Fig. 6
Altered distribution of MICOS in Miro DKO cells due to the loss of cytoskeletal anchorage. a Distribution of MICOS (Mic19/CHCHD3) clusters along the mitochondrial membrane in WT and DKO cells. Pseudo-colored representation of reconstructed dSTORM images of MICOS complexes (scale bar: 0.5 μm). b DBSCAN cluster map of Mic19/CHCHD3 clusters. DKO cells show the areas with depletion of clusters in comparison with WT cells (scale bar: 0.5 μm). c Quantification of nearest neighbor distances between MICOS clusters present in WT and DKO cells. Histogram of all the observed distances between MICOS clusters are shown. (WT: 8 cells, DKO: 11 cells comprising > 30,000 NND; p < 0.001, Kolmogorov–Smirnov’s test). d Endogenous immunoprecipitation experiment in WT and DKO cells using antibodies against the core-forming MICOS components (Mic19/CHCHD3, Mic60/Mitofilin, and Sam50) and antibodies against the TRAK motor adaptors. TRAK proteins interact with critical MICOS components in a Miro-dependent manner. e Mic19/CHCHD3-positive MICOS clusters in WT distribute homogeneously in the mitochondrial population in WT MEFs, while the loss of Miro proteins correlates with an increase in heterogeneity in the distribution, with cells showing mitochondrial units almost devoid of Mic19/CHCHD3 signal (white arrows), (scale bar: 10 μm; insets: 5 μm). f Overexpressing TRAK1 and KIF5C induces the redistribution of the mitochondria to the periphery. In WT cells, this redistribution correlates with increased Mic19/CHCHD3 signal in the periphery (cyan arrows), (scale bar: 10 μm; insets: 5 μm). In DKO cells, TRAK1/KIF5C redistribution enhances the heterogeneity of Mic19/CHCHD3 staining, indicating a transport-mediated uncoupling of OMM and IMM in Miro DKO cells. Mitochondria with low Mic19/CHCHD3 signal concentrates in the TRAK1/KIF5C anterogradely transported mitochondria (white arrows), while Mic19/CHCHD3 signal accumulate in the proximal—not transported—mitochondria (cyan arrows)
