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dc.contributor.authorDerivery, Emmanuel
dc.contributor.authorFink, Jenny
dc.contributor.authorMartin, Davy
dc.contributor.authorHoudusse, Anne
dc.contributor.authorPiel, Matthieu
dc.contributor.authorStradal, Theresia E
dc.contributor.authorLouvard, Daniel
dc.contributor.authorGautreau, Alexis
dc.date.accessioned2009-01-21T15:28:19Z
dc.date.available2009-01-21T15:28:19Z
dc.date.issued2008
dc.identifier.citationFree Brick1 is a trimeric precursor in the assembly of a functional wave complex. 2008, 3 (6):e2462 PLoS ONEen
dc.identifier.issn1932-6203
dc.identifier.pmid18560548
dc.identifier.doi10.1371/journal.pone.0002462
dc.identifier.urihttp://hdl.handle.net/10033/47809
dc.description.abstractBACKGROUND: The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membrane ruffles. The Wave complex is composed of five subunits, the smallest of which, Brick1/Hspc300 (Brk1), is the least characterized. We previously reported that, unlike the other subunits, Brk1 also exists as a free form. PRINCIPAL FINDINGS: Here we report that this free form of Brk1 is composed of homotrimers. Using a novel assay in which purified free Brk1 is electroporated into HeLa cells, we were able to follow its biochemical fate in cells and to show that free 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 membrane ruffles were normally emitted. In Brk1 depleted cells, the electroporation of free Brk1 was sufficient to restore Wave complex assembly and to rescue the blebbing phenotype. CONCLUSION: Together these results establish that the free form of Brk1 is an essential precursor in the assembly of a functional Wave complex.
dc.language.isoenen
dc.relation.urlhttp://www.plosone.org/article/info:doi/10.1371/journal.pone.0002462en
dc.subject.meshAmino Acid Sequenceen
dc.subject.meshBiopolymersen
dc.subject.meshCell Lineen
dc.subject.meshCytoskeletal Proteinsen
dc.subject.meshElectrophoresis, Polyacrylamide Gelen
dc.subject.meshElectroporationen
dc.subject.meshHumansen
dc.subject.meshImmunoprecipitationen
dc.subject.meshMolecular Sequence Dataen
dc.subject.meshTransfectionen
dc.titleFree Brick1 is a trimeric precursor in the assembly of a functional wave complex.en
dc.typeArticleen
dc.contributor.departmentInstitut Curie, Centre de Recherche, Morphogenesis and Cell Signaling laboratory, Paris, France.en
dc.identifier.journalPLoS ONEen
refterms.dateFOA2018-06-12T22:02:10Z
html.description.abstractBACKGROUND: The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membrane ruffles. The Wave complex is composed of five subunits, the smallest of which, Brick1/Hspc300 (Brk1), is the least characterized. We previously reported that, unlike the other subunits, Brk1 also exists as a free form. PRINCIPAL FINDINGS: Here we report that this free form of Brk1 is composed of homotrimers. Using a novel assay in which purified free Brk1 is electroporated into HeLa cells, we were able to follow its biochemical fate in cells and to show that free 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 membrane ruffles were normally emitted. In Brk1 depleted cells, the electroporation of free Brk1 was sufficient to restore Wave complex assembly and to rescue the blebbing phenotype. CONCLUSION: Together these results establish that the free form of Brk1 is an essential precursor in the assembly of a functional Wave complex.


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