Fig. 1: Poly(PR) and poly(GR) enhance FUS aggregation and phase separation.
A Schematic of the fibrillization and imaging assay. At 0 h, GST-TEV-FUS was incubated with 10 µM DPRs (i.e., (GA)20, (GR)20, or (PR)20) in the presence of TEV protease. Turbidity measurement at 395 nm was used to assess fibrillization. At the endpoint, 20 nM of TAMRA-(GR)20 or TAMRA-(PR)20 were added for visualization and image quantification purpose. Images were taken in both the DIC channel and the fluorescence channel. B GST-TEV-FUS (5 μM) was incubated with buffer, 10 μM of (GA)20, (GR)20, or (PR)20, in the absence or presence of TEV protease (16 μg/ml). Aggregation was assessed by turbidity measured at 395 nm. Solid lines are normalized mean. Dotted lines of corresponding colors represent ± SEM (n = 3 independent experiments). C Representative images of FUS aggregates formed in the presence or absence of the indicated R-DPR at T = 0 and T = 3 h of aggregation. Samples were supplemented with 20 nM of TAMRA-(GR)20 or TAMRA-(PR)20 before imaging for visualization and quantification. Scale bars, 5 μm. D Quantification of the images collected in (C) showing the integrated area of FUS aggregates. Data shown are mean ± SEM. N = 10 images per condition. An unpaired Student’s t-tests were used to compare different conditions, ****p ≤ 0.0001. E GST-TEV-FUS (5 μM) was incubated with different concentrations (1 μM, 5 μM, and 10 μM) of (GR)8 or (PR)8 in the absence or presence of TEV (16 μg/ml). Turbidity at 395 nm was used to assess aggregation. Solid lines are normalized mean. Data shown are mean ± SEM (n = 3 independent experiments). F Representative images of FUS droplets acquired using DIC and fluorescence microscopy. GST-TEV-FUS (10 μM) was incubated with buffer or 10 μM (GR)20 or (PR)20 in the absence of TEV protease for 3 h. Samples were supplemented with 20 nM of TAMRA-(GR)20 or (TAMRA)-(PR)20 for quantification of the fluorescence channel. Scale bars, 5 μm. G Quantification of the fluorescence images collected in (F) showing the distribution of FUS droplet size. Number of droplets quantified in each condition (n) is indicated in the figure. The bar represents the average droplet size ± SEM. An unpaired Student’s t-tests were used to compare different conditions, ****p ≤ 0.0001. H Quantification of the fluorescence images collected in (F) showing the integrated area of the FUS droplets in each condition. Data shown are mean ± SEM. N = 10 images per condition. An unpaired Student’s t-tests were used to compare different conditions; *p ≤ 0.05, **p ≤ 0.01. I Change of TAMRA anisotropy when 100 nM TAMRA-(GR)20 or TAMRA-(PR)20 binds to increasing concentrations of GST-TEV-FUS (0 μM to 7 μM) in assembly buffer (50 mM Tris-HCl pH 8, 20 mM Trehalose, 1 mM DTT, and 20 mM glutathione). Values represent means ± SEM (n = 4 (TAMRA-(GR)20), and 6 (TAMRA-(PR)20) independent experiments). Binding curves were fitted by Prism. The solid line represents the fit, and the fitted Kd was reported in the figure.
