Figure 1Domain structure of the longest human Tau isoform (2N4R) used in this study: the positively charged C-terminal half of Tau contains four repeats (R1-R4) and two proline rich regions (P1, P2), and comprises the microtubule binding region of Tau. This region binds to polyanionic macromolecules such as RNA through electrostatic interactions. The N-terminal ~200 amino acids (aa) project from the microtubule surface and contain two negatively charged N-terminal inserts (N1, N2). The homogenous charge distribution (sliding window of 10 aa) along its aa sequence assigns Tau an amphiphilic character. Addition of polyA RNA, PEG (PEG8000), or both induces the de-mixing of Tau at physiological concentrations (5 μM Tau in 25 mM HEPES, 1 mM DTT, pH 7.4). Scale bars=10 μm. Confocal fluorescence microscopy shows co-condensation of polyA-Cy3 (10:1, polyA:polyA-Cy3) with Tau (5 µM Tau with 1% Tau-DyLight488). PEG8000-Dylight647 is excluded from Tau condensates. Scale bar=10 μm. Representative size distribution of molecular Tau assemblies during Tau LLPS measured by time-resolved DLS (trDLS). At 25 μM Tau in low salt (10 mM NaCl) buffer, trDLS detects Tau monomers (radii ~10 nm) and mesoscopic Tau clusters (radii ~100-200 nm). Addition of PEG, polyA, or both after ~15 min induced the formation of Tau condensates with a radius of >103 nm, at the cost of monomers and mesoscopic clusters in the solution. Data point bubble sizes correspond to DLS amplitudes, which are proportional to the intensity of scattered light of the respective particles. Data shown are the same as in Figure 2D for 045 min. Kinetics of Tau:PEG, Tau:RNA, and Tau:PEG:RNA condensate growth assessed through power law, R=a*tb, fitting to trDLS data starting at the time of LLPS inducer addition. Kinetics of cluster growth in low salt (25 mM HEPES, 10 mM NaCl) at 30 μM and 50 μM Tau using the same fitting model as in (E). trDLS of Tau:PEG condensation at increasing NaCl concentrations. Radius of Tau:PEG condensates at different NaCl concentrations. N=3 experiments, 10 last data points per experiments shown, data shown as mean±SEM. Data information: Data have been compared by one-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 Radius of Tau clusters at different NaCl concentrations. N=2-3 experiments, 3 last data points per experiments shown, data shown as mean±SEM. Radius of Tau monomers at different NaCl concentrations. N=2-3 experiments, 3 last data points per experiments shown, data shown as mean±SEM. Data information: Data have been compared by one-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 Model of Tau condensation: Tau monomers (grey circles) and clusters coacervate with RNA (pink) to form condensates that grow through droplet fusion (Oswald ripening). Molecular crowding agents, like PEG (blue), are excluded from the dense phase. Inset: excluded volume effects exerted by PEG force individual Tau molecules to interact, thereby trigger LLPS and stabilize Tau:RNA coacervates against the electrostatic shielding of molecular interactions inside the dense phase. As a result, Tau:PEG:RNA condensates can form at cytosol-like ion concentrations. R0=radius of hydration.