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First published online 27 May 2008
doi: 10.1242/jcs.025130

Journal of Cell Science 121, 1981-1989 (2008)
Published by The Company of Biologists 2008
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Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration

Julia Meller1, Luis Vidali2 and Martin Alexander Schwartz1,3,*

1 Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA
2 Biology Department, University of Massachusetts, Amherst, MA 01003, USA
3 Departments of Biomedical Engineering and Cell Biology, Cardiovascular Research Center and Mellon Prostate Cancer Institute, University of Virginia, Charlottesville, VA 22908, USA


Figure 1
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  		Fig. 1. DN mutants of RhoG and ELMO inhibit cell spreading. (A) MEFs expressing GFP-RhoG-T17N, GFP-ELMOT625 or GFP alone were plated on 10 µg/ml fibronectin-coated coverslips in serum-free medium for 25 minutes, fixed and stained with Alexa-Fluor-568-conjugated phalloidin. Phalloidin staining of representative GFP-positive cells is shown. Scale bar: 10 µm. (B) Spread area of cells was calculated from 15 randomly selected GFP-positive cells for each condition. Values are means ± s.e.m.

 

Figure 2
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  		Fig. 2. Characterization of anti-RhoG monoclonal antibody. Cell lysates expressing GFP-RhoG, GFP-Rac or GFP-Cdc42 were resolved by SDS-PAGE and western blots probed for RhoG, Rac or Cdc42.

 

Figure 3
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  		Fig. 3. Effects of RhoG, Dock180 and Rac knockdowns on cell spreading. (A) MEFs transiently transfected with pSuper knockdown vectors plus GFP (ratio 40:1) were suspended in serum-free medium for 30 minutes then replated on coverslips coated with 10 µg/ml fibronectin (FN). At the indicated times, cells were fixed and stained for F-actin. Upper panels: left, representative blot of RhoG and Rac knockdowns; right, Alexa-Fluor-568–phalloidin staining of GFP-positive cells replated on FN for 25 minutes. Scale bar: 10 µm. Lower panel: quantitation of cell spreading. Average spread area of GFP-positive cells from randomly selected fields was measured and normalized to the size of unspread cells. Values are means ± s.e.m.; n>30 cells from two separate experiments. (B) Left panel: quantification of cell area after RhoG, Dock180 or Rac1 knockdown at 15 minutes after replating; *P<0.01, **P<0.001 (Student's t-test). Right panel: representative blot of Dock180 knockdowns. (C) Left panel: HeLa cells were transiently transfected and replated on FN-coated coverslips as described above. Spread area of GFP-positive cells was quantified from F-actin staining. Values are means ± s.e.m.; n>30 cells from two separate experiments. Right panel: representative blot of RhoG knockdown in HeLa cells. (D) Cell-adhesion assay. MEFs transfected with pSuper or RhoG shRNA were replated for 10 minutes in wells coated with the indicated amounts of FN or poly-L-lysine (pLL). Bound cells were quantified as described in the Materials and Methods. Values are means ± s.d.; n=6 from two independent experiments.

 

Figure 4
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  		Fig. 4. RhoG and Rac1 in cell migration. MEFs were transfected with pSuper, RhoG shRNA or Rac shRNA plus GFP (40:1 ratio). Time-lapse movies of randomly migrating cells were made as described in the Materials and Methods. Migration of at least 20 GFP-positive cells was analyzed from four independent experiments for each condition. Values are means for cell velocity (A) and directionality of migration (B) ± s.e.m. *P<0.001 (Student's t-test).

 

Figure 5
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  		Fig. 5. Role of RhoG and Dock180 in Rac activation by integrins. (A,B) MEFs transfected with RhoG (A) or Dock180 (B) shRNA were replated on fibronectin and Rac activation measured as described in the Materials and Methods. Left panels: Rac pulldown assays. S, suspended cells; R, replated. Right panels: active Rac was quantified and normalized to total Rac. Values are means ± s.d.; n=3.*P<0.05 (Student's t-test).

 

Figure 6
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  		Fig. 6. Effect of adhesion on RhoG activity. (A) MEF cell lysate was loaded with GDP or GTP{gamma}S as described in the Materials and Methods, and binding of GTP-RhoG to GST-ELMO was measured. (B) Left panel: MEFs were replated on fibronectin-coated dishes for the indicated times and RhoG activity was assayed using the ELMO pulldown assay. Transient expression of the first Trio DH-PH domain was used as a control for activated RhoG. Right panel: quantification of RhoG pulldown assay normalized to total RhoG. Values are means ± s.d.; n=3.

 

Figure 7
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  		Fig. 7. RhoG-induced ruffling via Rac-dependent and -independent pathways. `Floxed' Rac1 MEFs were infected with Cre adenovirus and assayed after 6 days. (A) Representative immunoblot for Rac. (B) At 5 days after the infection with Cre, cells were transfected with GFP–RhoG-Q61L. After 24 hours, cells were fixed and stained for F-actin. (C) Ruffling area was quantified and is plotted against levels of GFP–RhoG-Q61L. Values are means ± s.e.m.; n>10 cells for each condition.

 

Figure 8
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  		Fig. 8. RhoG in Rac-independent migration and cell spreading. Two clones (3 and 6) of `floxed' Rac1 MEFs in which RhoG was stably suppressed and a control (pSuper) clone were established. (A) Western blots. Upper panel: RhoG; lower panel: Rac1. (B) Stable clones were infected with GFP-Cre adenovirus (CRE) or control adenovirus (GFP). At 6-8 days after the infection, random cell migration was assayed by time-lapse imaging. Values are means ± s.e.m.; n>20 cells from three separate experiments for each condition. *P<0.005, **P<0.0005 (Student's t-test). (C) Re-expression of human RhoG in clone 3. At 5 days after infection with GFP-Cre adenovirus, cells were transfected with human GFP-RhoG (hRhoG) or control vector (RFP). Migration was assayed as before. Cells expressing GFP-RhoG were identified based on strong perinuclear fluorescence. Values are means ± s.e.m.; n>15 cells from three separate experiments. *P<0.005. (D) Cells prepared as in A were plated on fibronectin and spreading was analyzed as described in the Materials and Methods. Values are means ± s.e.m.; n>20 cells normalized to the size of unspread cells (5 minutes after adhesion).

 

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© The Company of Biologists Ltd 2008