Understanding Membrane-actin Coupling by Ezrin in Cellular Mechano-protection

Biswas, Ananya (2025) Understanding Membrane-actin Coupling by Ezrin in Cellular Mechano-protection. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Ananya Biswas (19RS099)) 19RS099.pdf - Submitted Version Restricted to Repository staff only Download (11MB)

Abstract

Cellular mechano-protection has majorly been attributed to be mediated by caveoale (Sinha et al., 2011; Sotodosos-Alonso et al., 2023a) but, other players such as cytoskeleton (Pegoraro et al., 2017) and various mechanosensitive channels like TRPV4, Piezo, (M. Zhang et al., 2022) TREK (Herrera-Pérez & Lamas, 2023)or signalling molecules like RhoA (Acharya et al., 2018)etc. are also likely to promote mechano-protection by either functioning as transducers or affectors of the external mechanical cues.. This study highlights the importance and functional significance of one such less highlighted molecule, ezrin, in mechano-protection. Primarily studied under the context of membrane-actin linker (Gautreau et al., 1999; Osawa et al., 2009) , we show that ezrin plays an important role in mechano-protection in a caveolaeindependent manner. In order to assess ezrin’s role when cells are mechanically challenged, we have developed a Calcein AM-mediated hypo-osmotic shock-based rupture assay which enables us to identify single cell ruptures. Hypo-osmotic shock is known to threaten membrane integrity (Bremer & Krämer, 2019) thus making it a useful tool to study mechano-protective measures. By quantifying and comparing the rupture characteristics on perturbing ezrin- both by using small molecule inhibitors of ezrin (Rizvi et al., 2009) and knockdown using siRNA (Osawa et al., 2009), we show that ezrin confers mechano-protection in a time-dependent manner (utmost dependency observed between 5-10 min of administering hypo-osmotic shock). In order to establish the caveolae-independent nature of ezrin’s mechano-protective function, we have further looked into the state of actin structures (cortex and stress fibres) in response to ezrin perturbation. Using TIRF microscopy and assaying actin-based retraction potential as a parameter, we demonstrate that ezrin acts as a membrane buffer during hypoosmotic shock by dissipating the hydrostatic pressure acting on the membrane to the underlying actomyosin network thus reducing the strain on cell membrane. Further investigating into the pathways involved in conferring ezrin-mediated mechano-protection, we show that by maintaining rigid links between the membrane and actin cortex, ezrin maintains a balance between the cortical actin and basal stress fibres leading to mechanical stability. Using cholesterol depletion, our study also demonstrates that although ezrin acts in tandem with caveoale to provide mechano-protection, in absence of caveolae, ezrin’s mechano-protective function is not hindered. We have further corroborated our findings in three different cell lines, namely, HEK293 (mimicking a physiologically relevant condition), wild type HeLa (mimicking a pathological condition) and HeLa Cav1 KO (mimicking a system devoid of caveolae). In both HEK293 and HeLa Cav1 KO, we observe an increased dependency on ezrin for mechano-protection when subjected to hypo-osmotic shock.

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