Tau liquid-liquid phase separation is modulated by the Ca²⁺ -switched chaperone activity of the S100B protein

Aggregation of the microtubule-associated protein tau is implicated in several neurodegenerative tauopathies including Alzheimer's disease (AD). Recent studies evidenced tau liquid-liquid phase separation (LLPS) into droplets as an early event in tau pathogenesis with the potential to enhance aggregation. Tauopathies like AD are accompanied by sustained neuroinflammation and the release of alarmins at early stages of inflammatory responses encompass protective functions. The Ca²⁺ -binding S100B protein is an alarmin augmented in AD that was recently implicated as a proteostasis regulator acting as a chaperone-type protein, inhibiting aggregation and toxicity through interactions of amyloidogenic clients with a regulatory surface exposed upon Ca²⁺ -binding. Here we expand the regulatory functions of S100B over protein condensation phenomena by reporting its Ca²⁺ -dependent activity as a modulator of tau LLPS induced by crowding agents (PEG) and metal ions (Zn²⁺ ). We observe that apo S100B has a negligible effect on PEG-induced tau demixing but that Ca²⁺ -bound S100B prevents demixing, resulting in a shift of the phase diagram boundary to higher crowding concentrations. Also, while incubation with apo S100B does not compromise tau LLPS, addition of Ca²⁺ results in a sharp decrease in turbidity, indicating that interactions with S100B-Ca²⁺ promote transition of tau to the mixed phase. Further, electrophoretic analysis and FLIM-FRET studies revealed that S100B incorporates into tau liquid droplets, suggesting an important stabilizing and chaperoning role contributing to minimize toxic tau aggregates. Resorting to Alexa488-labeled tau we observed that S100B-Ca²⁺ reduces the formation of tau fluorescent droplets, without compromising liquid-like behavior and droplet fusion events. The Zn²⁺ -binding properties of S100B also contribute to regulate Zn²⁺ -promoted tau LLPS as droplets are decreased by Zn²⁺ buffering by S100B, in addition to the Ca²⁺ -triggered interactions with tau. Altogether this work uncovers the versatility of S100B as a proteostasis regulator acting on protein condensation phenomena of relevance across the neurodegeneration continuum.