Membranes et fonctions cellulaires

Publications

Année de publication : 2018

Begoña Ugarte-Uribe, Coline Prévost, Kushal Kumar Das, Patricia Bassereau, Ana J. García-Sáez (2018 Apr 19)

Drp1 polymerization stabilizes curved tubular membranes similar to those of constricted mitochondria.

Journal of Cell Science : 132 : jcs208603 : DOI : 10.1242/jcs.208603 En savoir plus
Résumé

Dynamin-related protein 1 (Drp1), an 80 kDa mechanochemical GTPase of the dynamin superfamily, is required for mitochondrial division in mammals. Despite the role of Drp1 dysfunction in human disease, its molecular mechanism remains poorly understood. Here, we examined the effect of Drp1 on membrane curvature using tubes pulled from giant unilamellar vesicles (GUVs). We found that GTP promoted rapid rearrangement of Drp1 from a uniform distribution to discrete foci, in line with the assembly of Drp1 scaffolds at multiple nucleation sites around the lipid tube. Polymerized Drp1 preserved the membrane tube below the protein coat, also in the absence of pulling forces, but did not induce spontaneous membrane fission. Strikingly, Drp1 polymers stabilized membrane curvatures similar to those of constricted mitochondria against pressure changes. Our findings support a new model for mitochondrial division whereby Drp1 mainly acts as a scaffold for membrane curvature stabilization, which sets it apart from other dynamin homologs.

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Guillaume Kulakowski, Hugo Bousquet Jean‐Baptiste Manneville, Patricia Bassereau, Bruno Goud, Lena K. Oesterlin (2018 Apr 6)

Lipid packing defects and membrane charge control RAB GTPase recruitment.

Traffic : 19 : 536-545 : DOI : 10.1111/tra.12568 En savoir plus
Résumé

Specific intracellular localization of RAB GTPases has been reported to be dependent on protein factors, but the contribution of the membrane physicochemical properties to this process has been poorly described. Here, we show that three RAB proteins (RAB1/RAB5/RAB6) preferentially bind in vitro to disordered and curved membranes, and that this feature is uniquely dependent on their prenyl group. Our results imply that the addition of a prenyl group confers to RAB proteins, and most probably also to other prenylated proteins, the ability to sense lipid packing defects induced by unsaturated conical-shaped lipids and curvature. Consistently, RAB recruitment increases with the amount of lipid packing defects, further indicating that these defects drive RAB membrane targeting. Membrane binding of RAB35 is also modulated by lipid packing defects but primarily dependent on negatively charged lipids. Our results suggest that a balance between hydrophobic insertion of the prenyl group into lipid packing defects and electrostatic interactions of the RAB C-terminal region with charged membranes tunes the specific intracellular localization of RAB proteins.

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Mijo Simunovic, Patricia Bassereau, Gregory A. Voth (2018 Mar 30)

Organizing membrane-curving proteins: the emerging dynamical picture.

Current Opinion in Structural Biology : 51 : 99-105 : DOI : 10.1016/j.sbi.2018.03.018 En savoir plus
Résumé

Lipid membranes play key roles in cells, such as in trafficking, division, infection, remodeling of organelles, among others. The key step in all these processes is creating membrane curvature, typically under the control of many anchored, adhered or included proteins. However, it has become clear that the membrane itself can mediate the interactions among proteins to produce highly ordered assemblies. Computer simulations are ideally suited to investigate protein organization and the dynamics of membrane remodeling at near-micron scales, something that is extremely challenging to tackle experimentally. We review recent computational efforts in modeling protein-caused membrane deformation mechanisms, specifically focusing on coarse-grained simulations. We highlight work that exposed the membrane-mediated ordering of proteins into lines, meshwork, spirals and other assemblies, in what seems to be a very generic mechanism driven by a combination of short and long-ranged forces. Modulating the mechanical properties of membranes is an underexplored signaling mechanism in various processes deserving of more attention in the near future.

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Eran Agmon, Jérôme Solon, Patricia Bassereau, Brent R. Stockwell (2018 Mar 26)

Modeling the effects of lipid peroxidation during ferroptosis on membrane properties.

Scientific Reports : 8 : 5155 : DOI : 10.1038/s41598-018-23408-0 En savoir plus
Résumé

Ferroptosis is a form of regulated cell death characterized by the accumulation of lipid hydroperoxides. There has been significant research on the pathways leading to the accumulation of oxidized lipids, but the downstream effects and how lipid peroxides cause cell death during ferroptosis remain a major puzzle. We evaluated key features of ferroptosis in newly developed molecular dynamics models of lipid membranes to investigate the biophysical consequences of lipid peroxidation, and generated hypotheses about how lipid peroxides contribute to cell death during ferroptosis.

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Année de publication : 2017

Coline Prévost, Feng-Ching Tsai, Patricia Bassereau, Mijo Simunovic (2017 Dec 7)

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles.

Journal of Visualized Experiments : 130 : DOI : 10.3791/56086. En savoir plus
Résumé

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

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Win Pin Ng, Kevin D. Webster, Caroline Stefani, Eva M. Schmid, Emmanuel Lemichez, Patricia Bassereau, Daniel A. Fletcher (2017 Oct 2)

Force-induced transcellular tunnel formation in endothelial cells

Molecular Biology of the Cell : 28 : 2650-2660 : DOI : 10.1091/mbc.E17-01-0080 En savoir plus
Résumé

The endothelium serves as a protective semipermeable barrier in blood vessels and lymphatic vessels. Leukocytes and pathogens can pass directly through the endothelium by opening holes in endothelial cells, known as transcellular tunnels, which are formed by contact and self-fusion of the apical and basal plasma membranes. Here we test the hypothesis that the actin cytoskeleton is the primary barrier to transcellular tunnel formation using a combination of atomic force microscopy and fluorescence microscopy of live cells. We find that localized mechanical forces are sufficient to induce the formation of transcellular tunnels in human umbilical vein endothelial cells (HUVECs). When HUVECs are exposed to the bacterial toxin called epidermal cell differentiation inhibitor (EDIN), which can induce spontaneous transcellular tunnels, less mechanical work is required to form tunnels due to the reduced cytoskeletal stiffness and thickness of these cells, similarly to the effects of a Rho-associated protein kinase (ROCK) inhibitor. We also observe actin enrichment in response to mechanical indentation that is reduced in cells exposed to the bacterial toxin. Our study shows that the actin cytoskeleton of endothelial cells provides both passive and active resistance against transcellular tunnel formation, serving as a mechanical barrier that can be overcome by mechanical force as well as disruption of the cytoskeleton.

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Caroline Stefani, David Gonzalez-Rodriguez, Yosuke Senju, Anne Doye, Nadia Efimova, Sébastien Janel, Justine Lipuma, Meng Chen Tsai, Daniel Hamaoui, Madhavi P. Maddugoda, Olivier Cochet-Escartin, Coline Prévost, Frank Lafont, Tatyana Svitkina, Pekka Lappalainen, Patricia Bassereau, Emmanuel Lemichez (2017 Jun 23)

Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation.

Nature Communications : 8 : 15839 : DOI : 10.1038/ncomms15839 En savoir plus
Résumé

Transendothelial cell macroaperture (TEM) tunnels control endothelium barrier function and are triggered by several toxins from pathogenic bacteria that provoke vascular leakage. Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. Here, by conducting high-resolution microscopy approaches we unveil the presence of an actomyosin cable encircling TEMs. We develop a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This establishes the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the upstream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favouring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring.

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Mijo Simunovic, Jean-Baptiste Manneville, Henri-François Renard, Emma Evergren, Krishnan Raghunathan, Dhiraj Bhatia, Anne K. Kenworthy, Gregory A. Voth, Jacques Prost, Harvey T. McMahon, Ludger Johannes, Patricia Bassereau*, Andrew Callan-Jones* (2017 Jun 22)

Friction mediates scission of tubular membranes scaffolded by BAR proteins

Cell : 170 : 172-184 : DOI : 10.1016/j.cell.2017.05.047 En savoir plus
Résumé

Membrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly.

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David Saletti, Jens Radzimanowski, Gregory Effantin, Daniel Midtvedt, Stéphanie Mangenot, Winfried Weissenhorn, Patricia Bassereau, Marta Bally (2017 Jan 26)

The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model.

Scientific reports : 40801 : DOI : 10.1038/srep40801 En savoir plus
Résumé

Matrix proteins from enveloped viruses play an important role in budding and stabilizing virus particles. In order to assess the role of the matrix protein M1 from influenza C virus (M1-C) in plasma membrane deformation, we have combined structural and in vitro reconstitution experiments with model membranes. We present the crystal structure of the N-terminal domain of M1-C and show by Small Angle X-Ray Scattering analysis that full-length M1-C folds into an elongated structure that associates laterally into ring-like or filamentous polymers. Using negatively charged giant unilamellar vesicles (GUVs), we demonstrate that M1-C full-length binds to and induces inward budding of membrane tubules with diameters that resemble the diameter of viruses. Membrane tubule formation requires the C-terminal domain of M1-C, corroborating its essential role for M1-C polymerization. Our results indicate that M1-C assembly on membranes constitutes the driving force for budding and suggest that M1-C plays a key role in facilitating viral egress.

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Weria Pezeshkian, Haifei Gao, Senthil Arumugam, Ulrike Becken, Patricia Bassereau, Jean-Claude Florent, John Hjort Ipsen, Ludger Johannes, Julian C Shillcock (2017 Jan 24)

Mechanism of Shiga Toxin Clustering on Membranes.

ACS Nano : 11 : 314-324 : DOI : 10.1021/acsnano.6b05706 En savoir plus
Résumé

The bacterial Shiga toxin interacts with its cellular receptor, the glycosphingolipid globotriaosylceramide (Gb3 or CD77), as a first step to entering target cells. Previous studies have shown that toxin molecules cluster on the plasma membrane, despite the apparent lack of direct interactions between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height, or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga toxin molecules. By contrast, in coarse-grained computer simulations, a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface. The most likely interpretation of these findings is that a membrane fluctuation-induced force generates an effective attraction between toxin molecules. Such force would be of similar strength to the electrostatic force at separations around 1 nm, remain strong at distances up to the size of toxin molecules (several nanometers), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a route for the targeting of nanoparticles to cells for biomedical applications.

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Garten M., Mosgaard L.D., Bornschlögl T., Dieudonné S., Bassereau P., Toombes G.E.S. (2017 Jan 1)

Whole-GUV patch-clamping

Proceedings of the National Academy of Sciences : 114 : 328-333 : DOI : 10.1073/pnas.1609142114 En savoir plus
Résumé

Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the “whole-GUV” patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

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Année de publication : 2016

Mijo Simunovic, Emma Evergren, Ivan Golushko, Coline Prévost, Henri-François Renard, Ludger Johannes, Harvey T McMahon, Vladimir Lorman, Gregory A Voth, Patricia Bassereau (2016 Oct 4)

How curvature-generating proteins build scaffolds on membrane nanotubes.

Proceedings of the National Academy of Sciences of the United States of America : 113 : DOI : 10.1073/pnas.1606943113 En savoir plus
Résumé

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein-membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30-40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes.

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Mijo Simunovic, Coline Prévost, Andrew Callan-Jones, Patricia Bassereau (2016 Jun 15)

Physical basis of some membrane shaping mechanisms.

Philosophical transactions. Series A, Mathematical, physical, and engineering sciences : DOI : 10.1098/rsta.2016.0034 En savoir plus
Résumé

In vesicular transportation pathways, membrane lipids and proteins are internalized, externalized or transported Within cells, not by bulk diffusion of single molecules, goal embedded in the membrane of small vesicles or thin tubules. The formation of These ‘transportation carriers’ Follows sequential events: bending membrane fission from the donor compartment, and transportation Eventually fusion with the acceptor membrane. A similar sequence is Involved During the internalization of drug or gene carriers inside cells. These membrane-shaping events are mediated by proteins Generally binding to membranes. The thesis Mechanisms behind biological processes are Actively Studied Both in the context of cell biology and biophysics. Bin / Amphiphysin / Rvs (BAR) domain proteins are Ideally suited for single Illustrating how soft matter principles can account for deformation by membrane proteins. We review here Some experimental methods and theoretical models to measure Corresponding thesis how proteins affect the mechanics and the shape of membranes. In more detail, we show how an experimental method Employing optical tweezers to pull a tube from a giant vesicle May give significant quantitative insights into the mechanism by which proteins sense and generate membrane curvature and the mechanism of membrane scission.This article is share of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’.

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Alice Berthaud, François Quemeneur, Maxime Deforet, Patricia Bassereau, Françoise Brochard-Wyart, Stéphanie Mangenot (2015 Dec 15)

Spreading of porous vesicles subjected to osmotic shocks: the role of aquaporins.

Soft matter : 12 : 1601-1609 : DOI : 10.1039/c5sm01654a En savoir plus
Résumé

Aquaporin 0 (AQP0) is a transmembrane protein specific to the eye lens, Involved as a water carrier across the lipid membranes. During maturation eye lens, AQP0s are truncated by proteolytic cleavage. We Investigate this work in the capability of truncated AQP0 to conduite water across membranes. We Developed a method to Accurately determine water permeability across lipid membranes and proteins across from the deflation under osmotic pressure of giant unilamellar vesicles (GUVs) Deposited adhesive substrate on year. Using reflection interference contrast microscopy (RICM), we measure the spreading area of ​​GUVs During deswelling. We interpret thesis results using a model based on hydrodynamic, binder diffusion Reviews towards the touch area, and Helfrich’s law for the membrane voltage, qui allows us to spread recounts the area to the internal vesicle volume. We first study the specific adhesion of vesicles coated with biotin was spreading streptavidin substrate. Then we determine the permeability of a single functional AQP0 and Demonstrate That truncated AQP0 is no more a water channel.

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Année de publication : 2015

Mijo Simunovic, Gregory A Voth, Andrew Callan-Jones, Patricia Bassereau (2015 Nov 2)

When Physics Takes Over: BAR Proteins and Membrane Curvature.

Trends in cell biology : 780-92 : DOI : 10.1016/j.tcb.2015.09.005 En savoir plus
Résumé

Cell membranes Become highly curved During membrane trafficking, cytokinesis, infection, immune response, or cell motion. Bin / Amphiphysin / Rvs (BAR) domain proteins Intrinsically With Their curved shape anisotropy and are Involved in Many of These processes, aim with a wide spectrum of modes of action. In vitro experiments and computer simulations multiscale-have Contributed in Identifying a minimal set of physical parameters derived derived, namely protein density on the membrane, membrane voltage and membrane shape, That control how bound BAR domain proteins behave on the membrane. In this review, we summarize the multifaceted BAR coupling of proteins to membrane mechanics and offers a single-phase diagram That Recapitulates the effects of These parameters.

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