Membranes and cellular functions

Keywords: Intracellular transport, Membrane nanotubes, Active membranes, Adhesion, Cell membrane mechanics

Read the scientific activity report. (pdf 860Ko, last update 26th january 2009)

Molecular biology is traditionally devoted to identify proteins involved in cellular functions, to understand their potential interactions with other proteins or types of molecules. A large number of these proteins either interact with lipid membranes or are inserted in membranes, which have yet been often considered as a passive template. However, the physics of membranes has been widely investigated by biophysicists for the last two decades. In particular, non-trivial effects related to material properties of membranes (especially at length scales larger than molecular sizes) were evidenced. This suggests that a purely molecular description of the membrane is not sufficient to achieve a quantitative understanding of its function.

Model bio-membranes

Fig. 1 

Our group is developing a multidisciplinary approach to understand the roles of membranes in important cellular functions such as intracellular transport and adhesion. We design simple model systems, most often based on giant unilamellar vesicles (GUVs) to mimic these functions. GUVs are useful model membranes because they are composed of a limited and controlled number of components, which can be varied (Figure 1 et 2). Since the size of GUVs typically ranges from 1-100 µm, the effects of adding a particular protein, or any other components to the system can be investigated directly. Optical microscopy can quantitatively evaluate shape changes, membrane deformations, redistribution of fluorescent species etc. We can also monitor the mechanical properties of membranes by using micropipette techniques, shape analysis or optical tweezers. Studies of these model membranes can then be compared with the behaviour of living cells.

We work in collaboration with biologists at the Institut Curie (B. Goud, J.-B. Manneville, S. Dufour, C. Lamaze and L. Johannes of the UMR 144), and with some theoreticians in our unit UMR 168 (J. Prost, J.-F. Joanny and F. Brochard).

Highlights

Our most significant projects and findings over the past few years include:

Active membranes

Our studies of GUVscontaining reconstituted ion pumps such as bacteriorhodopsin or the Ca2+-ATPase have demonstrated that membrane position fluctuations are amplified largely, due to the non-equilibrium activity of the ion pumps. More recently, we have shown that the protein activity induces a lowering of the membrane tension in the case of bacteriorhodopsin.

Intracellular traffic

• Effect of fusion: Negative tension and membrane instabilities can be induced by lipid uptake during membrane fusion, leading, ultimately, to collapse of the lipid bilayer.
• Membrane deformation by proteins: Viral proteins, like the vesicular stomatitis virus M protein, or toxins such as the Shiga- and cholera- toxins, induce invaginations of model membranes containing their lipid receptors (Figure 1), thus mimicking the biological situation.
• Membrane nanotubes: We have designed a biomimetic system in which molecular motors attached to GUVs move along microtubules (Figure 2 and film) ; analysis of this system shows that the motors associate with each other to pull membrane tubes collectively. Moreover, with optical tweezers, we have systematically measured the forces required to pull membrane tubes and the mechanism of coalescence of tubes.

Fig. 2

Movie 1

Adhesion: from single molecule to cells

Fig. 3 

We have developed a systematic approach to study cell adhesion by measuring, first, the rupture force of single or multiple ligand-receptor bonds, then, the spreading of adhesive GUVs on substrates coated with adhesion receptors and, finally, spreading of cells themselves (Figure 3).

Rheology and mechanics of biomembranes

Fig. 4 

By pulling tubes from cells by micromanipulation, we were able to probe the interaction of the membrane with the cytoskeleton, and identify domains regulating membrane tension (Figure 4).

Projects

Our current and future research directions include:

• Signal propagation in a ‘minimal neuron'
• Endocytosis (membrane deformation by coat proteins or by virus-mimicking particles, fission and sorting)
• Cell membrane organization studied by membrane mechanics
• “Cell-cell” adhesion and “extracellular matrix-cell” adhesion

Last update: January 2009

Key publications

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., published on line - 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.
  - Full version

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.
  - Full version
• 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.
  - Full version
• 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.
  - Full version

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.
  - Full version

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.
  - Full version
• 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.
  - Full version