Christophe Thibault
Hoffmann-La Roche
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Publication
Featured researches published by Christophe Thibault.
Proceedings of the National Academy of Sciences of the United States of America | 2010
Anna Labernadie; Christophe Thibault; Christophe Vieu; Isabelle Maridonneau-Parini; Guillaume M. Charrière
Podosomes are unique cellular entities specifically found in macrophages and involved in cell–matrix interactions, matrix degradation, and 3D migration. They correspond to a core of F-actin surrounded at its base by matrix receptors. To investigate the structure/function relationships of podosomes, soft lithography, atomic force microscopy (AFM), and correlative fluorescence microscopy were used to characterize podosome physical properties in macrophages differentiated from human blood monocytes. Podosome formation was restricted to delineated areas with micropatterned fibrinogen to facilitate AFM analyses. Podosome height and stiffness were measured with great accuracy in living macrophages (578 ± 209 nm and 43.8 ± 9.3 kPa) and these physical properties were independent of the nature of the underlying matrix. In addition, time-lapse AFM revealed that podosomes harbor two types of overlapping periodic stiffness variations throughout their lifespan, which depend on F-actin and myosin II activity. This report shows that podosome biophysical properties are amenable to AFM, allowing the study of podosomes in living macrophages at nanoscale resolution and the analysis of their intimate dynamics. Such an approach opens up perspectives to better understand the mechanical functionality of podosomes under physiological and pathological contexts.
Langmuir | 2010
Christophe Thibault; Gábor Molnár; Lionel Salmon; Azzedine Bousseksou; Christophe Vieu
Microtransfer molding has been used to fabricate homogeneous micropatterns and nanopatterns of spin crossover nanoparticles of [Fe(NH(2)trz)](tos)(2) over a large area. We show that the use of an aprotic solvent (n-octane) may lead to successful results. Very well organized micropatterns are obtained, showing spin crossover phenomenon. Dark field optical and AFM images and Raman microspectrometry results are reported.
Nature Communications | 2014
Anna Labernadie; Anaı̈s Bouissou; Patrick Delobelle; Stéphanie Balor; Raphaël Voituriez; Amsha Proag; Isabelle Fourquaux; Christophe Thibault; Christophe Vieu; Renaud Poincloux; Guillaume M. Charrière; Isabelle Maridonneau-Parini
Podosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force.
Nanotechnology | 2011
Etienne Dague; Eric Jauvert; Loïc Laplatine; B. Viallet; Christophe Thibault; Laurence Ressier
Immobilization of live micro-organisms on solid substrates is an important prerequisite for atomic force microscopy (AFM) bio-experiments. The method employed must immobilize the cells firmly enough to enable them to withstand the lateral friction forces exerted by the tip during scanning but without denaturing the cell interface. In this work, a generic method for the assembly of living cells on specific areas of substrates is proposed. It consists in assembling the living cells within the patterns of microstructured, functionalized poly-dimethylsiloxane (PDMS) stamps using convective/capillary deposition. This versatile approach is validated by applying it to two systems of foremost importance in biotechnology and medicine: Saccharomyces cerevisiae yeasts and Aspergillus fumigatus fungal spores. We show that this method allows multiplexing AFM nanomechanical measurements by force spectroscopy on S. cerevisiae yeasts and high-resolution AFM imaging of germinated Aspergillus conidia in buffer medium. These two examples clearly demonstrate the immense potential of micro-organism assembly on functionalized, microstructured PDMS stamps by convective/capillary deposition for performing rigorous AFM bio-experiments on living cells.
Nanotechnology | 2012
Sven Salomon; Thierry Leïchlé; Denis Dezest; Florent Seichepine; Samuel Guillon; Christophe Thibault; Christophe Vieu; Liviu Nicu
The biofunctionalization of nanoelectromechanical systems (NEMS) is critical for the development of new classes of biosensors displaying improved performance and higher levels of integration. In this paper we propose a modified microcontact process (μCP) in order to biofunctionalize arrays of NEMS with a probe molecule on the active sensing areas together with an anti-fouling layer on the passive areas in a single, self-aligned step. We demonstrate the adequate functionalization/anti-fouling of arrays of freestanding nanocantilevers as dense as 10(5) nanostructures cm(-2) by using both fluorescence microscopy and dynamic measurements of the structures resonant frequency. The proper bioactivity of an antibody deposited onto the cantilevers and the blocking property of a bovine serum albumin layer are both assessed by incubating specific and non-specific tagged secondary antibodies followed by fluorescence imaging. Furthermore, measurement of the resonant frequency of the nanocantilevers before and after functionalization and biological recognition demonstrate that using μCP for device functionalization does not damage the nanostructures and preserves the mechanical sensing capability of our NEMS.
ACS Nano | 2015
Amsha Proag; Anaïs Bouissou; Thomas Mangeat; Raphaël Voituriez; Patrick Delobelle; Christophe Thibault; Christophe Vieu; Isabelle Maridonneau-Parini; Renaud Poincloux
Podosomes are mechanosensitive adhesion cell structures that are capable of applying protrusive forces onto the extracellular environment. We have recently developed a method dedicated to the evaluation of the nanoscale forces that podosomes generate to protrude into the extracellular matrix. It consists in measuring by atomic force microscopy (AFM) the nanometer deformations produced by macrophages on a compliant Formvar membrane and has been called protrusion force microscopy (PFM). Here we perform time-lapse PFM experiments and investigate spatial correlations of force dynamics between podosome pairs. We use an automated procedure based on finite element simulations that extends the analysis of PFM experimental data to take into account podosome architecture and organization. We show that protrusion force varies in a synchronous manner for podosome first neighbors, a result that correlates with phase synchrony of core F-actin temporal oscillations. This dynamic spatial coordination between podosomes suggests a short-range interaction that regulates their mechanical activity.
international microprocesses and nanotechnology conference | 2007
Jerome Chalmeau; Christophe Thibault; F. Carcenac; Christophe Vieu
The localized deposition of molecules such as DNA or proteins at the micron or submicron scale on solid supports is a crucial step in the fabrication of advanced biochips and lab on chip devices. Among the different methods for delivering biomolecules along well defined patterns, micro-contact printing (muCP) is emerging due to its low implementation cost, its ability to produce sub-micron patterns and large surface processing. However, one severe limitation of this technique is that only one type of biomolecule can be printed in one step because generally the stamp is inked everywhere with the same biomolecule. When different biomolecules are to be patterned it is then necessary to sequentially print them which rises the problem of the alignment between the different levels. Aligning elastomeric stamps made of PolyDiMethylSiloxane-based (PDMS) materials is not easy due to the deformability of the stamp and its high adhesion on flat surfaces. In this work a new method for generating self-aligned patterns of different biomolecules using PDMS stamps is proposed, exhibiting several levels of topography.
Journal of Vacuum Science & Technology B | 2016
Angelo Accardo; Emmanuelle Trévisiol; Aline Cerf; Christophe Thibault; Henrik Laurell; Melissa Buscato; Françoise Lenfant; Coralie Fontaine; Christophe Vieu
Here we report the fabrication of lotus-leaves-like tailored SU8 micropillars and their application in the context of a multi-technique characterization protocol for the investigation of the structural properties of the two estrogen receptors (ERα66/ERα46). ER (α) expression is undoubtedly the most important biomarker in breast cancer, because it provides the index for sensitivity to endocrine treatment. Beside the well-characterized ERα66 isoform, a shorter one (ERα46) was reported to be expressed in breast cancer cell line. The superhydrophobic supports were developed by using a double step approach including an optical lithography process and a plasma reactive ion roughening one. Upon drying on the micropillars, the bio-samples resulted in stretched fibers of different diameters which were then characterized by synchrotron X-ray diffraction (XRD), Raman and FTIR spectroscopy. The evidence of both different spectroscopic vibrational responses and XRD signatures in the two estrogen receptors suggests the presence of conformational changes between the two biomarkers. The SU8 micropillar platform therefore represents a valid tool to enhance the discrimination sensitivity of structural features of this class of biocompunds by exploiting a multi-technique in-situ characterization approach.
ACS Nano | 2017
Anaïs Bouissou; Amsha Proag; Nicolas Bourg; Karine Pingris; Clément Cabriel; Stéphanie Balor; Thomas Mangeat; Christophe Thibault; Christophe Vieu; Guillaume Dupuis; Emmanuel Fort; Sandrine Lévêque-Fort; Isabelle Maridonneau-Parini; Renaud Poincloux
Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.
Journal of Visualized Experiments | 2018
Anaïs Bouissou; Amsha Proag; Marion Portes; Vanessa Soldan; Stéphanie Balor; Christophe Thibault; Christophe Vieu; Isabelle Maridonneau-Parini; Renaud Poincloux
In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.