Robo recruitment of the Wave regulatory complex plays an essential and conserved role in midline repulsion

• Published in: eLife 2021-04, Vol.10
• Main Authors: Chaudhari, Karina, Gorla, Madhavi, Chang, Chao, Kania, Artur, Bashaw, Greg J
• Format: Article
• Language: English
• Subjects: Actin; Actin Cytoskeleton - genetics; Actin Cytoskeleton - metabolism; Animal experimentation; Animals; Animals, Genetically Modified; Axon Guidance; Axons - metabolism; Binding sites; Chickens; CRISPR; Cytoskeleton; Developmental Biology; Drosophila melanogaster - embryology; Drosophila melanogaster - genetics; Drosophila melanogaster - metabolism; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Embryonic development; Fruit-flies; Genotype & phenotype; HEK293 Cells; Humans; Insects; Mice; Microfilament Proteins - genetics; Microfilament Proteins - metabolism; midline; Mutagenesis; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Nervous system; Neurons; Neuroscience; Polymerization; Protein Binding; Protein Interaction Domains and Motifs; Proteins; Receptors, Immunologic - genetics; Receptors, Immunologic - metabolism; Robo; Robo protein; Roundabout Proteins; Signal Transduction; Slit; Slit protein; Spinal cord; Spinal Cord - embryology; Spinal Cord - metabolism; wave regulatory complex; Wiskott-Aldrich Syndrome Protein Family - genetics; Wiskott-Aldrich Syndrome Protein Family - metabolism
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.64474

The Roundabout (Robo) guidance receptor family induces axon repulsion in response to its ligand Slit by inducing local cytoskeletal changes; however, the link to the cytoskeleton and the nature of these cytoskeletal changes are poorly understood. Here, we show that the heteropentameric Scar/Wave Regulatory Complex (WRC), which drives Arp2/3-induced branched actin polymerization, is a direct effector of Robo signaling. Biochemical evidence shows that Slit triggers WRC recruitment to the Robo receptor's WRC-interacting receptor sequence (WIRS) motif. In embryos, mutants of the WRC enhance Robo1-dependent midline crossing defects. Additionally, mutating Robo1's WIRS motif significantly reduces receptor activity in rescue assays in vivo, and CRISPR-Cas9 mutagenesis shows that the WIRS motif is essential for endogenous Robo1 function. Finally, axon guidance assays in mouse dorsal spinal commissural axons and gain-of-function experiments in chick embryos demonstrate that the WIRS motif is also required for Robo1 repulsion in mammals. Together, our data support an essential conserved role for the WIRS-WRC interaction in Robo1-mediated axon repulsion.