UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2017

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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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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Année de publication : 2016

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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Benjamin Brunel, Grégory Beaune, Usharani Nagarajan, Sylvie Dufour, Françoise Brochard-Wyart, Françoise M Winnik (2016 Oct 8)

Nanostickers for cells: a model study using cell-nanoparticle hybrid aggregates.

Soft matter : 7902-7907 En savoir plus
Résumé

We present direct evidence that nanoparticles (NPs) can stick together cells that are inherently non-adhesive. Using cadherin-depleted S180 murine cells lines, which exhibit very low cell-cell adhesion, we show that NPs can assemble dispersed single cells into large cohesive aggregates. The dynamics of aggregation, which is controlled by diffusion and collision, can be described as a second-order kinetic law characterized by a rate of collision that depends on the size, concentration, and surface chemistry of the NPs. We model the cell-cell adhesion induced by the « nanostickers » using a three-state dynamical model, where the NPs are free, adsorbed on the cell membrane or internalized by the cells. We define a « sticking efficiency parameter » to compare NPs and look for the most efficient type of NP. We find that 20 nm carboxylated polystyrene NPs are more efficient nanostickers than 20 nm silica NPs which were reported to induce fast wound healing and to glue soft tissues. Nanostickers, by increasing the cohesion of tissues and tumors, may have important applications for tissue engineering and cancer treatment.

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Année de publication : 2017

Duclos G., Erlenkämper C., Joanny J.-F., Silberzan P. (2016 Sep 12)

Topological defects in confined populations of spindle-shaped cells

Nature Physics : 13 : 58-62 : DOI : 10.1038/nphys3876 En savoir plus
Résumé

Most spindle-shaped cells (including smooth muscles and sarcomas) organize in vivo into well-aligned ‘nematic’ domains, creating intrinsic topological defects that may be used to probe the behaviour of these active nematic systems. Active non-cellular nematics have been shown to be dominated by activity, yielding complex chaotic flows. However, the regime in which live spindle-shaped cells operate, and the importance of cell–substrate friction in particular, remains largely unexplored. Using in vitro experiments, we show that these active cellular nematics operate in a regime in which activity is effectively damped by friction, and that the interaction between defects is controlled by the system’s elastic nematic energy. Due to the activity of the cells, these defects behave as self-propelled particles and pairwise annihilate until all displacements freeze as cell crowding increases. When confined in mesoscopic circular domains, the system evolves towards two identical +1/2 disclinations facing each other. The most likely reduced positions of these defects are independent of the size of the disk,the cells’ activity or even the cell type, but are well described by equilibrium liquid crystal theory. These cell-based systems thus operate in a regime more stable than other active nematics, which may be necessary for their biological function.

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Année de publication : 2016

Marina Casiraghi, Marjorie Damian, Ewen Lescop, Elodie Point, Karine Moncoq, Nelly Morellet, Daniel Levy, Jacky Marie, Eric Guittet, Jean-Louis Banères, Laurent J Catoire (2016 Aug 5)

Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer.

Journal of the American Chemical Society : 11170-5 : DOI : 10.1021/jacs.6b04432 En savoir plus
Résumé

Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.

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A Bertin, E Nogales (2016 Jul 31)

Preparing recombinant yeast septins and their analysis by electron microscopy.

Methods in cell biology : 21-34 : DOI : 10.1016/bs.mcb.2016.03.010 En savoir plus
Résumé

Septins are highly conserved and essential eukaryotic cytoskeletal proteins that interact with the inner plasma membrane. They are involved in essential functions requiring cell membrane remodeling and compartmentalization, such as cell division and dendrite morphogenesis, and have been implicated in numerous diseases. Depending on the organisms and on the type of tissue, a specific set of septins genes are expressed, ranging from 2 to 13. Septins self-assemble into linear, symmetric rods that can further organize into linear filaments several microns in length. Only a subset of human septins has been described at high resolution by X-ray crystallography (Sirajuddin et al., 2007). Electron microscopy (EM) has proven to be a method of choice for analyzing the molecular organization of septins. It is possible to localize each septin subunit within the rod complex using genetic tags, such as maltose-binding protein or green fluorescent protein, to generate a visible label of a specific septin subunit in EM images that are processed using single-particle EM methodology. In this chapter we present, in detail, the methods that we have used to analyze the molecular organization of budding yeast septins (Bertin et al., 2008). These methods include purification of septin complexes, sample preparation for EM, and image processing procedures. Such methods can be generalized to analyze the organization of septins from any organism.

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Caorsi V, Lemière J, Campillo C, Bussonnier M, Manzi J, Betz T, Plastino J, Carvalho K, Sykes C (2016 Jul 20)

Cell-sized liposome doublets reveal active tension build-up driven by acto-myosin dynamics

Soft Matter : 12 : 6223-31 : DOI : 10.1039/c6sm00856a En savoir plus
Résumé

Cells modulate their shape to fulfill specific functions, mediated by the cell cortex, a thin actin shell bound to the plasma membrane. Myosin motor activity, together with actin dynamics, contributes to cortical tension. Here, we examine the individual contributions of actin polymerization and myosin activity to tension increase with a non-invasive method. Cell-sized liposome doublets are covered with either a stabilized actin cortex of preformed actin filaments, or a dynamic branched actin network polymerizing at the membrane. The addition of myosin II minifilaments in both cases triggers a change in doublet shape that is unambiguously related to a tension increase. Preformed actin filaments allow us to evaluate the effect of myosin alone while, with dynamic actin cortices, we examine the synergy of actin polymerization and myosin motors in driving shape changes. Our assay paves the way for a quantification of tension changes triggered by various actin-associated proteins in a cell-sized system.

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Leïla Perié, Ken R Duffy (2016 Jul 13)

Retracing the in vivo haematopoietic tree using single-cell methods.

FEBS letters : DOI : 10.1002/1873-3468.12299 En savoir plus
Résumé

The dynamic process by which self-renewing stem cells and their offspring proliferate and differentiate to create the erythroid, myeloid and lymphoid lineages of the blood system has long since been an important topic of study. A range of recent single cell and family tracing methodologies such as massively parallel single-cell RNA-sequencing, mass cytometry, integration site barcoding, cellular barcoding and transposon barcoding are enabling unprecedented analysis, dissection and re-evaluation of the haematopoietic tree. In addition to the substantial experimental advances, these new techniques have required significant theoretical development in order to make biological deductions from their data. Here, we review these approaches from both an experimental and inferential point of view, considering their discoveries to date, their capabilities, limitations and opportunities for further development.

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Mijo Simunovic, Coline Prévost, Andrew Callan-Jones, Patricia Bassereau (2016 Jun 15)

Physical basis of some membrane shaping mechanisms.

Philosophical transactions. Series A, Mathematical, physical, and engineering sciences : DOI : 10.1098/rsta.2016.0034 En savoir plus
Résumé

In vesicular transportation pathways, membrane lipids and proteins are internalized, externalized or transported Within cells, not by bulk diffusion of single molecules, goal embedded in the membrane of small vesicles or thin tubules. The formation of These ‘transportation carriers’ Follows sequential events: bending membrane fission from the donor compartment, and transportation Eventually fusion with the acceptor membrane. A similar sequence is Involved During the internalization of drug or gene carriers inside cells. These membrane-shaping events are mediated by proteins Generally binding to membranes. The thesis Mechanisms behind biological processes are Actively Studied Both in the context of cell biology and biophysics. Bin / Amphiphysin / Rvs (BAR) domain proteins are Ideally suited for single Illustrating how soft matter principles can account for deformation by membrane proteins. We review here Some experimental methods and theoretical models to measure Corresponding thesis how proteins affect the mechanics and the shape of membranes. In more detail, we show how an experimental method Employing optical tweezers to pull a tube from a giant vesicle May give significant quantitative insights into the mechanism by which proteins sense and generate membrane curvature and the mechanism of membrane scission.This article is share of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’.

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Renaud Renault, Jean-Baptiste Durand, Jean-Louis Viovy, Catherine Villard (2016 May 27)

Asymmetric axonal edge guidance: a new paradigm for building oriented neuronal networks.

Lab on a chip : 2188-91 : DOI : 10.1039/c6lc00479b En savoir plus
Résumé

We present a novel kind of directional axon guides for brain-on-a-chip applications. Contrarily to previous works, the directionality in our design is created by rerouting axons growing in the unwanted direction back to their original compartment while leaving the other growth direction unaffected. This design yields state-of-the-art levels of directionality without the disadvantages of previously reported technologies.

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Evangelos Gogolides, Angeliki Tserepi, Gerhard Jobst, Jean-Michel Friedt, David Rabus, Bruno Dupuy, Zuzana Bilkova, Stephanie Descroix, Jean-Louis Viovy, George Papadakis, Electra Gizeli (2016 May 27)

Micro-Nano-Bio Diagnostic System for Food Pathogen Detection Revolutionizes Food Safety Management & Protects Consumers Health.

Studies in health technology and informatics : 67-72 En savoir plus
Résumé

The development of integrated, fast and affordable platforms for pathogen detection is an emerging area where a multidisciplinary approach is necessary for designing microsystems employing miniaturized devices; these new technologies promise a significant advancement of the current state of analytical testing leading to improved healthcare. In this work, the development of a lab-on-chip microsystem platform for the genetic analysis of Salmonella in milk samples is presented. The heart of the platform is an acoustic detection biochip, integrated with a microfluidic module. This detection platform is combined with a micro-processor, which, alongside with magnetic beads technology and a DNA micro-amplification module, are responsible for performing sample pre-treatment, bacteria lysis, nucleic acid purification and amplification. Automated, multiscale manipulation of fluids in complex microchannel networks is combined with novel sensing principles developed by some of the partners. This system is expected to have a significant impact in food-pathogen detection by providing for the first time an integrated detection test for Salmonella screening in a very short time. Finally, thanks to the low cost and compact technologies involved, the proposed set-up is expected to provide a competitive analytical platform for direct application in field settings.

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