Ezrin mediated membrane cytoskeleton coupling : studies in biomimetic systems – EzrinBiomim

The aim of this project is to understand at the molecular level the link between membrane structures and the actin cytoskeleton. Ezrin, member of the ezrin/radixin/moesin (ERM) family, is a membrane cytoskeleton linker involved in the cellular morphogenesis and motility. ERM proteins are localized in membrane protrusions (like pseudopodia and microvilli) and contribute to the anchoring of a cell in its environment. Ezrin switches dynamically between the cytosol where it is under a dormant state and the membrane, once activated (open conformation). The activation process requires its interaction with a specific lipid, phosphatidylinositol-4,5-bisphophate (PIP2) and/or phosphorylations at specific sites. The membrane-associated ezrin can then interact with the actin cytoskeleton. The N-terminal domain of ezrin contains the PIP2 binding site and the C-terminal domain of the protein contains an actin binding site. In addition, upon opening of the molecule, another cryptic actin binding site is unmasked. Most of these observations are derived from in cellulo studies and the molecular mechanisms underlying these biological effects need now to be biochemically detailed. On the other hand, studies aimed at elucidating the structure-function relation of ERM proteins in vitro with membranes have began to emerge only recently with the pioneering studies of Takeda et al. on Giant Unilamellar Vesicles (GUVs) (J. Mol. Biol. 2006), of Herrig et al. (Biochemistry 2006) on supported lipid bilayers and of our group on both large unilamellar vesicles (Blin et al Biophys J 2008) and giant unilamellar vesicles (Biophys J, revised manuscript send on feb 19th 2008). In addition, the structure of the full-length moesin was only recently unravelled (Tesmer et al., J Mol Biol 2007). Our goal is therefore to study in vitro the interplay between PIP2, ezrin and actin in biomimetic systems in order to get a better knowledge of the underlying important molecular processes. These constituents will be added step by step as purified constituents. The originality of this project resides in three principal aspects 1) the anchoring of a key protein between a cytoskeletal protein (actin) and the plasma membrane via a specific lipid (PIP2), In particular, by using full length ezrin, phosphomimetic constructs and individual ezrin domains, we will investigate the respective roles of the membrane binding domain and of the phosphomimetic form, as both PIP2 binding and phosphorylation are known to be important in vivo. In ezrin as well as in other ERM proteins, the binding site located in the C-terminal domain has been identified and is acknowledged by the scientific community. The presence of the second cryptic site could be at the origin of a weak nucleation activity of ezrin on actin. Also, already polymerized F-actin filaments may be stabilized by ezrin, due to ezrin/actin interactions along the sides of the actin filament. 2) the investigation of the interactions between specific lipids and proteins by means of biochemical and biophysical methods on different types of biomimetic objects, which will bring complementary information. Thus, beads coated with ezrin, and lipidic bilayers incorporating PIP2 and forming multilamellar vesicles, large and giant unilamellar vesicles as well as supported lipid bilayers, will all be employed to answer specific questions. In addition, a pyrene assay in solution will also be used to decorticate the effects of ezrin on actin polymerization, elongation and depolymerization. 3) the understanding of the coupling between membrane curvature, lateral heterogeneities in the membrane composition (if any), ezrin activation and actin polymerisation. To answer these questions we will use biomimetic objects of different curvatures, such as planar membranes (supported lipid bilayers and GUVs) and curved membranes (LUVs or various diameters and supported bilayers on microfabricated glass substrate). The length of unfolded ezrin (about 25 nm) could be very important in the linker role of the molecule and in a possible effect of membrane curvature. Reversibly, Forces will be applied to the membrane of GUVs to induce strong localized deformation, in order to investigate the coupling between curvature and actin polymerization In short, we want to understand how actin polymerization near a membrane structure can generate enough forces to modulate its organisation. We think that ezrin is a key player in this process. This proposal is largely interdisciplinary and requires expertise in biochemistry, biophysics, physics and theoretical modelling, brought by the three partners