Abstract

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.

Highlights

  • Ezrin, a member of the ERM family, serves as a cross-linker between the plasma membrane and the actin cytoskeleton [1]

  • We investigated the role of ezrin, a linker between the cell membrane and actin cytoskeleton, in the relationship between cell migration and cells’ biophysical properties

  • Our results show that cells transfected with active ezrin T567D display an enhanced migration speed and directionality, decreased cortical stiffness, and increased cytoskeleton stiffness (Supplementary Table S1)

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Summary

Introduction

A member of the ERM (ezrin, radixin, moesin) family, serves as a cross-linker between the plasma membrane and the actin cytoskeleton [1]. A plethora of molecular biology studies have focused on characterizing the monomeric or oligomeric configuration of ezrin when bound to the cellular membrane or its binding rates and unbinding forces to the membrane and/or the actin cortex, especially in terms of ezrin’s activation state [2,3,4,5,6,7]. Given the broad extent of its regulatory function as well as its connection to the cell’s cytoskeleton, it can be hypothesized that ezrin may play a wider role than that of a membrane linker and influence the cellular state and function

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