Figure 3.

CR point mutants of TDP-43 have a gradient of in vivo condensation properties

(A) Positions of the 5 point mutations in TDP-43 variants.

(B) Mutations modify condensation on a gradient from perturbing (red/orange) to maintaining (green) or promoting (blue) the condensation capacity.

(C) Maximum z-projection images of HEK293 Flp-In cell lines expressing the indicated dox-inducible GFP-TDP-43 variants.

(D) Quantification of foci counts in each segmented nucleus of confocal z stacks. Mean ± 95% CI is shown for n (cells): 316del346 = 30, A326P = 32, M337P = 35, Q331K = 32, G294A = 32, G335A = 30, WT = 30.

(E) FRAP experiments on GFP-TDP-43 CR mutant cell lines. The fluorescence recovery curve was obtained after bleaching a spot of predefined size in the nucleoplasm. Mean ± 95% CI is shown for n (cells): 316del346 = 37, A326P = 36, M337P = 36, Q331K = 36, G294A = 36, G335A = 36, WT = 48.

(F) Rate constant of fluorescence recovery. Mean ± 95% CI is shown for the same number of cells as in (E).

Significance for (D) and (F) was tested with Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Reported p values are for the individual comparisons (^∗∗p adj. < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001).

(G) As in (E) for FRAP analysis of GFP TDP-43 mobility in nucleoplasm versus nuclear regions centered on foci on 316del346, A326P, G335A, and WT GFP-TDP-43 cell lines. Mean ± 95% CI is shown for n (cells): 316del346 nucleoplasm = 8, 316del346 foci = 12, A326P nucleoplasm = 8, A326P foci = 12, G335A nucleoplasm = 8, G335A foci = 12, WT nucleoplasm = 8, WT foci = 10, 1 focus per independent cell.