RNA stores tau reversibly in complex coacervates

Abstract

Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils.

Author summary

Tau is a common neuronal protein that, under circumstances and conditions not well understood to date, self-assembles into intracellular aggregates in several neurodegenerative diseases including Alzheimer disease. These aggregates are formed of fibrous polymers. The mechanism by which this critical transition from a soluble protein to insoluble fibrous material occurs is unknown. We have discovered a novel state in which many tau molecules become compacted into a protein-rich droplet while maintaining their solubility and native-like protein conformations. Chemists refer to this dense liquid droplet state as a complex coacervate phase, and it is held together by the opposite charges of their constituents, ions, and water. In the case of the tau protein, the oppositely charged constituent is RNA. Indeed, we found that in human neuronal cell culture, tau selectively binds to a category of RNA known as tRNA. Interestingly, tau and RNA favorably condense to a complex coacervate phase when the charges between them are matched and at elevated temperatures, such that tau-RNA droplets could be observed at physiologically viable protein concentrations simply by increasing the temperature from room to physiological temperatures. When the tau-RNA–dense droplets are incubated together over time, tau transitions to a conformation similar to that found in pathological fibers. Our experiments therefore demonstrate physicochemical properties of tau that may predispose it to undergo changes associated with neurodegenerative disease.