Fig. 4: In the presence of R-DPRs, Kapβ2 activity against PY-NLS mutant FUS is abolished. | Communications Biology

Fig. 4: In the presence of R-DPRs, Kapβ2 activity against PY-NLS mutant FUS is abolished.

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

Fig. 4

A, B GST-TEV-FUSP525L (5 μM) fibrillization was initiated by adding TEV protease (16 μg/ml). Fibrilization reaction was incubated with 10 μM (GR)20 (A), or 10 μM (PR)20 (B), in the presence or absence of 5 μM WT Kapβ2. Aggregation was assessed by turbidity at 395 nm. Solid lines are normalized mean. Dotted lines of corresponding colors represent ± SEM (n = 3 independent experiments). C GST-TEV-FUSP525L (5 μM) fibrillization was initiated by adding TEV protease (16 μg/ml). Fibrilization reaction was incubated with buffer, 10 μM (GR)20, or 10 μM (PR)20, in the presence or absence of 5 μM WT Kapβ2. After 100 min 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 sedimentation gels in (C). The amount of FUS P525L (D), and R-DPR (E) in the pellet fraction was determined by densitometry. Values represent means ± SEM (n = 4 independent experiments). Unpaired Student’s t-tests were used to compare different conditions; ****p ≤ 0.0001. F FUS-GFP (3 μM) liquid droplets were formed in LLPS buffer (50 mM KCl, 50 mM Tris pH 7.4, 0.5% Glycerol, and 1 mM DTT) in the presence of 12 μM (GR)20. At 2 hours, 3 µM WT Kapβ2 was added, and the sample was then imaged continuously for 135 seconds. Representative images were shown. 100 nM TAMRA-(GR)20 was added for visualization of (GR)20. Scale bars, 5 μm. See also Supplementary Movie 1-3. G Quantification of images collected in (F) showing the changes in the enrichment factor (ratio of mean fluorescence intensity in liquid droplet to the mean fluorescence intensity in bulk) of FUS-GFP and TAMRA-(GR)20 after addition of Kapβ2. Values represent means ± SEM. 31 droplets were quantified. Change of anisotropy when 100 nM FUS-GFP (H), 100 nM TAMRA-(GR)20 (I), or 100 nM TAMRA-(PR)20 (J) binds to increasing concentrations of WT Kapβ2 or Kapβ2W460A:W730A (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 = 3-5 independent experiments). Binding curves were fitted by Prism. Solid line represents the fit and the fitted Kd was reported on the figure.

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