Extended Data Fig. 5: Lipoamide weakly increases liquidity of FUS condensates in vitro.
From: Small-molecule dissolution of stress granules by redox modulation benefits ALS models
a, NMR chemical shift deviations per residue for the FUS N-terminal PLD (residues 1 to 163) with 500 μM lipoamide compared to the drug solvent control (1% DMSO). Light gray bars indicate tyrosine residues and residues neighboring a tyrosine. b, Average 1H and 15N shifts across residues zero, one, two, three or more than three residues from a tyrosine in the presence of lipoamide. c, Top, fractions of FUS proteins condensed at indicated salt (KCl) concentrations in the presence of 300 µM lipoamide or the DMSO control (0.3% v/v). n = 16 image fields for each condition. Bottom, dilute phase concentrations (equivalent to saturation concentrations) of FUS–GFP at 150 mM KCl at different temperatures and lipoamide concentrations (errors are s.d.) d, Schematic illustrating the quantitation of condensate droplet liquidity using optical tweezers. Two droplets are brought into contact and begin to fuse: the time taken to relax to a single spherical droplet (once adjusted for the geometric mean radius as the characteristic droplet size) is a measure of the viscosity to surface tension ratio of the droplet – a proxy of liquidity. e, Droplet size-corrected relaxation times for droplet fusions with either 300 μM lipoamide (n = 93 independent fusion event) or equivalent DMSO solvent control (0.3%, n = 60). Box represents the 25th, 50th and 75th percentiles, whiskers represent 5th and 95th percentiles. p value by unpaired two-tailed t-test. Lipoamide reduces fusion time, indicating lower viscosity and/or greater surface tension. f–h, Effect of 30 μM lipoamide or lipoic acid on FUS G156E-GFP condensates ‘aging’, relative to an equivalent DMSO solvent control (0.3%). Condensates were formed under 50 mM of KCl while shaking. f, Representative images after 1 and 3 h aging, showing fiber formation in the DMSO sample in contrast to the lipoamide or lipoic acid samples. g, Representative fluorescence recovery after photobleaching (FRAP) time series of FUS condensates and fibers at corresponding time points. h, Mean ± s.d. of relative intensity of FUS–GFP FRAP in G. Aged (3 h) condensates treated with lipoamide or lipoic acid maintain large FUS-GFP mobile fraction. Both compounds delay fiber formation. i, Changes in intramolecular crosslinking due to lipoamide of FUS in in vitro low salt (80 mM KCl) condensates using the lysine-rich FUS K12 or FUS G156E. Significantly changed crosslinking sites with a change in intensity of more than two-fold and FDR ≤ 0.1; 3 independent experiments) are shown coloured in green (increased) or red (decreased). Other crosslinking sites are shown in gray. j, Dose-dependent effect of lipoamide on FUS K12, plotting absolute change in crosslink intensity relative to no lipoamide. Crosslinking sites with false discovery rate (FDR) > 0.1 are shown in blue, those with FDR ≤ 0.1 in orange (2 independent experiments). Two-fold change is indicated with a dashed red line.
