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Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching

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 mag...

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Autores principales: Dresser, Lara, Hunter, Patrick, Yendybayeva, Fatima, Hargreaves, Alex L., Howard, Jamieson A.L., Evans, Gareth J.O., Leake, Mark C., Quinn, Steven D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Academic Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336786/
https://www.ncbi.nlm.nih.gov/pubmed/32544592
http://dx.doi.org/10.1016/j.ymeth.2020.06.007
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author Dresser, Lara
Hunter, Patrick
Yendybayeva, Fatima
Hargreaves, Alex L.
Howard, Jamieson A.L.
Evans, Gareth J.O.
Leake, Mark C.
Quinn, Steven D.
author_facet Dresser, Lara
Hunter, Patrick
Yendybayeva, Fatima
Hargreaves, Alex L.
Howard, Jamieson A.L.
Evans, Gareth J.O.
Leake, Mark C.
Quinn, Steven D.
author_sort Dresser, Lara
collection PubMed
description 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 Ca(2+) 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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spelling pubmed-83367862021-09-01 Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching Dresser, Lara Hunter, Patrick Yendybayeva, Fatima Hargreaves, Alex L. Howard, Jamieson A.L. Evans, Gareth J.O. Leake, Mark C. Quinn, Steven D. Methods Article 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 Ca(2+) 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. Academic Press 2021-09 /pmc/articles/PMC8336786/ /pubmed/32544592 http://dx.doi.org/10.1016/j.ymeth.2020.06.007 Text en © 2020 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Dresser, Lara
Hunter, Patrick
Yendybayeva, Fatima
Hargreaves, Alex L.
Howard, Jamieson A.L.
Evans, Gareth J.O.
Leake, Mark C.
Quinn, Steven D.
Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching
title Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching
title_full Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching
title_fullStr Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching
title_full_unstemmed Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching
title_short Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching
title_sort amyloid-β oligomerization monitored by single-molecule stepwise photobleaching
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336786/
https://www.ncbi.nlm.nih.gov/pubmed/32544592
http://dx.doi.org/10.1016/j.ymeth.2020.06.007
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