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. 2022 Aug 30;25(9):1134–1148. doi: 10.1038/s41593-022-01140-3

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© The Author(s) 2022, corrected publication 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — ai, Schematic illustration showing how FRET sensor detects aggregation. aii, AF488-α-Syn and AF594-α-Syn monomers are applied to cells, and the FRET signal is detected. bi, Representative bright-field (BF) FRET images after 72-hour incubation with oligomers. bii, Application of 500 nM WT oligomeric α-Syn exhibits detectable FRET, which increases over time (n = 3 independent experiments). ci, Representative FRET images after 72-hour incubation with monomers. cii, Application of 500 nM monomeric α-Syn exhibits low FRET signal initially, followed by an increase in FRET over time (n = 3 independent experiments). di,dii, A53T monomer exhibits the highest intracellular accumulation of α-Syn and the highest intracellular FRET intensity over time (n = 3 or 4 independent experiments). ei–eiii, FRET efficiency was calculated and binned into histograms that were fit to two Gaussian distributions. After 3 hours, only a low-FRET-efficiency population (centered at E = 0.24) was present. After 3 days, a second higher-FRET-efficiency population appeared (E = 0.48), and the fraction of this increased over time. f, Fraction of the high-FRET-efficiency population (out of total FRET events) increases over time for the WT and all mutants. Fitting error is shown in Extended Data Fig. 2c (n = 3 or 4 independent experiments). gi, Single-molecule confocal microscopy under conditions of fast flow used to analyze cell lysates. gii, 2D contour plots of approximate oligomer size and FRET efficiency after application of the monomers and oligomers. Both the number of events and the size of the oligomers increase over time in all cases. giii, Number and type of oligomeric events in cell lysates from the monomer/oligomer-treated cells. Data are represented as data ± s.d., as fraction of coincident events (n = 2 independent samples). Fitting error is shown in Extended Data Fig. 3a. hi, Photobleaching step analysis for A53T-oligomer-treated cells. hii, Step-fit example of a single A53T oligomer (24 hours) intensity trace (intensity is plotted as analogue-to-digital units (ADU)). hiii, Each step indicates photobleaching of a single fluorophore, from which oligomer size can be estimated. Note: Data are represented as data ± s.e.m. (box) unless otherwise mentioned. Detailed statistical information is shown in Supplementary Table 1. See also Extended Data Figs. 1–4. a.u., arbitrary units.

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