Molecular Dissection of Neurodevelopmental Disorder-Causing Mutations in CYFIP2

Actin remodeling 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 different experimental contexts. Almost all CYFIP2-derived mutations (7 out of 8) promoted WRC activation, but to variable extent and with at least two independent mechanisms. The majority of mutations occurs in a conserved WAVE-binding region, required for WRC transinhibition. One mutation is positioned closely adjacent to the Rac-binding A site and appears to ease Rac-mediated WRC activation. As opposed to these gain-of-function mutations, a truncating mutant represented a loss-of-function variant and failed to interact with WRC components. Collectively, our data show that explored 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.