Extended Data Fig. 8 |. Mitochondrial morphology analyses for the WT, fusion loop, and disease mutants of OPA1.

a, Fluorescence microscopy images of wild-type (WT), membrane docking (K738E, R858E), membrane-inserting loop (W771A, W775A, L776A, K779E), and disease (R781E, R824E) mutants in mitochondrial morphology analyses. HeLa cells were transfected with empty vector (E.V.) or the indicated siRNA-resistant OPA1 constructs for 24h and then subjected to RNA interference using the indicated siRNAs for 72h. The mitochondrial network was visualized by staining for the outer membrane protein TOMM22 (green), and co-transfected mCherry-NLS (red) was used to identify transfected cells. Performed in biological triplicate. Scale bar, 20 μm. b, Expression and stability of the siRNA-resistant OPA1 proteins were assessed by immunoblot analysis of Triton X-100 extracts derived from HeLa cells transfected with either empty vector (E.V.) or the indicated siRNA-resistant OPA1 constructs 24h before treatment with the indicated siRNAs for 72h. 20 μg per lane (n = 3 independent experiments). c, Quantification of microscopy images of wild-type, empty vector (E.V.), and mutants. Mitochondrial phenotypes observed in HeLa cells transiently expressing human OPA1 variants as described in a. Cells expressing the control siRNA E.V. (n = 280), OPA1 siRNA E.V. (n = 269 cells), wild-type OPA1 (n = 238 cells), OPA1 K738E (n = 235 cells), OPA1 W771A (n = 281 cells), OPA1 W775A (n = 264 cells), OPA1 W776A (n = 255 cells), OPA1 K779E (n = 282 cells), OPA1 R858E (n = 245 cells), OPA1 R781E (n = 262 cells), and OPA1 R824E (n = 283 cells) over three experimental replicates. Data points represent the average percentage of cells across three experimental replicates. Error bars indicate s.e.m. d, Human OPA1 mutations associated with optic atrophy, cerebellar ataxia, and other diseases mapped onto the paddle domain of OPA1 as solid spheres and numbered. Back and side views of the molecular structure of human S-OPA1 LBD (coloured in green).