Narrow equilibrium window for complex coacervation of tau and RNA under cellular conditions

Abstract

The conditions that lead to the liquid-liquid phase separation (LLPS) of the tau protein, a microtubule associated protein whose pathological aggregation has been implicated in neurodegenerative disorders, are not well understood. Establishing a phase diagram that delineates the boundaries of phase co-existence is key to understanding its LLPS. Using a combination of EPR, turbidity measurements, and microscopy, we show that tau and RNA form complex coacervates with lower critical solution temperature (LCST) behavior. The coacervates are reversible, and the biopolymers can be driven to the supernatant phase or coacervate phase by varying the experimental conditions (temperature, salt concentration, tau:RNA charge ratio, total polymer concentration and osmotic stress). Furthermore, the coacervates can be driven to a fibrillar state through the addition of heparin. The equilibrium phase diagram of the tau/RNA complex coacervate system can be described by a Flory-Huggins model, augmented by an approximate Voorn Overbeek electrostatic term (FH-VO), after fitting the experimental data to an empirical Flory interaction parameter divided into an entropic and enthalpic term. However, a more advanced model in which tau and RNA are treated as discrete bead-spring chains with a temperature-dependent excluded volume interaction and electrostatic interactions between charged residues, investigated through field theoretic simulations (FTS), provided direct and unique insight into the thermodynamic driving forces of tau/RNA complexation. FTS corroborated the experimental finding that the complex coacervation of tau and RNA is has an entropy-driven contribution, with a transition temperature around the physiological temperature of 37 {degrees}C and salt concentrations around 100-150 mM. Together, experiment and simulation show that LLPS of tau can occur under physiological cellular conditions, but has a narrow equilibrium window over experimentally tunable parameters including temperature, salt and tau concentrations. Guided by our phase diagram, we show that tau can be driven towards LLPS under live cell coculturing conditions with rationally chosen experimental parameters.