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Fig. S1. Rab8 regulates transferrin receptor localization. HeLa cell were treated with control (A,B,C,D) or Rab8 (E,F) RNAis for 48 hours. The cells were fixed, permeabilized and stained with anti-Rab8 (B,D,F) and anti-transferrin receptor (A,C,E). In control cells the transferrin receptor (Tfn-R) is frequently localized to the pericentriolar region and to cell surface protrusion together with Rab8 (A,B,C,D), whereas Tfn-R localized randomly throughout the cytoplasm in Rab8-depleted cells (E,F). Cell surface distribution of Tfn-R in control (G) and Rab8-depleted (H) cells. Note the differences in cell shape and cell-cell contact. Quantitative analysis of Tfn-R localized to the pericentriolar region (I) and protrusion (J). A total of about 60 cells were counted per experimental condition. Values, given as percentage of cells exhibiting pericentriolar Tfn-R in each scoring category, from three separate experiments are shown as the mean ± s.e.m. Bars, 20 μm.
Fig. S2. Localization of ectopically expressed Rab8 in fibrosarcoma cells. HT1080 cells were transiently transfected with pIRES-Rab8-T22N (C) or pIRES-Rab8-Q67L (A,B), and analyzed for myc-Rab8 and β1 integrin co-localization by confocal microscopy (merge). Rab8-Q67L co-localizes with β1 integrins in tubular structures entering leading lamella, and on vesicular structures in the perinuclear region (arrows). Rab8-T22N expressing cells strongly accumulate β1 integrins in large vacuoles (arrowheads), but β1 integrins show little co-localization with Rab8-T22N (C) in these asymmetric cells. Bars, 5 μm.
Fig. S3. Co-localization of different markers with Rab8. HeLa cell were transfected with constructs encoding either Rab8-Q67L (A-C) or Rab8-T22N (D-F). Next day the cells were processed for immunofluorescence microscopy and double stained for Rab8 and Tfn-R. Note that Rab8-Q67L promotes localization of Tfn-R together with Rab8 in perinuclear structures, while Tfn-R is localized in the periphery in the presence of Rab8-T22N. HeLa cells were incubated with anti-MHCI antibodies (G-I) for 1 hour, fixed, stained for Rab8. MHCI-specific antibodies are clearly present in Rab8-specific tubular structures. HeLa cells were also incubated with Alexa594-CTxB for 15 minutes (J-L), and then stained for Rab8. Some vesicles and tubules were positive for both CTxB and Rab8 (inserts). Bars, 20 μm.
Fig. S4. Expression of activated RhoA, and depolymerization of microtubules affects Rab8 distribution. Sparsely grown HeLa cells were transfected with either pEGFP-C1 (A, B), pEGFP-RhoA-T19N (C, D) or pEGFP-RhoA-G14V (E, F). One day later they were fixed with paraformaldehyde, permeabilized, and stained with anti-Rab8 antibody (B, D). Rab8 distribution was normal in GFP and GFP-RhoA-T19N cells with Rab8 in cell protrusions (arrow heads), whereas RhoA-G14V promoted redistribution of Rab8 into the perinuclear region (arrows). The percentage of cells showing Rab8 in cell protrusions was calculated for cells expressing GFP, GFP-RhoA-T19N and GFP-RhoA-G14V (I). Values from three separate experiments are shown as mean ± s.e.m. 60 cells were counted per category and experiment. HeLa cells were pre-incubated for 30 minutes in control medium (G) or medium containing 2 μg/ml nocodazole (H). The cells obtained then 0.1 μM cytochalasin D for an additional 30 minutes. The cells were fixed and stained with anti-Rab8 to detect endogenous Rab8. Note that control cells (−Noc) contain prominent Rab8-positive tubular structures (arrow heads), whereas in the presence of nocodazole (+Noc) Rab8 is translocated to a perinuclear region (arrows) and tubular structures are absent. Bars, 20 μm.
Fig. S5. Expression of JFC1 affects the formation of Rab8-specific tubules. HeLa cells expressing GFP (A,) or GFP-JFC1 (C,D) were treated with cytochalasin D for 30 minutes. In GFP-expressing cells cytochalasin D induced Rab8 tubules (A,B), whereas in GFP-JFC1-expressing cells Rab8 tubules were replaced by vesicles and vacuoles (C,D). Quantitative analysis of Rab8 tubule formation in GFP and GFP-JFC1-expressing cells after cytochalasin D treatment (I). A total of about 50 cells were counted per experimental condition. Values are given as percentage of cells exhibiting Rab8 tubules and shown as the mean ± s.e.m. Increased Rab8 association with vesicles and tubules in GFP-JFC1-expressing cells (G,H) compared with GFP-expressing cells (E,F) was observed. Intensity of Rab8 fluorescence on vesicles and tubules was quantified by TINA software from six vesicles or tubules per cell from ten different cells (J). Values given are as OD/mm2/vesicle and shown as the mean ± s.e.m. Bars, 20 μm (A-D), bars 5 μm (E-H).
Movie 1. Macropinosome formation and internalization at the leading edge of motile HT1080 cells expressing GFP-Rab8-Q67L (arrow). Dynamic movement of tubular structures (arrowhead). Note the formation of both filopodia and lamellipodia, which is typical for GFP-Rab8-Q67L expressing cells. Total time sequence is 16 minutes. Time-lapse images were taken every 5 seconds, and shown at 15 frames/second. Corresponds to still images in Fig. 6A.
Movie 2. Dynamic membrane traffic of GFP-Rab8b-wt vesicles and tubules in a NIH3T3. Arrow heads indicate ruffling area, and arrows indicate vesicular and tubular structures. Also note a rapid traffic of smaller vesicles towards the cell periphery. Total time sequence represents 16 minutes. Time-lapse images were taken at 5-second intervals, and is shown at 15 frames/second. The video clip corresponds to still images in Fig. 6B.
Movie 3. Membrane recycling at the leading edge. Ruffling membrane (arrow head) is internalized, transported to a sorting vacuole (arrow) and recycled back to the leading edge. Time-lapse images were taken at 5-second intervals, and is shown at 15 frames/second. The video corresponds to still images in Fig. 6C.
Movie 4. Formation of a GFP-Rab8b-wt tubular network after administration of cytochalasin D in NIH3T3 cells. The tubules are formed at the cell periphery (arrow), and accumulate in the perinuclear region (arrowhead). Total time sequence is 30 minutes. Time-lapse images were taken every 15 seconds, and shown at 15 frames/second. The video corresponds to still images in Fig. 7E,F.
Movie 5. Dynamic formation of GFP-Rab8-wt tubules in HeLa cells after cytochalasin D treatment (arrows). Total time sequence is 30 minutes. Time-lapse images were taken every 10 seconds, and shown at 15 frames/second. The video corresponds to still images in Fig. 7G,H.
Movie 6. Bidirectional trafficking of GFP-Rab8-wt vesicles and tubules at the cell periphery. Formation of vesicles and tubules at the plasma membrane, and their transport to the cell center (arrow). Anterograde traffic of small vesicles to the cell periphery, and into forming and retracting filopodia (arrowhead). Total time sequence is 30 minutes. Time-lapse images were taken every 10 seconds, and shown at 15 frames/second. The video corresponds to still images in Fig. 7I,J.
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