Membranes et fonctions cellulaires

Mots clés : transport intracellulaire, nanotubes de membrane, membranes actives, adhésion, mécaniques des membranes des cellules

Consultez le rapport d'activité scientifique. (pdf 4,70Mo, mise à jour 26 mars 2010)

La biologie moléculaire se consacre généralement à l'identification des protéines impliquées dans les fonctions cellulaires, afin de comprendre leurs interactions potentielles avec d'autres protéines ou d'autres types de molécules. Un grand nombre de ces protéines interagissent avec des membranes lipidiques ou sont insérées dans des membranes. Celles-ci ont été longtemps considérées comme un support passif. Cependant, la physique des membranes a été largement étudiée par des biophysiciens au cours des vingt dernières années. En particulier, des effets non-triviaux liés aux propriétés des membranes en tant que matériaux (spécialement à des échelles de longueur plus grandes que les tailles moléculaires) ont été mis en évidence. Cela suggère q'une description purement moléculaire de la membrane n'est pas suffisante pour comprendre de façon quantitative sa fonction.

Bio-membranes modèles

Fig. 1 

Notre groupe développe une approche multi-disciplinaire afin de comprendre les rôles des membranes dans les fonctions cellulaires importantes, telles que le transport intracellulaire et l'adhésion. Nous concevons des systèmes modèles simples, la plupart du temps basés sur les vésicules unilamellaires géantes (GUVs) afin de reproduire ces fonctions. Les GUVs sont des membranes modèles intéressantes car elles se composent d'un nombre limité et contrôlé d'éléments, qu'on peut largement faire varier (Figure 1 et 2). Comme la taille des GUVs varie généralement entre 1 et 100 µm, les effets de l'ajout au système d'une protéine particulière ou de tout autre élément peuvent être directement étudiés. La microscopie optique permet d'évaluer de façon quantitative les modifications de forme, les déformations des membranes, la redistribution d'espèces fluorescentes, etc. Nous pouvons également mesurer les propriétés mécaniques des membranes en utilisant des techniques d'aspiration par micropipette, l'analyse de formes ou les pinces optiques. Les études sur ces membranes modèles peuvent alors être comparées avec le comportement des cellules vivantes.

Nous travaillons en collaboration avec des biologistes à l'Institut Curie (B. Goud, J.-B. Manneville, S. Dufour, C. Lamaze et L. Johannes de l'UMR 144), et avec des théoriciens de notre unité UMR 168 (J. Prost, J.-F. Joanny et F. Brochard).

Nos faits marquants

Nos projets et résultats les plus significatifs de ces dernières années comprennent :

Membranes actives

Nos études sur les GUVs comprenant des pompes ioniques reconstituées, telles que la bactériorhodopsine ou l'ATPase Ca²⁺ ont démontré que les fluctuations de position des membranes sont largement amplifiées, à cause de l'activité hors-équilibre de ces pompes. Plus récemment, nous avons montré, pour la bactériorhodopsine, que l'activité des protéines induit une réduction de la tension membranaire.

Transport intracellulaire

• Effet de fusion : l'addition de lipides au cours de la fusion membranaire peut induire une tension membranaire négative et des instabilités, conduisant finalement au flambage de la bicouche lipidique.
• Déformation membranaire par des protéines : les protéines virales, comme la protéine de matrice du virus de la stomatite vésiculaire, ou des toxines telles que les toxines de Shiga et du choléra, induisent des invaginations des membranes modèles contenant leurs récepteurs lipidiques (Figure 1), imitant ainsi le phénomène biologique.
• Nanotubes membranaires : nous avons conçu un système bio-mimétique dans lequel les moteurs moléculaires liés aux GUVs se déplacent le long des microtubules (Figure 2 et film) ; l'analyse de ce système montre que les moteurs s'associent pour tirer collectivement les tubes de membrane. De plus, avec des pinces optiques, nous avons systématiquement mesuré les forces nécessaires pour tirer des tubes de membrane et le mécanisme de coalescence des tubes.

Fig. 2

Film 1

Adhésion : de la molécule unique aux cellules

Fig. 3 

Nous avons développé une approche systématique pour étudier l'adhésion cellulaire en mesurant, tout d'abord, la force de rupture de liaisons ligand-récepteur à l'échelle de la liaison unique ou multiple, puis, l'étalement de GUVs adhésives sur des substrats couverts des récepteurs d'adhésion et, enfin, l'étalement de cellules (Figure 3).

Rhéologie et mécanique des biomembranes

Fig. 4 

En tirant des tubes de cellules par micromanipulation, nous avons pu étudier l'interaction de la membrane avec le cytosquelette et identifier des domaines régulant la tension des membranes (Figure 4).

Projets

Nos directions de recherches actuelles et futures comprennent :

• La propagation de signaux dans un « neurone minimal » ;
• L'endocytose (déformation de la membrane par des protéines de manteaux ou par des particules mimant des virus, fission et tri) ;
• L'organisation des membranes cellulaires étudiée par la mécanique membranaire ;
• L'adhésion « cellule-cellule » et l'adhésion « matrice extracellulaire-cellule ».

Dernière mise à jour : mars 2010

Publications clés

2010

• Roux A., Koster G., Lenz M., Sorre B., Manneville J.B., Nassoy P. and Bassereau P.
  Membrane curvature controls dynamin polymerization
  Proceedings of the National Academy of Science U.S.A., 107, 4141-4146 - Abstract
  The generation of membrane curvature in intracellular traffic involves
  many proteins that can curve lipid bilayers. Among these,
  dynamin-like proteins were shown to deform membranes into
  tubules, and thus far are the only proteins known to mechanically
  drive membrane fission. Because dynamin forms a helical coat
  circling a membrane tubule, its polymerization is thought to be
  responsible for this membrane deformation. Here weshow that the
  force generated by dynamin polymerization, 18 pN, is sufficient to
  deform membranes yet can still be counteracted by high membrane
  tension. Importantly, we observe that at low dynamin concentration,
  polymer nucleation strongly depends on membrane curvature.
  This suggests that dynamin may be precisely recruited to
  membrane buds' necks because of their high curvature. To understand
  this curvature dependence, we developed a theory based on
  the competition between dynamin polymerization and membrane
  mechanical deformation. This curvature control of dynamin polymerization
  is predicted for a specific range of concentrations (∼0.1-
  10 μM), which corresponds to our measurements. More generally,
  we expect that any protein that binds or self-assembles onto membranes
  in a curvature-coupled way should behave in a qualitatively
  similar manner, but with its own specific range of concentration.
  - Version complète

2009

• Mabrouk E., Cuvelier, D., Brochard-Wyart F., Nassoy P. and Li M.-H.
  Bursting of sensitive polymersomes induced by curling
  Proceedings of the National Academy of Sciences U.S.A., 106, 7294-7298 - Abstract
  Polymersomes, which are stable and robust vesicles made of block copolymer amphiphiles, are good candidates for drug carriers or micro/nanoreactors. Polymer chemistry enables almost unlimited molecular design of responsive polymersomes whose degradation upon environmental changes has been used for the slow release of active species. Here, we propose a strategy to remotely trigger instantaneous polymersome bursting. We have designed asymmetric polymer vesicles, in which only one leaflet is composed of responsive polymers. In particular, this approach has been successfully achieved by using a UV-sensitive liquid-crystalline copolymer. We study experimentally and theoretically this bursting mechanism and show that it results from a spontaneous curvature of the membrane induced by the remote stimulus. The versatility of this mechanism should broaden the range of applications of polymersomes in fields such as drug delivery, cosmetics and material chemistry.
  - Version complète
• Sorre B., Callan-Jones A., Manneville J.B., Nassoy P., Joanny J.F., Prost J., Goud B. and Bassereau P.
  Curvature-driven lipid sorting needs proximity to a demixing point and is aided by proteins
  Proceedings of the National Academy of Science U.S.A., 106; 5622-5626 - Abstract
  Sorting of lipids and proteins is a key process allowing eukaryotic cells to execute efficient and accurate intracellular transport and to maintain membrane homeostasis. It occurs during the formation of highly curved transport intermediates that shuttle between cell compartments. Protein sorting is reasonably well described, but lipid sorting is much less understood. Lipid sorting has been proposed to be mediated by a physical mechanism based on the coupling between membrane composition and high curvature of the transport intermediates. To test this hypothesis, we have performed a combination of fluorescence and force measurements on membrane tubes of controlled diameters pulled from giant unilamellar vesicles. A model based on membrane elasticity and nonideal solution theory has also been developed to explain our results. We quantitatively show, using 2 independent approaches, that a difference in lipid composition can build up between a curved and a noncurved membrane. Importantly, and consistent with our theory, lipid sorting occurs only if the system is close to a demixing point. Remarkably, this process is amplified when even a low fraction of lipids is clustered upon cholera toxin binding. This can be explained by the reduction of the entropic penalty of lipid sorting when some lipids are bound together by the toxin. Our results show that curvature-induced lipid sorting results from the collective behavior of lipids and is even amplified in the presence of lipid-clustering proteins. In addition, they suggest a generic mechanism by which proteins can facilitate lipid segregation in vivo.
  - Version complète
• El Alaoui Faris M.D., Lacoste D., Pécréaux J., Joanny J.F., Prost J. and Bassereau P.
  Lowering of membrane tension induced by protein activity
  Physical Review Letters, 102, 038102 - Abstract
  Using videomicroscopy we present measurements of the fluctuation spectrum of giant vesicles containing bacteriorhodopsin pumps. When the pumps are activated, we observe a significant increase of the fluctuations in the low wave vector region, which we interpret as due to a lowering of the effective tension of the membrane.
  - Version complète

2008

• Nassoy P., D. Cuvelier, R. Bruinsma et F. Brochard-Wyart
  Nanofluidics in cellular tubes under oscillatory extension
  Europhysics Letters, 84,18004 - Abstract
  Membrane nanotubes or tethers extruded from cells exhibit dynamic features that are
  believed to exhibit viscoelastic rheological properties. We have performed typical microrheology
  experiments on tethers pulled from red blood cells by measuring the force response to small
  oscillatory extensions or compressions. Our data, supported by a simple theoretical model,
  show that the force response does not reflect any intrinsic viscoelastic properties of the tethers
  themselves, but instead is dominated by the drainage of the internal cellular fluid into and out
  of the oscillating nanoconduit over a frequency-dependent penetration depth. The simplicity of
  tether rheology suggests its usage as a probe for measuring the local viscosity of the cytosol near
  the plasma membrane.
  - Version complète

2007

• Römer W., Berland L., Chambon V., Gaus K., Windschiegl B., Tenza D., Aly M., Fraisier V., Florent J.-C., Perrais D., Lamaze C., Raposo G., Steinem C., Sens P., Bassereau P., Johannes L.
  Shiga toxin induces tubular membrane invaginations for its uptake into cells
  Nature, 450, 670-675 - Abstract
  Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit-Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.
  - Version complète
• D. Cuvelier, M. Théry, Y.-S. Chu, J. P. Thiery, M. Bornens, P. Nassoy et L. Mahadevan
  The universal dynamics of cell spreading
  Current Biology, 17, 694-699 - Abstract
  Cell adhesion and motility depend strongly on the interactions between cells and extracellular matrix (ECM) substrates. When plated onto artificial adhesive surfaces, cells first flatten and deform extensively as they spread. At the molecular level, the interaction of membrane-based integrins with the ECM has been shown to initiate a complex cascade of signaling events [1], which subsequently triggers cellular morphological changes and results in the generation of contractile forces [2]. Here, we focus on the early stages of cell spreading and probe their dynamics by quantitative visualization and biochemical manipulation with a variety of cell types and adhesive surfaces, adhesion receptors, and cytoskeleton-altering drugs. Wefind that the dynamics of adhesion follows a universal power-law behavior. This is in sharp contrast with thecommonbelief that spreading is regulated by either the diffusion of adhesion receptors toward the growing adhesive patch [3-5] or by actin polymerization [6-8]. To explain this, we propose a simple quantitative and predictive theory that models cells as viscous adhesive cortical shells enclosing a less viscous interior. Thus, although cell spreading is driven by well-identified biomolecular interactions, it is dynamically limited by its mesoscopic structure and material properties.
  - Version complète