title: Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells.
abstract: Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3'-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3'-phosphoinositide accumulation.
authors.affiliation: Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK. sean.warren09@imperial.ac.uk.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Warren
authors.affiliation: Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK. a.margineanu@imperial.ac.uk.
authors.forename: Anca
authors.initials: A
authors.lastname: Margineanu
authors.affiliation: Structural and Molecular Biology, University College London, London WC1E 6BT, UK. m.katan@ucl.ac.uk.
authors.forename: Matilda
authors.initials: M
authors.lastname: Katan
authors.affiliation: Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK. christopher.dunsby@imperial.ac.uk.
authors.forename: Chris
authors.initials: C
authors.lastname: Dunsby
authors.affiliation: Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK. paul.french@imperial.ac.uk.
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
mesh_terms: D000818:Animals; D016880:Anisotropy; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D010716:Phosphatidylinositols; D049452:Green Fluorescent Proteins; D051057:Proto-Oncogene Proteins c-akt; D002118:Calcium
keywords: FRET; homo-FRET; multiplexed imaging; time resolved fluorescence anisotropy imaging (TR-FAIM)