title: Optimization of ERK activity biosensors for both ratiometric and lifetime FRET measurements.
abstract: Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i) fluorophores and (ii) linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties.
authors.affiliation: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. pauline.vandame@ed.univ-lille1.fr.
authors.forename: Pauline
authors.initials: P
authors.lastname: Vandame
authors.affiliation: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. corentin.spriet@iri.univ-lille1.fr.
authors.forename: Corentin
authors.initials: C
authors.lastname: Spriet
authors.affiliation: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. franck.riquet@iri.univ-lille1.fr.
authors.forename: Franck
authors.initials: F
authors.lastname: Riquet
authors.affiliation: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. dave.trinel@iri.univ-lille1.fr.
authors.forename: Dave
authors.initials: D
authors.lastname: Trinel
authors.affiliation: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France.
authors.forename: Katia
authors.initials: K
authors.lastname: Cailliau-Maggio
authors.affiliation: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. Jean-Francois.Bodart@univ-lille1.fr.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Bodart
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D010766:Phosphorylation; D015398:Signal Transduction
chemical_list: D005456:Fluorescent Dyes