| Eph receptors play pivotal roles in neural circuit development. Emerging evidence shows that Eph-mediated signaling sculpts neuronal morphology by modulating the reorganization of actin cytoskeleton through regulation of Rho GTPases. A number of Rho regulators have been identified to be involved in Eph-dependent synapse development. Nonetheless, the coordinated regulation of Rho GTPases by these regulators is not well understood. In this study, we demonstrate that two Rho GTPase regulators, alpha2-chimaerin and ephexin1, are essential players in EphA4-dependent synapse development. In the first part, we report that alpha2-chimaerin, a Rac GTPase activating protein that inactivates Rac1, is a new signal transducer in EphA4-dependent axon guidance. We show that alpha2-chimaerin interacts with EphA4. Activated EphA4 induces a rapid increase of tyrosine phosphorylation of alpha2-chimaerin and enhances its activity towards Rac1 inhibition. More importantly, alpha2-chimaerin is required for EphA4-mediated growth cone collapse through modulation of Rac1 activity. These findings demonstrate a previously unknown role of Rho GAPs in Eph signaling. In the second part, we identify ephexin1, a Rho guanine nucleotide exchange factor, as a key regulator in postsynaptic maturation of the neuromuscular junction (NMJ). Ephexin1 was originally identified as a signaling target downstream of EphA4. We show that ephexin1 is concentrated at the NMJ. Intriguingly, adult ephexin1-/- mice exhibit abnormal NMJ morphology including simplified AChR structure and imprecise synaptic apposition, associated with compromised motor ability. While ephexin1-/- NMJs display normal morphology at birth, mutant AChRs are abnormally enlarged and fail to develop into a mature shape during early postnatal weeks. We then demonstrate that ephexin1 negatively regulates the stability of AChR clusters through activation of RhoA. These findings reveal a critical role of ephexin1 in postsynaptic maturation of the NMJ. Taken together, this study provides new insights for understanding molecular mechanisms that underlie the regulation of actin cytoskeleton by Eph receptors. |
