Journal: bioRxiv

Article Title: High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation

doi: 10.1101/2021.01.14.426579

Figure Lengend Snippet: (A) Schematic of supervised machine learning (ML) mitochondrial morphology imaging and quantification pipeline. Fibroblasts plated in 384 well plates are stained for mitochondria (anti-TOM40, green), nuclei (DAPI, blue), and cell body (CellMask, blue). Supervised ML training performed on cells with fragmented ( OPA1 or YME1L siRNA), normal (non-targeting NT siRNA), and hypertubular ( DNM1L siRNA) mitochondria. Automatic single-cell trinary classification of control (CTL1, 2, 3) and OPA1 S545R patient fibroblasts by supervised ML. (B) Representative confocal images of control (CTL1, 2, 3) and DOA+ patient fibroblasts carrying indicated monoallelic mutations imaged as described in A. Scale bar=20μm. (C) Mitochondrial morphology quantification of B. Data represent mean ± SD of two independent experiments, (195-2496 cells per cell line), One-way ANOVA. (D) Representative confocal images of control (CTL1) and OPA1 S545R patient fibroblasts treated with OPA1 , DNM1L , or non-targeting (NT) siRNAs for 72 hours and imaged as described in A. Scale bar=20μm. (E) Mitochondrial morphology quantification of D. Data represent mean ± SD of three independent experiments, (3219-5857 cells per cell line), One-way ANOVA. (F) Representative confocal images of control (CTL1) and OPA1 S545R patient fibroblasts treated with 50μM cycloheximide (CHX) where indicated for 6 hours. Imaging as described in A. Scale bar=20μm (G) Mitochondrial morphology quantification of F. Data represent mean ± SD of two independent experiments, (879-4154 cells per cell line), One-way ANOVA.

Article Snippet: Complementary DNA (cDNA) encoding mouse Pgs1 with a C-terminal Myc tag and Opa1 with a C-terminal 9X Myc tag (pclbw-opa1(isoform 1)-myc; a gift from David Chan (Addgene plasmid # 62845) were cloned into pDONR-221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Imaging, Staining, Control

Journal: bioRxiv

Article Title: High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation

doi: 10.1101/2021.01.14.426579

Figure Lengend Snippet: (A) Schematic 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. (B) 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 for morphology show in (A). The defined threshold for positive hits was 68.9% and identified 22 candidate genes, including OPA1 , YME1L , and AMBRA1 . (C) Candidate siRNAs (purple) causing mitochondrial hypertubulation relative to grounds truths for hypertubulation ( DNM1L siRNA). Violin plot representing % hypertubular morphology of Mitome siRNAs (purple). Hits were selected with a univariate 3-components statistical model programmed in R using ground truths for morphology show in (A). The defined threshold for positive hits was 69.2% and identified 145 candidate genes, including DNM1L , MIEF1, and PGS1 . (D) Schematic of Mitome siRNA imaging screen in OPA1 S545R patient fibroblasts. Fibroblasts transfection and imaging as described in A. Supervised ML training performed on OPA1 S545R fibroblasts treated with siRNA for OPA1 (hyperfragmented) NT control (normal), and DNM1L (rescued) were applied to single-cell trinary classification of OPA1 S545R patient fibroblasts. (E) 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), rescued ( DNM1L siRNA), and hyperfragmented ( OPA1 siRNA). The defined threshold for positive rescued hits was 49.81% and identified 91 candidate genes. (E) 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 OPA1 S545R fibroblasts) and 53 genes that specifically rescue mitochondrial fragmentation in OPA1 S545R fibroblasts.

Article Snippet: Complementary DNA (cDNA) encoding mouse Pgs1 with a C-terminal Myc tag and Opa1 with a C-terminal 9X Myc tag (pclbw-opa1(isoform 1)-myc; a gift from David Chan (Addgene plasmid # 62845) were cloned into pDONR-221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Imaging, Control, Transfection, Staining

Journal: bioRxiv

Article Title: High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation

doi: 10.1101/2021.01.14.426579

Figure Lengend Snippet: (A) Schematic of cardiolipin (CL) biosynthesis pathway in mitochondria. Phosphatidic acid (PA) is transported to the inner membrane by PRELID1 where it is converted to CDP-diacylglycerol (CDP-DAG) and glycerol 3-phosphate (G3P) by TAMM41. Phosphatidylglycerol phosphate (PGP) is dephosphorylated to phosphatidylglycerol (PG) by PTPMT1. PG is either degraded to DAG or reacts with CDP-DAG to form CL in a reaction catalyzed by cardiolipin synthase (CLS1). Tafazzin (TAZ) catalyzes the remodeling of monolysocardiolipin (MLCL) to mature CL. CL is transported to the outer membrane and converted to PA by mitoPLD. PA is converted to DAG by Lipin1. PA can be supplied to the inner membrane from DAG conversion by Acylglycerol Kinase (AGK). (B) Representative confocal micrographs of MEFs WT and Opa1 Crispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. (C) Supervised ML mitochondrial morphology quantification of (B) using WT MEFs with fragmented ( Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubular ( Dnm1l siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (726-4236 cells per cell line), (% fragmented). (D) quantitative RT-PCR (qRT-PCR) measurement of Prelid1 , Tamm41 , Pgs1 , Ptpmt1 , and Cls1 expression in Opa1 Crispr and WT MEFs. Fold change is indicated relative to WT control. Data represent mean ± SD of three independent experiments, One-way ANOVA. (E) Whole cell phospholipidome of WT and Opa1 Crispr MEFs treated with NT (non-targeting), Tamm41 or Pgs1 siRNAs. Data represent mean ± SD of four independent experiments. (F) Representative confocal micrographs of MEFs WT, Pgs1 Crispr and Dnm1l Crispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. (G) Supervised ML mitochondrial morphology quantification of (G) using WT MEFs with fragmented ( Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubular ( Dnm1l siRNA) mitochondria. Data represent mean ± SD of >3 independent experiments, (3096-7238 cells per cell line), One-way ANOVA (% fragmented).

Article Snippet: Complementary DNA (cDNA) encoding mouse Pgs1 with a C-terminal Myc tag and Opa1 with a C-terminal 9X Myc tag (pclbw-opa1(isoform 1)-myc; a gift from David Chan (Addgene plasmid # 62845) were cloned into pDONR-221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Membrane, CRISPR, Quantitative RT-PCR, Expressing, Control

Journal: bioRxiv

Article Title: High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation

doi: 10.1101/2021.01.14.426579

Figure Lengend Snippet: (A) Representative confocal images of control (CTL-1) and OPA1 S545R patient fibroblasts treated with OPA1 , DNM1L , PGS1 , and non-targeting (NT) siRNAs or indicated combinations for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=20μm. (B) Mitochondrial morphology quantification of (A) using control fibroblasts with fragmented ( OPA1 siRNA), normal (non-targeting NT siRNA), and hypertubular ( DNM1L siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (905-3695 cells per cell line), (% fragmented). (C) Representative confocal images of wild type (WT) and OPA1 Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. (D) Mitochondrial morphology quantification of (C) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubular) ground truth training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (6613-8758 cells per cell line), (% fragmented). (E) Representative confocal images of WT, Opa1 Crispr MEFs complemented with pLenti-Opa1, Opa1 Crispr Pgs1 Crispr MEFs and Pgs1 Crispr MEFs complemented with pLenti-Pgs1 by lentiviral delivery. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. (F) Supervised ML mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubular) training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (691-3990 cells per cell line), (% fragmented). (H) Equal amounts of protein extracted from MEFs were separated by SDS-PAGE, immunoblotted with anti-OPA1 antibody, and quantified (I) by densitometry relative to Stain-Free. Data represent mean ± SD of three independent experiments, One-way ANOVA.

Article Snippet: Complementary DNA (cDNA) encoding mouse Pgs1 with a C-terminal Myc tag and Opa1 with a C-terminal 9X Myc tag (pclbw-opa1(isoform 1)-myc; a gift from David Chan (Addgene plasmid # 62845) were cloned into pDONR-221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Control, CRISPR, Imaging, SDS Page, Staining

Journal: bioRxiv

Article Title: High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation

doi: 10.1101/2021.01.14.426579

Figure Lengend Snippet: (A, B) (Top) MEFs of the indicated genotypes were subjected to 4μM Actinomycin D and 10μM ABT-737 in the presence or absence of the pan-caspase inhibitor qVD. Dead cells (PI+ nuclei, orange) and total cells (NucBlue, blue) were imaged every hour for 25 hours. PI+ nuclei number divided by the total nuclei number was then quantified over time. (Bottom) Representative confocal images of (Top). Scale bar=100μm. Data represent mean ± SD of four independent experiments, (1380-2157 cells per cell line), One-way ANOVA. (C) Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1 Crispr and Opa1 Crispr Pgs1 Crispr MEFs. (D) Quantification of (C) of mitochondrial ultrastructure; outer membrane/inner membrane ration (OMM/IMM) and cristae number per mitochondrion. Violin plot of >50 mitochondria per cell line, One-way ANOVA.

Article Snippet: Complementary DNA (cDNA) encoding mouse Pgs1 with a C-terminal Myc tag and Opa1 with a C-terminal 9X Myc tag (pclbw-opa1(isoform 1)-myc; a gift from David Chan (Addgene plasmid # 62845) were cloned into pDONR-221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Transmission Assay, CRISPR, Membrane

Journal: bioRxiv

Article Title: High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation

doi: 10.1101/2021.01.14.426579

Figure Lengend Snippet: (A) Mitochondrial respiration measured in adherent MEFs of the indicated genotypes using Seahorse FluxAnalyzer. Oxygen consumption rate (OCR) normalized to protein concentration. Following basal respiration, cells were treated sequentially with 1μM Oligomycin (Omy), 2μM CCCP, Antimycin A 1μM + 1μM Rotenone. Bar graphs of (A) representing basal (B) and maximum (C) respiration. Data represent mean ± SEM of 6-12 independent OCR measurements, One-way ANOVA. (D) Mitochondrial membrane potential measured by fluorescence microscopy in WT, Opa1 Crispr , Opa1 Crispr + pLenti-Opa1, Opa1 Crispr Pgs1 Crispr , Opa1 Crispr Pgs1 Crispr + pLenti-Pgs1, and Pgs1 Crispr MEFs + pLenti-Pgs1. Membrane potential is represented as the ratio between TMRE/mitoYFP. WT MEFs treated with 20μM CCCP serve as a negative control for TMRE. Number of analyzed cells indicated in inset. (E) mtDNA content in MEFs from (F) was quantified by amplification of MTTL2 , 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA. (F) mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL2 , 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA. (G) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS-PAGE, immunoblotted with indicated antibodies and quantified by densitometry (H) . Data represent mean ± SD of three independent experiments, One-way ANOVA.

Article Snippet: Complementary DNA (cDNA) encoding mouse Pgs1 with a C-terminal Myc tag and Opa1 with a C-terminal 9X Myc tag (pclbw-opa1(isoform 1)-myc; a gift from David Chan (Addgene plasmid # 62845) were cloned into pDONR-221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Protein Concentration, Membrane, Fluorescence, Microscopy, CRISPR, Negative Control, Amplification, Mutagenesis, SDS Page