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

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

Brisure de symétrie et Polarisation cellulaire imposée par un signal mécanique externe

Cell polarity establishment implies a symmetry-breaking event, resulting in an axis along which the cell reorganizes. Whether the initial event that triggers polarity arises spontaneously or requires an external cue remains controversial. We set up a dual-objective system that combines quantitative mechanical manipulation using optical tweezers and fluorescence three-dimensional imaging to monitor in real time the local and global cell response to the application of a single fibronectin-coated trapped bead, mimicking an adhesion site of weak rigidity. Here we show that single and weak mechanical cue applied on detached round fibroblasts is required to control the location and duration of symmetry breaking of cortical actomyosin gel instability. Furthermore, we demonstrate that cells respond at a macroscopic level since the induced asymmetric actomyosin flow triggers the 3D migratory polarization axis and determines its orientation. As an initial event, the microtubule-independent actomyosin flow polarizes towards the opposite pole of the cue and drives higher contractility at the rear of the cell that persists after the application of mechanical stress has been halted. Microtubules are further required for long-term growth of leading edge protrusion associated with the MTOC reorientation relative to the cue. These findings support a model whereby the stochastic and transient symmetry-breaking events occuring in detached oscillating cells are not able to drive cell polarity establishment, the mechanical stress being required to trigger processes of global auto organization necessary for long-term functional polarization.L'établissement de la polarité cellulaire implique une brisure de symétrie, résultant en la formation d'un axe suivant lequel la cellule s'organise.Que l'évènement initial déclencheur de la mise en place de la polarité cellulaire soit spontané ou nécessite un signal externe reste controversé. Nous avons mis en place un système à double objectif, consistant en un outil de micromanipulation par pinces optiques couplé à l'imagerie en fluorescence pour suivre en temps réel la réponse locale et globale des cellules à l'application d'une bille fonctionnalisée à la fibronectine, mimant ainsi un site d'adhésion de faible rigidité. Nous montrons que l'application de points de tension mécanique de faible rigidité est nécessaire pour contrôler la position et la durée de la brisure de symétrie du gel d'actomyosine; En outre, nous démontrons également que le flux asymétrique d'actomyosine induit par le point de tension déclenche et sous-tend la formation et l'orientation d'un axe de migration polarisée en trois dimensions. Initialement, le flux se polarise dans la direction opposée à la position de la bille, ce qui renforce l'activité contractile à un pôle de la cellule, et cela se poursuit après que la bille se soit échappée du piège optique. Les microtubules interviennent dans un second temps pour participer à la croissance d'une protrusion sous la bille, et à la réorientation du centrosome dans la direction de la bille. Ces résultats suggèrent un modèle dans lequel la brisure de symétrique doit être stabilisée par des contraintes mécaniques afin de déclencher un processus d'auto-organisation globale, conduisant à l'établissement et au maintien d'un nouvel axe de polarité.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Etude de la réponse cellulaire à un différentiel de tensions mécaniques (étude spatio-temporelle à l'aide de pinces optiques multiples)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Développements méthodologiques pour la quantification des interactions protéiques en cellules vivantes par microscopie de fluorescence

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

: Dual color FLCS

International audienceDual-color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross-correlation data. In this article, problems with crosstalk were overcome with an approach based on fluorescence lifetime correlation spectroscopy (FLCS). We show that FLCS applied to dual-color EGFP and mCherry cross-correlation allows the determination of protein-protein interactions in living cells without the need of spectral bleed through calibration. The methodology was validated by using EGFP-mCherry tandem in comparison with coexpressed EGFP and mCherry in live cell. The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during experiment is really very helpful to study quantitatively protein interactions in live sample

Time-Domain Fluorescence Lifetime Imaging Microscopy: A Quantitative Method to Follow Transient Protein–Protein Interactions in Living Cells

International audienc

Quantitative study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy.

International audienceDual-color FCS is a powerful method to monitor protein-protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentration. But, when performing these experiments, the spectral overlap in the emission of the two colors produces an artifact that corrupts the cross-correlation data: spectral bleed-through. We have shown that problems with cross talk are overcome with Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS applied to dual-color cross-correlation, utilizing for example eGFP and mCherry fluorescent proteins, allows the determination of protein-protein interactions in living cells without the need of spectral bleed-through calibration. Here, we present in detail how this methodology can be implemented using a commercial setup (Microtime from PicoQuant, SP8 SMD from Leica or any conventional confocal with PicoQuant TCSPC module, and also with a Becker and Hickl TCSPC module). The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during the experiment constitutes a very powerful technique to quantitatively study protein interactions in live samples