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. 2000 Apr 17;149(2):263-70.
doi: 10.1083/jcb.149.2.263.

Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase

S Wahl  1 , H Barth, T Ciossek, K Aktories, B K Mueller
Affiliations

Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase

S Wahl et al. J Cell Biol. .

Abstract

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. For the first time, we show that the ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5-treated retinal ganglion cell cultures, Rho was activated and Rac was downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduced the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway.

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Figures

Figure 1
Figure 1
Staining of retinal axons and GCs with antibodies directed against RhoA, ROCK, and C3-transferase. (A and B) RhoA (A) and ROCK (B) are present in retinal axons and GCs. (C) RGC axons growing on laminin were treated with C2-C3 fusion toxin and were fixed and stained with an antibody directed against the C3-transferase (C, fluorescent image). Inset shows a retinal GC stained with the C3-transferase antibody. Nearly all axons are stained (E, phase-contrast image) albeit at different levels. (D) In the absence of the fusion toxin, no staining is observed with the C3-transferase antibody (D, fluorescent image; F, phase-contrast image).
Figure 2
Figure 2
In RGCs, the C2-C3 fusion toxin and Y-27632 inactivate Rho and ROCK, respectively, and f-EA5 activates Rho and inactivates Rac. (A) Treatment of retinal cultures with fusion toxin reduced the level of Rho available for radioactive ADP ribosylation. (Lane 1) Control, no C2-C3 was added to RGC cultures. (Lanes 2–5) Time course of Rho ADP ribosylation in highly enriched RGC extracts. A nearly complete reduction of ADP-ribosylatable Rho is observed after 4 h (lane 4). (B) Treatment of retinal cultures with Y-27632 (10 μM) reduced the level of phosphorylated RMLC (arrow). Time course of ROCK inhibition, analyzed by immunodetection of pp2b-stained phosphorylated RMLC in highly enriched RGC extracts. No phosphorylated RMLC is detectable after a 1-h incubation in Y-27632 (lane 3). (C) Affinity precipitation of GTP-Rho using a GST fusion protein of the Rho-binding domain of Rhotekin. In f-EA5–treated RGC cultures, much higher levels of Rho are detectable (right lane) compared with control cultures (left lane). No difference is observed in the amount of total Rho (arrowhead) in lysates of control-treated and f-EA5–treated RGC cultures. (D) Affinity precipitation of GTP-Rac using the GST Pak1-CRIB domain. In f-EA5–treated RGC cultures, much lower levels of Rac are detectable (right lane) compared with control cultures (left lane). In lysates of control-treated and f-EA5–treated RGC cultures, no difference is observed in the amount of total Rac (arrowhead).
Figure 3
Figure 3
Incubation of RGCs in C2-C3 or Y-27632 reduced the f-EA5–induced collapse of RGC GCs. (A and B) Incubation of RGC GCs in the presence of C2-C3 (A) or Y-27632 (B) did not affect GC morphology. (C and D) Addition of f-EA5 induces collapse of retinal GCs. (E and F) The f-EA5–induced collapse is reduced by pretreatment of retinal GCs with 300 ng/ml of C2-C3 fusion toxin for 4 h. (G and H) The f-EA5–induced collapse is reduced by pretreatment of retinal GCs with 10 μM of Y-27632. Left and right panels in C, E, and G and in D, F, and H correspond to low- and high-power magnification, respectively.
Figure 4
Figure 4
Summary of data from retinal explant cultures treated with C2-C3 fusion toxin or with Y-27632. The single components of the fusion toxin (C2I, C2II, and C2IN-C3) did not affect the f-EA5–induced collapse of RGC GCs (data not shown). Data are from six independent experiments, and in each culture ∼100 GCs at the axon front has been counted. Asterisks indicate statistical significance, P < 0.01.
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