UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2015

Ricard Alert, Jaume Casademunt, Jan Brugués, Pierre Sens (2015 Apr 23)

Model for probing membrane-cortex adhesion by micropipette aspiration and fluctuation spectroscopy.

Biophysical journal : 1878-86 : DOI : 10.1016/j.bpj.2015.02.027 En savoir plus
Résumé

We propose a model for membrane-cortex adhesion that couples membrane deformations, hydrodynamics, and kinetics of membrane-cortex ligands. In its simplest form, the model gives explicit predictions for the critical pressure for membrane detachment and for the value of adhesion energy. We show that these quantities exhibit a significant dependence on the active acto-myosin stresses. The model provides a simple framework to access quantitative information on cortical activity by means of micropipette experiments. We also extend the model to incorporate fluctuations and show that detailed information on the stability of membrane-cortex coupling can be obtained by a combination of micropipette aspiration and fluctuation spectroscopy measurements.

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Renaud Renault, Nirit Sukenik, Stéphanie Descroix, Laurent Malaquin, Jean-Louis Viovy, Jean-Michel Peyrin, Samuel Bottani, Pascal Monceau, Elisha Moses, Maéva Vignes (2015 Apr 23)

Combining microfluidics, optogenetics and calcium imaging to study neuronal communication in vitro.

PloS one : e0120680 : DOI : 10.1371/journal.pone.0120680 En savoir plus
Résumé

In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.

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Ludger Johannes, Robert G Parton, Patricia Bassereau, Satyajit Mayor (2015 Apr 11)

Building endocytic pits without clathrin.

Nature reviews. Molecular cell biology : 311-21 : DOI : 10.1038/nrm3968 En savoir plus
Résumé

How endocytic pits are built in clathrin- and caveolin-independent endocytosis still remains poorly Understood. Recent insight Suggests That different forms of clathrin-independent endocytosis might Involve the actin-driven focusing of membrane constituents, the lectin-glycosphingolipid-dependent endocytic nanoenvironments of building, and Bin-Amphiphysin-Rvs (BAR) domain proteins serving as scaffolding modules. We need the Chat for the different kinds of internalization processes in the context of diverse cellular functions, the existence of clathrin-independent Mechanisms of cargo recruitment and membrane bending from a physical and biological perspective, and finally propose a generic scheme for the formation of clathrin- independent endocytic pits.

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Lemière J, Carvalho K, Sykes C (2015 Apr 8)

Cell-sized liposomes that mimic cell motility and the cell cortex

Methods in Cell Biology : 128 : 271-85 : DOI : 10.1016/bs.mcb.2015.01.013 En savoir plus
Résumé

Cells move and change shape by dynamically reorganizing their cytoskeleton next to the plasma membrane. In particular, actin assembly generates forces and stresses that deform the cell membrane. Cell-sized liposomes are designed to mimic this function. The activation of actin polymerization at their membrane is able to push the membrane forward, thus reproducing the mechanism of lamellipodium extension at the cell front. Moreover, the cell cortex, a submicrometer-thick actin shell right beneath the cell membrane can be reproduced; it contributes to cell tension with the action of molecular motors. We will describe experimental methods to prepare liposomes that mimic the inside geometry of a cell, and that reproduce actin-based propulsion of the liposome using an outside geometry. Such systems allow to study how actin-related proteins control and affect actin cortex assembly and can produce forces that drive cell shape changes.

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Matthias Garten, Coline Prévost, Clotilde Cadart, Romain Gautier, Luc Bousset, Ronald Melki, Patricia Bassereau, Stefano Vanni (2015 Apr 1)

Methyl-branched lipids promote the membrane adsorption of α-synuclein by enhancing shallow lipid-packing defects.

Physical chemistry chemical physics : PCCP : 15589-97 : DOI : 10.1039/c5cp00244c En savoir plus
Résumé

Alpha-synuclein (AS) is a synaptic protein That Is Directly Involved in Parkinson’s disease due to ict tendency to form protein aggregates. Since AS aggregation can be dependent on the interactions entre les protein and the cell plasma membrane, elucidating the membrane binding properties of AS is of crucial importance to the suit les molecular basis of AS aggregation into toxic fibrils. Using a combination of in vitro reconstitution experiments based on Giant Unilamellar Vesicles (GUVs), confocal microscopy and all-atom molecular dynamics simulations, we-have Investigated the membrane binding properties of AS, with a focus on the relative contribution of hydrophobic versus electrostatic interactions. In contrast with previous observations, We Did not observed Any binding of AS to membranes Containing the ganglioside GM1, GM1-even at high Relatively happy. AS we Reviews the other hand, Showed a stronger affinity for neutral flat membranes consistant en methyl-branched lipids. To rationalize thesis results, we used all-atom molecular dynamics simulations to Investigate the effect of methyl-branched lipids is membrane interfacial properties. We found That methyl-branched lipids Promote the membrane adsorption of AS by Creating shallow lipid-packing defects to a larger extent than polyunsaturated and monounsaturated lipids. Our Findings suggest methyl-branched lipids That May Constitute a substrate for adhesive Remarkably peripheral proteins adsorbed That is hydrophobic membranes via inserts.

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Almonacid M, Ahmed WW, Bussonnier M, Mailly P, Betz T, Voituriez R, Gov NS, Verlhac MH (2015 Apr 1)

Active diffusion positions the nucleus in mouse oocytes

Nature Cell Biology : 17 : 470-9 : DOI : 10.1038/ncb3131 En savoir plus
Résumé

In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.

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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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