High throughput screening identifies known and novel genetic modifiers of mitochondrial morphology in control and DOA+ patient-derived fibroblasts (A) Schematic of Mitome siRNA imaging screen for mitochondrial morphology in control human fibroblasts.Figure 2Schematic of Mitome siRNA imaging screen for mitochondrial morphology in control human fibroblasts. Fibroblasts were reverse-transfected with siRNAs directed against 1531 nuclear-encoded mitochondrial genes in 384 well plates and stained for mitochondria (anti-TOM40, green), nuclei (DAPI, blue), and cytoplasm (CellMask, blue). Supervised ML training performed on control fibroblasts treated with siRNAs for OPA1 or YME1L (fragmented) NT control (normal), and DNM1L (hypertubular) were applied to single-cell trinary classification of Mitome siRNA treated fibroblasts. Passage number P14. Candidate siRNAs (purple) causing mitochondrial fragmentation relative to grounds truths for fragmentation (OPA1 siRNA). Violin plot representing % fragmented morphology of Mitome siRNAs (purple). Hits were selected with a univariate 3-components statistical model programmed in R using ground truths (n=30) for morphology shown in (A). The defined threshold for positive hits (thick dotted line inset) was 68.9% (solid dash on the Y axis and thin dotted line in the inset) and identified 22 candidate genes, including OPA1, YME1L, and AMBRA1 from two independent experiments. Candidate siRNAs (purple) causing mitochondrial hypertubulation relative to grounds truths for hypertubulation (DNM1L siRNA). Violin plot representing % hypertubulated morphology of Mitome siRNAs (purple). Hits were selected with a univariate 3-components statistical model programmed in R using ground truths (n=30) for morphology shown in (A). The defined threshold for positive hits (thick dotted line inset) was 69.2% (solid dash on the Y axis and thin dotted line in the inset) and identified 145 candidate genes, including DNM1L, MIEF1, and PGS1 from two independent experiments. Schematic of Mitome siRNA imaging screen in OPA1S545R patient fibroblasts. Fibroblasts transfection and imaging as described in A. Supervised ML training performed on OPA1S545R fibroblasts treated with siRNA for OPA1 (hyperfragmented) NT control (normal), and DNM1L (rescued) were applied to single-cell trinary classification of OPA1S545R patient fibroblasts. Passages number P12. Violin plot representing % rescued morphology of Mitome siRNAs. The siRNA able to rescue mitochondrial fragmentation were selected with a univariate 3-components statistical model programmed in R using the following ground truths for morphology: fragmented (NT siRNA, n=30), rescued (DNM1L siRNA, n=30), and hyperfragmented (OPA1 siRNA, n=30). The defined threshold for positive rescued hits (thick dotted line inset) was 49.81% (solid dash on the Y axis and thin dotted line in the inset) and identified 91 candidate genes from one experiment. Overlap between 91 candidates identified in (E) and (C) identify 38 overlapping genes leading to mitochondrial elongation (hypertubulation in CTL-1, CTL-2 and rescued in OPA1S545R fibroblasts) and 53 genes that specifically rescue mitochondrial fragmentation in OPA1S545R fibroblasts.