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. 2020 Jan 27;130(3):1139–1155. doi: 10.1172/JCI130988

Figure 4. sTDP43 accumulates within the cytoplasm due to a putative NES.

Figure 4

(A) Rodent primary mixed cortical neurons were transfected with mApple and EGFP-tagged TDP43 isoforms, and then imaged by fluorescence microscopy. (B) Amino acid sequence of the sTDP43 tail showing the putative NES identified using NetNES 1.1. Light blue, polar; purple, positively charged; green, hydrophobic residues. (C) sTDP43-EGFP was significantly more cytoplasmic compared with flTDP43-EGFP, while mutation of the putative NES (mNES) restores nuclear localization. N/C, nuclear/cytoplasmic. EGFP n = 481, flTDP43-EGFP n = 385, sTDP43-EGFP n = 456, sTDP43(mNES)-EGFP n = 490, stratified among 3 replicates. ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test. (D) Rodent primary mixed cortical neurons were transfected with 1 of 3 constructs: EGFP alone, EGFP fused to the sTDP43 C-terminus, or EGFP fused to the sTDP43 C-terminal tail harboring a mutated NES (mNES). (E) The sTDP43 C-terminus mislocalizes EGFP to the cytoplasm, and mislocalization depends on the putative NES. Shuttle-RFP, a construct with a strong NES, serves as a positive control for a cytoplasmic protein. EGFP n = 2490, Shuttle-RFP n = 2073, EGFP-tail n = 1956, EGFP-tail(mNES) n = 2482, combined from 3 replicates. ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test. Scale bars: 20 μm (A and D).