Inhibiting ezrin triggers formin-mediated actin remodelling reducing cellular mechano-protection

Ezrin plays a crucial role in linking the cortical actin cytoskeleton to the plasma membrane in animal cells. Inactivated by small molecule ezrin inhibitors (EzrInh), ezrin is known to regulate cellular functions like adhesion and motility. However, whether ezrin plays any significant role in protecting cells against mechanical stresses is less explored. We use hypo-osmotic shock (HS) to challenge cells and study the mechano-protective role of ezrin. Inhibition of ezrin phosphorylation led to increased cell rupture rates on hypo-shock, with a greater impact at later time points (∼ 10 min). The time scales match with that of enhanced tension contrast at edges at the basal plane. We also observed a lowered relative retraction rate at protrusions created by hypo-shock and a less contractile mid-plane cortex – both indicate the role of a weakening of the cortex by EzrInh in compromising membrane integrity on hypo-shock. Concomitantly, EzrInh also increased stress fibres (SFs), traction forces and cell spreading clearly indicative of additional cellular pre-stress. Utilizing formin inhibitor (SMIFH2), we showed that formin-mediated actin polymerization was critical for the SF response to EzrInh while it also prevented the subsequent EzrInh-treatment from enhancing cell rupture rates. Together EzrInh reduced mechano-protection in cells by tilting of balance of actin’s organization from the cortex to SF enhancing formin-mediated SF assembly.