Détection de nano-objets individuels dans des cellules : étude de la structure dynamique de la synapse de neurones vivants et développement de l'imagerie par contraste interférentiel photothermique.

La circulation des récepteurs au glutamate entre les trois compartiments cellulaires, que sont les membranes synaptiques, extra-synaptiques, et le cytoplasme, joue un rôle crucial dans l'activité synaptique. Récemment encore, les études se concentraient sur les processus d'endo et d'exocytose, et sur la diffusion latérale des récepteurs extra-synaptiques. La microscopie de fluorescence de molécules uniques permet de suivre les récepteurs sur toute la surface des neurones. Le suivi des GluR2, sous unité des récepteurs au glutamate de type AMPA, à la surface de neurones vivants a montré que la structure synaptique est dynamique, et que les phénomènes de plasticité post-synaptique sont corrélés avec des modifications de l'état diffusif des récepteurs synaptiques. Afin d'accéder à la stoechiométrie du nombre de récepteurs assemblés, dans des zones telles que la synapse ou les vésicules, une méthode de quantification robuste et applicable à des assemblées de protéines de taille quelconque est mise au point. La durée de suivi des biomolécules, en microscopie de molécules uniques, est limitée par le photoblanchiment rapide des fluorophores. Pour y remédier, une nouvelle technique de visualisation, basée sur l'effet photothermique, a été développée et appliquée à l'imagerie de systèmes biologiques.The trafficking of the glutamate receptors between the three cellular compartments which are the synaptic membrane, the extra-synaptic membrane and the cytoplasm plays a major role in synaptic activity. Till recently, the studies were focused on endocytosis, exocytotis, and lateral diffusion of extra-synaptic receptors. The microscopy of single molecule has enabled us to follow the receptors all along the neurons surface. The tracking of the AMPA receptor subunit called GluR2 on the surface of living neurons lead us to conclude that the synaptic structure is dynamic, and that postsynaptic plasticity processes are correlated to changes on the diffusive state of the synaptic receptors. In order to quantify the number of receptors gathered in place like the synapses or the vesicles, a robust method applicable to proteins assemblies of any size was established. The duration of biomolecules tracking thanks to single molecule microscopy is limited by the fast photobleaching of the fluorophores. To overcome this problem, a new imaging technique based on the phothermal effect has been developed and applied to image biological samples

Dependence of DNA persistence length on ionic strength of solutions with monovalent and divalent salts: a joint theory-experiment study

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

Influence of the Experimental Set-Up on Single Molecule DNA Dynamics When Analyzed by Tethered Particle Motion

International audienc

A model for the molecular organisation of the IS911 transpososome

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

Probing DNA conformational changes with high temporal resolution by Tethered Particle Motion

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.Comment: Improved version, to appear in Physical Biology. 10 pages + 10 pages of supporting materia

Post-replicative pairing of sister ter regions in Escherichia coli involves multiple activities of MatP

Funder: Université Toulouse III - Paul Sabatier (University of Toulouse III Paul Sabatier); doi: https://doi.org/10.13039/501100009160Abstract: The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division