title: Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms.
abstract: Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.
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; Regulation of Signal Division Team, Structural and Functional Glycobiology Unit (UGSF), CNRS-UMR 8576, Lille 1 University, Villeneuve d'Ascq, France; Groupement de Recherche Microscopie Imagerie du Vivant, GDR2588 MIV-CNRS, Villeneuve d'Ascq, France.
authors.forename: François
authors.initials: F
authors.lastname: Sipieter
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; Groupement de Recherche Microscopie Imagerie du Vivant, GDR2588 MIV-CNRS, Villeneuve d'Ascq, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Cappe
authors.affiliation: Equipe Biophotonique Cellulaire Fonctionnelle, Laboratoire de Physique des Lasers, Atomes et Molécules (PhLAM), CNRS-UMR 8523, Villeneuve d'Ascq, France; Groupement de Recherche Microscopie Imagerie du Vivant, GDR2588 MIV-CNRS, Villeneuve d'Ascq, France.
authors.forename: Mariano
authors.initials: M
authors.lastname: Gonzalez Pisfil
authors.affiliation: TISBio, Structural and Functional Glycobiology Unit (UGSF), CNRS-UMR 8576, FR3688, Lille 1 University, Villeneuve d'Ascq, France.
authors.forename: Corentin
authors.initials: C
authors.lastname: Spriet
authors.affiliation: Regulation of Signal Division Team, Structural and Functional Glycobiology Unit (UGSF), CNRS-UMR 8576, Lille 1 University, Villeneuve d'Ascq, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Bodart
authors.affiliation: Regulation of Signal Division Team, Structural and Functional Glycobiology Unit (UGSF), CNRS-UMR 8576, Lille 1 University, Villeneuve d'Ascq, France.
authors.forename: Katia
authors.initials: K
authors.lastname: Cailliau-Maggio
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; Methusalem Program, Ghent University, Ghent, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Vandenabeele
authors.affiliation: Equipe Biophotonique Cellulaire Fonctionnelle, Laboratoire de Physique des Lasers, Atomes et Molécules (PhLAM), CNRS-UMR 8523, Villeneuve d'Ascq, France; Groupement de Recherche Microscopie Imagerie du Vivant, GDR2588 MIV-CNRS, Villeneuve d'Ascq, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Héliot
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; Structural and Functional Glycobiology Unit (UGSF), CNRS-UMR 8576, Lille 1 University, Villeneuve d'Ascq, France; Groupement de Recherche Microscopie Imagerie du Vivant, GDR2588 MIV-CNRS, Villeneuve d'Ascq, France.
authors.forename: Franck B
authors.initials: FB
authors.lastname: Riquet
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D017930:Genes, Reporter; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods*; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis / Q000196:embryology* / Q000378:metabolism
chemical_list: D029867:Xenopus Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1