FIGURE 5.

Drb1 with a double mutation in both the NLS and NES is prone to aggregate in the cytoplasm and to recruit TDP-43 into the aggregates. A, schematic of Clover-T7-fused Drb1 involving NLS/NES mutations. B, representative image of Clover-Drb1-mtLL/R470G in HeLa cells. Cells were transfected for 24 h, followed by fixation and permeabilization as described previously. Nuclei were stained by Hoechst 33258. Arrowheads indicate cytoplasmic aggregation of Clover-Drb1-mtLL/R470G. Scale bar = 10 μm. C, the percentages of cells with Clover-fused Drb1 aggregates in the cytoplasm were counted in more than 100 transfected cells. Three independent experiments were performed. Error bars show standard deviation. D, representative images of the Clover-Drb1-mtLL/R470G expression construct transiently transfected without (top panel) or with (bottom panel) the TDP-WT-mRuby2 expression construct in HeLa cells. Localization of endogenous TDP-43 was visualized by indirect immunofluorescence (b and c in red). Scale bars = 10 μm. E, co-IP of the Drb1 NLS/NES mutant and TDP-43. The Clover-tagged Drb1 construct (WT or mtLL/R470G mutant) was transfected with the TDP-43 expression construct in HeLa cells, and IP was performed with an anti-GFP antibody (for the Clover tag). The expression level of TDP-43 protein increased ∼1.8-fold by overexpression (supplemental Fig. S3A). TDP-43 was co-immunoprecipitated with Clover-Drb1 WT and mtLL/R470G but not with Clover. IB, immunoblot. HC, immunoglobulin heavy chain.