Fig 1: Mitochondrial dynamic and PMPCA distribution studies of fibroblasts from control and PMPCA mutated patients. (A): Representative fluorescent images of mitochondrial network structure overlaid on phase-contrast (on the left) showed a mitochondrial network hyperconnection in PMPCA fibroblasts. Mitochondrial volume (in purple on black background in the middle) was assessed using the Mitotracker Green fluorescent signal by Imaris software and color-coded on the right. The inset illustrates the classification code. To present the changes in mitochondrial morphology in patients’ cells, types of mitochondria were classified into 5 groups according to mitochondrial length: blobs < 1 µm; fragmented < 5 µm; tubular < 10 µm; filamentous < 20 µm; mitochondrial network > 20 um. Bar graphs show the distribution of the mitochondrial population of Control, P1 and P2. Mean ± SEM. Scale bar: 10 µm. (B): Western blots (left) against PMPCA, PMPCB, OPA1 and citrate synthase (CS) on control (C1) and two patients’ (P1 and P2) fibroblasts reveal decreased levels of PMPCA and equal levels of PMPCB and OPA1 in the pathological conditions, as shown on the histogram (right). (C): Enzymatic activities of the respiratory complexes (CI to CV) from the control and the PMPCA mutated fibroblast strains related to the citrate synthase (CS) enzymatic activity, did not reveal a significant difference between control and mutated fibroblasts. Biochemical data were generated using the two-tailed paired t-test. Results are Mean ± S.E.M. from four independent experiments. (D): Single-molecule localization microscopy dSTORM was used to analyze PMPCA distribution, correlated to total internal reflection fluorescence TIRF microscopy for mitochondrial staining. Using Imaris 8.0 ® software, the dSTORM PMPCA immunofluorescence signal was used to quantify their mitochondrial surface protein distribution.