UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2013

Sabrina Hocine, Di Cui, Marie-Noelle Rager, Aurélie Di Cicco, Jian-Miao Liu, Joanna Wdzieczak-Bakala, Annie Brûlet, Min-Hui Li (2013 Jan 9)

Polymersomes with PEG corona: structural changes and controlled release induced by temperature variation.

Langmuir : the ACS journal of surfaces and colloids : 1356-69 : DOI : 10.1021/la304199z En savoir plus
Résumé

Thermoresponsive behavior of different kinds of polymersomes was studied using small angle neutron scattering (SANS), transmission electron microscopy (TEM), and proton nuclear magnetic resonance ((1)H NMR). The polymersomes were made of block copolymers containing a 2000 Da polyethylene glycol (PEG) as a hydrophilic block and either a liquidlike polymer (e.g., PBA: polybutylacrylate), a solidlike polymer (PS: polystyrene), or a liquid crystalline (LC) polymer as a hydrophobic block. Structural changes in polymersomes are driven in all cases by the critical dehydration temperature of PEG corona, which is closely related to the chemical structure and chain mobility of the hydrophobic block. No structural changes occur upon heating from 25 to 75 °C in the liquidlike polymersomes where the critical dehydration temperature of PEG should be higher than 75 °C. In contrast, glassy PEG-b-PS polymersomes and LC polymersomes show structural changes around 55 °C, which corresponds to the critical dehydration temperature of PEG in those block copolymers. Furthermore, the structural changes depend on the properties of the hydrophobic layer. Glassy PEG-b-PS polymersomes aggregate together above 55 °C, but the bilayer membrane is robust enough to remain intact. This aggregation is reversible, and rather separate polymersomes are recovered upon cooling. However, LC polymersomes display drastic and irreversible structural changes when heated above ∼55 °C. These changes are dependent on the LC structures of the hydrophobic layer. Nematic LC polymersomes turn into thick-walled capsules, whereas smectic LC polymersomes collapse into dense aggregates. As these drastic and irreversible changes decrease or remove the inner compartment volume of the vesicle, LC polymersomes can be used for thermal-responsive controlled release, as shown by a study of calcein release. Finally, toxicity studies proved that LC polymersomes were noncytotoxic and had no effect on cell morphology.

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Année de publication : 2012

L Dinis, P Martin, J Barral, J Prost, J F Joanny (2012 Dec 11)

Fluctuation-response theorem for the active noisy oscillator of the hair-cell bundle.

Physical review letters : 160602 En savoir plus
Résumé

The hair bundle of sensory cells in the vertebrate ear provides an example of a noisy oscillator close to a Hopf bifurcation. The analysis of the data from both spontaneous and forced oscillations shows a strong violation of the fluctuation-dissipation theorem, revealing the presence of an underlying active process that keeps the system out of equilibrium. Nevertheless, we show that a generalized fluctuation-dissipation theorem, valid for nonequilibrium steady states, is fulfilled within the limits of our experimental accuracy and computational approximations, when the adequate conjugate degrees of freedom are chosen.

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Scott Atwell, Ludovic Disseau, Alicja Z Stasiak, Andrzej Stasiak, Axelle Renodon-Cornière, Masayuki Takahashi, Jean-Louis Viovy, Giovanni Cappello (2012 Nov 28)

Probing Rad51-DNA interactions by changing DNA twist.

Nucleic acids research : 11769-76 : DOI : 10.1093/nar/gks1131 En savoir plus
Résumé

In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.

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Sandrine Morlot, Valentina Galli, Marius Klein, Nicolas Chiaruttini, John Manzi, Frédéric Humbert, Luis Dinis, Martin Lenz, Giovanni Cappello, Aurélien Roux (2012 Oct 30)

Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction.

Cell : 619-29 : DOI : 10.1016/j.cell.2012.09.017 En savoir plus
Résumé

The GTPase dynamin polymerizes into a helical coat that constricts membrane necks of endocytic pits to promote their fission. However, the dynamin mechanism is still debated because constriction is necessary but not sufficient for fission. Here, we show that fission occurs at the interface between the dynamin coat and the uncoated membrane. At this location, the considerable change in membrane curvature increases the local membrane elastic energy, reducing the energy barrier for fission. Fission kinetics depends on tension, bending rigidity, and the dynamin constriction torque. Indeed, we experimentally find that the fission rate depends on membrane tension in vitro and during endocytosis in vivo. By estimating the energy barrier from the increased elastic energy at the edge of dynamin and measuring the dynamin torque, we show that the mechanical energy spent on dynamin constriction can reduce the energy barrier for fission sufficiently to promote spontaneous fission. :

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Daniel Levy, Pierre-Emmanuel Milhiet (2012 Oct 23)

Imaging of transmembrane proteins directly incorporated within supported lipid bilayers using atomic force microscopy.

Methods in molecular biology (Clifton, N.J.) : 343-57 : DOI : 10.1007/978-1-62703-137-0_19 En savoir plus
Résumé

Structural analysis of transmembrane proteins remains a challenge in biology, mainly due to their difficulty in being overexpressed and the required use of detergents that impair different steps of biochemistry classically used to obtain 3D crystals. In this context, we have developed a new technique for protein incorporation within supported lipid bilayers that only requires a few picomoles of protein per assay. Proteins are directly inserted into a detergent-destabilized bilayer that can be imaged in buffer with atomic force microscopy (AFM) allowing structural analysis down to sub-nanometer lateral resolution. In this chapter, we describe the main guidelines for this technique, from the choice of detergent to the requirements for AFM high-resolution imaging.

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Brian S Gentry, Stef van der Meulen, Philippe Noguera, Baldomero Alonso-Latorre, Julie Plastino, Gijsje H Koenderink (2012 Oct 12)

Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening.

European biophysics journal : EBJ : 979-90 : DOI : 10.1007/s00249-012-0861-1 En savoir plus
Résumé

Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP’s main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP’s flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP’s effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP’s ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP’s network stiffening activity may be tuned by the local concentration of monomeric actin.

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Edouard Hannezo, Jacques Prost, Jean-François Joanny (2012 Oct 4)

Mechanical instabilities of biological tubes.

Physical review letters : 018101 En savoir plus
Résumé

We study theoretically the morphologies of biological tubes affected by various pathologies. When epithelial cells grow, the negative tension produced by their division provokes a buckling instability. Several shapes are investigated: varicose, dilated, sinuous, or sausagelike. They are all found in pathologies of tracheal, renal tubes, or arteries. The final shape depends crucially on the mechanical parameters of the tissues: Young’s modulus, wall-to-lumen ratio, homeostatic pressure. We argue that since tissues must be in quasistatic mechanical equilibrium, abnormal shapes convey information as to what causes the pathology. We calculate a phase diagram of tubular instabilities which could be a helpful guide for investigating the underlying genetic regulation.

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Paolo Maiuri, Emmanuel Terriac, Perrine Paul-Gilloteaux, Timothée Vignaud, Krista McNally, James Onuffer, Kurt Thorn, Phuong A Nguyen, Nefeli Georgoulia, Daniel Soong, Asier Jayo, Nina Beil, Jürgen Beneke, Joleen Chooi Hong Lim, Chloe Pei-Ying Sim, Yeh-Shiu Chu, , Andrea Jiménez-Dalmaroni, Jean-François Joanny, Jean-Paul Thiery, Holger Erfle, Maddy Parsons, Timothy J Mitchison, Wendell A Lim, Ana-Maria Lennon-Duménil, Matthieu Piel, Manuel Théry (2012 Sep 15)

The first World Cell Race.

Current biology : CB : R673-5 : DOI : 10.1016/j.cub.2012.07.052 En savoir plus
Résumé

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Mohamed Lemine Youba Diakité, Jerôme Champ, Stephanie Descroix, Laurent Malaquin, François Amblard, Jean-Louis Viovy (2012 Sep 13)

A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR.

Lab on a chip : 4738-47 : DOI : 10.1039/c2lc40372b En savoir plus
Résumé

In order to evolve from a « chip in the lab » to a « lab on a chip » paradigm, there is still a strong demand for low-cost, portable detection technologies, notably for analytes at low concentrations. Here we report a new label-free DNA detection method with direct electronic read, and apply it to long-range PCR. This method uses a nonlinear electrohydrodynamic phenomenon: when subjected to high electric fields (typically above 100 V cm(-1)), suspensions of large polyelectrolytes, such as long DNA molecules, create « giant » dynamic concentration fluctuations. These fluctuations are associated with large conductivity inhomogeneities, and we use here a contact-mode local conductivity detector to detect these fluctuations. In order to decouple the detection electronics from the high voltage excitation one, an original « doubly symmetric » floating mode battery-operated detection scheme was developed. A wavelet analysis was then applied, to unravel from the chaotic character of the electohydrodynamic instabilities a scalar signal robustly reflecting the amplification of DNA. As a first proof of concept, we measured the products of the off-chip amplification of 10 kbp DNA from lambda phage DNA, achieving a sensitivity better than 100 fg DNA in the original 50 μl sample. This corresponds to the amplification products of less than 100 initial copies of target DNA. The companion enabling technologies developed to implement this new concept, i.e. the doubly symmetric contact conductivity detection and wavelet analysis, may also find various other applications in lab-on-chips.

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Kawska A, Carvalho K, Manzi J, Boujemaa-Paterski R, Blanchoin L, Martiel JL, Sykes C (2012 Sep 4)

How actin network dynamics control the onset of actin-based motility

Proceedings of the National Academy of Sciences USA : 109 : 14440-5 : DOI : 10.1073/pnas.1117096109 En savoir plus
Résumé

Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or « gels » are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein. We vary protein concentrations and follow experimentally and through simulations the shape and mechanical properties of the actin gel growing around beads. Actin gel morphology is controlled by elementary steps including « primer » contact, growth of the network, entanglement, mechanical interaction and force production. We show that varying the biochemical orchestration of these steps can lead to the loss of network cohesion and the lack of effective force production. We propose a predictive phase diagram of actin gel fate as a function of protein concentrations. This work unveils how, in growing actin networks, a tight biochemical and physical coupling smoothens initial primer-caused heterogeneities and governs force buildup and cell motility.

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Stephane Romero, Alessia Quatela, Thomas Bornschlögl, Stéphanie Guadagnini, Patricia Bassereau, Guy Tran Van Nhieu (2012 Aug 18)

Filopodium retraction is controlled by adhesion to its tip.

Journal of cell science : 4999-5004 : DOI : 10.1242/jcs.104778 En savoir plus
Résumé

Filopodia extensions are thin cell sensing the environment. They play an essential role During cell migration, cell-cell or cell-matrix adhesion, by initiating event contacts and Conveying signals to the cell cortex. Pathogenic microorganisms can hijack filopodia to invade cells by inducing Their retraction Reviews towards the cell body. Because Their dynamics depend on a discrete number of actin filaments, filopodia Provide a model of choice to study elementary events linked to membership and downstream signaling. HOWEVER, the determinants controlling filopodial sensing are not well caractérisé. In this study, we used beads functionalized with different ligands That triggered filopodial retraction When in touch with filopodia of epithelial cells. With optical tweezers, We Were reliable to measure strengths stalling the retraction of a single filopodium. We found que la filopodial stall depends on the strength of the coating bead. Stall strengths atteint 8 pN for beads coated with the β1 integrin ligand Yersinia Invasin, whereas retraction Was stopped with a Higher strength of 15 pN When Were beads functionalized with carboxyl groups. In all cases, stall Increased strength in relation to the density of ligands Contacting filopodial tips and Were independent of the optical trap stiffness. Unexpectedly, a small number of discrete and Shigella induced Type three secretion systems stall force of 10 pN. These results suggest que le number of receptor-ligand interactions at the tip filopodial determined the maximum retraction forces exerted by filopodia goal discrete number of clustered receptors is Sufficient to Induce stall high retraction forces.

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Julien Autebert, Benoit Coudert, François-Clément Bidard, Jean-Yves Pierga, Stéphanie Descroix, Laurent Malaquin, Jean-Louis Viovy (2012 Jul 17)

Microfluidic: an innovative tool for efficient cell sorting.

Methods (San Diego, Calif.) : 297-307 : DOI : 10.1016/j.ymeth.2012.07.002 En savoir plus
Résumé

At first mostly dedicated to molecular analysis, microfluidic systems are rapidly expanding their range of applications towards cell biology, thanks to their ability to control the mechanical, biological and fluidic environment at the scale of the cells. A number of new concepts based on microfluidics were indeed proposed in the last ten years for cell sorting. For many of these concepts, progress remains to be done regarding automation, standardization, or throughput, but it is now clear that microfluidics will have a major contribution to the field, from fundamental research to point-of-care diagnosis. We present here an overview of cells sorting in microfluidics, with an emphasis on circulating tumor cells. Sorting principles are classified in two main categories, methods based on physical properties of the cells, such as size, deformability, electric or optical properties, and methods based on biomolecular properties, notably specific surface antigens. We document potential applications, discuss the main advantages and limitations of different approaches, and tentatively outline the main remaining challenges in this fast evolving field.

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Natsuhiko Yoshinaga, Philippe Marcq (2012 Jul 13)

Contraction of cross-linked actomyosin bundles.

Physical biology : 046004 : DOI : 10.1088/1478-3975/9/4/046004 En savoir plus
Résumé

Cross-linked actomyosin bundles retract when severed in vivo by laser ablation, or when isolated from the cell and micromanipulated in vitro in the presence of ATP. We identify the timescale for contraction as a viscoelastic time τ, where the viscosity is due to (internal) protein friction. We obtain an estimate of the order of magnitude of the contraction time τ ≈ 10-100 s, consistent with available experimental data for circumferential microfilament bundles and stress fibers. Our results are supported by an exactly solvable, hydrodynamic model of a retracting bundle as a cylinder of isotropic, active matter, from which the order of magnitude of the active stress is estimated.

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Daniel Horák, Zuzana Svobodová, Julien Autebert, Benoit Coudert, Zdeněk Plichta, Karel Královec, Zuzana Bílková, Jean-Louis Viovy (2012 Jul 7)

Albumin-coated monodisperse magnetic poly(glycidyl methacrylate) microspheres with immobilized antibodies: application to the capture of epithelial cancer cells.

Journal of biomedical materials research. Part A : 23-32 : DOI : 10.1002/jbm.a.34297 En savoir plus
Résumé

Monodisperse (4 μm) macroporous crosslinked poly(glycidyl methacrylate) (PGMA) microspheres for use in microfluidic immunomagnetic cell sorting, with a specific application to the capture of circulating tumor cells (CTCs), were prepared by multistep swelling polymerization in the presence of cyclohexyl acetate porogen and hydrolyzed and ammonolyzed. Iron oxide was then precipitated in the microspheres to render them magnetic. Repeated precipitation made possible to raise the iron oxide content to more than 30 wt %. To minimize nonspecific adsorption of the microspheres in a microchannel and of cells on the microspheres, they were coated with albumin crosslinked with glutaraldehyde. Antibodies of epithelial cell adhesion molecule (anti-EpCAM) were then immobilized on the albumin-coated magnetic microspheres using the carbodiimide method. Capture of breast cancer MCF7 cells as a model of CTCs by the microspheres with immobilized anti-EpCAM IgG was performed in a batch experiment. Finally, MCF7 cells were captured by the anti-EpCAM-immobilized albumin-coated magnetic microspheres in an Ephesia chip. A very good rejection of lymphocytes was achieved. Thus, albumin-coated monodisperse magnetic PGMA microspheres with immobilized anti-EpCAM seem to be promising for capture of CTCs in a microfluidic device.

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Zuzana Svobodova, Mohamad Reza Mohamadi, Barbora Jankovicova, Hermann Esselmann, Romain Verpillot, Markus Otto, Myriam Taverna, Jens Wiltfang, Jean-Louis Viovy, Zuzana Bilkova (2012 Jun 20)

Development of a magnetic immunosorbent for on-chip preconcentration of amyloid β isoforms: Representatives of Alzheimer’s disease biomarkers.

Biomicrofluidics : 24126-2412612 : DOI : 10.1063/1.4722588 En savoir plus
Résumé

Determination of amyloid β (Aβ) isoforms and in particular the proportion of the Aβ 1-42 isoform in cerebrospinal fluid (CSF) of patients suspected of Alzheimer’s disease might help in early diagnosis and treatment of that illness. Due to the low concentration of Aβ peptides in biological fluids, a preconcentration step prior to the detection step is often necessary. This study utilized on-chip immunoprecipitation, known as micro-immunoprecipitation (μIP). The technique uses an immunosorbent (IS) consisting of magnetic beads coated with specific anti-Aβ antibodies organized into an affinity microcolumn by a magnetic field. Our goal was to thoroughly describe the critical steps in developing the IS, such as selecting the proper beads and anti-Aβ antibodies, as well as optimizing the immobilization technique and μIP protocol. The latter includes selecting optimal elution conditions. Furthermore, we demonstrate the efficiency of anti-Aβ IS for μIP and specific capture of 5 Aβ peptides under optimized conditions using various subsequent analytical methods, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), capillary electrophoresis, microchip electrophoresis, and immunoblotting. Synthetic Aβ peptides samples prepared in buffer and spiked in human CSF were analyzed. Finally, on-chip immunoprecipitation of Aβ peptides in human CSF sample was performed.

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