Figure 5. RRM unfolding drives FUS aggregation and is rescued by HspB8.
(A) Aging process of molecular condensates containing either FUSm or the FUSm∆RRM (∆AA285–371) variant in the presence or absence of HspB8 monitored by fluorescence microscopy over time. Scale bar is 10 µm. (B) Inter-link abundances from reconstituted FUSm:HspB condensates. Plotted are the relative enrichment (droplet vs. non-droplet) for each unique crosslinking site (y-axis) sorted according to the known domain structure within FUSm and HspB8 (x-axis). Shown are only high confidence crosslinking sites (see Materials and methods for details) from five biologically independent sets of experiments (n=5; circles in different shades of gray). Crosslinking sites that were consistently upregulated or downregulated twofold or more (log2ratio≥1 or ≤−1 and FDR≤0.05) in at least two out of five biological replicate sets of experiments and in addition contained no opposing regulation in any replicate set were considered significant and are highlighted with a green (enriched in droplets) or red background rectangle (decreased in droplets). All other changes in crosslinking abundances were considered insignificant and are shown on gray background. The significance threshold of twofold enrichment is indicated as dashed red line. (C) Fluorescence microscopy images of FUSm and FUSm∆RRM variant during and after incubation under heat shock conditions. Samples were heated to 55°C for 10 min followed by a 5 min cool-down step to 25°C. Scale bar is 10 µm. (D) FUSm:HspB8 condensates were crosslinked in the presence (black) or absence (white) of a customized RNA oligonucleotide previously shown to bind to FUS (Maharana et al., 2018) and analyzed by LC-MS/MS (n=3; FDR≤0.05). RRM, RNA recognition motif.
Figure 5—figure supplement 1. RRM unfolding drives FUS aging and is rescued by HspB8.
