FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

Fluorescence microscopy is widely used in biology to localize, to track, or to quantify proteins in single cells. However, following particular events in living cells with good spatio-temporal resolution is much more complex. In this context, Forster resonance energy transfer (FRET) biosensors are tools that have been developed to monitor various events such as dimerization, cleavage, elasticity, or the activation state of a protein. In particular, genetically encoded FRET biosensors are strong tools to study mechanisms of activation and activity of a large panel of kinases in living cells. Their principles are based on a conformational change of a genetically encoded probe that modulates the distance between a pair of fluorescent proteins leading to FRET variations. Recent advances in fluorescence microscopy such as fluorescence lifetime imaging microscopy (FLIM) have made the quantification of FRET efficiency easier. This review aims to address the different kinase biosensors that have been developed, how they allow specific tracking of the activity or activation of a kinase, and to give an overview of the future challenging methods to simultaneously track several biosensors in the same system

The truncated prelamin A in Hutchinson-Gilford progeria syndrome alters segregation of A-type and B-type lamin homopolymers.

Hutchinson-Gilford progeria syndrome (HGPS) is a dominant autosomal premature aging syndrome caused by the expression of a truncated prelamin A designated progerin. A-type and Btype lamins are intermediate filament proteins that polymerize to form the nuclear lamina network apposed to the inner nuclear membrane of vertebrate somatic cells. It is not known if in vivo both type of lamins assemble independently or coassemble. The blebbing and disorganization of the nuclear envelope and adjacent heterochromatin in cells from patients with HGPS is a hallmark of the disease, and the ex vivo reversal of this phenotype is considered important for the development of therapeutic strategies. Here we investigated the alterations in the lamina structure that may underlie the disorganization caused in nuclei by progerin expression. We studied the polymerization of EGFP- and DsRed-tagged wild-type and mutated lamins in the nuclear envelope of living cells by measuring fluorescence resonance energy transfer (FRET) that occurs between the two fluorophores when tagged lamins interact. Using time domain fluorescence lifetime imaging microscopy (tdFLIM) that allows a quantitative analysis of FRET signals, we show that wild-type lamins A and B1 polymerize in distinct homopolymers that further interact in the lamina. In contrast, expressed progerin coassembles with lamin B1 and lamin A to form a mixed heteropolymer in which A-type and B-type lamin segregation is lost. We propose that such structural lamina alterations may be part of the primary mechanisms leading to HGPS, possibly by impairing functions specific for each lamin type such as nuclear membrane biogenesis, signal transduction, nuclear compartmentalization and gene regulation

A high-throughput direct FRET-based assay for analysing apoptotic proteases using flow cytometry and fluorescence-lifetime measurements

International audienceCytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations, since apoptotic inducers are potent anti-cancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tuneable FRET-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis

Actin cytoskeleton differently regulates cell surface organization of GPI-anchored proteins in polarized epithelial cells and fibroblasts

The spatiotemporal compartmentalization of membrane-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell surface regulates their biological activities. These GPI-APs occupy distinct cellular functions such as enzymes, receptors, and adhesion molecules, and they are implicated in several vital cellular processes. Thus, unraveling the mechanisms and regulators of their membrane organization is essential. In polarized epithelial cells, GPI-APs are enriched at the apical surface, where they form small cholesterol-independent homoclusters and larger heteroclusters accommodating multiple GPI-AP species, all confined within areas of approximately 65–70 nm in diameter. Notably, GPI-AP homoclustering occurs in the Golgi apparatus through a cholesterol- and calcium-dependent mechanism that drives their apical sorting. Despite the critical role of Golgi GPI-AP clustering in their cell surface organization and the importance of cholesterol in heterocluster formation, the regulatory mechanisms governing GPI-AP surface organization, particularly in the context of epithelial polarity, remain elusive. Given that the actin cytoskeleton undergoes substantial remodeling during polarity establishment, this study explores whether the actin cytoskeleton regulates the spatiotemporal apical organization of GPI-APs in MDCK cells. Utilizing various imaging techniques (number and brightness, FRET/FLIM, and dSTORM coupled to pair correlation analysis), we demonstrate that the apical organization of GPI-APs, at different scales, does not rely on the actin cytoskeleton, unlike in fibroblastic cells. Interestingly, calcium chelation disrupts the organization of GPI-APs at the apical surface by impairing Golgi GPI-AP clustering, emphasizing the existence of an interplay among Golgi clustering, apical sorting, and surface organization in epithelial cells. In summary, our findings unveil distinct mechanisms regulating the organization of GPI-APs in cell types of different origins, plausibly allowing them to adapt to different external signals and different cellular environments in order to achieve specialized functions

Clinical Study A Reappraisal of Chemotherapy-Induced Liver Injury in Colorectal Liver Metastases before the Era of Antiangiogenics

Background and Aims. Chemotherapy of colorectal liver metastases can induce hepatotoxicity in noncancerous liver. We describe these lesions and assess risk factors and impacts on postresection morbidity and mortality in naive patients to chemotherapy before the era of bevacizumab. Methods. Noncancerous liver tissue lesions were analysed according to tumour, chemotherapy, surgery, and patient characteristics. Results. Fifty patients aged 62 ± 9.3 years were included between 2003 and 2007. Thirty-three (66%) received chemotherapy, with Folfox (58%), Folfiri (21%), LV5FU2 (12%), or Xelox (9%) regimens. Hepatotoxicity consisted of 18 (36%) cases of severe sinusoidal dilatation (SD), 13 (26%) portal fibrosis, 7 (14%) perisinusoidal fibrosis (PSF), 6 (12%) nodular regenerative hyperplasia (NRH), 2 (4%) steatosis >30%, zero steatohepatitis, and 16 (32%) surgical hepatitis. PSF was more frequent after chemotherapy (21% versus 0%, = 0.04), especially LV5FU2 ( = 0.02). SD was associated with oxaliplatin (54.5% versus 23.5%, = 0.05) and low body mass index ( = 0.003). NRH was associated with oxaliplatin ( = 0.03) and extensive resection ( = 0.04). No impact on mortality and morbidity was observed, apart postoperative elevation of bilirubin levels in case of PSF ( = 0.03), longer hospitalization in case of surgical hepatitis ( = 0.03), and greater blood loss in case of portal fibrosis ( = 0.03). Conclusions. Chemotherapy of colorectal liver metastases induces sinusoidal dilatation related to oxaliplatin and perisinusoidal fibrosis related to 5FU, without any impact on postoperative mortality

Imagerie des déclins de fluorescence pour l'étude de la dynamique et des interactions de macromolécules en cellules vivantes

The aim of our work was to develop fluorescence decay imaging and to demonstrate its utility in the study of macromolecular dynamics and the interactions between macromolecules in live cells. Our approach consisted of simultaneous time-correlated single photon counting (TCSPC) and characterisation of spatial localisation of emission area along a line. Monodirectional fluorescence decay imaging was obtained that was characteristic of fluorescence kinetics in different subcellular regions. Fluorescence anisotropy decays from a small subcellular volume (1 µm3) were also measured under a microscope in confocal mode. The results show that this technological approach is useful for investigating the biology of living cells. Endogenous fluorescent labelling of proteins was carried out by GFP or spectral mutants fusion. Protein-protein interactions were studied by measuring donor fluorescence lifetime diminution (heteroFRET) or fluorescence depolarisation kinetics (homoFRET). We demonstrated the formation of (i) heterodimers of the p45 protein of NF-E2 transcription factor with two partners in different subcellular compartments by heteroFRET, and (ii) herpes simplex type 1 thymidine kinase dimer. In the latter case, homoFRET was more efficient at demonstrating dimerisation than heteroFRET. Moreover, measurement of ethidium fluorescence anisotropy decay demonstrated that DNA torsional dynamics, are restricted inside non-perturbed chromatin. Further developments in bidimensional and tri-dimensional imaging are promising.Le but de notre travail est de développer une imagerie des déclins de fluorescence et d'en démontrer les potentialités pour l'étude de la dynamique macromoléculaire et des interactions entre macromolécules en cellules vivantes. Notre approche repose sur la mesure de la corrélation temporelle de photons uniques de fluorescence (TCSPC) simultanément à la détermination de la localisation spatiale (le long d'une ligne) de la région d'émission. Des images monodirectionnelles de déclins de fluorescence ont ainsi été obtenues représentant la cinétique de fluorescence en différentes régions subcellulaires. Nous avons également mis au point la mesure de déclins d'anisotropie de fluorescence provenant d'un petit volume subcellulaire (1 µm3) sous microscope en mode confocal. Les résultats présentés dans ce mémoire démontrent l'intérêt de cette approche technologique pour des problématiques de biologie cellulaire. Le marquage fluorescent endogène de protéines a été réalisé en les fusionant à la GFP ou un de ses variants spectraux. Les interactions protéine-protéine ont été étudiées soit par hétéroFRET en mesurant la diminution de la durée de vie de fluorescence du chromophore donneur, soit par homoFRET en mesurant la cinétique de dépolarisation de la fluorescence. Nous avons mis en évidence (i) la formation d'hétérodimères de p45 du facteur de transcription NF-E2 avec deux partenaires dans différents compartiments subcellulaires par hétéroFRET, et (ii) l'homodimérisation de la thymidine kinase du virus de l'herpès simplex type 1 de façon plus concluante par homoFRET que par hétéroFRET. Par ailleurs, la mesure des déclins d'anisotropie de fluorescence de l'éthidium comme sonde de la dynamique torsionnelle de l'ADN a révélé l'existence d'une très forte restriction de cette dynamique dans la chromatine non perturbée. Les développements supplémentaires de notre système pour une imagerie bi-dimensionnelle puis tri-dimensionnelle sont prometteurs

Insights into the non-mitotic functions of Aurora kinase A: more than just cell division

International audienc

IMAGERIE DES DECLINS DE FLUORESCENCE POUR L'ETUDE DE LA DYNAMIQUE ET DES INTERACTIONS DE MACROMOLECULES EN CELLULES VIVANTES

LE BUT DE NOTRE TRAVAIL EST DE DEVELOPPER UNE IMAGERIE DES DECLINS DE FLUORESCENCE ET D'EN DEMONTRER LES POTENTIALITES POUR L'ETUDE DE LA DYNAMIQUE MACROMOLECULAIRE ET DES INTERACTIONS ENTRE MACROMOLECULES EN CELLULES VIVANTES. NOTRE APPROCHE REPOSE SUR LA MESURE DE LA CORRELATION TEMPORELLE DE PHOTONS UNIQUES DE FLUORESCENCE (TCSPC) SIMULTANEMENT A LA DETERMINATION DE LA LOCALISATION SPATIALE (LE LONG D'UNE LIGNE) DE LA REGION D'EMISSION. DES IMAGES MONODIRECTIONNELLES DE DECLINS DE FLUORESCENCE ONT AINSI ETE OBTENUES REPRESENTANT LA CINETIQUE DE FLUORESCENCE EN DIFFERENTES REGIONS SUBCELLULAIRES. NOUS AVONS EGALEMENT MIS AU POINT LA MESURE DE DECLINS D'ANISOTROPIE DE FLUORESCENCE PROVENANT D'UN PETIT VOLUME SUBCELLULAIRE (1 M 3) SOUS MICROSCOPE EN MODE CONFOCAL. LES RESULTATS PRESENTES DANS CE MEMOIRE DEMONTRENT L'INTERET DE CETTE APPROCHE TECHNOLOGIQUE POUR DES PROBLEMATIQUES DE BIOLOGIE CELLULAIRE. LE MARQUAGE FLUORESCENT ENDOGENE DE PROTEINES A ETE REALISE EN LES FUSIONANT A LA GFP OU UN DE SES VARIANTS SPECTRAUX. LES INTERACTIONS PROTEINE-PROTEINE ONT ETE ETUDIEES SOIT PAR HETEROFRET EN MESURANT LA DIMINUTION DE LA DUREE DE VIE DE FLUORESCENCE DU CHROMOPHORE DONNEUR, SOIT PAR HOMOFRET EN MESURANT LA CINETIQUE DE DEPOLARISATION DE LA FLUORESCENCE. NOUS AVONS MIS EN EVIDENCE (I) LA FORMATION D'HETERODIMERES DE P45 DU FACTEUR DE TRANSCRIPTION NF-E2 AVEC DEUX PARTENAIRES DANS DIFFERENTS COMPARTIMENTS SUBCELLULAIRES PAR HETEROFRET, ET (II) L'HOMODIMERISATION DE LA THYMIDINE KINASE DU VIRUS DE L'HERPES SIMPLEX TYPE 1 DE FACON PLUS CONCLUANTE PAR HOMOFRET QUE PAR HETEROFRET. PAR AILLEURS, LA MESURE DES DECLINS D'ANISOTROPIE DE FLUORESCENCE DE L'ETHIDIUM COMME SONDE DE LA DYNAMIQUE TORSIONNELLE DE L'ADN A REVELE L'EXISTENCE D'UNE TRES FORTE RESTRICTION DE CETTE DYNAMIQUE DANS LA CHROMATINE NON PERTURBEE. LES DEVELOPPEMENTS SUPPLEMENTAIRES DE NOTRE SYSTEME POUR UNE IMAGERIE BI-DIMENSIONNELLE PUIS TRI-DIMENSIONNELLE SONT PROMETTEURS.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Fluorescence anisotropy imaging microscopy for homo-FRET in living cells.

International audienceIn this chapter, we present the basic physical principles of the fluorescence anisotropy imaging microscopy (FAIM) and its application to study FP-tagged protein dynamics and interaction in live cells. The F?er mechanism of electronic energy transfer can occur between like chromophores (homo-fluorescence resonance energy transfer, homo-FRET) inducing fluorescence depolarization and can be monitored by fluorescence anisotropy. The energy transfer rate is fast compared to the rotational time of proteins, and therefore its detection as a fast depolarization process in the fluorescence anisotropy can be easily discriminated from rotational motion. Quantitative analysis of fluorescence anisotropy decays provides information on structural parameters: distance between the two interacting chromophores and spatial orientation between the chromophores within dimeric proteins. Fluorescence anisotropy decay is not easy to measure in living cells under the microscope and the instrumentations are necessarily sophisticated. In contrast, any type of microscope can be used to measure the steady-state anisotropy. Interestingly, two-photon excitation steady-state FAIM is a powerful tool for qualitative analysis of macromolecule interactions in living cells and can be used easily for time-lapse homo-FRET