Distinct interaction sites of Rac GTPase with WAVE regulatory complex have non-redundant functions in vivo

Schaks, M., Singh, S. P. , Kage, F., Thomason, P., Klünemann, T., Steffen, A., Blankenfeldt, W., Stradal, T. E., Insall, R. H. and Rottner, K. (2018) Distinct interaction sites of Rac GTPase with WAVE regulatory complex have non-redundant functions in vivo. Current Biology, 28(22), 3674-3684.e6. (doi: 10.1016/j.cub.2018.10.002) (PMID:30393033) (PMCID:PMC6264382)

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Abstract

Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show 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 toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. 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, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that 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 is not mandatory for WRC accumulation in the lamellipodium.

Item Type:Articles
Additional Information:This work was supported in part by Deutsche Forschungsgemeinschaft (DFG) grants GRK2223/1 (to K.R. and W.B.) and RO2414/3-2 (to K.R.), Cancer Research UK (grant CRUK-A20017 to R.H.I.), and intramural core grants (to T.E.S. and R.H.I.).
Keywords:Arp2/3 complex, CRISPR/CAS9, Rho-GTPase, filopodium, lamellipodium, migration, protrusion.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Insall, Professor Robert and Thomason, Dr Peter and Singh, Dr Shashi
Authors: Schaks, M., Singh, S. P., Kage, F., Thomason, P., Klünemann, T., Steffen, A., Blankenfeldt, W., Stradal, T. E., Insall, R. H., and Rottner, K.
College/School:College of Medical Veterinary and Life Sciences > School of Cancer Sciences
College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Journal Name:Current Biology
Publisher:Elsevier (Cell Press)
ISSN:0960-9822
ISSN (Online):1879-0445
Published Online:01 November 2018
Copyright Holders:Copyright © 2018 The Authors
First Published:First published in Current Biology 28(22): 3674-3684.e6
Publisher Policy:Reproduced under a Creative Commons License

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