title: IQGAP1 interactome analysis by in vitro reconstitution and live cell 3-color FRET microscopy.
abstract: IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which then activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound Cdc42 or Rac1, which also bind IQGAP1. Here we report the use of purified proteins for in vitro binding and actin polymerization assays, and Förster (or fluorescence) resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions among IQGAP1, N-WASP, actin, and either Cdc42 or Rac1. In pyrene-actin assembly assays containing N-WASP and Arp2/3 complex, IQGAP1 plus either small G protein cooperatively stimulated actin filament nucleation by reducing the lag time before 50% maximum actin polymerization was reached. Similarly, Cdc42 and Rac1 modulated the binding of IQGAP1 to N-WASP in a dose-dependent manner, with Cdc42 enhancing the interaction and Rac1 reducing the interaction. These in vitro reconstitution results suggested that IQGAP1 interacts by similar, yet distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. The physiological relevance of these multi-protein interactions was substantiated by 3-color FRET microscopy of live MDCK cells expressing various combinations of fluorescent N-WASP, IQGAP1, Cdc42, Rac1, and actin. This study also establishes 3-color FRET microscopy as a powerful tool for studying dynamic intermolecular interactions in live cells.
authors.affiliation: Department of Biology, University of Virginia, Charlottesville, Virginia; Keck Center for Cellular Imaging; University of Virginia, Charlottesville, Virginia.
authors.forename: Horst
authors.initials: H
authors.lastname: Wallrabe
authors.forename: Ying
authors.initials: Y
authors.lastname: Cai
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Sun
authors.forename: Ammasi
authors.initials: A
authors.lastname: Periasamy
authors.forename: Rafael
authors.initials: R
authors.lastname: Luzes
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Fang
authors.forename: Ho-Man
authors.initials: HM
authors.lastname: Kan
authors.forename: Luiz-Claudio
authors.initials: LC
authors.lastname: Cameron
authors.forename: Dorothy A
authors.initials: DA
authors.lastname: Schafer
authors.forename: George S
authors.initials: GS
authors.lastname: Bloom
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008856:Microscopy, Fluorescence / Q000379:methods; D011485:Protein Binding; D011817:Rabbits; D015398:Signal Transduction; D020703:ras GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000199:Actins; C101203:IQ motif containing GTPase activating protein 1; D020703:ras GTPase-Activating Proteins
keywords: Arp2/3 complex; Cdc42; Förster resonance energy transfer; N-WASP; Rac1; actin