Publications

2013

• Calibrating optical tweezers with Bayesian inference
  
  • Richly Maximilian U.
  • Türkcan Silvan
  • Le Gall Antoine
  • Fiszman Nicolas
  • Masson Jean-Baptiste
  • Westbrook Nathalie
  • Perronet Karen
  • Alexandrou Antigoni
  Optics Express, Optical Society of America - OSA Publishing, 2013, 21 (25), pp.31578. We present a new method for calibrating an optical-tweezer setup that does not depend on input parameters and is less affected by systematic errors like drift of the setup. It is based on an inference approach that uses Bayesian probability to infer the diffusion coefficient and the potential felt by a bead trapped in an optical or magnetic trap. It exploits a much larger amount of the information stored in the recorded bead trajectory than standard calibration approaches. We demonstrate that this method outperforms the equipartition method and the power-spectrum method in input information required (bead radius and trajectory length) and in output accuracy. (10.1364/OE.21.031578)
  DOI : 10.1364/OE.21.031578
• Imagerie Quantitative du Collagène par Génération de Seconde Harmonique
  
  • Bancelin Stéphane
  , 2013. Le collagène est une protéine ubiquitaire qui joue un rôle central dans l'architecture et la tenue mécanique des tissus conjonctifs et est impliqué dans de nombreuses pathologies. Synthétisé sous forme de triples hélices, le collagène s'auto-assemble en fibrilles in vivo et in vitro pour former des réseaux tridimensionnels. La microscopie multiphoton basée sur la génération de seconde harmonique (SHG) est une technique très spécifique, permettant de visualiser, sans marquage, le collagène en profondeur dans des tissus, avec une résolution sub-micrométrique. Ce travail de thèse vise à développer des approches quantitatives en imagerie SHG du collagène, tant à l'échelle fibrillaire que tissulaire. Nous avons montré que la microscopie SHG permet de sonder la dynamique de formation du réseau collagénique jusqu'à l'échelle d'une fibrille unique. En outre, nous avons caractérisé la structuration de gels collagéniques contrôlée par ajout de nanoparticules de silice fonctionnalisées. Nous avons ensuite réalisé de l'imagerie corrélative électronique/SHG sur ces gels pour mesurer la sensibilité de notre microscope et calibrer la réponse d'une fibrille en fonction de son diamètre. De plus, nous avons pu évaluer l'hyperpolarisabilité d'une molécule et valider le modèle additif utilisé pour calculer la réponse d'une fibrille. Enfin, nous avons développé une analyse d'images spécifique permettant de quantifier l'organisation d'un tissu collagénique à l'échelle fibrillaire, dans le but d'explorer la relation fonction/structure d'un tissu. Ceci a été validé en étudiant la modification des propriétés biomécaniques de souris génétiquement modifiées modèle du syndrome d'Ehlers-Danlos.
• Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication.
  
  • Lestini Roxane
  • Laptenok Sergey P.
  • Kühn Joëlle
  • Hink Mark A
  • Schanne-Klein Marie-Claire
  • Liebl Ursula
  • Myllykallio Hannu
  Nucleic Acids Research, Oxford University Press, 2013, 41 (22), pp.10358-10370. Hef is an archaeal member of the DNA repair endonuclease XPF (XPF)/Crossover junction endonuclease MUS81 (MUS81)/Fanconi anemia, complementation group M (FANCM) protein family that in eukaryotes participates in the restart of stalled DNA replication forks. To investigate the physiological roles of Hef in maintaining genome stability in living archaeal cells, we studied the localization of Hef-green fluorescent protein fusions by fluorescence microscopy. Our studies revealed that Haloferax volcanii Hef proteins formed specific localization foci under regular growth conditions, the number of which specifically increased in response to replication arrest. Purification of the full-length Hef protein from its native host revealed that it forms a stable homodimer in solution, with a peculiar elongated configuration. Altogether our data indicate that the shape of Hef, significant physicochemical constraints and/or interactions with DNA limit the apparent cytosolic diffusion of halophilic DNA replication/repair complexes, and demonstrate that Hef proteins are dynamically recruited to archaeal eukaryotic-like chromatin to counteract DNA replication stress. We suggest that the evolutionary conserved function of Hef/FANCM proteins is to enhance replication fork stability by directly interacting with collapsed replication forks. (10.1093/nar/gkt816)
  DOI : 10.1093/nar/gkt816
• From single cells to tissues: interactions between the matrix and human breast cells in real time.
  
  • Barnes Clifford
  • Speroni Lucia
  • Quinn Kyle P
  • Montévil Maël
  • Saetzler Kurt
  • Bode-Animashaun Gbemisola
  • Mckerr George
  • Georgakoudi Irene
  • Downes C Stephen
  • Sonnenschein Carlos
  • Howard C Vyvyan
  • Soto Ana
  PLoS ONE, Public Library of Science, 2013, 9 (4), pp.e93325. Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma--epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis. The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology. Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs. (10.1371/journal.pone.0093325)
  DOI : 10.1371/journal.pone.0093325
• Internal dynamics of heme-based sensor proteins studied using advanced time-resolved optical spectroscopy
  
  • Lobato Laura
  , 2013. Heme-proteins are involved in a large range of biological functions, including respiration, oxygen transport and xenobiotic detoxification. Importantly, bacterial heme-based sensor proteins exploit the ligation properties of heme to sense environmental gases. This thesis focuses on internal dynamics studies of the 6-coordinate heme-based gas sensor proteins CooA, from Rhodospirillum rubrum and DNR from Pseudomonas aeruginosa that are involved in adaptation of the metabolism of the organism to their environment. CooA and DNR, belonging to the important family of catabolite gene activator proteins, are transcription factors that bind DNA upon gas activation, thus enabling transcription of specific genes. Both sensor proteins are thought to undergo a large and delocalized conformational change upon binding of the physiological ligand to the heme (CO for CooA and NO for DNR). Here advanced optical spectroscopy techniques are used to investigate the mechanism and molecular pathway of activation/desactivation in this class of proteins. DNA-protein interactions were studied with steady-state and femtosecond ultrafast time resolved fluorescence techniques, using labeled DNA substrates. Physiological ligand-sensitive DNA binding in the nanomolar affinity range was deduced from anisotropy experiments. Quenching of the fluorescence label by energy transfer to the native heme in the protein moiety of the complex was observed, and the rate of this process, reflecting the heme-substrate distance, was determined directly from the measured fluorescence decays. This observation opens the perspective of mapping out the global protein conformational changes using time-resolved FRET techniques. The primary processes in heme-based sensor switching mechanisms concern ligand binding and ligand dissociation from the heme. Femtosecond transient absorption experiments were performed in order to study the ligand dynamics in CooA and DNR in the vicinity of the heme. In DNR, upon photodissociation of NO, particularly fast and efficient geminate recombination was observed. This strongly strengthens the hypothesis that NO-sensors act as ligand traps. Also, the energetic barriers involved in CO migration have been determined in both sensor proteins by temperature dependence studies. All 6-coordinate heme-based sensor proteins that function via the exchange of an internal residue and the gas molecule as a heme ligand, display barrierless recombination and a thermally activated CO-escape out of the heme pocket. By contrast, the barrier for the CO-escape appears smaller or absent for 5-coordinate systems, as has been found for the mycobacterial heme-sensor DosT. These findings point to a general mechanism, where similar protein motions are required for both, ligand exchange and ligand escape. For reasons of comparison, the energetic barriers have also been studied in ligand binding variants of the electron transfer protein cytochrome c. Here, a more complex mechanism of multiple barriers in the ligand escape pathway was deduced. This feature is proposed to reflect the rather rigid nature of this non-sensor protein, which contains a 6-coordinate heme and is devoid of ligand entry pathways in the native state. Finally, the primary processes occurring in the wild type and mutant heme domains of the recently discovered oxygen sensor YddV from Escherichia coli were investigated. In particular, an important role in the ligand dynamics was elucidated for the distal tyrosine residue. This residue hydrogen bonds to heme-bound O2 and NO molecules and was found to have a remarkably discriminating effect: after respective dissociation from the heme, it strongly promotes O2 rebinding, but favors NO escape from the heme pocket.
• Ultrafast carbonyl motion of the Photoactive Yellow Protein chromophore probed by femtosecond circular dichroism
  
  • Mendonça Lucille
  • Hache François
  • Changenet-Barret Pascale
  • Plaza Pascal
  • Chosrowjan Haik
  • Taniguchi Seiji
  • Imamoto Yasushi
  Journal of the American Chemical Society, American Chemical Society, 2013, 135, pp.14637. Motions of the trans-p-coumaric acid carbonyl group following the photoexcitation of the R52Q mutant of Photoactive Yellow Protein (PYP) are investigated, for the first time, by ultrafast time-resolved circular dichroism (TRCD) spectroscopy. TRCD is monitored in the near ultraviolet, over a timescale of 10 ps. Immediately after excitation, TRCD is found to exhibit a large negative peak, which decays within a few picoseconds. A quantitative analysis of the signals shows that, upon excitation, the carbonyl group undergoes a fast (< (10.1021/ja404503q)
  DOI : 10.1021/ja404503q
• Phase noise investigation in terahertz time-domain spectroscopy measurements
  
  • Wojdyla A.
  • Gallot Guilhem
  , 2013, pp.1-1. (10.1109/IRMMW-THZ.2013.6665432)
  DOI : 10.1109/IRMMW-THZ.2013.6665432
• Multicolour excitation module for a multiphoton imaging system, and associated method and system (extension internationale du brevet FR20120050990)
  
  • Beaurepaire Emmanuel
  • Mahou Pierre
  • Débarre Delphine
  • Supatto Willy
  • Martin Jean-Louis
  , 2013. The present invention concerns a multicolour excitation module 1 for a multiphoton imaging system 100. Such a module is used for imaging a sample 7 comprising at least three chromophores. The module 1 comprises: a first femtosecond laser source 2, emitting a first excitation beam 20; a second femtosecond laser source 3 emitting a second excitation beam 30. The first excitation beam 20 comprises a so-called "pumping" portion, said pumping portion acting as a pump beam to synchronously excite the second laser source 3, and a so-called "excitation" portion. An optical delay line 4 is arranged to spatially and temporally superpose the second excitation beam 30 and the excitation portion of the first excitation beam, so as to excite at least a third of the chromophores by multiphoton absorption, said absorbed photons coming from the first and second excitation beams 20, 30. The invention also concerns the complete multiphoton imaging system 100, and a method implemented in this module 1.
• Ionic contrast terahertz near field imaging
  
  • Gallot Guilhem
  , 2013, pp.881202. (10.1117/12.2029604)
  DOI : 10.1117/12.2029604
• Pulse shaping with birefringent crystals: a tool for quantum metrology
  
  • Labroille Guillaume
  • Pinel Olivier
  • Treps Nicolas
  • Joffre Manuel
  , 2013. A method for time differentiation based on a Babinet-Soleil-Bravais compensator is introduced. The complex transfer function of the device is measured using polarization spectral interferometry. Time differentiation of both the pulse field and pulse envelope are demonstrated over a spectral width of about 100~THz with a measured overlap with the objective mode greater than 99.8%. This pulse shaping technique is shown to be perfectly suited to time metrology at the quantum limit.
• A Bottom-Up Approach to Build the Hyperpolarizability of Peptides and Proteins from their Amino-Acids
  
  • Duboisset Julien
  • Deniset-Besseau Ariane
  • Benichou Emmanuel
  • Russier-Antoine Isabelle
  • Lascoux Noëlle
  • Jonin Christian
  • Hache François
  • Schanne-Klein Marie-Claire
  • Brevet Pierre-Francois
  Journal of Physical Chemistry B, American Chemical Society, 2013, 117 (34), pp.9877-9881. We experimentally demonstrate that some peptides and proteins lend themselves to an elementary analysis where their first hyperpolarizability can be decomposed into the coherent superposition of the first hyperpolarizability of their elementary units. We then show that those elementary units can be associated with the amino acids themselves in the case of nonaromatic amino acids and nonresonant second harmonic generation. As a case study, this work investigates the experimentally determined first hyperpolarizability of rat tail Type I collagen and compares it to that of the shorter peptide [(PPG)10]3, where P and G are the one-letter code for Proline and Glycine, respectively, and that of the triamino acid peptides PPG and GGG. An absolute value of (0.16 ± 0.01) × 10-30 esu for the first hyperpolarizability of nonaromatic amino acids is then obtained by using the newly defined 0.087 × 10-30 esu reference value for water. By using a collagen like model, the microscopic hyperpolarizability along the peptide bond can be evaluated at (0.7 ± 0.1) × 10-30 esu. (10.1021/jp312574q)
  DOI : 10.1021/jp312574q
• Properties of water confined at the nanometric scale
  
  • Audonnet Fabrice
  • Brodie-Linder Nancy
  • Deschamps Johnny
  • Schoeffel Markus
  • Frick B.
  • Koza M.
  • Teixeira José
  • Alba-Simionesco Christiane
  , 2013.
• Dynamique conformationnelle des protéines étudiée par dichroïsme circulaire résolu en temps
  
  • Mendonca Lucille
  , 2013. Les protéines sont parmi les molécules les plus importantes du monde vivant. Ce sont en quelque sorte les abeilles ouvrières de tout organisme. Elles remplissent, entre autres, des fonction enzymatiques, nerveuses, motrices ; elles transportent les petites molécules qui permettent aux cellules de fonctionner et de s'alimenter. Au cours de ce travail de thèse nous nous sommes intéressés à l'étude de différents aspects des changements conformationnels pouvant avoir lieu dans les protéines. Tous ces phénomènes ont été approchés par une méthode commune : la mesure du dichroïsme circulaire résolu en temps. En effet le dichroïsme circulaire peut donner des informations quantitatives sur la position d'un chromophore ou le repliement d'une hélice alpha ce qui est un grand avantage par rapport à d'autres mesures. Ainsi nous avons étudié l'influence du solvant sur le dépliement de l'acide polyglutamique et observé des différences dynamiques et thermodynamiques entre les polypeptides dissouts dans l'eau et ceux placés dans l'eau lourde. Nous avons, par une méthode différente, élucidé une étape clef de la relaxation du chromophore dans la protéine PYP. Enfin nous avons abordé le mouvement des hélices alpha dans la bactériorhodopsine et ouvert la porte à la possibilité de changements conformationnels non prédits par d'autres mesures. Grâce à différentes méthodes de mesure du dichroïsme circulaire nous avons pu étudier des phénomènes à des échelles de temps variant de la picoseconde à la microseconde. Nous espérons que ces méthodes pourront être étendues à l'étude d'autres mécanismes de repliement.
• Receptor displacement in the cell membrane by hydrodynamic force amplification through nanoparticles.
  
  • Türkcan Silvan
  • Richly Maximilian
  • Bouzigues Cedric I.
  • Allain Jean-Marc
  • Alexandrou Antigoni
  Biophysical Journal, Biophysical Society, 2013, 105 (1), pp.116-126. We introduce an intrinsically multiplexed and easy to implement method to apply an external force to a biomolecule and thus probe its interaction with a second biomolecule or, more generally, its environment (for example, the cell membrane). We take advantage of the hydrodynamic interaction with a controlled fluid flow within a microfluidic channel to apply a force. By labeling the biomolecule with a nanoparticle that acts as a kite and increases the hydrodynamic interaction with the fluid, the drag induced by convection becomes important. We use this approach to track the motion of single membrane receptors, the Clostridium perfringens ε-toxin (CPεT) receptors that are confined in lipid raft platforms, and probe their interaction with the environment. Under external force, we observe displacements over distances up to 10 times the confining domain diameter due to elastic deformation of a barrier and return to the initial position after the flow is stopped. Receptors can also jump over such barriers. Analysis of the receptor motion characteristics before, during, and after a force is applied via the flow indicates that the receptors are displaced together with their confining raft platform. Experiments before and after incubation with latrunculin B reveal that the barriers are part of the actin cytoskeleton and have an average spring constant of 2.5 ± 0.6 pN/μm before vs. 0.6 ± 0.2 pN/μm after partial actin depolymerization. Our data, in combination with our previous work demonstrating that the ε-toxin receptor confinement is not influenced by the cytoskeleton, imply that it is the raft platform and its constituents rather than the receptor itself that encounters and deforms the barriers formed by the actin cytoskeleton. (10.1016/j.bpj.2013.05.045)
  DOI : 10.1016/j.bpj.2013.05.045
• Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening.
  
  • Magellan Hervé
  • Boccara Martine
  • Drujon Thierry
  • Soulié Marie-Christine
  • Guillou Catherine
  • Dubois Joëlle
  • Becker Hubert F.
  Bioorganic and Medicinal Chemistry, Elsevier, 2013, 21 (17), pp.4997–5003. : Chitin synthases polymerize UDP-GlcNAC to form chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, chitin synthases are desirable targets for fungicides since chitin is absent in plants and mammals. Two potent Botrytis cinerea chitin synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for chitin synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited chitin synthases in vitro with IC50 values of 1.8 and 10μM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100μM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of chitin synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties. (10.1016/j.bmc.2013.06.058)
  DOI : 10.1016/j.bmc.2013.06.058
• Polarization-resolved SHG microscopy of rat-tail tendon with controlled mechanical strain
  
  • Gusachenko Ivan
  • Goulam Houssen Yannick
  • Tran V.
  • Allain Jean-Marc
  • Schanne-Klein Marie-Claire
  , 2013, 8797, pp.SHG/THG/CARS/SRS I. (10.1117/12.2032627)
  DOI : 10.1117/12.2032627
• Second Harmonic Generation imaging of collagen fibrillogenesis
  
  • Bancelin S
  • Aimé Carole
  • Machairas Vaïa
  • Decencière Etienne
  • Albert Claire
  • Mosser G
  • Coradin T.
  • Schanne-Klein Marie-Claire
  , 2013, pp.1 - 1. Development of nonlinear optical microscopy has significantly improved three-dimensional (3D) imaging of biological tissues in recent years. In particular, collagen has been shown to exhibit endogenous Second Harmonic Generation (SHG) signals and SHG microscopy has proved to enable the visualization of collagen architecture in tissues with unequalled contrast and specificity [1, 2]. Type I collagen is a major structural protein in mammals and shows highly structured macromolecular organizations specific to each tissue. It is synthesized by cells as triple helices, which self-assemble outside the cells into fibrils that further form fibers, lamellae or other three-dimensional (3D) networks. This assembly mechanism depends critically on the collagen concentration, as well as on the temperature, pH and ionic strength of the solution in vitro. Thorough characterization of collagen fibrillogenesis is crucial to understand the biological mechanisms of tissue formation and tissue remodeling in response to a variety of pathologies. Booming of tissue engineering furthermore requires advanced in situ quantitative imaging techniques to verify whether the tissue substitutes display appropriate biomimetic 3D organization for cell culture scaffolds or functional implants. In this study, we continuously monitored the formation of collagen fibrils by time-lapse SHG microscopy [3]. Fibrillogenesis was triggered in a controlled way by increasing the pH in a dilute solution of collagen I. The fibril density was measured every 10 to 20 minutes as the number of voxels with significant SHG signal in 3D image stacks [1]. Our results showed reproducible dynamics of fibrillar collagen formation that could be changed by tuning the pH (see figure 1). We also monitored the growth of single fibrils and measured the length increase over time, which had never been reported before using an optical technique. We then correlated these SHG images to TEM images at nanometer-scale resolution by blocking the fibrillogenesis at early stages and drying the samples. It showed that SHG microscopy allows imaging of fibrils with a diameter down to 30-50 nm in our experimental conditions. We finally investigated surface-mediated fibrillogenesis by adding silica nanoparticles to the solution [4]. We used Two-Photon excited fluorescence (2PEF) microscopy to visualize the fluorescently-died nanoparticles and quantify the self-assembly of collagen around these nanoparticles. In conclusion, SHG microscopy enabled sensitive and well contrasted 3D visualization of collagen fibrillogenesis in a non invasive way. This work illustrates the potential of SHG microscopy for the rational design and characterization of collagen-based biomaterials. 0 200 400 600 800 0.0 0.5 1.0 1.5 2.0 2.5 Pixel fraction (%) Time (min) (d) (a) (c) (b) Fig 1. 3D reconstruction of SHG images of collagen fibrillogenesis (at pH=6.5) after a) 170 b) 410 and c) 730 minutes; d) Experimental kinetics of fibril density in the SHG images (black dots) with exponential fitting (red line). References [1] M. (10.1109/CLEOE-IQEC.2013.6801528)
  DOI : 10.1109/CLEOE-IQEC.2013.6801528
• Etude de l'activité de l'enzyme de réparation NucS à l'échelle de la molécule unique
  
  • Rezgui Rachid
  , 2013. Les enzymes de réparation de l'ADN sont des facteurs essentiels pour assurer l'intégrité du génome. Ainsi, la compréhension de la dynamique de leurs mécanismes d'interaction est capitale. NucS est une nucléase récemment découverte chez l'archée Pyrococcus abyssi, qui interagit avec des structures branchées de l'ADN à simple brin libre, appelées flaps. Sa caractérisation biochimique a mis en évidence une affinité nanomolaire pour l'ADN simple brin, avec l'existence de deux sites de liaison (site I et II) ainsi qu'une activité bidirectionnelle caractérisée par la capacité à cliver les flaps 5' et 3'. Les mécanismes qui sont à l'origine de cette activité, notamment ses modalités de chargement, de localisation et de dissociation, restent toutefois peu connus. Afin de sonder la dynamique des interactions de NucS avec son substrat, nous avons mis en place un microscope à illumination en onde évanescente permettant la détection et le suivi des événements d'interaction individuels entre la protéine marquée avec un fluorophore et un substrat d'ADN attaché à la surface. Nous avons ensuite développé une nouvelle technique de colocalisation qui permet de positionner une protéine individuelle par rapport à son substrat avec une précision de 50 nm pour des durées arbitraires. Nous avons alors étudié la cinétique d'association et de dissociation des complexes NucS-ADN individuels afin de résoudre les mécanismes qui régulent l'activité de l'enzyme dans différentes conditions. Dans des conditions où le clivage est inhibé, nous avons montré que l'association était dépendante du site I, bidirectionnelle et symétrique pour les flaps 3' et 5' et que la dissociation était orientée avec une affinité supérieure pour les flap 5'. Nous avons également montré que la dissociation suivait une cinétique du premier ordre indépendante de la diffusion de NucS sur le flap. Ceci indique que, dans ces conditions non clivantes, NucS s'associe et/ou se dissocie à des positions arbitraires sur le flap. Par la suite, nous avons étudié la cinétique d'association et de dissociation du complexe NucS-ADN à 45 ◦ C en présence du cofacteur de la réparation PCNA. Nous avons démontré que PCNA augmente la vitesse d'association de NucS avec les substrats 3' et 5'. Ceci indique que PCNA agit comme une plateforme qui facilite la reconnaissance de la lésion, avec un chargement ciblé de NucS à la jonction. De plus, nous avons montré que PCNA déstabilise le complexe NucS-ADN, vraisemblablement en exerçant une force pour plier l'ADN afin d'amener l'extrémité du flap vers le site II. Dans le cas de flaps 5', nous avons montré que la dissociation suit un mécanisme en deux étapes, qui est indépendant de la longueur du flap. Ceci nous a permis de proposer un modèle où NucS se charge par son site I directement à la jonction, courbe le substrat pour capturer l'extrémité simple brin par le site II pour le cliver. Dans le cas d'un flap 3', nous avons montré que la dissociation suit un mécanisme du premier ordre ce qui est probablement dû à la rapidité de la seconde étape dans le mécanisme proposé pour les flaps 5'. Nos résultats constituent ainsi une nouvelle contribution à la caractérisation intramoléculaire des interactions NucS-ADN. Les méthodes que nous avons développées constituent en outre un moyen original pour sonder la cinétique des interactions entre biomolécules et pourront être appliquées à de multiples systèmes.
• Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX.
  
  • Laptenok Sergey P.
  • Bouzhir-Sima Latifa
  • Lambry Jean-Christophe
  • Myllykallio Hannu
  • Liebl Ursula
  • Vos Marten H.
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2013, 110 (22), pp.8924-8929. In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes. (10.1073/pnas.1218729110)
  DOI : 10.1073/pnas.1218729110
• Multiphoton microscopy : an efficient tool for in situ study of historical artifacts
  
  • Latour Gaël
  • Echard Jean-Philippe
  • Didier Marie
  • Schanne-Klein Marie-Claire
  , 2013, 8790, pp.Topography and Tomography. (10.1117/12.2020221)
  DOI : 10.1117/12.2020221
• Structure-Based Discovery of the Novel Antiviral Properties of Naproxen against the Nucleoprotein of Influenza A Virus
  
  • Lejal Nathalie
  • Tarus Bogdan
  • Bouguyon Edwige
  • Chenavas Sylvie
  • Bertho Nicolas
  • Delmas Bernard
  • Ruigrok Rob W H
  • Di Primo Carmelo
  • Slama-Schwok Anny
  Antimicrobial Agents and Chemotherapy, American Society for Microbiology, 2013, 57 (5), pp.2231 - 2242. The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove. (10.1128/AAC.02335-12)
  DOI : 10.1128/AAC.02335-12
• Monomeric Nucleoprotein of Influenza A Virus
  
  • Chenavas Sylvie
  • Estrozi Leandro F.
  • Slama-Schwok Anny
  • Delmas Bernard
  • Di Primo Carmelo
  • Baudin Florence
  • Li Xinping
  • Crépin Thibaut
  • Ruigrok Rob W. H.
  PLoS Pathogens, Public Library of Science, 2013, 9 (3). Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection. (10.1371/journal.ppat.1003275)
  DOI : 10.1371/journal.ppat.1003275
• Multiscale analysis of polarization-resolved third-harmonic generation microscopy from ordered lipid assemblies
  
  • Zimmerley Maxwell
  • Mahou Pierre
  • Débarre Delphine
  • Schanne-Klein Marie-Claire
  • Beaurepaire Emmanuel
  , 2013, 8622, pp.86220O. (10.1117/12.2004175)
  DOI : 10.1117/12.2004175
• Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas.
  
  • Kowalczuk Laura
  • Latour Gaël
  • Bourges Jean-Louis
  • Savoldelli Michèle
  • Jeanny Jean-Claude
  • Plamann Karsten
  • Schanne-Klein Marie-Claire
  • Behar-Cohen Françine
  Translational vision science & technology, Association for Research in Vision and Ophthalmology (ARVO), 2013, 2 (2), pp.3. PURPOSE: This study aimed to highlight structural corneal changes in a model of type 2 diabetes, using in vivo corneal confocal microscopy (CCM). The abnormalities were also characterized by transmission electron microscopy (TEM) and second harmonic generation (SHG) microscopy in rat and human corneas. METHODS: Goto-Kakizaki (GK) rats were observed at age 12 weeks (n = 3) and 1 year (n = 6), and compared to age-matched controls. After in vivo CCM examination, TEM and SHG microscopy were used to characterize the ultrastructure and the three-dimensional organization of the abnormalities. Human corneas from diabetic (n = 3) and nondiabetic (n = 3) patients were also included in the study. RESULTS: In the basal epithelium of GK rats, CCM revealed focal hyper-reflective areas, and histology showed proliferative cells with irregular basement membrane. In the anterior stroma, extracellular matrix modifications were detected by CCM and confirmed in histology. In the Descemet's membrane periphery of all the diabetic corneas, hyper-reflective deposits were highlighted using CCM and characterized as long-spacing collagen fibrils by TEM. SHG microscopy revealed these deposits with high contrast, allowing specific detection in diabetic human and rat corneas without preparation and characterization of their three-dimensional organization. CONCLUSION: Pathologic findings were observed early in the development of diabetes in GK rats. Similar abnormalities have been found in corneas from diabetic patients. TRANSLATIONAL RELEVANCE: This multidisciplinary study highlights diabetes-induced corneal abnormalities in an animal model, but also in diabetic donors. This could constitute a potential early marker for diagnosis of hyperglycemia-induced tissue changes. (10.1167/tvst.2.2.3)
  DOI : 10.1167/tvst.2.2.3
• SHG imaging and quantization of fibrosis
  
  • Schanne-Klein Marie-Claire
  , 2013, pp.349-372.