Intracellular pH modulates vimentin remodeling in response to oxidants

Abstract

The intermediate filament protein vimentin plays key roles in the integration of cytoskeletal functions with impact on essential cellular processes including migration, mitosis and autophagy. Moreover, vimentin is involved in pathological processes such as cancer, fibrosis and interaction with pathogens. The vimentin network is finely tuned by posttranslational modifications, among which, those affecting its single cysteine residue (C328) play a critical role not only in vimentin function, but also in its interplay with actin. Interestingly, C328 exhibits a low pKa, which favors the presence of the thiol group in the thiolate form, and therefore its reactivity, at physiological pH. Therefore, C328 reactivity and modifications could be modulated by pH fluctuations in the physiological range. Here we show that indeed, vimentin cysteine oxidation and alkylation, and the subsequent vimentin remodeling, can be modulated as a function of pH, in vitro and in cells. Lowering intracellular pH by several means renders vimentin unresponsive to disruption by oxidants, whereas provoking an intracellular alkalinization exerts a sensitizing effect. The protective effect of low pH appears selective for vimentin since it does not preclude oxidant-elicited disruption of actin or tubulin structures. Importantly, a C328A vimentin mutant is resistant to disruption by oxidants under all pH conditions, highlighting the role of the thiol group at this position in the pH-dependent modulation of vimentin susceptibility to oxidants. Taken together, these results put forward intracellular pH as a key factor modulating redox-dependent vimentin remodeling.

Formation of vimentin biomolecular condensate-like structures under oxidative stress

“…Most dots displayed an area smaller than 0.1 µm 2 , and many of them were compatible with the size of a few ULF, indicating that filaments were dissociated into small segments. Although we do not know the precise structure of vimentin dots, we have previously reported that vimentin monomers appear to be intact in diamide-treated cells (26), thus supporting that the reorganization of filament into dots does not require protein cleavage. Interestingly, the effect of short term diamide treatment was reversible.…”

“…Diamide is an electrophilic compound that elicits the formation of disulfide bonds (18,42,43). This is actually the main modification of vimentin observed upon treatment with diamide in vitro (18,26). This modification is readily reversed under reducing conditions.…”

“…Vimentin possesses a single, conserved cysteine residue (C328) that is a key target for oxidative or electrophilic modifications that impact vimentin remodeling into morphologically distinct patterns (18, 20, 21, 24). In particular, certain electrophiles elicit the retraction of vimentin filaments towards the nucleus, whereas others can induce bundling or filament dissociation into dots (21, 25, 26). Given the interactions of vimentin with numerous cellular proteins and organelles, its remodeling likely affects the subcellular distribution of cellular components, as it has been evidenced during aggresome formation, or upon heat shock elicited filament retraction, which can provoke the concentration of associated proteins such as chaperones (8, 20, 27, 28).…”

“…The fast remodeling of vimentin in response to oxidative stress or to certain electrophiles constitutes an excellent example of the dynamicity and versatility of the vimentin network (18, 37, 38). Particularly intriguing is the capacity of vimentin filaments to disassemble into dots or droplet-like small particles in response to certain oxidants (20, 21, 26). Here, we have addressed the hypothesis that these structures could behave as condensates.…”

Type III intermediate filaments in redox interplay: key role of the conserved cysteine residue

“…Therefore, intracellular pH variations could affect the proportion of the thiolate, more reactive form of C328, and its susceptibility to modification. In fact, it has been recently reported that in several cell lines acidification of the intracellular pH provides selective protection against vimentin modification by oxidants, while its alkalinization favors oxidative disruption of this particular type of IFs in a manner dependent on the presence of C328 [ 76 ].…”