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IQGAP1 interactome analysis by in vitro reconstitution and live cell 3‐color FRET microscopy
Author(s) -
Wallrabe Horst,
Cai Ying,
Sun Yuansheng,
Periasamy Ammasi,
Luzes Rafael,
Fang Xiaolan,
Kan HoMan,
Cameron LuizClaudio,
Schafer Dorothy A.,
Bloom George S.
Publication year - 2013
Publication title -
cytoskeleton
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.95
H-Index - 86
eISSN - 1949-3592
pISSN - 1949-3584
DOI - 10.1002/cm.21146
Subject(s) - iqgap1 , förster resonance energy transfer , biology , wiskott–aldrich syndrome protein , microbiology and biotechnology , mdia1 , actin binding protein , fluorescence microscope , actin , actin remodeling , protein filament , biophysics , rac1 , confocal microscopy , actin cytoskeleton , cytoskeleton , biochemistry , fluorescence , cell , scaffold protein , signal transduction , physics , quantum mechanics
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. © 2013 Wiley Periodicals, Inc.