Distinct intracellular localization of splicing variants of Miro1. (A) Domain structure of human authentic Miro1 and three splicing variants of Miro1. EF hands, calcium-binding EF hand domains. Partial genome structure of the human MIRO1 gene encoding the C-terminal region of Miro1 variants is shown at the bottom. Pink and orange boxes indicate the insertions 1 and 2 generated by alternative splicing of exons 19 and 20, respectively. Primers for RT-PCR are shown by half-arrowheads at the top. (B) Expression of mRNA of MIRO1 splicing variants in HeLa cells. Human MIRO1 encoding the C-terminal variable region of Miro1 was amplified by semiquantitative RT-PCR with RNA from HeLa cells and a pair of primers shown in A. Size markers are shown on the left. (C) Intracellular localization of splicing variants of Miro1. HA2-Miro1 variants were assessed by transient expression in HeLa cells for 24 h and immunostaining with antibodies to HA (a, e, i, and m; green), Pex14p (b, f, j, and n; red), and Tom20 (c, g, k, and o; blue). Merged images are shown (d, h, l, and p), and the boxed areas were magnified 3.5-fold in insets. Representative images are shown. Bars: (main images) 10 µm; (insets) 2 µm. (D) Data in C were quantified for localization of respective Miro1 variants to mitochondria (Mt; white), peroxisomes (Ps; dark gray), and both (Mt+Ps; light gray). Data are shown as means ± SD. Transfected cells (n ≥ 100) for each condition were counted in three independent experiments.
