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.

行为像液滴的非膜结合细胞器广泛分布在真核细胞中。在几种神经退行性疾病中，它们的失调似乎是关键的一步。在这里，我们报道tau蛋白，阿尔茨海默氏症神经原纤维缠结的主要成分，可以形成液滴，因此具有必要的生物物理特性，可以在细胞中经历液-液相分离（LLPS）。与诱导LLPS的因素一致，tau是一种内在无序的蛋白质，与RNA复合形成液滴。独特的是，活细胞中tau结合的RNA池是tRNA。tau的这种相态在蛋白质和核酸成分之间保持大约1：1的电荷平衡，因此在不同的RNA：tau质量比下可以达到最大，取决于所涉及的生物聚合物成分。该特征是复杂凝聚的特征。我们进一步表明，LLPS过程是直接和敏感地通过盐浓度和温度进行调节的，这意味着所涉及的蛋白质和核酸成分之间的静电相互作用以及熵的净变化都对LLPS过程进行了调节。尽管在复杂的凝聚相中蛋白质浓度很高，但tau在局部自由翻滚，并能够通过液滴内部扩散。实际上，处于浓缩相状态的tau与稀释溶液状态下的tau相比，并未显示出局部蛋白质堆积，局部构象和局部蛋白质动力学的任何立即变化。相比之下，与肝素络合诱导的易于聚集的tau种群伴随着局部tau构象的巨大变化和不可逆的聚集。然而，根据硫黄素-T测定，在液滴状态下的长时间停留最终导致可检测到的β-折叠结构的出现。这些发现表明，液滴状态可以温育tau，并使蛋白质倾向于不溶性原纤维的形成。