Articles

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
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
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
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
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
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
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
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
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
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
Non-invasive monitoring of cell metabolism and lipid production in 3D engineered human adipose tissues using label-free multiphoton microscopy
- Chang Tyler
- Zimmerley Maxwell
- Quinn Kyle P.
- Lamarre-Jouenne Isabelle
- Kaplan David
- Beaurepaire Emmanuel
- Georgakoudi Irene
Biomaterials, Elsevier , 2013, 34 (34), pp.8607-8616 . Non-linear optical microscopy methods can characterize over time multiple functional properties of engineered tissues during development. Here, we demonstrate how the combined use of third-harmonic generation (THG) and two-photon excited fluorescence (2PEF) imaging can provide direct quantitative biomarkers of adipogenic stem cell differentiation and metabolic state, respectively. Specifically, we imaged over nine weeks silk scaffolds embedded with human mesenchymal stem cells and exposed to either propagation (PM) or adipogenic differentiation media (AM). THG was employed to visualize the formation of lipid droplets. 2PEF was used to assess the metabolic state of the cells through the redox ratio defined based on the endogenous FAD and NADH fluorescence. The redox ratio of cells in the AM scaffold was significantly lower than that in the PM scaffold during week 5 and 9, and correlated with significant increases in lipid-to-cell volume ratio, and number and size of lipid droplets in the AM scaffold. These findings indicate that the decrease in redox ratio during adipogenic differentiation is associated with fatty acid synthesis and lipid accumulation. Our methods therefore enabled us to identify and measure dynamic correlations between lipid droplet formation and cell metabolic state, while providing insight on the spatial heterogeneity of the observed signals. (10.1016/j.biomaterials.2013.07.066)
DOI : 10.1016/j.biomaterials.2013.07.066
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
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
Crystal structure of Cex1p reveals the mechanism of tRNA trafficking between nucleus and cytoplasm
- Nozawa Kayo
- Ishitani Ryuichiro
- Yoshihisa Tohru
- Sato Mamoru
- Arisaka Fumio
- Kanamaru Shuji
- Dohmae Naoshi
- Mangroo Dev
- Senger Bruno
- Becker Hubert F.
- Nureki Osamu
Nucleic Acids Research, Oxford University Press , 2013, 41 (6), pp.3901-3914 . In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1alpha. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 A resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal alpha-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p.Gsp1p) machinery. These results provide the structural basis of Los1p.Gsp1p.Cex1p.tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus. (10.1093/nar/gkt010)
DOI : 10.1093/nar/gkt010
Arbitrary-detuning asynchronous optical sampling pump-probe spectroscopy of bacterial reaction centers
- Antonucci Laura
- Bonvalet Adeline
- Solinas Xavier
- Jones Mickael R.
- Vos Marten H.
- Joffre Manuel
Optics Letters, Optical Society of America - OSA Publishing , 2013, 38 (17), pp.3322-3324 . A recently reported variant of asynchronous optical sampling compatible with arbitrary unstabilized laser repetition rates is applied to pump-probe spectroscopy. This makes possible the use of a 5.1 MHz chirped pulse oscillator as the pump laser, thus extending the available time window to almost 200 ns with a time resolution as good as about 320 fs. The method is illustrated with the measurement in a single experiment of the complete charge transfer dynamics of the reaction center from Rhodobacter sphaeroides. © 2013 Optical Society of America (10.1364/OL.38.003322)
DOI : 10.1364/OL.38.003322
Picosecond binding of the his ligand to four-coordinate heme in cytochrome c ': A one-way gate for releasing proximal NO
- Yoo Byung-Kuk
- Lamarre Isabelle
- Martin Jean-Louis
- Andrew Colin R.
- Negrerie Michel
Journal of the American Chemical Society, American Chemical Society , 2013, 135 (8), pp.3248-3254 . We provide a direct demonstration of a "kinetic trap" mechanism in the proximal 5-coordinate heme-nitrosyl complex (5c-NO) of cytochrome c' from Alcaligenes xylosoxidans (AXCP) in which picosecond rebinding of the endogenous His ligand following heme-NO dissociation acts as a one-way gate for the release of proximal NO into solution. This demonstration is based upon picosecond transient absorption changes following NO photodissociation of the proximal 5c-NO AXCP complex. We have determined the absolute transient absorption spectrum of 4-coordinate ferrous heme to which NO rebinds with a time constant tNO = 7 ps (kNO = 1.4 × 1011 s-1) and shown that rebinding of the proximal histidine to the 4-coordinate heme takes place with a time constant tHis = 100 ± 10 ps (kHis = 1010 s-1) after the release of NO from the proximal heme pocket. This rapid His reattachment acts as a one-way gate for releasing proximal NO by precluding direct proximal NO rebinding once it has left the proximal heme pocket and requiring NO rebinding from solution to proceed via the distal heme face. Cop. 2013 American Chemical Society. (10.1021/ja312140f)
DOI : 10.1021/ja312140f
Achievement of cornea-like organizations in dense collagen I solutions: clues to the physico-chemistry of cornea morphogenesis
- de Sa Peixoto Paulo
- Deniset-Besseau Ariane
- Schmutz Marc
- Anglo Anny
- Illoul Corinne
- Schanne-Klein Marie-Claire
- Mosser Gervaise
Soft Matter, Royal Society of Chemistry , 2013, 9 (47), pp.11241-11248 . Multiphoton and electron microscopic analyses show that acido-soluble collagen I prepared in 5 mM acetic acid (pH 3.5) at concentration above 45 mg mL−1 spontaneously generates liquid crystal phases mimicking plywood organization found in cornea tissues. Those organizations extend for several hundred micrometers. Transmission electron microscopy reveals the presence of small nanofibrils organized in a complex phase, coupling overall smectic and cholesteric organizations together with local order. Those nanofibrils could be the mesogen elements giving rise to this plywood organization. These data provide clues to physico-chemical events that may take place in cornea morphogenesis in vivo. This result is invaluable for bioengineering fields, as this liquid crystal organization paves the way for the generation of collagen based bio-mimetic cornea matrices. (10.1039/c3sm52097h)
DOI : 10.1039/c3sm52097h
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.
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
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
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