Tau is a microtubule-associated protein that is regulated by post-translational modifications. The most studied of these modifications is phosphorylation, which affects Tau’s aggregation and loss- and gain-of-functions, including the interaction with microtubules, in Alzheimer’s disease and primary tauopathies. However, little is known about how Tau’s phosphorylation state affects its dynamics and organisation at the single-molecule level. Here, using quantitative single-molecule localisation microscopy, we examined how mimicking or abrogating phosphorylation at 14 disease-associated serine and threonine residues through mutagenesis influences the behaviour of Tau in live Neuro-2a cells. We observed that both pseudohyperphosphorylated Tau (TauE14) and phosphorylation-deficient Tau (TauA14) exhibit a heterogeneous mobility pattern near the plasma membrane. Notably, we found that the mobility of TauE14 molecules was higher than wild-type Tau molecules, while TauA14 molecules displayed lower mobility. Moreover, TauA14 was organised in a filament-like structure resembling cytoskeletal filaments, within which TauA14 exhibited spatial and kinetic heterogeneity. Our study provides a direct visualisation of how the phosphorylation state of Tau affects its spatial and temporal organisation, presumably reflecting the phosphorylation-dependent changes in the interactions between Tau and its partners. We suggest that alterations in Tau dynamics resulting from aberrant changes in phosphorylation could be a critical step in its pathological dysregulation.

中文翻译：

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Tau 蛋白的单分子成像揭示了磷酸化如何影响其在活细胞中的运动和限制

Tau 是一种微管相关蛋白，受翻译后修饰调节。这些修饰中研究最多的是磷酸化，它会影响 Tau 的聚集以及功能的丧失和获得，包括在阿尔茨海默病和原发性 tau 病中与微管的相互作用。然而，人们对 Tau 的磷酸化状态如何影响其在单分子水平上的动力学和组织知之甚少。在这里，我们使用定量单分子定位显微镜，研究了通过诱变模拟或消除 14 个与疾病相关的丝氨酸和苏氨酸残基的磷酸化如何影响活 Neuro-2a 细胞中 Tau 的行为。我们观察到假超磷酸化 Tau (TauE14) 和磷酸化缺陷 Tau (TauA14) 在质膜附近表现出异质迁移模式。值得注意的是，我们发现 TauE14 分子的迁移率高于野生型 Tau 分子，而 TauA14 分子的迁移率较低。此外，TauA14 组织成类似于细胞骨架丝的丝状结构，其中 TauA14 表现出空间和动力学异质性。我们的研究提供了 Tau 磷酸化状态如何影响其空间和时间组织的直接可视化，大概反映了 Tau 与其伙伴之间相互作用的磷酸化依赖性变化。我们认为，磷酸化异常变化导致的 Tau 动力学变化可能是其病理失调的关键一步。