Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: Schematic of supervised machine learning (ML) mitochondrial morphology imaging and quantification pipeline. Fibroblasts plated in 384‐well plates are stained for mitochondria (anti‐TOMM40, 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 (CTL‐1, 2, 3) and OPA1 S545R patient fibroblasts by supervised ML. Representative confocal images of control (CTL‐1, 2, 3) and DOA+ patient fibroblasts carrying indicated mono‐allelic mutations imaged as described in (A). Scale bar = 20 μm. Passage number between P12–15. Mitochondrial morphology quantification of (B). Data represent mean ± SD of two independent experiments, (195–2,496 cells per cell line), One‐way ANOVA; ** P < 0.01, **** P < 0.0001, ns; not significant. Representative confocal images of control (CTL‐1) and OPA1 S545R patient fibroblasts treated with OPA1 , DNM1L , or non‐targeting (NT) siRNAs for 72 h and imaged as described in (A). Scale bar = 20 μm. Passage number between P12–14. Mitochondrial morphology quantification of (D). Data represent mean ± SD of three independent experiments (3,219–5,857 cells per cell line), One‐way ANOVA; **** P < 0.0001, ns; not significant. Representative confocal images of control (CTL‐1) and OPA1 S545R patient fibroblasts treated with 50 μM cycloheximide (CHX) where indicated for 6 h. Imaging as described in (A). Scale bar = 20 μm. Passage number between P14–P15. Mitochondrial morphology quantification of (F). Data represent mean ± SD of two independent experiments (879–4,154 cells per cell line), One‐way ANOVA; **** P < 0.0001, ns; not significant. Source data are available online for this figure.

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Imaging, Staining, Control

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: Clinical features of patients from which fibroblasts were derived.

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Derivative Assay, Variant Assay

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: A, B Representative confocal images of candidate genes (siRNAs) able to fragment (B) or hypertubulate (A) mitochondrial morphology identified in Mitome screen of control (CTL‐1) fibroblasts (Fig ) Scale bar = 50 µm. C, D Representative confocal images of candidate genes (siRNAs) able to rescue (C) or hyperfragment (D) mitochondrial morphology identified in Mitome screen of OPA1 S545R patient fibroblasts (Fig ) Scale bar = 50 µm. E Violin plot representing % Hyperfragmented morphology of ground truth and Mitome siRNAs. The siRNA able to hyperfragment mitochondrial morphology in OPA1 S545R patient fibroblasts were selected with a univariate 3‐components statistical model programmed in R using ground truths for morphology in Fig . The defined threshold for positive hits was 72.9% (dotted line) and identified 27 candidate genes (Dataset ) from the same experiment as shown in Fig . F, G (F) Representative confocal images and (G) mitochondrial morphology quantification of control (CTL‐1) fibroblasts and OPA1 S545R patient fibroblasts treated with indicated siRNAs for 72 h. Scale bar=50µm. Supervised ML training performed on cells with fragmented ( OPA1 siRNA), normal (non‐targeting NT siRNA), and hypertubulated ( DNM1L siRNA) mitochondria. Data represent mean ± SD of measurement of 419–1,783 cells performed once. H Functional classification of 91candidate genes that rescued mitochondrial fragmentation in OPA1 S545R fibroblasts upon depletion (Dataset ).

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Control, Functional Assay

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: Schematic of Mitome siRNA imaging screen for mitochondrial morphology in control human fibroblasts. Fibroblasts were reverse‐transfected with siRNAs directed against 1,531 nuclear‐encoded mitochondrial genes in 384‐well plates and stained for mitochondria (anti‐TOMM40, 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 three‐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 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. 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 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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Imaging, Control, Transfection, Staining

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: 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). Representative confocal micrographs of MEFs WT and Opa1 Crispr MEFs treated with indicated siRNAs for 72 h. Mitochondria (anti‐TOMM40, green) and nuclei (DAPI, blue). Scale bar = 10 μm. Supervised ML mitochondrial morphology quantification of (B) using WT MEFs 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 (726–4,236 cells per cell line), (% fragmented); *** P < 0.001, **** P < 0.0001, ns; not significant. 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. Whole cell phospholipidome of WT and Opa1 Crispr MEFs treated with NT (non‐targeting), Tamm41 or Pgs1 siRNAs. Data represent mean ± SD of five independent experiments; * P < 0.05, *** P < 0.001, **** P < 0.0001, ns; not significant. Representative confocal micrographs of MEFs WT, Pgs1 Crispr , and Dnm1l Crispr MEFs treated with indicated siRNAs for 72 h. Mitochondria (anti‐TOMM40, green) and nuclei (DAPI, blue). Scale bar = 10 μm. Supervised ML mitochondrial morphology quantification of (G) using WT MEFs with fragmented ( Opa1 siRNA), normal (non‐targeting NT siRNA), and hypertubulated ( Dnm1l siRNA) mitochondria. Data represent mean ± SD of >3 independent experiments (3,096–7,238 cells per cell line), One‐way ANOVA (% fragmented); * P < 0.05, ** P < 0.01, **** P < 0.0001, ns; not significant. Source data are available online for this figure.

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

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

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

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 h. Mitochondria (anti‐TOMM40, green) and nuclei (DAPI, blue). Scale bar = 20 μm. Passages number between P10–15. B Mitochondrial morphology quantification of (A) using control fibroblasts with fragmented ( OPA1 siRNA), normal (non‐targeting NT siRNA), and hypertubulated ( DNM1L siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One‐way ANOVA (905–3,695 cells per cell line), (% fragmented); **** P < 0.0001, ns; not significant. C Representative confocal images of wild‐type (WT) and Opa1 Crispr MEFs treated with NT or Pgs1 siRNA for 72 h. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar = 10 μm. D Mitochondrial morphology quantification of (C) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) ground truth training sets. Data represent mean ± SD of three independent experiments, One‐way ANOVA (6,613–8,758 cells per cell line), (% fragmented); **** P < 0.0001, ns; not significant. 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 = 10 μm. F Supervised ML mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Data represent mean ± SD of three independent experiments, One‐way ANOVA (691–3,990 cells per cell line), (% fragmented); **** P < 0.0001, ns; not significant. G, H (G) Equal amounts of protein extracted from MEFs were separated by SDS–PAGE, immunoblotted with anti‐OPA1 antibody, and quantified (H) by densitometry relative to Stain‐Free. Data represent mean ± SD of three independent experiments, One‐way ANOVA; * P < 0.05, *** P < 0.001, **** P < 0.0001, ns; not significant. Source data are available online for this figure.

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

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

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: Opa1 splice isoforms in MEFs. Proteolytic cleavage sites for MPP, OMA1, and YME1L are indicated. TM, transmembrane domain. Equal amounts of protein extracted from WT and Opa1 KO MEFs were separated by SDS–PAGE, immunoblotted with indicated antibodies. WT and Opa1 Crispr immunoblots from Fig are cropped and included for reference. Quantitative RT–PCR (qRT–PCR) measurement of Opa1 and Pgs1 expression in indicated Opa1 Crispr MEFs complemented with pLenti‐Opa1 and Opa1 Crispr Pgs1 Crispr MEFs complemented with pLenti‐Pgs1 relative to WT control. Data represent mean ± SD of two independent experiments, unpaired t ‐test; ** P < 0.01, **** P < 0.0001, ns; not significant. (left) quantitative RT–PCR (qRT–PCR) measurement of normalized Opa1 expression (relative to Gapdh ) WT MEFs treated with Opa1 siRNA for 72 h relative to NT (non‐targeting) control. Data represent mean ± SD of 4 independent experiments, unpaired t ‐test; *** P < 0.001. (right) Equal amounts of protein extracted from WT MEFs treated with indicated siRNAs for 72 h were separated by SDS–PAGE, immunoblotted for OPA1. Representative confocal images of WT, Opa1 Crispr , and Opa1 Crispr MEFs complemented with pLenti‐Opa1‐Myc. Mitochondria (mitoYFP, green) and nuclei (DAPI, blue) and Opa1‐Myc (anti‐Myc, red). Scale bar = 10 μm. Mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) ground truth training sets. Data represent mean ± SD of six independent experiments (1,693–3,200 cells per cell line), One‐way ANOVA (% fragmented); *** P < 0.001, ns; not significant. Equal amounts of protein extracted from MEFs were separated by SDS–PAGE, immunoblotted with indicated antibodies. Horizontal line denotes separate membranes. Mitochondrial membrane potential measured in WT, Opa1 Crispr , Opa1 Crispr + pLenti‐Opa1‐Myc MEFs labeled with TMRE and analyzed by flow cytometry. TMRE/mitoYFP was used to normalize membrane potential. Data represent mean ± SD of three independent experiments (> 10,000 cells/sample), 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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: SDS Page, CRISPR, Western Blot, Quantitative RT-PCR, Expressing, Control, Membrane, Labeling, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: A, B (A) Equal amounts of protein extracted from WT and mutant MEFs stably expressing pLenti‐Opa1 or pLenti‐Pgs1 where indicated were separated by SDS–PAGE, immunoblotted with indicated antibodies and quantified by densitometry (B) relative to tubulin. Data represent mean ± SD of four independent experiments. C Equal amounts of protein extracted from WT MEFs treated with 20 μM CCCP (30 min) or 16 μM 4Br‐A23187 (18 h) were separated by SDS–PAGE, immunoblotted with indicated antibodies. Tubulin was used as loading control. D Representative confocal images of MEFs knocked in for mTurquoise2‐Dnm1l by Crispr/Cas9 genome editing in WT (WT Drp1KI ) and Opa1 Crispr ( Opa1 Crispr‐DrpKI ) MEFs treated with non‐targeting (NT) or Pgs1 siRNA for 72 h. DRP1 (mTurquoise2, purple), mitochondria (mitoYFP, green). Scale bar = 20 µm. E Bar graph representation of DRP1 localized to mitochondria (blue) vs cytosol (green). Data represent mean ± SD of five replicates (193–1,062 cells per cell line), One‐way ANOVA. Source data are available online for this figure.

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Mutagenesis, Stable Transfection, Expressing, SDS Page, Control, CRISPR

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: Representative confocal micrographs of MEFs WT and Opa1 KO MEFs treated with indicated siRNAs for 72 h. Mitochondria (anti‐TOMM40, green) and nuclei (DAPI, blue). Scale bar = 10 μm. Mitochondrial morphology quantification of (a) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Data represent mean ± SD of two independent experiments, (185–2,689 cells per cell line), One‐way ANOVA (% fragmented); **** P < 0.0001. Representative confocal images of wild‐type (WT) and Yme1l −/− MEFs treated with indicated siRNAs for 72 h. Live imaging of mitochondria labeled with MitoTracker Deep Red (MTDR, green), TMRE (pink), and NucBlue (Nuclei, blue). Scale bar = 10 µm. Supervised ML mitochondrial morphology quantification of (c) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Opa1 KO MEFs (from Fig ) are represented for comparison. Quantification of mitochondrial membrane potential (TMRE/MTDR) of cells imaged in (C). Number of analyzed cells inset. Data represent mean ± SD of one independent experiments, One‐way ANOVA; *** P < 0.001, **** P < 0.0001.

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Imaging, Labeling, Comparison, Membrane

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: A, B (A) MEFs of the indicated genotypes were subjected to 4 μM Actinomycin D and 10 μM 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 h. PI + nuclei number divided by the total nuclei number was then quantified over time. (B) Representative confocal images of (A). Scale bar = 100 μm. Data represent mean ± SD of three independent experiments (1,380–2,157 cells per cell line), One‐way ANOVA; **** P < 0.0001, ns; not significant. 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. Scale bar = 200 nm. D Quantification of (C) of mitochondrial ultrastructure; outer membrane/inner membrane ration (IMM/OMM) and cristae number per mitochondrion. Violin plot of > 50 mitochondria per cell line, One‐way ANOVA; * P < 0.05, **** P < 0.0001, ns; not significant. Source data are available online for this figure.

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Transmission Assay, CRISPR, Membrane

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: A–C (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 1 μM Oligomycin (Omy), 2 μM CCCP, Antimycin A 1 μM + 1 μM Rotenone. Bar graphs of (A) representing basal (B) and maximum (C) respiration. Data represent mean ± SEM of 7–12 independent OCR measurements, One‐way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001, ns; not significant. 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, Pgs1 Crispr , and Pgs1 Crispr MEFs + pLenti‐Pgs1. Membrane potential is represented as the ratio between TMRE/mitoYFP. WT MEFs treated with 20 μM CCCP serve as a negative control for TMRE. Data represent mean ± SD of three independent experiments, number of analyzed cells indicated in inset, One‐way ANOVA; ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns; not significant. E mtDNA content in MEFs from (F) was quantified by amplification of Mtll1 , 16s, and Mt‐ nd1 genes relative to the Gapdh nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One‐way ANOVA; **** P < 0.0001, ns; not significant. F mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 h was quantified by amplification of Mttl1 , 16s, and Mt‐ nd1 genes relative to the GapdhH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One‐way ANOVA; ** P < 0.01, **** P < 0.0001, ns; not significant. G, H (G) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS–PAGE (horizontal line denotes separate membranes), immunoblotted with indicated antibodies, and quantified by densitometry (H). Data represent mean ± SD of three independent experiments, One‐way ANOVA; ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns; not significant. Source data are available online for this figure.

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]) (Mishra et al, ) 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

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: A Basal oxygen consumption (JO 2 flux) measured by O2k high‐resolution respirometry (Oroboros) in WT, Opa1 Crispr , Opa1 Crispr + pLenti‐Opa1, Opa1 Crispr Pgs1 Crispr , Opa1 Crispr Pgs1 Crispr + pLenti‐Pgs1, Pgs1 Crispr and Pgs1 Crispr MEFs + pLenti‐Pgs1. Measurements were made in pairwise fashion compared with WT MEFs. O 2 flux normalized to protein concentration. Data represent mean ± SD of at least three independent experiments, unpaired t ‐test; * P < 0.05, ** P < 0.01, ns, not significant. B, C 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 1 μM Oligomycin (Omy), 2 μM CCCP (maximal). Bar graphs of representing basal (B) and maximal (C) respiration. Data represent mean ± SEM of 22–24 parallel OCR measurement, One‐way ANOVA; **** P < 0.0001, ns, not significant.

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: CRISPR, Protein Concentration

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: Mitochondrial morphology quantification workflow using PhenoLOGIC Harmony 4.9 supervised machine learning (Dataset ). Fluorescence imaging of cells (1) followed by nuclei (2) and cell segmentation (3) using cytoplasm defined by mitochondria. Cells on the border of the field of view are excluded from downstream analyses (4). Mitochondrial signal intensity is transformed into various texture parameters (5). (6) Supervised ML is trained using cells harboring mitochondria that are fragmented ( Opa1 siRNA, red), hypertubulated ( Dnm1l siRNA, blue), or normal (NT siRNA, green). Debris cells (yellow) not included in the classification. (7) Class segregation represented by goodness of fit and then applied to unknown, non‐trained images (8).

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]) (Mishra et al, ) were cloned into pDONR‐221 and then pLenti6/Ubc (Invitrogen) using Gateway Technology (Invitrogen).

Techniques: Fluorescence, Imaging, Transformation Assay