non-mutated tumors in the T2–4 group (n = 63). The expression of the deregulated genes as a function of <i>MKI67</i> expression is shown in TaG1G2 <i>FGFR3</i>-mutated tumors (upper figures) and in T2–4 non-mutated tumors (lower figures). Only the plots for the correlated genes are presented (p<1%, which corresponds to a correlation coefficient, |r| above 0.479 for the <i>FGFR3</i>-mutated tumor group and above 0.323 for the <i>FGFR3</i>-non-mutated tumor group).</p

Flow chart of the different analysis steps.

<p>The first step is the identification through public data bases and expert knowledge of the genes of interest to study, here the Rabs and their effectors. The second step consists of selecting subgroups of tumors and analysing the expression of the different genes selected in the first step in these subgroups compared to the normal urothelium. The subgrouping here has been done taking into account the <i>FGFR3</i> mutation status, the stage and the grade, separating the tumors into two pathways. A comparison of the expression observed in bladder cancer cell lines and in cultured normal human urothelial cells allowed discarding of genes for which the expression could be possibly due to the presence of stroma (in comparison to normal cells, upregulation in bladder tumors but not in bladder tumor cell lines). Different types of analysis were then performed on the selected deregulated genes: 1) a comparison of the expression in <i>FGFR3</i>-mutated tumors and <i>FGFR3</i>-non-mutated tumors allowed the identification of genes specifically deregulated in one of the two pathways of bladder cancer pathogenesis; 2) by grouping genes into cluster of genes (here the Rab clusters), we identified clusters with deregulated expression; 3) by analysing the possible correlation between the expression of the deregulated genes and the expression of proliferation or differentiation marker genes, we identified the deregulated genes associated with proliferation or differentiation.</p