The mitochondria-associated membrane (MAM), a crucial interface between the endoplasmic reticulum (ER) and mitochondria, plays a pivotal role in cellular physiology. This study delves into the impact of electron transport chain (ETC) dysfunction on MAM function, particularly focusing on Soat1 Electron Transport Chain interactions and their consequences in human cells. We investigated phospholipid and cholesteryl ester (CE) synthesis, key MAM-related processes, in human ρ0 cells devoid of mitochondrial DNA (mtDNA) and cybrid cell lines harboring pathogenic mtDNA mutations, as well as fibroblasts with nuclear DNA mutations affecting ETC complex I. Our findings reveal a significant disruption of MAM function in cells with impaired ETC activity, manifested as altered phospholipid metabolism, reduced cholesteryl ester synthesis via acyl-CoA:cholesterol acyltransferase 1 (ACAT1), encoded by the SOAT1 gene, and changes in ER-mitochondrial contact. These disruptions correlate with altered mitochondrial membrane potential and contribute to cellular dysfunction and cell death.
We first investigated MAM function in ρ0 cells, a model of extreme bioenergetic failure lacking a functional respiratory chain. Phospholipid synthesis and transport, well-established indicators of MAM function, were assessed by measuring the incorporation of radiolabeled L-serine (3H-Ser) into phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn). PtdSer synthesis in ρ0 cells remained comparable to respiratory-competent ρ+ control cells. However, 3H-PtdEtn synthesis and the PtdEtn/PtdSer ratio, indicative of conversion efficiency, were significantly reduced in ρ0 cells (Fig. 1B). This reduction was attributed to MAM defects rather than altered enzyme levels, as Western blot analysis showed no changes in phosphatidylserine synthase 1 (PSS1), phosphatidylserine decarboxylase (PISD), or mitochondrial mass marker TOM20 (Fig. 1C).
Fig. 1: MAM function in ρ0 cells and the role of phospholipid transfer in the context of soat1 electron transport chain.
(A) Schematic depiction of phospholipid synthesis and transport at the MAM, highlighting the involvement of both ER and mitochondria. Key enzymes and molecules are labeled: PISD (phosphatidylserine decarboxylase), PSS1 (phosphatidylserine synthase 1), PS (phosphatidylserine), PE (phosphatidylethanolamine), and Mito (mitochondria). (B) Comparative analysis of 3H-Ser incorporation into 3H-PtdSer and 3H-PtdEtn in ρ0 cells relative to control ρ+ cells (dotted line) over time (n = 4 independent experiments). The graph illustrates the significant decrease in PtdEtn synthesis in ρ0 cells compared to ρ+ cells, while PtdSer synthesis remains unchanged. The lower panel quantifies the PtdEtn/PtdSer ratio, demonstrating reduced conversion of 3H-PtdSer to 3H-PtdEtn in ρ0 cells. (C) Representative Western blot showing protein levels of phospholipid synthesis-related proteins (PSS1, PISD) and the mitochondrial marker TOM20 in ρ+ and ρ0 cells, normalized to vinculin. Molecular weight markers are indicated in kDa. Quantification on the right demonstrates similar protein levels in both cell types. (D) Confocal microscopy images of MAM (MAMtracker-Green, green) in ρ+ and ρ0 cells, scale bars = 10 μm. The images show increased MAMtracker fluorescence intensity in ρ0 cells compared to ρ+ cells. (E) Quantification of MAMtracker-Green fluorescence intensity in transfected ρ+ and ρ0 cells (n = 4 independent experiments, 7-8 cells per experiment). Data are mean ± SD, analyzed by Student’s t-test (, p* < 0.05; *, p* < 0.01; *, p < 0.001; **, p < 0.0001).
Further investigation using MAMtracker-Green, a fluorescent reporter for ER-mitochondrial proximity, revealed a significant reduction (~50%) in MAMtracker fluorescence intensity in ρ0 cells compared to ρ+ cells (Fig. 1D, E). These findings suggest that while PtdSer synthesis remains intact, the diminished transfer of PtdSer to mitochondria for PtdEtn conversion in ρ0 cells results from a decreased physical association between the ER and mitochondria.
We then evaluated cholesteryl ester synthesis, another crucial MAM function linked to soat1 electron transport chain dynamics. Acyl-CoA:cholesterol acyltransferase 1 (ACAT1), encoded by SOAT1 and enriched in MAM, catalyzes the esterification of free cholesterol to cholesteryl esters (CEs). Incubation of ρ+ and ρ0 cells with 3H-cholesterol and subsequent quantitative thin layer chromatography (TLC) revealed a significant downregulation (~50%) of ACAT1 activity in ρ0 cells (Fig. 2B), indicating a potential link between soat1 electron transport chain components and cholesterol metabolism at the MAM. Free cholesterol levels remained unchanged (Supplementary Fig. 2C).
Fig. 2: MAM function in ρ0 cells and the role of cholesteryl ester synthesis in relation to soat1 electron transport chain.
(A) Schematic representation of cholesteryl ester (CE) synthesis at the MAM, emphasizing the exclusive involvement of MAM in this process. Key components are labeled: ACAT1 (acyl-CoA:cholesterol acyltransferase 1, product of the SOAT1 gene), CE (cholesteryl ester), and LD (lipid droplet). (B) Comparison of 3H-cholesterol to 3H-CE conversion in ρ0 cells relative to ρ+ cells (dotted line) over time (n = 3 independent experiments). The graph demonstrates a significant reduction in cholesterol conversion to CE in ρ0 cells. (C) Confocal microscopy images of lipid droplets stained with LipidTox Green (green) and nuclei labeled with DAPI (blue) in ρ+ and ρ0 cells, scale bars = 45 μm. Expanded images are shown in boxes. Quantification of LipidTox Green fluorescence intensity in ρ0 cells relative to ρ+ cells (dotted line) is shown on the right (n = 4 independent experiments, >50 cells per experiment), indicating increased lipid droplet formation in ρ0 cells. (D) Quantification of LipidTox Green fluorescence intensity in ρ0 cells relative to ρ+ cells (dotted line) by flow cytometry (n = 3 independent experiments, >20000 cells per experiment), confirming increased lipid droplet formation. (E) Analysis of 3H-oleic acid conversion to 3H-cholesteryl oleate (CE) and 3H-triglycerides (TGA) in ρ0 cells relative to ρ+ cells (dotted line) after 4 hours. The graph shows increased TGA levels and decreased CE levels in ρ0 cells, consistent with panel B (n = 3 independent experiments). (F) Quantification of lipid droplet synthesis-related proteins ACAT1 and DGAT2 relative to vinculin in ρ+ and ρ0 cells (n = 3). Protein levels are similar in both cell types. (G) Lipidomic analysis of isolated lipid droplets from ρ0 cells relative to ρ+ cells (dotted line), quantifying lipid content (n = 3 independent experiments). The analysis reveals increased TGA compared to CE in ρ0 cells. (H) Heatmap representation of lipidomic analysis of crude mitochondria (containing MAM) in ρ+ and ρ0 cells (n = 3), focusing on PtdEtn, free cholesterol (FC), cholesteryl esters (CE), diacylglycerides (DGA), and triglycerides (TGA). Results are Z-scores, demonstrating lipid profile changes in ρ0 cells.
The reduced ACAT1 activity correlated with altered lipid droplet (LD) formation. Confocal microscopy and flow cytometry revealed increased lipid droplet number and fluorescence intensity in ρ0 cells (Fig. 2C, D). Further investigation into lipid droplet composition showed an increase in triglyceride (TGA) synthesis in ρ0 cells, measured by 3H-oleic acid incorporation and diacyglycerol O-acyltransferase 2 (DGAT2) activity, while CE production remained reduced (Fig. 2E). Lipidomic analysis of isolated LDs confirmed a higher TGA content compared to CE in ρ0 cells (Fig. 2G). Lipidomic analysis of the crude mitochondrial fraction, including MAM, revealed decreased PtdEtn and CE levels, and increased diacylglycerides (DGA) and triglycerides (TGA) in ρ0 cells (Fig. 2H), indicating a disrupted lipid homeostasis in the context of soat1 electron transport chain dysfunction. These combined results highlight a disruption in MAM function in ρ0 cells, affecting both phospholipid and cholesterol metabolism.
To further explore the link between MAM function and mitochondrial dysfunction, we examined cybrid cell lines with pathogenic mtDNA mutations causing Kearns-Sayre syndrome (KSS) and maternally-inherited Leigh syndrome (MILS). KSS cybrids, harboring a large mtDNA deletion (Δ-mtDNA) and lacking respiratory chain function, showed increased PtdSer synthesis but unchanged PtdEtn levels, resulting in a significantly lower PtdEtn/PtdSer ratio (Fig. 3B). MAMtracker-Green intensity was also reduced in Δ-KSS cybrids (Fig. 3C), indicating impaired ER-mitochondrial connectivity, similar to ρ0 cells.
Fig. 3: Analysis of MAM function in KSS cybrids and its relation to soat1 electron transport chain.
(A) Schematic representation of respiratory chain (R.C.) complexes compromised in KSS (red X’s), indicating the impact of KSS mutations on the electron transport chain. (B) 3H-Ser incorporation into 3H-PtdSer and 3H-PtdEtn in Δ-KSS cybrids compared to WT-KSS cybrids (dotted line) at 4 hours (n = 3). The graph highlights the altered phospholipid synthesis. Quantification of the PtdEtn/PtdSer ratio in WT-KSS and Δ-KSS cybrids is shown on the right, demonstrating reduced conversion in Δ-KSS cybrids. (C) Confocal microscopy images of MAM (MAMtracker-Green, green) in WT-KSS and Δ-KSS cybrids, scale bars = 15 μm. Quantification of MAMtracker-Green fluorescence intensity in transfected WT-KSS and Δ-KSS cybrids (as in Fig. 1E) is shown on the right, indicating decreased MAM proximity in Δ-KSS cybrids. (D) Conversion of 3H-cholesterol to 3H-CE in Δ-KSS relative to WT-KSS cybrids (dotted line) at 4 hours (n = 3). The graph shows increased ACAT activity in Δ-KSS cybrids, in contrast to ρ0 cells. (E) Confocal microscopy images of lipid droplet formation stained with LipidTox Green (green) and nuclei with DAPI (blue) in WT-KSS and Δ-KSS cybrids, scale bars = 45 μm. Expanded images in boxes. Quantification of LipidTox Green fluorescence intensity (as in Fig. 2C) is shown on the right, demonstrating increased lipid droplets in Δ-KSS cybrids. (F) Quantification of LipidTox Green fluorescence intensity in Δ-KSS cybrids compared to WT-KSS cybrids (dotted line) by flow cytometry (as in Fig. 2D), confirming increased lipid droplets. (G) Conversion of 3H-oleic acid to 3H-cholesteryl oleate (CE) and 3H-triglycerides (TGA) in Δ-KSS cybrids compared to WT-KSS cybrids (dotted line) at 4 hours (n = 3). The graph shows increased levels of both lipid species in Δ-KSS cells. (H) Representative Western blot of phospholipid synthesis-related proteins (PSS1, PISD), LD-related proteins (ACAT1, DGAT2), and mitochondria marker (TOM20) in WT-KSS and Δ-KSS cybrids, normalized to vinculin. Protein levels remain unchanged in both cell types.
Interestingly, ACAT activity was significantly increased in Δ-KSS cybrids (Fig. 3D), opposite to the finding in ρ0 cells. Consistent with this, Δ-KSS cybrids exhibited numerous lipid droplets (Fig. 3E, F). Incorporation of 3H-oleic acid into both CE and TGAs was increased in Δ-KSS cells (Fig. 3G). Enzyme levels remained unchanged (Fig. 3H). These data suggest that despite similarities in phospholipid transport defects and MAM disruption, the impact on soat1 electron transport chain dysfunction and cholesterol metabolism differs between ρ0 and KSS cells.
In contrast, MILS cybrids with the T8993G mutation in ATPase6, affecting ATP synthesis but not the respiratory chain, showed increased PtdSer and PtdEtn synthesis and an enhanced PtdSer to PtdEtn conversion (Fig. 4B). MAMtracker-Green intensity was also increased (Fig. 4C), indicating enhanced ER-mitochondrial apposition. ACAT activity was decreased in MILS cybrids (Fig. 4D), similar to ρ0 cells, but lipid droplet formation was increased (Fig. 4E, F), likely due to TGA accumulation rather than CE (Fig. 4G). Enzyme levels were unaltered (Fig. 4H).
Fig. 4: Analysis of MAM function in MILS cybrids and its relation to soat1 electron transport chain.
(A) Schematic representation of the T8993G mutation in ATPase6 causing NARP and MILS, highlighting that in these cells, the respiratory chain remains intact, while ATP synthesis is compromised, contrasting with the soat1 electron transport chain defects in other models. (B) 3H-Ser incorporation into 3H-PtdSer and 3H-PtdEtn in MILS cybrids compared to WT-MILS cybrids (dotted line) at 4 hours (n = 4). The graph illustrates increased phospholipid synthesis in MILS cybrids. Quantification of the PtdEtn/PtdSer ratio is shown on the right, demonstrating enhanced conversion in MILS cybrids. (C) Confocal microscopy images of MAM (MAMtracker-Green, green) in WT-MILS and MILS cybrids, scale bars = 15 μm. Quantification of MAMtracker-Green fluorescence intensity in transfected WT-MILS and MILS cybrids (n = 4 independent experiments, 7-8 cells per experiment) is shown on the right, indicating increased MAM proximity in MILS cybrids. (D) Conversion of 3H-cholesterol to 3H-CE in MILS relative to WT-MILS cybrids (dotted line) at 4 hours (n = 3). The graph shows decreased ACAT activity in MILS cells, similar to ρ0 cells. (E) Confocal microscopy images of lipid droplet staining with LipidTox Green (green) and nuclei with DAPI (blue) in WT-MILS and MILS cybrids, scale bars = 45 μm. Expanded images in boxes. Quantification is shown on the right (as in Fig. 2C), demonstrating increased lipid droplets in MILS cybrids. (F) Quantification of LipidTox Green fluorescence intensity in MILS cybrids compared to WT-MILS cybrids (dotted line) by flow cytometry (as in Fig. 2D), confirming increased lipid droplets. (G) Conversion of 3H-oleic acid to 3H-cholesteryl oleate (CE) and 3H-triglycerides (TGA) in MILS cybrids compared to WT-MILS cybrids (dotted line) at 4 hours (n = 3). The graph shows increased TGA accumulation but not CE in MILS cells. (H) Representative Western blot of phospholipid- and LD-related proteins in WT-MILS and MILS cybrids (as in Fig. 3H). Protein levels remain unchanged in both cell types.
Fibroblasts with a mutation in the nucleus-encoded NDUFS4 subunit of complex I, also affecting ATP production, showed decreased PtdSer and PtdEtn synthesis and reduced MAMtracker-Green intensity (Fig. 5B, C). ACAT activity was increased in NDUFS4 fibroblasts (Fig. 5D), similar to KSS cybrids, but lipid droplet formation was decreased (Fig. 5E, F). 3H-oleic acid incorporation into TGA decreased, while CE incorporation increased (Fig. 5G). Enzyme levels were unchanged (Fig. 5H).
Fig. 5: Analysis of MAM function in NDUFS4-mutant fibroblasts and the context of soat1 electron transport chain.
(A) Schematic representation of the mutation in the nucleus-encoded NDUFS4 subunit of complex I, illustrating a different genetic origin of soat1 electron transport chain dysfunction. (B) 3H-Ser incorporation into 3H-PtdSer and 3H-PtdEtn in control fibroblasts (C1, C2, C3) and NDUFS4 fibroblasts at 4 hours (n = 3). The graph shows decreased phospholipid synthesis in mutant fibroblasts. Quantification of the PtdEtn/PtdSer ratio is shown on the right, indicating no change in conversion efficiency. (C) Confocal microscopy images of MAM (MAMtracker-Green, green) in control and NDUFS4 fibroblasts, scale bars = 15 μm. Quantification is shown on the right (as in Fig. 2C), demonstrating decreased MAM proximity in NDUFS4 fibroblasts. (D) Conversion of 3H-cholesterol to 3H-CE in NDUFS4 fibroblasts relative to control (dotted line) at 4 hours (n = 3). The graph shows increased ACAT activity in NDUFS4 fibroblasts. (E) Confocal microscopy images of lipid droplet staining with LipidTox Green (green) and nuclei with DAPI (blue) in control and NDUFS4 fibroblasts, scale bars = 45 μm. Quantification is shown on the right (as in Fig. 2C), demonstrating decreased lipid droplets in NDUFS4 fibroblasts. (F) Quantification of LipidTox Green fluorescence intensity in NDUFS4 fibroblasts relative to control (dotted line) (as in Fig. 2D). (G) Conversion of 3H-oleic acid into 3H-cholesteryl oleate (CE) and 3H-triglycerides (TGA) in mutant NDUFS4 fibroblasts compared to control (dotted line) at 4 hours (n = 3). The graph shows increased CE accumulation but decreased TGA in mutant fibroblasts. (H) Representative Western blot (as in Fig. 3H) in control and NDUFS4 fibroblasts. Protein levels remain unchanged.
Pharmacological inhibition of ETC complexes in ρ+ cells revealed that rotenone (CI inhibitor), antimycin (CIII inhibitor), and cyanide (CIV inhibitor) reduced PtdSer to PtdEtn conversion, suggesting disrupted ER-mitochondrial connectivity. Atpenin A5 (CII inhibitor) had no effect (Fig. 6B). Oligomycin (CV inhibitor) increased PtdSer levels but not PtdEtn, while uncouplers FCCP and BAM15 decreased PtdEtn synthesis (Fig. 6D).
Fig. 6: MAM function in R.C. complexes and the role of membrane potential in relation to soat1 electron transport chain.
(A) Schematic representation of specific R.C. inhibitors used in the study, highlighting their targets within the electron transport chain. (B) 3H-Ser incorporation into 3H-PtdSer and 3H-PtdEtn in ρ+ cells exposed to R.C. inhibitors compared to untreated cells (dotted line) at 6 hours (n = 3). Quantification of PtdEtn/PtdSer is shown on the right, demonstrating reduced phospholipid transport with CI, CIII, and CIV inhibition, but not CII. (C) Schematic representation of OxPhos inhibitors (oligomycin for CV) and uncouplers (FCCP, BAM15) used. (D) 3H-Ser incorporation into 3H-PtdSer and 3H-PtdEtn in ρ+ cells exposed to oligomycin and uncouplers (dotted line) at 6 hours (n = 3). Quantification of PtdEtn/PtdSer is shown on the right, indicating varied effects on phospholipid transport. (E) Quantification of ATP-linked OCR in WT-MILS and mut-MILS cybrids (n = 3), showing decreased ATP production in mut-MILS cybrids. (F) Mitochondrial membrane potential (MMP) quantification using TMRM in ρ+ cells exposed to R.C. inhibitors (n = 3). The graph shows reduced MMP with CI, CIII, and CIV inhibition, but minimal effect with CII. (G) MMP quantification in ρ+ cells exposed to oligomycin and uncouplers (n = 3). Oligomycin increases MMP, while uncouplers depolarize mitochondria. (H) MMP quantification in ρ0 cells, KSS cybrids, MILS cybrids, and their respective controls (n = 3). The graph demonstrates lower MMP in ρ0 and KSS cells, and higher MMP in MILS cybrids. (I) MMP quantification in control and NDUFS4 fibroblasts (n = 3), showing decreased MMP in NDUFS4 fibroblasts. (J) 3H-Ser incorporation into 3H-PtdSer and 3H-PtdEtn in mut-MILS cybrids exposed to uncouplers compared to untreated mut-MILS cybrids at 6 hours (n = 3). Quantification of PtdEtn/PtdSer is shown on the right, demonstrating reduced phospholipid transport with uncoupler treatment. (K) Confocal microscopy images of MAM (MAMtracker-Green, green) in mut-MILS cells untreated or treated with FCCP, scale bars = 15 μm. Quantification is shown on the right (as in Fig. 2E), demonstrating decreased MAM proximity with uncoupler treatment.
Mitochondrial membrane potential (MMP) measurements revealed that inhibitors of complexes I, III, and IV reduced MMP, while complex II inhibition had little effect. Oligomycin increased MMP, and uncouplers decreased MMP (Fig. 6F, G). ρ0 cells and KSS cybrids showed lower MMP, MILS cybrids showed higher MMP, and NDUFS4 fibroblasts showed lower MMP compared to controls (Fig. 6H, I). Uncoupler treatment of MILS cybrids reduced both phospholipid synthesis and MMP, decreasing ER-mitochondrial apposition (Fig. 6J, K). These results suggest a strong correlation between MMP and ER-mitochondrial connectivity, linking soat1 electron transport chain activity to MAM function.
Overexpression of Mitofusin 2 (Mfn2), an ER-mitochondria tether, in ρ0 cells and Δ-KSS cybrids restored MMP and PtdEtn synthesis to near-normal levels (Fig. 7A, B, D, E). MAMtracker-Green intensity also increased upon Mfn2 overexpression (Fig. 7C, F), further confirming the link between ER-mitochondrial connectivity, MMP, and MAM function in the context of soat1 electron transport chain disruptions.
Fig. 7: Increased MMP reverses deficiencies in ER-mitochondrial communication, highlighting the soat1 electron transport chain connection.
(A) MMP quantification in ρ0 mock-transfected or Mfn2-transfected cells compared to ρ+ cells (dotted line) (n = 3). The graph shows increased MMP in Mfn2-expressing ρ0 cells. (B) 3H-Ser incorporation into 3H-PtdSer and 3H-PtdEtn in Mfn2-expressing ρ0 cells compared to mock-transfected ρ0 cells (n = 3). The graph demonstrates restored PtdEtn synthesis in Mfn2-expressing ρ0 cells. (C) Confocal microscopy images of MAM (MAMtracker-Green, green) in mock-transfected and Mfn2-transfected ρ0 cells, scale bars = 20 μm. Quantification is shown on the right (as in Fig. 2E), demonstrating increased MAM proximity in Mfn2-expressing ρ0 cells. (D) MMP quantification in Δ-KSS cybrids mock-transfected or Mfn2-transfected compared to WT-KSS cells (dotted line) (n = 3). The graph shows increased MMP in Mfn2-expressing Δ-KSS cybrids. (E) 3H-Ser incorporation into 3H-PtdSer and 3H-PtdEtn in Mfn2-expressing Δ-KSS cells compared to mock-transfected Δ-KSS cells (n = 3). The graph demonstrates increased phospholipid synthesis in Mfn2-expressing Δ-KSS cells. (F) Confocal microscopy images of MAM (MAMtracker-Green, green) in mock-transfected and Mfn2-transfected Δ-KSS cells, scale bars = 20 μm. Quantification is shown on the right (as in Fig. 2E), demonstrating increased MAM proximity in Mfn2-expressing Δ-KSS cells.
Finally, we investigated the impact of MAM disruption on cell survival. ρ0 cells, Δ-KSS, and MILS cybrids showed reduced cell viability (Fig. 8A). Cytotoxicity was increased in ρ0 and Δ-KSS cells (Fig. 8B). Caspase activity was reduced in ρ0 cells but increased in Δ-KSS and MILS cybrids (Fig. 8C). NDUFS4 fibroblasts also showed reduced viability and increased apoptosis (Fig. 8D, F). Mfn2 overexpression in ρ0 cells and Δ-KSS cybrids restored cell viability and reduced cytotoxicity (Fig. 8G, H, J, K). Apoptosis was also modulated by Mfn2 overexpression (Fig. 8I, L).
Fig. 8: Alterations in MAM have consequences for cell survivability, potentially mediated by soat1 electron transport chain dysfunction.
(A) Cell viability quantification in ρ0, Δ-KSS, and MILS cells relative to WT counterparts (dotted line) (n = 3 independent experiments), showing reduced viability in mutant cells. (B) Cytotoxicity quantification in ρ0, Δ-KSS, and MILS cells relative to WT counterparts (dotted line) (n = 3 independent experiments), showing increased cytotoxicity in ρ0 and Δ-KSS cells. (C) Apoptosis quantification in ρ0, Δ-KSS, and MILS cells relative to WT counterparts (dotted line) (n = 3 independent experiments), showing reduced apoptosis in ρ0 and increased apoptosis in Δ-KSS and MILS cybrids. (D) Cell viability quantification in NDUFS4 and control fibroblasts (n = 3 independent experiments), showing reduced viability in NDUFS4 fibroblasts. (E) Cytotoxicity quantification in NDUFS4 and control fibroblasts (n = 3 independent experiments), showing no significant change. (F) Apoptosis quantification in NDUFS4 and control fibroblasts, showing increased apoptosis in NDUFS4 fibroblasts. (G) Cell viability quantification in mock-transfected and Mfn2-transfected ρ0 cells compared to ρ+ cells (dotted line) (n = 3), showing restored viability in Mfn2-expressing ρ0 cells. (H) Cytotoxicity quantification in mock-transfected and Mfn2-transfected ρ0 cells compared to ρ+ cells (dotted line) (n = 3), showing restored cytotoxicity levels. (I) Apoptosis quantification in mock-transfected and Mfn2-transfected ρ0 cells compared to ρ+ cells (dotted line) (n = 3), showing restored apoptosis levels. (J) Cell viability quantification in mock-transfected and Mfn2-transfected Δ-KSS cybrids compared to WT-KSS cells (n = 3), showing restored viability in Mfn2-expressing Δ-KSS cybrids. (K) Cytotoxicity quantification in mock-transfected and Mfn2-transfected Δ-KSS cybrids compared to WT-KSS cells (n = 3), showing restored cytotoxicity levels. (L) Apoptosis quantification in mock-transfected and Mfn2-transfected Δ-KSS cybrids compared to WT-KSS cells (n = 3), showing restored apoptosis levels.
In conclusion, our findings demonstrate a critical link between soat1 electron transport chain function, mitochondrial membrane potential, and MAM integrity. Disruption of the ETC leads to MAM dysfunction, impacting phospholipid and cholesterol metabolism, ER-mitochondrial communication, and ultimately cell survival. The modulation of ER-mitochondrial connectivity via Mfn2 highlights the therapeutic potential of targeting MAM function in mitochondrial disorders. Further research is warranted to fully elucidate the molecular mechanisms underlying the soat1 electron transport chain – MAM axis and its implications for human health and disease.
