Quantitative Analyse der Ezrin-Aktin Wechselwirkung an Membranen
Zusammenfassung der Projektergebnisse
Ezrin, a member of the ezrin-radixin-moesin (ERM) protein family, regulates the cell membrane architecture by connecting the plasma membrane with the actin cytoskeleton. In the inactive or closed state, ezrin is conformationally autoinhibited by self-association of the N- and C-terminal domains that harbor binding sites for membrane lipids and proteins and F actin, respectively. Ezrin activation is thought to rely on a conformational change induced by binding to Lα-phosphatidylinositol-4,5bisphosphate (PI(4,5)P2) and phosphorylation of a conserved threonine residue, thus rendering the binding site for filamentous actin (F-actin) accessible. Moreover, the Ca2+regulated binding of S100P to ezrin is discussed as an alternative regulator. The contribution of the individual activation factors is still controversially discussed. It was the aim of this project to unravel the molecular processes that lead to the activation of ezrin and thus regulate plasma membrane-F-actin contacts in cells. To investigate the activation state of ezrin as a function of the different factors in vitro, supported lipid bilayers were used as a model system. These enabled us to quantitatively analyze the conformation (open/closed) and lateral organization (monomeric/oligomeric) of ezrin as well as its capability to bind F-actin. Based on the results obtained in our project, we conclude that the mode of ezrin activation strongly depends on binding to the lipid PI(4,5)P2 and phosphorylation at Thr-567 (as a surrogate for this phosphorylation we used a pseudophosphorylated mutant protein carrying an aspartate at position 567). Only if the pseudophosphorylated protein is bound to PI(4,5)P 2, a loosely packed protein arrangement is observed on the membrane surface. This arrangement provides sufficient access to the C-terminal F-actin binding site of ezrin. Force spectroscopy experiments demonstrated that the individual interaction between the F-actin binding site of ezrin and F-actin is weak. Instead of strong individual interactions, ezrin activation means that the number of ezrin molecules in the open conformation, in which the Factin binding site is accessible, increases in case of the pseudophosphorylated ezrin mutant (slightly) and in the presence of PI(4,5)P2 (significantly). This modular activation process is key to a dynamic regulation of the cell cortex. Only if multiple weak bonds are formed between the plasma membrane and the actin cortex, the cell can dynamically control cell adhesion and migration as well as regulate its morphology.
Projektbezogene Publikationen (Auswahl)
- (2011) Activation of F-actin binding capacity of ezrin: synergism of PIP2 interaction and phosphorylation. Biophys. J. 100, 1708-1717
Bosk, S., Braunger, J., Gerke, V., Steinem, C.
- (2013) Ezrin activation in vitro: Investigation of ezrin's conformation and interaction between ezrin and F-actin, PhD thesis, Göttingen
Braunger, J.
- (2013) Solid supported membranes doped with PIP2 – influence of ionic strength and pH on bilayer formation and membrane organization. Langmuir 29, 14204-14213
Braunger, J. A., Kramer, C., Morick, D., Steinem, C.
(Siehe online unter https://doi.org/10.1021/la402646k) - (2014) Phosphatidylinositol 4,5-bisphosphate alters the number of attachment sites between ezrin and actin filaments: a colloidal probe study. J. Biol. Chem. 289, 9833-9843
Braunger, J. A., Brückner, B. R., Nehls, S., Pietuch, A., Gerke, V., Mey, I., Janshoff, A., Steinem, C.
(Siehe online unter https://doi.org/10.1074/jbc.M113.530659) - (2015) Conformational changes in ezrin analyzed by Förster resonance energy transfer. PhD thesis, Münster
Shabardina, V.
- (2015) Ezrin interacts with the scaffold protein IQGAP1 and affects its cortical localization. Biochim. Biophys. Acta Mol. Cell Res. 1853, 2086-2094
Nammalwar, R.C., Heil, A., Gerke, V.
(Siehe online unter https://doi.org/10.1016/j.bbamcr.2014.12.026) - (2016) Mode of ezrin-membrane interaction as a function of PIP2 binding and pseudophosphorylation. Biophys. J. 110, 2710-2719
Shabardina, V., Kramer, C., Gerdes, B., Braunger, J., Cordes, A., Schäfer, J., Mey, I., Grill, D., Gerke, V., Steinem, C.
(Siehe online unter https://doi.org/10.1016/j.bpj.2016.05.009) - (2016) Verknüpfung zwischen Plasmamembran und Zytoskelett: Charakterisierung der Organisation von Ezrin und F-Aktin an artifiziellen Lipidmembranen. PhD thesis, Göttingen
Kramer, C.
