UMR168 – Laboratoire Physico-Chimie Curie

Publications de l’UMR 168

Année de publication : 2003

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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Cyril Rauch, Anne-Christine Brunet, Julie Deleule, Emmanuel Farge (2002 Jun 11)

C2C12 myoblast/osteoblast transdifferentiation steps enhanced by epigenetic inhibition of BMP2 endocytosis.

American journal of physiology. Cell physiology : C235-43 En savoir plus
Résumé

We investigated the modulation of critical transcriptional steps of C2C12 myoblast/osteoblast transdifferentiation triggered by the bone morphogenetic protein 2 (BMP2) signaling protein, in response to epigenetic inhibition of the endocytotic internalization of exogenous BMP2. BMP2 endocytosis was inhibited chemically with polyethylene glycol-50 (PEG-Chol) and cyclodextrin and mechanically by mild hyposmotic treatment. BMP2-dependent nuclear translocation of the mother against Dpp (Smad1) transcription factor was ten times faster if BMP2 endocytosis was inhibited. Smad1-dependent expression of the JunB gene, the first transcriptional step in myoblast dedifferentiation, was increased by a factor of three to four. JunB-dependent levels of myogenin repression, one of the critical markers of terminal myoblastic differentiation, was amplified by a factor of three. Smad1-dependent levels of alkaline phosphatase expression, one of the C2C12 osteoblast differentiation markers, were 3.5 to 5 times higher. The same behavior was observed for osteopontin, the other C2C12 osteoblast differentiation marker. These results suggest that the cell genome could « sense » tissue mechanical deformations by mechanical inhibition of signaling protein endocytosis, thereby translating mechanical strains into transcription events involved in cell differentiation.

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Anne Bernheim-Groswasser, Sebastian Wiesner, Roy M Golsteyn, Marie-France Carlier, Cécile Sykes (2002 May 17)

The dynamics of actin-based motility depend on surface parameters.

Nature : 308-11 En savoir plus
Résumé

In cells, actin polymerization at the plasma membrane is induced by the recruitment of proteins such as the Arp2/3 complex, and the zyxin/VASP complex. The physical mechanism of force generation by actin polymerization has been described theoretically using various approaches, but lacks support from experimental data. By the use of reconstituted motility medium, we find that the Wiskott Aldrich syndrome protein (WASP) subdomain, known as VCA, is sufficient to induce actin polymerization and movement when grafted on microspheres. Changes in the surface density of VCA protein or in the microsphere diameter markedly affect the velocity regime, shifting from a continuous to a jerky movement resembling that of the mutated ‘hopping’ Listeria. These results highlight how simple physical parameters such as surface geometry and protein density directly affect spatially controlled actin polymerization, and play a fundamental role in actin-dependent movement.

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Aurélien Roux, Giovanni Cappello, Jean Cartaud, Jacques Prost, Bruno Goud*, Patricia Bassereau* (2002 Apr 18)

A minimal system allowing tubulation with molecular motors pulling on giant liposomes.

Proceedings of the National Academy of Sciences of the United States of America : 5394-9 : DOI : 10.1073/pnas.082107299 En savoir plus
Résumé

The elucidation of physical and molecular mechanisms by which a membrane tube is generated from a membrane reservoir is central to the understanding of the structure and dynamics of intracellular organelles and of transport intermediates in eukaryotic cells. Compelling evidence exists that molecular motors of the dynein and kinesin families are involved in the tubulation of organelles. Here, we show that lipid giant unilamellar vesicles (GUVs), to which kinesin molecules have been attached by means of small polystyrene beads, give rise to membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. Similar tubes and networks are obtained with GUVs made of purified Golgi lipids, as well as with Golgi membranes. No tube formation was observed when kinesins were directly bound to the GUV membrane, suggesting that it is critical to distribute the load on both lipids and motors by means of beads. A kinetic analysis shows that network growth occurs in two phases: a phase in which membrane-bound beads move at the same velocity than free beads, followed by a phase in which the tube growth rate decreases and strongly fluctuates. Our work demonstrates that the action of motors bound to a lipid bilayer is sufficient to generate membrane tubes and opens the way to well controlled experiments aimed at the understanding of basic mechanisms in intracellular transport.

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Année de publication : 2001

P Martin, A J Hudspeth (2001 Nov 29)

Compressive nonlinearity in the hair bundle’s active response to mechanical stimulation.

Proceedings of the National Academy of Sciences of the United States of America : 14386-91 En savoir plus
Résumé

The auditory system’s ability to interpret sounds over a wide range of amplitudes rests on the nonlinear responsiveness of the ear. Whether measured by basilar-membrane vibration, nerve-fiber activity, or perceived loudness, the ear is most sensitive to small signals and grows progressively less responsive as stimulation becomes stronger. Seeking a correlate of this behavior at the level of mechanoelectrical transduction, we examined the responses of hair bundles to direct mechanical stimulation. As reported by the motion of an attached glass fiber, an active hair bundle from the bullfrog’s sacculus oscillates spontaneously. Sinusoidal movement of the fiber’s base by as little as +/-1 nm, corresponding to the application at the bundle’s top of a force of +/-0.3 pN, causes detectable phase-locking of the bundle’s oscillations to the stimulus. Although entrainment increases as the stimulus grows, the amplitude of the hair-bundle movement does not rise until phase-locking is nearly complete. A bundle is most sensitive to stimulation at its frequency of spontaneous oscillation. Far from that frequency, the sensitivity of an active hair bundle resembles that of a passive bundle. Over most of its range, an active hair bundle’s response grows as the one-third power of the stimulus amplitude; the bundle’s sensitivity declines accordingly in proportion to the negative two-thirds power of the excitation. This scaling behavior, also found in the response of the mammalian basilar membrane to sound, signals the operation of an amplificatory process at the brink of an oscillatory instability, a Hopf bifurcation.

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P Martin, A J Hudspeth, F Jülicher (2001 Nov 29)

Comparison of a hair bundle’s spontaneous oscillations with its response to mechanical stimulation reveals the underlying active process.

Proceedings of the National Academy of Sciences of the United States of America : 14380-5 En savoir plus
Résumé

Hearing relies on active filtering to achieve exquisite sensitivity and sharp frequency selectivity. In a quiet environment, the ears of many vertebrates become unstable and emit one to several tones. These spontaneous otoacoustic emissions, the most striking manifestation of the inner ear’s active process, must result from self-sustained mechanical oscillations of aural constituents. The mechanoreceptive hair bundles of hair cells in the bullfrog’s sacculus have the ability to amplify mechanical stimuli and oscillate spontaneously. By comparing a hair bundle’s spontaneous oscillations with its response to small mechanical stimuli, we demonstrate a breakdown in a general principle of equilibrium thermodynamics, the fluctuation-dissipation theorem. We thus confirm that a hair bundle’s spontaneous movements are produced by energy-consuming elements within the hair cell. To characterize the dynamical behavior of the active process, we introduce an effective temperature that, for each frequency component, quantifies a hair bundle’s deviation from thermal equilibrium. The effective temperature diverges near the bundle’s frequency of spontaneous oscillation. This behavior, which is not generic for active oscillators, can be accommodated by a simple model that characterizes quantitatively the fluctuations of the spontaneous movements as well as the hair bundle’s linear response function.

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J.-B. Manneville, P. Bassereau, S. Ramaswamy, J. Prost (2001 Jul 24)

Active membrane fluctuations studied by micropipet aspiration.

Physical Review E : 64 : 021908 : DOI : 10.1103/PhysRevE.64.021908 En savoir plus
Résumé

We present a detailed analysis of the micropipet experiments recently reported by J-B. Manneville et al., [Phys. Rev. Lett. 82, 4356 (1999)], including a derivation of the expected behavior of the membrane tension as a function of the areal strain in the case of an active membrane, i.e., containing a nonequilibrium noise source. We give a general expression, which takes into account the effect of active centers both directly on the membrane and on the embedding fluid dynamics, keeping track of the coupling between the density of active centers and the membrane curvature. The data of the micropipet experiments are well reproduced by our expressions. In particular, we show that a natural choice of the parameters quantifying the strength of the active noise explains both the large amplitude of the observed effects and its remarkable insensitivity to the active-center density in the investigated range.

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Année de publication : 2000

A J Hudspeth, Y Choe, A D Mehta, P Martin (2000 Oct 26)

Putting ion channels to work: mechanoelectrical transduction, adaptation, and amplification by hair cells.

Proceedings of the National Academy of Sciences of the United States of America : 11765-72 En savoir plus
Résumé

As in other excitable cells, the ion channels of sensory receptors produce electrical signals that constitute the cellular response to stimulation. In photoreceptors, olfactory neurons, and some gustatory receptors, these channels essentially report the results of antecedent events in a cascade of chemical reactions. The mechanoelectrical transduction channels of hair cells, by contrast, are coupled directly to the stimulus. As a consequence, the mechanical properties of these channels shape our hearing process from the outset of transduction. Channel gating introduces nonlinearities prominent enough to be measured and even heard. Channels provide a feedback signal that controls the transducer’s adaptation to large stimuli. Finally, transduction channels participate in an amplificatory process that sensitizes and sharpens hearing.

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P Martin, A D Mehta, A J Hudspeth (2000 Oct 12)

Negative hair-bundle stiffness betrays a mechanism for mechanical amplification by the hair cell.

Proceedings of the National Academy of Sciences of the United States of America : 12026-31 En savoir plus
Résumé

Hearing and balance rely on the ability of hair cells in the inner ear to sense miniscule mechanical stimuli. In each cell, sound or acceleration deflects the mechanosensitive hair bundle, a tuft of rigid stereocilia protruding from the cell’s apical surface. By altering the tension in gating springs linked to mechanically sensitive transduction channels, this deflection changes the channels’ open probability and elicits an electrical response. To detect weak stimuli despite energy losses caused by viscous dissipation, a hair cell can use active hair-bundle movement to amplify its mechanical inputs. This amplificatory process also yields spontaneous bundle oscillations. Using a displacement-clamp system to measure the mechanical properties of individual hair bundles from the bullfrog’s ear, we found that an oscillatory bundle displays negative slope stiffness at the heart of its region of mechanosensitivity. Offsetting the hair bundle’s position activates an adaptation process that shifts the region of negative stiffness along the displacement axis. Modeling indicates that the interplay between negative bundle stiffness and the motor responsible for mechanical adaptation produces bundle oscillation similar to that observed. Just as the negative resistance of electrically excitable cells and of tunnel diodes can be embedded in a biasing circuit to amplify electrical signals, negative stiffness can be harnessed to amplify mechanical stimuli in the ear.

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C Rauch, E Farge (2000 May 29)

Endocytosis switch controlled by transmembrane osmotic pressure and phospholipid number asymmetry.

Biophysical journal : 3036-47 En savoir plus
Résumé

The dynamics of endocytosis in living K562 cells was investigated after the osmotic pressure of the external medium was decreased and the transmembrane phospholipid number asymmetry was increased. When the external pressure was decreased by a factor of 0.54, a sudden inhibition of endocytosis was observed. Under these conditions, the endocytosis suddenly recovered after the phospholipid number asymmetry was increased. The phospholipid asymmetry was generated by the addition of exogenous phosphatidylserine, which is translocated by the endogenous flippase activity to the inner layer of the membrane. The recovery of endocytosis is thus consistent with the view that the phospholipid number asymmetry can act as a budding force for endocytosis. Moreover, we quantitatively predict both the inhibition and recovery of endocytosis as first-order phase transitions, using a general model that assumes the existence of a transmembrane surface tension asymmetry as the budding driving force. In this model, the tension asymmetry is considered to be elastically generated by the activity of phospholipid pumping. We finally propose that cells may trigger genetic transcription responses after the internalization of cytokine-receptor complexes, which could be controlled by variations in the cytosolic or external pressure.

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V Noireaux, R M Golsteyn, E Friederich, J Prost, C Antony, D Louvard, C Sykes (2000 Feb 29)

Growing an actin gel on spherical surfaces.

Biophysical journal : 1643-54 En savoir plus
Résumé

Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement. On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a « comet-like » tail of F-actin is produced. We show experimentally that the stationary thickness of the gel depends on the radius of the beads. Moreover, the actin gel is not formed if the ActA surface density is too low. To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel. Our model also takes into account treadmilling of actin. We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN. Finally, we estimate from our theoretical model possible conditions for developing actin comet tails.

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