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First published online 14 November 2006
doi: 10.1242/jcs.03275

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Characterization of the Rab8-specific membrane traffic route linked to protrusion formation

¹ Institute of Biotechnology, PO Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland
² Molecular Cancer Biology Research Program, Biomedicum Helsinki, P.O. Box 63 (Haartmaninkatu 8), FIN-00014 University of Helsinki, Finland

View larger version (76K): [in this window] [in a new window]   Fig. 1. Rab8 modulates cell shape changes. (A) A control stable cell line expressing GFP (green fluorescent protein) form semi-ordered contacts, while (B) a stable cell line expressing Rab8-Q67L (pEGFP-Rab8-Q67L) have lost contact inhibition and (C) a stable cell line expressing Rab8-T22N (pEGFP-Rab8-T22N) shows close cell-cell contacts. (D) Expression of EGFP-Rab8-T22N, EGFP-RabQ67L (GFP-Rab8) and endogenous Rab8 (Rab8) in these stable cell lines was monitored by western blotting. (E-K) Depletion of Rab8 from HT1080 fibrosarcoma cells by RNAi. HT1080 cells were transfected with control RNAi (E,G,I) or with Rab8-specific RNAi (F,H,J). Note that Rab8 depletion promotes cell-cell adhesion (F,H). Arrows indicated cells with lamellar structures (I), whereas cells containing actin stress fibers are indicated by arrowheads (J). (K) Total lysates from cells transfected with control or Rab8-specific RNAi were analyzed by western blotting by anti-actin and anti-Rab8 antibodies. Bars, 200 µM (A-C,E-H), 20 µM (I,J).

View larger version (116K): [in this window] [in a new window]   Fig. 2. Rab8 colocalizes with Rab11, but not with Rab4 or Rab5. HT1080 cells expressing EGFP-Rab4, EGFP-Rab5 and EGFP-Rab11 (A-C; green) and endogenous Rab8 (red) were analyzed by confocal microscopy (merge pictures). Endogenous Rab8 (D,G) in HeLa cells expressing EGFP-Rab11 (E,H) colocalizes sometimes on vesicular structures (arrows). F and I are merge pictures. Arrowheads indicate Rab8-positive vacuoles, vesicles and tubules at the leading edge. A HT1080 cell expressing GFP-Rab11 (K) and myc-Rab8-Q67L (J) show a prominent protrusion with Rab8 on tubules (arrow), the plasma membrane and in ruffles (arrowhead). Colocalization is seen on vesicles (L). Bars, 20 µM (A-D,J), 5 µM (G).

View larger version (76K): [in this window] [in a new window]   Fig. 3. A role in transferrin recycling for Rab8. Hela cells were transfected with control (A,B,E,F) or Rab8-specific (C,D,G,H) RNAis for 48 hours. Alexa-transferrin was applied to serum starved cells for 30 minutes at 37° (Pulse) and then chased for 30 minutes at 37°C. Note that transferrin (Tfn) is localized to the perinuclear region in Rab8 expressing cells (A), but randomly in Rab8 depleted cells (C). (I) Total lysates from HeLa cells transfected with control or Rab8-specific RNAis were analyzed by western blotting by anti-actin and anti-Rab8 antibodies. Bars, 20 µM.

View larger version (59K): [in this window] [in a new window]   Fig. 4. Rab8 affects cholera toxin B trafficking. Hela cell were treated with control (A,C,E) or Rab8-specific (B,D,F) RNAis for 48 hours. Alexa-595 cholera toxin were bound to the cells on ice for 30 minutes, and then CTxB was allowed to progress to the Golgi apparatus for 30 minutes at 37°C. The cells were fixed and stained for anti-GM130 (C,D) and anti-Rab8 (E,F). The percentage of cells showing CTxB-staining in Golgi was calculated for the cells treated with control RNAi contra Rab8-specific RNAi (G). Values from three separate experiments are shown as the mean ±s.e.m. 60 cells were counted per category and experiment. Bars, 20 µM.

View larger version (75K): [in this window] [in a new window]   Fig. 5. Rab8 localizes to a tubular compartment. Confocal localization of endogenous Rab8 with ß1 integrin and transferrin receptor in three different human cell lines; HeLa carcinoma (A), HT1080 fibrosarcoma (B), and Paju neuroblastoma cells (C). HeLa cells contained Rab8-specific tubular structures that were negative for the transferrin receptor (A). In the HT1080 cells, Rab8 colocalized with ß1 integrins on vacuolar structures in the leading lamella (arrow), and sometimes also in the tail region (arrowhead) (B). Paju cells showed prominent Rab8-specific tubules that were positive for ß1 integrins (C). Bars, 10 µm.

View larger version (156K): [in this window] [in a new window]   Fig. 6. Rab8-specific membrane turnover at the leading edge. (A) HT1080 cells expressing GFP-Rab8-67L vesicles/vacuoles (arrows) are formed at the leading edge and move toward the cell center. Dynamic tubular structures (arrowheads) are also seen to attach to and retract from the plasma membrane (supplementary material Movie 1). (B) In stably GFP-Rab8b-wt expressing NIH3T3 cells, tubular membrane structures containing GFP-Rab8b-wt are seen forming behind active ruffling lamellipodia (arrowhead), and the tubules are transported to a perinuclear region (arrows) (supplementary material Movie 2). (C) An example of membrane turnover at the leading edge of NIH3T3. GFP-Rab8b-wt positive membranes taken in from a ruffling area (arrow) fuse with a sorting vacuole (arrowhead) that turn into a tubular structure that fuses with the plasma membrane behind the lamellipodia (supplementary material Movie 3). Bars, 10 µM.

View larger version (40K): [in this window] [in a new window]   Fig. 7. The formation of Rab8-specific tubular structures is actin- and RhoA-dependent. HeLa cells were transfected overnight with plasmids containing either pEGFP-RhoA-T19N (A,B) or pEGFP-RhoA-G14V (C,D) at 37°C. The transfectants were then incubated in 0.1 µM cytochalasin D (CD) for 30 minutes at 37°C. The cells were fixed, processed for immunofluorescence and stained to detect endogenous Rab8. RhoA proteins were detected through GFP fluorescence. Note that expression of RhoA-T19N did not inhibit CD induced formation of Rab8-specific tubules, whereas RhoA-G14V did (arrow). (K) Quantification of cells containing Rab8-specific tubular structures after CD-treatment, in the absence or presence of GFP-RhoA-G14V and GFP-RhoA-T19N. CD indicates untransfected cells that obtained cytochalasin D. C indicates cells in the absence of CD. A total of about 50 cells were counted per experimental condition. Values, given as percentage of cells (n=50) exhibiting Rab8-specific tubules in each scoring category, from three separate experiments are shown as the mean ±s.e.m. Formation of GFP-Rab8b-wt (NIH3T3; E,F) and GFP-Rab8-wt (Hela; G-J) tubular structures (arrows) were seen after cytochalasin D treatment (30 minutes) (supplementary material Movies 4-6). GFP-Rab8 and GFP-Rab8b vesicles formed at the cell periphery (arrows) and fused to a tubular network in the cell center (arrowhead). There is also an anterograde transport of small Rab8-specific vesicles to the outermost region of the cell, and into forming and retracting filopodia (arrowhead) (I and J; supplementary material Movie 6). Bars, 20 µM (A-F), 10 µM (G-J).

View larger version (50K): [in this window] [in a new window]   Fig. 8. Arf6 is linked to the formation of Rab8-tubules. HeLa cells were transfected with constructs expressing Arf6-wt (A,B), Arf6-T27N (C,D) or Arf6-Q67L (E,F). Next day the transfectants were incubated in 0.1 µM cytochalasin D (CD) for 30 minutes at 37°C. The cells were fixed, processed for immunofluorescence and stained to detect endogenous Rab8 and recombinant Arf6. Note the absence of tubules in the Arf6-Q67L expressing cell (arrow) (E,F). (G) Quantification of cells containing Rab8-specific tubular structures after CD-treatment, in the presence of Arf6-wt, Arf6-T22N and Arf6-Q67L were done. A total of about 50 cells were counted per experimental condition. Values, given as percentage of cells (n=50) exhibiting Rab8-specific tubules in each scoring category, from three separate experiments are shown as the mean ±s.e.m. (H) The specificity of the anti-Arf6 antibody was tested by immnuoblotting against mock-transfected and Arf6-Q67L transfected total cell extracts. Bars, 20 µM.

View larger version (34K): [in this window] [in a new window]   Fig. 9. (A) Arf6 and Rab8 co-operate in inducing cell protrusions. Co-expression studies of Rab8-Q67L and Arf6-wt, Rab8-Q67L and Arf6-T27N, and Rab8-Q67L and Arf6-Q67L in HT1080 cells. Confocal microscopy showed that Rab8-Q67L/Arf6-wt expressing cells contain cell extensions, with Arf6/Rab8-specific tubular structures entering ruffling lamella. Rab8-Q67L and Arf6-T27N colocalized on vesicular and tubular structures. Co-expression of Arf6-Q67L and Rab8-Q67L resulted in the formation of spherical cells, where Arf6-Q67L was localized on large vacuolar structures, and Rab8-Q67L in the perinuclear region. (B) Arf6 cross-talks with Rab8 in the formation of cell surface extensions. HT1080 cells were mock-transfected or transfected with a construct encoding Rab8-Q67L. Co-transfection was done with constructs expressing Arf6-wt/Rab8-Q67L, Arf6-T27N/Rab8-Q67L and Arf6-Q67L/Rab8-Q67L. Cells showing neurite-like extensions were calculated as extension-positive. A total of about 50 cells were counted per experimental condition. Values, given as percentage of cells exhibiting cell surface extension in each scoring category, from three separate experiments are shown as the mean ±s.e.m. Representative fields of these cells are seen in the lowest row of A. Bars, 10 µM, 20 µM (lower panel).

View larger version (35K): [in this window] [in a new window]   Fig. 10. JFC1 binds specifically to Rab8-GTP. (A) Table showing interactions of JFC1 in the two-hybrid system. Positive interaction (+) is indicated by growth in the absence of leucine and by enhanced ß-galactosidase activity using XGal as substrate. (B) Hela cells were transfected by a myc-JFC1 encoding construct, and then the cells were incubated with cytochalasin D for 30 minutes, and stained by anti-myc and anti-Rab8. Endogenous Rab8 colocalizes with myc-JFC1 on tubular structues. (C) In vitro translated Rab2, Rab8, Rab27a and laminin proteins were incubated with GST-JFC1 beads, and bound proteins were analyzed by SDS-PAGE. (D) Cell lysates from cells transfected with contructs encoding GFP-Rab8-Q67L, GFP-Rab8-T22N and mock control were incubated with GST-JFC. Bound proteins were eluted and analyzed by western blotting using anti-Rab8. Note that GST-JFC1 binds GFP-Rab8-Q67L and endogenous Rab8, but not GFP-Rab8-T22N. (E) In vivo binding of JFC1 to Rab8. The construct encoding myc-JFC1 was co-transfected with constructs encoding GST-Rab8-Q67L, GST-Rab8-T22N or GST. Cell lysates from these cells were bound to glutathione-Sepharose beads, and bound proteins were analyzed by westerm blotting with anti-JFC1 and anti-GST antibodies. Only GST-Rab8-Q67L bound JFC1 in vivo. Bar, 10 µM.