UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2013

Aurélie Bertin, Eva Nogales (2013 Jun 7)

Septin filament organization in Saccharomyces cerevisiae.

Communicative & integrative biology : 503-5 : DOI : 10.4161/cib.21125 En savoir plus
Résumé

Septins are a family of GTP-binding, membrane-interacting cytoskeletal proteins, highly conserved and essential in all eukaryotes (with the exception of plants). Septins play important roles in a number of cellular events that involve membrane remodeling and compartmentalization. One such event is cytokinesis, the last stage of cell division. While cytokinesis is ultimately achieved via the mechanical contraction of an actomyosin ring at the septum, determination of the location where cytokinesis will take place, and recruitment of factors involved in signaling events leading to septation requires the activity of septins. We are working towards dissecting the properties of septins from the budding yeast Saccharomyces cerevisiae, where they were first discovered as cell cycle mutants. In our studies we have employed several complementary electron microscopy techniques to describe the organization and structure of septins both in vitro and in situ.

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Bruno Teste, Anaïs Ali-Cherif, Jean Louis Viovy, Laurent Malaquin (2013 May 4)

A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

Lab on a chip : 2344-9 : DOI : 10.1039/c3lc50353d En savoir plus
Résumé

Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

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Morgan Delarue, Fabien Montel, Ouriel Caen, Jens Elgeti, Jean-Michel Siaugue, Danijela Vignjevic, Jacques Prost, Jean-François Joanny, Giovanni Cappello (2013 Apr 16)

Mechanical control of cell flow in multicellular spheroids.

Physical review letters : 138103 En savoir plus
Résumé

Collective cell motion is observed in a wide range of biological processes. In tumors, physiological gradients of nutrients, growth factors, or even oxygen give rise to gradients of proliferation. We show using fluorescently labeled particles that these gradients drive a velocity field resulting in a cellular flow in multicellular spheroids. Under mechanical stress, the cellular flow is drastically reduced. We describe the results with a hydrodynamic model that considers only convection of the particles by the cellular flow.

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Winfried Weissenhorn, Emilie Poudevigne, Gregory Effantin, Patricia Bassereau (2013 Apr 16)

How to get out: ssRNA enveloped viruses and membrane fission.

Current opinion in virology : 159-67 : DOI : 10.1016/j.coviro.2013.03.011 En savoir plus
Résumé

Enveloped viruses ACQUIRE Their membrane from the host cell and accordingly need to separate Their envelope from cellular membranes via membrane fission. ALTHOUGH Reviews some of the enveloped viruses recruit the endosomal sorting complex required for transport (ESCRT) to catalyze the final fission reaction, Many enveloped viruses sccm to bud in an ESCRT-independent Manner. Here we describe the principles That Govern membrane fission reactions in general and review progress in the understanding of ESCRT-mediated membrane fission. We ESCRT function relates to budding of single stranded RNA viruses and the Chat alternative ways to mediate membrane fission That May Govern ESCRT-independent budding.

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Manuela Dezi, Aurelie Di Cicco, Patricia Bassereau, Daniel Lévy (2013 Apr 15)

Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents.

Proceedings of the National Academy of Sciences of the United States of America : 7276-81 : DOI : 10.1073/pnas.1303857110 En savoir plus
Résumé

Giant unilamellar vesicles (GUVs) biomimetic systems are convenient size of the Sami have cells That are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. HOWEVER, current Approaches to Incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature of gold proteins. Here, we report a method to Incorporate transmembrane proteins in GUVs, based on concepts Developed for detergent-mediated reconstitution in wide unilamellar vesicles. Reconstitution is Performed Either live by incorporation from purified proteins in detergent micelles or by fusion of native purified vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show That unidirectionally proteins are incorporated in the GUVs and keep Their functions on. We-have successfully tested our method with three kinds of transmembrane proteins. Containing GUVs bacteriorhodopsin, a proton pump photoactivable, can generate wide transmembrane potential and pH gradients That are light-switchable and steady for hours. GUVs with FhuA, a bacterial porin, Were used to follow the DNA injection by phage T5 upon binding to receptor transmembrane icts. Incorporating GUVs BMRC / BmrD, a bacterial heterodimeric ATP-binding cassette efflux transport, Were used to Demonstrate the protein-dependent translocation of drugs and Their interactions with encapsulated DNA. Our method shoulds THUS apply to a wide variety of peripheral membrane proteins or for Producing more complex biomimetic GUVs.

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Campillo C, Sens P, Köster D, Pontani LL, Lévy D, Bassereau P, Nassoy P, Sykes C. (2013 Mar 19)

Unexpected membrane dynamics unveiled by membrane nanotube extrusion

Biophysical Journal : 104 : 1248-1256 : DOI : 10.1016/j.bpj.2013.01.051 En savoir plus
Résumé

In cell mechanics, distinguishing the respective roles of the plasma membrane and of the cytoskeleton is a challenge. The difference in the behavior of cellular lipid membranes and pure is usually Attributed To the presence of the cytoskeleton have Explored by nanotube membrane extrusion. Here we revisit this prevalent picture by unveiling unexpected strength responses of plasma membrane and cytoskeleton spheres devoid of synthetic liposomes. We show That a tiny variation in the content of synthetic membranes Does not Affect Their static mechanical properties, purpose is enough to reproduce the dynamic behavior of Their Counterparts cellular. This effect is amplified year Attributed To Intramembrane friction. Reconstituted actin cortices inside liposomes Induce additional year, but not dominant, contribution to the effective friction membrane. Our work Underlines the necessity of a careful consideration of the role of membrane proteins on cell membrane rheology in addition to the role of the cytoskeleton.

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Olivier Preux, Dominique Durand, Alexis Huet, James F Conway, Aurélie Bertin, Claire Boulogne, Jeannine Drouin-Wahbi, Didier Trévarin, Javier Pérez, Patrice Vachette, Pascale Boulanger (2013 Mar 19)

A two-state cooperative expansion converts the procapsid shell of bacteriophage T5 into a highly stable capsid isomorphous to the final virion head.

Journal of molecular biology : 1999-2014 : DOI : 10.1016/j.jmb.2013.03.002 En savoir plus
Résumé

Capsids of double-stranded DNA (dsDNA) bacteriophages initially assemble into compact procapsids, which undergo expansion upon the genome packaging. This shell remodeling results from a structural rearrangement of head protein subunits. It is a critical step in the capsid maturation pathway that yields final particles capable to withstand the huge internal pressure generated by the packed DNA. Here, we report on the expansion process of the large capsid (T=13) of bacteriophage T5. We purified the intermediate prohead II form, which is competent for packaging the 121-kbp dsDNA genome, and we investigated its morphology and dimensions using cryo-electron microscopy and small-angle X-ray scattering. Decreasing the pH or the ionic strength triggers expansion of prohead II, converting them into thinner and more faceted capsids isomorphous to the mature virion particles. At low pH, prohead II expansion is a highly cooperative process lacking any detectable intermediate. This two-state reorganization of the capsid lattice per se leads to a remarkable stabilization of the particle. The melting temperature of expanded T5 capsid is virtually identical with that of more complex shells that are reinforced by inter-subunit cross-linking (HK97) or by additional cementing proteins (T4). The T5 capsid with its « simple » two-state conversion thus appears to be a very attractive model for investigating the mechanism of the large-scale allosteric transition that takes place upon the genome packaging of dsDNA bacteriophages.

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Néstor Sepúlveda, Laurence Petitjean, Olivier Cochet, Erwan Grasland-Mongrain, Pascal Silberzan, Vincent Hakim (2013 Mar 7)

Collective cell motion in an epithelial sheet can be quantitatively described by a stochastic interacting particle model.

PLoS computational biology : e1002944 : DOI : 10.1371/journal.pcbi.1002944 En savoir plus
Résumé

Modelling the displacement of thousands of cells that move in a collective way is required for the simulation and the theoretical analysis of various biological processes. Here, we tackle this question in the controlled setting where the motion of Madin-Darby Canine Kidney (MDCK) cells in a confluent epithelium is triggered by the unmasking of free surface. We develop a simple model in which cells are described as point particles with a dynamic based on the two premises that, first, cells move in a stochastic manner and, second, tend to adapt their motion to that of their neighbors. Detailed comparison to experimental data show that the model provides a quantitatively accurate description of cell motion in the epithelium bulk at early times. In addition, inclusion of model « leader » cells with modified characteristics, accounts for the digitated shape of the interface which develops over the subsequent hours, providing that leader cells invade free surface more easily than other cells and coordinate their motion with their followers. The previously-described progression of the epithelium border is reproduced by the model and quantitatively explained.

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Emmanuel Lemichez, David Gonzalez-Rodriguez, Patricia Bassereau, Françoise Brochard-Wyart (2013 Feb 27)

Transcellular tunnel dynamics: Control of cellular dewetting by actomyosin contractility and I-BAR proteins.

Biology of the Cell : 105 : 109-117 : DOI : 10.1111/boc.201200063 En savoir plus
Résumé

Dewetting is the spontaneous withdrawal of a liquid from the film has non-wettable area by nucleation and growth of dry patches. Two recent reports now offers que la principles of dewetting explain the physical phenomena underpinning the opening of transendothelial cell macroaperture (TEM) tunnels, Referred to as cellular dewetting. Reviews This was Discovered by studying a group of bacterial toxins endowed with the property of corrupting actomyosin cytoskeleton contractility. For Both liquid and cellular dewetting, the growth of holes is-governed by a competition entre area strengths and line voltage. We also how the dynamics of the Chat TEM opening and closure systems to Investigate remarkable Represent actin cytoskeleton regulation by sensors of plasma membrane curvature and Investigate the impact on membrane voltage and the role of TEM in vascular dysfunctions.

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Marie Girardot, Fanny d'Orlyé, Stéphanie Descroix, Anne Varenne (2013 Jan 22)

Aptamer-conjugated nanoparticles: preservation of targeting functionality demonstrated by microchip electrophoresis in frontal mode.

Analytical biochemistry : 150-2 : DOI : 10.1016/j.ab.2012.12.022 En savoir plus
Résumé

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.

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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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