Influence of ezrin's actin binding properties on the formation of minimal actin cortices
von Tim Heißenberg
Datum der mündl. Prüfung:2023-11-14
Erschienen:2024-02-16
Betreuer:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Sarah Koester
Gutachter:Prof. Dr. Silvio Rizzoli
Dateien
Name:Doktorthesis.pdf
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Diese Datei ist bis 13.11.2024 gesperrt.
Zusammenfassung
Englisch
The cytoskeleton is a pivotal structure within cells, crucial for their motion and life-indispensable processes. The actin cortex is a dense actin network that is part of the cytoskeleton and linked to the plasmamembrane. ERM (ezrin, radixin and moesin) proteins are central for the connection between the actin cortex and plasmamembrane. Ezrin recruits filamentous actin (f-actin) to the plasmamembrane resulting in the formation of actin cortex structures. In vivo, ezrin is activated in a two step mechanism. First, ezrin binds with its N-terminal domain (ezrin-FERM) to L-α -phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the plasmamembrane. This induces a conformational change, that allows the C-terminal domain of ezrin to bind f-actin. A phosphorylation of threonine at position 567 stabilises the open conformation of ezrin and makes it fully active. In vitro a phosphomimetic mutation is inserted to circumvent the phosphorylation (ezrin T567D). In this project, mutations were inserted into the actin binding site of ezrin T567D, to alter its actin binding properties. The mutations R569A, I580A, I571M and K577A were selected and compared to the unmodified actin binding site of ezrin T567D. The mutations were selected on a literature review and displayed different co-sedimentation ratios with f-actin. Since ezrin is activated by binding to PtdIns(4,5)P2, it was verified that the binding affinity of this interaction was not changed upon the inserted mutations. The ezrin-actin binding strength was measured by means of atomic force microscopy measurements with the colloidal probe technique (CPT). Force-distance curves between the with the ezrin mutants funtionalised colloidal probe and an f-actin layer were recorded and the ezrin-actin binding properties were determined. The mutation R569A and I580A resulted in a stronger actin binding compared to ezrin T567D, whereas I571M and K577A decreased the binding strength. Minimal actin cortices (MACs) formed by the ezrin mutants were investigated by means of fluorescence microscopy. Hereby the architecture and dynamics of the bound f-actin were analysed. The mutation I571M and K577A were not able to form stable MACs and could only bind f-actin transiently. Ezrin with the additional R569A or I580A mutation was able to attach more f-actin to the surface with less PtdIns(4,5)P2 in the membrane compared to ezrin T567D. The adhesion strength revealed a limit, at which ezrin is not able to bind f-actin steadily. The ezrin T567D passes this limit within the observed PtdIns(4,5)P2 content regime that is comparable to physiological PtdIns(4,5)P2 concentration. These results demonstrate the importance of ezrin's weak actin binding strength, that enables the control of f-actin recruitment via increased PtdIns(4,5)P2 concentrations within the membrane.
Keywords: Ezrin; Minimal actin cortices