Fig. 4

Nup62 co-aggregates with TDP-43 PrLD and recruits KPNB1 to facilitate reduction of aggregation. a Immunofluorescence (IF) of HEK293T cells co-expressing GFP-tagged TDP-CTF, sTDP or TDP-43^mNLS with mCherry or mCherry-KPNB1. KPNB1 abolished TDP-CTF and TDP-43^mNLS aggregates, whereas sTDP aggregates were only reduced in size. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. b Western blot analysis and quantification of insoluble GFP-tagged TDP-CTF, sTDP and TDP-43^mNLS in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 strongly reduced insoluble TDP-CTF and TDP-43^mNLS protein levels, and sTDP levels to a lesser extent. β-actin was used as a loading control. Statistical analysis was performed using two-way ANOVA and Bonferroni’s post hoc test (*p < 0.05, ***p < 0.001, n = 3). c IF of HEK293T cells co-expressing mCherry or Nup62-mCherry with GFP or GFP-tagged TDP-CTF, TDP-43^mNLS, TDP-43^mNLS 1–265, TDP-PrLD or sTDP. TDP-CTF and TDP-43^mNLS strongly colocalize with Nup62 via their PrLD, whereas sTDP lacking this domain does not. Arrowheads indicate co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. d Lysates from HEK293T cells expressing GFP, GFP-TDP-CTF or GFP-sTDP 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. Unlike sTDP, TDP-CTF strongly interacts with endogenous Nup62 and KPNB1. e IF of HEK293T cells co-expressing mCherry/Nup62-mCherry/mCherry-Nup85, GFP-TDP-CTF/GFP-sTDP and FLAG-KPNB1. Nup62 recruits KPNB1 to TDP-CTF but not sTDP aggregates. Nup85 which lacks FG repeats does not recruit KPNB1 to either TDP-CTF or sTDP, although it colocalizes with both aggregates. Arrowheads indicate co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. f Dissolution assays for fluorophore-labeled recombinant His₆-tagged TDP-CTF (ATTO488) and Nup62FG (ATTO610) co-aggregates in the presence of BSA or GST-tagged KPNB1 WT, KPNB1 mNIS, H1–9^WT or H1–9^mNIS. KPNB1 and H1–9 WT strongly dissolved TDP-CTF–Nup62 aggregates, whereas NIS mutations abolished this activity. g Quantification of dissolution of pre-formed aggregates (top) and prevention of aggregation (bottom) assays of TDP-CTF in the presence or absence of Nup62FG and different KPNB1 constructs. Optical density at 395 nm was measured to assess turbidity. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (*p < 0.05, **p < 0.005, ***p < 0.001, n = 3). h IF of HEK293T cells co-expressing Nup62-mCherry with GFP or GFP-tagged KPNB1^WT, KPNB1^mNIS, KPNB1 H1–8^WT or H1–8^mNIS. KPNB1^WT associated with Nup62 aggregates and strongly reduced their size, whereas KPNB1 H1–8^WT rendered Nup62 completely soluble. Arrowheads indicate co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. i IF of HEK293T cells co-expressing Nup62-mCherry with GFP or GFP-tagged IPO13, TNPO1, XPO1, XPO7 or RANBP17. IPO13 associated with Nup62 aggregates and strongly reduced their size. TNPO1 and exportins co-aggregated with Nup62 but only TNPO1 slightly reduced individual aggregate size. Arrowheads indicate co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm