Deficient tRNA posttranscription modification dysregulated the mitochondrial quality controls and apoptosis

Summary Mitochondria are dynamic organelles in cellular metabolism and physiology. Mitochondrial DNA (mtDNA) mutations are associated with a broad spectrum of clinical abnormalities. However, mechanisms underlying mtDNA mutations regulate intracellular signaling related to the mitochondrial and cellular integrity are less explored. Here, we demonstrated that mt-tRNAMet 4435A>G mutation-induced nucleotide modification deficiency dysregulated the expression of nuclear genes involved in cytosolic proteins involved in oxidative phosphorylation system (OXPHOS) and impaired the assemble and integrity of OXPHOS complexes. These dysfunctions caused mitochondrial dynamic imbalance, thereby increasing fission and decreasing fusion. Excessive fission impaired the process of autophagy including initiation phase, formation, and maturation of autophagosome. Strikingly, the m.4435A>G mutation upregulated the PARKIN dependent mitophagy pathways but downregulated the ubiquitination-independent mitophagy. These alterations promoted intrinsic apoptotic process for the removal of damaged cells. Our findings provide new insights into mechanism underlying deficient tRNA posttranscription modification regulated intracellular signaling related to the mitochondrial and cellular integrity.


INTRODUCTION
2][3] Mitochondrial damages due to mitochondrial DNA (mtDNA) mutations are associated with a wide spectrum of clinical abnormalities. 1 Health mitochondria in the cells are maintained by the processes of biogenesis, dynamics of fusion and fission as well as targeted degradation. 4,56][17][18][19] In fact, the organelles have their own defense pathways to respond to these genetic and environmental stressors and maintain their quality control via fusion and fission processes as well as mitophagy. 7,20,21In particular, these mitochondria dysfunctions due to mt-tRNA mutations may dysregulate the nuclear gene expression to cell function such as autophagy and apoptosis through mitochondrial retrograde signal pathways. 2,22However, the mechanism underlying deficient mt-tRNA posttranscription modification regulates the OXPHOS biogenesis, mitochondrial quality controls, and apoptosis is far less understood.
][25] In particular, the mt-tRNA Met 4435A>G mutation-induced deficient nucleotide modification altered the mt-tRNA metabolism, codon-anticodon interactions, and led to faulty mitochondrial translation, especially in the fidelity and efficiency. 24,25The impairment of mitochondrial translation impeded the activities of OXPHOS complexes, diminished mitochondrial ATP levels, reduced membrane potential, and elevated the production of reactive oxygen species (ROS). 25The m.4435A>G mutation-induced deficiencies may dysregulate the expression of nucleus-encoding mitochondrial proteins involved in mitochondrial biogenesis, autophagy, and apoptosis.In this study, we demonstrated that the m.4435A>G mutation dysregulated the expression of nuclear genes encoding OXPHOS subunits and affected the assembly and biogenesis of OXPHOS complexes.We assessed if the instability and dysfunction of OXPHOS due to the m.4435A>G mutation then mediated the mitochondrial quality control processes via fission and fusion.We then examined if the dysregulation of mitochondrial quality control process regulated the signaling pathways involved in selective degradation of damaged mitochondria and cell death machinery intrinsic apoptosis.
To test whether the m.4435A>G mutation affected the assembly of OXPHOS complexes, we measured the levels of assembly components (FOXRED1 for complex I, UQCC2 for complex III, COX16 for complex IV and ATPAF1 for complex V) in the mutant and control cell lines. 26,27As shown in Figures 1D and 1E, the levels of FOXRED1, UQCC2, COX16, and ATPAF1 in the mutant cell lines were 169%, 193%, 156, and 181% of those in the control cell lines, respectively.These indicated that m.4435A>G mutation dysregulated nucleus-encoding subunits of OXPHOS and impaired the assembly of OXPHOS complexes.

Defective assembly and activity of OXPHOS complexes
We examined the consequence of the m.4435A>G mutation-induced deficiency on the assembly and activities of OXPHOS complexes.Mitochondria isolated from various cell lines were analyzed by BN-PAGE and western blot analysis. 28,29As shown in Figure 2A, mutant cell lines bearing the m.4435A>G mutation displayed aberrant assembly of complexes I, III, IV, and V but not II.In particular, the average levels of complexes I, II, III, IV, and V in three mutant cell lines were 60%, 97%, 75%, 43%, and 67%, relative to the mean values measured in three control cell lines, respectively (Figure 2B).
We then assessed the stability and activities of complexes I, II, IV, and V using the in-gel activity assay.Mitochondrial membrane proteins isolated from various cell lines were separated by BN-PAGE and stained with specific substrates of complexes I, II, IV, and V. 29,30 Defective assembly of complexes I, IV and V were further confirmed in the mutant cell lines, as compared with control cell lines (Figures 2C and 2D).In particular, the in-gel activities of complexes I, IV and V in mutant cell lines were 62%, 56%, and 75%, relative to the average values of control cell lines, respectively.In contrast, the in-gel activities of complexes II in the mutant cell lines were comparable with those of control cell lines.

Imbalance of mitochondrial dynamics
The m.4435A>G mutation-induced mitochondrial dysfunctions may affect the mitochondrial integrity and homeostasis, which is achieved through constant fusion and fission.We used immunofluorescence to assess the effect of m.4435A>G mutation on mitochondrial morphology and dynamics using cells staining with mitochondrial dye MitoTracker and labeling with antibody of fission-related protein Drp1.As shown in Figures 3A and 3B, mutant cells bearing the m.4435A>G mutation revealed abnormal mitochondrial morphologies, including markedly increased fragments and reduced elongated network of mitochondria, as compared with control cells.As shown in Figures 3A and 3C, the immunofluorescence patterns of double-labeled cells with rabbit monoclonal antibody specific for Drp1 and Mitotracker revealed 57% increases in the levels of Drp1 in the mutant cells, compared with control cells.This indicated that the m.4435A>G mutation promoted mitochondrial fission.The levels of Drp1 in mutant and control cell lines were further confirmed by western blot analysis (Figures 3D and 3F).Furthermore, we examined the levels of two fission-related proteins (MFF and FIS1) and three fusion-related proteins (OPA1, MFN1, and MFN2) in mutant and control cell lines by western blot analysis. 31,32As shown in Figures 3D-3F, mutant cell lines displayed elevating levels of fission-related proteins but reduced levels of fusion proteins.In particular, the average levels of MFF and FIS1 in three mutant cell lines were 171% and 144% of the average values measured in three control cell lines, respectively (Figure 3E).In contrast, the average levels of OPA1, MFN1, and MFN2 in three mutant cell lines were 50%, 46%, and 58% of the mean values measured in three control cell lines, respectively (Figure 3F).These data indicated that the m.4435A>G mutation led to the mitochondrial dynamic imbalance toward promoting fission.

Promoted autophagy
Increased mitochondrial fission by the m.4435A>G mutation may facilitate autophagy.We assessed the autophagic states of mutant and control cell lines using immunoblotting and immunofluorescence assays.As shown in Figure 4A, mutant cell lines displayed markedly increases in the levels of lysosome-associated membrane glycoprotein 1 (LAMP1) and microtubule-associated protein 1A/1B light chain 3 (LC3), indicating that the m.4435A>G mutation significantly increased autophagy activity.The increasing levels of LC3 in the mutant cybirds were further confirmed by the autophagy flux using live cell imaging techniques (Figure S1).The effect of m.4435A>G mutation on autophagy was then examined by western blot analysis using three markers: LC3, P62, and Beclin-1, which involved in the initiation phase of autophagy, in mutant and control cell lines.During autophagy, the cytoplasmic form (LC3-I) is processed into a cleaved and lipidated membrane-bound form (LC3-II), which is recruited to autophagosomal membranes.The amount of LC3-II is clearly correlated with the number of autophagosomes. 33The sequestosome 1/P62 protein (SQSTM1, hereafter referred to P62), is an autophagy substrate that colocalizes with ubiquitinated protein aggregates. 34As shown in Figures 5B  and 5C, increased levels of Beclin-1 and LC3, but decreased levels of P62 were observed in mutant cell lines carrying the m.4435A>G mutation, compared with control cell lines.In particular, the average levels of Beclin-1, LC3-II, and P62 in the three mutant cell lines carrying the m.4435A>G mutation were 215% (p = 0.0061), 213% (p = 0.0026), and 73% (p = 0.0079) of the mean values measured in three control cell lines, respectively.
To examine the effect of m.4435A>G mutation on the autophagosome formation and maturation, we measured the levels of ATG5, ATG7, ATG16L1, and ATG12 in the mutant and control cell lines using western blot analysis.In fact, the activity of the conserved Atg12-Atg5-Atg16 complex is essential for autophagosome formation and membrane elongation, while ATG7 plays a central role in autophagosome biogenesis by conjugating ATG5 to ATG12. 33As illustrated in Figures 4D and 4E, various increases in the levels of ATG5, ATG7, ATG16L1, and ATG12-ATG5 were observed in the mutant cybrids, as compared with those in control cybrids.In particular, the levels of ATG5, ATG7, ATG16L1, and ATG12-ATG5 in the mutant cybrids were 148%, 143%, 158%, and 158%, respectively.These results implicated that the m.4435A>G mutation promoted the formation and maturation of autophagosome.
To further investigate the process of autophagy, transmission electron microscopy was used to monitor the autophagy and quantification of autophagic accumulation in mutant and control cells.As shown in Figure 4F, the mutant cells exhibited predominant accumulations of matured late autophagic vacuoles than the formations of early autophagic vacuoles containing morphologically intact cytoplasm, as compared with those in the control cells.These data suggest that the m.4435A>G mutation impaired autophagy, especially the defects in the formation of autolysosome.The steady-state levels of five OXPHOS complexes by blue native gel electrophoresis.Twenty micrograms of mitochondrial proteins from various cell lines were electrophoresed through a Blue Native gel, electroblotted, and hybridized with antibodies specific for subunits of five OXPHOS complexes (NDUFS2 for complex I, SDHB for complex II, UQCRC2 for complex III, COX5A for complex IV, and ATP5A a for complex V), and with TOM20 as a loading control.(B) Quantification of levels of complexes I, II, III, IV, and V in mutant and control cell lines.The calculations were based on three independent experiments.(C) In-gel activity of complexes I, II, IV, and V.The activities of OXPHOS complexes from various cell lines after BN-PAGE were measured in the presence of specific substrates [NADH and NTB for complex I, sodium succinate, phenazine methosulfate, and NTB for complex II, DAB and cytochrome c for complex IV, glycine, MgSO4, ATP, and Pb(NO3)2 for complex V]. 16D) Quantification of in-gel activities of complexes I, II, IV, and V.The calculations were based on three independent determinations in each cell line.Graph details and symbols are explained in the legend to Figure 1.

Impaired mitophagy
5][36] Mitochondrial fragmentation resulting from decreased fusion and increased fission in mitochondria is necessary for mitophagy since smaller mitochondria are more easily engulfed by autophagosomes than larger ones and require less energy to be autophagocytosed.Based on the targeting signals on damaged or superfluous mitochondria that initiate mitophagy, this process can be grouped into ubiquitination-dependent mitophagy such as PARKIN dependent and ubiquitination-independent or receptor based mitophagy including apoptosis related proteins as mitophagy receptors or inhibitor. 34To investigate the effect of the m.4435A>G mutation on dynamics of mitophagy, we performed the immunofluorescence assays using live cell imaging under normal or energy starved (absence and presence of   5A and 5B, mutant cell lines displayed markedly increased or reduced levels of LC3 in mitochondria under normal and energy starved conditions, respectively, indicating that the m.4435A>G mutation impaired the mitophagy activity.To further investigate the impact of m.4435A>G mutation on mitophagy, we evaluated the mitophagic states of mutant and control cell lines using immunofluorescence and western blot assays with proteins involved in PARKIN dependent mitophagy and ubiquitination-independent mitophagy.Upon acute mitochondrial dysfunction, PARKIN dependent mitophagy (PINK1-PARKIN Pathway) is activated by PARKIN recruitment from the cytosol to the mitochondrial surface, ultimately leading to mitophagy. 36,37Furthermore, ubiquitination-independent mitophagy was regulated by pro-apoptotic proteins BNIP3 and BNIP3-like (BNIP3L) or Nip3-like protein X (NIX) belonging to the BCL2 family. 34mmunofluorescence assays revealed increasing levels of PARKIN but decreased levels of BNIP3L in mitochondria in the mutant cybrids, as compared with those in the control cybrids, respectively (Figures 5C and 5D).The levels of PARKIN, PINK1, BNIP3, and BNIP3L in mutant and control cell lines were further evaluated by western blot analysis under normal and energy starved conditions.As shown in Figures 5E and  S2), the levels of PARKIN and PINK1 in mutant cybrids were markedly increased under normal condition but drastically decreased under energy starved condition, relative to the average values in three control cybrids, respectively.By contrast, the levels of BNIP3L in the mutant cybrids were markedly reduced in the mutant cybrids under normal and energy starved conditions (Figures 5F and S2).These data indicated that the m.4435A>G mutation upregulated the PARKIN dependent mitophagy but downregulated the ubiquitination-independent mitophagy.

Upregulated intrinsic apoptosis
Mitochondrial fission machinery actively participates in the process of intrinsic apoptosis. 38The impact of m.4435A>G mutation-induced fission on apoptotic process was examined by Annexin V/PI-based flow cytometry for cellular apoptosis, immunofluorescence and western blot assays.As shown in Figures 6A and 6B, the average ratios of Annexin V-positive cells in the mutant cell lines carrying the m.4435A>G mutation were 188% of the mean values measured in the control cell lines.As shown in Figures 6C and 6D, the immunofluorescence patterns of double-labeled cells with rabbit monoclonal antibody specific for the cytochrome c and MitoTracker displayed markedly increased levels of cytochrome c in the mutant cells, compared with control cells.The levels of cytochrome c in cytosol in mutant and control cell lines were further evaluated by fractioning the cells into mitochondrial and cytosolic fractions and western blot analysis.As shown in Figures 6D-6F, the levels of cytochrome c in the mutant cell lines were markedly increased in the total cellular, mitochondrial and cytosolic fractions, as compared with those in control cell lines.Furthermore, we examined the levels of one apoptosis inhibited protein (Bcl-xL) and eight activated proteins (BAD, BAX, uncleaved/cleaved caspases 3, 7, and 9) in mutant and control cell lines by western blot analysis. 39,40As shown in Figures 6E and 6F, the average levels of Bcl-xL, BAD, BAX, uncleaved/cleaved-caspases 3, 7, and 9 in three mutant cell lines were 35%, 183%, 141%, 125%, 180%, 141%, 175%, 134%, and 129% of the mean values measured in three control cell lines, respectively.These results strongly indicated that the m.4435A>G mutation promoted apoptotic process.

DISCUSSION
The mechanism underlying mitochondrial tRNA mutations regulated the expression of nuclear genes to maintain healthy organelle populations for cellular function is far less understood.In this study, we investigated the effect of mt-tRNA Met 4435A>G mutation on mitochondrial OXPHOS integrity and activity, and intracellular signaling related to mitochondrial morphology, autophagy and cell death.The m 1 G37 modification of mt-tRNA Met created by m.4435A>G mutation caused the instability, deficient aminoacylation and faulty codon-anticodon interactions, and thereby impaired the synthesis of 13 mtDNA-encoding OXPHOS subunits, especially in the fidelity and efficiency. 24,25As a result, the mt-tRNA Met 4435A>G mutation led to significant decline of mitochondrial functions associated with abnormalities in the respiratory chain and ATP synthesis, diminished membrane potentials and increased oxidative stress. 25In particular, the altered quality and quantity of these mtDNA encoding proteins may result in the imbalances between the increased levels of de novo protein synthesis and decreased folding capacity for the mtDNA-and nucleus-encoded OHPHOS subunits. 42In this study, mutant cells bearing the m.4435A>G mutation displayed various reductions in the nucleus encoding OXPHOS subunits (NDUFA10, NDUFC2, NDUFB8, NDUFS5, UQCRFS1, COX4, and ATP5C1 but not in other 8 subunits including 2 complex II subunits) and assemble factors of complexes I, III, IV, and V.These deficiencies gave rise to the abnormal assemble and instability of complexes I, III, IV, and V as well as intact supercomplexes observed in the mutant cell lines bearing the m.4435A>G mutation.Indeed, these mtDNA-encoded subunits appear to act as seeds for building new complexes, which requires nucleusencoding subunit import and assembly with the assistance of assembly factors and assembly factors for complexes I, III, IV, and V. 26,43 These defects yielded the reduced activities of complexes I, III, IV, and V but not complex II.Therefore, the m.4435A>G mutation dysregulated the expression of nucleus-encoding OXPHOS subunits and thereby impaired the assemble, stability, and activity of OXPHOS.(C and E) Quantification of markers of autophagy (C) and autophagosome formation and maturation associated proteins (E).Three independent determinations were done in each cell line.(F) Cells from mutant and control cybrids were examined by transmission electron microscopy of initial autophagic vacuoles (white), degradative autophagic vacuole (black); M: mitochondria; N: nucleus; P: phagophore; scale bar: 500 nm.Ultrathin sections were stained with uranyl acetate and alkaline lead citrate.50,0003 magnifications were used.Graph details and symbols are explained in the legend to Figure 1.
The m.4435A>G mutation-induced dysfunction of OXPHOS makes mitochondria undergo constant fission and fusion to repair damaged OXPHOS components, which allows segregation of damaged mitochondria via the fission process, exchange of materials between healthy mitochondria via the fusion process, and finally the elimination of damaged mitochondria via mitophagy. 37Electron microscopy studies showed the abnormalities of mitochondrial structures, including markedly increased fragments and reduced elongated network, and an imbalance between mitochondrial dynamics-related proteins in the mutant cells bearing the m.4435A>G mutation.The mutant cells carrying the m.4435A>G mutation revealed markedly increased expression of nucleus-encoding fission-related genes DRP1, FIS1, and MFN than these control cells lacking the mutation.By contrast, marked reductions in the levels of three fusion-related proteins (OPA1, MFN1, and MFN2) were observed in mutant cells harboring the m.4435A>G mutation.These strongly indicated that the m.4435A>G mutation dysregulated mitochondrial dynamics through promoting fission and reducing fusion.
Imbalance of mitochondrial dynamics and impaired membrane potentials by the m.4435A>G mutation regulated mitophagy which is a mitochondria-specific type of autophagy to dispose of damaged mitochondria.In this study, the cells bearing the m.4435A>G mutation exhibited predominant accumulations of matured late autophagic vacuoles and marked increases in the levels of LAMP1 and LC3, 44,45 The m.4435A>G mutation impaired the process of autophagy, including the initiation phase, supported by increased levels of Beclin-1 but decreased levels of P62, formation and maturation of autophagosome, evidenced by raising levels of ATG5, ATG7, ATG16L1, and ATG12-ATG5 in the cells carrying  the m.4435A>G mutation. 36,37,45The m.4435A>G mutation-induced autophagy dysregulation was consistent with increased autophagy in the cells carrying the mt-tRNA Ile 4295A>G and mt-tRNA Ala 5587T>C mutations, 18,46 suggesting that altered quality and quantity of 13 mitochondrial proteins caused by mitochondrial tRNA mutations upregulated autophagy pathway.By contrast, reduced levels of only ND1 or ND6 protein due to m.3460G>A and m.14484T>C mutations downregulated autophagy process. 41,47,48In fact, mitophagy regulatory pathways are classified as ubiquitin-dependent or -independent pathways. 49Mitochondrial depolarization activates PINK1 to recruit ubiquitin ligase, PARKIN, to mitochondria, leading to clearance, while excessive mitochondrial fission leads to depolarization-independent mitophagy. 36,45Strikingly, the m.4435A>G mutation upregulated the PARKIN dependent mitophagy, evidenced by markedly increased levels in PARKIN and PINK1 in cells carrying the m.4435A>G mutation, in contrast with reduced levels in PARKIN and PINK1 in the cells m.3460G>A or m.14484T>C mutation. 41,47urthermore, the m.4435A>G mutation downregulated the ubiquitination-independent mitophagy, suggested by the significant decreases in the BNIP3 and BNIP3L acting as mitophagy receptors and pro-apoptotic proteins in the mutant cell lines. 34,49These suggested that the m.4435A>G mutation upregulated mitophagy, especially ubiquitination-dependent pathway.
The m.4435A>G mutation-induced elevation of mitochondrial fusion and ROS production and diminished membrane potential promoted intrinsic apoptotic process for the removal of damaged cells.In this study, we showed that the m.4435A>G mutation conferred defects in the apoptosis, evidenced by 88% increased levels of Annexin V intensity.The elevated levels of pro-apoptotic proteins Bax and Bak, which translocate to mitochondria, raised the release of cytochrome c into the cytosol, evidenced by immunofluorescence and western blot assays in the cybrids.The release of cytochrome c promotes the activation of caspase-3, -7, and -9, which subsequently initiates cell death. 38,39The impact of m.4435A>G mutation on intrinsic apoptotic process was further supported by decreased levels of anti-apoptotic members including Bcl-xL and BNIP3L/NIX. 50These findings demonstrated that mitochondria dysfunctions due to the m.4435A>G mutation dysregulated the signaling pathways for mitochondrial dynamics, selective degradation of damaged mitochondria and intrinsic apoptosis.
In summary, we demonstrated that the mt-tRNA Met 4435A>G mutation dysregulated the expression of nuclear genes encoding cytosolic proteins involved in OXPHOS and impaired the assembly and activities of OXPHOS complexes.These mitochondrial dysfunctions led to the mitochondrial dynamic imbalance toward fission, elevated mitophagy via ubiquitination-dependent pathway and finally promoted intrinsic apoptosis.The broad effect of the m.44534A>G mutation on cytosolic signaling related to the mitochondrial and cellular integrity may regulate various aspects of vascular function, thereby being critical for the pathogenesis of hypertension.Thus, our findings may provide new insights into the understanding of the pathophysiology of maternally inherited hypertension.

Figure 1 .
Figure 1.Western blot analysis of mitochondrial proteins (A and D) Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with antibodies for 17 subunits (CO3 encoded by mtDNA and 16 encoded by nuclear genes) (A) 4 assembly factors (D) of OXPHOS, and TOM20 as a loading control, respectively.(B and E) Quantification of 17 subunits (B) and 4 assembly factors (E) of OXPHOS.Average relative each polypeptide content per cell was normalized to the average content per cell of TOM20 in each cell line.The values for the latter are expressed as percentages of the average values for the control cell line.(C) Average levels of subunits from each complex of OXPHOS (6 of complexes I, 2 of II, 3 of III, 3 of IV, and 3 of V).The calculations were based on three independent determinations.The error bars indicate two standard error of the mean (SEM) of the means.p indicates the significance, according to the t test, of the differences between mutant and control cell lines.*p < 0.05; **p < 0.001; ***p < 0.0001; #, not significant.

Figure 2 .
Figure 2. Defective assembly and activity of OXPHOS complexes (A)The steady-state levels of five OXPHOS complexes by blue native gel electrophoresis.Twenty micrograms of mitochondrial proteins from various cell lines were electrophoresed through a Blue Native gel, electroblotted, and hybridized with antibodies specific for subunits of five OXPHOS complexes (NDUFS2 for complex I, SDHB for complex II, UQCRC2 for complex III, COX5A for complex IV, and ATP5A a for complex V), and with TOM20 as a loading control.(B) Quantification of levels of complexes I, II, III, IV, and V in mutant and control cell lines.The calculations were based on three independent experiments.(C) In-gel activity of complexes I, II, IV, and V.The activities of OXPHOS complexes from various cell lines after BN-PAGE were measured in the presence of specific substrates [NADH and NTB for complex I, sodium succinate, phenazine methosulfate, and NTB for complex II, DAB and cytochrome c for complex IV, glycine, MgSO4, ATP, and Pb(NO3)2 for complex V].16 (D) Quantification of in-gel activities of complexes I, II, IV, and V.The calculations were based on three independent determinations in each cell line.Graph details and symbols are explained in the legend to Figure1.

Figure 3 .
Figure 3. Assessment of mitochondrial dynamics (A) Immunofluorescence analysis.The distributions of cytochrome c from mutant II-9.5 and control C59.8 cybrids were visualized by immunofluorescent staining with mitochondrial dye MitoTracker and labeling with DRP1 antibody conjugated to Alex Fluor 488 (green) analyzed by confocal microscopy.Scale bars: 10 mm.(B) Quantification of mitochondrial morphology.Mitochondrial morphology was scored as follows: fragmented, mainly small and round; normal, mixture of round and shorter tubulated; and elongated, long and higher interconnectivity.The percentage of cells with indicated mitochondrial morphologies was determined as a percentage of the total number of cells counted (R100 cells per experiment).n = 3 independent experiments.(C) Quantification of levels of DRP1 fluorescence intensity.Three independent determinations were done in each cell line.(D) Western blot analysis of mitochondrial fission-associated proteins (DRP1, MFF, and FIS1) and fusion-associated proteins (OPA1, MFN1, and MFN2) in six cell lines with b-actin as a loading control.(E) Quantification of mitochondrial fission-associated proteins (DRP1, MFF, and FIS1) and fusion-associated proteins (OPA1, MFN1, and MFN2).Three independent experiments were made for each cell line.Graph details and symbols are explained in the legend to Figure 1.

Figure 4 .
Figure 4. Analysis of autophagy (A) Immunofluorescence analysis.The distributions of LAMP1 from cybrids (C59.12 and II.9-4) were visualized by immunofluorescent labeling with LAMP1 antibody conjugated to Alex Fluor 488 (green) and LC3 antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy.DAPI-stained nuclei were shown by the blue fluorescence.Scale bars: 10 mm.

Figure 4 .
Figure 4. Continued (B and D) Western blot analysis of autophagy-associated proteins (B) and autophagosome formation and maturation associated proteins (D) in six cell lines with bactin as a loading control.(Cand E) Quantification of markers of autophagy (C) and autophagosome formation and maturation associated proteins (E).Three independent determinations were done in each cell line.(F) Cells from mutant and control cybrids were examined by transmission electron microscopy of initial autophagic vacuoles (white), degradative autophagic vacuole (black); M: mitochondria; N: nucleus; P: phagophore; scale bar: 500 nm.Ultrathin sections were stained with uranyl acetate and alkaline lead citrate.50,0003 magnifications were used.Graph details and symbols are explained in the legend to Figure1.

Figure 5 .
Figure 5. Mitophagy assays (A and B) Immunofluorescence assays using live cell imaging under normal or energy starved conditions in the absence (A) and presence (B) of HBSS.The distributions of LC3 from mutant and control cybrids were visualized by GFP-LC3 (green) and immunofluorescent staining with mitochondrial dye MitoTracker (red) analyzed by confocal microscopy.Scale bars: 10 mm.(C and D) Immunofluorescence analysis.The distributions of PARKIN and BNIP3L from mutant and control cybrids were visualized by immunofluorescent staining with mitochondrial dye MitoTracker (red) and labeling with PARKIN (C), and BNIP3L (D) antibody conjugated to Alex Fluor 488 (green) analyzed by confocal microscopy.(E and F) Western blot analysis of PARKINdependent mitophagy proteins (E) and ubiquitination-independent mitophagy proteins (F) in six cell lines with b-actin as a loading control.

Figure 6 .
Figure 6.Upregulated intrinsic apoptosis (A) Annexin V/PI apoptosis assay by flow cytometry.Cells were harvested and stained with Annexin V and 1 mL of propidium iodide.The percentage of Annexin V-positive cells were assessed.(B) Relative Annexin V-positive cells from various cell lines.Three independent determinations were done in each cell line.(C) Immunofluorescence analysis.The distributions of cytochrome c from mutant II-9.2 and control C59.8 cybrids were visualized by immunofluorescent staining with mitochondrial dye MitoTracker and labeling with cytochrome c antibody conjugated to Alex Fluor 488 (green) analyzed by confocal microscopy.DAPI stained nuclei were identified by their blue fluorescence.Scale bars: 10 mm.(D) The levels of cytochrome c in cytosol in mutant and control cell lines were measured by fractioning the cells into mitochondrial and cytosolic fractions and western blot analysis using cytochrome c, Tom20 for mitochondrial protein and Vinculin for cytosolic protein.Total, total cell lysate; Cyto, cytosol; Mito, mitochondria.(E) Western blotting analysis of apoptosis-associated proteins.Total cellular proteins (20 mg) from various cell lines were electrophoresed, electroblotted and hybridized with several apoptosis-associated protein antibodies: cytochrome c, Bcl-xL, BAD, BAX, uncleaved caspases 9, caspases 3 and caspases 7, and cleaved caspases 3, 7, and 9, with b-actin as a loading control.(F) Quantification of apoptosis-associated proteins: cytochrome c, Bcl-xL, BAD, BAX, uncleaved caspases 3, 7, and 9, and cleaved caspases 3, 7, and 9.The levels of apoptosis-associated proteins in various cell lines were determined as described elsewhere. 41Three independent experiments were made for each cell line.Graph details and symbols are explained in the legend to Figure 1.