Ezrin, a member of the ERM (ezrin/radixin/moesin) family of proteins, serves as a crosslinker between the plasma membrane and the actin cytoskeleton. By doing so, it provides structural links to strengthen the connection between the cell cortex and the plasma membrane, acting also as a signal transducer in multiple pathways during migration, proliferation, and endocytosis. In this study, we investigated the role of ezrin phosphorylation and its intracellular localization on cell motility, cytoskeleton organization, and cell stiffness, using fluorescence live-cell imaging, image quantification, and atomic force microscopy (AFM). Our results show that cells expressing constitutively active ezrin T567D (phosphomimetic) migrate faster and in a more directional manner, especially when ezrin accumulates at the cell rear. Similarly, image quantification results reveal that transfection with ezrin T567D alters the cell’s gross morphology and decreases cortical stiffness. In contrast, constitutively inactive ezrin T567A accumulates around the nucleus, and although it does not impair cell migration, it leads to a significant buildup of actin fibers, a decrease in nuclear volume, and an increase in cytoskeletal stiffness. Finally, cell transfection with the dominant negative ezrin FERM domain induces significant morphological and nuclear changes and affects actin, microtubules, and the intermediate filament vimentin, resulting in cytoskeletal fibers that are longer, thicker, and more aligned. Collectively, our results suggest that ezrin’s phosphorylation state and its intracellular localization plays a pivotal role in cell migration, modulating also biophysical properties, such as membrane–cortex linkage, cytoskeletal and nuclear organization, and the mechanical properties of cells.
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