Fig. 3
KPNB1-mediated reduction of TDP-CTF and TDP-43 aggregates depends on its FG-Nup interaction domain. a Schematic domain structure of KPNB1. KPNB1 is comprised of 19 HEAT repeats (H1–19). RanGTP and importin-α interact with H1–8 and H8–19, respectively. FG-Nups bind to two regions of KPNB1, H5–7 and H14–16. b Lysates from HEK293T cells expressing GFP, GFP-KPNB1 full-length (FL), N-terminal fragments of KPNB1 (H1–9…H1) or the C-terminal fragment of KPNB1 (H10–19) were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. KPNB1 H1–8, the smallest fully active KPNB1 fragment in reducing TDP-43 aggregation, strongly interacts with FG-Nups and Ran, and weakly with importin-α1 in comparison with full-length KPNB1. c (Top) Schematic domain structure of KPNB1 H1–8mNIS harboring four missense mutations (I178A, F217A, Y255A, I263R) in the nucleoporin-interacting site (NIS). (Bottom) Lysates from HEK293T cells expressing GFP, GFP-KPNB1 H1–8WT or H1–8mNIS were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. KPNB1 H1–8mNIS shows strongly reduced interaction with FG-Nups. d Immunofluorescence (IF) of SH-SY5Y cells co-expressing mCherry or mCherry-TDP-CTF with GFP-KPNB1 H1–8WT or H1–8mNIS. KPNB1 H1–8mNIS does not reduce cytoplasmic TDP-CTF aggregates. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. e Western blot analysis and quantification of insoluble mCherry-TDP-CTF in SH-SY5Y cells expressing GFP, GFP-KPNB1 H1–8WT or H1–8mNIS. KPNB1 H1–8WT strongly decreased insoluble TDP-CTF levels, whereas KPNB1 H1–8mNIS did not. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (**p < 0.01, ***p < 0.001, n = 4). f IF of HEK293T cells co-expressing GFP-(GR)100 with an empty plasmid (ctrl), FLAG-tagged KPNB1 H1–8WT or H1–8mNIS and stained for endogenous TDP-43. Unlike KPNB1 H1–8mNIS, KPNB1 H1–8WT prevents the sequestration of endogenous TDP-43 in cytoplasmic GR aggregates. Arrowheads point to cytoplasmic GR aggregates. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. g Quantification of the percentage of cells with cytoplasmic GR aggregates positive for endogenous TDP-43. KPNB1 H1–8WT significantly reduced the number of TDP-43-positive GR aggregates, while KPNB1 H1–8mNIS had no effect. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (three independent experiments; *p < 0.05, ***p < 0.001, n = 50–52 cells per group). h IF of HEK293T cells co-expressing GFP-(GR)100 with an empty plasmid (ctrl), FLAG-tagged KPNB1 H1–8WT or H1–8mNIS and stained for endogenous Nup62. Unlike KPNB1 H1–8mNIS, KPNB1 H1–8WT prevents the sequestration of endogenous Nup62 in cytoplasmic GR aggregates. Arrowheads point to cytoplasmic GR aggregates. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. i Quantification of the percentage of cells with cytoplasmic GR aggregates positive for endogenous Nup62. KPNB1 H1–8WT significantly reduced the number of Nup62-positive GR aggregates, while KPNB1 H1–8mNIS had no effect. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (three independent experiments; ***p < 0.001, n = 50–51 cells per group)