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dc.contributor.authorSchaks, Matthias
dc.contributor.authorSingh, Shashi P
dc.contributor.authorKage, Frieda
dc.contributor.authorThomason, Peter
dc.contributor.authorKlünemann, Thomas
dc.contributor.authorSteffen, Anika
dc.contributor.authorBlankenfeldt, Wulf
dc.contributor.authorStradal, Theresia E
dc.contributor.authorInsall, Robert H
dc.contributor.authorRottner, Klemens
dc.date.accessioned2018-11-20T13:29:37Z
dc.date.available2018-11-20T13:29:37Z
dc.date.issued2018-10-25
dc.identifier.issn1879-0445
dc.identifier.pmid30393033
dc.identifier.doi10.1016/j.cub.2018.10.002
dc.identifier.urihttp://hdl.handle.net/10033/621575
dc.description.abstractCell 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.en_US
dc.rightsAttribution-NonCommercial-ShareAlike 3.0 United States*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/3.0/us/*
dc.subjectArp2/3 complexen_US
dc.subjectCRISPR/CAS9en_US
dc.subjectRho-GTPaseen_US
dc.subjectfilopodiumen_US
dc.subjectlamellipodiumen_US
dc.subjectmigrationen_US
dc.subjectprotrusionen_US
dc.titleDistinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.en_US
dc.typeArticleen_US
dc.contributor.departmentHZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.en_US
refterms.dateFOA2018-11-20T13:29:37Z
dc.source.journaltitleCurrent biology : CB


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