title: Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.
abstract: Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells.
authors.affiliation: Department of Biology, W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, Virginia.|Department of Biology, University of Virginia, Charlottesville, Virginia.
authors.forename: Horst
authors.initials: H
authors.lastname: Wallrabe
authors.affiliation: Department of Biology, W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, Virginia.
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Department of Biology, University of Virginia, Charlottesville, Virginia.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Fang
authors.affiliation: Department of Biology, W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, Virginia.|Department of Biology, University of Virginia, Charlottesville, Virginia.
authors.forename: Ammasi
authors.initials: A
authors.lastname: Periasamy
authors.affiliation: Department of Biology, University of Virginia, Charlottesville, Virginia.|Department of Cell Biology, University of Virginia, Charlottesville, Virginia.
authors.forename: George S
authors.initials: GS
authors.lastname: Bloom
mesh_terms: D008841:Actin Cytoskeleton / Q000502:physiology; D000818:Animals; D002460:Cell Line; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D061985:Madin Darby Canine Kidney Cells; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D015398:Signal Transduction; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*
chemical_list: C101203:IQ motif containing GTPase activating protein 1; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020703:ras GTPase-Activating Proteins; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein
keywords: Cdc42; E%: energy-transfer efficiency; IQGAP1; Key terms: FRET: Förster (or fluorescence) resonance energy transfer; N-WASP; PFRET: processed FRET or spectral bleedthrough-corrected FRET; ROI: region of interest; Rac1; actin nucleation; quantitative FRET analysis