Figure 9.

Non-toxic ataxin-1 mutant S776A shows increased toxicity and decreased NB exchange dynamics similar to ataxin-1[85Q] level following arsenite stress. Neuro-2a cells were transfected with GFP, GFP-ataxin-1[85Q], or GFP-ataxin-1[85Q]S776A as indicated. At 24 h post-transfection, cells were left untreated or treated with arsenite (0.3 mM, 1 h). (A) Cell death percentage for the transfected cell population was assessed by staining with the SYTOX Red dead cell stain followed by flow cytometry analysis. (B,C) Cells were fixed and stained with DAPI before CLSM imaging. Representative images are shown from 3 independent experiments. (C) Average sizes of ataxin-1 NBs corresponding to the conditions as per (B) were measured using CellProfiler. Results represent the mean ± SEM (Significance values calculated by ANOVA, n > 70, ****p < 0.0001, n.s. = not significant). (D–G). cClls were incubated in an imaging chamber equilibrated with 5% CO₂ at 37 °C prior to FRAP using CLSM. (D) Representative images are shown from 3 independent experiments for quantitative assessments of GFP-ataxin-1[85Q] or S776A exchange dynamics. A small ROI (indicated by white open arrowhead for dynamic NB or white solid arrowhead for non-dynamic NB; S776A ROI with multiple small NBs was indicated also by the white circle) was photobleached and the fluorescence recovery was subsequently monitored as post-bleach at 5 s intervals for 250 s. Scale bars = 10 μm. (E) Plot of the percentage recovery of fluorescence from experiments as shown in (D). Each symbol represents fluorescence measured at the indicated time. (D–E) Pooled data from FRAP experiments measuring recovery of fluorescence for GFP-ataxin-1[85Q] or GFP-ataxin-1[85Q] S776A over time. Each symbol represents a single data point. The results are also represented as the mean ± SEM for (F) fluorescence maximum recovery percentage in the bleached area (where 100% represents full recovery) and (F) initial rates (average percentage recovery of fluorescence in the first 15 s) (Fn%/s). Significance were values calculated by ANOVA, n > 7, ^***p < 0.0001, ^****p < 0.001, n.s. = not significant.