Figure 8.
Figure 8. ER-targeted Mff facilitates mitochondrial division. (A) Schematic cartoon of rapamycin-induced Mff recruitment to either OMM (left) or ER (right). “Mff” refers to the cytoplasmic portion of Mff-S. (B) Dynamics of GFP-Mff-FRB translocation to mitochondria upon rapamycin treatment in Mff KO cells. Live-cell images of cell transfected with AKAP-FKBP12 (red), GFP-Mff-Cyto-FRB (green), eBFP2-peroxisome (peroxisomes, blue), and mitoBFP (mitochondria, blue). Rapamycin (final concentration: 10 µM) added at time 0. (C) Dynamics of GFP-Mff-Cyto translocation to ER upon rapamycin in rapamycin treatment in Mff KO cells. Live-cell images of cells transfected with Sac1-FKBP12 (ER, blue), GFP-Mff-Cyto-FRB (green), and mCherry-mito7 (mitochondria, red). The lower green panel represents GFP-MFF-CytoFRB signal that has been thresholded to remove the cytoplasmic signal. Rapamycin (final concentration: 10 µM) added at time 0. (D) Rapamycin-induced mitochondrial division rates in control U2OS cells (16 ROIs from 15 cells); Mff KO cells (21 ROIs from 21 cells; ***, P = 0.00001); Mff KO cells transfected with mitochondria-targeted Mff (34 ROIs from 30 cells; *, P = 0.0179); Mff KO cells transfected with ER-targeted Mff (20 ROIs from 17 cells; ***, P = 0.0049); or Mff KO cells transfected with both mitochondria- and ER-targeted Mff (34 ROIs from 30 cells; P = 0.4181). Statistical analysis based on comparison to control cells by Student’s t test. N.S., not significant. Error bars represent SD. (E) Western blot showing Mff and Drp1 expression levels in WT cells, Mff KO cells, and Mff KO cells transfected with either the Mff-FRB construct + the mitochondrially targeted FKBP12 construct (Mff KO + Mff-mito) or the Mff-FRB construct + the mitochondrially targeted FKBP12 construct + the ER-targeted FKBP12 construct (Mff KO + Mff-ER & Mff-mito). Tubulin and myosin IIA are loading controls. Endogenous Mff runs as a doublet less than 37 kD, whereas the Mff-FRB construct runs at the 37-kD marker. Bars: (whole-cell images) 10 µm; (insets) 2 µm. Time in seconds.

ER-targeted Mff facilitates mitochondrial division. (A) Schematic cartoon of rapamycin-induced Mff recruitment to either OMM (left) or ER (right). “Mff” refers to the cytoplasmic portion of Mff-S. (B) Dynamics of GFP-Mff-FRB translocation to mitochondria upon rapamycin treatment in Mff KO cells. Live-cell images of cell transfected with AKAP-FKBP12 (red), GFP-Mff-Cyto-FRB (green), eBFP2-peroxisome (peroxisomes, blue), and mitoBFP (mitochondria, blue). Rapamycin (final concentration: 10 µM) added at time 0. (C) Dynamics of GFP-Mff-Cyto translocation to ER upon rapamycin in rapamycin treatment in Mff KO cells. Live-cell images of cells transfected with Sac1-FKBP12 (ER, blue), GFP-Mff-Cyto-FRB (green), and mCherry-mito7 (mitochondria, red). The lower green panel represents GFP-MFF-CytoFRB signal that has been thresholded to remove the cytoplasmic signal. Rapamycin (final concentration: 10 µM) added at time 0. (D) Rapamycin-induced mitochondrial division rates in control U2OS cells (16 ROIs from 15 cells); Mff KO cells (21 ROIs from 21 cells; ***, P = 0.00001); Mff KO cells transfected with mitochondria-targeted Mff (34 ROIs from 30 cells; *, P = 0.0179); Mff KO cells transfected with ER-targeted Mff (20 ROIs from 17 cells; ***, P = 0.0049); or Mff KO cells transfected with both mitochondria- and ER-targeted Mff (34 ROIs from 30 cells; P = 0.4181). Statistical analysis based on comparison to control cells by Student’s t test. N.S., not significant. Error bars represent SD. (E) Western blot showing Mff and Drp1 expression levels in WT cells, Mff KO cells, and Mff KO cells transfected with either the Mff-FRB construct + the mitochondrially targeted FKBP12 construct (Mff KO + Mff-mito) or the Mff-FRB construct + the mitochondrially targeted FKBP12 construct + the ER-targeted FKBP12 construct (Mff KO + Mff-ER & Mff-mito). Tubulin and myosin IIA are loading controls. Endogenous Mff runs as a doublet less than 37 kD, whereas the Mff-FRB construct runs at the 37-kD marker. Bars: (whole-cell images) 10 µm; (insets) 2 µm. Time in seconds.

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