Abstract：
Tau is an intrinsically disordered protein that binds and stabilizes axonal microtubules (MTs) in neurons of the central nervous system. The binding of Tau to MTs is mediated by its repeat domain and flanking proline-rich domains. The positively charged (basic) C-terminal half of Tau also mediates the assembly Tau into fibrillar aggregates in Alzheimer\'s disease (AD) and tauopathy brains. In recent years, another assembly form of Tau has been identified: Tau can undergo liquid-liquid phase separation (LLPS), which leads to its condensation into liquid-dense phases, either by complex coacervation with polyanions like heparin or RNA or through \"self-coacervation\" at high Tau concentrations. Condensation of Tau in the absence of polyanions can be enhanced by the presence of molecular crowding agents in a dilute Tau solution. In vitro experiments using recombinant purified Tau are helpful to study the physicochemical determinants of Tau LLPS, which can then be extrapolated into the cellular context. Tau condensation is a new aspect of Tau biology that may play a role for the initiation of Tau aggregation, but also for its physiological function(s), for example, the binding to microtubules. Here we describe how to study the condensation of Tau in vitro using light microscopy, including fluorescence recovery after photobleaching (FRAP), to assess the shape and molecular diffusion in the condensates, a proxy for the degree of condensate percolation. We also describe turbidity measurements of condensate-containing solutions to assess the overall amount of LLPS and time-resolved dynamic light scattering (trDLS) to study the formation and size of Tau condensates.
摘要：
Tau是一种内在无序的蛋白质，可结合并稳定中枢神经系统神经元中的轴突微管（MT）。Tau与MT的结合由其重复结构域和侧翼富含脯氨酸的结构域介导。Tau的带正电（碱性）C末端的一半也将组装Tau介导为阿尔茨海默氏病（AD）和tau病大脑中的纤维状聚集体。近年来,已经确定了Tau的另一种组装形式：Tau可以进行液-液相分离（LLPS），导致其冷凝成液体致密相，通过与肝素或RNA等聚阴离子的复合凝聚或在高Tau浓度下通过“自凝聚”。在稀释的Tau溶液中存在分子拥挤剂可以增强不存在聚阴离子的Tau的缩合。使用重组纯化Tau的体外实验有助于研究TauLLPS的物理化学决定因素，然后可以外推到细胞环境中。Tau凝聚是Tau生物学的一个新方面，可能对Tau聚集的启动起作用，但也因为它的生理功能，例如,与微管的结合。在这里，我们描述如何使用光学显微镜研究体外Tau的凝结，包括光漂白后的荧光恢复（FRAP），评估冷凝物的形状和分子扩散，凝析油渗滤程度的代表。我们还描述了含冷凝物的溶液的浊度测量，以评估LLPS的总量和时间分辨动态光散射（trDLS），以研究Tau冷凝物的形成和大小。
