UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2004

Hakim Boukellal, Otger Campás, Jean-François Joanny, Jacques Prost, Cécile Sykes (2004 Jul 13)

Soft Listeria: actin-based propulsion of liquid drops.

Physical review. E, Statistical, nonlinear, and soft matter physics : 061906 En savoir plus
Résumé

We study the motion of oil drops propelled by actin polymerization in cell extracts. Drops deform and acquire a pearlike shape under the action of the elastic stresses exerted by the actin comet, a tail of cross-linked actin filaments. We solve this free boundary problem and calculate the drop shape taking into account the elasticity of the actin gel and the variation of the polymerization velocity with normal stress. The pressure balance on the liquid drop imposes a zero propulsive force if gradients in surface tension or internal pressure are not taken into account. Quantitative parameters of actin polymerization are obtained by fitting theory to experiment.

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Philippe Girard, Jacques Pécréaux, Guillaume Lenoir, Pierre Falson, Jean-Louis Rigaud, Patricia Bassereau (2004 Jul 9)

A new method for the reconstitution of membrane proteins into giant unilamellar vesicles.

Biophysical journal : 419-29 : DOI : 10.1529/biophysj.104.040360 En savoir plus
Résumé

In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.

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Damien Cuvelier, Cyrille Vezy, Annie Viallat, Patricia Bassereau, Pierre Nassoy (2004 Jun 18)

Mimicking cell/extracellular matrix adhesion with lipid membranes and solid substrates: requirements, pitfalls and proposals.

Journal of Physics: Condensed Matter : 16 : S2427-S2437 : DOI : 10.1088/0953-8984/16/26/016 En savoir plus
Résumé

The interest in physical approaches to the study of cell adhesion has generated numerous recent works on the development of substrates mimicking the extracellular matrix and the use of giant synthetic liposomes, commonly considered as basic models of living cells. The use of well-characterized bioactive substrates and artificial cells should allow us to gain new insight into the cell–extracellular matrix interactions, provided that their biomimetic relevance has been really proved. The aim of this paper is to define some minimal requirements for effective biomimetic features and to propose simple adhesion assays. We show, for instance, that immobilization of specific ligands is sometimes not sufficient to ensure specific adhesion of cells expressing the corresponding receptors. By investigating comparatively the adhesive behaviour of decorated erythrocytes and vesicles, we also discuss the potentialities and limitations of synthetic vesicles as test cells.

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Tatsiana Lobovkina, Paul Dommersnes, Jean-Francois Joanny, Patricia Bassereau, Mattias Karlsson, Owe Orwar (2004 May 14)

Mechanical tweezer action by self-tightening knots in surfactant nanotubes.

Proceedings of the National Academy of Sciences of the United States of America : 101 : 7949-7953 : DOI : 10.1073/pnas.0401760101 En savoir plus
Résumé

Entanglements and trefoil knots on surfactant nanotubes in the liquid phase were produced by a combination of network self-organization and micromanipulation. The resulting knots are self-tightening, and the tightening is driven by minimization of surface free energy of the surfactant membrane material. The formation of the knot and the steady-state knot at quasi-equilibrium can be directly followed and localized by using fluorescence microscopy. Knots on nanotubes can be used as nanoscale mechanical tweezers for trapping and manipulation of single nano- and micrometer-sized high-aspect ratio objects. Furthermore, we demonstrate that by controlling the surface tension, objects captured by a knot can be transported along given trajectories defined by the nanotube axes.

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J. Pécréaux, H.-G. Döbereiner, J. Prost, J.-F. Joanny, P. Bassereau (2004 Apr 21)

Refined contour analysis of giant unilamellar vesicles.

The European Physical Journal E : 13 : 277-290 : DOI : 10.1140/epje/i2004-10001-9 En savoir plus
Résumé

The fluctuation spectrum of giant unilamellar vesicles is measured using a high-resolution contour detection technique. An analysis at higher q vectors than previously achievable is now possible due to technical improvements of the experimental setup and of the detection algorithm. The global fluctuation spectrum is directly fitted to deduce the membrane tension and the bending modulus of lipid membranes. Moreover, we show that the planar analysis of fluctuations is valid for spherical objects, even at low wave vectors. Corrections due to the integration time of the video camera and to the section of a 3D object by the observation plane are introduced. A precise calculation of the error bars has been done in order to provide reliable error estimate. Eventually, using this technique, we have measured bending moduli for EPC, SOPC and SOPC: CHOL membranes confirming previously published values. An interesting application of this technique can be the measurement of the fluctuation spectra for non-equilibrium membranes, such as « active membranes ».

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Aurélie Bertin, Amélie Leforestier, Dominique Durand, Françoise Livolant (2004 Apr 21)

Role of histone tails in the conformation and interactions of nucleosome core particles.

Biochemistry : 4773-80 En savoir plus
Résumé

The goal of this work was to test the role of the histone tails in the emergence of attractive interactions between nucleosomes above a critical salt concentration that corresponds to the complete tail extension outside the nucleosome [Mangenot, S., et al (2002) Biophys. J. 82, 345-356; Mangenot, S., et al (2002) Eur. Phys. J. E 7, 221-231]. Small angle X-ray scattering experiments were performed in parallel with intact and trypsin tail-deleted nucleosomes with 146 +/- 3 bp DNA. We varied the monovalent salt concentration from 10 to 300 monovalent salt concentration and followed the evolution of (i) the second virial coefficient that characterizes the interactions between particles and (ii) the conformation of the particle. The attractive interactions do not emerge in the absence of the tails, which validates the proposed hypothesis.

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Yann Marcy, Jacques Prost, Marie-France Carlier, Cécile Sykes (2004 Apr 14)

Forces generated during actin-based propulsion: a direct measurement by micromanipulation.

Proceedings of the National Academy of Sciences of the United States of America : 5992-7 En savoir plus
Résumé

Dynamic actin networks generate forces for numerous types of movements such as lamellipodia protrusion or the motion of endocytic vesicles. The actin-based propulsive movement of Listeria monocytogenes or of functionalized microspheres have been extensively used as model systems to identify the biochemical components that are necessary for actin-based motility. However, quantitative force measurements are required to elucidate the mechanism of force generation, which is still under debate. To directly probe the forces generated in the process of actin-based propulsion, we developed a micromanipulation experiment. A comet growing from a coated polystyrene bead is held by a micropipette while the bead is attached to a force probe, by using a specially designed « flexible handle. » This system allows us to apply both pulling and pushing external forces up to a few nanonewtons. By pulling the actin tail away from the bead at high speed, we estimate the elastic modulus of the gel and measure the force necessary to detach the tail from the bead. By applying a constant force in the range of -1.7 to 4.3 nN, the force-velocity relation is established. We find that the relation is linear for pulling forces and decays more weakly for pushing forces. This behavior is explained by using a dimensional elastic analysis.

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Année de publication : 2003

Julie Plastino, Ioannis Lelidis, Jacques Prost, Cécile Sykes (2003 Dec 10)

The effect of diffusion, depolymerization and nucleation promoting factors on actin gel growth.

European biophysics journal : EBJ : 310-20 En savoir plus
Résumé

In eukaryotic cells, localized actin polymerization is able to deform the plasma membrane and push the cell forward. Depolymerization of actin filaments and diffusion of actin monomers ensure the availability of monomers at sites of polymerization, and therefore these processes must play an active role in cellular actin dynamics. Here we reveal experimental evidence that actin gel growth can be limited by monomer diffusion, consistent with theoretical predictions. We study actin gels formed on beads coated with ActA (and ActA fragments), the bacterial factor responsible for actin-based movement of Listeria monocytogenes. We observe a saturation of gel thickness with increasing bead radius, the signature of diffusion control. Data analysis using an elastic model of actin gel growth gives an estimate of 2×10(-8) cm(-2) s(-1) for the diffusion coefficient of actin monomers through the gel, ten times less than in buffer, and in agreement with literature values in bulk cytoskeleton, providing corroboration of our model. The depolymerization rate of actin filaments and the elastic modulus of the gel are also evaluated. Furthermore, we qualitatively examine the different actin gels produced when ActA fragments interact with either VASP or the Arp2/3 complex.

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Jean-Louis Rigaud, Daniel Lévy (2003 Nov 13)

Reconstitution of membrane proteins into liposomes.

Methods in enzymology : 65-86 En savoir plus
Résumé

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Simon Scheuring, Francesco Francia, Johan Busselez, Bruno Andrea Melandri, Jean-Louis Rigaud, Daniel Lévy (2003 Oct 29)

Structural role of PufX in the dimerization of the photosynthetic core complex of Rhodobacter sphaeroides.

The Journal of biological chemistry : 3620-6 En savoir plus
Résumé

Monomeric and dimeric PufX-containing core complexes have been purified from membranes of wild-type Rhodobacter sphaeroides. Reconstitution of both samples by detergent removal in the presence of lipids leads to the formation of two-dimensional crystals constituted of dimeric core complexes. Two-dimensional crystals were further analyzed by cryoelectron microscopy and atomic force microscopy. A projection map at 26-A resolution reveals that core complexes assemble in an « S »-shaped dimeric complex. Each core complex is composed of one reaction center, 12 light-harvesting 1 alpha/beta-heterodimers, and one PufX protein. The light-harvesting 1 assemblies are open with a gap of density of approximately 30-A width and surround oriented reaction centers. A maximum density is found at the dimer junction. Based on the projection map, a model is proposed, in which the two PufX proteins are located at the dimer junction, consistent with the finding of dimerization of monomeric core complexes upon reconstitution. This localization of PufX in the core complex implies that PufX is the structural key for the dimer complex formation rather than a channel-forming protein for the exchange of ubiquinone/ubiquinol between the reaction center and the cytochrome bc1 complex.

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Peter Lenz, Jean-François Joanny, Frank Jülicher, Jacques Prost (2003 Oct 4)

Membranes with rotating motors.

Physical review letters : 108104 En savoir plus
Résumé

We study collections of rotatory motors confined to two-dimensional manifolds. These systems show a nontrivial collective behavior since the rotational motion leads to a repulsive hydrodynamic interaction between motors. While for high rotation speed motors might exhibit crystalline order, they form at low speed a disordered phase where diffusion is enhanced by velocity fluctuations. These effects should be experimentally observable for motors driven by external fields and for dipolar biological motors embedded into lipid membranes in a viscoelastic solvent.

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Giovanni Cappello, Mathilde Badoual, Albrecht Ott, Jacques Prost, Lorenzo Busoni (2003 Oct 4)

Kinesin motion in the absence of external forces characterized by interference total internal reflection microscopy.

Physical review. E, Statistical, nonlinear, and soft matter physics : 021907 En savoir plus
Résumé

We study the motion of the kinesin molecular motor along microtubules using interference total internal reflection microscopy. This technique achieves nanometer scale resolution together with a fast time response. We describe the first in vitro observation of kinesin stepping at high ATP concentration in the absence of an external load, where the 8-nm step can be clearly distinguished. The short-time resolution allows us to measure the time constant related to the relative motion of the bead-motor connection; we deduce the associated bead-motor elastic modulus.

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Emmanuel Farge (2003 Aug 23)

Mechanical induction of Twist in the Drosophila foregut/stomodeal primordium.

Current biology : CB : 1365-77 En savoir plus
Résumé

Morphogenetic movements are closely regulated by the expression of developmental genes. Here I examine whether developmental gene expression can in turn be mechanically regulated by morphogenetic movements. I have analyzed the effects of mechanical stress on the expression of Twist, which is normally expressed only in the most ventral cells of the cellular blastoderm embryo under the control of the Dorsal morphogen gradient. At embryogenesis gastrulation (stage 7), Twist is also expressed in the anterior foregut and stomodeal primordia.

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Damien Cuvelier, Olivier Rossier, Patricia Bassereau, Pierre Nassoy (2003 Jul 10)

Micropatterned « adherent/repellent » glass surfaces for studying the spreading kinetics of individual red blood cells onto protein-decorated substrates.

European Biophysics Journal : 32 : 342-354 : DOI : 10.1007/s00249-003-0282-2 En savoir plus
Résumé

We report in this paper two simple and effective methods to decorate glass surfaces that enable protein micropatterning and subsequent spatially controlled adhesion of cells. The first method combines simultaneously the potentialities of two existing techniques, namely microcontact printing (muCP) and microfluidic networks (muFN) to achieve dual protein patterning in a single step. The second method is mainly based on the well-known property of poly(ethylene glycol) (PEG) to resist against protein adsorption. Both approaches were used to produce heterogeneous surfaces on which micron-size or submicronic streptavidin-coated lines alternate with cell-repellent areas. We first describe the implementation of the two methods and discuss the main pitfalls to avoid. Then, using these templates, we have monitored the kinetics of attachment of individual biotinylated (i.e. « attractant » towards streptavidin) red blood cells by directly measuring the propagation velocity of the adhesion front. Depending on the surface density of biotin, we found two distinct regimes, in agreement with existing theoretical models.

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Pascal Martin, D Bozovic, Y Choe, A J Hudspeth (2003 Jun 14)

Spontaneous oscillation by hair bundles of the bullfrog’s sacculus.

The Journal of neuroscience : the official journal of the Society for Neuroscience : 4533-48 En savoir plus
Résumé

One prominent manifestation of mechanical activity in hair cells is spontaneous otoacoustic emission, the unprovoked emanation of sound by an internal ear. Because active hair bundle motility probably constitutes the active process of nonmammalian hair cells, we investigated the ability of hair bundles in the bullfrog’s sacculus to produce oscillations that might underlie spontaneous otoacoustic emissions. When maintained in the normal ionic milieu of the ear, many bundles oscillated spontaneously through distances as great as 80 nm at frequencies of 5-50 Hz. Whole-cell recording disclosed that the positive phase of movement was associated with the opening of transduction channels. Gentamicin, which blocks transduction channels, reversibly arrested oscillation; drugs that affect the cAMP phosphorylation pathway and might influence the activity of myosin altered the rate of oscillation. Increasing the Ca 2+ concentration rendered oscillations faster and smaller until they were suppressed; lowering the Ca 2+ concentration moderately with chelators had the opposite effect. When a bundle was offset with a stimulus fiber, oscillations were transiently suppressed but gradually resumed. Loading a bundle by partial displacement clamping, which simulated the presence of the accessory structures to which a bundle is ordinarily attached, increased the frequency and diminished the magnitude of oscillation. These observations accord with a model in which oscillations arise from the interplay of the hair bundle’s negative stiffness with the activity of adaptation motors and with Ca 2+-dependent relaxation of gating springs.

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