Miro1-var4 induces accumulation of peroxisomes in the cell periphery. (A) HeLa cells expressing HA2–Miro1-var4 were immunostained with antibodies to HA (a, green), Pex14p (b, red), and Tom20 (c, blue) as in Fig. 1 C. The merged image (d) and 3.5-fold magnified views of the boxed areas (insets) were shown. Note that HA2–Miro1-var4 was often colocalized with Pex14p-positive accumulates of peroxisomes in the cell periphery (arrowheads). (B) Nocodazole treatment abrogates Miro1-var4–induced accumulation of peroxisomes at the cell periphery. HeLa cells expressing HA2–Miro1-var4 as in A were treated with DMSO (vehicle; a–c) or 20 µM nocodazole for 1 h (d–f) and immunostained with antibodies to HA, Pex14p, and α-tubulin. Circles indicate peroxisome-enriched regions in cell periphery. Representative images are shown. Bars: (main images) 10 µm; (insets) 2 µm. (C) HeLa cells transfected with an empty vector (mock) or WT and respective GTPase mutants, P13V and T18N of HA2–Miro1-var4 and HA2–Miro1-var1 were treated with DMSO (−) or nocodazole (+) and immunostained as in B. Percentages of cells exhibiting normal distribution of peroxisomes throughout the cytoplasm (normal, open bar) and accumulation of peroxisomes in the cell periphery (accumulated, solid bar) were represented as the means. Transfected cells (n ≥ 100) for each condition repeated three times.
