Intracellular pH modulates vimentin remodeling in response to oxidants

Abstract

Vimentin plays key roles in cell mechanosensing, cytoskeletal crosstalk and stress responses, and is finely tuned by posttranslational modifications. The vimentin single cysteine residue, C328, is a hotspot for modification and essential for filament remodeling by oxidants and electrophiles. With a pKa near physiological pH, C328 reactivity could be sensitive to cellular pH fluctuations. Here, we show that C328 modifications and vimentin reorganization by various reactive agents are modulated in response to pH variations. Lowering intracellular pH prevents, whereas intracellular alkalinization potentiates vimentin network disruption by oxidative and electrophilic species, including diamide, hydrogen peroxide and hydroxynonenal. The protective effect associated with low pH is selective for vimentin since it does not preclude oxidant-elicited disruption of actin or tubulin structures. Vimentin C328A and C328H mutants are resistant to disruption under all pH conditions, which highlights the importance of the thiol group at this position for the sensitizing effect of alkaline pH. Chemogenetic and optogenetic modulation of cellular pH allow spatiotemporal tuning of vimentin susceptibility to oxidants, suggesting the potential role of pH in the regulation of vimentin organization at precise locations. Alkalinization and generation of reactive oxygen species cooperate at the cell front during migration. We show that vimentin disassembly at cell edges of migrating fibroblasts, and lamellipodia formation, are affected by pH changes and the presence of C328. We propose that vimentin C328 could behave as a coincidental pH and redox responsive element, contributing to the precise regulation of vimentin assembly by the concert of these factors, illustrating the pH dependence of cysteine-mediated redox signaling.