p97 and p47 promote membrane tethering during mitotic Golgi reassembly in cooperation with FTCD (2020)
Figure 8

Figure 8. FTCD works as a tethering factor in cooperation with the p97/p47 complex in living cells.

(A) The expression of FTCDwt-HA-MAO causes the aggregation of mitochondria. HeLa Tet-off cells inducibly expressing either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO were cultured in DOX-free medium for 48 hrs. Cells which were cultured in the DOX-containing medium were used as a control. The cells were fixed and stained with a monoclonal antibody to mitochondria and a polyclonal antibody to HA, followed by observation by confocal microscopy. Panels a-j display representative images. Scale bar = 10 μm.

(B) The results of quantification of (A). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method).

(C) Representative EM images of a cell expressing FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were cultured for 48 hrs in the absence of DOX, and used for EM observation. Panel b displays the inset in panel a. Arrowheads show narrow gaps between mitochondria. Scale bar = 0.2 μm. The low magnification image is presented in Figure EV2C.

(D) Endogenous p47 and p97 are necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with either mock, p47 siRNA or p97 siRNA duplexes and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCDwt-HA-MAO, and then analyzed as in (A). Panels a-l display representative images. Scale bar = 10 μm.

(E) The results of quantification of (D). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method).

(F) Binding between FTCD and p47 and between p47 and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with mammalian expression constructs of siRNA-insensitive Flag-tagged p47wt/mutants at the same time as the treatment of p47 siRNA, and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCD-HA-MAO. After fixation, the cells were visualized with a monoclonal antibody to mitochondria and polyclonal antibodies to HA and Flag. Panels a-l display representative images. Scale bar = 10 μm.

(G) Binding between FTCD and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with the mammalian expression construct of siRNA-insensitive Flag-tagged p97wt/mutant at the same time as treatment with p97 siRNA. The following procedures were the same as in (F). Panels a-i display representative images. Scale bar = 10 μm.

(H) The results of quantification of (F) and (G). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with siRNA treatment alone ('none') and compared with mutant expression (Bonferroni method).