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

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

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

Instrumentation biophotonique pour la mesure d'interactions moléculaires dans la cellule

La cellule est un ensemble tridimensionnel complexe et dynamique. L'ensemble de ses fonctions est régulé par la formation de complexes multi-protéiques. L'étude de la formation de ces complexes dans la cellule est donc une question rencontrée de façon ubiquitaire en biologie. Différentes techniques permettent d'observer ces interactions moléculaires. Nous avons choisi d'utiliser l'étude FRET par mesure de FLIM, cette technique étant le meilleur compromis entre invasivité et précision des résultats. En particulier, notre approche repose sur le comptage de photon unique corrélé dans le temps (TCSPC), cette technique étant la plus sensible et la plus précise. Ce manuscrit présente les différentes optimisations que nous avons apportées aux systèmes de mesure de temps de vie classiquement utilisés en microscopie. Pour cela, nous avons tout d'abord optimisé l'instrumentation liée à cette technologie en implémentant de nouvelles solutions d'excitation et de mesures. Après une caractérisation de ces éléments, nous avons testé différentes améliorations de l'analyse des données, en particulier pour tenir compte de l'hétérogénéité des interactions lors d'expériences d'imagerie de temps vie de fluorescence réalisées sur cellule. Malgré ce système d'acquisition, différentes questions restent complexes à résoudre en utilisant les mesures de FLIM seules. Nous avons donc développé et caractérisé un système de mesures corrélées de spectre et de temps de vie de fluorescence (SLiM). L'exploitation de ces données dans les études de FRET sera également discutée. Chacun de ces systèmes de mesure de FRET a été appliqué a différentes problématiques biologiques que nous détaillerons.LILLE1-BU (590092102) / SudocSudocFranceF

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

FRET-SLiM on native autofluorescence : A fast and reliable method to study interactions between fluorescent probes and lignin in plant cell wall

Background: Lignocellulosic biomass is a complex network of polymers making the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that necessitates some pretreatments and several types of catalysts to be transformed efficiently. In particular, enzymes degrading lignocellulose can become inactivated due to their binding to lignin through non-specific interactions, leading to a loss in catalytic efficiency of industrial processes. Gaining more knowledge in the strength of interactions would allow optimizing enzymes and selecting appropriate pretreatments. Results: Measuring interactions directly in plant cell wall can theoretically be performed using confocal fluorescence techniques by evaluating fluorescence resonance energy transfer (FRET) between compatible fluorophores. In this study, autofluorescence of plant cell wall, mainly originating from lignin, was considered as a donor fluorophore while the acceptor was a common rhodamine-based fluorescent probe. To overcome complex plant cell wall fluorescence, which limits FRET analysis by standard techniques, we have developed an original approach, combining spectral and lifetime measurements. It consists in (1) dissecting autofluorescence signal in each spectral channel, (2) optimizing spectral channel choice for lifetime measurements and (3) achieving an unambiguous FRET signature with an autofluorescent donor fluorophore. Interactions between rhodamine-based probes of various sizes and untreated or pretreated wheat sample were evaluated, showing it was possible to discriminate interactions at the nano-scale, revealing some accessibility differences and the effect of pretreatment. Conclusions: SLiM measurement allows precise estimation of the optimal spectral range for FRET measurement. SLiM response allows for the first time doubtless FRET measurements between lignin as a donor, and an acceptor fluorophore with high accuracy and sensitivity related to lifetime decrease studies. As demonstrated, it thus becomes possible to measure interactions of fluorescent probes directly inside plant cell wall samples. This approach can thus be applied to various fields such as lignocellulose deconstruction to optimize the action of enzymes or plant cell wall development to assay in situ the biosynthesis of lignin