Abstract
Actin remodelling is frequently regulated by antagonistic activities driving protrusion and contraction downstream of Rac and Rho small GTPases, respectively. WAVE regulatory complex (WRC), which primarily operates downstream of Rac, plays pivotal roles in neuronal morphogenesis. Recently, two independent studies described de novo mutations in the CYFIP2 subunit of WRC, which caused intellectual disability (ID) in humans. Although mutations had been proposed to effect WRC activation, no experimental evidence for this was provided. Here, we made use of CRISPR/Cas9-engineered B16-F1 cell lines that were reconstituted with ID-causing CYFIP variants in the context of compromised WRC activation with or without reduced Rac activities, which established that the majority of CYFIP2 mutations (5 out of 8) indeed cause constitutive WRC activation. Strikingly, activating mutations are positioned in a conserved WAVE- binding region mediating WRC transinhibition. As opposed to such gain-of-function mutations, a truncating mutant represented a loss-of-function variant, because it failed to interact with WRC components, and two mutants displayed no or at best a moderate increase of WRC activation. Collectively, our data show that CYFIP2 mutations frequently but not always coincide with WRC activation and suggest that normal brain development requires a delicate and precisely tuned balance of neuronal WRC activity.