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Figures
- Fig. 1.
Ephrin stimulation induces retraction and rounding of COS cells. (A) Still images from time-lapse movies of COS cells stimulated with ephrin-A1 Fc or ephrin-B1 Fc. (B) Photographs of cells stimulated with ephrin-A1 Fc or ephrin-B1 Fc for the indicated times (in minutes), or with Fc for 45 minutes as a control, and stained with rhodamine-phalloidin to label filamentous actin. (C) Endogenous EphA2 and EphB2 receptors become tyrosine-phosphorylated in COS cells stimulated with ephrin-A1 Fc or ephrin-B1 Fc, respectively. Immunoprecipitated EphA2 and EphB2 were probed with anti-phosphotyrosine antibodies and reprobed with anti-EphA2 or anti-EphB2 antibodies. (D) The YSA peptide causes EphA2 tyrosinephosphorylation. COS cells were left untreated or stimulated with the YSA EphA2-activating peptide, a control (ctrl) peptide, or ephrin-A1 Fc. Lysates were used for immunoprecipitation with monoclonal anti-EphA2 antibodies. The immunoprecipitates were probed with anti-phosphotyrosine antibodies and reprobed with polyclonal anti-EphA2 antibodies. (E) Activation of EphA2 induces COS cell retraction. COS cells stimulated as in D were stained with rhodamine-phalloidin. (F) Histogram showing the percentage of cells that have spikes at the periphery, indicating cell retraction, in a representative experiment as in D and E. Bars, 20 μm.
- Fig. 2.
GTP-bound R-Ras inhibits ephrin-induced COS cell retraction. (A,B) COS cells were transiently transfected with the indicated EGFP-tagged R-Ras constructs or the EGFP vector as a control. Cells were stimulated with ephrin-A1 Fc, ephrin-B1 Fc or Fc control and stained with rhodamine-phalloidin (red). (C,D) Histogram showing the percentage of transfected (EGFP-positive) cells that have spikes at the periphery, indicating cell retraction, in representative experiments. Cells expressing R-Ras38V or R-Ras38VY66F, which cannot hydrolyze GTP, do not retract in response to ephrin stimulation. (E) GTP-bound H-Ras does not inhibit ephrin-induced COS cell retraction. COS cells were transiently transfected with EGFP together with either constitutively active H-RasV12 or pcDNA3 as a control. Cells were stimulated and stained as in A and B. (F) Histogram as in C and D. Cells expressing H-RasV12 undergo peripheral retraction similar to control cells, in response to ephrin stimulation. Bars, 20 μm.
- Fig. 3.
Ephrin stimulation decreases the level of GTP-bound R-Ras. COS cells stably transfected with R-Ras were stimulated with ephrin-A1 Fc (A), ephrin-B1 Fc (B) or Fc as a control. GTP-bound R-Ras was isolated with GST-Raf1 RBD and detected with anti-R-Ras antibodies. The histograms show the mean levels of GTP-bound R-Ras relative to control from three experiments; the bars represent standard errors. Levels of GTP-bound R-RAS in ephrin Fc-treated cells were compared with those in Fc-treated cells by one-way ANOVA and Tukey's post-hoc test. *P<0.05; **P<0.01.
- Fig. 4.
A dominant negative form of p120RasGAP prevents the decrease in R-RasGTP levels and COS cell retraction induced by ephrins. (A,B) A COS cell line stably transfected with R-Ras was transiently transfected with a dominant negative form of p120RasGAP (GAP-N) or pcDNA3 vector control and stimulated with ephrin-A1 Fc, ephrin-B1 Fc or Fc as a control. GTP-bound R-Ras was isolated with GST-Raf1 RBD and detected with anti-R-Ras antibodies in duplicate samples for the GAP-N transfected cells. (C) The histogram shows the relative levels of R-Ras GTP quantified from the experiment in A and B and normalized to the R-Ras levels in the lysates. (D) COS cells were transiently transfected with EGFP-tagged GAP-N or EGFP vector as a control. Cells were stimulated with ephrin-A1 Fc, ephrin-B1 Fc or Fc as a control and stained with rhodamine-phalloidin (red). Bar, 20 μm. (E) Histogram showing the mean percentage of cells that have spikes at the periphery; bars represent standard errors from three experiments. GAP-N-transfected cells treated with ephrin-A1 Fc or ephrin-B1 Fc were compared with similarly treated control-transfected cells by one-way ANOVA and Tukey's post-hoc test, **P<0.01 and ***P<0.001.
- Fig. 5.
GTP-bound R-Ras that cannot be phosphorylated on tyrosine 66 prevents the repulsive effects of ephrin-B1 Fc on COS cell migration. (A-C) Equal numbers of COS cells transfected with the indicated constructs were seeded on Transwell filters coated on the underside with fibronectin. Cells were allowed to migrate through the filters towards medium containing ephrin-B1 Fc or Fc as a control. The histograms show the mean number of transfected (EGPF-positive) cells that migrated through a filter in 3 hours. Bars indicate the standard deviations from measurements from three different filters. For each transfection, ephrin-B1 Fc-treated samples were compared with Fc-treated samples by one-way ANOVA and Tukey's post-hoc test, **P<0.01 and ***P<0.001. (D) R-Ras is tyrosine phosphorylated in ephrin-B1 Fc-stimulated COS cells. Cells transiently transfected with Myc-tagged R-Ras were treated with ephrin-B1 Fc for the indicated times or with Fc for 30 minutes as a control. Tyrosine-phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine antibodies and probed with anti-Myc antibodies to detect phosphorylated Myc-R-Ras.
- Fig. 6.
Effects of R-Ras mutants on ephrin-induced growth cone collapse in hippocampal and retinal neurons. (A) R-Ras is expressed in hippocampal neurons and in various P10 brain regions. Lysates of primary rat hippocampal (HC) neurons cultured for 14 days and lysates from various mouse brain regions were probed by immunoblotting for R-Ras and actin as a loading control. Probing of lysates from intestine of wild-type (WT) mice, but not R-Ras knockout (KO) mice (Komatsu and Ruoslahti, 2005), shows the presence of the R-Ras band. (B,C) Expression of R-Ras38V, R-RasY66F or R-Ras38VY66F prevents ephrin-induced growth collapse in primary hippocampal neurons. Primary rat hippocampal neurons were transfected with the indicated EGFP-tagged R-Ras constructs and stimulated with ephrin-A1 Fc, ephrin-B1 Fc or Fc as a control. Neurons were labeled with anti-tubulin antibodies (blue, digitally converted to green) and phalloidin (red). Bar, 20 μm. Growth cones from transfected (EGFP-positive) neurons were classified as `collapsed' when the actin staining did not extend beyond the tubulin staining. For each condition, approximately 500-600 total growth cones from three experiments were scored blindly. The histogram in C shows the mean percentages of collapsed growth cones under the indicated conditions; bars represent the standard error from three experiments. Although 40% of growth cones in control-transfected neurons had a non-spread collapsed appearance, ephrin treatment caused significant collapse (P<0.01 for eprin-A1 Fc-treated versus Fc-treated and P<0.001 for ephrin-B1 Fc-treated versus Fc-treated by oneway ANOVA and Tukey's post-hoc test). Growth cone collapse was also significant in neurons transfected with wild-type R-Ras (P<0.001 for both ephrins). Growth cones transfected with the indicated plasmids and treated with ephrin-A1 Fc or ephrin-B1 Fc were also compared with similarly treated R-Ras-transfected growth cones by one-way ANOVA and Tukey's post-hoc test, *P<0.05, **P<0.01 and ***P<0.001. (D) Expression of R-Ras38VY66F, but not R-Ras38V, prevents ephrin-A5-induced growth collapse in dissociated cultures of chicken retinal neurons. Growth cones from transfected (EGFP-positive) neurons were classified as `collapsed' based on a spread or collapsed morphology after phalloidin labeling. For each condition, ∼100-400 total growth cones from several experiments were counted. The histogram shows the percentage of collapsed growth cones under the indicated conditions; bars represent the standard error. Transfected growth cones treated with ephrin-A5 Fc were compared with similarly treated control-transfected growth cones by one-way ANOVA and Tukey's post-hoc test, ***P<0.001.
Tables
- Table 1.
Raf1 binding and effects of R-Ras mutants on ephrin-mediated repulsive responses
R-Ras construct RBD binding* Inhibits cell retraction†‡ Inhibits cell migration† Inhibits growth-cone collapse† WT + No n.d. No 38V ++++§ Yes No Yes/no 38VY66F ++§ Yes Yes Yes 38VY66E −§ No No n.d. Y66F +¶ No No Yes Y66E −¶ No†† n.d. n.d. 43N −** No†† n.d. n.d. -
n.d., not determined.
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↵* Independent of phosphorylation on tyrosine 66 because orthovanadate was not included in the buffers; RBD, Ras-binding domain of Raf1.
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↵† Induced by ephrin stimulation.
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↵‡ The R-Ras Y66E and 43N mutants do not cause cell retraction in the absence of ephrin, suggesting that R-Ras inactivation is not sufficient to cause cell retraction.
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↵§ See supplementary material Fig. S5.
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↵¶ Zou et al., 1999.
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↵** Based on the GTP-binding deficiency of this mutant.
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↵†† Data not shown.
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