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. 2015 Aug 11;6:8015. doi: 10.1038/ncomms9015

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(a) Miro is necessary for Cenp-F mitochondrial recruitment. Localization of Cenp-F (green) and mitochondria (red) in the presence and absence of Miro1, Miro2 or both. Top: Cenp-F signal only. Middle: overlay. Bottom: magnification of the boxed areas. R: Pearson coefficient colocalization analysis±s.e.m. Scale bar, 10 μm. Two to four regions were selected per cell. Number of selected regions: Miro1^CRISPR: 98; siMiro2: 36; Miro1^CRISPR-siMiro2: 34. The experiment was repeated at least four times. (b) Miro is sufficient for Cenp-F recruitment. Immunofluorescence of Miro-less cells overexpressing FLAG-Miro1 (blue). Green: Cenp-F, red: mitochondria. Scale bar, 10 μm. (c) Cenp-F recruitment to mitochondria by Miro1 mutants. Miro-less cells were transfected as in b with plasmids encoding the indicated mutants of Miro1. Recruitment was scored using the Pearson's R coefficient. Ø: untransfected cells. *P value <10⁻⁷ (compared with untransfected cells) from a Mann–Whitney–Wilcoxon U-test. NS, non-significant. Quantifications as in a. Number of selected regions: WT: 29; T18N: 17; E208K: 33; E328K: 28; E208K+E328K: 28; S432N: 31; φ: 27. The experiment was repeated at least four times. (d) Cytosolic calcium withdrawal does not prevent Cenp-F mitochondrial recruitment. Cells treated with EGTA and BAPTA-AM to chelate extra- and intracellular calcium were imaged as in a. (e) Mapping of the Miro-interaction domain on Cenp-F. KERMIT cells were cotransfected with FLAG-Miro and GFP fusion of indicated Cenp-F fragments. Green fragments are recruited to mitochondria. Red ones are not. (f) Co-immunoprecipitation of transiently transfected Cenp-F fragment (amino acids 2375–3144) and wild-type (wt) and calcium-binding mutant (m) Miro1. * IgG shedding from the beads. (g) Yeast two-hybrid mapping of the Miro-interaction domain on Cenp-F. Green fragments are positive for interaction. Red ones are not. (h) Alignment of the Miro-binding domain of Cenp-F in chordates. The phylogenetic tree is generated by neighbour joining using percentage identity.