Figure 1
From: Nuclear egress of TDP-43 and FUS occurs independently of Exportin-1/CRM1
Predicted NESs of TDP-43 and FUS are non-functional. (A) Schematic diagrams of TDP-43 and FUS. TDP-43 contains an internal classical bipartite nuclear localization signal (cNLS), whereas FUS contains a proline-tyrosine (PY)-NLS at the C-terminus (in red). For each protein, two putative CRM1-dependent NESs (in green, key hydrophobic amino acids underlined) were predicted using bioinformatic NES prediction tools. They are localized within RNA-recognition motif (RRM) domains of TDP-43 and FUS. (B) Heterokaryon assay performed to analyze nuclear export of TDP-43 and FUS. HeLa cells expressing the indicated V5-tagged TDP-43 or HA-tagged FUS constructs were fused with mouse embryonic fibroblasts and the resulting heterokaryons were incubated for 2 h (TDP-43) and 5 h (FUS), respectively, in the presence of cycloheximide. Cells were stained with a V5- or HA-specific antibody (green), a human hnRNP-C-specific antibody to visualize a non-shuttling control protein in human nuclei (magenta) and DAPI as a nucleic acid stain (blue). The appearance of TDP-43 and FUS in the murine nucleus (marked with an asterisk in the DAPI channel) indicates that both proteins undergo nuclear export, in contrast to the non-shuttling control protein hnRNP-C. Mutation of key hydrophobic amino acids in one or both predicted NESs (mNES or double mNES) does not abrogate nuclear export, demonstrating that the predicted NESs are not necessary for TDP-43 or FUS export. Scale bars: 20 μm. (C) HeLa cells were transiently transfected with V5-tagged TDP-43 or HA-tagged FUS constructs carrying a mutated NLS (mNLS) or both a mutated NLS and mutations in the predicted NESs (mNLS-double mNES). Cells were stained with a V5- or HA-specific antibody (green) and DAPI (blue) and localization of mutant proteins was examined by fluorescence microscopy. Scale bars: 20 μm.
