Dynamische Strukturen am Zellcortex: Aktivierbarkeit und Akkumulation von Ezrin in Abhängigkeit von PIP2

Abstract

Ezrin, a member of the ezrin/radixin/moesin (ERM) protein family, acts as a membrane-cytoskeleton linker, thereby participating in a variety of cellular events. The N-terminal region of ezrin specifically binds to the lipid L-á-phosphatidylinositol-4,5-bisphosphate (PIP2) while the C-terminal region harbors a binding site for filamentous actin (F-actin). In monomeric ezrin, the respective terminal domains are tightly associated leaving ezrin in an inactive, so-called dormant state, in which the F-actin binding site is masked. Several means of regulation of ezrin s activation have been described including binding to PIP2 and phosphorylation of threonine 567 in the C-terminal domain of ezrin. To date, it is not known to what extent these two molecular factors contribute to a full activation of ezrin. The aim of this study was to design an in vitro assay that permits a detailed investigation into the processes leading to ezrin activation. In this respect, we were especially interested in identifying the relative contributions and possible synergy of PIP2 binding and phosphorylation. For that purpose, the wild type of ezrin and two mutants mimicking the unphosphorylated and the phosphorylated state, respectively, were used. Solid-supported lipid bilayers (SLB) doped with either DOGS-NTA-Ni or PIP2 allowed for specific immobilization of the proteins. Quartz crystal microbalance (QCM) experiments showed a high binding affinity in the range of 10 nM of all three proteins and very high membrane coverage. From variations of the lipid composition of the lipid membranes we inferred that ezrin binds to several PIP2 molecules. Fluorescence microscopy experiments and a detailed analysis of ezrin binding on the membrane revealed that attractive protein-protein interactions lead to protein clustering on the membrane surface. Experiments employing a C-terminally truncated version of ezrin indicate that this attractive interaction involves C-terminal domains. Fluorescence microscopy on ezrin-decorated SLBs showed that, dependent on the mode of binding and phosphorylation state, ezrin is capable of binding F-actin. A clear synergism between phosphorylation and PIP2 as binding partner of ezrin was observed suggesting a conformational switch from dormant to active state. As this interaction was only observed on planar surfaces, but not in solution, we conclude that effective F-actin binding is accomplished by cooperative binding of ezrin clusters. Based on the experimental results, a detailed activation model of ezrin is presented that provides new insights into the regulation of the membrane-cytoskeleton connection.