UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2017

Caroline Stefani, David Gonzalez-Rodriguez, Yosuke Senju, Anne Doye, Nadia Efimova, Sébastien Janel, Justine Lipuma, Meng Chen Tsai, Daniel Hamaoui, Madhavi P. Maddugoda, Olivier Cochet-Escartin, Coline Prévost, Frank Lafont, Tatyana Svitkina, Pekka Lappalainen, Patricia Bassereau, Emmanuel Lemichez (2017 Jun 23)

Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation.

Nature Communications : 8 : 15839 : DOI : 10.1038/ncomms15839 En savoir plus
Résumé

Transendothelial cell macroaperture (TEM) tunnels control endothelium barrier function and are triggered by several toxins from pathogenic bacteria that provoke vascular leakage. Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. Here, by conducting high-resolution microscopy approaches we unveil the presence of an actomyosin cable encircling TEMs. We develop a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This establishes the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the upstream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favouring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring.

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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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Karla Perez-Toralla, Guillaume Mottet, Ezgi Tulukcuoglu-Guneri, Jérôme Champ, François-Clément Bidard, Jean-Yves Pierga, Jerzy Klijanienko, Irena Draskovic, Laurent Malaquin, Jean-Louis Viovy, Stéphanie Descroix (2017 Jan 4)

FISH-in-CHIPS: A Microfluidic Platform for Molecular Typing of Cancer Cells.

Methods in molecular biology (Clifton, N.J.) : 211-220 : DOI : 10.1007/978-1-4939-6734-6_16 En savoir plus
Résumé

Microfluidics offer powerful tools for the control, manipulation, and analysis of cells, in particular for the assessment of cell malignancy or the study of cell subpopulations. However, implementing complex biological protocols on chip remains a challenge. Sample preparation is often performed off chip using multiple manually performed steps, and protocols usually include different dehydration and drying steps that are not always compatible with a microfluidic format.Here, we report the implementation of a Fluorescence in situ Hybridization (FISH) protocol for the molecular typing of cancer cells in a simple and low-cost device. The geometry of the chip allows integrating the sample preparation steps to efficiently assess the genomic content of individual cells using a minute amount of sample. The FISH protocol can be fully automated, thus enabling its use in routine clinical practice.

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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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Garten M., Mosgaard L.D., Bornschlögl T., Dieudonné S., Bassereau P., Toombes G.E.S. (2017 Jan 1)

Whole-GUV patch-clamping

Proceedings of the National Academy of Sciences : 114 : 328-333 : DOI : 10.1073/pnas.1609142114 En savoir plus
Résumé

Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the “whole-GUV” patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

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Année de publication : 2016

Valentino F, Sens P, Lemière J, Allard A, Betz T, Campillo C, Sykes C (2016 Nov 28)

Fluctuations of a membrane nanotube revealed by high-resolution force measurements

Soft Matter : 12 : 9429-9435 : DOI : 10.1039/c6sm02117d En savoir plus
Résumé

Pulling membrane nanotubes from liposomes presents a powerful method to gain access to membrane mechanics. Here we extend classical optical tweezers studies to infer membrane nanotube dynamics with high spatial and temporal resolution. We first validate our force measurement setup by accurately measuring the bending modulus of EPC membrane in tube pulling experiments. Then we record the position signal of a trapped bead when it is connected, or not, to a tube. We derive the fluctuation spectrum of these signals and find that the presence of a membrane nanotube induces higher fluctuations, especially at low frequencies (10-1000 Hz). We analyse these spectra by taking into account the peristaltic modes of nanotube fluctuations. This analysis provides a new experimental framework for a quantitative study of the fluctuations of nanotubular membrane structures that are present in living cells, and now classically used for in vitro biomimetic approaches.

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Laura Devis, Cristian P Moiola, Nuria Masia, Elena Martinez-Garcia, Maria Santacana, Tomita Vasilica Stirbat, Françoise Brochard-Wyart, Ángel García, Francesc Alameda, Silvia Cabrera, Jose Palacios, Gema Moreno-Bueno, Miguel Abal, William Thomas, Sylvie Dufour, Xavier Matias-Guiu, Anna Santamaria, Jaume Reventos, Antonio Gil-Moreno, Eva Colas (2016 Nov 23)

Activated leukocyte cell adhesion molecule (ALCAM) is a marker of recurrence and promotes cell migration, invasion and metastasis in early stage endometrioid endometrial cancer.

The Journal of pathology : DOI : 10.1002/path.4851 En savoir plus
Résumé

Endometrial cancer is the most common gynaecological cancer in western countries, being the most common subtype of endometrioid tumours. Most patients are diagnosed at an early stage and present an excellent prognosis. However, a number of those continue to suffer recurrence, without means of identification by risk classification systems. Thus, finding a reliable marker to predict recurrence becomes an important unmet clinical issue. ALCAM is a cell-cell adhesion molecule and member of the Immunoglobulin superfamily that has been associated with genesis of many cancers. Here, we first determined the value of ALCAM as marker of recurrence in endometrioid endometrial cancer by conducting a retrospective multicentre study of 174 primary tumours. In early stage patients (N = 134), recurrence-free survival was poorer in patients with ALCAM-positive compared to ALCAM-negative tumours (HR 4.237; 95%CI 1.01-17.76). This difference was more significant in patients with early stage moderately-poorly differentiated tumours (HR 9.259; 95%CI 2.12-53.47). In multivariate analysis, ALCAM-positivity was an independent prognostic factor in early stage disease (HR 6.027, 95% CI 1.41-25.74). Then, we demonstrated in vitro a role for ALCAM in cell migration and invasion by using a loss-of-function model in two endometrial cancer cell lines. ALCAM depletion resulted in a reduced primary tumour size and reduced metastatic local spread in an orthotopic murine model. Gene expression analysis of ALCAM-depleted cell lines supported that motility, invasiveness, cellular assembly and organization were the most deregulated functions. Finally, we assessed some of the downstream effector genes that are involved in ALCAM mediated cell migration; specifically FLNB, TXNRD1 and LAMC2 were validated at the mRNA and protein level. In conclusion, our results highlight the potential of ALCAM as a recurrent biomarker in early stage endometrioid endometrial cancer and point to ALCAM as an important molecule in endometrial cancer dissemination by regulating cell migration, invasion and metastasis.

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Lemière J, Valentino F, Campillo C, Sykes C (2016 Nov 1)

How cellular membrane properties are affected by the actin cytoskeleton.

Biochimie : 130 : 33-40 : DOI : 10.1016/j.biochi.2016.09.019 En savoir plus
Résumé

Lipid membranes define the boundaries of living cells and intracellular compartments. The dynamic remodelling of these membranes by the cytoskeleton, a very dynamic structure made of active biopolymers, is crucial in many biological processes such as motility or division. In this review, we present some aspects of cellular membranes and how they are affected by the presence of the actin cytoskeleton. We show that, in parallel with the direct study of membranes and cytoskeleton in vivo, biomimetic in vitro systems allow reconstitution of biological processes in a controlled environment. In particular, we show that liposomes, or giant unilamellar vesicles, encapsulating a reconstituted actin network polymerizing at their membrane are suitable models of living cells and can be used to decipher the relative contributions of membrane and actin on the mechanical properties of the cellular interface.

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Année de publication : 2017

Ayako Yamada, Renaud Renault, Aleksandra Chikina, Bastien Venzac, Iago Pereiro, Sylvie Coscoy, Marine Verhulsel, Maria Carla Parrini, Catherine Villard, Jean-Louis Viovy, Stéphanie Descroix (2016 Nov 1)

Transient microfluidic compartmentalization using actionable microfilaments for biochemical assays, cell culture and organs-on-chip.

Lab on a chip : DOI : 10.1039/C6LC01143H En savoir plus
Résumé

We report here a simple yet robust transient compartmentalization system for microfluidic platforms. Cylindrical microfilaments made of commercially available fishing lines are embedded in a microfluidic chamber and employed as removable walls, dividing the chamber into several compartments. These partitions allow tight sealing for hours, and can be removed at any time by longitudinal sliding with minimal hydrodynamic perturbation. This allows the easy implementation of various functions, previously impossible or requiring more complex instrumentation. In this study, we demonstrate the applications of our strategy, firstly to trigger chemical diffusion, then to make surface co-coating or cell co-culture on a two-dimensional substrate, and finally to form multiple cell-laden hydrogel compartments for three-dimensional cell co-culture in a microfluidic device. This technology provides easy and low-cost solutions, without the use of pneumatic valves or external equipment, for constructing well-controlled microenvironments for biochemical and cellular assays.

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