UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2005

Ewa Paluch, Cécile Sykes, Jacques Prost, Michel Bornens (2005 Dec 6)

Dynamic modes of the cortical actomyosin gel during cell locomotion and division.

Trends in cell biology : 5-10 En savoir plus
Résumé

Tight regulation of the contractility of the actomyosin cortex is essential for proper cell locomotion and division. Enhanced contractility leads, for example, to aberrations in the positioning of the mitotic spindle or to anomalous migration modes that allow tumor cells to escape anti-dissemination treatments. Spherical membrane protrusions called blebs occasionally appear during cell migration, cell division or apoptosis. We have shown that the cortex ruptures at sites where actomyosin cortical contractility is increased, leading to the formation of blebs. Here, we propose that bleb formation, which releases cortical tension, can be used as a reporter of cortical contractility. We go on to analyze the implications of spontaneous cortical contractile behaviors on cell locomotion and division and we particularly emphasize that variations in actomyosin contractility can account for a variety of migration modes.

Replier
Jérôme Solon, Olivier Gareil, Patricia Bassereau*, Yves Gaudin* (2005 Nov 22)

Membrane deformations induced by the matrix protein of vesicular stomatitis virus in a minimal system.

The Journal of General Virology : 86 : 3357-63 : DOI : 10.1099/vir.0.81129-0 En savoir plus
Résumé

The matrix (M) protein of vesicular stomatitis virus plays a key role in both assembly and budding of progeny virions. In vitro experiments have shown a strong propensity of M protein to bind to vesicles containing negatively charged phospholipids. In vivo, it has also been demonstrated that recruitment of some cellular proteins by M protein is required for efficient virus budding and release of newly synthesized virions in the extracellular medium. The ability of M protein to deform target membranes in vitro was investigated in this study. It was shown that incubation of purified M protein with giant unilamellar vesicles results in the formation of patches of M protein at their surface, followed by deformations of the membrane toward the inside of the vesicle, which could be observed in phase-contrast microscopy. This provides the first evidence that M protein alone is able to impose the correct budding curvature on the membrane. Using confocal microscopy, patches of M protein that colocalized with negatively charged lipid domains a few minutes after vesicle injection were observed. After a longer incubation period, membrane deformations appeared in these domains. At this time, a strict colocalization of M protein, negatively charged lipids and membrane deformation was observed. The influence on this process of the basic N-terminal part of the protein and of the previously identified hydrophobic loop has also been investigated. Interestingly, the final fission event has never been observed in our experimental system, indicating that other partners are required for this step.

Replier
Alexandre Saez, Axel Buguin, Pascal Silberzan, Benoît Ladoux (2005 Oct 7)

Is the mechanical activity of epithelial cells controlled by deformations or forces?

Biophysical journal : L52-4 En savoir plus
Résumé

The traction forces developed by cells depend strongly on the substrate rigidity. In this letter, we characterize quantitatively this effect on MDCK epithelial cells by using a microfabricated force sensor consisting in a high-density array of soft pillars whose stiffness can be tailored by changing their height and radius to obtain a rigidity range from 2 nN/microm up to 130 nN/microm. We find that the forces exerted by the cells are proportional to the spring constant of the pillars meaning that, on average, the cells deform the pillars by the same amount whatever their rigidity. The relevant parameter may thus be a deformation rather than a force. These dynamic observations are correlated with the reinforcement of focal adhesions that increases with the substrate rigidity.

Replier
Arnold J Storm, Cornelis Storm, Jianghua Chen, Henny Zandbergen, Jean-François Joanny, Cees Dekker (2005 Sep 24)

Fast DNA translocation through a solid-state nanopore.

Nano letters : 1193-7 En savoir plus
Résumé

We report experiments and modeling of translocation of double-strand DNA through a siliconoxide nanopore. Long DNA molecules with different lengths ranging from 6500 to 97000 base pairs have been electrophoretically driven through a 10 nm pore. We observe a power-law caling of the translocation time with the length, with an exponent of 1.27. This nonlinear scaling is strikingly different from the well-studied linear behavior observed in similar experiments performed on protein pores. We present a theoretical model where hydrodynamic drag on the ection of the polymer outside the pore is the dominant force counteracting the electrical driving force. We show that this applies to our experiments, and we derive a power-law scaling with an exponent of 1.22, in good agreement with the data.

Replier
Max Chabert, Kevin D Dorfman, Jean-Louis Viovy (2005 Sep 2)

Droplet fusion by alternating current (AC) field electrocoalescence in microchannels.

Electrophoresis : 3706-15 En savoir plus
Résumé

We present a system for the electrocoalescence of microfluidic droplets immersed in an immiscible solvent, where the undeformed droplet diameters are comparable to the channel diameter. The electrodes are not in direct contact with the carrier liquid or the droplets, thereby minimizing the risk of cross-contamination between different coalescence events. Results are presented for the coalescence of buffered aqueous droplets in both quiescent and flowing fluorocarbon streams, and on-flight coalescence is demonstrated. The capillary-based system presented here is readily amenable to further miniaturization to any lab-on-a-chip application where the conductivity of the droplets is much greater than the conductivity of the stream containing them, and should aid in the further application of droplet microreactors to biological analyses.

Replier
D. Cuvelier, N. Chiaruttini, P. Bassereau and P. Nassoy (2005 Sep 1)

Pulling long tubes from firmly adhered vesicles

Europhysics Letters : 71 : 1015-1021 : DOI : 10.1209/epl/i2005-10173-4 En savoir plus
Résumé

We used optical tweezers to measure the force-extension curve for the elongation of nanotubes from adhered giant vesicles. We show that the force increases significantly with the length of the tube, which is drastically different from what is observed when the membrane tension is kept constant, e.g. by pipette aspiration. The absence of any force plateau is quantitatively analysed in the framework of the material model of membranes. In particular, we rationalize a counter-intuitive weaker force rise for long tubes and demonstrate that the measured force-length trace allows us to probe both the entropic regime (characterised by the bending rigidity) and the elastic regime (characterised by the area expansion modulus) of the lipid membrane.

Replier
Jing Yang, Daniel Lévy, Wei Deng, Patrick Keller, Min-Hui Li (2005 Aug 23)

Polymer vesicles formed by amphiphilic diblock copolymers containing a thermotropic liquid crystalline polymer block.

Chemical communications (Cambridge, England) : 4345-7 En savoir plus
Résumé

New amphiphilic diblock copolymers composed of poly(ethylene glycol) and a thermotropic liquid crystalline polymer have been synthesized and demonstrated to form well-defined unilamellar vesicles in water by cryo-electron microscopy.

Replier
Marcela Slovakova, Nicolas Minc, Zuzana Bilkova, Claire Smadja, Wolfgang Faigle, Claus Fütterer, Myriam Taverna, Jean-Louis Viovy (2005 Aug 16)

Use of self assembled magnetic beads for on-chip protein digestion.

Lab on a chip : 935-42 En savoir plus
Résumé

The use of grafted trypsin magnetic beads in a microchip for performing protein digestion is described. The PDMS device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the center of the chip channel. This allows for the formation of a low-hydrodynamic resistance plug of magnetic trypsin beads that serves as a matrix for protein digestion. This device represents an inexpensive way of fabricating a multi open-tubular-like column with an appropriate pore size for proteins. Kinetics studies of the hydrolysis of a model peptide show a 100-fold increase in digestion speed obtained by the microsystem when compared to a batch wise system. This system also offers the great advantage of easy replacement, as the bead matrix is easily washed out and replaced. High performance and reproducibility for digesting recombinant human growth hormone are confirmed by analysing the digest products in both CE and MALDI-TOF MS. Similar sequence coverage (of about 44%) is obtained from MS analysis of products after 10 minutes on-chip and 4 h with soluble trypsin in bulk.

Replier
Thomas Risler, Jacques Prost, Frank Jülicher (2005 Aug 11)

Universal critical behavior of noisy coupled oscillators: a renormalization group study.

Physical review. E, Statistical, nonlinear, and soft matter physics : 016130 En savoir plus
Résumé

We show that the synchronization transition of a large number of noisy coupled oscillators is an example for a dynamic critical point far from thermodynamic equilibrium. The universal behaviors of such critical oscillators, arranged on a lattice in a d -dimensional space and coupled by nearest-neighbors interactions, can be studied using field-theoretical methods. The field theory associated with the critical point of a homogeneous oscillatory instability (or Hopf bifurcation of coupled oscillators) is the complex Ginzburg-Landau equation with additive noise. We perform a perturbative renormalization group (RG) study in a (4-epsilon)-dimensional space. We develop an RG scheme that eliminates the phase and frequency of the oscillations using a scale-dependent oscillating reference frame. Within Callan-Symanzik’s RG scheme to two-loop order in perturbation theory, we find that the RG fixed point is formally related to the one of the model A dynamics of the real Ginzburg-Landau theory with an O2 symmetry of the order parameter. Therefore, the dominant critical exponents for coupled oscillators are the same as for this equilibrium field theory. This formal connection with an equilibrium critical point imposes a relation between the correlation and response functions of coupled oscillators in the critical regime. Since the system operates far from thermodynamic equilibrium, a strong violation of the fluctuation-dissipation relation occurs and is characterized by a universal divergence of an effective temperature. The formal relation between critical oscillators and equilibrium critical points suggests that long-range phase order exists in critical oscillators above two dimensions.

Replier
Nicolas Minc, Jean-Louis Viovy, Kevin D Dorfman (2005 Aug 11)

Non-markovian transport of DNA in microfluidic post arrays.

Physical review letters : 198105 En savoir plus
Résumé

We present an analytically solvable model for the transport of long DNA through microfluidic arrays of posts. The motion is a repetitive three-part cycle: (i) collision with the post and extension of the arms; (ii) rope-over-pulley post disengagement; and (iii) a random period of uniform translation before the next collision. This cycle, inspired by geometration, is a nonseparable (Scher-Lax) continuous-time random walk on a lattice defined by the posts. Upon adopting a simple model for the transition probability density on the lattice, we analytically compute the mean DNA velocity and dispersivity in the long-time limit without any adjustable parameters. The results compare favorably with the limited amount of experimental data on separations in self-assembled arrays of magnetic beads. The Scher-Lax formalism provides a template for incorporating more sophisticated microscale models.

Replier
Michal Tokarz, Björn Akerman, Jessica Olofsson, Jean-Francois Joanny, Paul Dommersnes, Owe Orwar (2005 Jun 18)

Single-file electrophoretic transport and counting of individual DNA molecules in surfactant nanotubes.

Proceedings of the National Academy of Sciences of the United States of America : 9127-32 En savoir plus
Résumé

We demonstrate a complete nanotube electrophoresis system (nanotube radii in the range of 50 to 150 nm) based on lipid membranes, comprising DNA injection, single-molecule transport, and single-molecule detection. Using gel-capped electrodes, electrophoretic single-file transport of fluorescently labeled dsDNA molecules is observed inside nanotubes. The strong confinement to a channel of molecular dimensions ensures a detection efficiency close to unity and identification of DNA size from its linear relation to the integrated peak intensity. In addition to constituting a nanotechnological device for identification and quantification of single macromolecules or biopolymers, this system provides a method to study their conformational dynamics, reaction kinetics, and transport in cell-like environments.

Replier
Pierre Douette, Rachel Navet, Pascal Gerkens, Moreno Galleni, Daniel Lévy, Francis E Sluse (2005 Jun 18)

Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL.

Biochemical and biophysical research communications : 686-93 En savoir plus
Résumé

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein.

Replier
Kevin D Dorfman, Max Chabert, Jean-Hugues Codarbox, Gilles Rousseau, Patricia de Cremoux, Jean-Louis Viovy (2005 Jun 1)

Contamination-free continuous flow microfluidic polymerase chain reaction for quantitative and clinical applications.

Analytical chemistry : 3700-4 En savoir plus
Résumé

We present a method for performing polymerase chain reaction (PCR) using isolated droplets flowing in an immiscible fluorinated solvent system. Thanks to an optimized control of interfacial properties, we could achieve in this capillary-based system reproducible amplification factors, without any detectable contamination between neighboring droplets. The system is readily amenable to further miniaturization and automation and serves as the first step toward a clinically viable, high-throughput, quantitative continuous flow PCR apparatus.

Replier
Anne Bernheim-Groswasser, Jacques Prost, Cécile Sykes (2005 Jun 1)

Mechanism of actin-based motility: a dynamic state diagram.

Biophysical journal : 1411-9 En savoir plus
Résumé

Cells move by a dynamical reorganization of their cytoskeleton, orchestrated by a cascade of biochemical reactions directed to the membrane. Designed objects or bacteria can hijack this machinery to undergo actin-based propulsion inside cells or in a cell-like medium. These objects can explore the dynamical regimes of actin-based propulsion, and display different regimes of motion, in a continuous or periodic fashion. We show that bead movement can switch from one regime to the other, by changing the size of the beads or the surface concentration of the protein activating actin polymerization. We experimentally obtain the state diagram of the bead dynamics, in which the transitions between the different regimes can be understood by a theoretical approach based on an elastic force opposing a friction force. Moreover, the experimental characteristics of the movement, such as the velocity and the characteristic times of the periodic movement, are predicted by our theoretical analysis.

Replier
Simon Scheuring, Daniel Lévy, Jean-Louis Rigaud (2005 May 28)

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy.

Biochimica et biophysica acta : 109-27 En savoir plus
Résumé

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function. Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 A lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.

Replier