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. 2020 Jan 31;10:1557. doi: 10.1038/s41598-020-57994-9

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© The Author(s) 2020

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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Fluorescence Fluctuation Spectroscopy (FFS) analysis of ataxin-1 NB fusion reveals liquid droplet-like behavior. Neuro-2a cells were transfected to express GFP-ataxin-1[85Q] for live cell imaging. (A) A time series acquisition (1000 frames) of ataxin-1 NBs enables the fluctuation in GFP-ataxin-1[85Q] fluorescence intensity to be acquired in each pixel of an image. (B,C) Estimate of coordinated GFP-ataxin-1[85Q] mobility in each pixel location. (B) Calculation of the ratio of the first and second moment (variance/mean) of the fluorescence fluctuation recorded in each pixel, directly corresponding to the image presented in (C). (C) The spatial map of a low versus high ratio of the variance to the mean demonstrates coordinated GFP-ataxin-1[85Q] mobility is at the NB boundary. (D) Time series acquisition (1000 frames) of two ataxin-1 NBs under control (Ctrl) conditions undergoing fusion, segmented into 10 s intervals (80 frames): the top row presents the intensity maps of GFP-ataxin-1[85Q] localization during each time segment and the bottom row presents the maps of GFP-ataxin-1[85Q] coordinated movement during each time segment. (E) Time series acquisition (1000 frames) of two ataxin-1 NBs not undergoing fusion in arsenite-treated cells, segmented into 10 s intervals (80 frames): the top row presents intensity maps of GFP-ataxin-1[85Q] localization during each time segment and the bottom row presents maps of GFP-ataxin-1[85Q] coordinated movement during each time segment. (F,G) A comparison of the degree of coordinated GFP-ataxin-1[85Q] movement (variance/mean) within (F) and across the axis (G) of GFP-ataxin-1[85Q] NBs undergoing fusion in Ctrl cells versus NBs not undergoing fusion in arsenite-treated cells. All scale bars in (C–E) = 2 μm. Images presented in (D–G) are representative of n = 3 cells under each condition, with average data presented in histograms in (F) and (G). Results in (G) represent mean ± SEM (n = 3 cells).