Fig. 2: Hypo- and hyper phosphomimetic mutants significantly demix with wild-type TDP-43.
From: TDP-43 nuclear retention is antagonized by hypo-phosphorylation of its C-terminus in the cytoplasm
a Presentation of the automatic detection scheme allowing to measure fluorescence intensities in spots along the microtubules network in HeLa cells co-expressing wild-type and mutant TDP-43 fused to a microtubule binding-domain (MBD) by using the Harmony software. In each “microtubule” spot, the intensity of RFP and GFP was measured as well as in the surrounding cytoplasm. Scale bar: 50 μm. Zoom scale bar: 20 μm. b Left panel: Scatter plot of the relative GFP and RFP fluorescence intensity (spots versus cytoplasm) detected in all HeLa cells present in a single well under indicated conditions. The R-squared (R2) value indicates to which extent the scatter plot distribution can be fitted using linear regression (R² = 1: perfect mixing between the two proteins). Right panel: images of Hela cells showing that TDP-43 is well mixed with itself (R² = 0.96) but not with G3BP-1 (R² = 0.47). Scale bar: 50 μm. c Representative images of HeLa cell co-expressing indicated proteins used to measure their mixing along the microtubule network. Scale bar: 50 μm. Zoom scale bar: 30 μm. Lower panel: Mixing score (R² values) measured in HeLa cells expressing wild-type TDP-43 and the indicated phosphomimetic mutants (see Fig. 1a). Error bars indicate SEM and the asterisks indicate statistical significance with ***p < 0.001, ****p < 0.0001, ns. non-significant, as measured by ANOVA test from n = 4 wells (each dot represents one well). d Images of HeLa cells expressing indicated HA-tagged protein and immune-stained with an anti-phospho TDP-43 antibody (Ser409/410). Note the co-localization of the hyper-phosphomimetic TDP-43 mutant and the anti-phospho TDP-43 in the nucleoplasm and some nuclear granules. Scale bars: 20 μm. Lower panel: Relative increase in cellular anti-phospho TDP-43 (Ser409/410) fluorescence intensity with respect to anti-HA intensity in HeLa cells expressing indicated HA-tagged TDP-43 phosphomimetic mutant. Note the non-recognition of the 5E mutant by the anti-phospho TDP-43 since Ser409/410 were not mutated into Glu residues in this mutant. Error bars indicate SEM and the asterisks indicate statistical significance with ****p < 0.0001, ns non-significant, as measured by ANOVA test from n = 4 wells (each dot represents one well).
