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

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

Annexe 5. Éléments de datation du rempart R3 du faubourg des Petites Caisses

1. L’occupation du faubourg à la fin de l’âge du Fer Le sondage 2001.1 a mis en évidence à proximité du rempart deux habitations superposées, qui correspondent, d’après l’étude de leurs céramiques, à deux moments différents de l’occupation du faubourg. La première habitation, construite selon la technique du solin de pierre surmonté par une élévation en adobes, possédait un sol d’argile jaune permettant de rattraper les irrégularités du substrat rocheux (Us 0102). La batterie de cuisine étai..

Sentiers de randonnée et Plan départemental des itinéraires de promenade et de randonnée.

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Annexe 3. Datation de la construction et de l’occupation de l’espace habité près du rempart dans la zone Z06 à la fin du deuxième âge du Fer

Il me semble nécessaire de rappeler au préalable certaines des données archéologiques qui m’ont été communiquées par Yves Marcadal, responsable de la fouille. Dans l’histoire de l’occupation de la zone Z06, la fin du deuxième âge du Fer est marquée par la construction d’un nouveau rempart (Période 4, IIe s. av. J.-C.), et plus tard par l’établissement d’un nouvel habitat à côté de lui (Période 5, deuxième moitié du Ier s. av. J.-C.). Après plusieurs campagnes de recherche dans cette zone, une..

Annexe 6. Éléments de datation du rempart R5

L’étude des céramiques a permis de proposer une fourchette chronologique pour les différentes phases mises en évidence lors de la fouille. 1. Phase 1 : Un habitat antérieur au rempart (125-100/75 av. J.-C.) La période de fabrication de nombreux vases est trop large pour permettre des datations précises. C’est le cas par exemple de 7 fragments de bord correspondant à des urnes CNT-PRO U7 (125-1 av. J.-C.) et d’1 gobelet à parois fines PAR-FIN 2 (150-20 av. J.-C.). Il en est de même pour les cé..

Chromatin condensation fluctuations rather than steady-state predict chromatin accessibility

Chromatin accessibility to protein factors is critical for genome activities. However, the dynamic properties of chromatin higher-order structures that regulate its accessibility are poorly understood. Here, we took advantage of the microenvironment sensitivity of the fluorescence lifetime of EGFP-H4 histone incorporated in chromatin to map in the nucleus of live cells the dynamics of chromatin condensation and its direct interaction with a tail acetylation recognition domain (the double bromodomain module of human TAFII250, dBD). We reveal chromatin condensation fluctuations supported by mechanisms fundamentally distinct from that of condensation. Fluctuations are spontaneous, yet their amplitudes are affected by their sub-nuclear localization and by distinct and competing mechanisms dependent on histone acetylation, ATP and both. Moreover, we show that accessibility of acetylated histone H4 to dBD is not restricted by chromatin condensation nor predicted by acetylation, rather, it is predicted by chromatin condensation fluctuations