Fig 2: TDP-43 and other disease-linked RNA-binding proteins form nuclear inclusions in GA-Nes mice. a Neuronal nuclear inclusions of TDP-43 (arrow) are detected by immunofluorescence in hippocampus and frontotemporal cortex in GA-Nes end-stage mice, but not Nes littermate controls. No TDP-43 inclusions are detected in affected or asymptomatic PR-Nes mice, or a 68-week Nes control, even in cells with abundant poly-PR (green) inclusions. Scale bar = 10 µm. b Schematic drawing of the distribution of the rare nuclear TDP-43 aggregates in GA-Nes mice. Aggregates were found in the lateral frontal cortex (at least 100 µm from the midline) and hippocampal CA regions marked with red x. c Regional overview of TDP-43 nuclear aggregates (arrows) in the frontal cortex and hippocampus CA3 region of end-stage GA-Nes mice, scale bar = 100 µm. d Nuclear TDP-43 aggregates (arrows) in end-stage GA-Nes mice colocalize with hnRNP H, and weakly with hnRNP A1, but not with Fus, Sfpq or Alyref. No nuclear aggregates of any marker are seen in age-matched Nes mice. Scale bar = 10 µm

Fig 3: Molecular Dissection of FUS Toxicity(A) Scheme illustrating different domain deletions tested.(B) Deletion RGG2 and NLS domains rescues FUS toxicity on both eclosion rates and the appearance of the immature phenotype, whereas deletion of QGSY only rescues eclosion rates. Kruskal-Wallis, Dunn’s multiple comparison.****p < 0.0001.

Fig 4: RGG Domains Are Required for Efficient Binding of the FUS C-Terminal Domain to Phase-Separated QGSY Hydrogels and Liquid Droplets(A) Scheme illustrating the C-terminal deletion constructs tested for QGSY droplet and hydrogel binding.(B) The RGG domains are the main domains affecting binding of GFP fusion constructs to mCherry-QGSY hydrogels or QGSY droplets. Asterisk indicates higher exposure setting to show that there is still some residual binding of single ?RGG mutants to the hydrogel.(C) Partitioning of the C-terminal deletion constructs in the QGSY droplets and hydrogels in vitro correlates with the toxicity of the corresponding full-length deletion construct in fly. The red fit takes into account all values; the blue fit considers ?RGG1 as an outlier.

Fig 5: N- and C-Terminal LCDs Are Sufficient to Replicate Full-Length Toxicity(A) Comparison between caz and FUS. Both proteins share a similar domain architecture, protein disorder, and amino acid composition, but caz lacks the QGSY domain. Amino acid composition was calculated for each domain.(B) Humanizing caz shows that addition of the FUS QGSY domain to the benign caz NLS mutant makes the chimeric protein toxic again.(C) Drosophilizing FUS shows that deleting QGSY generates a protein with similar properties to the benign caz NLS mutant.(D and E) Schematic representation of the constructs containing different regions of FUS that were used to create transgenic flies (D) and the effect on toxicity (E). Only when fused together do the N- and C-terminal LCDs cause toxicity.For (B)–(E), Kruskal-Wallis, Dunn’s multiple comparison. **p < 0.01, ****p < 0.0001.