Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Nonnredundant Functions in Vivo

Abstract

Cell migration often involves the formation of sheet‐like lamellipodia, generated by actin filament branching through Arp2/3 complex. In these structures, the latter is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family. Recent structural studies defined two independent Rac1 binding sites on WRC within the Sra‐1/PIR121 subunit of the pentameric WRC, but the functions of these sites in vivo have remained unknown. Here we used CRISPR/Cas9‐mediated gene disruption of Sra‐1 and its paralogue PIR121 in murine B16‐F1 cells combined with Sra‐1 mutant rescue to dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra‐1. We found that the A site, positioned adjacent to the binding region of WAVE‐WCA mediating actin‐ and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site towards the C‐terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium amoebas. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants , we conclude these residues are important for Rac/D site interaction. Finally, lamellipodia formation was observable even after disruption of both Rac interaction sites in constitutively active WRC, showing Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but not mandatory for WRC accumulation in the lamellipodium.