Novel reporter for faithful monitoring of ERK2 dynamics in living cells and model organisms

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

FRET-Based Enzyme Activity Reporter: Practical Hints for Kinases as Indicators of Virulence

Modulation of protein kinases activity is often requested for pathogenicity or virulence. This chapter provides several hints for one who might be interested in using FRET-based kinase activity reporters. The archetypes of these reporters, which are now within the arsenal of biosensors, were devoted to the detection and characterization of the activity of the cAMP-Protein kinase A pathway. Based on the principle of this biosensor, other FRET-based kinase activity reporters emerged. Here, the choice of the kinase to be monitored, the artifacts that might be met, and the flexibility and amenability of the FRET-based kinase activity reporters both for high-throughput analysis and dissection of protein kinase functions are discussed

Mythologie cellulaire

Rapport de collaboration Art-ScienceLe projet « Mythologie cellulaire » confère une dimension imaginaire à des éléments microscopiques en s’appuyant sur des faits scientifiques. C’est une invitation à rêver la science, à laisser l’imagination envahir le domaine du scientifique. La cellule fascine les imaginaires des artistes et des scientifiques. Le développement des technologies d’observation à l’échelle cellulaire permet de produire des corpus de matériaux avec des caractéristiques esthétiques

Mapping eGFP Oligomer Mobility in Living Cell Nuclei

Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers

Beyond boundaries: unveiling the art of blockchain-enshrined molecules through xSublimatio

In 2023, the xSublimatio project showcased a fusion of art and science, presenting an interactive platform where molecules were transformed into digital artworks within the blockchain. This innovative concept leveraged advanced artificial intelligence predictions to bridge empirical precision with creative expression, offering a unique exploration of scientific data through artistic interpretation.The creation of xSublimatio involved meticulous selection and representation of molecules, blending scientific accuracy with aesthetic appeal. Through AlphaFold-inspired insights, the project reimagined molecular design, transcending traditional boundaries.During its presentation at the GDR ChemBio conference in Strasbourg, xSublimatio sparked insightful discussions within the French chemistry community. This article explores its technical implementation, its potential for introducing blockchain and non-fungible token concepts to diverse communities, and its broader implications for interdisciplinary collaboration and decentralized science

Interleukin-7 Regulates Adipose Tissue Mass and Insulin Sensitivity in High-Fat Diet-Fed Mice through Lymphocyte-Dependent and Independent Mechanisms

Although interleukin (IL)-7 is mostly known as a key regulator of lymphocyte homeostasis, we recently demonstrated that it also contributes to body weight regulation through a hypothalamic control. Previous studies have shown that IL-7 is produced by the human obese white adipose tissue (WAT) yet its potential role on WAT development and function in obesity remains unknown. Here, we first show that transgenic mice overexpressing IL-7 have reduced adipose tissue mass associated with glucose and insulin resistance. Moreover, in the high-fat diet (HFD)-induced obesity model, a single administration of IL-7 to C57BL/6 mice is sufficient to prevent HFD-induced WAT mass increase and glucose intolerance. This metabolic protective effect is accompanied by a significant decreased inflammation in WAT. In lymphocyte-deficient HFD-fed SCID mice, IL-7 injection still protects from WAT mass gain. However, IL-7-triggered resistance against WAT inflammation and glucose intolerance is lost in SCID mice. These results suggest that IL-7 regulates adipose tissue mass through a lymphocyte-independent mechanism while its protective role on glucose homeostasis would be relayed by immune cells that participate to WAT inflammation. Our observations establish a key role for IL-7 in the complex mechanisms by which immune mediators modulate metabolic functions

Entretien avec Corentin Spriet

International audienceLe programme de recherche" Images, sciences et technologie" construit des dispositifs qui amènent des artistes à travailler en laboratoire. En 2016/2017, la plateforme de microscopie photonique TISBio et l'Ecole Supérieure d'Art du Nord-pas-de-calais ont entrepris une collaboration dont l'objectif était de produire une exposition dans le cadre élargi d'une recherche sur les relations entre l'imagerie scientifique et les pratiques artistiques contemporaines. Comment un ingénieur de recherche (CNRS) est-il en mesure de répondre aux demandes artistiques et qu'en est-il des rapports entre art et imagerie scientifique

Entretien avec Corentin Spriet

International audienceLe programme de recherche" Images, sciences et technologie" construit des dispositifs qui amènent des artistes à travailler en laboratoire. En 2016/2017, la plateforme de microscopie photonique TISBio et l'Ecole Supérieure d'Art du Nord-pas-de-calais ont entrepris une collaboration dont l'objectif était de produire une exposition dans le cadre élargi d'une recherche sur les relations entre l'imagerie scientifique et les pratiques artistiques contemporaines. Comment un ingénieur de recherche (CNRS) est-il en mesure de répondre aux demandes artistiques et qu'en est-il des rapports entre art et imagerie scientifique