Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching.
Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching.
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通过单分子逐步光漂白监测的β-淀粉样蛋白寡聚化
DOI：
10.1016/j.ymeth.2020.06.007
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发表时间：
2021-09
期刊：
影响因子：
--
通讯作者：
Quinn SD
中科院分区：
文献类型：
--
作者：
Dresser L;Hunter P;Yendybayeva F;Hargreaves AL;Howard JAL;Evans GJO;Leake MC;Quinn SD
该方法无需外源性荧光探针即可研究淀粉样β寡聚体的化学计量比。
在体外使用单分子逐步光漂白技术。
揭示了寡聚体群体内的异质性。
检测寡聚体在活细胞中诱导的细胞内Ca2 +稳态失调。
阿尔茨海默病的一个主要特征是淀粉样β肽（Aβ）的错误折叠和聚集。虽然早期研究指出大型纤维状和斑块状聚集体是毒性最强的种类，但最近的证据表明，小的可溶性Aβ寡聚体的危害程度要高出几个数量级。因此，能够表征寡聚体化学计量比和组装的技术对于更深入地了解神经退行性变的早期阶段以及合理测试下一代寡聚体抑制剂至关重要。虽然诸如硫黄素 - T等外源性荧光探针的荧光反应已成为表征溶液中大型Aβ聚集体的常用工具，但人们普遍认为这些方法存在许多重要缺陷，包括对寡聚体种类不敏感。在此，我们整合了几种生物物理技术，以便在单分子水平上对寡聚体的形成有新的认识。我们展示了荧光染料分子的单分子逐步光漂白是一种强大的方法，可绕过许多传统限制，并提供了在体外实施该技术的分步指南。通过宽场全内反射荧光（TIRF）成像收集用HiLyte Fluor 555在N端标记的单个Aβ（1 - 42）肽的荧光发射，我们演示了如何表征每个单个固定寡聚体的肽数量，并揭示样本群体内的异质性。重要的是，使用衍射受限的光学显微镜工具很难研究Aβ寡聚体发出的荧光。为了检测寡聚体的活性，我们还展示了另一种生物物理方法的实施，该方法涉及对加载了Fura - 2 - AM的细胞进行比率成像，可量化寡聚体诱导的细胞内Ca2 +稳态失调的速率。我们预计，这里强调的综合单分子生物物理方法将进一步发展，并且原则上可扩展到在受控实验条件下对其他蛋白质聚集系统的研究。
Method enables investigation of amyloid-β oligomer stoichiometry without requiring extrinsic fluorescent probes. Uses single-molecule stepwise photobleaching in vitro. Unveils heterogeneity within populations of oligomers. Assays oligomer-induced dysregulation of intracellular Ca2+ homeostasis in living cells. A major hallmark of Alzheimer’s disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1–42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.
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DOI：
10.1002/cphc.201600750
发表时间：
2016-11-04
期刊：
Chemphyschem : a European journal of chemical physics and physical chemistry
影响因子：
--
作者：
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DOI：
10.1016/j.bbadis.2008.06.003
发表时间：
2008-09-01
影响因子：
6.2
作者：
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Scheper, Wiep
DOI：
10.1073/pnas.0904532106
发表时间：
2010-02-02
影响因子：
11.1
作者：
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通讯作者：
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DOI：
10.1016/j.mcn.2014.05.002
发表时间：
2014-07
期刊：
Molecular and cellular neurosciences
影响因子：
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作者：
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通讯作者：
Baillie GS
影响因子：
6.8
作者：
Fitzpatrick, Anthony W. P.;Saibil, Helen R.
通讯作者：
Saibil, Helen R.