Fig. 9: Characteristics of TDP-43 C-terminal fragments in nematodes.

a Confocal fluorescence and bright field images of nematodes expressing GFP-C220, GFP-C233, and GFP monomers (Mo) on 4 or 8 days after synchronization by bleaching (Day 1 and 5 of adulthood, respectively). The two images of the same sample are from the same nematode. The low magnification images of the nematodes displayed here are shown in Supplementary Fig. 9. Bars = 20 μm. b Western blot of nematode lysates in 4 or 8 days after synchronization by bleaching (Day 1 and 5, respectively) using an anti-GFP and anti-α-tubulin antibody. Arrows show the monomer-corresponding bands for GFP-C220, GFP-C233, and GFP monomers. Oligomers indicate high molecular weight species. The asterisk shows a non-specific staining band by anti-GFP antibody. c Fluorescence recovery after photobleaching (FRAP) analysis of GFP monomers as a control and condensates of C220 or C233 tagged with GFP in live nematodes on 4 or 8 days after synchronization by bleaching (Days 1 and 5 of adulthood, respectively) Left: Typical fluorescence images of condensates in nematodes before and after the photobleaching. Arrowheads indicate photobleached positions. Bars = 0.5 μm. Pseudocolor indicates the fluorescence intensities. Right: Plots of the fluorescence recovery curve after the photobleaching of GFP monomers, GFP-C220, and -C233 in live nematodes (mean ± 95% CI; n = 7 independent experiments). The Y-axis represents the mobile fraction (normalized relative fluorescence intensity; nRFI) of C220 and C233 in the condensates and GFP monomers. Student’s t-test for comparison among GFP monomers, C220, and C233 at the same day: ***p < 0.001. Statistical comparison just after the photobleaching (t = 0 s) is represented in Supplementary Fig. 10. The individual data points are available in Supplemental Data 1. d, e Fluorescence correlation spectroscopy (FCS) analysis of GFP-C220, -C233, and GFP monomers (Mo) in nematode lysates on 4 or 8 days after synchronization by bleaching (Day 1 and 5). Counts per particle (CPP) in d and diffusion time in e. Bars indicate mean and 95% CI (n = 3 independent experiments). p-values above the bars and lines: one-way ANOVA with Tukey’s test compared to GFP monomers as control and comparison between the lines (###p < 0.001 and ***p < 0.001), respectively. NS with a cyan color indicates p > 0.05. f The number of bends per 30 seconds of nematodes expressing GFP-C220, -C233, and GFP monomers on 4 or 8 days after synchronization by bleaching (Day 1 and 5 of adulthood, respectively) in M9 buffer. Bars indicate the mean and 95% CI of 36 nematodes. p-values above the bars and lines: one-way ANOVA with Tukey’s test compared to GFP monomers as control and comparison between the lines (###p < 0.001 and ***p < 0.001), respectively. NS with cyan color indicates p > 0.05. g Survival curve of synchronized nematode expressing GFP-C220, -C233, or GFP monomers (magenta, green, and dark gray, respectively) (n = 75 nematodes). The x-axis shows the day after the synchronization by bleaching (i.e., its day 4 corresponds to day 1 of adulthood). p-values were obtained from Gehan’s generalized Wilcoxon test: ***p < 0.001. NS indicates p > 0.05. h Survival curve of synchronized nematode expressing GFP-C220, -C233, or GFP monomers (magenta, green, and dark gray, respectively) when 270 μM Juglone were treated (n = 200 nematodes). The x-axis shows time on Juglone-containing plates. p-values were obtained from Gehan’s generalized Wilcoxon test: ***p < 0.001.