Mimicking two posttranslational modifications associated with oxidative stress affords phase separation of vimentin

Biomolecular condensates are membraneless compartments critical for the functional organization of cellular macromolecules in essential processes such as cell division, gene transcription or stress responses. We previously reported that vimentin filaments remodel into phase separated biomolecular condensates upon oxidative stress. This process requires vimentin single cysteine, C328, suggesting the involvement of oxidative modifications of this residue. Here, we aimed to generate vimentin condensates by inserting mutations mimicking posttranslational modifications associated with oxidative stress. In vimentin deficient cells, a cysteine oxidation mimetic mutant, vimentin C328D, formed only elongated particles or short filaments that evolved towards droplets upon serum deprivation or treatment with the oxidant diamide. Among vimentin posttranslational modifications rapidly responding to these stimuli, glycosylation confers filament stability whereas phosphorylation promotes disassembly. We observed that the O-deglycosylation inhibitor thiamet G, and the kinase inhibitors staurosporine and H-89, attenuated diamide-elicited vimentin C328D droplet formation, suggesting a potential glycosylation/phosphorylation interplay in this effect. Indeed, introducing phosphomimetic residues at certain single vimentin glycosylation and/or phosphorylation sites induced the formation of droplets, only if combined with the C328D mutation. In particular, the vimentin S49D,C328D mutant formed condensates that were reversibly dispersed by dilution through hypotonic shock. Therefore, mimicking C328 oxidation and S49 phosphorylation was sufficient to elicit vimentin phase separation. In vitro, purified vimentin S49D,C328D polymerized into a mixture of aberrant filaments and aggregates, which, in the presence of crowders, evolved towards paracrystals or clusters of beaded assemblies depending on pH. These findings highlight the role of C328 perturbations in the formation of biomolecular condensates and suggest a modulatory role of glycosylation/phosphorylation, thus shedding light on the processes regulating vimentin phase separation.