Journal: Science advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress.

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: Fig. 3. Mitochondrial translation hubs are dynamic. (A) Top: HPG labeling time course with variable chase times. Bottom: IMR90 cells pulse labeled for 15 min with HPG and chased with unlabeled methionine for 5, 15, 30, or 60 min. (B) Density of thresholded HPG domains in each condition. (C) Average size of HPG-labeled domains in each condition. (D) Average above-threshold fluorescence intensity of HPG-labeled domains in each condition (5-min chase: n = 10 fields of view; 39 cells; 15-min chase: n = 10 fields of view 40 cells; 30-min chase: n = 10 fields of view; 30 cells; 1-hour chase: n = 10 fields of view; 28 cells). (E) Top: HPG labeling time course with variable pulse times. Bottom: IMR90 cells pulse labeled for 15, 30, or 60 min with HPG followed by a constant chase in unlabeled methionine for 5 min, relative to control cells incubated in HPG for 15 min concurrent with CAP. (F to H) Quantification of the number of thresholded HPG objects in each condition. (CAP: n = 10 fields of view; 49 cells; 15-min pulse: n = 10 fields of view; 39 cells; 30-min pulse: n = 10 fields of view; 34 cells; 1-hour pulse: n = 10 fields of view; 28 cells). Scale bars, 1 μm. (I) IMR90 cells that were transiently transfected with mCherry-DRP1K38A (grayscale), pulse labeled for 15 min with HPG (green), immunolabeled with an antibody against TOM20 (blue), and RNR2- FISH (magenta). Scale bars, 5 μm; 1 μm in zoom. (J) Average size of thresholded RNR2 signal intensity. (K) Average size of thresholded HPG signal intensity. (L) Average fluorescence intensity of RNR2 signals per thresholded mitochondrion. (M) Average fluorescence intensity of HPG per thresholded mitochondrion (control: n = 22 cells; mCherry-DRP1K38A: n = 16 cells) (**P < 0.01 and *P < 0.05, Mann-Whitney test). OE, over expression.

Article Snippet: Plasmids encoding SUV3- HA, SUV3G207V- HA, and mCherry- DRP1K38A have been deposited to Addgene.

Techniques: Labeling, Fluorescence, Control, Incubation, Transfection, Immunolabeling, MANN-WHITNEY, Over Expression

Journal: iScience

Article Title: Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

doi: 10.1016/j.isci.2023.107180

Figure Lengend Snippet: Figure 1. Multiple mtDNA copies accumulate within mitobulbs in DRP1 mutant fibroblasts (A) Representative live cell images showing the mitochondrial marker TMRM (magenta) and PicoGreen-stained DNA (green) in control and DRP1 mutant human fibroblasts. The zoomed images show the enlarged nucleoids present in mitobulbs (arrowheads). (B) Quantification of the number of mitobulbs per cell. Each point represents an individual cell, with 45 cells quantified in 3 independent experiments. Bars show the average GSD. (C) Representative images of mCherry-expressing DRP1 mutants labeled for EdU (green) and TOM20 (mitochondria, magenta). Arrowheads indicate EdU-positive mtDNA in mitobulbs. Scale bar 10 mm. (D) Quantification of number of EdU foci/mitobulb. Each point represents an individual cell, with 45 cells quantified in 3 independent experiments. Bars show the average GSD. (E) Quantification of the number of EdU foci found in mitobulbs relative to those found outside of mitobulbs in the cells quantified in (D). (F) qPCR quantification of mtDNA levels in controls cells and two patient fibroblast lines (P1, P2). Each point represents one independent experiment. Bars show the average GSD. (G) Quantification of EdU foci in control and DRP1 mutants cells labeled with EdU for 4 h. Each point represents one cell, with at least 44 cells quantified in 3 independent experiments. Bars show the average GSD. ***p < 0.001 two-sided t-test.

Article Snippet: MEF cells were transiently transfected with DRP1 K38A (gift from Alexander van der Bliek & Richard Youle,52 Addgene, 45161) using Metafectene Pro (Biontex, T040) for 24 hours.

Techniques: Mutagenesis, Marker, Staining, Control, Expressing, Labeling

Journal: iScience

Article Title: Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

doi: 10.1016/j.isci.2023.107180

Figure Lengend Snippet: Figure 2. Increased contact sites between rough ER and mitochondria in DRP1 mutant fibroblasts (A) Representative images of control fibroblasts showing the PLA for Calnexin and TOM20 (white), along with Calnexin (ER, green), and TOM20 (mitochondria, magenta). Arrowheads denote PLA foci. Scale bar 2 mm. Full image in Figure S1. (B) Quantification of Calnexin-TOM20 PLA. Each data point represents one cell. Bars represent the average of 40 cells per genotype in 3 independent experiments GSD **p < 0.01 two-sided t-test. (C) Quantification of IP3R-VDAC PLA. Each data point represents one cell. Bars represent the average of 30 cells per genotype in 3 independent experiments GSD **p < 0.01 two-sided t-test. (D) Representative TEM images of control and DRP1 mutant fibroblasts. Arrowheads denote ER-mitochondria contact sites (ERMCS). Scale bar 1 mm. (E) Quantification of the ERMCs length (mm) per mitochondrial perimeter (Left) and number of ERMCS per mitochondrial perimeter (Right) in TEM images of smooth ER. Each data point represents one cell. Bars represent the average of 15 cells per genotype GSD. (F) Quantification of the ERMCs length (mm) per mitochondrial perimeter (Left) and number of ERMCS per mitochondrial perimeter (Right) in TEM images of rough ER. Each data point represents one cell. Bars represent the average of 15 cells per genotype GSD *p < 0.05, **p < 0.01 two-sided t-test. (G) FIB-SEM images of a DRP1 mutant mitochondria showing its association with rough ER. Left, FIB-SEM image, middle and right, 3D reconstruction. Scale bar 500 nm.

Article Snippet: MEF cells were transiently transfected with DRP1 K38A (gift from Alexander van der Bliek & Richard Youle,52 Addgene, 45161) using Metafectene Pro (Biontex, T040) for 24 hours.

Techniques: Mutagenesis, Control

Journal: iScience

Article Title: Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

doi: 10.1016/j.isci.2023.107180

Figure Lengend Snippet: Figure 3. Increased contact sites between CLIMP63-labeled ER and mitochondria in DRP1 mutant fibroblasts (A) Representative images of control and DRP1 mutant fibroblasts showing the PLA for CLIMP63 and TOM20 (White), along with CLIMP63 (ER sheets, green), TOM20 (mitochondria, magenta) and nuclei (Hoechst, blue). Scale bar 10 mm. Full image in Figure S2A. (B) Quantification of CLIMP63-TOM20 PLA. Each data point represents one cell. Bars represent the average of 50 control and 48 mutant cells in 3 independent experiments GSD ***p < 0.001 two-sided t-test. (C) Representative SIM images of control and DRP1 mutant fibroblasts stained for CLIMP63 (ER sheets, green) and MitoTracker Orange (mitochondria, magenta). Scale bar 10 mm. Full image in Figure S2B. (D) Manders’ coefficients calculated from the SIM images in (C). Left, M1 (relative to mitochondria); Right, M2 (relative to the ER). In the turned condition, the CLIMP63 images were rotated 90 to represent a random distribution. Each data point represents one cell. Bars represent the average of 11 cells per genotype GSD *p < 0.05 One-way ANOVA. (E) Fraction of overlapping signal between ER and mitochondria normalized to mitochondria (Left) or the ER area (Right) in SIM images (C). Each data point represents one cell. Bars represent the average of 11 cells per genotype GSD *p < 0.05 two-sided t-test.

Article Snippet: MEF cells were transiently transfected with DRP1 K38A (gift from Alexander van der Bliek & Richard Youle,52 Addgene, 45161) using Metafectene Pro (Biontex, T040) for 24 hours.

Techniques: Labeling, Mutagenesis, Control, Staining

Journal: iScience

Article Title: Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

doi: 10.1016/j.isci.2023.107180

Figure Lengend Snippet: Figure 4. Decreased RRPB1-SYNJ2BP contact sites in DRP1 mutant fibroblasts (A) Representative images of control and DRP1 mutant fibroblasts showing the PLA for RRBP1 and SYNJ2BP (white), along with RRBP1 (ER sheets, green) and SYNJ2BP (mitochondria, magenta). Scale bar 10 mm. Full image in Figure S4A. (B) Quantification of RRBP1-SYNJ2BP PLA. Each data point represents one cell. Bars represent the average of 50 control and 48 mutant cells in 3 independent experiments GSD ***p < 0.001 two-sided t-test. (C) representative SIM images of control and DRP1 mutant fibroblasts stained for RRBP1 (ER sheets, green) and MitoTracker Orange (mitochondria, magenta) Scale bar 10 mm. Full image in Figure S4B. (D) Manders’ coefficients calculated from the SIM images in (C). Left, M1 (relative to mitochondria); Right, M2 (relative to the ER). In the turned condition, the RRBP1 images were rotated 90 to represent a random distribution. Each data point represents one cell. Bars represent the average of 11 cells per genotype GSD *p < 0.05 One-way ANOVA. (E) Fraction of overlapping signal between ER and mitochondria normalized to mitochondria (Left) or the ER area (Right) in SIM images (C). Each data point represents one cell. Bars represent the average of 11 cells per genotype GSD *p < 0.05 two-sided t-test.

Article Snippet: MEF cells were transiently transfected with DRP1 K38A (gift from Alexander van der Bliek & Richard Youle,52 Addgene, 45161) using Metafectene Pro (Biontex, T040) for 24 hours.

Techniques: Mutagenesis, Control, Staining

Journal: iScience

Article Title: Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

doi: 10.1016/j.isci.2023.107180

Figure Lengend Snippet: Figure 5. ER sheets are associated with mitobulbs (A and B) Colocalization between ER sheets and mitobulbs. Colocalization was measured in cells immunolabeled for CLIMP63 (ER sheets, red) and ATP5a (mitochondria, green). (A) Representative image (Top) and Line scan analysis (Bottom) along the line shown in the image. (B) Quantification of the percent of mitobulbs that are associated with CLIMP63-positive ER sheets in two independent DRP1 mutant lines (P1 and P2). Each data point represents one cell. Bars represent the average of 45 control and 45 mutant mitochondria GSD. (C and D) Interaction between ER sheets and mitobulbs as measured by PLA for TOM20 and CLIMP63 or Calnexin. (C) Representative image of TOM20-CLIMP63 PLA. Arrowheads denote PLA foci (White) on mitobulbs (TOM20, Green) at sites where they contact ER sheets (CLIMP63, Red). (D) Quantification of mitobulbs associated with PLA foci for TOM20- CLIMP63 and TOM20-Calnexin. Each data point represents one cell. Bars represent the average of 48 (CLIMP63 PLA) and 40 cells (Calnexin PLA) in 3 independent experiments GSD. Scale bars 2 mm. (E) Representative image of 3D rendering of an SIM image (middle, right) showing a mitobulb (magenta) in association with ER sheets (green) in a DRP1 mutant cell, original image on the left. Contact sites (golden yellow) as identified by the Imaris software. The asterisk denotes a mitobulb.

Article Snippet: MEF cells were transiently transfected with DRP1 K38A (gift from Alexander van der Bliek & Richard Youle,52 Addgene, 45161) using Metafectene Pro (Biontex, T040) for 24 hours.

Techniques: Immunolabeling, Mutagenesis, Control, Software

Journal: iScience

Article Title: Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

doi: 10.1016/j.isci.2023.107180

Figure Lengend Snippet: Figure 6. Altered ER sheet structure in DRP1 mutants (A) Representative images of CLIMP63 staining in control and DRP1 mutant fibroblasts. The white lines denote the edge of the cell as determined by DIC. Full image in Figure S5. Scale bars 10 mm. (B) Quantification of total area covered by ER sheets. Each data point represents one cell. Bars represent the average of 41 cells in 3 independent experiments GSD ***p < 0.001 two-sided t-test. (C) Representative images of RRBP1 staining in control and DRP1 mutant fibroblasts. The white lines denote the edge of the cell as determined by DIC. Scale bars 10 mm. (D) Quantification of ER sheet structure as Structured (Blue) or Altered (red; presence of punctate structures and thick ER sheet patches). Each point represents one independent experiment, with at least 20 cells quantified per experiment. Bars show the average GSD. *p < 0.05, **p < 0.01 One-way ANOVA using the data for Structured ER. (E) Representative SIM images of CLIMP63 (Top) or RRBP1 (Bottom)- labeled ER sheets in Control and DRP1 mutant fibroblasts. (F) Quantification of the average size of individual CLIMP63-labeled (right), RRBP1-labeled (left) ER sheets from SIM images in (E). Each data point represents one cell. Bars represent the average of 15 cells in 3 independent experiments GSD ***p < 0.001 two-sided t-test.

Article Snippet: MEF cells were transiently transfected with DRP1 K38A (gift from Alexander van der Bliek & Richard Youle,52 Addgene, 45161) using Metafectene Pro (Biontex, T040) for 24 hours.

Techniques: Staining, Control, Mutagenesis, Labeling

Journal: iScience

Article Title: Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

doi: 10.1016/j.isci.2023.107180

Figure Lengend Snippet: Figure 8. Modulation of ER sheets-mitochondria contact sites rescues nucleoid numbers in DRP1 mutant fibroblasts (A and B) Quantification of PLA foci (CLIMP63-TOM20) in mCherry and mCherrry-CLIMP63 expressing control and DRP1 mutant cells. Total PLA foci (A) and mitobulbs associated with PLA foci (B) were quantified. Each point represents one cell, with at least 43 cells quantified per condition in 3 independent experiments. Bars show the average GSD. **p < 0.01, ***p < 0.001, ns not significant. One-way ANOVA. (C) Representative images of mCherry and mCherrry-CLIMP63 expressing control and DRP1 mutant cells stained for the mitochondrial marker TOM20 (mitochondria, magenta) and the nucleoid marker TFAM (nucleoids, Green). Full images in Figure S9A. Scale bar 10 mm. (D) Quantification of nucleoid area in mCherry and mCherry-CLIMP63 expressing control and DRP1 mutants from images in Figure S9A. Each point represents one cell, with at least 32 cells quantified per condition in 3 independent experiments. Bars show the average GSD. ***p < 0.001, ns not significant. One-way ANOVA. (E–G) Rescue of nucleoid numbers in CLIMP63-expressing DRP1 mutant cells. Quantification of total nucleoids (TFAM- positive, E), mitobulbs containing nucleoids (TFAM-positive, F) and mitochondrial bulb-like structures (independently of the presence of nucleoids, G) in mCherry and mCherrry-CLIMP63 expressing control and DRP1 mutant cells. Each point represents one cell, with 40 mCherry (mch) and 47 mCherry-CLIMP63 cells quantified in 3 independent experiments. Bars show the average GSD. One-way ANOVA (E), two-sided t-test (F, G). *p < 0.05, ***p < 0.001, ns, not significant. (H) Quantification of nucleoid area in mCherry-Fis1 and mCherry-SYNJ2BP expressing control and DRP1 mutants from images in Figure S9B. Each point represents one cell, with at least 30 cells quantified per condition in 3 independent experiments. Bars show the average GSD. ***p < 0.001, ns not significant. One-way ANOVA. (I–K) Nucleoid numbers in mCherry-SYNJ2BP-expressing DRP1 mutant cells. Quantification of total nucleoids (TFAM- positive, I), mitobulbs containing nucleoids (TFAM-positive, J) and mitochondrial bulb-like structures (independently of

Article Snippet: MEF cells were transiently transfected with DRP1 K38A (gift from Alexander van der Bliek & Richard Youle,52 Addgene, 45161) using Metafectene Pro (Biontex, T040) for 24 hours.

Techniques: Mutagenesis, Expressing, Control, Staining, Marker

Journal: iScience

Article Title: Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

doi: 10.1016/j.isci.2023.107180

Figure Lengend Snippet: Figure 9. CLIMP63 expression rescues nucleoid replication in DRP1 mutant fibroblasts (A) Representative images of mCherry and mCherrry-CLIMP63 expressing control and DRP1 mutant cells stained for EdU (Green) and the mitochondrial marker TOM20 (mitochondria, magenta). Full images in Figure S10. Scale bar 10 mm. (B) Quantification of EdU foci in control and DRP1 mutants expressing mCherry or mCherry-CLIMP63 in cells labeled with EdU for 4 h. Each point represents one cell, with at least 43 cells quantified in 3 independent experiments. Bars show the average GSD. One-way ANOVA. *p < 0.05, ***p < 0.001, ns not significant. (C) Quantification of EdU-positive mitobulbs in EdU-labeled DRP1 mutants expressing mCherry or mCherry-CLIMP63. Cells were pulsed with EdU as in (A) then the EdU was chased for 24 h where indicated. Each point represents one cell, with at least 44 cells quantified in 3 independent experiments. Bars show the average GSD. One-way ANOVA. ***p < 0.001. (D) EdU foci ratio (chase/no chase) from the experiments in (C). Each point represents an independent experiment (n = 3). Bars show the average GSD. Two-sided t-test. ns not significant.

Article Snippet: MEF cells were transiently transfected with DRP1 K38A (gift from Alexander van der Bliek & Richard Youle,52 Addgene, 45161) using Metafectene Pro (Biontex, T040) for 24 hours.

Techniques: Expressing, Mutagenesis, Control, Staining, Marker, Labeling