|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
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.
|
