UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2004

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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Jean-François Joanny, Frank Jülicher, Jacques Prost (2003 May 7)

Motion of an adhesive gel in a swelling gradient: a mechanism for cell locomotion.

Physical review letters : 168102 En savoir plus
Résumé

Motivated by the motion of nematode sperm cells, we present a model for the motion of an adhesive gel on a solid substrate. The gel polymerizes at the leading edge and depolymerizes at the rear. The motion results from a competition between a self-generated swelling gradient and the adhesion on the substrate. The resulting stress provokes the rupture of the adhesion points and allows for the motion. The model predicts an unusual force-velocity relation which depends in significant ways on the point of application of the force.

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Simon Scheuring, Jérôme Seguin, Sergio Marco, Daniel Lévy, Bruno Robert, Jean-Louis Rigaud (2003 Feb 8)

Nanodissection and high-resolution imaging of the Rhodopseudomonas viridis photosynthetic core complex in native membranes by AFM. Atomic force microscopy.

Proceedings of the National Academy of Sciences of the United States of America : 1690-3 En savoir plus
Résumé

In photosynthesis, highly organized multiprotein assemblies convert sunlight into biochemical energy with high efficiency. A challenge in structural biology is to analyze such supramolecular complexes in native membranes. Atomic force microscopy (AFM) with high lateral resolution, high signal-to-noise ratio, and the possibility to nanodissect biological samples is a unique tool to investigate multiprotein complexes at molecular resolution in situ. Here we present high-resolution AFM of the photosynthetic core complex in native Rhodopseudomonas viridis membranes. Topographs at 10-A lateral and approximately 1-A vertical resolution reveal a single reaction center (RC) surrounded by a closed ellipsoid of 16 light-harvesting (LH1) subunits. Nanodissection of the tetraheme cytochrome (4Hcyt) subunit from the RC allows demonstration that the L and M subunits exhibit an asymmetric topography intimately associated to the LH1 subunits located at the short ellipsis axis. This architecture implies a distance distribution between the antenna and the RC compared with a centered location of the RC within a circular LH1, which may influence the energy transfer within the core complex. The LH1 subunits rearrange into a circle after removal of the RC from the core complex.

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Année de publication : 2002

Simon Scheuring, Jerome Seguin, Sergio Marco, Daniel Lévy, Cécile Breyton, Bruno Robert, Jean-Louis Rigaud (2002 Dec 25)

AFM characterization of tilt and intrinsic flexibility of Rhodobacter sphaeroides light harvesting complex 2 (LH2).

Journal of molecular biology : 569-80 En savoir plus
Résumé

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 microm and, as revealed by a 10 A cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 A and gamma = 90 degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 A. Further, the high vertical resolution of approximately 1 A, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 A on one side and 3.8 A on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2 degree tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to approximately 15 A.

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Xavier Hagnerelle, Célia Plisson, Olivier Lambert, Sergio Marco, Jean Louis Rigaud, Ludger Johannes, Daniel Lévy (2002 Oct 31)

Two-dimensional structures of the Shiga toxin B-subunit and of a chimera bound to the glycolipid receptor Gb3.

Journal of structural biology : 113-21 En savoir plus
Résumé

The B-subunit of Shiga toxin has been demonstrated as a powerful vector for carrying attached peptides into cells for intracellular transport studies and for medical research. We have investigated the structure of the B-subunit and of a chimera bearing a peptide extension, bound to the membranous lipidic receptor, the globotriaosylceramide (Gb3). Two-dimensional crystals of both B-subunits have been obtained by the lipid layer method and projection maps have been calculated at 8.5A resolution from ice-embedded samples. The B-subunits as the chimera are organized in a pentameric form similar to the X-ray structure of the B-subunit not bound to Gb3. A difference map of both proteins has been calculated in which no density could be attributed to the peptide extension. Cross-correlations with projections of the B-subunit X-ray structure revealed that pentamers in the 2D crystals were oriented with their binding sites pointing to the lipid layer. Thus, it is likely that the peptide extension was disordered and confined to the surface of the pentamer opposite to the Gb3 binding sites. This location confirms the hypothesis that addition of peptide extension to the C-terminus conserves the ability of the modified B-subunit to bind the membranous receptor Gb3.

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Imre Derényi, Frank Jülicher, Jacques Prost (2002 Jun 13)

Formation and interaction of membrane tubes.

Physical review letters : 238101 En savoir plus
Résumé

We show that the formation of membrane tubes (or membrane tethers), which is a crucial step in many biological processes, is highly nontrivial and involves first-order shape transitions. The force exerted by an emerging tube is a nonmonotonic function of its length. We point out that tubes attract each other, which eventually leads to their coalescence. We also show that detached tubes behave like semiflexible filaments with a rather short persistence length. We suggest that these properties play an important role in the formation and structure of tubular organelles.

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