title: WAVE regulatory complex activation.
abstract: The WAVE regulatory complex (WRC) is critical to control of actin polymerization at the eukaryotic cell membrane. By reconstituting WAVE-dependent actin assembly on silica microspheres coated with phospholipid bilayers in mammalian brain extracts, we discovered that membrane recruitment and activation of WRC require the cooperative action of two mammalian GTPases, Arf and Rac. Here, we describe detailed methods to generate phospholipid-coated microspheres and porcine brain extract and outline conditions necessary to reconstitute WRC-dependent motility. In addition, we describe how to generate acylated recombinant GTPases, anchor them to lipid-coated microspheres, and reconstitute GTPase activation of WRC.
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hume
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Humphreys
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom. Electronic address: vk103@cam.ac.uk.
authors.forename: Vassilis
authors.initials: V
authors.lastname: Koronakis
mesh_terms: D000199:Actins / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D004867:Equipment Design; D005374:Filtration / Q000295:instrumentation; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D008863:Microspheres; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D013552:Swine; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D014981:Xenopus
chemical_list: D000199:Actins; D055767:Immobilized Proteins; D010743:Phospholipids; D051300:Wiskott-Aldrich Syndrome Protein Family; D012822:Silicon Dioxide; D020558:GTP Phosphohydrolases
keywords: Actin; Comet tail; GTPase; Membrane; Motility; Reconstitution; SCAR; WAVE regulatory complex