UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2017

Win Pin Ng, Kevin D. Webster, Caroline Stefani, Eva M. Schmid, Emmanuel Lemichez, Patricia Bassereau, Daniel A. Fletcher (2017 Oct 2)

Force-induced transcellular tunnel formation in endothelial cells

Molecular Biology of the Cell : 28 : 2650-2660 : DOI : 10.1091/mbc.E17-01-0080 En savoir plus
Résumé

The endothelium serves as a protective semipermeable barrier in blood vessels and lymphatic vessels. Leukocytes and pathogens can pass directly through the endothelium by opening holes in endothelial cells, known as transcellular tunnels, which are formed by contact and self-fusion of the apical and basal plasma membranes. Here we test the hypothesis that the actin cytoskeleton is the primary barrier to transcellular tunnel formation using a combination of atomic force microscopy and fluorescence microscopy of live cells. We find that localized mechanical forces are sufficient to induce the formation of transcellular tunnels in human umbilical vein endothelial cells (HUVECs). When HUVECs are exposed to the bacterial toxin called epidermal cell differentiation inhibitor (EDIN), which can induce spontaneous transcellular tunnels, less mechanical work is required to form tunnels due to the reduced cytoskeletal stiffness and thickness of these cells, similarly to the effects of a Rho-associated protein kinase (ROCK) inhibitor. We also observe actin enrichment in response to mechanical indentation that is reduced in cells exposed to the bacterial toxin. Our study shows that the actin cytoskeleton of endothelial cells provides both passive and active resistance against transcellular tunnel formation, serving as a mechanical barrier that can be overcome by mechanical force as well as disruption of the cytoskeleton.

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Shunsuke Yabunaka, Philippe Marcq (2017 Sep 28)

Cell growth, division, and death in cohesive tissues: A thermodynamic approach.

Physical review. E : 022406 : DOI : 10.1103/PhysRevE.96.022406 En savoir plus
Résumé

Cell growth, division, and death are defining features of biological tissues that contribute to morphogenesis. In hydrodynamic descriptions of cohesive tissues, their occurrence implies a nonzero rate of variation of cell density. We show how linear nonequilibrium thermodynamics allows us to express this rate as a combination of relevant thermodynamic forces: chemical potential, velocity divergence, and activity. We illustrate the resulting effects of the nonconservation of cell density on simple examples inspired by recent experiments on cell monolayers, considering first the velocity of a spreading front, and second an instability leading to mechanical waves.

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Shuji Ishihara, Philippe Marcq, Kaoru Sugimura (2017 Sep 28)

From cells to tissue: A continuum model of epithelial mechanics.

Physical review. E : 022418 : DOI : 10.1103/PhysRevE.96.022418 En savoir plus
Résumé

A two-dimensional continuum model of epithelial tissue mechanics was formulated using cellular-level mechanical ingredients and cell morphogenetic processes, including cellular shape changes and cellular rearrangements. This model incorporates stress and deformation tensors, which can be compared with experimental data. Focusing on the interplay between cell shape changes and cell rearrangements, we elucidated dynamical behavior underlying passive relaxation, active contraction-elongation, and tissue shear flow, including a mechanism for contraction-elongation, whereby tissue flows perpendicularly to the axis of cell elongation. This study provides an integrated scheme for the understanding of the orchestration of morphogenetic processes in individual cells to achieve epithelial tissue morphogenesis.

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Plastino J, Blanchoin L (2017 Sep 25)

Adaptive actin networks

Developmental Cell : 42 : 565-566 : DOI : 10.1016/j.devcel.2017.09.005 En savoir plus
Résumé

Despite their fundamental importance in the regulation of cell physiology, the mechanisms that confer cell adaptability to changes in the microenvironment are poorly understood. A recent study in Cell (Mueller et al., 2017) examines the capability of branched actin networks to respond and adapt to mechanical load in vivo.

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Rückerl F, Lenz M, Betz T, Manzi J, Martiel J-L, Safouane M, Paterski-Boujemaa R, Blanchoin L, Sykes C (2017 Sep 5)

Adaptive response of actin bundles under mechanical stress

Biophysical Journal : 113 : 1072-1079 : DOI : 10.1016/j.bpj.2017.07.017 En savoir plus
Résumé

Actin is one of the main components of the architecture of cells. Actin filaments form different polymer networks with versatile mechanical properties that depend on their spatial organization and the presence of cross-linkers. Here, we investigate the mechanical properties of actin bundles in the absence of cross-linkers. Bundles are polymerized from the surface of mDia1-coated latex beads, and deformed by manipulating both ends through attached beads held by optical tweezers, allowing us to record the applied force. Bundle properties are strikingly different from the ones of a homogeneous isotropic beam. Successive compression and extension leads to a decrease in the buckling force that we attribute to the bundle remaining slightly curved after the first deformation. Furthermore, we find that the bundle is solid, and stiff to bending, along the long axis, whereas it has a liquid and viscous behavior in the transverse direction. Interpretation of the force curves using a Maxwell visco-elastic model allows us to extract the bundle mechanical parameters and confirms that the bundle is composed of weakly coupled filaments. At short times, the bundle behaves as an elastic material, whereas at long times, filaments flow in the longitudinal direction, leading to bundle restructuring. Deviations from the model reveal a complex adaptive rheological behavior of bundles. Indeed, when allowed to anneal between phases of compression and extension, the bundle reinforces. Moreover, we find that the characteristic visco-elastic time is inversely proportional to the compression speed. Actin bundles are therefore not simple force transmitters, but instead, complex mechano-transducers that adjust their mechanics to external stimulation. In cells, where actin bundles are mechanical sensors, this property could contribute to their adaptability.

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Shunsuke Yabunaka, Philippe Marcq (2017 Aug 30)

Emergence of epithelial cell density waves.

Soft matter : DOI : 10.1039/c7sm01172e En savoir plus
Résumé

Epithelial cell monolayers exhibit traveling mechanical waves. We rationalize this observation thanks to a hydrodynamic description of the monolayer as a compressible, active and polar material. We show that propagating waves of the cell density, polarity, velocity and stress fields may be due to a Hopf bifurcation occurring above threshold values of active coupling coefficients.

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Breau M, Bonnet I, Stoufflet J, Xie J, De Castro S, Schneider-Maunoury S (2017 Aug 21)

Extrinsic mechanical forces mediate retrograde axon extension in a developing neuronal circuit

Nature Communications : 8 : 282 : DOI : 10.1038/s41467-017-00283-3 En savoir plus
Résumé

To form functional neural circuits, neurons migrate to their final destination and extend axons towards their targets. Whether and how these two processes are coordinated in vivo remains elusive. We use the zebrafish olfactory placode as a system to address the underlying mechanisms. Quantitative live imaging uncovers a choreography of directed cell movements that shapes the placode neuronal cluster: convergence of cells towards the centre of the placodal domain and lateral cell movements away from the brain. Axon formation is concomitant with lateral movements and occurs through an unexpected, retrograde mode of extension, where cell bodies move away from axon tips attached to the brain surface. Convergence movements are active, whereas cell body lateral displacements are of mainly passive nature, likely triggered by compression forces from converging neighbouring cells. These findings unravel a previously unknown mechanism of neuronal circuit formation, whereby extrinsic mechanical forces drive the retrograde extension of axons.

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Cáceres R, Plastino J (2017 Apr 17)

Cytoskeleton dynamics: actin in cell invasion

Encyclopedia of Life Sciences : DOI : 10.1002/9780470015902.a0001254.pub2 En savoir plus
Résumé

Basement membrane (BM) is a dense sheet of specialised extracellular matrix that separates epithelial layers of cells from the underlying tissue. The penetration of cells through BM barriers, called ‘invasion’, is an important process during normal tissue development and in cancer metastasis. To enable invasion, the cell adopts different shapes and creates different protrusive structures powered mainly by actin cytoskeleton dynamics. However, the exact cytoskeletal strategy that the cell uses to cross the physical BM barrier depends on the physiological context and the physical environment, as observed by examining actin structures in invading cancer and immune cells, and in cells that invade during developmental processes such as angiogenesis and anchor cell invasion in Caenorhabditis elegans.

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Thuan Beng Saw, Amin Doostmohammadi, Vincent Nier, Leyla Kocgozlu, Sumesh Thampi, Yusuke Toyama, Philippe Marcq, Chwee Teck Lim, Julia M Yeomans, Benoit Ladoux (2017 Apr 14)

Topological defects in epithelia govern cell death and extrusion.

Nature : 212-216 : DOI : 10.1038/nature21718 En savoir plus
Résumé

Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling, oncogenic transformation and overcrowding of cells. Despite the important linkage of cell extrusion to developmental, homeostatic and pathological processes such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity, caspase-3-mediated cell death, and extrusions. Importantly, the defect-driven extrusion mechanism depends on intercellular junctions, because the weakening of cell-cell interactions in an α-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting extrusion hotspots and dynamics in vivo, with potential applications to tissue regeneration and the suppression of metastasis. Moreover, we anticipate that the analogy between the epithelium and active nematic liquid crystals will trigger further investigations of the link between cellular processes and the material properties of epithelia.

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M E Dolega, M Delarue, F Ingremeau, J Prost, A Delon, G Cappello (2017 Jan 28)

Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression.

Nature communications : 14056 : DOI : 10.1038/ncomms14056 En savoir plus
Résumé

The surrounding microenvironment limits tumour expansion, imposing a compressive stress on the tumour, but little is known how pressure propagates inside the tumour. Here we present non-destructive cell-like microsensors to locally quantify mechanical stress distribution in three-dimensional tissue. Our sensors are polyacrylamide microbeads of well-defined elasticity, size and surface coating to enable internalization within the cellular environment. By isotropically compressing multicellular spheroids (MCS), which are spherical aggregates of cells mimicking a tumour, we show that the pressure is transmitted in a non-trivial manner inside the MCS, with a pressure rise towards the core. This observed pressure profile is explained by the anisotropic arrangement of cells and our results suggest that such anisotropy alone is sufficient to explain the pressure rise inside MCS composed of a single cell type. Furthermore, such pressure distribution suggests a direct link between increased mechanical stress and previously observed lack of proliferation within the spheroids core.

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David Saletti, Jens Radzimanowski, Gregory Effantin, Daniel Midtvedt, Stéphanie Mangenot, Winfried Weissenhorn, Patricia Bassereau, Marta Bally (2017 Jan 26)

The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model.

Scientific reports : 40801 : DOI : 10.1038/srep40801 En savoir plus
Résumé

Matrix proteins from enveloped viruses play an important role in budding and stabilizing virus particles. In order to assess the role of the matrix protein M1 from influenza C virus (M1-C) in plasma membrane deformation, we have combined structural and in vitro reconstitution experiments with model membranes. We present the crystal structure of the N-terminal domain of M1-C and show by Small Angle X-Ray Scattering analysis that full-length M1-C folds into an elongated structure that associates laterally into ring-like or filamentous polymers. Using negatively charged giant unilamellar vesicles (GUVs), we demonstrate that M1-C full-length binds to and induces inward budding of membrane tubules with diameters that resemble the diameter of viruses. Membrane tubule formation requires the C-terminal domain of M1-C, corroborating its essential role for M1-C polymerization. Our results indicate that M1-C assembly on membranes constitutes the driving force for budding and suggest that M1-C plays a key role in facilitating viral egress.

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Démosthène Mitrossilis, Jens-Christian Röper, Damien Le Roy, Benjamin Driquez, Aude Michel, Christine Ménager, Gorky Shaw, Simon Le Denmat, Laurent Ranno, Frédéric Dumas-Bouchiat, Nora M Dempsey, Emmanuel Farge (2017 Jan 24)

Mechanotransductive cascade of Myo-II-dependent mesoderm and endoderm invaginations in embryo gastrulation.

Nature communications : 13883 : DOI : 10.1038/ncomms13883 En savoir plus
Résumé

Animal development consists of a cascade of tissue differentiation and shape change. Associated mechanical signals regulate tissue differentiation. Here we demonstrate that endogenous mechanical cues also trigger biochemical pathways, generating the active morphogenetic movements shaping animal development through a mechanotransductive cascade of Myo-II medio-apical stabilization. To mimic physiological tissue deformation with a cell scale resolution, liposomes containing magnetic nanoparticles are injected into embryonic epithelia and submitted to time-variable forces generated by a linear array of micrometric soft magnets. Periodic magnetically induced deformations quantitatively phenocopy the soft mechanical endogenous snail-dependent apex pulsations, rescue the medio-apical accumulation of Rok, Myo-II and subsequent mesoderm invagination lacking in sna mutants, in a Fog-dependent mechanotransductive process. Mesoderm invagination then activates Myo-II apical accumulation, in a similar Fog-dependent mechanotransductive process, which in turn initiates endoderm invagination. This reveals the existence of a highly dynamic self-inductive cascade of mesoderm and endoderm invaginations, regulated by mechano-induced medio-apical stabilization of Myo-II.

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M Serra, I Pereiro, A Yamada, J-L Viovy, S Descroix, D Ferraro (2017 Jan 24)

A simple and low-cost chip bonding solution for high pressure, high temperature and biological applications.

Lab on a chip : 629-634 : DOI : 10.1039/c6lc01319h En savoir plus
Résumé

The sealing of microfluidic devices remains a complex and time-consuming process requiring specific equipment and protocols: a universal method is thus highly desirable. We propose here the use of a commercially available sealing tape as a robust, versatile, reversible solution, compatible with cell and molecular biology protocols, and requiring only the application of manually achievable pressures. The performance of the seal was tested with regards to the most commonly used chip materials. For most materials, the bonding resisted 5 bars at room temperature and 1 bar at 95 °C. This method should find numerous uses, ranging from fast prototyping in the laboratory to implementation in low technology environments or industrial production.

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Bruno Teste, Jerome Champ, Arturo Londono-Vallejo, Stéphanie Descroix, Laurent Malaquin, Jean-Louis Viovy, Irena Draskovic, Guillaume Mottet (2017 Jan 17)

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

Lab on a chip : 530-537 : DOI : 10.1039/c6lc01535b En savoir plus
Résumé

Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

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Davide Ferraro, Jérôme Champ, Bruno Teste, M Serra, Laurent Malaquin, Stéphanie Descroix, Patricia de Cremoux, Jean-Louis Viovy (2017 Jan 4)

Droplet Microfluidic and Magnetic Particles Platform for Cancer Typing.

Methods in molecular biology (Clifton, N.J.) : 113-121 : DOI : 10.1007/978-1-4939-6734-6_9 En savoir plus
Résumé

Analyses of nucleic acids are routinely performed in hospital laboratories to detect gene alterations for cancer diagnosis and treatment decision. Among the different possible investigations, mRNA analysis provides information on abnormal levels of genes expression. Standard laboratory methods are still not adapted to the isolation and quantitation of low mRNA amounts and new techniques needs to be developed in particular for rare subsets analysis. By reducing the volume involved, time process, and the contamination risks, droplet microfluidics provide numerous advantages to perform analysis down to the single cell level.We report on a droplet microfluidic platform based on the manipulation of magnetic particles that allows the clinical analysis of tumor tissues. In particular, it allows the extraction of mRNA from the total-RNA sample, Reverse Transcription, and cDNA amplification, all in droplets.

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