UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2014

Olivier Cochet-Escartin, Jonas Ranft, Pascal Silberzan, Philippe Marcq (2013 Jul 20)

Border forces and friction control epithelial closure dynamics.

Biophysical journal : 65-73 : DOI : 10.1016/j.bpj.2013.11.015 En savoir plus
Résumé

We study the closure dynamics of a large number of well-controlled circular apertures within an epithelial monolayer, where the collective cell migration responsible for epithelization is triggered by the removal of a spatial constraint rather than by scratching. Based on experimental observations, we propose a physical model that takes into account border forces, friction with the substrate, and tissue rheology. Border protrusive activity drives epithelization despite the presence of a contractile actomyosin cable at the periphery of the wound. The closure dynamics is quantified by an epithelization coefficient, defined as the ratio of protrusive stress to tissue-substrate friction, that allows classification of different phenotypes. The same analysis demonstrates a distinct signature for human cells bearing the oncogenic RasV12 mutation, demonstrating the potential of the approach to quantitatively characterize metastatic transformations.

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Année de publication : 2013

Zi Liang Wu, Renbo Wei, Axel Buguin, Jean-Marie Taulemesse, Nicolas Le Moigne, Anne Bergeret, Xiaogong Wang, Patrick Keller (2013 Jul 16)

Stimuli-responsive topological change of microstructured surfaces and the resultant variations of wetting properties.

ACS applied materials & interfaces : 7485-91 : DOI : 10.1021/am4017957 En savoir plus
Résumé

It is now well established that topological microstructures play a key role in the physical properties of surfaces. Stimulus-induced variations of topological microstructure should therefore lead to a change in the physical properties of microstructured responsive surfaces. In this paper, we demonstrate that roughness changes alter the wetting properties of responsive organic surfaces. Oriented nematic liquid crystalline elastomers (LCEs) are used to construct the microstructured surfaces via a replica molding technique. The topological microstructure of the surfaces covered with micropillars changes with temperature, due to the reversible contraction of the LCE pillars along the long axis at the nematic-to-isotropic phase transition. This is directly observed for the first time under environmental scanning electron microscopy (E-SEM). A high boiling point liquid, glycerol, is used to continuously monitor the contact angle change with temperature. The glycerol contact angle of the microstructured surfaces covered with small pillars decreases from 118° at room temperature to 80° at 140 °C, corresponding to a transition from Cassie state to Wenzel state.

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Thibaut Brunet, Adrien Bouclet, Padra Ahmadi, Démosthène Mitrossilis, Benjamin Driquez, Anne-Christine Brunet, Laurent Henry, Fanny Serman, Gaëlle Béalle, Christine Ménager, Frédéric Dumas-Bouchiat, Dominique Givord, Constantin Yanicostas, Damien Le-Roy, Nora M Dempsey, Anne Plessis, Emmanuel Farge (2013 Jun 21)

Evolutionary conservation of early mesoderm specification by mechanotransduction in Bilateria.

Nature communications : 2821 : DOI : 10.1038/ncomms3821 En savoir plus
Résumé

The modulation of developmental biochemical pathways by mechanical cues is an emerging feature of animal development, but its evolutionary origins have not been explored. Here we show that a common mechanosensitive pathway involving β-catenin specifies early mesodermal identity at gastrulation in zebrafish and Drosophila. Mechanical strains developed by zebrafish epiboly and Drosophila mesoderm invagination trigger the phosphorylation of β-catenin-tyrosine-667. This leads to the release of β-catenin into the cytoplasm and nucleus, where it triggers and maintains, respectively, the expression of zebrafish brachyury orthologue notail and of Drosophila Twist, both crucial transcription factors for early mesoderm identity. The role of the β-catenin mechanosensitive pathway in mesoderm identity has been conserved over the large evolutionary distance separating zebrafish and Drosophila. This suggests mesoderm mechanical induction dating back to at least the last bilaterian common ancestor more than 570 million years ago, the period during which mesoderm is thought to have emerged.

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Natalia de Val, Michael A McMurray, Lisa H Lam, Chris C-S Hsiung, Aurélie Bertin, Eva Nogales, Jeremy Thorner (2013 Jun 19)

Native cysteine residues are dispensable for the structure and function of all five yeast mitotic septins.

Proteins : 1964-79 : DOI : 10.1002/prot.24345 En savoir plus
Résumé

Budding yeast septins assemble into hetero-octamers and filaments required for cytokinesis. Solvent-exposed cysteine (Cys) residues provide sites for attaching substituents useful in assessing assembly kinetics and protein interactions. To introduce Cys at defined locations, site-directed mutagenesis was used, first, to replace the native Cys residues in Cdc3 (C124 C253 C279), Cdc10 (C266), Cdc11 (C43 C137 C138), Cdc12 (C40 C278), and Shs1 (C29 C148) with Ala, Ser, Val, or Phe. When plasmid-expressed, each Cys-less septin mutant rescued the cytokinesis defects caused by absence of the corresponding chromosomal gene. When integrated and expressed from its endogenous promoter, the same mutants were fully functional, except Cys-less Cdc12 mutants (which were viable, but exhibited slow growth and aberrant morphology) and Cdc3(C124V C253V C279V) (which was inviable). No adverse phenotypes were observed when certain pairs of Cys-less septins were co-expressed as the sole source of these proteins. Cells grew less well when three Cys-less septins were co-expressed, suggesting some reduction in fitness. Nonetheless, cells chromosomally expressing Cys-less Cdc10, Cdc11, and Cdc12, and expressing Cys-less Cdc3 from a plasmid, grew well at 30°C. Moreover, recombinant Cys-less septins–or where one of the Cys-less septins contained a single Cys introduced at a new site–displayed assembly properties in vitro indistinguishable from wild-type.

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Manos Mavrakis, Yannick Azou-Gros, Feng-Ching Tsai, José Alvarado, Aurélie Bertin, Francois Iv, Alla Kress, Sophie Brasselet, Gijsje H Koenderink, Thomas Lecuit (2013 Jun 10)

Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles.

Nature cell biology : 322-34 : DOI : 10.1038/ncb2921 En savoir plus
Résumé

Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

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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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