title: Determination of in vivo dissociation constant, KD, of Cdc42-effector complexes in live mammalian cells using single wavelength fluorescence cross-correlation spectroscopy.
abstract: The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membrane-bound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (K(D)) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine K(D) of Cdc42-effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured K(D) for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nm, respectively. The determination of K(D) for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells.
authors.affiliation: Institute of Medical Biology, 8A Biomedical Grove, Immunos, Singapore 138665.
authors.forename: Thankiah
authors.initials: T
authors.lastname: Sudhaharan
authors.affiliation: Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore.
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.affiliation: Institute of Medical Biology, 8A Biomedical Grove, Immunos, Singapore 138665; Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore.
authors.forename: Yong Hwee
authors.initials: YH
authors.lastname: Foo
authors.affiliation: Institute of Medical Biology, 8A Biomedical Grove, Immunos, Singapore 138665.
authors.forename: Wenyu
authors.initials: W
authors.lastname: Bu
authors.affiliation: Institute of Medical Biology, 8A Biomedical Grove, Immunos, Singapore 138665.
authors.forename: Kim Buay
authors.initials: KB
authors.lastname: Lim
authors.affiliation: Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wohland
authors.affiliation: Institute of Medical Biology, 8A Biomedical Grove, Immunos, Singapore 138665. Electronic address: sohail.ahmed@imb.a-star.edu.sg.
authors.forename: Sohail
authors.initials: S
authors.lastname: Ahmed
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D051316:Wiskott-Aldrich Syndrome Protein; D049452:Green Fluorescent Proteins; D020764:cdc42 GTP-Binding Protein