Fig. 5: Kapβ2W460A:W730A selectively extracts R-DPR from co-aggregates and co-condensates of FUS/R-DPR. | Communications Biology

Fig. 5: Kapβ2W460A:W730A selectively extracts R-DPR from co-aggregates and co-condensates of FUS/R-DPR.

From: NLS-binding deficient Kapβ2 reduces neurotoxicity via selective interaction with C9orf72-ALS/FTD dipeptide repeats

Fig. 5

A, B GST-TEV-FUS (5 μM) fibrillization was initiated by adding TEV protease (16 μg/ml). Fibrilization reactions were incubated with buffer, 10 μM (GR)20 (A), or 10 μM (PR)20 (B), in the presence or absence of 5 μM Kapβ2W460A:W730A. After 100 minutes of incubation, fibrillization was assessed by sedimentation assay. Pellet and supernatant fractions were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. A representative gel is shown. Quantification of the gel images in (A) and (B). The amount of FUS (C), or R-DPR (D) in the pellet fraction was determined by densitometry. Values represent means ± SEM (n = 3 independent experiments). Unpaired Student’s t-tests were used to compare different conditions. E FUS-GFP LLPS was initiated by adding 3 C protease (18 μg/ml) into MBP-FUS-GFP (3 μM) incubated with buffer (150 mM KCl, 50 mM Tris pH 7.4, 0.5% Glycerol, and 1 mM DTT), 3 μM (GR)20, or 3 μM (PR)20, in the presence or absence of 6 μM indicated Kapβ2 variant. All R-DPRs containing samples were supplemented with 100 nM TAMRA-R-DPR. Droplets were visualized after 2 hours of incubation. Scale bars, 5 μm. F Quantification of the GFP channel of the fluorescence images collected in (E) shows the integrated area of FUS droplets in each condition. Data shown are mean ± SEM. n = 29 images per condition. Unpaired Student’s t-tests were used to compare different conditions. (G) Quantification of the GFP channel of the fluorescence images collected in (E) shows the size of the droplets in each condition. Data shown are mean ± SEM. The number of droplets (n) quantified in each condition is indicated in the figure. Conditions with WT Kapβ2 were N/A because there were not enough droplets to be quantified. (H) Quantification of images collected in (E) showing the enrichment factor (ratio of mean fluorescence intensity in liquid droplets to the mean fluorescence intensity in bulk) of FUS-GFP and TAMRA-(GR)20 in each droplet. Data shown are mean ± SEM. The number of images (n) quantified in each condition is indicated in the figure. Conditions with WT Kapβ2 were N/A because there were not enough droplets to be quantified. Unpaired Student’s t-tests were used to compare different conditions; ****p ≤ 0.0001.

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