Membranes et fonctions cellulaires

Publications

Année de publication : 2004

Cécile Leduc, Otger Campàs, Konstantin B Zeldovich, Aurélien Roux, Pascale Jolimaitre, Line Bourel-Bonnet, Bruno Goud, Jean-François Joanny, Patricia Bassereau*, Jacques Prost* (2004 Dec 1)

Cooperative extraction of membrane nanotubes by molecular motors.

Proceedings of the National Academy of Sciences of the United States of America : 17096-101 : DOI : 10.1073/pnas.0406598101 En savoir plus
Résumé

In eukaryotic cells, nanotubes represent a substantial fraction of transport intermediates between organelles. They are extracted from membranes by molecular motors walking along microtubules. We previously showed that kinesins fixed on giant unilamellar vesicles in contact with microtubules are sufficient to form nanotubes in vitro. Motors were attached to the membrane through beads, thus facilitating cooperative effects. Koster et al. proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. We demonstrate, in a recently designed experimental system, the existence of an accumulation of motors allowing tube extraction. We determine the motor density along a tube by using fluorescence intensity measurements. We also perform a theoretical analysis describing the dynamics of motors and tube growth. The only adjustable parameter is the motor binding rate onto microtubules, which we measure to be 4.7 +/- 2.4 s(-1). In addition, we quantitatively determine, for a given membrane tension, the existence of a threshold in motor density on the vesicle above which nanotubes can be formed. We find that the number of motors pulling a tube can range from four at threshold to a few tens away from it. The threshold in motor density (or in membrane tension at constant motor density) could be important for the understanding of membrane traffic regulation in cells.

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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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Année de publication : 2003

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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Année de publication : 2002

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

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 : 1999

M. C. Fauré, P. Bassereau, L. T. Lee, A. Menelle, C. Lheveder (1999 Nov 24)

Phase Transitions in Monolayers of PS−PEO Copolymer at the Air−Water Interface.

Macromolecules : 32 : 8538-8550 : DOI : 10.1021/ma9900840 En savoir plus
Résumé

The structures and phase transitions occurring during the compression of polystyrene−poly(ethylene oxide) (PS−PEO) diblock copolymer monolayers at the air−water interface have been studied by surface pressure isotherms, neutron reflectivity, and Brewster angle microscopy. At low coverage, the EO monomers adsorb at the air−water interface, but the PEO layer is not purely bidimensional. At high coverage, the EO−interface interaction becomes repulsive. A brush structure has been observed with differences between the long and the short PEO chains attributed to the difference in the accessible range of surface concentration. For the longest chain, the PEO concentration profiles exhibit a superposition of a depletion layer near the interface and discontinuous profile characteristics of a two-phase brush described by the “n-cluster” theory. For the shortest chains, the profiles are “pseudoparabolic”. Finally, Brewster angle microscopy shows that, for the long chains, the transition between the adsorbed and the brush structures is first order.

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J.-B. Manneville, P. Bassereau, D. Lévy, and J. Prost (1999 May 24)

Activity of transmembrane proteins induces magnification of shape fluctuations of lipid membranes.

Phys. Rev. Lett. : 82 : 4356 : DOI : 10.1103/PhysRevLett.82.4356 En savoir plus
Résumé

We report the first experimental evidence of the effect of the activity of transmembrane proteins on shape fluctuations of a lipid membrane. We incorporate a light-driven proton pump, the bacteriorhodopsin (BR), inside the phospholipid bilayer of fluctuating giant vesicles. Using the micropipet technique, we measure the excess surface area due to the fluctuations of the vesicles. The excess surface area is larger when the BR pumps protons than when it is not activated.

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Année de publication : 1998

M.C. Fauré, P. Bassereau, M. A. Carignano, I. Szleifer, Y. Gallot, D. Andelman (1998 Jun 1)

Monolayers of diblock copolymer at the air-water interface: the attractive monomer-surface case.

The European Physical Journal B - Condensed Matter and Complex Systems : 3 : 365-375 : DOI : 10.1007/s100510050324 En savoir plus
Résumé

We have studied both experimentally and theoretically the surface pressure isotherms of copolymers of polystyrene-polyethyleneoxide (PS-PEO) at the air-water interface. The SCMF (single chain mean-field) theory provides a very good agreement with the experiments for the entire range of surface densities and is consistent with the experiments if an adsorption energy per PEO monomer at the air-water interface of about one kB T is taken. In addition, the chain density profile has been calculated for a variety of surface densities, from the dilute to the very dense ones. The SCMF approach has been complemented by a mean-field approach in the low density regime, where the PEO chains act as a two-dimensional layer. Both theoretical calculations agree with the experiments in this region.

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Année de publication : 1997

Patricia Bassereau, Frédéric Pincet (1997 Dec 24)

Quantitative Analysis of Holes in Supported Bilayers Providing the Adsorption Energy of Surfactants on Solid Substrate.

Langmuir : 13 : 7003-7007 : DOI : 10.1021/la970515c En savoir plus
Résumé

We investigated the topography of mixed bilayers consisting of a first monolayer of DMPE (dimyristoylphosphatidylethanolamine) and of a second monolayer of DOPC (dioleoylphosphatidylcholine) that were Langmuir−Blodgett deposited on mica. Using transfer ratio measurements and tapping mode atomic force microscopy experiments, we show that the subnanometric holes in the bilayers result from the desorption of lipids of the first monolayer during the transfer of the second monolayer. We present a new simple technique based on the quantitative analysis of these holes that allows determination of the adsorption energy of amphiphilic molecules on solid surfaces. This technique is valid for relatively low adsorption energies in the range 1 to 10 kBT.

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Année de publication : 1995

P. Bassereau, T. P. Russell (1995 Feb 19)

Thermal expansion of thin diblock copolymer films.

Isreal Journal of Chemistry : 35 : 13-19 : DOI : 10.1002/ijch.199500004 En savoir plus
Résumé

The thermal expansion of thin films of symmetric diblock copolymers of polystyrene (PS) and poly(methyl methácrylate) (PMMA) was investigated by X‐ray reflectivity. The confinement of the copolymer to the substrate, coupled with the multilayering of the copolymer where PS and PMMA layers are oriented parallel to the substrate, gives rise to unusual thermal expansion characteristics. The total thickness of the film increases as 3αL, where αL is the linear thermal expansion coefficient of the copolymer. Unlike homopolymer films, the thermal expansion of an ordered block copolymer film results in an excessive stretching of the copolymer chains at the interface between the PS and PMMA layers. This excess stretching is a result of the confinement of the junction points of the copolymer chains to the interfaces and the suppression of the lateral expansion of the copolymer. When the stretching of the chains becomes too high, relaxation occurs by transporting copolymer chains to the surface. This is evidenced by a reduction in the period of the multilayer. After the copolymer chains have relaxed, the change in the multilayer period with temperature closely follows αL.

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