Publications

2014

• Multifunctional rare-Earth vanadate nanoparticles: luminescent labels, oxidant sensors, and MRI contrast agents.
  
  • Abdesselem Mouna
  • Schoeffel Markus
  • Maurin Isabelle
  • Ramodiharilafy Rivo
  • Autret Gwennhael
  • Clément Olivier
  • Tharaux Pierre-Louis
  • Boilot Jean-Pierre
  • Gacoin Thierry
  • Bouzigues Cedric
  • Alexandrou Antigoni
  ACS Nano, American Chemical Society, 2014, 8 (11), pp.11126-37. Collecting information on multiple pathophysiological parameters is essential for understanding complex pathologies, especially given the large interindividual variability. We report here multifunctional nanoparticles which are luminescent probes, oxidant sensors, and contrast agents in magnetic resonance imaging (MRI). Eu(3+) ions in an yttrium vanadate matrix have been demonstrated to emit strong, nonblinking, and stable luminescence. Time- and space-resolved optical oxidant detection is feasible after reversible photoreduction of Eu(3+) to Eu(2+) and reoxidation by oxidants, such as H2O2, leading to a modulation of the luminescence emission. The incorporation of paramagnetic Gd(3+) confers in addition proton relaxation enhancing properties to the system. We synthesized and characterized nanoparticles of either 5 or 30 nm diameter with compositions of GdVO4 and Gd0.6Eu0.4VO4. These particles retain the luminescence and oxidant detection properties of YVO4:Eu. Moreover, the proton relaxivity of GdVO4 and Gd0.6Eu0.4VO4 nanoparticles of 5 nm diameter is higher than that of the commercial Gd(3+) chelate compound Dotarem at 20 MHz. Nuclear magnetic resonance dispersion spectroscopy showed a relaxivity increase above 10 MHz. Complexometric titration indicated that rare-earth leaching is negligible. The 5 nm nanoparticles injected in mice were observed with MRI to concentrate in the liver and the bladder after 30 min. Thus, these multifunctional rare-earth vanadate nanoparticles pave the way for simultaneous optical and magnetic resonance detection, in particular, for in vivo localization evolution and reactive oxygen species detection in a broad range of physiological and pathophysiological conditions.
• Map the intracellular concentration of reactive oxygen species
  
  • Bouzigues Cédric
  • Alexandrou Antigoni
  Médecine/Sciences, EDP Sciences, 2014, 30 (10), pp.848 - 850. L’organisation dans le temps et l’espace des voies de signalisation est un élément essentiel dans le façonnage de la réponse cellulaire. Ceci est vrai notamment dans les processus chimiotactiques, où l’apparition d’une organisation intracellulaire asymétrique est physiologiquement indispensable. Dans ce contexte, la signalisation par les espèces oxygénées réactives, ou ROS, est singulière. En effet, ces molécules, comme le peroxyde d’hydrogène H2O2, sont connues principalement pour leur activité bactéricide et les dommages induits par le stress oxydant. Elles jouent cependant en parallèle un rôle essentiel dans plusieurs voies de signalisation physiologiques contrôlant des réponses variées – contraction, prolifération, migration – dans de nombreux tissus (systèmes nerveux, hépatique, vasculaire, etc.). L’homéostasie locale des ROS est donc un processus vital dans la vie cellulaire pour préserver l’action physiologique sans effet nuisible. (10.1051/medsci/20143010010)
  DOI : 10.1051/medsci/20143010010
• Spectroscopie résolue en temps de la femtoseconde à la milliseconde
  
  • Antonucci Laura
  , 2014. L'objet de ce travail a été le développement d'un système de mesure pompe-sonde résolue en temps permettant d'atteindre des fenêtres temporelles allant de la centaine de femtosecondes à la milliseconde. Une méthode de balayage optique asynchrone originale, appelée AD-ASOPS, a été développée et a donné lieu à un dépôt de brevet avec valorisation industrielle. La méthode peut être appliquée avec deux lasers de fréquences de répétition arbitraires, ce qui est une nouveauté et ouvre de nombreuses possibilités expérimentales. Une mise en œuvre spécifique a été réalisée selon que les systèmes lasers utilisés soient deux oscillateurs (AD-ASOPS MHz) ou bien deux lasers amplifiés (AD-ASOPS kHz). Une étude de faisabilité a été faite pour confirmer les larges possibilités d’applications. Une caractérisation complète a été menée avec plusieurs tests qui ont démontré une résolution sub-picoseconde d’environ 400 fs pour des balayages temporels réalisés sur des fenêtres temporelles uniquement limitées par la période du laser pompe. Grâce à la flexibilité d’utilisation introduite par l’AD-ASOPS une expérience pompe-sonde sur échantillon biologique (centre réactionnel de Rhodobacter Sphaeroides) a été réalisée en utilisant comme sonde un oscillateur Ti:Saphire conventionnel à 74.5 MHz et comme pompe un oscillateur CPO à 5.1 MHz. La dynamique de retour à l’état fondamental, se développant sur plusieurs ordres de grandeur, de la picoseconde à la cinquantaine de nanosecondes, a pu être enregistrée en seulement quelques minutes d’acquisitions sur un banc de test unique. Les résultats obtenus sont en accord avec les résultats de la littérature.Enfin une expérience de démonstration de la variante AD-ASOPS kHz a été effectuée sur deux systèmes laser amplifiés, par comparaison avec la méthode d’interférométrie spectrale par transformée de Fourier. Une résolution sub-picoseconde a été confirmée sur une fenêtre de balayage d’une milliseconde.
• Determination of collagen fiber orientation in histological slides using Mueller microscopy and validation by second harmonic generation imaging.
  
  • Bancelin Stéphane
  • Nazac André
  • Ibrahim Bicher Haj
  • Dokládal Petr
  • Decencière Etienne
  • Teig Benjamin
  • Haddad Huda
  • Fernandez Hervé
  • Schanne-Klein Marie-Claire
  • de Martino Antonello
  Optics Express, Optical Society of America - OSA Publishing, 2014, 22 (19), pp.22561-22574. We studied the azimuthal orientations of collagen fibers in histological slides of uterine cervical tissue by two different microscopy techniques, namely Mueller polarimetry (MP) and Second Harmonic Generation (SHG). SHG provides direct visualization of the fibers with high specificity, which orientations is then obtained by suitable image processing. MP provides images of retardation (among other polarimetric parameters) due to the optical anisotropy of the fibers, which is enhanced by Picrosirius Red staining. The fiber orientations are then assumed to be those of the retardation slow axes. The two methods, though fully different from each other, provide quite similar maps of average fiber orientations. Overall, our results confirm that MP microscopy provides reliable images of dominant fiber orientations at a much lower cost that SHG, which remains the "gold standard" for specific imaging of collagen fibers using optical microscopy. (10.1364/OE.22.022561)
  DOI : 10.1364/OE.22.022561
• Dynamics of the Heme-binding Bacterial Gas-sensing Dissimilative Nitrate Respiration Regulator (DNR) and Activation Barriers for Ligand Binding and Escape.
  
  • Lobato Laura
  • Bouzhir-Sima Latifa
  • Yamashita Taku
  • Wilson Michael T
  • Vos Marten H
  • Liebl Ursula
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2014, 289 (38), pp.26514-24. DNR (dissimilative nitrate respiration regulator) is a heme-binding transcription factor that is involved in the regulation of denitrification in Pseudomonas aeruginosa. In the ferrous deoxy state, the heme is 6-coordinate; external NO and CO can replace an internal ligand. Using fluorescence anisotropy, we show that high-affinity sequence-specific DNA binding occurs only when the heme is nitrosylated, consistent with the proposed function of DNR as NO sensor and transcriptional activator. This role is moreover supported by the NO "trapping" properties revealed by ultrafast spectroscopy that are similar to those of other heme-based NO sensor proteins. Dissociated CO-heme pairs rebind in an essentially barrierless way. This process competes with migration out of the heme pocket. The latter process is thermally activated (Ea ∼7 kJ/mol). This result is compared with other heme proteins, including the homologous CO sensor/transcription factor CooA, variants of the 5-coordinate mycobacterial sensor DosT and the electron transfer protein cytochrome c. This comparison indicates that thermal activation of ligand escape from the heme pocket is specific for systems where an external ligand replaces an internal one. The origin of this finding and possible implications are discussed. (10.1074/jbc.M114.571398)
  DOI : 10.1074/jbc.M114.571398
• Determination of collagen fibril size via absolute measurements of second-harmonic generation signals.
  
  • Bancelin Stéphane
  • Aimé Carole
  • Gusachenko Ivan
  • Kowalczuk Laura
  • Latour Gaël
  • Coradin Thibaud
  • Schanne-Klein Marie-Claire
  Nature Communications, Nature Publishing Group, 2014, septembre (5), pp.4920. The quantification of collagen fibril size is a major issue for the investigation of pathological disorders associated with structural defects of the extracellular matrix. Second-harmonic generation microscopy is a powerful technique to characterize the macromolecular organization of collagen in unstained biological tissues. Nevertheless, due to the complex coherent building of this nonlinear optical signal, it has never been used to measure fibril diameter so far. Here we report absolute measurements of second-harmonic signals from isolated fibrils down to 30 nm diameter, via implementation of correlative second-harmonic-electron microscopy. Moreover, using analytical and numerical calculations, we demonstrate that the high sensitivity of this technique originates from the parallel alignment of collagen triple helices within fibrils and the subsequent constructive interferences of second-harmonic radiations. Finally, we use these absolute measurements as a calibration for ex vivo quantification of fibril diameter in the Descemet's membrane of a diabetic rat cornea. (10.1038/ncomms5920)
  DOI : 10.1038/ncomms5920
• Optical tweezers calibration with Bayesian inference (Orale)
  
  • Türkcan Silvan
  • Richly Maximilian U.
  • Le Gall Antoine
  • Fiszman Nicolas
  • Masson Jean-Baptiste
  • Westbrook Nathalie
  • Perronet Karen
  • Alexandrou Antigoni
  , 2014, 9164, pp.UNSP 916415.
• Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0–1.2 mm Wavelength Range
  
  • Débarre Delphine
  • Olivier Nicolas
  • Supatto Willy
  • Beaurepaire Emmanuel
  PLoS ONE, Public Library of Science, 2014, pp.0104250. Light-induced toxicity is a fundamental bottleneck in microscopic imaging of live embryos. In this article, after a review of photodamage mechanisms in cells and tissues, we assess photo-perturbation under illumination conditions relevant for point-scanning multiphoton imaging of live Drosophila embryos. We use third-harmonic generation (THG) imaging of developmental processes in embryos excited by pulsed near-infrared light in the 1.0–1.2 mm range. We study the influence of imaging rate, wavelength, and pulse duration on the short-term and long-term perturbation of development and define criteria for safe imaging. We show that under illumination conditions typical for multiphoton imaging, photodamage in this system arises through 2-and/or 3-photon absorption processes and in a cumulative manner. Based on this analysis, we derive general guidelines for improving the signal-to-damage ratio in two-photon (2PEF/SHG) or THG imaging by adjusting the pulse duration and/or the imaging rate. Finally, we report label-free time-lapse 3D THG imaging of gastrulating Drosophila embryos with sampling appropriate for the visualisation of morphogenetic movements in wild-type and mutant embryos, and long-term multiharmonic (THG-SHG) imaging of development until hatching. Citation: Débarre D, Olivier N, Supatto W, Beaurepaire E (2014) Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0– 1.2 mm Wavelength Range. PLoS ONE 9(8): e104250. (10.1371/journal.pone.0104250)
  DOI : 10.1371/journal.pone.0104250
• Firefly bioluminescence: a spectroscopic perspective
  
  • Naumov P.
  • Solntsev K.M.
  • Laptenok S.
  • Rebarz M.
  • Marko-Kukovec B.
  • Maltsev O.
  • Ruckebusch C.
  • Sliwa M.
  • Hintermann L.
  , 2014.
• Fibrillogenesis from nanosurfaces: multiphoton imaging and stereological analysis of collagen 3D self-assembly dynamics
  
  • Bancelin Stéphane
  • Decencière Etienne
  • Machairas Vaïa
  • Albert Claire
  • Coradin Thibaud
  • Schanne-Klein Marie-Claire
  • Aimé Carole
  Soft Matter, Royal Society of Chemistry, 2014, pp.6651-6657. The assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects. (10.1039/c4sm00819g)
  DOI : 10.1039/c4sm00819g
• Multicolor two-photon light-sheet microscopy
  
  • Mahou Pierre
  • Vermot Julien
  • Beaurepaire Emmanuel
  • Supatto Willy
  Nature Methods, Nature Publishing Group, 2014, 11, pp.600-601. Two-photon microscopy is the most effective approach for deep-tissue fluorescence cellular imaging; however, its application to high-throughput or high-content imaging is often hampered by low pixel rates, challenging multicolor excitation and potential cumulative photodamage. To overcome these limitations, we extended our prior work and combined two-photon scanned light-sheet... (10.1038/nmeth.2963)
  DOI : 10.1038/nmeth.2963
• Keto or Enol-That is the Question: Recent Progress in Spectra-Structure Correlations of Firefly Oxyluciferin and its Derivatives
  
  • Naumov P.
  • Solntsev K.M.
  • Laptenok Sergey P.
  • Rebarz Mateusz
  • Kukovec B.M.
  • Maltsev Oleg
  • Ruckebusch Cyril
  • Sliwa Michel
  • Hintermann Lukas
  Luminescence, Wiley, 2014, 29 (S1), pp.O0047.
• Regulation of the ROS Response Dynamics and Organization to PDGF Motile Stimuli Revealed by Single Nanoparticle Imaging.
  
  • Bouzigues Cedric I.
  • Nguyên Thanh-Liêm
  • Ramodiharilafy Rivo
  • Claeson Amy
  • Tharaux Pierre-Louis
  • Alexandrou Antigoni
  Chemistry and Biology, Elsevier, 2014, 21 (5), pp.647-56. Although reactive oxygen species (ROS) are better known for their harmful effects, more recently, H2O2, one of the ROS, was also found to act as a secondary messenger. However, details of spatiotemporal organization of specific signaling pathways that H2O2 is involved in are currently missing. Here, we use single nanoparticle imaging to measure the local H2O2 concentration and reveal regulation of the ROS response dynamics and organization to platelet-derived growth factor (PDGF) signaling. We demonstrate that H2O2 production is controlled by PDGFR kinase activity and EGFR transactivation, requires a persistent stimulation, and is regulated by membrane receptor diffusion. This temporal filtering is impaired in cancer cells, which may determine their pathological migration. H2O2 subcellular mapping reveals that an external PDGF gradient induces an amplification-free asymmetric H2O2 concentration profile. These results support a general model for the control of signal transduction based only on membrane receptor diffusion and second messenger degradation. (10.1016/j.chembiol.2014.02.020)
  DOI : 10.1016/j.chembiol.2014.02.020
• Comparative Study of the Folding/Unfolding Dynamics of Poly(glutamic acid) in Light and Heavy Water
  
  • Mendonça Lucille
  • Steinbacher Andreas
  • Bouganne Raphaël
  • Hache François
  Journal of Physical Chemistry B, American Chemical Society, 2014, 118 (20), pp.5350-5356. The folding/unfolding equilibrium is investigated in poly(glutamic acid) (PGA) by two complementary sets of experiments: temperature-dependent steady-state circular dichroism spectra on the one hand and time-resolved circular dichroism measurements coupled with a T-jump experiment on the other hand. The experiments are performed for PGA dissolved in water for various pH values, as well as in heavy water. The kinetic and thermodynamic parameters extracted from these measurements are shown to be markedly different between light and heavy water, which is assigned to the difference in hydrogen bond energies in both solvents. (10.1021/jp501282z)
  DOI : 10.1021/jp501282z
• T-cell therapy using a bank of EBV-specific cytotoxic T cells: lessons from a phase I/II feasibility and safety study.
  
  • Gallot Géraldine
  • Vollant Solène
  • Saïagh Soraya
  • Clémenceau Béatrice
  • Vivien Régine
  • Cerato Evelyne
  • Bignon Jean-D
  • Ferrand Christophe
  • Jaccard Arnaud
  • Vigouroux Stéphane
  • Choquet Sylvain
  • Dalle Jean-Hugues
  • Frachon Irène
  • Bruno Bénédicte
  • Mothy Mohamad
  • Mechinaud Françoise
  • Leblond Véronique
  • Milpied Noël
  • Vié Henri
  Journal of Immunotherapy, Lippincott, Williams & Wilkins, 2014, 37 (3), pp.170-9. We report herein the results we obtained and the limitations we experienced during the production and use of a bank of Epstein-Barr virus (EBV)-transformed human cytotoxic T lymphocytes (EBV-CTLs). To assess the feasibility and toxicity of this strategy, we selected and stored, in liquid nitrogen, 4 billion EBV-CTLs from each of the 13 selected donors. Subsequently, in a multicenter phase I/II study, 11 patients with EBV-associated lymphoma resistant to conventional treatments received 1-3 doses of 5 million EBV-CTLs/kg with 1-3 and 0-4 compatibilities for human leukocyte antigen (HLA)-I and HLA-II, respectively. Except for one event of fever after injection, no immediate or delayed toxicity, no graft versus host disease, and no graft rejection attributable to CTL infusion were observed. Three patients presented complete remission and 1 partial remission after treatment. Considering the clinical options currently available, and the constrains associated with CTL preparation and implementation, we conclude that CTL banks should consist of a reasonably small number of cell lines with documented specificities. This objective could be more easily achieved if the few homozygous donors for the most frequent HLA alleles of the targeted population could be made available for such a project. (10.1097/CJI.0000000000000031)
  DOI : 10.1097/CJI.0000000000000031
• Calibrating optical tweezers with Bayesian inference (Poster)
  
  • Richly Maximilian U.
  • Türkcan Silvan
  • Le Gall Antoine
  • Fiszman Nicolas
  • Masson Jean-Baptiste
  • Westbrook Nathalie
  • Perronet Karen
  • Alexandrou Antigoni
  , 2014.
• Multiplex Cell and Lineage Tracking with Combinatorial Labels
  
  • Loulier Karine
  • Barry Raphaëlle
  • Mahou Pierre
  • Le franc Yann
  • Supatto Willy
  • Matho Katherine s.
  • Ieng Siohoi
  • Fouquet Stéphane
  • Dupin Elisabeth
  • Benosman Ryad
  • Chédotal Alain
  • Beaurepaire Emmanuel
  • Morin Xavier
  • Livet Jean
  Neuron, Elsevier, 2014, 81 (3), pp.505–520. (10.1016/j.neuron.2013.12.016)
  DOI : 10.1016/j.neuron.2013.12.016
• Substrate interaction dynamics and oxygen control in the active site of thymidylate synthase ThyX
  
  • Becker Hubert f.
  • Djaout Kamel
  • Lamarre Isabelle
  • Ulmer Jonathan e.
  • Schaming Delphine
  • Balland Véronique
  • Liebl Ursula
  • Myllykallio Hannu
  • Vos Marten h.
  Biochemical Journal, Portland Press, 2014, 459 (1), pp.37-45. Thymidylate synthase ThyX, required for DNA synthesis in many pathogenic bacteria, is considered a promising antimicrobial target. It binds FAD and three substrates, producing dTMP (2′-deoxythymidine-5′-monophosphate) from dUMP (2′-deoxyuridine-5′-monophosphate). However, ThyX proteins also act as NADPH oxidase by reacting directly with O2. In the present study we investigated the dynamic interplay between the substrates and their role in competing with this wasteful and potentially harmful oxidase reaction in catalytically efficient ThyX from Paramecium bursaria Chlorella virus-1. dUMP binding accelerates the O2-insensitive half-reaction between NADPH and FAD by over four orders of magnitude to ~30 s−1. Thus, although dUMP does not have a direct role in FAD reduction, any turnover with molecular O2 requires its presence. Inversely, NADPH accommodation accelerates dUMP binding ~3-fold and apparently precedes dUMP binding under physiological conditions. In the oxidative half-reaction, excess CH2H4folate (N5,N10-methylene-5,6,7,8-tetrahydrofolate) was found to re-oxidize FADH2 within 1 ms, thus very efficiently competing with FADH2 oxidation by O2 (1.5 s−1 under aerobic conditions). The resulting reaction scheme points out how the interplay between the fast reactions with the native substrates, although not rate-limiting for overall catalysis, avoids NADPH oxidase activity in aerobic micro-organisms, including many pathogens. These observations also explain why ThyX proteins are also present in aerobic micro-organisms (10.1042/BJ20131567)
  DOI : 10.1042/BJ20131567
• Applications des impulsions femtosecondes infrarouges : façonnage programmable et spectroscopie bidimensionnelle
  
  • Vieille Thibault
  , 2014. La mise au point des lasers femtosecondes, en particulier dans le moyen-infrarouge, a ouvert une voie d’étude des systèmes biologiques, où les couplages et les fluctuations impliquent des déphasages ultra-rapides. Ce travail participe à l’élaboration d’outils pour l’étude de systèmes vibrationnels, ici la carboxy-hémoglobine HbCO. L’approche du contrôle optimal, d’une part, détermine empiriquement le profil optimal d’une impulsion en vue d’un objectif, nécessitant la programmation d’impulsions de formes arbitraires. Nous avons étudié un prototype d’une technologie acousto-optique de façonnage d’impulsions infrarouges, le Dazzler, conçu par la société FASTLITE. Pour cela, une méthode adaptée de mesure du champ, effectuant la mesure homodyne d’une réplique de l’impulsion dans le domaine visible, a été mise au point. Puis, la production et la mesure d’impulsions infrarouges de formes complexes comprenant un saut de phase spectrale, ont été réalisées, montrant l’adéquation du Dazzler pour des expériences de contrôle cohérent dans les hémoprotéines. D’autre part, la spectroscopie multidimensionnelle mesure la réponse non-linéaire d’un échantillon, constituant un outil puissant d’étude de la surface de potentiel. Un spectromètre bidimensionnel a donc été finalisé, qui combine une géométrie simplifiée, une excellente résolution, une procédure d’auto-calibration itérative et précise, et une solution simple pour éliminer la principale source de bruit. Des mesures de la fonction de corrélation à deux temps de la fréquence de résonance du CO ont enfin été comparées à des résultats préliminaires issus d’avancées théoriques récentes, montrant un accord prometteur pour leur validation.
• Single YVO4:Eu nanoparticle emission spectra using direct Eu3+ ion excitation with a sum-frequency 465-nm solid-state laser
  
  • Nguyên Thanh-Liêm
  • Castaing Marc
  • Gacoin Thierry
  • Boilot Jean-Pierre
  • Balembois François
  • Georges Patrick
  • Alexandrou Antigoni
  Optics Express, Optical Society of America - OSA Publishing, 2014, 22 (17), pp.20542. We report emission spectrum measurements on single YxEu1-xVO4 nanoparticles. The inhomogeneous widths of the emission peaks are identical for single nanoparticles and for ensembles of nanoparticles, while being broader than those of the bulk material. This indicates that individual nanoparticles are identical in terms of the distribution of different local Eu3+ sites due to crystalline defects and confirms their usability as identical, single-particle oxidant biosensors. Moreover, we report a 465 nm solid-state laser based on sum-frequency mixing that provides a compact, efficient solution for direct Eu3+ excitation of these nanoparticles. Both these two aspects should broaden the scope of Eu-doped nanoparticle applications. (10.1364/OE.22.020542)
  DOI : 10.1364/OE.22.020542
• Differential Interaction Kinetics of a Bipolar Structure- Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps
  
  • Rezgui Rachid
  • Lestini Roxane
  • Künh Joëlle
  • Fave Xenia
  • Mcleod Lauren
  • Myllykallio Hannu
  • Alexandrou Antigoni
  • Bouzigues Cedric
  PLoS ONE, Public Library of Science, 2014 (november 20), pp.0113493. As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 59 and 39-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 59 or 39 extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases. (10.1371/journal.pone.0113493)
  DOI : 10.1371/journal.pone.0113493
• Investigating the Cell Membrane via Single Particle Tracking, Bayesian Inference and Hydrodynamic Force Application
  
  • Richly Maximilian
  • Türkcan Silvan
  • Bouzigues Cédric
  • Popoff Michel R.
  • Masson Jean-Baptiste
  • Allain Jean-Marc
  • Alexandrou Antigoni
  Biophysical Journal, Biophysical Society, 2014, 106 (2), pp.p633a. We investigate the potential felt by membrane receptors inside membrane microdomains via tracking of single receptors labelled with rare-earth doped luminescent nanoparticles and Bayesian inference analysis of the recorded trajectories. We demonstrated that the potential felt by peptidic toxin receptors confined in lipid rafts is well described by a second-order polynomial potential, possibly due to an inhomogeneous lipid and protein distribution [Türkcan et al., Biophys. J. 2012] In contrast, the potential experienced by transferrin receptors in cytoskeleton-delimited microdomains is localized at the border of the confinement domain. (10.1016/j.bpj.2013.11.3504)
  DOI : 10.1016/j.bpj.2013.11.3504
• Subpicosecond Kerr-Gate Spectrofluorometry
  
  • Laptenok Sergey P.
  • Nuernberger Patrick
  • Lukacs Andras
  • Vos Marten H.
  , 2014, pp.321-336. This chapter describes an experimental layout for time and spectrally resolved fluorescence measurements with femtosecond time resolution based on Kerr gating. The combination of data recorded using different Kerr media allows a temporal dynamic range from ~100 fs to several nanoseconds. Simultaneous analysis of multiple datasets is described. (10.1007/978-1-62703-649-8_13)
  DOI : 10.1007/978-1-62703-649-8_13
• Advances in whole-embryo imaging: a quantitative transition is underway
  
  • Pantazis Periklis
  • Supatto Willy
  Nature Reviews Molecular Cell Biology, Nature Publishing Group, 2014, 15, pp.327-339. With the advent of imaging probes and live microscopy, developmental biologists have markedly extended our understanding of the molecular and cellular details of embryonic development. To fully comprehend the complex mechanistic framework that forms the developing organism, quantitative studies with high fidelity in space and time are now required. We discuss how integrating established, newly introduced and future imaging tools with quantitative analysis will ensure that imaging can fulfil its promise to elucidate how new life begins. (10.1038/nrm3786)
  DOI : 10.1038/nrm3786