Figure 4.
REEP5–MFN1/2 interaction is involved in mitochondrial distribution. (A) Comparison of mitochondrial aspect ratio between mEmerald-REEP5 or mEmerald expressing MEFs. (B) Time-lapse images of mitochondrial dynamics in MEFs co-transfected with mito-Dendra2 and mTagBFP-REEP5 or mTagBFP. mito-Dendra2 was photo-switched by illuminating the indicated region once. Bars: 20 µm (whole cell view, left); 5 µm (zoomed-in images). Numbered boxes, enlarged area; dash-line boxes, illuminated regions. (C and D) Mitochondrial distribution upon REEP5 knockdown. Endogenous mitochondria and REEP5 were immunostained with anti-Tom20 and anti-REEP5 in U2OS cells in C. The radial distance to the center of mitochondrial network was quantified in D. Bars: 20 µm (whole cell view); 5 µm (zoomed-in images). (E and F) Comparison of mitochondrial distribution in U2OS cells transiently expressing mEmerald, siRNA-resistant mEmerald tagged REEP5 wt or mutant #2 after REEP5 depletion in E. MitoTracker labeled mitochondria. The radial distance to the center of the mitochondrial network was quantified in F. Bars: 10 µm (whole cell view); 5 µm (zoomed-in images). (G and H) Mitochondrial morphology from cells expressing mEmerald or mEmerald-REEP5 C-terminus (CT). MitoTracker labeled mitochondria. The radial distance to the center of mitochondrial network was quantified in H. Bars: 10 µm (whole cell view); 5 µm (zoomed-in images). (I) Schematic imaging workflow of doxycycline-induced REEPs expression on mitochondrial dynamics. (J–L) Long-term time-lapse images of ER-mitochondria movements as the accumulation of REEP5 wt (J) or REEP5 mutant #2 (K) or REEP6 (L) over time in U2OS cells. 200 nM doxycycline and 5 µM Trolox were added before imaging. Yellow arrowheads indicate the dynamic correlation between MFN2 and REEP5. Bars: 10 µm. (M) Quantification of mitochondrial directional movement within 10 min fast live imaging window, as indicated in I, before and after REEPs expression (also see Videos 10, 11, and 12). (N and O) U2OS cells were transfected with FKBP-HaloTag-MFN1/2, FRB-mEmerald-REEP5 and mito-RFP. Cells were treated with or without 20 nM rapamycin, subregions of mito-RFP network within the dash-line boxes were repeatedly photobleached (also see Videos 15 and 16). Dash-line boxes, illuminated regions. Bars: 10 µm. Values shown are means ± SEM, n = 35 for A; n = 15 for D; n = 15 for F; n = 17 for H; n = 10 for M.

REEP5–MFN1/2 interaction is involved in mitochondrial distribution. (A) Comparison of mitochondrial aspect ratio between mEmerald-REEP5 or mEmerald expressing MEFs. (B) Time-lapse images of mitochondrial dynamics in MEFs co-transfected with mito-Dendra2 and mTagBFP-REEP5 or mTagBFP. mito-Dendra2 was photo-switched by illuminating the indicated region once. Bars: 20 µm (whole cell view, left); 5 µm (zoomed-in images). Numbered boxes, enlarged area; dash-line boxes, illuminated regions. (C and D) Mitochondrial distribution upon REEP5 knockdown. Endogenous mitochondria and REEP5 were immunostained with anti-Tom20 and anti-REEP5 in U2OS cells in C. The radial distance to the center of mitochondrial network was quantified in D. Bars: 20 µm (whole cell view); 5 µm (zoomed-in images). (E and F) Comparison of mitochondrial distribution in U2OS cells transiently expressing mEmerald, siRNA-resistant mEmerald tagged REEP5 wt or mutant #2 after REEP5 depletion in E. MitoTracker labeled mitochondria. The radial distance to the center of the mitochondrial network was quantified in F. Bars: 10 µm (whole cell view); 5 µm (zoomed-in images). (G and H) Mitochondrial morphology from cells expressing mEmerald or mEmerald-REEP5 C-terminus (CT). MitoTracker labeled mitochondria. The radial distance to the center of mitochondrial network was quantified in H. Bars: 10 µm (whole cell view); 5 µm (zoomed-in images). (I) Schematic imaging workflow of doxycycline-induced REEPs expression on mitochondrial dynamics. (J–L) Long-term time-lapse images of ER-mitochondria movements as the accumulation of REEP5 wt (J) or REEP5 mutant #2 (K) or REEP6 (L) over time in U2OS cells. 200 nM doxycycline and 5 µM Trolox were added before imaging. Yellow arrowheads indicate the dynamic correlation between MFN2 and REEP5. Bars: 10 µm. (M) Quantification of mitochondrial directional movement within 10 min fast live imaging window, as indicated in I, before and after REEPs expression (also see Videos 10, 11, and 12). (N and O) U2OS cells were transfected with FKBP-HaloTag-MFN1/2, FRB-mEmerald-REEP5 and mito-RFP. Cells were treated with or without 20 nM rapamycin, subregions of mito-RFP network within the dash-line boxes were repeatedly photobleached (also see Videos 15 and 16). Dash-line boxes, illuminated regions. Bars: 10 µm. Values shown are means ± SEM, n = 35 for A; n = 15 for D; n = 15 for F; n = 17 for H; n = 10 for M.

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