Fig. 4
ALS-associated protein mutants colocalize with ORF1p in cytoplasmic aggregates of HEK 293T and 2102Ep cells. a Exogenously expressed wild-type 3XFLAG-tagged FUS protein is nuclear and only rarely colocalizes with ORF1-EGFP in the cytoplasm, but some mutants strongly overlap in cytoplasmic granules of unstressed HEK 293T cells. b GFP-tagged wild-type TDP-43 is mostly nuclear with only faint colocalization with endogenous ORF1p in cytoplasic foci of some unstressed cells (arrows, top panels). However, mutations to the TDP-43 NLS (K82/84A and A90V) and some ALS-associated mutations (for example, A315T) show colocalization with ORF1p in cytoplasmic granules. c Cherry tomato-tagged wild-type SOD1 protein is diffusely cytoplasmic (top), but some ALS mutants are present with ORF1p in cytoplasmic foci of 293T cells. d Endogenous TDP-43 strongly colocalizes with endogenous ORF1p in cytoplasmic granules of Na-arsenite stressed but not untreated 2102Ep cells. ORF1p foci are much increased in size in stressed cells. e Red fluorescent protein (RFP)-tagged hnRNPA1 colocalizes with ORF1-EGFP cytoplasmic granules in unstressed HEK 293T cells. f, g Cytoplasmic granules formed by exogenously expressed OPTN or ANG do not colocalize with ORF1-EGFP granules. h) ORF1p is generally excluded from GFP-(GA)31 dipeptide aggregates. The full-length L1 construct pc-L1-1FH expresses ORF1p with HA-FLAG tags. Size bars are 10 μm