Université PSL



Laboratoire :
Auteur :
Revue :
Année :
ViSP: tool for visualizing 3D super-resolution data
M. El Beheiry and M. Dahan
Nat. Methods - 10 :689–690 - doi: 10.1093/bioinformatics/btu720 - 2013
Quantitative nanoscopy at inhibitory synapses: counting gephyrin molecules and receptor binding
Christian G. Specht, Ignacio Izeddin, Pamela C. Rodriguez, Mohamed El Beheiry, Philippe Rostaing, Xavier Darzacq, Maxime Dahan and Antoine Triller
Neuron - 79(2) :308-21 - DOI:10.1016/j.neuron.2013.05.013 - 2013
The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. However, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted three-dimensional and quantitative nanoscopic imaging based on single-molecule detections to characterize the ultrastructure of inhibitory synapses and to count scaffold proteins and receptor binding sites. We observed a close correspondence between the spatial organization of gephyrin scaffolds and glycine receptors at spinal cord synapses. Endogenous gephyrin was clustered at densities of 5,000–10,000 molecules/μm2. The stoichiometry between gephyrin molecules and receptor binding sites was approximately 1:1, consistent with a two-dimensional scaffold in which all gephyrin molecules can contribute to receptor binding. The competition of glycine and GABAA receptor complexes for synaptic binding sites highlights the potential of single-molecule imaging to quantify synaptic plasticity on the nanoscopic scale.
Subcellular control of Rac signalling by magnetogenetic manipulation in living cells
F. Etoc, D. Lisse, Y. Bellaiche, J. Piehler, M. Coppey and M. Dahan
Nat. Nanotechnol. - 8(3) :193-8 - DOI:10.1038/nnano.2013.23 - 2013
Many cell functions rely on the coordinated activity of signalling pathways at a subcellular scale. However, there are few tools capable of probing and perturbing signalling networks with a spatial resolution matching the intracellular dimensions of their activity patterns. Here we present a generic magnetogenetic approach based on the self-assembly of signalling complexes on the surface of functionalized magnetic nanoparticles inside living cells. The nanoparticles act as nanoscopic hot spots that can be displaced by magnetic forces and trigger signal transduction pathways that bring about a cell response. We applied this strategy to Rho-GTPases, a set of molecular switches known to regulate cell morphology via complex spatiotemporal patterns of activity. We demonstrate that the nanoparticle-mediated activation of signalling pathways leads to local remodelling of the actin cytoskeleton and to morphological changes.
Amplification and temporal filtering during gradient sensing by nerve growth cones probed with a microfluidic assay
M. Morel, V. Shynkar, J.-C. Galas, I. Dupin, C. Bouzigues, V. Studer and M. Dahan
Biophys. J. - 103(8) :1648-56 - DOI:10.1016/j.bpj.2012.08.040 - 2013
Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. To ensure a sensitive and robust response to directional signals in complex and dynamic chemical landscapes, GCs are presumably able to amplify and filter external information. How these processing tasks are performed remains however poorly known. Here, we probe the signal-processing capabilities of single GCs during γ-Aminobutyric acid (GABA) directional sensing with a shear-free microfluidic assay that enables systematic measurements of the GC output response to variable input gradients. By measuring at the single molecule level the polarization of GABA(A) chemoreceptors at the GC membrane, as a function of the external GABA gradient, we find that GCs act as i), signal amplifiers over a narrow range of concentrations, and ii), low-pass temporal filters with a cutoff frequency independent of stimuli conditions. With computational modeling, we determine that these systems-level properties arise at a molecular level from the saturable occupancy response and the lateral dynamics of GABA(A) receptors.
Surface functionalization of COC microfluidic materials by plasma and click chemistry processes
Y. Ladner, F. D’orlye, C. Perrard, B. Da Silva, C. Guyon, M. Tatoulian, S. Griveau, F. Bedioui & A. Varenne
Plasma Process - 10(11) :959-69 - DOI:10.1002/ppap.201300066 - 2013
A robust method for COC surfaces functionalization was developed for the first time by plasma polymerization. 1-bromopropane in the vapor phase allowed the formation of a brominated deposit on COC surfaces, which stability, homogeneity and chemical nature were evaluated for different experimental conditions. The analysis of vapor phase was achieved by mass spectrometry to control brominated precursor fragmentation. Following nucleophilic exchange in the presence of NaN3 to convert the brominated surface to azide-bearing surface, these modified COC were further functionalized with a fluorescent alkyne, via the copper-catalyzed azide–alkyne cycloaddition reaction, i.e. “click” reaction. Surface modifications were characterized by water contact angle measurement, ellipsometry, electrochemical microscopy, XPS, IR and fluorescence microscopy. This new process was proved to be efficient and stable in time up to 7 days.
Catkin liked nano-Co3O4 catalyst built-in organic microreactor by PEMOCVD method for trace CO oxidation at room temperature
G. L. Chen,C. Guyon,Z. X. Zhang,B. Da Silva,P. Da Costa,S. Ognier,D. Bonn,M. Tatoulian
Microfluidics and Nanofluidics - 16(1-2) :141-148 - DOI:10.10.1007/s10404-013-1220-y - 2013
In this paper, tricobalt tetraoxide (Co3O4) catalyst was coated on the polydimethylsiloxane microchannel by the plasma-enhanced metal-organic chemical vapor deposition technology. The obtained Co3O4 film was characterized by SEM, XRD, XPS, and TEM, and the results show that the as-deposited Co3O4 film was initially composed of many cauliflowers-shaped microclusters. Also, the microcauliflower was transformed from an amorphous phase to a crystal phase when the Co3O4 film was treated by Ar and O2 plasma for more than 20 min, and the crystal lattice line occurred on the surface of nano-sized-Co3O4 particles. Meanwhile, the interface of Co3O4 particles with diameter between 3 and 12 nm became obvious and some nano-catkin structures were also formed on the Co3O4 film. The ratio of Co3+/Co2+ in the spinel-type Co3O4 was nearly 2, and the nano-particles predominantly expose their {311}, {111}, and {220} planes. These morphologies and structure characteristics were found to be ideal for increasing the catalytic activity efficiency of Co3O4 for CO oxidation, and the catalytic stability of Co3O4 coated on the organic microreactor lasted nearly 85 h for trace CO oxidation at room temperature.
he different structure characteristics of nanosized Co3O 4 film crystallized by the annealing and plasma techniques
G.L. Chen, C. Guyon, Z.X., Zhang, S. Ognier, J. Beem, M.Tatoulian
Microfluidics and Nanofluidics - 107 :1111 – 114 - DOI:10.1016/j.matlet.2013.05.071 - 2013
In this study, we deposited nano-Co3O4 film on silicon substrate using plasma-enhanced metal—organic chemical vapor deposition (PEMOCVD), and the structure difference of Co3O4 crystallized by the annealing and the Ar/O2 plasma techniques were explored by SEM, TEM, XRD, and XPS. Compared to the net morphology of Co3O4 film treated with high calcinations temperature, the cauliflowers-shaped micro-clusters were changed to nano-catkin when the sample was treated with Ar and O2 plasma for 40 min. Additionally, both samples (annealed and plasma-treated) showed the formation of both the {311} and {220} planes. The surface richness of active Co3+ sites on the exposed {220} plane indicated that the as-deposited nano-Co3O4 films have potential catalytic properties for CO and hydrocarbon oxidation.
Binding parameters between an aptamer and its target by frontal analysis continuous microchip electrophoresis (FACMCE): a comprehensive study of buffer composition and thermal treatment in the case of lysozyme
M. Girardot, H-Y. Li, S. Descroix, A. Varenne
Chromatographia - 76 305-312 - DOI: 10.1007/s10337-012-2346-x - 2013
An original and simple methodology based on microchip electrophoresis (MCE) in a continuous frontal analysis mode (named frontal analysis continuous microchip electrophoresis, FACMCE) was developed for the simultaneous determination of the binding parameters, i.e. ligand-site dissociation constant (k(d)) and number of binding sites on the substrate (n). This simultaneous determination was exemplified with the interaction between an aptamer and its target. The selected target is a strongly basic protein, lysozyme, as its quantification is of great interest due to its antimicrobial and allergenic properties. A glass microdevice equipped with a fluorescence detection system was coated with hydroxypropylcellulose, reducing the electroosmotic flow and adsorption onto the channel walls. This microdevice allowed the continuous electrokinetic injection of a mixture of fluorescently labelled aptamer and non-labelled lysozyme. By determining the concentration of the free fluorescently labelled aptamer thanks to its corresponding plateau height, mathematical linearization methods allowed to determine a k(d) value of 48.4±8.0 nM, consistent with reported results (31 nM), while the average number of binding sites n on lysozyme, never determined before, was 0.16±0.03. These results seem to indicate that the buffer nature and the SELEX process should influence the number and affinity of the binding sites. In parallel it has been shown that the binding between lysozyme and its aptamer presents two sites of different binding affinities.
Aptamer-conjugated nanoparticles: Preservation of targeting functionality demonstrated by microchip electrophoresis in frontal mode
M. Girardot, F. d'Orlye, S. Descroix, A. Varenne.
Analytical Biochemistry - 435 150-152 - PMID:23333271 - 2013
Aptamer-conjugated nanoparticles (Apt-NPs) are increasingly being developed for biomedical purposes and especially for diagnosis and therapy. However, there is no quantitative study of the targeting functionality of such grafted aptamers compared with free aptamers. Thus, we report the first determination of binding parameters for Apt-NP/target complexes, thanks to a continuous frontal analysis in a microchip electrophoresis format (named FACMCE), based on a methodology previously developed by our group. As a model system, the targeting ability of a lysozyme-binding aptamer conjugated to fluorescent maghemite nanoparticles was evaluated and showed evidence that the conjugation does not alter the affinity of this aptamer.
Electrokinetic characterization of superparamagnetic nanoparticle–aptamer conjugates: design of new highly specific probes for miniaturized molecular diagnostics
M. Girardot, F. d'Orlye, A. Varenne
Anal. Bioanal. Chem - 406(4) :1089-98 - DOI:10.1007/s00216-013-7265-7 - 2013
With the view of designing new nanoparticle (NP)-aptamer conjugates and proving their suitability as biorecognition tools for miniaturized molecular diagnostics, new maghemite-silica core-shell NP-aptamer conjugates were characterized for the first time in terms of grafting rate and colloidal stability under electrophoretic conditions using capillary electrophoresis. After the grafting rate (on the order of six to 50) of the lysozyme-binding aptamer had been estimated, the electrophoretic stability and peak dispersion of the resulting oligonucleotide-NP conjugates were estimated so as to determine the optimal separation conditions in terms of buffer pH, ionic strength and nature, as well as temperature and electric field strength. The effective surface charge density of the NPs was close to zero for pH lower than 5, which led to some aggregation. The NPs were stable in the pH range from 5 to 9, and an increase in electrophoretic mobility was evidenced with increasing pH. Colloidal stability was preserved at physiological pH for both non-grafted NPs and grafted NPs in the 10-100 mM ionic strength range and in the 15-60 °C temperature range. A strong influence of the nature of the buffer counterion on NP electrophoretic mobility and peak dispersion was evidenced, thus indicating some interactions between buffer components and NP-aptamer conjugates. Whereas an electric field effect (50-900 V cm(-1)) on NP electrophoretic mobility was evidenced, probably linked to counterion dissociation, temperature seems to have an appreciable effect on the zeta potential and aptamer configuration as well. This information is crucial for estimating the potentialities of such biorecognition tools in electrophoretic systems.

405 publications.