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. 2023 Jun 19;26(7):107180.
doi: 10.1016/j.isci.2023.107180. eCollection 2023 Jul 21.

Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation

Hema Saranya Ilamathi  1   2   3 , Sara Benhammouda  1   2   3 , Amel Lounas  4 , Khalid Al-Naemi  5 , Justine Desrochers-Goyette  1   2   3 , Matthew A Lines  6 , François J Richard  4 , Jackie Vogel  5 , Marc Germain  1   2   3
Affiliations

Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation

Hema Saranya Ilamathi et al. iScience. .

Abstract

Mitochondria are multifaceted organelles crucial for cellular homeostasis that contain their own genome. Mitochondrial DNA (mtDNA) replication is a spatially regulated process essential for the maintenance of mitochondrial function, its defect causing mitochondrial diseases. mtDNA replication occurs at endoplasmic reticulum (ER)-mitochondria contact sites and is affected by mitochondrial dynamics: The absence of mitochondrial fusion is associated with mtDNA depletion whereas loss of mitochondrial fission causes the aggregation of mtDNA within abnormal structures termed mitobulbs. Here, we show that contact sites between mitochondria and ER sheets, the ER structure associated with protein synthesis, regulate mtDNA replication and distribution within mitochondrial networks. DRP1 loss or mutation leads to modified ER sheets and alters the interaction between ER sheets and mitochondria, disrupting RRBP1-SYNJ2BP interaction. Importantly, mtDNA distribution and replication were rescued by promoting ER sheets-mitochondria contact sites. Our work identifies the role of ER sheet-mitochondria contact sites in regulating mtDNA replication and distribution.

Keywords: Biochemistry; Biological sciences; Cell biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
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 ±SD. (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 μm. (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 ±SD. (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 ±SD. (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 ±SD. ∗∗∗p < 0.001 two-sided t-test.
Figure 2
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 μm. 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 ±SD ∗∗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 ±SD ∗∗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 μm. (E) Quantification of the ERMCs length (μm) 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 ±SD. (F) Quantification of the ERMCs length (μm) 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 ±SD ∗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.
Figure 3
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 μm. 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 ±SD ∗∗∗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 μm. 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 ±SD ∗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 ±SD ∗p < 0.05 two-sided t-test.
Figure 4
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 μm. 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 ±SD ∗∗∗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 μm. 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 ±SD ∗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 ±SD ∗p < 0.05 two-sided t-test.
Figure 5
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 ±SD. (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 ±SD. Scale bars 2 μm. (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.
Figure 6
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 μm. (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 ±SD ∗∗∗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 μm. (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 ±SD. ∗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 ±SD ∗∗∗p < 0.001 two-sided t-test. (G) Quantification of the average ER sheet (Left) and ER tubule (Right) area in TEM images (Figure 2D). Each data point represents one cell. Bars represent the average of 10 cells ±SD ∗∗p < 0.01 two-sided t-test. (H) WB showing ER (CLIMP63, RTN4, RRBP1, calnexin) and mitochondrial proteins (TOM20, ATP5a) in control and DRP1 mutant human fibroblasts. Quantification is shown in the panel on the right, with data point representing expression level of the indicated proteins in DRP1 mutant cells relative to control cells for each experiment. The dashed line shows expression level in control. Bars represent the average of 3–6 experiments ±SD ∗∗∗p < 0.001 two-sided t-test relative to control cells.
Figure 7
Figure 7
ER sheets are required for proper nucleoid maintenance (A) Representative images of U2OS cells knocked down for the indicated protein and stained for the MitoTracker (mitochondria, magenta) and PicoGreen (DNA, Green). Western blots showing altered protein expression in Figure S6. (B) Quantification of the number of mitobulbs present in cells with the indicated protein knocked down by siRNA as in (A). To identify mitobulbs, cells were stained with TOM20 and TFAM. Each data point represents one cell. Bars represent the average of 30 cells in 3 independent experiments ±SD. One-way ANOVA. ∗∗∗p < 0.001. (C and D). Quantification of nucleoid size (C) and number (D) in cells as in (A). Each data point represents one cell. Bars represent the average of 30 cells in 3 independent experiments ±SD. One-way ANOVA. ∗p < 0.05, ∗∗∗p < 0.001.
Figure 8
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 ±SD. ∗∗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 μm. (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 ±SD. ∗∗∗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 ±SD. 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 ±SD. ∗∗∗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 the presence of nucleoids, K) in mCherry-Fis1 and mCherrry-SYNJ2BP expressing control and DRP1 mutant cells. Each point represents one cell, with 30 cells quantified in 3 independent experiments. Bars show the average ±SD. One-way ANOVA (I), two-sided t-test (J, K). ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 9
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 μm. (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 ±SD. 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 ±SD. 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 ±SD. Two-sided t-test. ns not significant.
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