UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2015

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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Havrylenko S, Mezanges X, Batchelder E, Plastino J (2014 Oct 1)

Extending the molecular clutch beyond actin-based cell motility

New Journal of Physics : 16 : 105012 : DOI : 10.1088/1367-2630/16/10/105012 En savoir plus
Résumé

Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the « molecular clutch » description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of Major Sperm Protein (MSP), which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton.

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Pascal Martin (2014 Aug 21)

All that jazz coming out of my ears.

Biophysical journal : 800-2 : DOI : 10.1016/j.bpj.2014.07.011 En savoir plus
Résumé

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Bussonnier M, Carvalho K, Lemière J, Joanny JF, Sykes C, Betz T (2014 Aug 19)

Mechanical detection of a long-range actin network emanating from a biomimetic cortex

Biophysical Journal : 107 : 854-62 : DOI : 10.1016/j.bpj.2014.07.008 En savoir plus
Résumé

Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the « actin cloud » and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.

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Philippe Bun, JunJun Liu, Hervé Turlier, ZengZhen Liu, Karen Uriot, Jean-François Joanny, Maïté Coppey-Moisan (2014 Jul 17)

Mechanical checkpoint for persistent cell polarization in adhesion-naive fibroblasts.

Biophysical journal : 324-35 : DOI : 10.1016/j.bpj.2014.05.041 En savoir plus
Résumé

Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.

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Ayako Yamada, Sungyon Lee, Patricia Bassereau, Charles N Baroud (2014 Jun 17)

Trapping and release of giant unilamellar vesicles in microfluidic wells.

Soft matter : 5878-85 : DOI : 10.1039/c4sm00065j En savoir plus
Résumé

We describe the trapping and release of giant unilamellar vesicles (GUVs) in a thin and wide microfluidic channel, As They cross indentations etched in the channel ceiling. This trapping results from the reduction of the membrane elastic energy, qui est Stored in the GUV as it squeezes to enter into the thin channel. We Demonstrate That GUVs Whose diameter is Slightly larger than the channel height can be trapped and That They Can be untrapped by the outer fluid flowing beyond a critical velocity. GUVs smaller than the flow channel height undisturbed while much larger Those can not squeeze into the thin regions. Within the ranks That allows trapping, larger GUVs are anchored more than smaller Strongly GUVs. The Ability to trap vesicles Provides access to the optical GUVs for extended Periods of time; this allows the observation of recirculation flow on the surface area of ​​the GUVs, in the direction of forward near the mid-plane of the channel and in the reverse direction of elsewhere. We also obtenir the shape of GUVs under different flow requirements through confocal microscopy. This geometric information is used to drift a mechanical model of the power balance That equates the viscous effects from the outer flow with the elastic effects based on the variation of the membrane stretching energy. This model yields Good agreement with the experimental data values ​​When stretching of the moduli are taken from the scientific literature. This microfluidic approach Provides a New Way of Storing a wide number of GUVs at specific locations, with or without the presence of an outer flow. As Such, it was deriving their high-throughput alternative to micropipette manipulation of individual chemical or biological GUVs for applications.

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Jana Kucerova, Zuzana Svobodova, Petr Knotek, Jiri Palarcik, Milan Vlcek, Miloslav Kincl, Daniel Horak, Julien Autebert, Jean-Louis Viovy, Zuzana Bilkova (2014 May 27)

PEGylation of magnetic poly(glycidyl methacrylate) microparticles for microfluidic bioassays.

Materials science & engineering. C, Materials for biological applications : 308-15 : DOI : 10.1016/j.msec.2014.04.011 En savoir plus
Résumé

In this study, magnetic poly(glycidyl methacrylate) microparticles containing carboxyl groups (PGMA-COOH) were coated using highly hydrophilic polymer poly(ethylene glycol) (PEG). PEG was used to reduce nonspecific interactions with proteins and cells while decreasing adhesion of particles to the walls of a microfluidic devices from poly(dimethylsiloxane) (PDMS) and cyclic olefin copolymer (COC). Zeta potential measurement, infrared spectroscopy, scanning electron microscopy, anti-PEG ELISA assay, and bioaffinity interactions between biotin and streptavidin-HRP successfully proved the presence of PEG on the surface of microspheres. Both neat and PEGylated microspheres were then incubated with the inert protein bovine serum albumin or cells to evaluate the rate of nonspecific adsorption (NSA). PEG with Mr of 30,000 Da was responsible for 45% reduction in NSA of proteins and 74% for cells compared to neat particles. The microspheres’ behavior in PDMS and COC microchannels was then evaluated. Aggregation and adhesion of PEGylated microspheres significantly decreased compared to neat particles. Finally, the model enzyme horseradish peroxidase was immobilized on the microspheres through the heterobifunctional PEG chain. The possibility for subsequent covalent coupling of the ligand of interest was confirmed. Such PEGylated microparticles can be efficiently used in PDMS microchips as a carrier for bioaffinity separation or of enzyme for catalysis.

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C Blanch-Mercader, J Casademunt, J F Joanny (2014 May 24)

Morphology and growth of polarized tissues.

The European physical journal. E, Soft matter : 41 : DOI : 10.1140/epje/i2014-14041-2 En savoir plus
Résumé

We study and classify the time-dependent morphologies of polarized tissues subject to anisotropic but spatially homogeneous growth. Extending previous studies, we model the tissue as a fluid, and discuss the interplay of the active stresses generated by the anisotropic cell division and three types of passive mechanical forces: viscous stresses, friction with the environment and tension at the tissue boundary. The morphology dynamics is formulated as a free-boundary problem, and conformal mapping techniques are used to solve the evolution numerically. We combine analytical and numerical results to elucidate how the different passive forces compete with the active stresses to shape the tissue in different temporal regimes and derive the corresponding scaling laws. We show that in general the aspect ratio of elongated tissues is non-monotonic in time, eventually recovering isotropic shapes in the presence of friction forces, which are asymptotically dominant.

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Lin Jia, Di Cui, Jérôme Bignon, Aurelie Di Cicco, Joanna Wdzieczak-Bakala, Jianmiao Liu, Min-Hui Li (2014 May 19)

Reduction-responsive cholesterol-based block copolymer vesicles for drug delivery.

Biomacromolecules : 2206-17 : DOI : 10.1021/bm5003569 En savoir plus
Résumé

We developed a new robust reduction-responsive polymersome based on the amphiphilic block copolymer PEG-SS-PAChol. The stability and robustness were achieved by the smectic physical cross-linking of cholesterol-containing liquid crystal polymer PAChol in the hydrophobic layer. The reduction-sensitivity was introduced by the disulfide bridge (-S-S-) that links the hydrophilic PEG block and the hydrophobic PAChol block. We used a versatile synthetic strategy based on atom transfer radical polymerization (ATRP) to synthesize the reduction-responsive amphiphilic block copolymers. The reductive cleavage of the disulfide bridge in the block copolymers was first evidenced in organic solution. The partial destruction of PEG-SS-PAChol polymersomes in the presence of a reducing agent was then demonstrated by cryo-electron microscopy. Finally, the calcein release from PEG-SS-PAChol polymersomes triggered by glutathione (GSH) was observed both in PBS suspension and in vitro inside the macrophage cells. High GSH concentrations (≥35 mM in PBS or artificially enhanced in macrophage cells by GSH-OEt pretreatment) and long incubation time (in the order of hours) were, however, necessary to get significant calcein release. These polymersomes could be used as drug carriers with very long circulation profiles and slow release kinetics.

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Laura Picas, Julien Viaud, Kristine Schauer, Stefano Vanni, Karim Hnia, Vincent Fraisier, Aurélien Roux, Patricia Bassereau, Frédérique Gaits-Iacovoni, Bernard Payrastre, Jocelyn Laporte, Jean-Baptiste Manneville, Bruno Goud (2014 May 19)

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin.

Nature communications : 5647 : DOI : 10.1038/ncomms6647 En savoir plus
Résumé

Phosphoinositide play a central role in physiological processes by Many Assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How is this recruitment Coordinated in space and time is not well Understood. Here we show That BIN1 / M-Amphiphysin2, a protein Involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns (4,5) P2 to recruit ict downstream partner dynamin. By using Several mutants associated with centronuclear myopathies, we find that N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin we membranes, respectively. We that show phosphoinositide Clustering is a mechanism shared by –other That proteins interact with PtdIns (4,5) P2, do not aim to contain a BAR domain. Our numerical simulations Point Out That clustering is a diffusion-driven process in which molecules are not sequestered phosphoinositide. We propose That this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

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