title: High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein.
abstract: We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.
authors.affiliation: Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), The University of Edinburgh, King's Buildings, Edinburgh EH9 3JZ, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Millington
authors.forename: G Joan
authors.initials: GJ
authors.lastname: Grindlay
authors.forename: Kirsten
authors.initials: K
authors.lastname: Altenbach
authors.forename: Robert K
authors.initials: RK
authors.lastname: Neely
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
authors.forename: Mojca
authors.initials: M
authors.lastname: Bencina
authors.forename: Nick D
authors.initials: ND
authors.lastname: Read
authors.forename: Anita C
authors.initials: AC
authors.lastname: Jones
authors.forename: David T F
authors.initials: DT
authors.lastname: Dryden
authors.forename: Steven W
authors.initials: SW
authors.lastname: Magennis
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins