title: Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells.
abstract: Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.
authors.affiliation: Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA.
authors.forename: Emilia
authors.initials: E
authors.lastname: Galperin
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
authors.forename: Alexander
authors.initials: A
authors.lastname: Sorkin
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology*