UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2005

Jasper van der Gucht, Ewa Paluch, Julie Plastino, Cécile Sykes (2005 May 25)

Stress release drives symmetry breaking for actin-based movement.

Proceedings of the National Academy of Sciences of the United States of America : 7847-52 En savoir plus
Résumé

By using a simple assay composed of purified proteins, we studied the spontaneous polarization of actin networks polymerizing on spherical beads, which subsequently undergo movement. We show evidence that this symmetry breaking is based on the release of elastic energy, analogous to the fracture of polymer gels. The dynamics of this process and the thickness at which it occurs depend on the growth rate and mechanical properties of the actin gel. We explain our experimental results with a model based on elasticity theory and fracture mechanics.

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Ewa Paluch, Matthieu Piel, Jacques Prost, Michel Bornens, Cécile Sykes (2005 May 10)

Cortical actomyosin breakage triggers shape oscillations in cells and cell fragments.

Biophysical journal : 724-33 En savoir plus
Résumé

Cell shape and movements rely on complex biochemical pathways that regulate actin, microtubules, and substrate adhesions. Some of these pathways act through altering the cortex contractility. Here we examined cellular systems where contractility is enhanced by disassembly of the microtubules. We found that adherent cells, when detached from their substrate, developed a membrane bulge devoid of detectable actin and myosin. A constriction ring at the base of the bulge oscillated from one side of the cell to the other. The movement was accompanied by sequential redistribution of actin and myosin to the membrane. We observed this oscillatory behavior also in cell fragments of various sizes, providing a simplified, nucleus-free system for biophysical studies. Our observations suggest a mechanism based on active gel dynamics and inspired by symmetry breaking of actin gels growing around beads. The proposed mechanism for breakage of the actomyosin cortex may be used for cell polarization.

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Aurélien Roux, Damien Cuvelier, Pierre Nassoy, Jacques Prost, Patricia Bassereau*, Bruno Goud* (2005 Mar 26)

Role of curvature and phase transition in lipid sorting and fission of membrane tubules.

The EMBO journal : 1537-45 : DOI : 10.1038/sj.emboj.7600631 En savoir plus
Résumé

We have recently developed a minimal system for generating long tubular nanostructures that resemble tubes observed in vivo with biological membranes. Here, we studied membrane tube pulling in ternary mixtures of sphingomyelin, phosphatidylcholine and cholesterol. Two salient results emerged: the lipid composition is significantly different in the tubes and in the vesicles; tube fission is observed when phase separation is generated in the tubes. This shows that lipid sorting may depend critically on both membrane curvature and phase separation. Phase separation also appears to be important for membrane fission in tubes pulled out of giant liposomes or purified Golgi membranes.

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Renaud Fulconis, Marie Dutreix, Jean-Louis Viovy (2005 Mar 8)

Numerical investigation of sequence dependence in homologous recognition: evidence for homology traps.

Biophysical journal : 3770-9 En savoir plus
Résumé

During the initial phase of RecA-mediated recombination, known as the search for homology, a single-stranded DNA coated by RecA protein and a homologous double-stranded DNA have to perfectly align and pair. We designed a model for the homology search between short molecules, and performed Monte Carlo Metropolis computer simulations of the process. The central features of our model are 1), the assumption that duplex DNA longitudinal thermal fluctuations are instrumental in the binding; and 2), the explicit consideration of the nucleotide sequence. According to our results, recognition undergoes a first slow nucleation step over a few basepairs, followed by a quick extension of the pairing to adjacent bases. The formation of the three-stranded complex tends to be curbed by heterologies but also by another possible obstacle: the presence of partially homologous stretches, such as mono- or polynucleotide repeats. Actually, repeated sequences are observed to trap the molecules in unproductive configurations. We investigate the dependence of the phenomenon on various energy parameters. This mechanism of homology trapping could have a strong biological relevance in the light of the genomic instability experimentally known to be triggered by repeated sequences.

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Adeline Giganti, Julie Plastino, Bassam Janji, Marleen Van Troys, Delphine Lentz, Christophe Ampe, Cécile Sykes, Evelyne Friederich (2005 Mar 3)

Actin-filament cross-linking protein T-plastin increases Arp2/3-mediated actin-based movement.

Journal of cell science : 1255-65 En savoir plus
Résumé

Increasing evidence suggests that actin cross-linking or bundling proteins might not only structure the cortical actin cytoskeleton but also control actin dynamics. Here, we analyse the effects of T-plastin/T-fimbrin, a representative member of an important actin-filament cross-linking protein by combining a quantitative biomimetic motility assay with biochemical and cell-based approaches. Beads coated with the VCA domain of the Wiskott/Aldrich-syndrome protein (WASP) recruit the actin-nucleating Arp2/3 complex, polymerize actin at their surface and undergo movement when placed in cell-free extracts. T-Plastin increased the velocity of VCA beads 1.5 times, stabilized actin comets and concomitantly displaced cofilin, an actin-depolymerizing protein. T-Plastin also decreased the F-actin disassembly rate and inhibited cofilin-mediated depolymerization of actin filaments in vitro. Importantly, a bundling-incompetent variant comprising the first actin-binding domain (ABD1) had similar effects. In cells, this domain induced the formation of long actin cables to which other actin-regulating proteins were recruited. Altogether, these results favor a mechanism in which binding of ABD1 controls actin turnover independently of cross-link formation. In vivo, this activity might contribute to the assembly and maintenance of the actin cytoskeleton of plasma-membrane protrusions.

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Girard P, Prost J, Bassereau P (2005 Mar 1)

Passive or active fluctuations in membranes containing proteins.

Phys. Rev. Lett. : 94 : 088102 : DOI : 10.1103/PhysRevLett.94.088102 En savoir plus
Résumé

We have experimentally investigated the effect of a transmembrane protein, the Ca2+-ATPase, on shape fluctuations of giant vesicles. By using the micropipette method, we have measured a substantial renormalization of the bending modulus due to the presence of proteins in the membrane. Moreover, we have produced the first quantitative measurement of the active force dipole associated with the amplification of the fluctuations when the proteins are activated by adenosine 5′-triphosphate (ATP).

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Kevin D Dorfman, Renaud Fulconis, Marie Dutreix, Jean-Louis Viovy (2005 Feb 9)

Model of RecA-mediated homologous recognition.

Physical review letters : 268102 En savoir plus
Résumé

We consider theoretically the homology search between a long double-stranded DNA and a RecA-single-stranded DNA nucleofilament, emphasizing the polymeric nature of the search and the ability of double-stranded DNA to overcome the difference in pitch between itself and the nucleofilament by thermally activated stretching from the canonical B state to the metastable, stretched S state. Our analytical first-passage-time analysis agrees well with experimental data, predicts new dependencies on the intracellular fluid viscosity and ionic strength, and strongly suggests that initial homologous recognition involves a three base-pair seed.

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Laetitia Cohen-Tannoudji, Emanuel Bertrand, Lydie Bressy, Cécile Goubault, Jean Baudry, Jacob Klein, Jean-François Joanny, Jérôme Bibette (2005 Feb 9)

Polymer bridging probed by magnetic colloids.

Physical review letters : 038301 En savoir plus
Résumé

Superparamagnetic particles offer a new way to probe the kinetics of adhesive processes. Two different scenarios of physical adhesion are studied. The thermal activation of van der Waals adhesion is well described by an Arrhenius model. In contrast, it is necessary to go beyond the Arrhenius description to understand the thermal activation of bridging between colloidal particles by a polymer at equilibrium adsorbance. We show that polymer bridging requires some removal of adsorbed polymer and is strongly influenced by the proximity of a glass transition within the adsorbed polymer.

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Damien Cuvelier, Imre Derényi, Patricia Bassereau, Pierre Nassoy (2005 Feb 8)

Coalescence of membrane tethers: experiments, theory, and applications.

Biophysical journal : 88 : 2714-26 : DOI : 10.1529/biophysj.104.056473 En savoir plus
Résumé

Tethers are nanocylinders of lipid bilayer membrane, arising in situations ranging from micromanipulation experiments on synthetic vesicles to the formation of dynamic tubular networks in the Golgi apparatus. Relying on the extensive theoretical and experimental works aimed to understand the physics of individual tethers formation, we addressed the problem of the interaction between two nanotubes. By using a combination of micropipette manipulation and optical tweezers, we quantitatively studied the process of coalescence that occurred when the separation distance between both vesicle-tether junctions became smaller than a threshold length. Our experiments, which were supported by an original theoretical analysis, demonstrated that the measurements of the tether force and angle between tethers at coalescence directly yield the bending rigidity, kappa, and the membrane tension, sigma, of the vesicles. Contrary to other methods used to probe the bending rigidity of vesicles, the proposed approach permits a direct measurement of kappa without requiring any control of the membrane tension. Finally, after validation of the method and proposal of possible applications, we experimentally investigated the dynamics of the coalescence process.

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Olivia du Roure, Alexandre Saez, Axel Buguin, Robert H Austin, Philippe Chavrier, Pascal Silberzan, Pascal Siberzan, Benoit Ladoux (2005 Feb 1)

Force mapping in epithelial cell migration.

Proceedings of the National Academy of Sciences of the United States of America : 2390-5 En savoir plus
Résumé

We measure dynamic traction forces exerted by epithelial cells on a substrate. The force sensor is a high-density array of elastomeric microfabricated pillars that support the cells. Traction forces induced by cell migration are deduced from the measurement of the bending of these pillars and are correlated with actin localization by fluorescence microscopy. We use a multiple-particle tracking method to estimate the mechanical activity of cells in real time with a high-spatial resolution (down to 2 microm) imposed by the periodicity of the post array. For these experiments, we use differentiated Madin-Darby canine kidney (MDCK) epithelial cells. Our data provide definite information on mechanical forces exerted by a cellular assembly. The maximum intensity of the forces is localized on the edge of the epithelia. Hepatocyte growth factor promotes cell motility and induces strong scattering activity of MDCK cells. Thus, we compare forces generated by MDCK cells in subconfluent epithelia versus isolated cells after hepatocyte growth factor treatment. Maximal-traction stresses at the edge of a monolayer correspond to higher values than those measured for a single cell and may be due to a collective behavior.

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Aurélien Bancaud, Gaudeline Wagner, Kevin D Dorfman, Jean-Louis Viovy (2005 Feb 1)

Measurement of the surface concentration for bioassay kinetics in microchannels.

Analytical chemistry : 833-9 En savoir plus
Résumé

We present a simple and versatile method, based on fluorescence microscopy, to reliably measure the concentration of advected molecules in the vicinity of surfaces in microchannels. This tool is relevant to many microfluidic applications such as immunoassays and single-molecule experiments, where one probes the kinetics of a reaction between an immobilized target and a reactant carried by the bulk flow. The characterization of the surface concentration highlights the dominant role of transverse diffusion, which generates an apparent diffusivity at the surface 3-4 orders of magnitude greater than molecular diffusion alone, even close to the point of injection. We directly measure the effects of the longitudinal position along the channel and of the flow rate on the concentration front and develop a simple analytical model that compares well with the data. Finally, we propose a method to properly account for concentration fronts in single-molecule measurements and use it to directly access the kinetics parameters of protamine-induced condensation of DNA.

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Pascale Jolimaître, Aurélien Roux, Annick Blanpain, Cécile Leduc, Patricia Bassereau, Line Bourel-Bonneta (2005 Feb 1)

Synthesis and preliminary physical applications of a rhodamin-biotin phosphatidylethanolamine, an easy attainable lipid double probe.

Chemistry and Physics of Lipids : 133 : 215-233 : DOI : 10.1016/j.chemphyslip.2004.10.005 En savoir plus
Résumé

In route to a physical study aimed at understanding lipids and proteins sorting in cells, we designed a rhodamin-labelled biotinylated phosphatidylethanolamine (PE), as a useful and easy-attainable lipid double probe. The target compound was successfully engaged in preliminary physical experiments.

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Max Davidson, Paul Dommersnes, Martin Markström, Jean-Francois Joanny, Mattias Karlsson, Owe Orwar (2005 Jan 27)

Fluid mixing in growing microscale vesicles conjugated by surfactant nanotubes.

Journal of the American Chemical Society : 1251-7 En savoir plus
Résumé

This work addresses novel means for controlled mixing and reaction initiation in biomimetic confined compartments having volume elements in the range of 10(-12) to 10(-15) L. The method is based on mixing fluids using a two-site injection scheme into growing surfactant vesicles. A solid-state injection needle is inserted into a micrometer-sized vesicle (radius 5-25 microm), and by pulling on the needle, we create a nanoscale surfactant channel connecting injection needle and the vesicle. Injection of a solvent A from the needle into the nanotube results in the formation of a growing daughter vesicle at the tip of the needle in which mixing takes place. The growth of the daughter vesicle requires a flow of surfactants in the nanotube that generates a flow of solvent B inside the nanotube which is counterdirectional to the pressure-injected solvent. The volume ratio psi between solvent A and B inside the mixing vesicle was analyzed and found to depend only on geometrical quantities. The majority of fluid injected to the growing daughter vesicle comes from the pressure-based injection, and for a micrometer-sized vesicle it dominates. For the formation of one daughter vesicle (conjugated with a 100-nm radius tube) expanded from 1 to 200 microm in radius, the mixing ratios cover almost 3 orders of magnitude. We show that the system can be expanded to linear strings of nanotube-conjugated vesicles that display exponential dilution. Mixing ratios spanning 6 orders of magnitude were obtained in strings of three nanotube-conjugated micrometer-sized daughter vesicles.

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Zuzana Bílková, Annalisa Castagna, Gianluigi Zanusso, Alessia Farinazzo, Salvatore Monaco, Eugen Damoc, Michael Przybylski, Milan Benes, Jirí Lenfeld, Jean-Louis Viovy, Pier Giorgo Righetti (2005 Jan 26)

Immunoaffinity reactors for prion protein qualitative analysis.

Proteomics : 639-47 En savoir plus
Résumé

The cellular prion protein (PrPc) represents the substrate for generation of conformational aberrant PrP isoforms which occur in human and animal prion diseases. The published two-dimensional maps of human PrPc show a vast microheterogeneity of this glycoprotein. The main goal of this project was to develop a highly specific immunoaffinity reactor for qualitative analysis of PrP cellular isoforms isolated from brain homogenate, cerebrospinal fluid and other tissues. New techniques for affinity proteomics, carriers and immobilization chemistry were applied. The choice of matrix (chemical and magnetic properties, particle size and distribution, porosity) was the key factor that influenced the quality of the reactor and the nature of final applications. Mouse anti-prion IgGs directed to N-terminal and C-terminal epitopes (residues 23-40 and 147-165) were grafted in different manners to magnetic micro- and nanoparticles particularly developed for micro-CHIP application. High operational and storage stability of affinity reactors with minimized nonspecific absorption were achieved. The quality of the immunoreactors was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by two-dimensional electrophoresis.

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Julie Plastino, Cécile Sykes (2005 Jan 22)

The actin slingshot.

Current opinion in cell biology : 62-6 En savoir plus
Résumé

Actin polymerization generates the force that deforms the cell membrane, pulls the cell forward and propels endosomes and bacteria within the cell. The mechanism of force generation has been probed using experimental biomimetic systems where force generation and movement occur by the same actin-polymerization processes observed in cells. The advantage of such systems over living cells is that their physical properties can be changed, such as the size of the load, its composition and its deformability, in order to respond to specific questions. Recent experimental developments and associated theoretical models have provided us with a better understanding of motility based on actin polymerization. This paves the way towards a better comprehension of cell motility.

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