Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching.
Amyloid oligomerization
Calcium sensing
Fluorescence
Photobleaching
Single-molecule TIRF
Journal
Methods (San Diego, Calif.)
ISSN: 1095-9130
Titre abrégé: Methods
Pays: United States
ID NLM: 9426302
Informations de publication
Date de publication:
09 2021
09 2021
Historique:
received:
10
03
2020
revised:
02
06
2020
accepted:
10
06
2020
pubmed:
17
6
2020
medline:
5
1
2022
entrez:
17
6
2020
Statut:
ppublish
Résumé
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
Identifiants
pubmed: 32544592
pii: S1046-2023(20)30084-0
doi: 10.1016/j.ymeth.2020.06.007
pmc: PMC8336786
pii:
doi:
Substances chimiques
Amyloid beta-Peptides
0
Fluorescent Dyes
0
Peptide Fragments
0
Protein Aggregates
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
80-95Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/R001235/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/P000746/1
Pays : United Kingdom
Informations de copyright
Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.
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