Journal
PLOS ONE
Volume 10, Issue 10, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0140924
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Funding
- Lille 1 University
- Centre National de la Recherche Scientifique (CNRS)
- Fonds Wetenschappelijk Onderzoek [FWO G.0875.11]
- Methusalem grant (Bijzonder Onderzoeksfonds) [BOF09/01M00709]
- Belgian grants (Interuniversity Attraction Poles) [IAP 7/32]
- Flemish grant [FWO G.0973.11, FWO G.0A45.12N, FWO G.0172.12, FWO G.0787.13N, FWO G.0C31.14N]
- Ghent University grant
- Foundation against Cancer [2012-188]
- Vlaams Instituut voor Biotechnologie (VIB)
- Agence Nationale pour la Recherche (ANR) [ANR-13-BSV2-0016-02]
- contrat Plan Etat-Region Campus Intelligent
- ANR Multimodal and DynamIC
- Leica Microsystems
- Agence Nationale de la Recherche (ANR) [ANR-13-BSV2-0016] Funding Source: Agence Nationale de la Recherche (ANR)
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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.
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