Fig. 1

SUMOylation dynamically regulates nuclear TDP-43 in a stress-responsive manner. A Schematic of immunoprecipitation assays to detect TDP-43 SUMOylation. B Representative SUMOylation Assay western blot in HEK293T cells detecting TDP-43 SUMOylation specifically in response to 1 h sodium arsenite (250 µM) stress. * = SUMOylated TDP-43, ** = PolySUMOylated TDP-43. C Schematic of TDP-43-GFP Nuclear Localization Sequence (NLS) variants and representative florescent microscopy analysis of their subcellular localization. (Scale bar = 10 µm). Yellow amino acids represent those critical for PY-NLS function. Teal arginine residue was mutated from the native HNRNPA1. D Representative GFP-trap SUMOylation assay in HEK293T cells detecting loss of SUMOylation in response to TDP-43 mislocalization in response to 1 h sodium arsenite stress (250 µM). (N = 3) RM One-Way ANOVA with Fisher’s LSD test. Data presented as mean ± SEM relative to stressed TDP-43-GFP (WT) condition, * p < 0.05, ** p < 0.005, *** p < 0.0005. Grey bar = Unstressed, Cyan bar = Stressed. E Representative image and quantification of relative proximity ligation signal between TDP-43 and SUMO2/3 in murine primary cortical neuron cultures (7 DIV) in response to 1 h sodium arsenite (250 µM) treatment. Scale bar = 20 μm. (N = 4 per condition) Unpaired T-test data presented as mean ± SEM, *** p < 0.0005 F Representative SUMOylation Assay in HEK293T cells of TDP-43 SUMOylation dynamics during stress (250 µM sodium arsenite) and recovery demonstrating a dependency on the ubiquitin proteosome for clearance of polySUMOylated TDP-43 by treatment with proteasome inhibitor MG132 (2 µM). (N = 3) RM One-Way ANOVA with Fisher’s LSD test. Data presented as mean ± SEM relative to 1 h stressed condition, * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001. Grey Bar = Unstressed, Cyan Bar = Stressed, Yellow Bar = Post stress recovery, Red Bar = MG132 inhibitor added. G Schematic and representative GFP-Trap SUMOylation assay in HEK293T cells screening for potential E3 ligases regulating TDP-43 SUMOylation in response to 1 h sodium arsenite stress (250 µM). (N = 3) RM One-Way ANOVA with Fisher’s LSD test. Data presented as mean ± SEM relative to stressed control without sgRNA, *** p < 0.0005, **** p < 0.0001. Light Grey Bar = Control or insignificant change from stressed condition, Dark Grey Bar = Positive control, Cyan Bar = Significantly different than stressed condition