Figure 6. A disease-related mutation interferes with HspB8 activity.

(A) Location of the disease-related K141E mutation inside the αCD of HspB8. (B) Partitioning of 5 µM HspB8-K141E (1:20 mix of labeled:unlabeled) into condensates formed by 5 µM FUS_m. Scale bar is 10 µm. (C) Aging assay of 5 µM FUS_m condensates in the absence (ctrl) and presence of HspB8-WT, HspB8-K141E, or a 1:1 mix of WT and K141 mutant. Final chaperone concentrations in all reactions are 125 nM. Scale bar is 10 µm. (D) Kinetics of the aging process. Plotted are the initial slopes of the FRAP recovery curves for FUS_m condensates in the absence (ctrl) and presence of HspB8-WT (red), HspB8-K141E (blue), or a 1:1 mix of WT and K141E mutant (cyan). (E) Equal amounts of FUS_m:HspB8-K141E (left) or FUS_m:HspB8-WT (right) were crosslinked under condensate inducing low salt conditions (75 mM NaCl). Experiments were carried out in triplicates and crosslinks were only considered, if they were identified in two out of three replicates with a deltaS<0.95, a minimum Id score≥20, and an ld score≥25 in at least one replicate (filtering was done on the level of the unique crosslinking site) and an FDR<0.05. The mutated site in HspB8-K141E is shown in red. Inter-links are shown in black and intra-links are shown in violet. Mono-links are shown with a flag, loop links with a pointed triangle, and homo-dimeric links with a loop. (F) Quantitative comparison of crosslinking patterns from HspB8-K141E and HspB8-WT condensates. Crosslinking sites that were upregulated or downregulated twofold or more (log2ratio≥1 or ≤−1 and FDR≤0.05) were considered significant and are highlighted in green (i.e., relative enrichment in FUS_m:HspB8-K141E condensates) or red (i.e., relative decrease in FUS_m:HspB8-K141E condensates). All other changes in crosslinking abundances were considered insignificant and are shown in gray background. αCD, α-crystallin domain; FRAP, fluorescence recovery after photobleaching.