Catherine Tardin

IPBS - Institut de Pharmacologie et de Biologie Structurale, Tolosa de Llenguadoc, Midi-Pyrénées, France

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Publications (16)70.16 Total impact

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    ABSTRACT: Using high-throughput Tethered Particle Motion single molecule experiments, the double-stranded DNA persistence length, $L_p$, is measured in solutions with Na$^+$ and Mg$^{2+}$ ions of various ionic strengths, $I$. Several theoretical equations for $L_p(I)$ are fitted to the experimental data, but no decisive theory is found which fits all the $L_p$ values for the two ion valencies. Properly extracted from the particle trajectory using simulations, $L_p$ varies from 30~nm to 55~nm, and is compared to previous experimental results. For the Na$^+$ only case, $L_p$ is an increasing concave function of $I^{-1}$, well fitted by Manning's electrostatic stretching approach, but not by classical Odjik-Skolnick-Fixman theories with or without counter-ion condensation. With added Mg$^{2+}$ ions, $L_p$ shows a marked decrease at low $I$, interpreted as an ion-ion correlation effect, with an almost linear law in $I^{-1}$, fitted by a proposed variational approach.
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    ABSTRACT: Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high-throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven 6A-tracts, we find that the sequence CA6CGG induces a bend angle of 19° ± 4°. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Nucleic Acids Research 03/2015; DOI:10.1093/nar/gkv201 · 8.81 Impact Factor
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    Serge Mazeres, Etienne Joly, Andre Lopez, Catherine Tardin
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    ABSTRACT: Microdomains corresponding to localized partition of lipids between ordered and less ordered environments are the subject of intensive investigations, because of their putative participation in modulating cellular responses. One popular approach in the field consists in labelling membranes with solvatochromic fluorescent probes such as laurdan and C-laurdan. In this report, we describe a high-yield procedure for the synthesis of laurdan, C-laurdan and two new fluorophores, called MoC-laurdan and M-laurdan, as well as their extensive photophysical characterization. We find that the latter probe, M-laurdan, is particularly suited to discriminate lipid phases independently of the chemical nature of the lipids, as measured by both fluorescence Generalized Polarization (GP) and anisotropy in large unilamellar vesicles made of various lipid compositions. In addition, staining of live cells with M-laurdan shows a good stability over time without any apparent toxicity, as well as a wider distribution in the various cell compartments than the other probes.
    11/2014; 3:172. DOI:10.12688/f1000research.4805.2
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    ABSTRACT: Circular chromosomes can form dimers during replication and failure to resolve those into monomers prevents chromosome segregation, which leads to cell death. Dimer resolution is catalysed by a highly conserved site-specific recombination system, called XerCD-dif in Escherichia coli. Recombination is activated by the DNA translocase FtsK, which is associated with the division septum, and is thought to contribute to the assembly of the XerCD-dif synapse. In our study, direct observation of the assembly of the XerCD-dif synapse, which had previously eluded other methods, was made possible by the use of Tethered Particle Motion, a single molecule approach. We show that XerC, XerD and two dif sites suffice for the assembly of XerCD-dif synapses in absence of FtsK, but lead to inactive XerCD-dif synapses. We also show that the presence of the γ domain of FtsK increases the rate of synapse formation and convert them into active synapses where recombination occurs. Our results represent the first direct observation of the formation of the XerCD-dif recombination synapse and its activation by FtsK.
    Nucleic Acids Research 11/2013; 42(3). DOI:10.1093/nar/gkt1024 · 8.81 Impact Factor
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    Biophysical Journal 01/2013; 104(2):76-. DOI:10.1016/j.bpj.2012.11.455 · 3.83 Impact Factor
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    ABSTRACT: The invention relates to biochips 1 comprising a substrate 2, wherein said substrate comprises at the surface thereof isolated regions 3 for the anchoring of a nucleic acid molecule, said isolated regions having an area of less than 1 μm2, and the space 4 between two isolated regions being at least equal to the square root of the value of said area of said isolated regions.
    World Patent Information 07/2012;
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    ABSTRACT: Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA-protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.
    Nucleic Acids Research 03/2012; 40(12):e89. DOI:10.1093/nar/gks250 · 8.81 Impact Factor
  • Biophysical Journal 01/2012; 102(3):383-. DOI:10.1016/j.bpj.2011.11.2095 · 3.83 Impact Factor
  • ChemPhysChem 11/2011; 12(16):2915-9. DOI:10.1002/cphc.201100616 · 3.36 Impact Factor
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    ABSTRACT: The tethered particle motion (TPM) technique informs about conformational changes of DNA molecules, e.g. upon looping or interaction with proteins, by tracking the Brownian motion of a particle probe tethered to a surface by a single DNA molecule and detecting changes of its amplitude of movement. We discuss in this context the time resolution of TPM, which strongly depends on the particle-DNA complex relaxation time, i.e. the characteristic time it takes to explore its configuration space by diffusion. By comparing theory, simulations and experiments, we propose a calibration of TPM at the dynamical level: we analyze how the relaxation time grows with both DNA contour length (from 401 to 2080 base pairs) and particle radius (from 20 to 150 nm). Notably we demonstrate that, for a particle of radius 20 nm or less, the hydrodynamic friction induced by the particle and the surface does not significantly slow down the DNA. This enables us to determine the optimal time resolution of TPM in distinct experimental contexts which can be as short as 20 ms.
    Physical Biology 12/2010; 7(4):046003. DOI:10.1088/1478-3975/7/4/046003 · 3.14 Impact Factor
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    ABSTRACT: Tight regulation of transposition activity is essential to limit damage transposons may cause by generating potentially lethal DNA rearrangements. Assembly of a bona fide protein-DNA complex, the transpososome, within which transposition is catalysed, is a crucial checkpoint in this regulation. In the case of IS911, a member of the large IS3 bacterial insertion sequence family, the transpososome (synaptic complex A; SCA) is composed of the right and left inverted repeated DNA sequences (IRR and IRL) bridged by the transposase, OrfAB (the IS911-encoded enzyme that catalyses transposition). To characterise further this important protein-DNA complex in vitro, we used different tagged and/or truncated transposase forms and analysed their interaction with IS911 ends using gel electrophoresis. Our results allow us to propose a model in which SCA is assembled with a dimeric form of the transposase. Furthermore, we present atomic force microscopy results showing that the terminal inverted repeat sequences are probably assembled in a parallel configuration within the SCA. These results represent the first step in the structural description of the IS911 transpososome, and are discussed in comparison with the very few other transpososome examples described in the literature.
    Mobile DNA 06/2010; 1(1):16. DOI:10.1186/1759-8753-1-16 · 2.43 Impact Factor
  • Biophysical Journal 01/2010; 98(3). DOI:10.1016/j.bpj.2009.12.985 · 3.83 Impact Factor
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    ABSTRACT: Using single-molecule microscopy, we present a method to quantify the number of single autofluorescent proteins when they cannot be optically resolved. This method relies on the measurement of the total intensity emitted by each aggregate until it photobleaches. This strategy overcomes the inherent problem of blinking of green fluorescent proteins. In the case of small protein aggregates, our method permits us to describe the mean composition with a precision of one protein. For aggregates containing a large number of proteins, it gives access to the average number of proteins gathered and a signature of the inhomogeneity of the aggregates' population. We applied this methodology to the quantification of small purified citrine multimers.
    Journal of Biomedical Optics 05/2008; 13(3):031216. DOI:10.1117/1.2940600 · 2.75 Impact Factor
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    ABSTRACT: We performed a visualization of membrane proteins labeled with 10-nm gold nanoparticles in cells, using an all-optical method based on photothermal interference contrast. The high sensitivity of the method and the stability of the signals allows 3D imaging of individual nanoparticles without the drawbacks of photobleaching and blinking inherent to fluorescent markers. A simple analytical model is derived to account for the measurements of the signal amplitude and the spatial resolution. The photothermal interference contrast method provides an efficient, reproducible, and promising way to visualize low amounts of proteins in cells by optical means.
    Proceedings of the National Academy of Sciences 10/2003; 100(20):11350-5. DOI:10.1073/pnas.1534635100 · 9.81 Impact Factor
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    ABSTRACT: Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra-synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single-molecule fluorescence microscopy. Inside individual synapses, we found immobile and mobile receptors, which display restricted diffusion. Extra-synaptic receptors display free diffusion. Receptors could also exchange between these membrane compartments through lateral diffusion. Glutamate application increased both receptor mobility inside synapses and the fraction of mobile receptors present in a juxtasynaptic region. Block of inhibitory transmission to favor excitatory synaptic activity induced a transient increase in the fraction of mobile receptors and a decrease in the proportion of juxtasynaptic receptors. Altogether, our data show that rapid exchange of receptors between a synaptic and extra-synaptic localization occurs through regulation of receptor diffusion inside synapses.
    The EMBO Journal 10/2003; 22(18):4656-65. DOI:10.1093/emboj/cdg463 · 10.75 Impact Factor
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Publication Stats

463 Citations
70.16 Total Impact Points

Institutions

  • 2011–2014
    • IPBS - Institut de Pharmacologie et de Biologie Structurale
      Tolosa de Llenguadoc, Midi-Pyrénées, France
  • 2012
    • Arizona State University
      Phoenix, Arizona, United States
  • 2003–2012
    • French National Centre for Scientific Research
      • Laboratoire de Microbiologie et Génétique Moléculaires
      Lutetia Parisorum, Île-de-France, France
  • 2008
    • University of Bordeaux
      Burdeos, Aquitaine, France