Fluorescence Self-Quenching from Reporter Dyes Informs on the Structural Properties of Amyloid Clusters Formed in Vitro and in Cells.
Fluorescence Self-Quenching from Reporter Dyes Informs on the Structural Properties of Amyloid Clusters Formed in Vitro and in Cells.
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DOI：
10.1021/acs.nanolett.6b03686
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发表时间：
2017-01-11
期刊：
影响因子：
10.8
通讯作者：
Kaminski CF
中科院分区：
文献类型：
--
作者：
Chen W;Young LJ;Lu M;Zaccone A;Ströhl F;Yu N;Kaminski Schierle GS;Kaminski CF
淀粉样蛋白聚集动力学的表征和随后聚集物的结构特性对于研究许多神经退行性疾病的发病机制和发现治疗靶点至关重要。在这篇文章中，我们展示了与淀粉样蛋白共价结合的合成染料的荧光寿命影响了在体外和细胞中形成的淀粉样蛋白簇的结构特性。我们证明了这种蛋白质聚集的“寿命传感器”背后的机制是基于荧光自猝灭的，并且它提供了一个很好的动态范围来报告聚集的各个阶段，而不会显着干扰正在调查的过程。我们表明，传感器以高通量和定量的方式通知淀粉样蛋白簇的结构密度，并且在这些方面，传感器优于超分辨率成像技术。我们展示了该方法的力量和速度，例如，在监测生物自组装的治疗筛选中提供了能力。我们在阿尔茨海默病相关蛋白tau的K18蛋白片段及其体外形成的淀粉样蛋白聚集体的研究中探讨了寿命传感器的机制和优势。最后，我们通过在细胞中进行相关的荧光寿命成像显微镜和结构照明显微镜实验，证明了传感器在聚谷氨酰胺蛋白聚集体研究中的应用，这是一种用于亨廷顿病研究的模型。
The characterization of the aggregation kinetics of protein amyloids and the structural properties of the ensuing aggregates are vital in the study of the pathogenesis of many neurodegenerative diseases and the discovery of therapeutic targets. In this article, we show that the fluorescence lifetime of synthetic dyes covalently attached to amyloid proteins informs on the structural properties of amyloid clusters formed both in vitro and in cells. We demonstrate that the mechanism behind such a “lifetime sensor” of protein aggregation is based on fluorescence self-quenching and that it offers a good dynamic range to report on various stages of aggregation without significantly perturbing the process under investigation. We show that the sensor informs on the structural density of amyloid clusters in a high-throughput and quantitative manner and in these aspects the sensor outperforms super-resolution imaging techniques. We demonstrate the power and speed of the method, offering capabilities, for example, in therapeutic screenings that monitor biological self-assembly. We investigate the mechanism and advantages of the lifetime sensor in studies of the K18 protein fragment of the Alzheimer’s disease related protein tau and its amyloid aggregates formed in vitro. Finally, we demonstrate the sensor in the study of aggregates of polyglutamine protein, a model used in studies related to Huntington’s disease, by performing correlative fluorescence lifetime imaging microscopy and structured-illumination microscopy experiments in cells.