Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 26 Qiuyue Road, Shanghai 201210, China.
Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, University of the Chinese Academy of Sciences, 19 A Yuquan Road, Shijingshan District, Beijing 100049, China.
Int J Mol Sci. 2018 Dec 26;20(1):90. doi: 10.3390/ijms20010090.
The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer's disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated the conformations of two different isoforms of Tau, Tau40 and k19, in mammalian cells. Combined with immunofluorescence imaging and western blot analyses, we found that the isotope-enriched Tau, which was delivered into the cultured mammalian cells by electroporation, is partially colocalized with MT and actin filaments (F-actin). We acquired the NMR spectrum of Tau in human embryonic kidney 293 (HEK-293T) cells, and compared it with the NMR spectra of Tau added with MT, F-actin, and a variety of crowding agents, respectively. We found that the NMR spectrum of Tau in complex with MT best recapitulates the in-cell NMR spectrum of Tau, suggesting that Tau predominantly binds to MT at its MT-binding repeats in HEK-293T cells. Moreover, we found that disease-associated phosphorylation of Tau was immediately eliminated once phosphorylated Tau was delivered into HEK-293T cells, implying a potential cellular protection mechanism under stressful conditions. Collectively, the results of our study reveal that Tau utilizes its MT-binding repeats to bind MT in mammalian cells and highlight the potential of using in-cell NMR to study protein structures at the residue level in mammalian cells.
天然无序蛋白 Tau 在神经元中含量丰富，有助于微管 (MT) 和肌动蛋白网络的调节，而其细胞内异常聚集与阿尔茨海默病密切相关。在这里，我们使用细胞内核磁共振 (NMR) 光谱法研究了 Tau 的两种不同异构体 Tau40 和 k19 在哺乳动物细胞中的构象。结合免疫荧光成像和 Western blot 分析，我们发现通过电穿孔递送到培养的哺乳动物细胞中的同位素标记 Tau 部分与 MT 和肌动蛋白丝 (F-actin) 共定位。我们获得了人胚肾 293 (HEK-293T) 细胞中 Tau 的 NMR 谱，并将其与添加 MT、F-actin 和各种拥挤剂的 Tau 的 NMR 谱进行了比较。我们发现与 MT 复合的 Tau 的 NMR 谱最好地再现了 Tau 的细胞内 NMR 谱，表明 Tau 主要在 HEK-293T 细胞中通过其 MT 结合重复与 MT 结合。此外，我们发现一旦磷酸化 Tau 被递送到 HEK-293T 细胞中，与疾病相关的 Tau 磷酸化就立即被消除，这暗示了在应激条件下细胞的潜在保护机制。总之，我们的研究结果表明 Tau 在哺乳动物细胞中利用其 MT 结合重复来结合 MT，并强调了使用细胞内 NMR 在哺乳动物细胞中研究残基水平的蛋白质结构的潜力。
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