Fig 2: (A) Immortalized fibroblasts were transfected either with the SPLICS-long, or with the SPLICS-short construct. After 12 h, cells were stained with MitoTracker deep red FM for live cell imaging, using a 63x objective; scale bars indicate 20 µm. (B) Quantification of wide MERCs (from SPLICS-long signal) and (C) narrow MERCs (from SPLICS-short signal) per cell. Fibroblasts transfected with (D) SPLICS-long or (E) SPLICS-short constructs were fixed and stained with an antibody against Miro1. Afterwards, cells were imaged with a 63× objective and SPLICS signals with and without Miro1 were quantified. All data indicated as mean ± SEM. Significance was assessed using a Kruskal-Wallis test (n = 3; ~16 cells analyzed per fibroblast line per experiment). * p < 0.05; *** p < 0.0001; **** p < 0.00001.

Fig 3: Down-regulating MICAL2PV promotes mitochondrial infiltration and mitochondrial localization of mitochondrial transport proteins Down-regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton. Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). Scale bar: 25 µm.Down-regulating MICAL2PV enhances mitochondrial infiltration. Mitochondrial infiltration index was quantified as the ratio of the radial distance from the nucleus to the furthest mitochondria and the radial distance from the nucleus to the plasma membrane. Fifty cells were quantified for each group.Down-regulating MICAL2PV enhances the mitochondrial localization of Miro1 and Miro2. Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting. The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic ß-tubulin protein.The band intensity of target proteins in the mitochondrial fraction was determined [gels shown in (C)] and expressed relative to the Control. Data information: In (A), nuclei were stained with Hoechst 33342 (blue). In (B, D), data were analyzed using one-way ANOVA with Bonferroni post-test and shown as mean ± SEM; *P < 0.05, ***P < 0.001. Three independent experiments were conducted. Source data are available online for this figure.

Fig 4: (A) Sanger sequencing result for the mutations c.1290A > G and c.2067A > G (NM_001033568), leading to the amino acid exchanges T351A or T610A (NP_001028740), respectively. (B) Schematic overview of Miro1 protein structure, showing the two newly identified mutations in Miro1: T351A is located within the second EF-hand domain and T610A within the C-terminus. (C) Homology model of human Miro1 based on the 3D structure of Drosophila Miro. The 3D structure shows both EF-hand domains, the C-terminal GTPase domain and the C-terminus. The amino acid T351 is highlighted with a green circle, (D) while the amino acid position T610 is highlighted with an orange circle. (E) Overview of the mutation effects on protein stability and functionality as predicted by SDM, mCSB, DUET, SIFT, Polyphen2, and SNAP2. (Red is “destabilizing”, “deleterious” or “possibly damaging”; Green is “stabilizing”, “tolerated” or “benign”. As indicated in the table.) (F) Representative Western blot image of Miro1 protein in immortalized fibroblasts with the mutations Miro1-T351A or Miro1-T610A. (G) Densitometry of Western blot analysis from (F) for Miro1 protein levels normalized to ß-Actin. Data indicated as mean ± SEM (n = 6). (H) Representative Western blot image of Tom20 protein in immortalized fibroblasts. (I) Densitometry of Western blot analysis of Tom20 protein levels normalized to ß-Actin. Data indicated as mean ± SEM (n = 3).

Fig 5: Correlation Analysis of Miro1 Ratio in Fibroblasts. (A) One-Way Anova is used. (B) Two-Way Anova is used to determine the interaction between sex and genetic background with Miro1 ratio as the response variable. (C, D) Multivariable regression is used to determine the interaction between age and genetic background (C), or age and sex (D), with Miro1 ratio as the response variable. All P values are calculated by linear fit.