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First published online 8 December 2005
doi: 10.1242/jcs.02720

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Activation of Rac1 by RhoG regulates cell migration

Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

View larger version (36K): [in a new window]   Fig. 1. RhoG knockdown in HeLa cells. (A) Cell lysates from HEK 293T cells transiently transfected with the indicated plasmids were analyzed by immunoblotting with anti-Myc (left) and anti-Rac1 (right) antibodies. (B) Cell lysates from stable HeLa cell clones expressing pSilencer-RhoGa (RhoG RNAi) and pSilencer-RhoGb (Control RNAi), or cell lysate from parental HeLa cells were analyzed by immunoblotting with anti-Rac1 (top), anti-Dock180 (middle) and anti-Crk (bottom) antibodies. (C) The levels of endogenous RhoG and ELMO2 mRNAs in parental, control RNAi and RhoG-RNAi HeLa cells were shown by semiquantitative RT-PCR.

View larger version (55K): [in a new window]   Fig. 2. Knockdown of RhoG suppresses cell spreading. (A) Parental, control RNAi and RhoG RNAi HeLa cells were plated on fibronectin-coated coverslips for 15 minutes, and cells were examined by phase contrast (left panels), or fixed and stained with Alexa Fluor 594-conjugated phalloidin to visualize F-actin (right panels). Bars, 50 µm (left) and 20 µm (right). (B) Quantitative analyses of cell spreading. Cells were plated on fibronectin-coated coverslips for 15 minutes. Average cell area was calculated from the phase contrast images of nine randomly selected fields (top). The number of cells that had fully spread (cell area >1000 µm2) was expressed as a percentage of the total number of cells (bottom). About 100 cells were assessed in one experiment, and data are the means ± s.e.m. of three independent experiments. (C) Control RNAi and RhoG RNAi HeLa cells were plated onto fibronectin-coated dishes, and they were examined by phase contrast at 15, 30 and 60 minutes after plating. Bar, 20 µm. (D) Cell lysates from HEK 293T cells transiently transfected with the indicated plasmids were analyzed by immunoblotting with anti-GFP antibody: GFP-hRhoG, GFP-tagged human RhoG; GFP-mRhoG, GFP-tagged mouse RhoG. (E) RhoG RNAi HeLa cells transiently transfected with GFP or GFP-tagged mouse RhoG (GFP-mRhoG) were plated on fibronectin-coated coverslips. At 15 minutes after plating, they were fixed and stained with Alexa Fluor 594-conjugated phalloidin to visualize F-actin (left panels). The transfected cells were shown by the fluorescence of GFP (right panels). Bar, 20 µm.

View larger version (57K): [in a new window]   Fig. 3. Knockdown of RhoG reduces cell migration. (A) Motility of parental, control RNAi and RhoG RNAi HeLa cells was evaluated by Transwell migration assays. Cells were plated in the upper chamber of the filters that had been coated with fibronectin on the underside. At 4 hours after plating, cells that had migrated to the underside of the filters were fixed and stained with crystal violet. Bar, 200 µm. Relative cell migration was determined by the number of the migrated cells normalized to the total number of the cells adhering to fibronectin, and the value from parental cells was arbitrarily set at 100%. (B) Control RNAi and RhoG RNAi HeLa cells were transiently transfected with GFP or GFP-tagged mouse RhoG (GFP-mRhoG), and motility of the transfected cells was evaluated by Transwell migration assays. Relative cell migration was determined by the number of GFP-positive cells that had migrated to the underside of the filter normalized to the total number of GFP-positive cells adhering to fibronectin, and the value from control RNAi HeLa cells transfected with GFP was arbitrarily set at 100%. Expression of GFP and GFP-tagged mouse RhoG was determined by immunoblotting with anti-GFP antibody (bottom). (C) Parental HeLa cells were transiently transfected with GFP or GFP-tagged N-terminal region of ELMO (GFP-ELMO-NT), and motility of the transfected cells was evaluated by Transwell migration assays. For relative cell migration, the value from GFP-transfected cells was arbitrarily set at 100%. Expression of GFP and GFP-ELMO-NT was determined by immunoblotting with anti-GFP antibody (bottom). (D) Control RNAi and RhoG RNAi HeLa cells were transiently transfected with GFP alone or cotransfected with GFP, Flag-tagged Dock180 (Flag-Dock180) and HA-tagged ELMO (HA-ELMO), and motility of the transfected cells was evaluated by Transwell migration assays. Relative cell migration was determined by the number of the GFP-positive cells that had migrated to the underside of the filter normalized to the total number of GFP-positive cells adhering to fibronectin, and the value from control RNAi HeLa cells transfected with GFP alone was arbitrarily set at 100%. Expression of Flag-Dock180 and HA-ELMO were determined by immunoblotting with anti-Flag and anti-HA antibodies (bottom). For each experiment, the number of migrated cells was counted from the images of nine randomly selected fields on the underside of the filter. About 100 cells were assessed in one experiment, and data are the means ± s.e.m. of three independent experiments.

View larger version (78K): [in a new window]   Fig. 4. Migration-induced activation of Rac1 is reduced in RhoG RNAi HeLa cells. (A) Phase contrast images of wound-healing assays of parental, control RNAi and RhoG RNAi HeLa cells at 0 and 9 hours after wounding. Cells were plated in complete medium at a confluent density and scratched with a micropipette tip. Average rates of wound closure were calculated from three independent experiments. Bar, 300 µm. (B) Cells were fixed 2 hours after wounding and stained with Alexa Fluor 594-conjugated phalloidin to examine cell morphology at the wound edge. Bar, 20 µm. Cells extending lamellipodia into the open wound area were scored as a percentage of the total number of cells at the wound edge, and data are the means ± s.e.m. of three independent experiments. (C) RhoG RNAi HeLa cells transiently transfected with GFP or GFP-tagged mouse RhoG (GFP-mRhoG) were fixed 2 hours after wounding and stained with Alexa Fluor 594-conjugated phalloidin to examine cell morphology at the wound edge. Bar, 20 µm. (D) Multiple scratch wounds were made in confluent cells, and GTP-bound Rac1 in the cells at indicated times after wounding were precipitated with the GST-fused CRIB domain of Pak. The amounts of GTP-bound Rac1 and total Rac1 were detected by immunoblotting with anti-Rac1 antibody. Relative Rac1 activity was determined by the amount of GTP-bound Rac1 bound to GST-CRIB normalized to the amount of Rac1 in cell lysates analyzed by NIH Image software, and the value from control RNAi HeLa cells at 0 minute after wounding was arbitrarily set at 1. Data are the means ± s.e.m. of three independent experiments.

View larger version (33K): [in a new window]   Fig. 5. RhoG promotes cell migration through ELMO and Dock180. (A) Motility of HEK 293T cells transiently transfected with the indicated plasmids was evaluated by Transwell migration assays. Cells were plated in the upper chamber of the filters that had been coated with fibronectin on the underside. At 6 hours after plating, cells that had migrated to the underside of the filters were fixed, and transfected cells were shown by the fluorescence of GFP. Bar, 200 µm. Expression of Myc-tagged RhoG-V12 and RhoG-A37 was determined by immunoblotting with anti-Myc antibody. (B) HEK 293T cells were transiently transfected with the indicated plasmids, and motility of the transfected cells was evaluated by Transwell migration assays. Expression of Flag-tagged Dock180, HA-tagged ELMO and Myc-tagged RhoG was determined by immunoblotting with antibodies against Flag, HA and Myc, respectively. Relative cell migration was determined by the number of GFP-positive cells that had migrated to the underside of the filter normalized to the total number of GFP-positive cells adhering to fibronectin, and the value from cells transfected with GFP alone was arbitrarily set at 100%. For each experiment, the number of migrated cells was counted from the images of nine randomly selected fields on the underside of the filter. About 100 cells were assessed in one experiment, and data are the means ± s.e.m. of three independent experiments.

View larger version (36K): [in a new window]   Fig. 6. Interaction of Dock180 with Crk is not required for Dock180-dependent Rac1 activation by RhoG. (A) The Dock180 constructs used in this study (Docker, Dock180 homology domain involved in exchange on Rac; Pro, proline-rich region; ISP, specific amino acid residues required for the activation of Rac within Docker domain; numbers indicate amino acid position within the sequence). (B) Flag-tagged Dock180 or Flag-tagged Dock180-T1657 expressed in HEK 293T cells was used in a pull-down assay with GST or GST-fused CrkII. Bound proteins and total cell lysates were analyzed by immunoblotting with anti-Flag antibody. (C) Lysates from HEK 293T cells transfected with the indicated plasmids were immunoprecipitated with anti-Myc antibody, and bound proteins and total cell lysates were analyzed by immunoblotting with antibodies against Flag, HA and Myc. (D) Cellular homogenates from HEK 293T cells transfected with the indicated plasmids were separated into cytosolic (C) and membrane (M) fractions, and were then analyzed by immunoblotting. (E) Cell lysates from HEK 293T cells transfected with the indicated plasmids were incubated with GST-CRIB, and bound Rac1 was detected with anti-Rac1 antibody. Relative Rac1 activity was determined by the amount of GTP-bound Rac1 bound to GST-CRIB normalized to the amount of Rac1 in cell lysates analyzed by NIH Image software, and the value from untransfected cells was arbitrarily set at 1. Data are the means ± s.e.m. of three independent experiments. WT, wild-type.

View larger version (31K): [in a new window]   Fig. 7. The interaction of Dock180 with Crk is not required for the RhoG-induced promotion of cell migration. Motility of HEK 293T cells transiently transfected with the indicated plasmids was evaluated by Transwell migration assays. Cells were plated in the upper chamber of the filters that had been coated with fibronectin on the underside. At 6 hours after plating, cells that had migrated to the underside of the filters were fixed, and GFP-positive cells were counted. Relative cell migration was determined by the number of the GFP-positive cells that had migrated to the underside of the filter normalized to the total number of the GFP-positive cells adhering to fibronectin, and the value from cells transfected with GFP alone was arbitrarily set at 100%. (A,B) Expression of Myc-tagged RhoG, Flag-tagged Dock180 and CrkII was determined by immunoblotting with antibodies against Myc, Flag and Crk, respectively. For each experiment, the number of migrated cells was counted from the images of nine randomly selected fields on the underside of the filter. About 100 cells were assessed in one experiment, and data are the means ± s.e.m. of three independent experiments.

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