The Distance between N and C Termini of Tau and of FTDP-17 Mutants Is Modulated by Microtubule Interactions in Living Cells
The Distance between N and C Termini of Tau and of FTDP-17 Mutants Is Modulated by Microtubule Interactions in Living Cells
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DOI：
10.3389/fnmol.2017.00210
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发表时间：
2017-06-30
影响因子：
4.8
通讯作者：
Cattaneo, Antonino
中科院分区：
文献类型：
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作者：
Di Primio, Cristina;Quercioli, Valentina;Cattaneo, Antonino
微管（MT）相关蛋白Tau是一种天然未折叠的蛋白质，涉及许多神经退行性疾病，统称为tau蛋白病，聚集在神经元缠结（NFT）中。从MT结合分子到易于聚集的Tau种类的转化如何发生，以及Tau蛋白病相关突变是否以及如何影响其在细胞中的行为，这是一个悬而未决的问题。为了解决这些问题，我们开发了一种基于全长Tau蛋白的基因编码的FRET传感器，以在活细胞中不同条件下真实的时间监测Tau构象变化。通过研究FRET信号，我们发现从MT上分离的可溶性Tau分子显示出未折叠的结构。相反，我们观察到与MT结合的Tau单体产生的FRET信号增加，表明与MT的缔合诱导Tau蛋白折叠，减少其N和C末端之间的距离。我们利用FRET传感器来研究FTDP-17突变和磷酸化位点突变对活细胞中Tau折叠和流动性的影响。我们证明了FTDP-17 Tau突变减弱了Tau与细胞MT的相互作用，将平衡向可溶性池转移，而相反，磷酸化位点突变将Tau的平衡向MT结合状态和更封闭的构象转移。
The microtubule (MT)-associated protein Tau is a natively unfolded protein, involved in a number of neurodegenerative disorders, collectively called tauopathies, aggregating in neurofibrillary tangles (NFT). It is an open question how the conversion from a MT bound molecule to an aggregation-prone Tau species occurs and, also, if and how tauopathy-related mutations affect its behavior in the cell. To address these points, we exploited a genetically encoded FRET sensor based on the full length Tau protein, to monitor in real time Tau conformational changes in different conditions in live cells. By studying the FRET signal we found that soluble Tau molecules, detached from MTs, display an unfolded structure. On the contrary, we observed an increased FRET signal generated by Tau monomers bound to MT, indicating that the association with MTs induced a folding of Tau protein, decreasing the distance between its N and C termini. We exploited the FRET sensor to investigate the impact of FTDP-17 mutations and of phosphorylation-site mutations on Tau folding and mobility in live cells. We demonstrated that the FTDP-17 Tau mutations weaken the interaction of Tau with cellular MTs, shifting the equilibrium towards the soluble pool while, conversely, phosphorylation site mutations shift the equilibrium of Tau towards the MT-bound state and a more closed conformation.