Articles

Probing ordered lipid assemblies with polarized third-harmonic-generation microscopy
- Zimmerley Maxwell
- Mahou Pierre
- Débarre Delphine
- Schanne-Klein Marie-Claire
- Beaurepaire Emmanuel
Physical Review X, American Physical Society , 2013, 3 (1), pp.11002 . Ordered lipid assemblies are responsible for important physiological functions including skin barrier and axon conductivity. However, techniques commonly used to probe molecular order such as X-ray scattering and nuclear magnetic resonance are not suited for in-situ tissue studies. Here, we identify and characterize a novel contrast mechanism in nonlinear optical microscopy which is sensitive to molecular ordering in multilamellar lipid vesicles (MLVs) and in samples obtained from human skin biopsy: polarized third-harmonic generation (P-THG). We develop a multiscale theoretical framework to calculate the anisotropic, nonlinear optical response of lipid arrays as a function of molecular order. This analysis reveals that conserved carbon-carbon bond and aliphatic tail directionality are the atomic- and molecular-scale sources of the observed P-THG response, respectively. Agreement between calculations and experiments on lipid droplets and MLVs validates the use of P-THG as a probe of lipid ordering. Finally, we show that P-THG can be used to map molecular ordering in the multilamellar, intercorneocyte lipid matrix of the stratum corneum of human skin. These results provide the foundation for the use of P-THG in probing molecular order and highlight a novel biomedical application of multiphoton microscopy in an optically accessible tissue relevant to monitoring lipid-related disorder. (10.1103/PhysRevX.3.011002)
DOI : 10.1103/PhysRevX.3.011002
Probing membrane protein interactions with their lipid raft environment using single-molecule tracking and Bayesian inference analysis.
- Türkcan Silvan
- Richly Maximilian U.
- Alexandrou Antigoni
- Masson Jean-Baptiste
PLoS ONE, Public Library of Science , 2013, 8 (1), pp.e53073 . The statistical properties of membrane protein random walks reveal information on the interactions between the proteins and their environments. These interactions can be included in an overdamped Langevin equation framework where they are injected in either or both the friction field and the potential field. Using a Bayesian inference scheme, both the friction and potential fields acting on the ε-toxin receptor in its lipid raft have been measured. Two types of events were used to probe these interactions. First, active events, the removal of cholesterol and sphingolipid molecules, were used to measure the time evolution of confining potentials and diffusion fields. Second, passive rare events, de-confinement of the receptors from one raft and transition to an adjacent one, were used to measure hopping energies. Lipid interactions with the ε-toxin receptor are found to be an essential source of confinement. ε-toxin receptor confinement is due to both the friction and potential field induced by cholesterol and sphingolipids. Finally, the statistics of hopping energies reveal sub-structures of potentials in the rafts, characterized by small hopping energies, and the difference of solubilization energy between the inner and outer raft area, characterized by higher hopping energies. (10.1371/journal.pone.0053073)
DOI : 10.1371/journal.pone.0053073
Attenuated internal reflection terahertz imaging
- Wojdyla Antoine
- Gallot Guilhem
Optics Letters, Optical Society of America - OSA Publishing , 2013, 38 (2), pp.112-114 . We present a terahertz (THz) imaging technique based on attenuated internal reflection, which is ideally suited for the analysis of liquid and biological samples. Inserted in a THz time-domain system, and using a high-resistivity low loss silicon prism to couple the THz wave into the sample, the detection scheme is based on the relative differential spectral phase of two orthogonal polarizations. Biological sample imaging as well as subwavelength (?/16) longitudinal resolution are demonstrated. Cop. 2013 Optical Society of America.
Upregulation of Adhesion Molecules on Leukemia Targets Improves the Efficacy of Cytotoxic T Cells Transduced With Chimeric Anti-CD19 Receptor
- Laurin David
- Marin Virna
- Biagi Ettore
- Pizzitola Irene
- Agostoni Valentina
- Gallot Géraldine
- Vié Henri
- Christine Jacob Marie
- Chaperot Laurence
- Aspord Caroline
- Plumas Joël
Journal of Immunotherapy, Lippincott, Williams & Wilkins , 2013, 36 (3), pp.181-189 . (10.1097/CJI.0b013e318288f8c1)
DOI : 10.1097/CJI.0b013e318288f8c1
Parallel measurements of reaction kinetics using ultralow-volumes
- Fradet Etienne
- Abbyad Paul
- Vos Marten H.
- Baroud Charles N.
Lab on a Chip, Royal Society of Chemistry , 2013, 13 (22), pp.4326-4330 . We present a new platform for the production and manipulation of microfluidic droplets in view of measuring the evolution of a chemical reaction. Contrary to existing approaches, our device uses gradients of confinement to produce a single drop on demand and guide it to a pre-determined location. In this way, two nanoliter drops containing different reagents can be placed in contact and merged together, in order to trigger a chemical reaction. The reaction rate is extracted from an analysis of the observed reaction-diffusion front. We show that the results obtained using this platform are in excellent agreement with stopped-flow measurements, while decreasing the sample consumption 5000 fold. We also show how the device operation can be parallelized in order to react an initial sample with a range of compounds or concentrations, on a single integrated chip. This integrated chip thus further reduces sample consumption while reducing the time required for the experimental runs from hours to minutes. (10.1039/c3lc50768h)
DOI : 10.1039/c3lc50768h
Primary processes in heme-based sensor proteins
- Liebl Ursula
- Lambry Jean-Christophe
- Vos Marten H.
Biochimica et Biophysica Acta Proteins and Proteomics, Elsevier , 2013, 1834 (9), pp.1684-1692 . A wide and still rapidly increasing range of heme-based sensor proteins has been discovered over the last two decades. At the molecular level, these proteins function as bistable switches in which the catalytic activity of an enzymatic domain is altered mostly by binding or dissociation of small gaseous ligands (O2, NO or CO) to the heme in a sensor domain. The initial "signal" at the heme level is subsequently transmitted within the protein to the catalytic site, ultimately leading to adapted expression levels of specific proteins. Making use of the photolability of the heme-ligand bond that mimics thermal dissociation, early processes in this intra-protein signaling pathway can be followed using ultrafast optical spectroscopic techniques; they also occur on timescales accessible to molecular dynamics simulations. Experimental studies performed over the last decade on proteins including the sensors FixL (O2), CooA (CO) and soluble guanylate cyclase (NO) are reviewed with an emphasis on emerging general mechanisms. After heme-ligand bond breaking, the ligand can escape from the heme pocket and eventually from the protein, or rebind directly to the heme. Remarkably, in all sensor proteins the rebinding, specifically of the sensed ligand, is highly efficient. This "ligand trap" property possibly provides means to smoothen the effects of fast environmental fluctuations on the switching frequency. For 6-coordinate proteins, where exchange between an internal heme-bound residue and external gaseous ligands occurs, the study of early processes starting from the unliganded form indicates that mobility of the internal ligand may facilitate signal transfer. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. Cop. 2013 Elsevier B.V. All rights reserved. (10.1016/j.bbapap.2013.02.025)
DOI : 10.1016/j.bbapap.2013.02.025
Configurational fluctuations and flavin-substrate interactions in the flavoenzyme ThyX studied by time- and spectrally resolved fluorescence
- Laptenok Sergey P.
- Bouzhir-Sima Latifa
- Myllykallio Hannu
- Liebl Ursula
- Vos Marten H.
EPJ Web of Conferences, EDP Sciences , 2013, 41, pp.07011 . Femtosecond-resolved fluorescence of bacterial thymidilate synthase using a Kerr-gate based setup identifies a close-by tyrosine involved in flavin fluorescence quenching, shows that the substrate dUMP acts as a strong quencher itself and highlights functional configurational flexibility. © Owned by the authors, published by EDP Sciences, 2013 (10.1051/epjconf/20134107011)
DOI : 10.1051/epjconf/20134107011
Numerical simulation of polarization-resolved second harmonic microscopy in birefringent media
- Gusachenko Ivan
- Schanne-Klein Marie-Claire
Physical Review A : Atomic, molecular, and optical physics [1990-2015], American Physical Society , 2013, 88 (5), pp.053811 . Polarization-resolved second-harmonic microscopy has recently emerged as a valuable technique for in situ imaging of collagen structure in tissues. Nevertheless, collagen-rich tissues such as tendon, ligament, skin dermis, bone, cornea, or artery exhibit a heterogeneous and anisotropic architecture that results in complex optical properties. While experimental evidence of polarization distortions has been reported in various tissues, the physics of second-harmonic imaging within such tissues is not fully understood yet. In this work, we performed numerical simulations of polarization-resolved second-harmonic generation in a strongly focused regime within a birefringent tissue. We show that vectorial components due to strong focusing have a rather small effect on the measurement of the second-harmonic tensorial response, while birefringence and optical dispersion may affect these measurements dramatically. We show indeed that a difference in the focal field distribution for ordinary and extraordinary waves results in different phase-matching conditions, which strongly affects the relative efficacy of second-harmonic generation for different polarizations. These results are of great interest for extracting reliable quantitative parameters from second-harmonic images. ©2013 American Physical Society (10.1103/PhysRevA.88.053811)
DOI : 10.1103/PhysRevA.88.053811
Microscopie multiphoton illuminée par nappe : imagerie de fluorescence rapide et en profondeur dans les tissus vivants
- Supatto Willy
Photoniques, EDP Sciences , 2012 (62), pp.33-37 . (10.1051/photon/20126233)
DOI : 10.1051/photon/20126233
Third-harmonic generation microscopy with Bessel beams: a numerical study
- Olivier Nicolas
- Débarre Delphine
- Mahou Pierre
- Beaurepaire Emmanuel
Optics Express, Optical Society of America - OSA Publishing , 2012, 20 (22), pp.24886-24902 . We study theoretically and numerically third-harmonic generation (THG) from model geometries (interfaces, slabs, periodic media) illuminated by Bessel beams produced by focusing an annular intensity profile. Bessel beams exhibit a phase and intensity distribution near focus different from Gaussian beams, resulting in distinct THG phase matching properties and coherent scattering directions. Excitation wave vectors are controlled by adjusting the bounding aperture angles of the Bessel beam. In addition to extended depth-of-field imaging, this opens interesting perspectives for coherent nonlinear microscopy, such as extracting sample spatial frequencies in the λ/8 - λ range in the case of organized media. © 2012 OSA (10.1364/OE.20.024886)
DOI : 10.1364/OE.20.024886
In situ 3D characterization of historical coatings and wood using multimodal nonlinear optical microscopy
- Latour Gaël
- Echard Jean-Philippe
- Didier Marie
- Schanne-Klein Marie-Claire
Optics Express, Optical Society of America - OSA Publishing , 2012, 20 (22), pp.24623-24635 . We demonstrate multimodal nonlinear optical imaging of historical artifacts by combining Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (2PEF) microscopies. We first identify the nonlinear optical response of materials commonly encountered in coatings of cultural heritage artifacts by analyzing one- and multi-layered model samples. We observe 2PEF signals from cochineal lake and sandarac and show that pigments and varnish films can be discriminated by exploiting their different emission spectral ranges as in luminescence linear spectroscopy. We then demonstrate SHG imaging of a filler, plaster, composed of bassanite particles which exhibit a non centrosymmetric crystal structure. We also show that SHG/2PEF imaging enables the visualization of wood microstructure through typically 60 µm-thick coatings by revealing crystalline cellulose (SHG signal) and lignin (2PEF signal) in the wood cell walls. Finally, in situ multimodal nonlinear imaging is demonstrated in a historical violin. SHG/2PEF imaging thus appears as a promising non-destructive and contactless tool for in situ 3D investigation of historical coatings and more generally for wood characterization and coating analysis at micrometer scale. © 2012 OSA (10.1364/OE.20.024623)
DOI : 10.1364/OE.20.024623
In situ three-dimensional monitoring of collagen fibrillogenesis using SHG microscopy.
- Bancelin Stéphane
- Aimé Carole
- Coradin Thibaud
- Schanne-Klein Marie-Claire
Biomedical optics express, Optical Society of America - OSA Publishing , 2012, 3 (6), pp.1446-54 . We implemented in situ time-lapse Second Harmonic Generation (SHG) microscopy to monitor the three-dimensional (3D) self-assembly of collagen in solution. As a proof of concept, we tuned the kinetics of fibril formation by varying the pH and measured the subsequent exponential increase of fibril volume density in SHG images. We obtained significantly different time constants at pH = 6.5 ± 0.3 and at pH = 7.5 ± 0.3. Moreover, we showed that we could focus on the growth of a single isolated collagen fibril because SHG microscopy is sensitive to well-organized fibrils with diameter below the optical resolution. This work illustrates the potential of SHG microscopy for the rational design and characterization of collagen-based biomaterials. (10.1364/BOE.3.001446)
DOI : 10.1364/BOE.3.001446
Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels.
- Creze Christophe
- Ligabue Alessio
- Laurent Sébastien
- Lestini Roxane
- Laptenok Sergey P.
- Khun Joelle
- Vos Marten H.
- Czjzek Mirjam
- Myllykallio Hannu
- Flament Didier
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2012, 287 (19), pp.15648-60 . Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. (10.1074/jbc.M112.346361)
DOI : 10.1074/jbc.M112.346361
Polarization-resolved second-harmonic generation in tendon upon mechanical stretching
- Gusachenko Ivan
- Tran Viet
- Goulam Houssen Yannick
- Allain Jean-Marc
- Schanne-Klein Marie-Claire
Biophysical Journal, Biophysical Society , 2012, 102 (9), pp.2220-2229 . Collagen is a triple-helical protein that forms various macromolecular organizations in tissues and is responsible for the biomechanical and physical properties of most organs. Second-harmonic generation (SHG) microscopy is a valuable imaging technique to probe collagen fibrillar organization. In this article, we use a multiscale nonlinear optical formalism to bring theoretical evidence that anisotropy of polarization-resolved SHG mostly reflects the micrometer-scale disorder in the collagen fibril distribution. Our theoretical expectations are confirmed by experimental results in rat-tail tendon. To that end, we report what to our knowledge is the first experimental implementation of polarization-resolved SHG microscopy combined with mechanical assays, to simultaneously monitor the biomechanical response of rat-tail tendon at macroscopic scale and the rearrangement of collagen fibrils in this tissue at microscopic scale. These experiments bring direct evidence that tendon stretching corresponds to straightening and aligning of collagen fibrils within the fascicle. We observe a decrease in the SHG anisotropy parameter when the tendon is stretched in a physiological range, in agreement with our numerical simulations. Moreover, these experiments provide a unique measurement of the nonlinear optical response of aligned fibrils. Our data show an excellent agreement with recently published theoretical calculations of the collagen triple helix hyperpolarizability. Copyright © 2012 Biophysical Society (10.1016/j.bpj.2012.03.068)
DOI : 10.1016/j.bpj.2012.03.068
Methodology for reconstructing early zebrafish development from in vivo multiphoton microscopy.
- Luengo-Oroz Miguel Angel
- Rubio-Guivernau José L.
- Faure Emmanuel
- Savy Thierry
- Duloquin Louise
- Olivier Nicolas
- Pastor-Escuredo David
- Ledesma-Carbayo María Jesús
- Debarre Delphine
- Bourgine Paul
- Beaurepaire Emmanuel
- Peyriéras Nadine
- Santos Andrés
IEEE Transactions on Image Processing, Institute of Electrical and Electronics Engineers , 2012, 21 (4), pp.2335-40 . Investigating cell dynamics during early zebrafish embryogenesis requires specific image acquisition and analysis strategies. Multiharmonic microscopy, i.e., second- and third-harmonic generations, allows imaging cell divisions and cell membranes in unstained zebrafish embryos from 1- to 1000-cell stage. This paper presents the design and implementation of a dedicated image processing pipeline (tracking and segmentation) for the reconstruction of cell dynamics during these developmental stages. This methodology allows the reconstruction of the cell lineage tree including division timings, spatial coordinates, and cell shape until the 1000-cell stage with minute temporal accuracy and micrometer spatial resolution. Data analysis of the digital embryos provides an extensive quantitative description of early zebrafish embryogenesis. (10.1109/TIP.2011.2177911)
DOI : 10.1109/TIP.2011.2177911
Ultrafast heme-ligand recombination in truncated hemoglobin HbO from Mycobacterium tuberculosis: A ligand cage
- Jasaitis Audrius
- Ouellet Hugues
- Lambry Jean-Christophe
- Martin Jean-Louis
- Friedman Joel M.
- Guertin Michel
- Vos Marten H.
Chemical Physics, Elsevier , 2012, 396, pp.10-16 . Truncated hemoglobin HbO from Mycobacterium tuberculosis displays very slow exchange of diatomic ligands with its environment. Using femtosecond spectroscopy, we show that upon photoexcitation, ligands rebind with unusual speed and efficiency. Only ∼1% O2 can escape from the heme pocket and less than 1% NO. Most remarkably, CO rebinding occurs for 95%, predominantly in 1.2 ns. The general CO rebinding properties are unexpectedly robust against changes in the interactions with close by aromatic residues Trp88 (G8) and Tyr36 (CD1). Molecular dynamics simulations of the CO complex suggest that interactions of the ligand with structural water molecules as well as its rotational freedom play a role in the high reactivity of the ligand and the heme. The slow exchange of ligands between heme and environment may result from a combination of hindered ligand access to the heme pocket by the network of distal aromatic residues, and low escape probability from the pocket. (10.1016/j.chemphys.2011.04.003)
DOI : 10.1016/j.chemphys.2011.04.003
Accuracy of correction in modal sensorless adaptive optics.
- Facomprez Aurélie
- Beaurepaire Emmanuel
- Débarre Delphine
Optics Express, Optical Society of America - OSA Publishing , 2012, 20 (3), pp.2598-612 . We investigate theoretically and experimentally the parameters governing the accuracy of correction in modal sensorless adaptive optics for microscopy. On the example of two-photon fluorescence imaging, we show that using a suitable number of measurements, precise correction can be obtained for up to 2 radians rms aberrations without optimising the aberration modes used for correction. We also investigate the number of photons required for accurate correction when signal acquisition is shot-noise limited. We show that only 10(4) to 10(5) photons are required for complete correction so that the correction process can be implemented with limited extra-illumination and associated photoperturbation. Finally, we provide guidelines for implementing an optimal correction algorithm depending on the experimental conditions.
Quaternary Structure Controls Ligand Dynamics in Soluble Guanylate Cyclase
- Yoo Byung-Kuk
- Lamarre Isabelle
- Martin Jean-Louis
- Negrerie Michel
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2012, 287, pp.6851-6859 . Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor. The mechanisms of activation and deactivation of this heterodimeric enzyme are unknown. For deciphering them, functional domains can be overexpressed. We have probed the dynamics of the diatomic ligands NO and CO within the isolated heme domain β1(190) of human sGC by piconanosecond absorption spectroscopy. After photo-excitation of nitrosylated sGC, only NO geminate rebinding occurs in 7.5 ps. In β1(190), both photo-dissociation of 5c-NO and photo-oxidation occur, contrary to sGC, followed by NO rebinding (7 ps) and back-reduction (230 ps and 2 ns). In full-length sGC, CO geminate rebinding to the heme does not occur. In contrast, CO geminately rebinds to β1(190) with fast multiphasic process (35, 171, and 18 ns). We measured the bimolecular association rates kon = 0.075 ± 0.01 × 106 M−1*s−1 for sGC and 0.83 ± 0.1 × 106 M−1*s−1 for β1(190). These different dynamics reflect conformational changes and less proximal constraints in the isolated heme domain with respect to the dimeric native sGC. We concluded that the α-subunit and the β1(191-619) domain exert structural strains on the heme domain. These strains are likely involved in the transmission of the energy and relaxation toward the activated state after Fe2+-His bond breaking. This also reveals the heme domain plasticity modulated by the associated domains and subunit. (10.1074/jbc.M111.299297)
DOI : 10.1074/jbc.M111.299297
Mechanistic and structural basis for inhibition of thymidylate synthase ThyX
- Basta Tamara
- Boum Yap
- Briffotaux Julien
- Becker Hubert F.
- Lamarre-Jouenne Isabelle
- Lambry Jean-Christophe
- Skouloubris Stéphane
- Liebl Ursula
- Graille Marc
- van Tilbeurgh Herman
- Myllykallio Hannu
Open Biology, Royal Society , 2012, 2 (10), pp.120120 . Nature has established two mechanistically and structurally unrelated families of thymidylate synthases that produce de novo thymidylate or dTMP, an essential DNA precursor. Representatives of the alternative flavin-dependent thymidylate synthase family, ThyX, are found in a large number of microbial genomes, but are absent in humans. We have exploited the nucleotide binding pocket of ThyX proteins to identify non-substrate-based tight-binding ThyX inhibitors that inhibited growth of genetically modified Escherichia coli cells dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. We also solved the crystal structure of a viral ThyX bound to 2-hydroxy-3-(4-methoxybenzyl)-1,4-naphthoquinone at a resolution of 2.6 angstrom. This inhibitor was found to bind within the conserved active site of the tetrameric ThyX enzyme, at the interface of two monomers, partially overlapping with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis. As essential ThyX proteins are found e. g. in Mycobacterium tuberculosis and Helicobacter pylori, our studies have also potential to pave the way towards the development of new anti-microbial compounds. (10.1098/rsob.120120)
DOI : 10.1098/rsob.120120
Adaptive Optics for Biomedical Microscopy
- Booth Martin J.
- Débarre Delphine
- Jesacher Alexander
Optics and photonics news, Optical Society of America - OSA Publishing , 2012, 23 (1), pp.22-29 . Over the last decade, researchers have applied adaptive optics--a technology that was originally conceived for telescopes--to high-resolution microscopy in order to overcome the problems caused by specimen-induced aberrations. © 2012 Optical Society of America (10.1364/OPN.23.1.000022)
DOI : 10.1364/OPN.23.1.000022
Photoinduced dynamics of oxyluciferin analogues: Unusual enol "super"photoacidity and evidence for keto-enol isomerization
- Solntsev K.M.
- Laptenok Sergey P.
- Naumov P.
Journal of the American Chemical Society, American Chemical Society , 2012, 134 (40), pp.16452-16455 . The first systematic pico-nanosecond time-resolved spectroscopic study of the firefly emitter oxyluciferin and two of its chemically modified analogues revealed that in the excited state the enol group is more acidic than the phenol group. The 6'-dehydroxylated derivative, in which only the 4-enolic hydroxyl proton is acidic, has an experimentally determined pK a* of 0.9 in dimethyl sulfoxide and an estimated pK a* of -0.3 in water. Moreover, this compound provided direct evidence that in a nonpolar, basic environment the keto form in the excited state can tautomerize into the enol, which subsequently undergoes excited-state proton transfer (ESPT) to produce enolate ion. This observation presents the first experimental evidence of excited-state keto-enol tautomerization of a firefly fluorophore, and it could be important in resolving the enol-keto conundrum related to the color-tuning mechanism of firefly bioluminescence. The 6'-dehydroxylated form of oxyluciferin adds a very rare case of a stable enol to the family of "super"photoacids. Cop. 2012 American Chemical Society. (10.1021/ja3045212)
DOI : 10.1021/ja3045212
Picosecond to second dynamics reveals a structural transition in clostridium botulinum no-sensor triggered by the activator BAY-41-2272
- Yoo Byung-Kuk
- Lamarre Isabelle
- Rappaport Fabrice
- Nioche Pierre
- Raman C.S.
- Martin Jean-Louis
- Negrerie Michel
ACS Chemical Biology, American Chemical Society , 2012, 7 (12), pp.2046-2054 . Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor that synthesizes cGMP upon NO activation. In synergy with the artificial allosteric effector BAY 41-2272 (a lead compound for drug design in cardiovascular treatment), sGC can also be activated by carbon monoxide (CO), but the structural basis for this synergistic effect are unknown. We recorded in the unusually broad time range from 1 ps to 1 s the dynamics of the interaction of CO binding to full length sGC, to the isolated sGC heme domain ß1(200) and to the homologous bacterial NO-sensor from Clostridium botulinum. By identifying all phases of CO binding in this full time range and characterizing how these phases are modified by BAY 41-2272, we show that this activator induces the same structural changes in both proteins. This result demonstrates that the BAY 41-2272 binding site resides in the ß1(200) sGC heme domain and is the same in sGC and in the NO-sensor from Clostridium botulinum. Cop. 2012 American Chemical Society. (10.1021/cb3003539)
DOI : 10.1021/cb3003539
Observing the Confinement Potential of Bacterial Pore-Forming Toxin Receptors Inside Rafts with Nonblinking Eu3+-Doped Oxide Nanoparticles
- Türkcan Silvan
- Masson Jean-Baptiste
- Casanova Didier
- Mialon Geneviève
- Gacoin Thierry
- Boilot Jean-Pierre
- Popoff Michel R.
Biophysical Journal, Biophysical Society , 2012, 102 (10), pp.2299-2308 . We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: α-toxin of Clostridium septicum and -toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30 nm for acquisition times of 51.3 ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 0.05 m2. Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 0.01 m2/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 0.03 pN/m at 37C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family. (10.1016/j.bpj.2012.03.072)
DOI : 10.1016/j.bpj.2012.03.072
Imaging and 3D morphological analysis of collagen fibrils
- Altendorf Hellen
- Decencière Etienne
- Jeulin Dominique
- de Sa Peixoto Paolo
- Deniset-Besseau Ariane
- Angelini E.
- Mosser G.
- Schanne-Klein Marie-Claire
Journal of Microscopy, Wiley , 2012, 247 (2), pp.161-175 . The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants. (10.1111/j.1365-2818.2012.03629.x)
DOI : 10.1111/j.1365-2818.2012.03629.x
Genomic binding of Pol III transcription machinery and relationship with TFIIS transcription factor distribution in mouse embryonic stem cells
- Carrière Lucie
- Graziani Sébastien
- Alibert Olivier
- Ghavi-Helm Yad
- Boussouar Fayçal
- Humbertclaude Hélène
- Jounier Sylvie
- Aude Jean-Christophe
- Keime Celine
- Murvai Janos
- Foglio Mario
- Gut Marta
- Gut Ivo
- Lathrop Mark
- Soutourina Julie
- Gérard Matthieu
- Werner Michel
Nucleic Acids Research, Oxford University Press , 2012, 40 (1), pp.270-283 . (10.1093/nar/gkr737)
DOI : 10.1093/nar/gkr737