Fig. 4: Hyper-phosphomimetic TDP-43 mutant is not recruited in stress granules but only under acute oxidative stress.

a Left panel: Representative images of HeLa cells expressing HA-tagged phosphomimetic mutants and G3BP1-GFP with or without arsenite (300 µM, 1 h). Right panel: Automatic quantification of the recruitment of HA-tagged TDP-43 mutants in stress granules. Each dot represents the mean slope of the SG/Cytoplasm enrichment of HA versus GFP fluorescence measured over all stress granules detected in a single well (96-well plate). When the slope value is 0, there is no HA enrichment in SGs. Error bars indicate SEM and the asterisks indicate statistical significance with *p < 0.05, **p < 0.01, ****p < 0.0001, ns non-significant, as measured by Student’s t-test from n  =  4 wells. Scale bar: 50 μm. b Left panel: Representative images of HeLa cells expressing the indicated TDP-43 mutants fused to microtubule-binding domain to analyze the recruitment of mRNA along microtubules in the presence or absence of arsenite (300 µM, 1 h). Right panel: Relative recruitment of mRNA along microtubules for indicated proteins used as baits. Each dot represents the mean value over all stress granules detected in a single well (96-well plate). Note the absence of mRNA along microtubules with the hyper-phosphomimetic mutant (18E) after arsenite treatment. Error bars indicate SEM and the asterisks indicate statistical significance with **p < 0.01, ***p < 0.001, ****p < 0.0001, ns non-significant, as measured by Student’s t-test from n  =  4 wells. Scale bar: 50 μm. mRNA was detected by in situ hybridization with a fluorescent poly-dT probe. c Left panel: Representative images of HeLa cells expressing indicated HA-tagged TDP-43 mutants and wild-type TDP-43 in the presence of arsenite. Scale bar: 50 μm. Center panel: Measurement of the recruitment of HA-tagged TDP-43 mutant and wild-type TDP-43 in stress granules. Right panel: Measurement of the SG/Cytoplasm enrichment of anti-HA or GFP fluorescence in HeLa cells were co-transfected with GFP-tagged wild-type TDP-43 and HA-tagged wild-type or phosphomimetic mutants, as indicated. Wild-type TDP-43-GFP is no longer recruited in stress granules in arsenite-treated HeLa cells expressing the hyper-phosphomimetic mutant (18E). 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. d Schematic view of the differential recruitment of TDP-43 in stress granules regulated by the phosphorylation status of TDP-43 C-terminus domain and the level of oxidative stress.