A Proline-Rich-Domain-Binding Single Domain Antibody Selectively Inhibits RNA-Induced Phase Separation of Tau

Phase separation mediates the formation of biomolecular condensates, which organize cellular processes such as synaptic plasticity in neurons. The neuronal protein tau undergoes phase separation under specific conditions, regulating synaptic vesicle clustering and microtubule dynamics. In vitro, tau phase separation is induced by cofactors such as polyethylene glycol (PEG) or RNA, mainly via weak multivalent electrostatic interactions. However, the molecular mechanisms governing the formation of tau phase separation, including domain specific contribution, remain unclear. In this study, we used eight single-domain antibodies (VHHs), targeting six distinct short sequences of tau, to explore the mechanisms of tau phase separation in vitro. By combining several biophysical methods, we evaluated the effect of each anti-tau VHH on tau phase separation. With PEG as an inducer, VHHs A31 and Z70 targeting the tau microtubule binding domain enhanced tau phase separation by increasing the droplet size. With RNA as an inducer, the effect of the VHHs was mixed: VHH F8-2 targeting the C-terminal domain promoted condensation, while VHH B1-1, which binds the proline-rich domain (PRD), abolished droplet formation. Nuclear magnetic resonance and surface plasmon resonance spectroscopies confirmed 1 to 1 binding of VHH B1-1 to the PRD, and competition assays with a PRD peptide restored phase separation, demonstrating mechanistic specificity. Our findings provide domain-resolved insights into the regulation of tau phase separation and introduce VHHs as selective probes to modulate biomolecular condensates in physiological and pathological contexts.