Membranes et fonctions cellulaires

Publications

Année de publication : 2011

Madhavi P. Maddugoda, Caroline Stefani, David Gonzalez-Rodriguez, Juha Saarikangas, Stéphanie Torrino, Sebastien Janel, Patrick Munro, Anne Doye, François Prodon, Michel Aurrand-Lions, Pierre L. Goossens, Frank Lafont, Patricia Bassereau, Pekka Lappalainen, Françoise Brochard, Emmanuel Lemichez (2011 Nov 17)

cAMP signaling by anthrax edema toxin induces transendothelial cell tunnels, which are resealed by MIM via Arp2/3-driven actin polymerization.

Cell Host & Microbe : 10 : 464-474 : DOI : 10.1016/j.chom.2011.09.014 En savoir plus
Résumé

RhoA-inhibitory bacterial toxins, such as Staphylococcus aureus EDIN toxin, induce large transendothelial cell macroaperture (TEM) tunnels that rupture the host endothelium barrier and promote bacterial dissemination. Host cells repair these tunnels by extending actin-rich membrane waves from the TEM edges. We reveal that cyclic-AMP signaling produced by Bacillus anthracis edema toxin (ET) also induces TEM formation, which correlates with increased vascular permeability. We show that ET-induced TEM formation resembles liquid dewetting, a physical process of nucleation and growth of holes within a thin liquid film. We also identify the cellular mechanisms of tunnel closure and reveal that the I-BAR domain protein Missing in Metastasis (MIM) senses de novo membrane curvature generated by the TEM, accumulates at the TEM edge, and triggers Arp2/3-dependent actin polymerization, which induces actin-rich membrane waves that close the TEM. Thus, the balance between ET-induced TEM formation and resealing likely determines the integrity of the host endothelium barrier.

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Sophie Aimon, John Manzi, Daniel Schmidt, Jose Antonio Poveda Larrosa, Patricia Bassereau, Gilman E S Toombes (2011 Oct 15)

Functional reconstitution of a voltage-gated potassium channel in giant unilamellar vesicles.

PloS one : e25529 : DOI : 10.1371/journal.pone.0025529 En savoir plus
Résumé

Ion channels voltage-gated are key players in cellular excitability. Recent studies suggest That Their behavior can depend on the lipid membrane Strongly composition and physical state. In vivo studies of membrane / channel and channel / channel interactions are challenging as membrane properties are Actively regulated in living cells, and are difficulty to control in experimental settings. We Developed method to reconstitute a functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) qui in membrane composition, geometry and voltage can be controlled. First, a voltage-gated potassium channel, KvAP, Was purified, fluorescently Labeled and reconstituted into proteoliposomes small. Small Proteoliposomes Were Then converted into GUVs via electroformation. GUVs Could Be FORMED using different lipid compositions and buffers Containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs Was caractérisé with quantitative confocal microscopy, and the protein density of GUVs Was comparable to the small proteoliposomes from They Were qui FORMED. Furthermore, patch clamp measurements confirmed que la reconstituted channels Retained selectivity and potassium voltage-gated activation. Containing GUVs functional voltage-gated ion channels will allow the study of channel activity, while controlling distribution and diffusion membrane state, and shoulds Prove Powerful tool for understanding how the diaphragm modulates cellular excitability.

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Marianne Renner, Yegor Domanov, Fanny Sandrin, Ignacio Izeddin, Patricia Bassereau*, Antoine Triller* (2011 Sep 29)

Lateral Diffusion on Tubular Membranes: Quantification of Measurements Bias.

PLoS One : 6 : e25731 : DOI : 10.1371/journal.pone.0025731 En savoir plus
Résumé

Single Particle Tracking (SPT) is a powerful technique for the analysis of the lateral diffusion of the lipid and protein components of biological membranes. In neurons, SPT allows the study of the real-time dynamics of receptors for neurotransmitters that diffuse continuously in and out synapses. In the simplest case where the membrane is flat and is parallel to the focal plane of the microscope the analysis of diffusion from SPT data is relatively straightforward. However, in most biological samples the membranes are curved, which complicates analysis and may lead to erroneous conclusions as for the mode of lateral diffusion. Here we considered the case of lateral diffusion in tubular membranes, such as axons, dendrites or the neck of dendritic spines. Monte Carlo simulations allowed us to evaluate the error in diffusion coefficient (D) calculation if the curvature is not taken into account. The underestimation is determined by the diameter of the tubular surface, the frequency of image acquisition and the degree of mobility itself. We found that projected trajectories give estimates that are 25 to 50% lower than the real D in case of 2D-SPT over the tubular surface. The use of 3D-SPT improved the measurements if the frequency of image acquisition was fast enough in relation to the mobility of the molecules and the diameter of the tube. Nevertheless, the calculation of D from the components of displacements in the axis of the tubular structure gave accurate estimate of D, free of geometrical artefacts. We show the application of this approach to analyze the diffusion of a lipid on model tubular membranes and of a membrane-bound GFP on neurites from cultured rat hippocampal neurons.

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Yegor A Domanov, Sophie Aimon, Gilman E S Toombes, Marianne Renner, François Quemeneur, Antoine Triller, Matthew S Turner, Patricia Bassereau (2011 Jul 20)

Mobility in geometrically confined membranes.

Proceedings of the National Academy of Sciences of the United States of America : 12605-10 : DOI : 10.1073/pnas.1102646108 En savoir plus
Résumé

Lipid and protein lateral mobility is essential for biological function. Our theoretical understanding of this mobility can be traced to the seminal work of Saffman and Delbrück, Who Predicted a logarithmic dependence of the protein diffusion coefficient (i) on the reverse of the size of the protein and (ii) the « membrane size » for membranes of finite size [P Saffman, Delbrück M (1975) Proc Natl Acad Sci USA 72: 3111-3113]. ALTHOUGH the experimental proof of the first prediction is a matter of debate, the second Has not beens Previously thought to be Experimentally accessible. Here, we just construct Such a geometrically confined by forming membrane lipid bilayer nanotubes of controlled radii connected to giant liposomes. We Followed the distribution of individual molecules in the tubular membrane using single particle tracking of quantum dots coupled to lipids voltage-gated potassium channels KvAP gold, while changing the membrane tube radius from Approximately 250 to 10 nm. We found That Both lipid and protein diffusion Was Slower in tubular membranes with smaller radii. The protein diffusion coefficient Decreased as much as 5-fold Compared To Effectively broadcast on the giant flat membrane of the liposomes. Both lipid and protein diffusion data are consistent with the predictions of a hydrodynamic theory That extends the work of Saffman and Delbrück to cylindrical geometries. The Therefore this study Provides strong experimental backing for the ubiquitous Saffman-Delbrück theory and elucidates the role of membrane geometry and size in regulating lateral diffusion.

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Ellen L Batchelder, Gunther Hollopeter, Clément Campillo, Xavier Mezanges, Erik M Jorgensen, Pierre Nassoy, Pierre Sens, Julie Plastino (2011 Jun 29)

Membrane tension regulates motility by controlling lamellipodium organization.

Proceedings of the National Academy of Sciences of the United States of America : 11429-34 : DOI : 10.1073/pnas.1010481108 En savoir plus
Résumé

Many cell movements proceed via a crawling mechanism, Where polymerization of the cytoskeletal protein actin pushes out the leading edge membrane. In this model, membrane tension has-been seen as an impediment to filament growth and cell motility. Here we use a single model of cell motility, the Caenorhabditis elegans sperm cell, to test how membrane voltage affects movement and cytoskeleton dynamics. To enable thesis analyzes, we create transgenic worm strains carrying sperm with a fluorescently Labeled cytoskeleton. Via osmotic shock and deoxycholate treatments, we relaxed or tense the cell membrane and membrane voltage QUANTIFY apparent exchange membrane by the technical tether. Surprisingly, we find That membrane voltage reduction is correlated with a Decrease in cell displacement speed, whereas an Increase in membrane tension Enhances motility. We further Top Demonstrate That apparent polymerization rates follow the trends Sami. We observed That membrane voltage reduction leads to an unorganized, rough lamellipodium, Composed of short filaments angled away from the management of movement. On Reviews the other hand, an Increase in tension Reduces lateral membrane protrusions in the lamellipodium, and filaments are oriented along and more Toward the management of movement. Overall we propose That tension membrane optimizes motility by streamlining polymerization in the management of movement, THUS Adding a layer of complexity to our current understanding of how membrane voltage Enters into the equation motility.

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Andrew Callan-Jones, Benoit Sorre, Patricia Bassereau (2011 Mar 23)

Curvature-driven lipid sorting in biomembranes.

Cold Spring Harbor perspectives in biology : DOI : 10.1101/cshperspect.a004648 En savoir plus
Résumé

It has beens Often suggéré que la high curvature of transportation intermediates in cells May be a Sufficient means clustering to segregate different lipid populations based on the relative energy costs of forming bent membranes. In this review, we present in vitro experiments That highlight the essential physics of lipid sorting at thermal equilibrium: It is driven by a trade-off entre bending energy, mixing entropy, and species interactions entre. We collect evidence That lipid sorting Strongly depends on lipid-lipid and protein-lipid interactions, and hence on the composition of the Underlying membrane and on the presence of bound proteins.

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Année de publication : 2010

Mahassine Safouane, Ludwig Berland, Andrew Callan-Jones, Benoit Sorre, Winfried Römer, Ludger Johannes, Gilman E S Toombes, Patricia Bassereau (2010 Oct 5)

Lipid cosorting mediated by shiga toxin induced tubulation.

Traffic (Copenhagen, Denmark) : 1519-29 : DOI : 10.1111/j.1600-0854.2010.01116.x En savoir plus
Résumé

To Maintain cell membrane homeostasis, lipids must be dynamically Redistributed During the formation of transportation intermediates, the goal Mechanisms driving lipid sorting are not yet fully Understood. Lowering sphingolipid concentration can Reduce the bending energy of a membrane, and this effect Could account for sphingolipid depletion along the retrograde pathway. HOWEVER, sphingolipids and cholesterol are enriched along the anterograde pathway, implying That Other lipid sorting Mechanisms, Such As protein-mediated sorting, can dominate. To Characterize the effect of protein binding on the lipid composition of highly curved membranes, we Studied the interaction of the B-subunit of Shiga toxin (STxB) with giant unilamellar vesicles Containing ict glycosphingolipid receptor [globotriaosylceramide (Gb 3)]. STxB binding induced the formation of tubular membrane invaginations, and fluorescence microscopy pictures of These highly curved membranes Were consistent with co-enrichment of Gb3 and sphingolipids. In agreement with theory, sorting Was stronger for membrane compositions close to demixing. These results Strongly supporting the hypothesis That Indirectly proteins can mediate the sorting of lipids into highly curved transportation via intermediates interactions entre lipids and the membrane receptor of the protein.

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Jan Brugués, Benoit Maugis, Jaume Casademunt, Pierre Nassoy, François Amblard, Pierre Sens (2010 Aug 16)

Dynamical organization of the cytoskeletal cortex probed by micropipette aspiration.

Proceedings of the National Academy of Sciences of the United States of America : 15415-20 : DOI : 10.1073/pnas.0913669107 En savoir plus
Résumé

Bleb-based cell motility proceeds by the successive inflation and retraction of large spherical membrane protrusions (« blebs ») coupled with substrate adhesion. In addition to their role in motility, cellular blebs constitute a remarkable illustration of the dynamical interactions between the cytoskeletal cortex and the plasma membrane. Here we study the bleb-based motions of Entamoeba histolytica in the constrained geometry of a micropipette. We construct a generic theoretical model that combines the polymerization of an actin cortex underneath the plasma membrane with the myosin-generated contractile stress in the cortex and the stress-induced failure of membrane-cortex adhesion. One major parameter dictating the cell response to micropipette suction is the stationary cortex thickness, controlled by actin polymerization and depolymerization. The other relevant physical parameters can be combined into two characteristic cortex thicknesses for which the myosin stress (i) balances the suction pressure and (ii) provokes membrane-cortex unbinding. We propose a general phase diagram for cell motions inside a micropipette by comparing these three thicknesses. In particular, we theoretically predict and experimentally verify the existence of saltatory and oscillatory motions for a well-defined range of micropipette suction pressures.

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Cécile Leduc, Otger Campàs, Jean-François Joanny, Jacques Prost, Patricia Bassereau (2010 Jul 1)

Mechanism of membrane nanotube formation by molecular motors.

Biochimica et biophysica acta : 1798 : 1418-1426 : DOI : 10.1016/j.bbamem.2009.11.012 En savoir plus
Résumé

Membrane nanotubes are ubiquitous in eukaryotic cells due to Their involvement in the communication entre Many different compartments membrane. They are very dynamical structures, qui Generally are extended along the microtubule network. One feasible mechanism of tube formation Involves the share of molecular motors, can generate qui The Necessary strength to pull the tubes along the cytoskeleton tracks. HOWEVER, It has not so far-been feasible to image in living organisms Simultaneously Both tube formation and the molecular motors Involved in the process. The Reasons for this are technological Mainly. To Overcome thesis limitations and to elucidate in detail the mechanism of tube formation, Many experiments-have beens Developed over the last years in cell-free environments. In the present review, we present the results, qui-have-been therefor obtained in vitro in cell extracts Either or with purified and artificial components. In Particular, we will focus on a biomimetic system, qui Involves Unilamellar Giant Vesicles, kinesin-1 motors and microtubules in the presence of ATP. We present Both theoretical and experimental results based on fluorescence microscopy That elucidate the dynamics of membrane tube formation, growth and stalling.

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Winfried Römer, Léa-Laetitia Pontani, Benoît Sorre, Carles Rentero, Ludwig Berland, Valérie Chambon, Christophe Lamaze, Patricia Bassereau, Cécile Sykes, Katharina Gaus, Ludger Johannes (2010 Feb 19)

Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis.

Cell : 140 : 540-553 : DOI : 10.1016/j.cell.2010.01.010 En savoir plus
Résumé

Nascent transport intermediates detach from donor membranes by scission. This process can take place in the absence of dynamin, notably in clathrin-independent endocytosis, by mechanisms that are yet poorly defined. We show here that in cells scission of Shiga toxin-induced tubular endocytic membrane invaginations is preceded by cholesterol-dependent membrane reorganization and correlates with the formation of membrane domains on model membranes, suggesting that domain boundary forces are driving tubule membrane constriction. Actin triggers scission by inducing such membrane reorganization process. Tubule occurrence is indeed increased upon cellular depletion of the actin nucleator component Arp2, and the formation of a cortical actin shell in liposomes is sufficient to trigger the scission of Shiga toxin-induced tubules in a cholesterol-dependent but dynamin-independent manner. Our study suggests that membranes in tubular Shiga toxin-induced invaginations are poised to undergo actin-triggered reorganization leading to scission by a physical mechanism that may function independently from or in synergy with pinchase activity.

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Aurélien Roux, Gerbrand Koster, Martin Lenz, Benoît Sorre, Jean-Baptiste Manneville, Pierre Nassoy, Patricia Bassereau (2010 Feb 18)

Membrane curvature controls dynamin polymerization.

Proceedings of the National Academy of Sciences of the United States of America : 4141-6 : DOI : 10.1073/pnas.0913734107 En savoir plus
Résumé

The generation of membrane curvature in intracellular traffic Involves Many proteins That curve can lipid bilayers. Among these, dynamin-like proteins Were shown to deform into membrane tubules, Thus Far and are the only proteins Known to mechanically drive membrane fission. Because dynamin forms a helical membrane coat circling a tubule, ict polymerization is thought to be responsible for this membrane deformation. Here we show que la Force Generated by dynamin polymerization, 18 pN, is Sufficient to deform membranes yet can still be counteracted by high membrane tension. Importantly, we observed That dynamin at low concentration, polymer nucleation Strongly depends on membrane curvature. This Suggests That dynamin May be PRECISELY or recruited to membrane buds’ necks Because of Their high curvature. To Understand This curvature dependence, we Developed a theory based on the competition entre dynamin polymerization and membrane mechanical deformation. This curvature control of dynamin polymerization is Predicted for a specific Range of concentrations (0.1-10 microM Approximately) qui corresponds to our measurements. More Generally, we expect That Any protein That Bind self-assembled gold onto membranes in a curvature-coupled way shoulds behave in a qualitatively similar Manner, with purpose icts own specific ranks of concentration.

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Ernesto Ambroggio, Benoît Sorre, Patricia Bassereau, Bruno Goud, Jean-Baptiste Manneville, Bruno Antonny (2010 Jan 20)

ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions.

The EMBO Journal : 29 : 292-303 : DOI : 10.1038/emboj.2009.341 En savoir plus
Résumé

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1-GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1-GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R approximately 35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1-GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1-GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1-GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.

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Année de publication : 2009

Pia Streicher, Pierre Nassoy, Michael Bärmann, Aurélien Dif, Valérie Marchi-Artzner, Françoise Brochard-Wyart, Joachim Spatz, Patricia Bassereau (2009 Aug 12)

Integrin reconstituted in GUVs: a biomimetic system to study initial steps of cell spreading.

Biochimica et biophysica acta : 2291-300 : DOI : 10.1016/j.bbamem.2009.07.025 En savoir plus
Résumé

A novel in vitro membrane system mimicking the first steps of integrin-mediated cell spreading has-been Developed and caractérisé. We-have reconstituted the transmembrane alpha (IIb) beta (3) integrin into giant unilamellar vesicles (GUVs). The rebuilding process has-been validated by Analyzing protein incorporation and biological activity by checking the specific interaction of GUVs Containing integrin with quantum dots (QD) or surfaces coated with the integrin receptor tri-peptide RGD. (1) The spreading dynamics of integrin-functionalized GUVs onto fibrinogen-coated surfaces has-been monitored by Reflection Interference Contrast Microscopy (RICM). Our results are quantitatively consistent with a theoretical model based on a dewetting process coupled to binder distribution and Provide a comprehensive description Of the following sequence: i) nucleation and growth of adhesive patches coupled to the diffusion of the adhesive proteins to adhesive thesis areas ii) merger and formation of patches of adhesive year ring iii) Complete spreading of the GUV by dewetting of the central liquid movie from the border to form an adhesive patch circular That is not Significantly enriched in integrins, as Compared to the unbound membrane. This finding is consistent with the reconnu role of the actin cytoskeleton in stabilizing focal complexes and focal adhesions in a cell-extracellular matrix contacts. These very large unilamellar integrin-containing vesicles Provide a single artificial system, qui Could Be Developed further Top Towards realistic mimic cell and used to study the complexity of integrin-mediated cell spreading.

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Barbara Windschiegl, Alexander Orth, Winfried Römer, Ludwig Berland, Bahne Stechmann, Patricia Bassereau, Ludger Johannes, Claudia Steinem (2009 Jul 17)

Lipid reorganization induced by Shiga toxin clustering on planar membranes.

PloS one : e6238 : DOI : 10.1371/journal.pone.0006238 En savoir plus
Résumé

The homopentameric B-subunit of Shiga toxin bacterial protein (STxB) binds to the glycolipid Gb (3) in plasma membranes, qui is the initial step for Reviews entering cells by a clathrin-independent mechanism. It has-been suggéré That clustering protein and lipid reorganization determined toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb (3) and clustering for the Proposed reorganization in planar lipid membranes. The effect of binding site III of the B-subunit as well as the Gb (3) Was Investigated lipid structure by means clustering of high resolution methods Such As fluorescence microscopy and scanning strength. STxB Was found to form one homogenous protein clusters 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) / cholesterol / Gb (3) (65: 30: 5) bilayers. In contrast, membranes Composed of DOPC / cholesterol / sphingomyelin / Gb (3) (40: 35: 20: 5) into a separate phase of liquid ordered and disordered liquid phase. Dependent on the fatty acid composition of Gb (3) STxB-Gb (3) complex organizes dans le ordered liquid-phase protein upon binding. Our Findings suggest That STxB is able of forming a new stage membrane by lipid That Is caractérisé compaction. The significance of this finding is Discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.

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Otger Campàs, Cécile Leduc, Patricia Bassereau, Jean-François Joanny, Jacques Prost (2009 Apr 1)

Collective oscillations of processive molecular motors.

Biophysical Reviews and Letters : 4 : 163-178 : DOI : 10.1142/S1793048009000971 En savoir plus
Résumé

We present both a theoretical and an experimental study of the long time behavior of membrane nanotubes pulled from giant unilamellar vesicles by molecular motors. Experimentally, two types of behaviors are observed, either tubes stall at a finite length or they undergo periodic oscillations. Theoretically we write the equations for the tube dynamics as a two-dimensional dynamical system where the variables are the tube length (or the force required to pull the tube at a given length) and the number of motors at the tip pulling the tube. We construct stability diagrams showing the stalling and oscillating states and present an example of oscillations in a non-linear regime. These results can explain the membrane tube retractions and oscillations observed in living cells.

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