Figure 7

mSigR1 accumulation leads to altered RNA-binding protein homeostasis. (a) Co-staining of SigR1 and TDP-43 in MCF-7 cells transfected with either wtSigR1 or mSigR1. Note the minor cytoplasmic TDP-43 accumulations (white arrow) without co-localization with SigR1, in contrast to the more pronounced TDP-43 aggregates co-localizing with SigR1 in E102Q-SigR1 fALS lymphoblastoid cell depicted in e. Scale bar, 15 μm. (b) Translocation of FUS from the nucleus (yellow arrow) to the cytoplasm (white arrow) and co-localization with SigR1 aggregates in MCF-7 cells expressing mSigR1 compared to wtSigR1-transfected cells. Scale bar, 10 μm. (c) Immunofluorescence staining of SigR1 and matrin-3 in MCF-7 cells expressing wtSigR1 or mSigR1. Note the cytoplasmic accumulations (white arrow) along with the loss of nuclear matrin-3 (yellow arrow) corresponding to the higher amount of globular mSigR1 aggregates. Scale bar, 10 μm. (d) Quantification (n=3 each) of the experiments illustrated in (a–c). *P<0.05. (e–g) Nuclear loss of TDP43 (e), FUS (f) and matrin-3 (g) and their co-localization with mSigR1 aggregates in E102Q-SigR1 fALS lymphoblastoid cells compared to healthy controls. Scale bar, 10 μm. (h) Immunoblot analysis of subcellular fractions obtained from E102Q-SigR1 patients' lymphoblastoid cell lines compared to healthy controls (n=2 for fALS; n=3 for control). Matrin-3, FUS and TDP-43 distributions are shown in the cytoplasmic (Cyto.), membrane (Memb.) and nuclear (Nucl.) fractions. Note the translocation of matrin-3 from the nucleus to the cytoplasm in E102Q-SigR1 patients' lymphoblastoid cells. Tubulin is used as a loading control and Lamin A as a positive control for the nuclear fraction. (i) Immunohistochemical analysis of lumbar α-MNs using matrin-3 antibody in sALS and fALS compared to normal controls. Cytoplasmic accumulation (arrowheads, middle) of matrin-3 in α-MNs of fALS patients harbouring FUS and C9ORF72 mutations. Strong nuclear immunoreactivity of matrin-3 (arrows) is evident in sALS α-MNs (right), whereas the control shows an overall weaker nuclear matrin-3 signal (arrows, left). Paraffin sections, DAB-immunohistochemistry; scale bars, 20 μm. (j) Quantification of immunohistochemical data illustrated in I; for the immunohistochemical analysis n=12 sALS; n=8 (C9ORF72); n=4 (FUS) and n=4 control cases were examined. *P<0.05, while # denotes absence of significance (k) loss of nuclear Tia1 (yellow arrows) and accumulation of cytoplasmic Tia-1 (white arrow in the middle panel) positive stress granules co-localized with mSigR1 aggregates in MCF-7 cells. Scale bar, 15 μm. (l) Quantification of data shown in (k). *P<0.05