Free Brick1 Is a Trimeric Precursor in the Assembly of a Functional Wave Complex 英文参考文献.docVIP

FreeBrick1IsaTrimericPrecursorintheAssemblyofa FunctionalWaveComplex EmmanuelDerivery1,4¤,JennyFink2,4,DavyMartin3,4,AnneHoudusse3,4,MatthieuPiel2,4,TheresiaE. Stradal5,DanielLouvard1,4,AlexisGautreau1,4¤ * 1InstitutCurie,CentredeRecherche,‘‘MorphogenesisandCellSignaling’’laboratory,Paris,France,2InstitutCurie,CentredeRecherche,‘‘SystemsCellBiologyofCell PolarityandCellDivision’’ laboratory,Paris, France, 3Institut Curie,CentredeRecherche,‘‘StructuralMotility’’laboratory,Paris,France,4CNRSUMR144,Paris,France, 5HZI-HelmholzCenterforInfectionResearch,Braunschweig,Germany Abstract Background: The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membraneruffles.TheWavecomplexiscomposedoffivesubunits,thesmallestofwhich,Brick1/Hspc300(Brk1),istheleast characterized.Wepreviouslyreportedthat,unliketheothersubunits,Brk1alsoexistsasafreeform. PrincipalFindings:HerewereportthatthisfreeformofBrk1iscomposedofhomotrimers.Usinganovelassayinwhich purifiedfreeBrk1iselectroporatedintoHeLacells,wewereabletofollowitsbiochemicalfateincellsandtoshowthatfree Brk1 becomes incorporated into the Wave complex. Importantly, incorporation of free Brk1 into the Wave complex was blocked upon inhibition of protein synthesis and incorporated Brk1 was found to associate preferentially with neosynthesized subunits. Brk1 depleted HeLa cells were found to bleb, as were Nap1, Wave2 or ARPC2 depleted cells, suggesting that this blebbing phenotype of Brk1 depleted cells is due to an impairment of the Wave complex function rather than a specific function of free Brk1. Blebs of Brk1 depleted cells were emitted at sites where lamellipodia and membraneruffleswerenormallyemitted.InBrk1depletedcells,theelectroporationoffreeBrk1wassufficienttorestore Wavecomplexassemblyandtorescuetheblebbingphenotype. Conclusion: Together these results establish that the free form of Brk1 is an essential precursor in the assembly of a functionalWavecomplex. Citation:DeriveryE,FinkJ