UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2013

Carvalho K, Tsai FC, Lees E, Voituriez R, Koenderink GH, Sykes C. (2013 Oct 8)

Cell-sized liposomes reveal how actomyosin cortical tension drives shape change

Proceedings of the National Academy of Sciences USA : 110 : 16456-61 : DOI : 10.1073/pnas.1221524110 En savoir plus
Résumé

Animal cells actively generate contractile stress in the actin cortex, a thin actin network beneath the cell membrane, to facilitate shape changes during processes like cytokinesis and motility. On the microscopic scale, this stress is generated by myosin molecular motors, which bind to actin cytoskeletal filaments and use chemical energy to exert pulling forces. To decipher the physical basis for the regulation of cell shape changes, here, we use a cell-like system with a cortex anchored to the outside or inside of a liposome membrane. This system enables us to dissect the interplay between motor pulling forces, cortex-membrane anchoring, and network connectivity. We show that cortices on the outside of liposomes either spontaneously rupture and relax built-up mechanical stress by peeling away around the liposome or actively compress and crush the liposome. The decision between peeling and crushing depends on the cortical tension determined by the amount of motors and also on the connectivity of the cortex and its attachment to the membrane. Membrane anchoring strongly affects the morphology of cortex contraction inside liposomes: cortices contract inward when weakly attached, whereas they contract toward the membrane when strongly attached. We propose a physical model based on a balance of active tension and mechanical resistance to rupture. Our findings show how membrane attachment and network connectivity are able to regulate actin cortex remodeling and membrane-shape changes for cell polarization.

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Bibiana Peralta, David Gil-Carton, Daniel Castaño-Díez, Aurelie Bertin, Claire Boulogne, Hanna M Oksanen, Dennis H Bamford, Nicola G A Abrescia (2013 Oct 3)

Mechanism of membranous tunnelling nanotube formation in viral genome delivery.

PLoS biology : e1001667 : DOI : 10.1371/journal.pbio.1001667 En savoir plus
Résumé

In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.

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Karla Perez-Toralla, Jérôme Champ, Mohamad Reza Mohamadi, Olivier Braun, Laurent Malaquin, Jean-Louis Viovy, Stéphanie Descroix (2013 Sep 25)

New non-covalent strategies for stable surface treatment of thermoplastic chips.

Lab on a chip : 4409-18 : DOI : 10.1039/c3lc50888a En savoir plus
Résumé

In order to be more extensively used outside of research laboratories, lab-on-chip technologies must be mass-produced using low-cost materials such as thermoplastics. Thermoplastics, however, are generally hydrophobic in their native state, which makes them unsuitable for direct use with biological samples in aqueous solution, and thus require surface coating. This coating should be robust, inexpensive and simple to implement, in order not to hinder the industrial advantage of thermoplastic chips. Cyclic Olefin Copolymer (COC) is a particularly appealing polymer, but it is also difficult to functionalize due to its chemical inertness. Here we introduce and compare the performance of two new approaches for COC coating. One relies on the use of a commercial triblock copolymer, Pluronic® F127. The second approach uses new copolymers synthesized by radical polymerization, and consisting of a dimethylacrylamide (DMA) backbone carrying aliphatic side chains (C22). Two DMA-C22 copolymers were synthesized with various C22/DMA ratios: DMA-S at 0.175% and DMA-M at 0.35%. Different physicochemical properties of the polymers such as critical micellar concentration (CMC), water contact angle, electroosmosis were investigated. Coated COC chips were then tested for their ability to reduce the adsorption of proteins, microparticles, and for protein electrophoresis. For each application we found an optimal treatment protocol to considerably improve the performance of the thermoplastic chip. These treatments use physisorption in situ which requires no photografting or chemical reaction and can be performed by a simple incubation either after chip production, or just prior to use.

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Carvalho K, Lemière J, Faqir F, Manzi J, Blanchoin L, Plastino J, Betz T, Sykes C. (2013 Sep 23)

Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking

Philosophical Transactions of the Royal Society London : 368 : 20130005 : DOI : 10.1098/rstb.2013.0005 En savoir plus
Résumé

Cells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an ‘outside geometry’. The actin shell is either grown from an activator of actin polymerization immobilized at the membrane by a biotin-streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications.

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Hervé Turlier, Basile Audoly, Jacques Prost, Jean-François Joanny (2013 Sep 10)

Furrow constriction in animal cell cytokinesis.

Biophysical journal : 114-23 : DOI : 10.1016/j.bpj.2013.11.014 En savoir plus
Résumé

Cytokinesis is the process of physical cleavage at the end of cell division; it proceeds by ingression of an acto-myosin furrow at the equator of the cell. Its failure leads to multinucleated cells and is a possible cause of tumorigenesis. Here, we calculate the full dynamics of furrow ingression and predict cytokinesis completion above a well-defined threshold of equatorial contractility. The cortical acto-myosin is identified as the main source of mechanical dissipation and active forces. Thereupon, we propose a viscous active nonlinear membrane theory of the cortex that explicitly includes actin turnover and where the active RhoA signal leads to an equatorial band of myosin overactivity. The resulting cortex deformation is calculated numerically, and reproduces well the features of cytokinesis such as cell shape and cortical flows toward the equator. Our theory gives a physical explanation of the independence of cytokinesis duration on cell size in embryos. It also predicts a critical role of turnover on the rate and success of furrow constriction. Scaling arguments allow for a simple interpretation of the numerical results and unveil the key mechanism that generates the threshold for cytokinesis completion: cytoplasmic incompressibility results in a competition between the furrow line tension and the cell poles’ surface tension.

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Bérangère Deleglise, Benjamin Lassus, Vaneyssa Soubeyre, Aurélie Alleaume-Butaux, Johannes J Hjorth, Maéva Vignes, Benoit Schneider, Bernard Brugg, Jean-Louis Viovy, Jean-Michel Peyrin (2013 Aug 27)

Synapto-protective drugs evaluation in reconstructed neuronal network.

PloS one : e71103 : DOI : 10.1371/journal.pone.0071103 En savoir plus
Résumé

Chronic neurodegenerative syndromes such as Alzheimer’s and Parkinson’s diseases, or acute syndromes such as ischemic stroke or traumatic brain injuries are characterized by early synaptic collapse which precedes axonal and neuronal cell body degeneration and promotes early cognitive impairment in patients. Until now, neuroprotective strategies have failed to impede the progression of neurodegenerative syndromes. Drugs preventing the loss of cell body do not prevent the cognitive decline, probably because they lack synapto-protective effects. The absence of physiologically realistic neuronal network models which can be easily handled has hindered the development of synapto-protective drugs suitable for therapies. Here we describe a new microfluidic platform which makes it possible to study the consequences of axonal trauma of reconstructed oriented mouse neuronal networks. Each neuronal population and sub-compartment can be chemically addressed individually. The somatic, mid axon, presynaptic and postsynaptic effects of local pathological stresses or putative protective molecules can thus be evaluated with the help of this versatile « brain on chip » platform. We show that presynaptic loss is the earliest event observed following axotomy of cortical fibers, before any sign of axonal fragmentation or post-synaptic spine alteration. This platform can be used to screen and evaluate the synapto-protective potential of several drugs. For instance, NAD⁺ and the Rho-kinase inhibitor Y27632 can efficiently prevent synaptic disconnection, whereas the broad-spectrum caspase inhibitor zVAD-fmk and the stilbenoid resveratrol do not prevent presynaptic degeneration. Hence, this platform is a promising tool for fundamental research in the field of developmental and neurodegenerative neurosciences, and also offers the opportunity to set up pharmacological screening of axon-protective and synapto-protective drugs.

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Sébastien Magnifico, Laure Saias, Bérangère Deleglise, Eric Duplus, Devrim Kilinc, Marie-Christine Miquel, Jean-Louis Viovy, Bernard Brugg, Jean-Michel Peyrin (2013 Aug 27)

NAD+ acts on mitochondrial SirT3 to prevent axonal caspase activation and axonal degeneration.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology : 4712-22 : DOI : 10.1096/fj.13-229781 En savoir plus
Résumé

In chronic degenerative syndromes, neuronal death occurs over long periods, during which cells progressively lose their axons and, ultimately, their cell bodies. Although apoptosis is recognized as a key event in neuronal death, the molecular mechanisms involved in CNS axons degeneration are poorly understood. Due to the highly polarized phenotypes of CNS neurons, the different neuronal subcompartments are likely to be targeted by light repetitive and localized aggression. Such locally initiated deleterious signal transduction pathways could theoretically spread through the cytoplasm. However, where axon-degenerative signals initiate, what these early signals are, and how they lead to axon degeneration are unanswered questions that limit our understanding of neurodegenerative diseases and our ability to identify novel therapeutic targets. Using a microfluidic culture device adapted to CNS primary neurons, allowing specific access to the axonal and somatodendritic compartments, we analyzed the molecular pathways involved in axonal degeneration of differentiated neurons. We show here that local application of proapoptotic stimuli on the somatodentritic compartment triggers a dying-back pattern involving caspase-dependent axonal degeneration. Using complementary pharmacological and genetic approaches, we further demonstrate that NAD(+) and grape wine polyphenols prevent axonal apoptosis and act via mitochondrial SirT3 activation in axons.

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Pierre Frederic Fribourg, Mohamed Chami, Carlos Oscar S Sorzano, Francesca Gubellini, Roberto Marabini, Sergio Marco, Jean-Michel Jault, Daniel Lévy (2013 Aug 7)

3D cryo-electron reconstruction of BmrA, a bacterial multidrug ABC transporter in an inward-facing conformation and in a lipidic environment.

Journal of molecular biology : 2059-69 : DOI : 10.1016/j.jmb.2014.03.002 En savoir plus
Résumé

ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether or not their nucleotide-binding domains (NBDs) are physically disengaged in the resting state. To settle this controversy, we obtained structural data on BmrA, a bacterial multidrug homodimeric ABC transporter, in a membrane-embedded state. BmrA in the apostate was reconstituted in lipid bilayers forming a mixture of ring-shaped structures of 24 or 39 homodimers. Three-dimensional models of the ring-shaped structures of 24 or 39 homodimers were calculated at 2.3 nm and 2.5 nm resolution from cryo-electron microscopy, respectively. In these structures, BmrA adopts an inward-facing open conformation similar to that found in mouse P-glycoprotein structure with the NBDs separated by 3 nm. Both lipidic leaflets delimiting the transmembrane domains of BmrA were clearly resolved. In planar membrane sheets, the NBDs were even more separated. BmrA in an ATP-bound conformation was determined from two-dimensional crystals grown in the presence of ATP and vanadate. A projection map calculated at 1.6 nm resolution shows an open outward-facing conformation. Overall, the data are consistent with a mechanism of drug transport involving large conformational changes of BmrA and show that a bacterial ABC exporter can adopt at least two open inward conformations in lipid membrane.

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Andrew Callan-Jones, Patricia Bassereau (2013 Aug 1)

Curvature-driven membrane lipid and protein distribution.

Current Opinion in Solid State and Materials Science : 17 : 143-150 : DOI : 10.1016/j.cossms.2013.08.004 En savoir plus
Résumé

Cellular transport requires that membranes have the ability to recruit specific lipids and proteins to particular positions and at specific times. Here, we review recent work showing that lipids and proteins can be redistributed by spatially varying membrane curvature, without necessarily the need for biochemical targeting signals. We present here an emerging understanding of the various mechanisms by which membrane curvature can sort lipids and proteins, providing the experimental methods in addition to the supporting theoretical concepts.

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Chaigne A, Campillo C, Gov NS, Voituriez R, Azoury J, Umaña-Diaz C, Almonacid M, Queguiner I, Nassoy P, Sykes C, Verlhac MH, Terret ME (2013 Aug 1)

A soft cortex is essential for asymmetric spindle positioning in mouse oocytes

Nature Cell Biology : 15 : 958-66 : DOI : 10.1038/ncb2799 En savoir plus
Résumé

At mitosis onset, cortical tension increases and cells round up, ensuring correct spindle morphogenesis and orientation. Thus, cortical tension sets up the geometric requirements of cell division. On the contrary, cortical tension decreases during meiotic divisions in mouse oocytes, a puzzling observation because oocytes are round cells, stable in shape, that actively position their spindles. We investigated the pathway leading to reduction in cortical tension and its significance for spindle positioning. We document a previously uncharacterized Arp2/3-dependent thickening of the cortical F-actin essential for first meiotic spindle migration to the cortex. Using micropipette aspiration, we show that cortical tension decreases during meiosis I, resulting from myosin-II exclusion from the cortex, and that cortical F-actin thickening promotes cortical plasticity. These events soften and relax the cortex. They are triggered by the Mos-MAPK pathway and coordinated temporally. Artificial cortex stiffening and theoretical modelling demonstrate that a soft cortex is essential for meiotic spindle positioning.

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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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