Figure 5. Miro1-var2 and Miro1-var4 are specifically involved in fast directional movement of peroxisomes in a manner dependent on its GTPase activity and microtubules. (A) Live imaging of peroxisome movement. Time-lapse images of peroxisomes in HeLa cells stably expressing EGFP-SKL (HeLa/EGFP-SKL) were acquired by confocal fluorescence microscopy at one picture per second. Arrowheads (yellow) indicate a peroxisome showing fast and long-distance movement. Bars, 5 µm. (B) HeLa/EGFP-SKL cells were transfected with an empty vector (mock), mCherry–Miro1-var4–WT (WT), or T18N (T18N) encoding a dominant-negative mutant of the first GTPase domain. At 24 h after transfection, DMF of peroxisomes in mCherry-positive cells was quantified as described in the Live imaging of peroxisomes section of Materials and methods. *, P < 0.05 versus mock-treated cells (Student’s t test). (C) HeLa/EGFP-SKL cells expressing WT mCherry–Miro1-var4 were treated for 1 h with DMSO (vehicle) or 20 µM nocodazole as in Fig. 4 B. DMF of peroxisomes was analyzed as in B. **, P < 0.01 versus vehicle-treated cells (Student’s t test). (D) Expression of mCherry-Miro1 variants. HeLa/EGFP-SKL cells were treated for 72 h with control siRNA (lane 1) or Miro1 siRNA against all of the Miro1 variants (lanes 2–6). Cells were then transfected with an empty vector (mock, lanes 1 and 2) or plasmids encoding an siRNA-resistant version of mCherry-Miro1 variants (mCherry-Miro1 rescue variants, lanes 3–6) as indicated at the top. After further 24-h culture, cells were lysed and analyzed by SDS-PAGE and immunoblotting with anti–Miro1-ins1 antibody (top) and anti-RFP antibody (bottom). The arrowhead indicates endogenous Miro1-var2. (E) HeLa/EGFP-SKL cells were transfected with Miro1 siRNA and plasmids encoding mCherry-Miro1 rescue variants as in D. At 24 h after transfection of plasmids, DMF of peroxisomes was analyzed as in B. Data are shown as means ± SD (error bars). Cells (n ≥ 6) for each condition in three independent experiments. *, P < 0.05 (one-way ANOVA followed by a Dunnett’s test). (F) Accumulation of peroxisomes at the cell periphery requires Pex14p and Miro1-var4. Normal fibroblasts (a–d) and fibroblasts from a PEX14-deficient patient (PBD-K01; e–h) were cotransfected with EGFP-PEX26 and HA2–Miro1-var4 for 48 h and immunostained with antibodies to HA (b and f; red) and Pex14p (c and g; blue). EGFP fluorescence of EGFP-Pex26p (a and e; green) and merged views (d and h) are shown. Arrowheads indicate accumulated peroxisomes in the cell periphery (a–d). Representative images are shown. Bars, 10 µm. (G) Percentages of cells showing accumulation of peroxisomes in the cell periphery in F are represented as means ± SD (error bars). Cells (n ≥ 20) for each condition in three independent experiments.
Figure 5.

Miro1-var2 and Miro1-var4 are specifically involved in fast directional movement of peroxisomes in a manner dependent on its GTPase activity and microtubules. (A) Live imaging of peroxisome movement. Time-lapse images of peroxisomes in HeLa cells stably expressing EGFP-SKL (HeLa/EGFP-SKL) were acquired by confocal fluorescence microscopy at one picture per second. Arrowheads (yellow) indicate a peroxisome showing fast and long-distance movement. Bars, 5 µm. (B) HeLa/EGFP-SKL cells were transfected with an empty vector (mock), mCherry–Miro1-var4–WT (WT), or T18N (T18N) encoding a dominant-negative mutant of the first GTPase domain. At 24 h after transfection, DMF of peroxisomes in mCherry-positive cells was quantified as described in the Live imaging of peroxisomes section of Materials and methods. *, P < 0.05 versus mock-treated cells (Student’s t test). (C) HeLa/EGFP-SKL cells expressing WT mCherry–Miro1-var4 were treated for 1 h with DMSO (vehicle) or 20 µM nocodazole as in Fig. 4 B. DMF of peroxisomes was analyzed as in B. **, P < 0.01 versus vehicle-treated cells (Student’s t test). (D) Expression of mCherry-Miro1 variants. HeLa/EGFP-SKL cells were treated for 72 h with control siRNA (lane 1) or Miro1 siRNA against all of the Miro1 variants (lanes 2–6). Cells were then transfected with an empty vector (mock, lanes 1 and 2) or plasmids encoding an siRNA-resistant version of mCherry-Miro1 variants (mCherry-Miro1 rescue variants, lanes 3–6) as indicated at the top. After further 24-h culture, cells were lysed and analyzed by SDS-PAGE and immunoblotting with anti–Miro1-ins1 antibody (top) and anti-RFP antibody (bottom). The arrowhead indicates endogenous Miro1-var2. (E) HeLa/EGFP-SKL cells were transfected with Miro1 siRNA and plasmids encoding mCherry-Miro1 rescue variants as in D. At 24 h after transfection of plasmids, DMF of peroxisomes was analyzed as in B. Data are shown as means ± SD (error bars). Cells (n ≥ 6) for each condition in three independent experiments. *, P < 0.05 (one-way ANOVA followed by a Dunnett’s test). (F) Accumulation of peroxisomes at the cell periphery requires Pex14p and Miro1-var4. Normal fibroblasts (a–d) and fibroblasts from a PEX14-deficient patient (PBD-K01; e–h) were cotransfected with EGFP-PEX26 and HA2–Miro1-var4 for 48 h and immunostained with antibodies to HA (b and f; red) and Pex14p (c and g; blue). EGFP fluorescence of EGFP-Pex26p (a and e; green) and merged views (d and h) are shown. Arrowheads indicate accumulated peroxisomes in the cell periphery (a–d). Representative images are shown. Bars, 10 µm. (G) Percentages of cells showing accumulation of peroxisomes in the cell periphery in F are represented as means ± SD (error bars). Cells (n ≥ 20) for each condition in three independent experiments.

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