UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2015

Marco Biondini, Guillaume Duclos, Nathalie Meyer-Schaller, Pascal Silberzan, Jacques Camonis, Maria Carla Parrini (2015 Mar 31)

Akirin specifies NF-κB selectivity of Drosophila innate immune response via chromatin remodeling

Scientific reports : 11759 : DOI : 10.1038/srep11759 En savoir plus
Résumé

RalA and RalB proteins are key mediators of oncogenic Ras signaling in human oncogenesis. Herein we investigated the mechanistic contribution of Ral proteins to invasion of lung cancer A549 cells after induction of epithelial-mesenchymal transition (EMT) with TGFβ. We show that TGFβ-induced EMT promotes dissemination of A549 cells in a 2/3D assay, independently of proteolysis, by activating the Rho/ROCK pathway which generates actomyosin-dependent contractility forces that actively remodel the extracellular matrix, as assessed by Traction Force microscopy. RalB, but not RalA, is required for matrix deformation and cell dissemination acting via the RhoGEF GEF-H1, which associates with the Exocyst complex, a major Ral effector. Indeed, uncoupling of the Exocyst subunit Sec5 from GEF-H1 impairs RhoA activation, generation of traction forces and cell dissemination. These results provide a novel molecular mechanism underlying the control of cell invasion by RalB via a cross-talk with the Rho pathway.

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Julien Autebert, Benoit Coudert, Jérôme Champ, Laure Saias, Ezgi Tulukcuoglu Guneri, Ronald Lebofsky, François-Clément Bidard, Jean-Yves Pierga, Françoise Farace, Stéphanie Descroix, Laurent Malaquin, Jean-Louis Viovy (2015 Mar 28)

High purity microfluidic sorting and analysis of circulating tumor cells: towards routine mutation detection.

Lab on a chip : 2090-101 : DOI : 10.1039/c5lc00104h En savoir plus
Résumé

A new generation of the Ephesia cell capture technology optimized for CTC capture and genetic analysis is presented, characterized in depth and compared with the CellSearch system as a reference. This technology uses magnetic particles bearing tumour-cell specific EpCAM antibodies, self-assembled in a regular array in a microfluidic flow cell. 48,000 high aspect-ratio columns are generated using a magnetic field in a high throughput (>3 ml h(-1)) device and act as sieves to specifically capture the cells of interest through antibody-antigen interactions. Using this device optimized for CTC capture and analysis, we demonstrated the capture of epithelial cells with capture efficiency above 90% for concentrations as low as a few cells per ml. We showed the high specificity of capture with only 0.26% of non-epithelial cells captured for concentrations above 10 million cells per ml. We investigated the capture behavior of cells in the device, and correlated the cell attachment rate with the EpCAM expression on the cell membranes for six different cell lines. We developed and characterized a two-step blood processing method to allow for rapid processing of 10 ml blood tubes in less than 4 hours, and showed a capture rate of 70% for as low as 25 cells spiked in 10 ml blood tubes, with less than 100 contaminating hematopoietic cells. Using this device and procedure, we validated our system on patient samples using an automated cell immunostaining procedure and a semi-automated cell counting method. Our device captured CTCs in 75% of metastatic prostate cancer patients and 80% of metastatic breast cancer patients, and showed similar or better results than the CellSearch device in 10 out of 13 samples. Finally, we demonstrated the possibility of detecting cancer-related PIK3CA gene mutation in 20 cells captured in the chip with a good correlation between the cell count and the quantitation value Cq of the post-capture qPCR.

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M Leoni, P Sens (2015 Mar 14)

Polarization of cells and soft objects driven by mechanical interactions: consequences for migration and chemotaxis.

Physical review. E, Statistical, nonlinear, and soft matter physics : 022720 En savoir plus
Résumé

We study a generic model for the polarization and motility of self-propelled soft objects, biological cells, or biomimetic systems, interacting with a viscous substrate. The active forces generated by the cell on the substrate are modeled by means of oscillating force multipoles at the cell-substrate interface. Symmetry breaking and cell polarization for a range of cell sizes naturally « emerge » from long range mechanical interactions between oscillating units, mediated both by the intracellular medium and the substrate. However, the harnessing of cell polarization for motility requires substrate-mediated interactions. Motility can be optimized by adapting the oscillation frequency to the relaxation time of the system or when the substrate and cell viscosities match. Cellular noise can destroy mechanical coordination between force-generating elements within the cell, resulting in sudden changes of polarization. The persistence of the cell’s motion is found to depend on the cell size and the substrate viscosity. Within such a model, chemotactic guidance of cell motion is obtained by directionally modulating the persistence of motion, rather than by modulating the instantaneous cell velocity, in a way that resembles the run and tumble chemotaxis of bacteria.

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Coline Prévost, Hongxia Zhao, John Manzi, Emmanuel Lemichez, Pekka Lappalainen, Andrew Callan-Jones*, Patricia Bassereau* (2015 Feb 26)

IRSp53 senses negative membrane curvature and phase separates along membrane tubules.

Nature communications : 8529 : DOI : 10.1038/ncomms9529 En savoir plus
Résumé

BAR domain proteins to membrane deformation contribute in diverse cellular processes. The inverted-BAR (I-BAR) IRSp53 protein, for instance, is found on the inner leaflet of the tubular membrane of filopodia; HOWEVER icts role in the formation of These structures is incompletely Understood. Here we Develop an original assay in which proteins are encapsulated in unilamellar giant vesicles connected to membrane nanotubes. Our results Demonstrate That I-BAR dimers sense negative curvature membrane. Experiment and theory reveal that the I-BAR displays a non-monotonic sorting with curvature, and Expands the tube at high voltage Imposed while constricting it at low voltage. Strikingly, at low protein density and pressure, protein-rich domains APPEAR along the tube. This peculiar behavior is due to the shallow intrinsic curvature of I-BAR dimers. It allows constriction of weakly curved membranes coupled to local protein enrichment at biologically relevant conditions. This might explain how IRSp53 Contributes to the in vivo induction of filopodia.

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Senthil Arumugam, Patricia Bassereau (2015 Feb 7)

Membrane nanodomains: contribution of curvature and interaction with proteins and cytoskeleton.

Essays in biochemistry : 109-19 : DOI : 10.1042/bse0570109 En savoir plus
Résumé

The understanding of lipid membranes and Their organization HAS Undergone significant development with better technology and more The Therefore resolved experiments. Many new factoring and organizing principles-have-been Discovered, and interplay entre thesis factoring is expected to result in rich functional Behaviours. The major factoring regulating the lateral heterogeneity membrane, apart from the well-Studied stage separation, are pinning cytoskeleton, clustering of lipids and curvature. These are factoring effective means clustering to create rich membrane domains That Provide biological functions on. We review the recent advances and concepts of heterogeneity membrane curvature by organization, clustering and cytoskeleton proteins.

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Matthias Garten, Sophie Aimon, Patricia Bassereau, Gilman E S Toombes (2015 Feb 5)

Reconstitution of a transmembrane protein, the voltage-gated ion channel, KvAP, into giant unilamellar vesicles for microscopy and patch clamp studies.

Journal of visualized experiments : JoVE : 52281 : DOI : 10.3791/52281 En savoir plus
Résumé

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. HOWEVER, Commonly used protocols to grow GUVs must be modified in order to form GUVs Containing functional transmembrane proteins. This Article Describes two dehydration-rehydration methods – electroformation and gel-assisted swelling – to form GUVs Containing the voltage-gated potassium channel, KvAP. In Both methods, a solution of protein-containing small unilamellar vesicles is Partially dehydrated to form a stack of membranes, qui est Then allowed to swell in a rehydration buffer. For the electroformation method, the movie is Deposited on platinum electrodes so That year AC field Can Be Applied During rehydration movie. In contrast, the gel-assisted swelling method uses an agarose gel substrate to Enhance movie rehydration. Both methods can Produce GUVs in low (eg, 5 mM) and physiological (eg, 100 mM) salt concentrations. The resulting and GUVs are caractérisé via fluorescence microscopy, and the function of reconstituted channels Measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) Gives a high yield of defect-free GUVs, the gel-assisted method swelling Produces a more homogeneous protein distribution and requires no special equipment.

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Anagha Datar, Thomas Bornschlögl, Patricia Bassereau, Jacques Prost, Pramod A Pullarkat (2015 Feb 5)

Dynamics of membrane tethers reveal novel aspects of cytoskeleton-membrane interactions in axons.

Biophysical journal : 489-97 : DOI : 10.1016/j.bpj.2014.11.3480 En savoir plus
Résumé

Mechanical properties of cell membranes are Known to be Significantly Influenced by the Underlying cortical cytoskeleton. The art of pulling membrane tethers from cells is one of the MOST effective ways of studying the mechanics membrane and the membrane-cortex interaction. In this article, we show That axon membranes make an interesting system to exhibit explores As They Both free membrane-like behavior Where the tether-junction membrane is movable on the area of ​​the axons (Unlike Many –other cell membranes) as well as cell- like behavior Where There are transient and spontaneous eruptions in the strength tether That vanish When F-actin is depolymerized. We analyze the passive and spontaneous responses of axonal membrane tethers and offers theoretical models to explain the Observed behavior.

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Renard HF, Simunovic M, Lemière J, Boucrot E, Garcia-Castillo MD, Arumugam S, Chambon V, Lamaze C, Wunder C, Kenworthy AK, Schmidt AA, McMahon HT, Sykes C*, Bassereau P*, Johannes L (2015 Jan 22)

Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis

Nature : 517 : 493-6 : DOI : 10.1038/nature14064 En savoir plus
Résumé

During endocytosis, energy is Invested to narrow necks of the cargo-containing plasma membrane invaginations to radii at the Opposing segments That coalesce spontaneously, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN / Amphiphysin / Rvs (BAR) domain-containing protein Endophilin. Clathrin-independent endocytic events are less connecting Often we dynamin, and whether in contention boxes BAR domain proteins Such As Endophilin contribuer to split HAS Remained unexplored. Here we show, in human and mammalian cell lines –other, That Endophilin-A2 (endoA2) SPECIFICALLY and functionally associates with very early uptake structures are induced by the bacterial Shiga toxins and cholera That, qui Both are clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before split. Furthermore, we Demonstrate That endoA2, dynamin and actin contribuer in parallel to the split of Shiga-toxin-induced tubules. Our results Follows has the novel function of endoA2 in clathrin-independent endocytosis. They separate paper That split factoring operate in an additive Manner, and predict That specificity Within a Given uptake process Arises from defined combinations of universal modules. Our Findings highlight a link entre Previously unnoticed by endoA2 membrane scaffolding and pulling-force-driven dynamic split.

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Chaigne A, Campillo C, Gov NS, Voituriez R, Sykes C, Verlhac MH, Terret ME (2015 Jan 19)

A narrow window of cortical tension guides asymmetric spindle positioning in the mouse oocyte

Nature Communications : 6 : 6027 : DOI : 10.1038/ncomms7027 En savoir plus
Résumé

Cell mechanics control the outcome of cell division. In mitosis, external forces applied on a stiff cortex direct spindle orientation and morphogenesis. During oocyte meiosis on the contrary, spindle positioning depends on cortex softening. How changes in cortical organization induce cortex softening has not yet been addressed. Furthermore, the range of tension that allows spindle migration remains unknown. Here, using artificial manipulation of mouse oocyte cortex as well as theoretical modelling, we show that cortical tension has to be tightly regulated to allow off-center spindle positioning: a too low or too high cortical tension both lead to unsuccessful spindle migration. We demonstrate that the decrease in cortical tension required for spindle positioning is fine-tuned by a branched F-actin network that triggers the delocalization of myosin-II from the cortex, which sheds new light on the interplay between actin network architecture and cortex tension.

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Havrylenko S, Noguera P, Abou-Ghali M, Manzi J, Faqir F, Lamora A, Guérin C, Blanchoin L, Plastino J (2015 Jan 1)

WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly

Molecular Biology of the Cell : 26 : 55-65 : DOI : 10.1091/mbc.E14-07-1200 En savoir plus
Résumé

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex-based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP’s ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.

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Année de publication : 2014

Morgan Delarue, Jean-François Joanny, Frank Jülicher, Jacques Prost (2014 Dec 9)

Stress distributions and cell flows in a growing cell aggregate.

Interface focus : 20140033 : DOI : 10.1098/rsfs.2014.0033 En savoir plus
Résumé

We discuss the short-time response of a multicellular spheroid to an external pressure jump. Our experiments show that 5 min after the pressure jump, the cell density increases in the centre of the spheroid but does not change appreciably close to the surface of the spheroid. This result can be explained if the cells are polarized which we show to be the case. Motivated by the experimental results, we develop a theory for polarized spheroids where the cell polarity is radial (except in a thin shell close to the spheroid surface). The theory takes into account the dependence of cell division and apoptosis rates on the local stress, the cell polarity and active stress generated by the cells and the dependence of active stress on the local pressure. We find a short-time increase of the cell density after a pressure jump that decays as a power law from the spheroid centre, which is in reasonable agreement with the experimental results. By comparing our theory to experiments, we can estimate the isotropic compression modulus of the tissue.

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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 (2014 Dec 5)

FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool.

Lab on a chip : 811-22 : DOI : 10.1039/c4lc01059k En savoir plus
Résumé

Microfluidic systems bear promise to provide new powerful tools for the molecular characterization of cancer cells, in particular for the routine detection of multiple cancer biomarkers using a minute amount of the sample. However, taking miniaturized cell-based assays into the clinics requires the implementation and validation of complex biological protocols on chip, as well as the development of disposable microdevices produced at a low cost. Based on a recently developed microfluidic chip made of Cyclic Olefin Copolymer for cell immobilization with minimal dead volume and controlled shear stress, we developed a protocol performed entirely in the liquid phase, allowing the immobilization and fixation of cells and their quantitative characterization by fluorescence in situ hybridization. We demonstrated first in cell lines and then in two clinical case studies the potential of this method to perform quantitative copy number measurement and clinical scoring of the amplification of the ERBB2 gene, a decisive biomarker for the prescription of HER2+ related targeted therapies. This validation was performed in a blind protocol in two clinical case studies, in reference to the gold standard and clinically used method based on glass slides. We obtained a comparable reproducibility and a minor difference in apparent amplification, which can be corrected by internal calibration. The method thus reaches the standard of robustness needed for clinical use. The protocol can be fully automated, and its consumption of samples and DNA probes is reduced as compared to glass slide protocols by a factor of at least 10. The total duration of the assay is divided by two.

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Guillaume van Niel, Ptissam Bergam, Aurelie Di Cicco, Ilse Hurbain, Alessandra Lo Cicero, Florent Dingli, Roberta Palmulli, Cecile Fort, Marie Claude Potier, Leon J Schurgers, Damarys Loew, Daniel Levy, Graça Raposo (2014 Nov 13)

Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells.

Cell reports : 43-51 : DOI : 10.1016/j.celrep.2015.08.057 En savoir plus
Résumé

Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

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Leïla Perié, Shalin H Naik (2014 Nov 11)

Toward defining a ‘lineage’–The case for dendritic cells.

Seminars in cell & developmental biology : 3-8 : DOI : 10.1016/j.semcdb.2015.02.004 En savoir plus
Résumé

The immune system consists of a heterogeneous ensemble of cell types that immunologists have tried to classify and order for decades. This classification has relied on varying criteria, resulting in major debates in the immunology community. Discovered in the late 1970s [1], dendritic cells (DCs) are no exception, and their membership to a distinct immune lineage is still vividly debated [2-6]. Here, we review recent work on the origin of DCs and discuss the possible definition of a separate ‘DC lineage’.

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Charles-Edouard Leroux, Sylvain Monnier, Irène Wang, Giovanni Cappello, Antoine Delon (2014 Nov 1)

Fluorescent correlation spectroscopy measurements with adaptive optics in the intercellular space of spheroids.

Biomedical optics express : 3730-8 : DOI : 10.1364/BOE.5.003730 En savoir plus
Résumé

In this study we demonstrate the use of adaptive optics to correct the biasing effects of optical aberrations when measuring the dynamics of molecules diffusing between cells in multicellular spheroids. Our results indicate that, on average, adaptive optics leads to a reduction of the 3D size of the point spread function that is statistically significant in terms of measured number of molecules and diffusion time. The sensorless approach, which uses the molecular brightness as optimization metric, is validated in a complex, highly heterogeneous, biological environment. This work paves the way towards the design of accurate diffusion measurements of molecules in thick biological specimens.

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