title: Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM.
abstract: Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM.
authors.affiliation: CNRS, UMR 6290, Rennes, France.|Université de Rennes 1, Institut de Génétique et Développement de Rennes, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Demeautis
authors.affiliation: Molecular Signaling and Cell Death Unit, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.|Molecular Signaling and Cell Death Unit, Inflammation Research Center (IRC), VIB, Ghent, Belgium.|Equipe Biophotonique Cellulaire Fonctionnelle, Laboratoire de Physique des Lasers, Atomes et Molécules (PhLAM), CNRS-UMR 8523, Villeneuve d'Ascq, France.
authors.forename: François
authors.initials: F
authors.lastname: Sipieter
authors.affiliation: CNRS, UMR 6290, Rennes, France.|Université de Rennes 1, Institut de Génétique et Développement de Rennes, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Roul
authors.affiliation: CNRS, UMR 6290, Rennes, France.|Université de Rennes 1, Institut de Génétique et Développement de Rennes, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Chapuis
authors.affiliation: Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford OX3 7BN, UK.|Cellular Imaging Core, Wellcome Trust Centre for Human Genetics, University of Oxford, UK.
authors.forename: Sergi
authors.initials: S
authors.lastname: Padilla-Parra
authors.affiliation: Molecular Signaling and Cell Death Unit, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.|Molecular Signaling and Cell Death Unit, Inflammation Research Center (IRC), VIB, Ghent, Belgium.|Unité de Glycobiologie Structurale et Fonctionnelle, CNRS-UMR 8576, Université Lille1, Villeneuve d'Ascq, France.
authors.forename: Franck B
authors.initials: FB
authors.lastname: Riquet
authors.affiliation: CNRS, UMR 6290, Rennes, France.|Université de Rennes 1, Institut de Génétique et Développement de Rennes, France.|Microscopy Rennes Imaging Centre, Biosit, Université de Rennes 1, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Tramier
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis*; D003584:Cytological Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis*; D048052:Mitogen-Activated Protein Kinase 3 / Q000032:analysis*; D061848:Optical Imaging / Q000379:methods*; D015398:Signal Transduction
chemical_list: D005456:Fluorescent Dyes; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3