title: FRET-FLIM for Visualizing and Quantifying Protein Interactions in Live Plant Cells.
abstract: Proteins are the workhorses that control most biological processes in living cells. Although proteins can accomplish their functions independently, the vast majority of functions require proteins to interact with other proteins or biomacromolecules. Protein interactions can be investigated through biochemical assays such as co-immunoprecipitation (co-IP) or Western blot analysis, but such assays lack spatial information. Here we describe a well-developed imaging method, Förster resonance energy transfer (FRET) analyzed by fluorescence lifetime imaging microscopy (FLIM), that can be used to visualize protein interactions with both spatial and temporal resolution in live cells. We demonstrate its use in plant developmental research by visualizing in vivo dimerization of AUXIN RESPONSE FACTOR (ARF) proteins, mediating auxin responses.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703HA, Wageningen, The Netherlands.
authors.forename: Alejandra Freire
authors.initials: AF
authors.lastname: Rios
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703HA, Wageningen, The Netherlands.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Radoeva
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703HA, Wageningen, The Netherlands.|Department of Plant Systems Biology, Flemish Institute of Biotechnology, VIB, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.
authors.forename: Bert
authors.initials: B
authors.lastname: De Rybel
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703HA, Wageningen, The Netherlands.
authors.forename: Dolf
authors.initials: D
authors.lastname: Weijers
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703HA, Wageningen, The Netherlands. janwillem.borst@wur.nl.|Microspectroscopy Center, Wageningen University, Dreijenlaan 3, 6703HA, Wageningen, The Netherlands. janwillem.borst@wur.nl.
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: Borst
mesh_terms: D055592:Biophysical Phenomena / Q000502:physiology; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D061848:Optical Imaging / Q000379:methods; D010944:Plants / Q000378:metabolism*; D060066:Protein Interaction Maps / Q000502:physiology*
chemical_list: D049452:Green Fluorescent Proteins
keywords: FLIM; FRET; Fluorescent proteins; Protein interactions