Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms

PLoS One. 2015 Oct 30;10(10):e0140924. doi: 10.1371/journal.pone.0140924. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation
  • Genes, Reporter*
  • MAP Kinase Kinase 1 / genetics
  • MAP Kinase Kinase 1 / metabolism
  • MAP Kinase Signaling System
  • Mice
  • Mitogen-Activated Protein Kinase 1 / genetics
  • Mitogen-Activated Protein Kinase 1 / metabolism*
  • NIH 3T3 Cells
  • Phosphorylation
  • Signal Transduction
  • Single-Cell Analysis / methods*
  • Xenopus Proteins / metabolism
  • Xenopus laevis / embryology*
  • Xenopus laevis / metabolism

Substances

  • Xenopus Proteins
  • Mitogen-Activated Protein Kinase 1
  • MAP Kinase Kinase 1

Grants and funding

FS is a joint PhD student between Lille1 University and Ghent University, and is funded by Lille 1 University, the Centre National de la Recherche Scientifique (CNRS), and Vandenabeele’s group research funding: Fonds Wetenschappelijk Onderzoek (FWO G.0875.11) and Methusalem grant (Bijzonder Onderzoeksfonds, BOF09/01M00709). PV is senior full professor at Ghent University. FR is a visiting research professor at Ghent University and full associate professor at Lille 1 University. Research in the Vandenabeele group is further supported by Belgian grants (Interuniversity Attraction Poles, IAP 7/32), Flemish grants (FWO G.0973.11, FWO G.0A45.12N, FWO G.0172.12, FWO G.0787.13N, FWO G.0C31.14N), Ghent University grants (Multidisciplinary Research Platforms (MRP), Ghent Researchers On Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium), grant from the Foundation against Cancer, 2012-188) and grants from Vlaams Instituut voor Biotechnologie (VIB). This research is supported by the Agence Nationale pour la Recherche (ANR): G2Progress program (ANR-13-BSV2-0016-02). This work was funded by grants from contrat Plan Etat-Région “Campus Intelligent” and ANR “Multimodal” and DynamIC and supported by contrat with Leica Microsystems.