Funded projects

Combining theory of membrane deformations and force measurements to study the effects of local mechanical perturbations on plasma membrane organization.

2015 - C. Lamaze (UMR3666/U1143), P. Sens (team Joanny/Prost UMR168), D. Köster (NCBS Bangalore)

Living cells change their shape constantly and rapidly (within seconds) due to external or internally originating perturbations like stretch induced by attached neighboring cells, remodeling of the cytoskeleton during cell movement or filopodia formation. These changes in cell shape imply deformations and reorganizations of the plasma membrane, a complex mixture of lipids and proteins delimiting the cell from its environment and hub for many cell processes like cargo uptake/release and signaling. In this project we will address the question of how specialized membrane structures called caveolae, affect the behavior of the plasma membrane on short time (seconds) and length scales (μm) during a local change in cell membrane shape? For this we will extrude thin membrane tethers using optical tweezers and monitor the force as well as visualizing individual caveolae using fluorescence microscopy. Findings of this study will give us an insight whether local perturbations of the cell plasma membrane result also in local changes of its organization or affect it globally.

Combining theory of membrane deformations and force measurements to study the effects of local mechanical perturbations on plasma membrane organization.

The cell plasma membrane is a complex heterogeneous mixture of hundreds of lipid species and membrane embedded or associated proteins which can more or less freely move in the plane of the asymmetric lipid bilayer. In addition, the plasma membrane interacts strongly with the actin cytoskeleton that either leads to membrane deformations (e.g. during the formation of filopodia) or affect the mobility of membrane proteins that bind to actin (e.g. ezrin). Given this complex scenario, understanding the dynamics and nature of lipid-lipid and lipid-protein configurations in the cell plasma membrane is of central interest, as most signaling events are transmitted into the cell through specific protein complexes at the plasma membrane and uptake (endocytosis) and release (exocytosis) of cargo need the recruitment of a specific set of lipids and proteins. Models like the fluid mosaic model by Singer and Nicolson1, the lipid raft hypothesis by Simons2 or Kusumi’s picket fence model3 were important and ground breaking to advance the understanding of membrane organization, but do not account well for the dynamic changes in the system.

In previous work, we showed that specialized plasma membrane invaginations called caveolae flatten out under mechanical stress to prevent rupture of the plasma membrane and to buffer the plasma membrane tension4,5. Caveolae are membrane invaginations of 50-80 nm diameter, build up mainly by the proteins caveolin-1 (caveolin-3 in case of muscle cells) and cavins, and enriched in cholesterol and sphingolipids6. It is suggested that the unfolding of caveolae releases surface area to the plasma membrane which then relaxes the membrane tension. This unfolding is independent of cellular energy (ATP) and is triggered by mechanical perturbation (here osmotic shock or cell stretch), whereas reformation of caveolae is energy dependent and depends on the actin cytoskeleton as actin perturbing drugs like cytochalasin D increased the new formation of caveolae4.

In contrast to the earlier work, where the mechanical perturbation was delivered through global means like hypo-osmotic shock or stretching of cells on PDMS, we want now to turn to local mechanical perturbations, to address the question of what happens locally with caveolae at the site of mechanical perturbation and how do the mechanical perturbations propagate in the cell membrane. This question is based on recent discussions between Pierre Sens, Pierre and caveolin-1 knock out form that Christophe Lamaze ’s lab is currently using for other studies. In addition, we will employ drugs to perturb the actin cytoskeleton (cytochalasin D, latrunculin A), motor activity (blebbistatin) or the membrane cholesterol levels (M-beta cyclodextrin) to disconnect plasma membrane and cytoskeleton.

In addition, we plan to also measure f(v) just after global membrane perturbation through hypo- osmotic shock, in order to quantify the mechanical properties of the stressed membrane state and to study the dynamics of relaxation towards the steady (homeostatic) state. This will give us insight on the relevance of caveolae to the mechanical homeostasis of the plasma membrane.

Besides the tether force measurements, we also plan to follow the fate of single caveolae using fluorescence microscopy. For this, we will use the optical tweezers set up combined with a confocal microscope that is present in the lab of P. Bassereau, and to identify caveolae in cells expressing caveolin-1-EGFP and Cavin-1-mCherry by spots where both proteins co-localize.

Given the fact that all the material and cells are currently ready and available at Institut Curie, we anticipate that a preliminary set of experiments on this project can be performed in a relatively short time. We would like to ask the LabEx to support this project and to fund the travel for Darius Köster from Bangalore, India, to Paris and his stay for six weeks, which would allow us to run first tests and to decide whether to pursue this project in form of a PhD between

Pierre Sens in UMR 168 and Christophe Lamaze in INSERM U1143 – CNRS UMR 3666.

References

  1. Singer, S. J. & Nicolson, G. L. The Fluid Mosaic Model of the Structure of Cell Membranes. Science 175, 720–731 (1972).
  2. Lingwood, D. & Simons, K. Lipid rafts as a membrane-organizing principle. Science 327, 46–50 (2010).
  3. Kusumi, A. et al. Paradigm shift of the plasma membrane concept from the two- dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. Annu. Rev. Biophys. Biomol. Struct. 34, 351–78 (2005).
  4. Sinha, B. et al. Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144, 402–13 (2011).
  5. Sens, P. & Turner, M. S. Budded membrane microdomains as tension regulators. Phys. Rev. E – Stat. Nonlinear, Soft Matter Phys. 73, 1–4 (2006).
  6. Parton, R. G. & del Pozo, M. A. Caveolae as plasma membrane sensors, protectors and organizers. Nat. Rev. Mol. Cell Biol. 14, 98–112 (2013).
  7. Brochard-Wyart, F., Borghi, N., Cuvelier, D. & Nassoy, P. Hydrodynamic narrowing of tubes extruded from cells. Proc. Natl. Acad. Sci. U. S. A. 103, 7660–3 (2006).