Mitochondrial nucleoid trafficking regulated by the inner-membrane AAA-ATPase ATAD3A modulates respiratory complex formation

Significance

Mitochondria are dynamic organelles that continually undergo fusion and fission, processes that are critical for mitochondrial function. Mitochondria also contain their own DNA (mtDNA), which is assembled into many punctate structures called “nucleoids.” We previously reported that the dynamic features of nucleoids are regulated in cooperation with mitochondrial fission; however, the molecular details of nucleoid dynamics and their role in mitochondrial function remain poorly understood. Here, we show a mechanism for mitochondrial activation via nucleoid dynamics. ATAD3A is essential for nucleoid movement, which determines nucleoid morphology in collaboration with mitochondrial fission. The distribution of nucleoids throughout the mitochondrial network activates mtDNA expression and increases respiratory complex formation, which may enable the development of new approaches to activate mitochondria.

Abstract

Mitochondria have their own DNA (mtDNA), which encodes essential respiratory subunits. Under live imaging, mitochondrial nucleoids, composed of several copies of mtDNA and DNA-binding proteins, such as mitochondrial transcription factor A (TFAM), actively move inside mitochondria and change the morphology, in concert with mitochondrial membrane fission. Here we found the mitochondrial inner membrane-anchored AAA-ATPase protein ATAD3A mediates the nucleoid dynamics. Its ATPase domain exposed to the matrix binds directly to TFAM and mediates nucleoid trafficking along mitochondria by ATP hydrolysis. Nucleoid trafficking also required ATAD3A oligomerization via an interaction between the coiled-coil domains in intermembrane space. In ATAD3A deficiency, impaired nucleoid trafficking repressed the clustered and enlarged nucleoids observed in mitochondrial fission-deficient cells resulted in dispersed distribution of small nucleoids observed throughout the mitochondrial network, and this enhanced respiratory complex formation. Thus, mitochondrial fission and nucleoid trafficking cooperatively determine the size, number, and distribution of nucleoids in mitochondrial network, which should modulate respiratory complex formation.

Mitochondria are believed to be derived from the endosymbiosis of bacteria and contain their own DNA (mtDNA) within the mitochondrial matrix (1, 2). Under fluorescence microscopy, more than 1,000 copies per cell of mtDNA are observed as hundreds of dot-like structures known as mitochondrial nucleoids (3–7), which might represent a type of microdomain inside mitochondria. Proteomic analysis has identified various nucleoid-related proteins—such as mitochondrial transcription factor A (TFAM), DNA polymerase γ, twinkle, mtSSB, and M19 (8–10)—which have roles in mtDNA replication, transcription, and translation. TFAM was originally identified as a homolog of bacteria-type transcriptional factor A. In TFAM-repressed cells, not only mitochondrial transcription but also mtDNA copy number and nucleoid structures are impaired, leading to respiratory dysfunction. Purified TFAM is packaged with mtDNA to form nucleoid structures in vitro (10). Crystal structure analysis of the TFAM-DNA complex revealed that two HMG boxes of TFAM bind to and bend DNA (11), which affects the secondary structure of mtDNA, although the detailed roles of TFAM in terms of nucleoid dynamics in living cells are less well understood.

ATPase family AAA domain-containing protein 3A (ATAD3A) is a mitochondrial inner-membrane (IM)–anchored protein and a causative gene product of mitochondrial disease (12). ATAD3A was also identified as a mitochondrial nucleoid protein (13–15). A previous report showed that when ATAD3A was repressed by antisense RNA treatment, visible nucleoids disappeared without affecting mtDNA copy number, suggesting that ATAD3A is essential for the formation of nucleoid structures (14). ATAD3A is a multifunctional protein that plays roles in tumor progression, lipid homeostasis, assembly of respiratory complexes, and cristae formation (16–21). However, the detailed molecular function of ATAD3A in nucleoid morphology remains unclear.

Mitochondria are highly dynamic organelles that undergo active fusion and fission and have various important roles in mitochondrial and cellular signaling (22, 23). Live imaging has also revealed that nucleoids dynamically move along mitochondria (24–26). We previously reported that nucleoids in cells deficient for the mitochondrial fission factor dynamin-related protein 1 (Drp1) actively move in elongated mitochondria, and many nucleoids cluster together, resulting in the enlargement of nucleoids (24). Fluorescence microscopy analysis of nucleoids also revealed that mitochondrial fission factor (Mff), an outer-membrane (OM) receptor that recruits Drp1 to mitochondrial fission sites, is located in the vicinity of nucleoids; thus, mitochondrial fission tends to occur next to nucleoids, which should prevent nucleoid interactions and clustering, thereby maintaining many small nucleoids throughout mitochondria. However, it is also unknown how Drp1 outside the OM recognizes mitochondrial nucleoids beyond the mitochondrial double membrane. We also found that the nucleoid clustering in mitochondrial fission-deficient cells leads to the formation of bulb-like enlarged mitochondria with developed cristae, named mito-bulbs (24). Furthermore, in tissue sections of Drp1 knockout (KO) cardiomyocytes, as well as in Drp1 KO HeLa cells, nucleoids accumulate locally in the enlarged mito-bulbs, and in these cells, we frequently observed the respiratory impaired mitochondria that often lost nucleoids (27, 28). However, the physiological roles of the dynamics and distribution of nucleoids in mitochondrial and cellular functions are not well understood.

To understand the molecular mechanisms of nucleoid morphogenesis, we analyzed ATAD3A and found that the dynamic movements of nucleoids are regulated by ATAD3A via direct interaction of its ATPase domain with TFAM. We also found that the nucleoid clustering observed in mitochondrial fission-deficient cells occurs by the active trafficking of nucleoids, which is dependent on the ATPase and coiled-coil domains of ATAD3A. Regulation of nucleoid dynamics is crucial for the maintenance of respiratory complexes on the mitochondrial IM, and thus, this report of a role for ATAD3A in mitochondrial nucleoid dynamics is unique.

Results

IM-Anchored ATAD3A Interacts with Mff in the OM and TFAM in the Mitochondrial Matrix.

We previously reported that mitochondrial fission tends to occur next to mtDNA nucleoids, although how cytoplasmic Drp1 recognizes mtDNA inside the mitochondrial double membrane is less well understood. Here, we focused on ATAD3A because it is reported to be anchored to the mitochondrial IM and exposed to the intermembrane space (IMS) and matrix, and functions in mitochondrial fission and nucleoid morphology (20, 29). To investigate the relationship of ATAD3A with nucleoids and mitochondrial fission, we performed coimmunoprecipitation experiments (model in Fig. 1A). HeLa cells were cotransfected with myc-tagged human ATAD3A and TFAM, Drp1, or Mff, and these cells were chemically cross-linked, immuno-isolated using antibodies, and analyzed by immunoblotting. Exogenously expressed ATAD3A-myc was coprecipitated with TFAM (Fig. 1B) and Mff (Fig. 1C), but not with Drp1 (Fig. 1C). Endogenous Mff and TFAM were also coprecipitated with ATAD3A-myc (Fig. 1D). These results suggest that ATAD3A has the potential to form a link between mitochondrial fission factors on the OM and mtDNA nucleoids in the mitochondrial matrix beyond the IM (model in Fig. 1A).

Fig. 1.

AAA-ATPase domain of ATAD3A interacts with nucleoids in the mitochondrial matrix. (A) Schematic representation of ATAD3A, nucleoids, and mitochondrial fission factors. (B–D) Binding of ATAD3A and TFAM, Mff, or Drp1 in HeLa cells that were transfected with the indicated plasmids. The cells were treated with 1% formaldehyde for 10 min and immunoprecipitation was performed using the cell lysates. The precipitates were analyzed by immunoblotting. (E) HeLa cells were treated with siRNA for Drp1 or control for 96 h. Confocal images of nucleoids and endogenous ATAD3A by immunofluorescence staining of each cell. Insets show a magnified image of each panel. Red, ATAD3A; green, mtDNA. (Scale bars: 10 μm in large panels and 2 μm in Insets.) (F) Confocal images of HA and Tom20 by immunofluorescence staining of HeLa cells transiently expressing C-terminally tagged ATAD3A under conditions in which only the OM was permeabilized (0.04% digitonin) or the OM and IM were both permeabilized (0.2% digitonin). (Scale bar, 10 μm.) (G) Confocal images of the C terminus of ATAD3A, N terminus of ATAD3A, and Tom22 were visualized by immunofluorescence staining in the presence of 0.04% or 0.2% digitonin. (Scale bars, 10 μm.) (H and I) Schematic representation of ATAD3A (H) and TFAM (I) constructs. (J and K) The purified GST-fused proteins and the purified mCherry-fused proteins were separated by SDS/PAGE and stained with Coomassie Brilliant blue (CBB). (L and M) Interaction between ATAD3A and TFAM constructs measured by an on-beads Halo assay. See SI Appendix, Fig. S1C. The signals of mCherry-fused TFAM on GST-ATAD3A (245–586) beads in the presence of ATP or ADP were quantified (M). Steel-Dwass test was performed using Statcel4 software. **P < 0.01. Data are presented as mean ± SD.

Next, we confirmed the localization of ATAD3A by fluorescence microscopy. Immunostaining revealed that ATAD3A was dispersed in all mitochondria, but were not enriched at mitochondrial fission sites with Drp1 (SI Appendix, Fig. S1A) or with nucleoids (Fig. 1E, “cont”). Furthermore, in Drp1-deficient cells, ATAD3A did not colocalize with enlarged nucleoids inside mito-bulbs (24) (Fig. 1E, “Drp1 KD”), suggesting that ATAD3A was not concentrated inside cristae but instead should localize mainly to the inner boundary membrane next to the OM. These data suggest that ATAD3A is not a major stable component of nucleoids. ATAD3A has a central hydrophobic domain that is anchored in the IM (20). From structural prediction according to the amino acid sequence of ATAD3A, there are two coiled-coil domains at the N terminus and an AAA-ATPase domain at the C terminus (Fig. 1 A, F, and G) (20, 29). To examine the membrane topology of ATAD3A in the IM, we performed immunofluorescent staining using an antigen-tag or specific antibodies against its N or C terminus under OM-permeabilized conditions with digitonin, and confirmed that the N terminus containing the coiled-coil domains of ATAD3A was located outside the IM, while the C terminus containing the ATPase domain was located inside the IM (Fig. 1 F and G). Thus, ATAD3A has the potential to interact with matrix proteins such as TFAM and OM proteins such as Mff (model in Fig. 1A).

To analyze the direct interaction of ATAD3A with TFAM biochemically and to examine the domains required for this interaction, we expressed truncated forms of ATAD3A and TFAM in bacteria, and their association was analyzed in vitro (Fig. 1 H–M). TFAM contains two HMG box domains (HMG box A and B) for mtDNA binding and packaging, and a C-terminal domain that enhances transcription (9, 11). Full-length or several truncated forms of TFAM fused with a His-tag and mCherry (model in Fig. 1I) were expressed in Escherichia coli and purified using Ni-chelating beads (Fig. 1K). Full-length TFAM, TFAM without the C-terminal domain, and with HMG box A, but not HMG box B, interacted with DNA and formed higher molecular weight complexes (SI Appendix, Fig. S1B), suggesting these constructs have the potential to package mtDNA to form nucleoids. From human ATAD3A cDNA, the N-terminal half (IMS domain: 1 to 250) or C-terminal half (matrix domain: 245 to 586) was expressed in E. coli as GST-fusion proteins (model in Fig. 1H). These constructs were successfully expressed (Fig. 1J) and recovered with glutathione beads. However, these proteins were unstable and prone to aggregation after extraction from the beads. Thus, we examined their interactions under fluorescence microscopy to observe mCherry fluorescence fused with TFAM variants on glutathione beads coated with the GST-fused ATAD3A variants.

Using these proteins, we found that the C-terminal AAA domain of ATAD3A interacted efficiently with TFAM (Fig. 1L and SI Appendix, Fig. S1C). TFAM without the C-terminal domain slightly but clearly interacted with the C-terminal AAA domain of ATAD3A, suggesting that the C-terminal domain of TFAM enhances not only transcription but also its interaction with ATAD3A. These results indicate that ATAD3A interacts directly with nucleoids via its C-terminal AAA-ATPase domain, consistent with the membrane topology of ATAD3A; the AAA-ATPase domain is exposed in the mitochondrial matrix, where mtDNA is located (Fig. 1 F and G).

ATAD3A Regulates the Size and Number of mtDNA Nucleoids.

Next, we examined the effect of ATAD3A knockdown (KD) by RNA interference (RNAi) on the morphology and distribution of mitochondria and nucleoids. Of the three human ATAD3 genes (ATAD3A, -B, and -C), ATAD3A is the major variant expressed in HeLa cells (SI Appendix, Fig. S2A). We transfected HeLa cells with small interfering RNA (siRNA) designed to target both ATAD3A and -B. In ATAD3 KD HeLa cells, immunoblotting showed that the protein levels of ATAD3 variants were severely repressed (SI Appendix, Fig. S2A). ATAD3A repression was reported to cause the loss of mtDNA nucleoids (14); however, when we examined mtDNA nucleoids carefully using anti-DNA (Fig. 2A) or anti-TFAM antibodies (SI Appendix, Fig. S2B), we observed remarkably smaller signals of nucleoids in ATAD3 KD cells under fluorescence microscopy. Interestingly, the number of nucleoids in ATAD3 KD cells was increased compared with control cells (Fig. 2A and SI Appendix, Fig. S2B). The copy number of mtDNA was comparable to that of control cells (Fig. 2D), suggesting that ATAD3A is not essential for the maintenance of mtDNA copy number, consistent with a previous report (14), but is essential to maintain the size and number of nucleoids.

Fig. 2.

ATAD3A has a role in mitochondrial nucleoid trafficking, and is critical for the regulation of the size and number of mtDNA nucleoids. (A and B) Confocal images of nucleoids and mitochondria by immunofluorescence staining. Insets show a magnified image of each panel. Red, cytochrome c; green, mtDNA. The proportion of cells containing nucleoid clustering was measured by mtDNA staining (n > 100). (Scale bar: 10 μm in large panels and 2 μm in Insets.) (C) HeLa cells were treated with Drp1 siRNA and/or ATAD3 siRNA for 96 h. Protein levels were determined by immunoblotting using the indicated antibodies. (D) mtDNA content was determined by quantitative PCR. The relative amount of mtDNA (317–381, 65 bp) per nuclear gene (β2M, 95 bp) is shown. (E–H) HeLa cells stably expressing mitochondria-targeted DsRed (mitRFP:red) were treated with the indicated siRNA and stained with SYBR Green I (green) for 5 min and live-cell images were obtained by spinning-disk confocal fluorescence microscopy. Images were taken every 5 s for 10 min. Arrowheads in E–H indicate randomly selected and tracked nucleoids in a part of live cell imaging shown in Movies S1–S4. The color of each arrowhead corresponds to the color of each trajectory in I. (Scale bar, 1 μm.) (I) Trajectories of nucleoids in E–H are shown. The color of each trajectory corresponds to the arrowhead of each color in E–H. (Scale bar, 1 μm.) Also see Movie S5. (J) The moving distance migrated in 10 min of each randomly selected nucleoid is shown. Tukey–Kramer multiple comparisons test was performed using Statcel4 software. *P < 0.05, **P < 0.01. Data are represented as mean ± SD in all graphs.

When we analyzed mitochondrial morphology in ATAD3 KD cells, the mitochondria formed a fine mesh network with more branches, compared with the simpler network seen in control KD cells (Fig. 2A and SI Appendix, Fig. S2 B and D). Next, we focused on mitochondrial dynamics by fusion and fission because we previously reported that impaired mitochondrial dynamics causes nucleoid deformation (24, 28). To analyze mitochondrial dynamics quantitatively, we measured the diffusion rates of photoactivatable GFP (PAGFP) localized in mitochondria after photoactivation (SI Appendix, Fig. S2C) (30). As previously reported, the fluorescence intensity of mito-PAGFP was highly maintained in mitochondrial fusion-deficient cells (optic atrophy 1 [OPA1] KD). In contrast, the diffusion of mito-PAGFP fluorescence was increased in mitochondrial fission-deficient cells (Drp1 KD). However, the diffusion rate was almost comparable between ATAD3 KD cells and control cells. Furthermore, the protein levels of fusion and fission factors were not affected in ATAD3 KD cells (SI Appendix, Fig. S2A). These data suggest that ATAD3A is indispensable for mitochondrial fusion and fission, inconsistent with a previous report (29); thus, the presence of deformed nucleoids in ATAD3 KD cells is not caused by impaired mitochondrial dynamics through alternation of the balance of fusion and fission. Because ATAD3A interacts with TFAM and Mff (Fig. 1 B–D), it has the potential to link nucleoids and mitochondrial fission. However, in ATAD3A-repressed cells, most of the Drp1 and Mff puncta were observed next to nucleoids (SI Appendix, Fig. S3). These data suggest that ATAD3A is not essential for marking mitochondrial fission sites beside mtDNA nucleoids.

ATAD3A Is Required for Nucleoid Clustering in Drp1-Deficient Cells.

We further investigated how ATAD3A affects nucleoid morphology and distribution. It was previously reported that nucleoids accumulate and are highly enlarged in bulb-like mitochondria in mitochondrial fission-deficient cells (24) (Fig. 2 B and C, “Drp1 KD”), and corepression with a mitochondrial fusion factor should rescue the deformed nucleoid structures in Drp1-deficient cells (SI Appendix, Fig. S2D, “Drp1+mitofusin 1 [Mfn1]+Mfn2 triple KD [TKD]”), suggesting that the size and distribution of nucleoids are controlled under a balance between fusion and fission. Here, we found that repression of ATAD3A rescued the nucleoid enlargement observed in Drp1-deficient cells and resulted in the formation of many small nucleoids, as observed in control cells (Fig. 2 B and C, “Drp1+ATAD3 double KD [DKD]”). However, mitochondrial membrane dynamics were not affected by codepletion of ATAD3 and Drp1, as analyzed by photoactivation experiments (SI Appendix, Fig. S2C). In fusion/fission double-deficient cells (Drp1+Mfn1+Mfn2 TKD), mitochondrial morphology was normal, as in control cells (SI Appendix, Fig. S2D). In contrast, in Drp1+ATAD3 DKD cells, small and dispersed nucleoids were observed on highly elongated mitochondria (Fig. 2B). Thus, ATAD3A has an essential role in nucleoid enlargement in Drp1-deficient cells, without affecting mitochondrial fusion and fission. We also found that mito-bulbs were lost in Drp1+ATAD3 DKD cells, to form elongated mitochondria, suggesting that mito-bulb formation requires nucleoid clustering in mitochondrial fission-deficient cells, as we previously reported (24).

ATAD3A Mediates Nucleoid Trafficking in Mitochondria.

Under live imaging, most nucleoids move dynamically in mitochondria (24, 26). To investigate the significance of ATAD3A in nucleoid dynamics, we performed high-speed live imaging of nucleoids using spinning-disk confocal microscopy (25) (Fig. 2 E–I and Movies S1–S4), and measured the speed of nucleoid movement. We found that most of nucleoids moved very slowly in ATAD3 KD cells (Fig. 2 F, I, and J) compared with control cells (Fig. 2 E, I, and J), suggesting that ATAD3A is crucial for nucleoid trafficking. The diffusion rates of mitochondrial content in ATAD3 KD cells were comparable to those of control cells (SI Appendix, Fig. S2C), suggesting that ATAD3A specifically affects nucleoid trafficking, but not mitochondrial dynamics in the cytoplasm. These data indicate that ATAD3A accelerates the speed of nucleoid trafficking inside mitochondria.

Next, we examined nucleoid trafficking in Drp1-deficient conditions. Live imaging of Drp1 KD cells revealed that nucleoids moved in elongated mitochondria at a similar speed as in control cells (Fig. 2 G, I, and J and Movie S5), which resulted in nucleoid clustering (Fig. 2G). However, live imaging of Drp1+ATAD3 DKD cells demonstrated that nucleoid trafficking was severely impaired in elongated mitochondria (Fig. 2 H–J), similar to that observed in ATAD3 KD cells. From these results, we conclude that ATAD3A has a critical role in nucleoid trafficking inside mitochondria, and that its disruption can suppress nucleoid clustering in Drp1-deficient cells. Thus, the size and number of nucleoids are determined under a balance between mitochondrial fusion and fission and the movement of nucleoids inside mitochondria.

ATAD3A ATPase Domain in the Mitochondrial Matrix and Coiled-Coil Domain Are Both Required for Nucleoid Trafficking.

Next, we examined the molecular mechanism by which nucleoid dynamics are regulated by ATAD3A. To further analyze the roles of ATAD3A domains in nucleoid morphology and trafficking in cultured cells, we established HeLa cell lines that stably expressed either WT or mutated ATAD3A variants, followed by Drp1+ATAD3 KD by RNAi (Fig. 3A). We confirmed that the protein levels of all exogenously expressed proteins were comparable (SI Appendix, Fig. S4). As shown above, many small nucleoids were dispersed in elongated mitochondria in Drp1+ATAD3 DKD cells (Fig. 3C). By exogenous expression of WT ATAD3A in Drp1+ATAD3 DKD cells, clustered nucleoids were observed in mito-bulbs at the same level as in Drp1 KD cells (Fig. 3 C and D). As mentioned above, nucleoids moved slower in ATAD3 KD cells compared with control KD cells (Fig. 3 E and F and Movie S6), but exogenous expression of WT ATAD3A rescued the impaired movement of nucleoids in ATAD3 KD cells to the level observed in control cells, suggesting that the altered nucleoid movement induced by ATAD3 repression is reversible.

Fig. 3.

ATPase domain and coiled-coil domains for ATAD3A oligomerization are required for nucleoid trafficking. (A) Schematic representation of ATAD3A mutants. (B) Binding between WT or coiled-coil mutant (LPAP) ATAD3A in HeLa cells transfected with the indicated plasmids. The cells were harvested and immunoprecipitation was performed using the cell lysates. The precipitates were analyzed by immunoblotting. (C) Rescue experiments on nucleoid enlargement and clustering. HeLa cells stably expressing mitRFP and WT or mutant ATAD3A were treated with the indicated siRNA or control for 96 h. Confocal images of nucleoids and mitochondria by immunofluorescence staining are shown. Red, mitRFP; green, mtDNA. (Scale bar, 2 μm.) (D) Quantification of cells with clustered nucleoids in C. Tukey–Kramer multiple comparisons test was performed using Statcel4 software. **P < 0.01 (vs. Drp1 RNAi). Data are represented as mean ± SD. (E) Rescue experiments of nucleoid dynamics. HeLa cells stably expressing mitRFP and WT or mutant ATAD3A were treated with siRNA for ATAD3 or control for 96 h. The cells were stained with SYBR Green I for 5 min and live-cell images were obtained by spinning-disk confocal fluorescence microscopy. The images were obtained every 5 s for 10 min. Red, mitRFP; green, mtDNA. (Right) Trajectories of randomly selected nucleoids. Also see Movie S6. (Scale bar, 5 μm.) (F) The moving distance migrated in 10 min for each randomly selected nucleoid is shown. Tukey–Kramer multiple comparisons test was performed using Statcel4 software, **P < 0.01 (vs. cont RNAi). Box-and-whisker plots were represented minimum to maximum. (G) Schematic representation of nucleoid trafficking is shown. Nucleoid movement requires an active ATPase domain in the mitochondrial matrix and coiled-coil domains in the IMS of ATAD3A.

Next, we investigated the ATAD3A domains required for nucleoid trafficking. Following the exogenous expression of ATAD3A (E412Q), which had a mutation in the conserved Walker motif B in the ATPase domain, in Drp1+ATAD3 DKD cells the nucleoids remained small and dispersed (Fig. 3 C and D). The nucleoids moved slowly in ATAD3 KD cells expressing ATAD3A (E412Q) (Fig. 3 E and F), suggesting that an active ATPase domain is required for nucleoid trafficking. Using an in vitro assay, we showed that the purified AAA-ATPase domain of ATAD3A interacted with TFAM in the presence and absence of ATP or ADP (Fig. 1M), suggesting that an active ATPase domain is required for nucleoid trafficking without affecting the interaction between ATAD3A and TFAM.

Next, we examined the roles of the predicted coiled-coil domains of ATAD3A (Fig. 3A), which should be located in the IMS (Fig. 1 F and G). ATAD3A formed homo-oligomeric complexes because differentially tagged ATAD3A coexpressed in HeLa cells could be corecovered by immunoprecipitation (Fig. 3B). However, when we constructed and expressed a mutated ATAD3A (ATAD3A [LPAP]), in which four leucine and alanine residues in the coiled-coil domains were substituted with helix-breaking proline residues, they failed to form ATAD3A oligomers (Fig. 3B) and could not rescue nucleoid clustering in Drp1+ATAD3 DKD cells (Fig. 3 C and D). The speed of nucleoid movement was also still slow (Fig. 3 E and F). In conclusion, the coiled-coil domains in the IMS and AAA-ATPase domain in the mitochondrial matrix are all required for nucleoid trafficking and to regulate the size and number of nucleoids. ATAD3A forms a homo-oligomeric complex via the N-terminal coiled-coil domains in the IMS, and the C-terminal AAA-ATPase domain interacts directly with TFAM in the mitochondrial matrix, and nucleoids should move in an ATP hydrolysis-dependent manner (model in Fig. 3G).

Nucleoid Distribution Is Crucial for Respiratory Complex Biogenesis.

We previously showed that Drp1 KO HeLa cells (28) and Drp1 KO cardiomyocytes (27) have a respiratory defect and highly clustered nucleoids. However, the physiological meaning of nucleoid morphology and dynamics, as well as the relationship between nucleoid morphology and respiratory function, are less well understood.

To investigate respiratory complex formation in nucleoid morphology-deficient cells, we analyzed the protein levels of respiratory subunits by immunoblotting. We previously established Drp1 KO HeLa cells (31) in which nucleoids are extremely enlarged, even though mtDNA copy number is normal (28) (Fig. 2D). The protein levels of respiratory chain subunits were considerably decreased in Drp1 KO HeLa cells (Fig. 4 B and C), similar to the previous report (28). However, when ATAD3A was further suppressed by RNAi in Drp1 KO cells, the nucleoids became smaller and dispersed (Fig. 4A), and the respiratory subunits of complexes III and IV were significantly recovered (Fig. 4 B and C). We further analyzed the protein levels of respiratory subunits in ATAD3 KD cells and found that the levels of COX I in complex IV was highly increased (Fig. 4 D and E). These data suggest that respiratory complex formation is enhanced by ATAD3A inactivation. In conclusion, nucleoids move along mitochondria in a manner that is dependent on the active ATPase domain and coiled-coil domains of ATAD3A oligomers on the IM. The large number of dispersed nucleoids on mitochondria should support the efficient formation of respiratory complexes, and we unveiled a role for nucleoid structures in respiratory complex formation in cultured mammalian cells (model in Fig. 4F).

Fig. 4.

Nucleoid distribution affects the stabilization of respiratory subunits. (A) Drp1 KO HeLa cells were treated with siRNA for ATAD3 or control for 96 h. The cells were stained with MitoTracker Red for 15 min and SYBR Green I for 5 min. The cells were observed by fluorescence microscopy. (Scale bar, 20 μm in large panels and 10 μm in Insets.) (B) Protein levels of respiratory subunits were determined by immunoblotting using the indicated antibodies. Drp1 KO HeLa cells were treated with siRNA for ATAD3 or control for 144 h. Cells treated with 10 μM 2′,3′-Dideoxycytidine (ddC) for 168 h were used as controls. (C) The graph shows the relative protein level of each OXPHOS component at ATAD3 RNAi for 96 h against control. Multiple comparison with Tukey’s test was performed using GraphPad Prism version 6.00. *P < 0.05, **P < 0.01, ***P < 0.001 (vs. each subunit of cont RNAi). Data are represented as mean ± SD. (D) HeLa cells treated as described in C were determined by immunoblotting using the indicated antibodies. (E) The relative protein level of each OXPHOS component is shown graph as described in C. Unpaired t test with Welch’s collection was performed using GraphPad Prism v6.00. *P < 0.05, **P < 0.01 (vs. each subunit of cont RNAi). (F) Schematic representation of the mechanism and the role of mtDNA nucleoid distribution regulated by ATAD3A and Drp1. The accumulation of clustered nucleoids in mitochondrial fission-deficient cells formed by active nucleoid trafficking mediated by ATAD3A leads to the reduced respiratory complexes.

Discussion

ATAD3A is reported to be associated with nucleoids (13–15); however, the roles of nucleoid morphogenesis by ATAD3A are not well understood. In this report, detailed microscopic analysis including live imaging revealed that ATAD3A is involved in nucleoid trafficking along tubular mitochondria, which affects the size and number of nucleoids (Figs. 2 and 3). A previous report showed that nucleoids, observed by PicoGreen staining, are lost in ATAD3A-repressed cells without any change in mtDNA copy number (14). However, we found a large number of smaller nucleoids in ATAD3A-repressed cells (Fig. 2 and SI Appendix, Fig. S2) because we stained nucleoids using a more sensitive dye SYBR Green I or specific antibodies. This is an unique report detecting smaller nucleoids without a loss of mtDNA copy number, which leads to the finding of a physiological role of nucleoid morphology and distribution in respiratory complex formation. It has been shown that each nucleoid in normal mammalian cells contains one or at most several mtDNA genomes (32). It is possible that the smaller nucleoids found in ATAD3A KD cells contain only one genome each, although we need further detailed analysis to know the composition and character of the smaller nucleoids.

We previously demonstrated by live imaging that mitochondrial fission often occurs in the vicinity of nucleoids (24), and it was reported that mitochondrial fission sites are marked by the endoplasmic reticulum (26, 33). It was also reported that ATAD3A interacts with Drp1 in Huntington’s disease models (29). However, the mechanism by which cytoplasmic Drp1 recognizes nucleoids beyond the mitochondrial double membrane remains to be clarified. Because ATAD3A is anchored to the IM by its centrally located transmembrane domain and is exposed to the IMS and mitochondrial matrix, we found that ATAD3A bound to not only TFAM in the matrix but also to the OM-anchored protein Mff, which recruits Drp1 to mitochondrial fission sites (Figs. 1 and 3), suggesting that ATAD3A has the potential to connect mitochondrial fission factors and nucleoids. However, it is not clear whether IM-anchored ATAD3A could directly interact with OM-anchored Mff in the IMS, since only two amino acid residues (-Arg-Arg) at the C terminus of human Mff are exposed to the IMS (34). ATAD3A is a multifunctional protein and that associated with various mitochondrial proteins (16, 18, 35, 36), and Mff has also been reported to interact with anther OM proteins, such as MAVS (37), suggesting that ATAD3A may indirectly bind Mff via interaction with another OM-anchored protein. The orientation of the N terminus of ATAD3A is also controversial, as reported that the N terminus penetrates the OM and is exposed to the cytoplasm (20, 35, 38, 39). Further analysis of the membrane topology and complex formation of ATAD3A should lead to the detailed understanding of the role in mtDNA distribution and mitochondrial fission. However, even in ATAD3A-deficient cells, Drp1 was often observed in the vicinity of nucleoids (SI Appendix, Fig. S3), suggesting that ATAD3A is not essential for the localization of Drp1 next to nucleoids, as we first expected. It might be a possibility that another factor possessing redundant function with ATAD3A recruits Drp1 to the vicinity of nucleoids, and how mitochondrial fission factors recognize nucleoids beyond the mitochondrial double membrane remains an open question.

Under live imaging, most nucleoids move dynamically in mitochondria (24, 26). However, no skeleton or motor proteins are found within mitochondria, and the mechanism of nucleoid movement along mitochondria is not well understood. Here, from further extensive analysis of nucleoid morphology and dynamics, we demonstrated that ATAD3A is essential for the rapid movement of nucleoids within mitochondria (Fig. 2). Although ATP was dispensable for binding between ATAD3A and TFAM in vitro (Fig. 1), the active ATPase domain was required for nucleoid movement (Fig. 3). ATAD3A homo-oligomers formed in the IM supported nucleoid movement, possibly using energy from the ATP hydrolysis (model in Fig. 3G). It is also possible that ATAD3A could affect the characteristics of the IM, including lipid composition, lateral flux of membrane components, and cristae structures, which would in turn affect nucleoid motility because nucleoids are believed to interact with the IM (18). Further studies are required for a deeper understanding of the molecular mechanisms underlying nucleoid trafficking in mitochondria.

We previously reported that Drp1 repression leads to clustered and enlarged nucleoids, showing that mitochondrial fission suppresses nucleoid clustering (24), and here we further showed that ATAD3A repression resulted in small and numerous nucleoids dispersed throughout the mitochondrial network. The present study further clarified the fundamental principle of the morphology and distribution of mtDNA within a cell; rapid nucleoid movement along elongated mitochondria enhances frequent encountering of nucleoids to each other, leading to the clustering and enlargement of nucleoids (model in Fig. 4F). It might also be possible that proper nucleoid trafficking is related to the quality control and inheritance of mtDNA and further extensive analysis of nucleoid trafficking will help clarify the pathophysiological roles of nucleoid dynamics within mitochondria.

We also previously showed that Drp1 KO in cardiomyocytes and HeLa cells caused nucleoid clustering and declined respiratory subunits (27, 28). However, the detailed role of nucleoid morphology and dynamics in respiration was not well understood. In ATAD3A-repressed Drp1 KO HeLa cells, the nucleoid clustering and diminished respiratory subunits were both restored (Fig. 4), suggesting that nucleoid clustering destabilizes respiratory subunits. For the assembly of functional respiratory complexes, proteins encoded by the nuclear and mitochondrial genomes are synthesized in the cytoplasm and mitochondrial matrix, respectively, and subsequently integrated into and assembled in the IM. Many copies of mtDNA are distributed widely in mitochondria as a large number of nucleoid structures because mtDNA should be only locally effective. We speculate that the presence of dispersed nucleoids around the mitochondrial network is necessary for the efficient assembly of respiratory subunits encoded by the mitochondrial and nuclear genomes. It has also been reported that ATAD3A oligomerization promotes mitochondrial fragmentation via interaction with Drp1, and that inhibition of this interaction improves mitochondrial function in Huntington’s disease models (29). However, in our condition, respiratory activity, as measured by oxygen consumption, was not efficiently restored by ATAD3A repression, and cell growth rate was also greatly decreased in ATAD3A-deficient cells, even when the respiratory subunits were stabilized (Fig. 4). Previous reports showed that ATAD3A is a multifunctional protein (16, 18–21) that should cause respiratory dysfunction even these cells have more respiratory subunits. To further elucidate the physiological function of nucleoid distribution and morphology, we have to identify more specific regulators of nucleoid dynamics. By analyzing those candidate factors, we will be able to examine the direct role of the nucleoid morphology in the respiratory activity and the cellular functions.

In conclusion, here we found that nucleoid trafficking in mitochondria, regulated by ATAD3A-ATPase, should have critical roles in the regulation of size and number of nucleoids, for the efficient respiratory complex formation. The nucleoid trafficking may represent a new therapeutic target to prevent mitochondrial dysfunction in various diseases.

Materials and Methods

Materials.

Rabbit polyclonal anti-TFAM antibodies were prepared by injecting rabbits with purified mouse TFAM protein expressed in E. coli and used for immunoblotting. Rabbit polyclonal anti–ATAD3A-N and anti–ATAD3A-C antibodies were generous gifts from O. Kuge, Kyushu University, Fukuoka, Japan (17). SYBR Green I was purchased from Molecular Probes; MitoTracker Red CMXRos was purchased from Invitrogen. The mito-PAGFP expression plasmid was a kind gift from R. Youle, NIH, MD, USA (30). The primary and secondary antibodies used for immunoblotting or immunostaining are listed in SI Appendix, Table S1, while the primers and plasmids are in SI Appendix, Table S2.

Cell Culture and Transfection.

HeLa cells, HeLa cells expressing mitochondria-targeted DsRed (mitRFP), HeLa cells expressing WT or mutated ATAD3A variants, and Drp1 KO HeLa cells (24, 25, 28) were grown in Dulbecco’s modified Eagle’s medium (Wako) supplemented with 10% fetal bovine serum (Invitrogen and Sigma-Aldrich). Transient transfection of plasmids was performed using Lipofectamine 2000 (Invitrogen). To create mitRFP-HeLa cell lines expressing WT or mutated ATAD3A variants, 24 h after transfection of each plasmid, cells were selected with Blasticidin.

For RNAi, cultured HeLa cells were transfected with siRNAs by Lipofectamine RNAiMAX (Invitrogen). After 2 d, the cells were transfected again and cultured for another 2 d. Luciferase siRNA and negative control siRNA were mixed and used as a control. ATAD3A siRNA and ATAD3B siRNA were mixed and used as ATAD3 RNAi, (all ATAD3 variants were repressed) (SI Appendix, Fig. S2A). siRNA oligonucleotides were purchased from Bonac based on the following sequences as the targets: ATAD3A siRNA sense 5′-GCAACCAACCAGAGCAGUUtt-3′, ATAD3B siRNA sense 5′-CCAAGGACAAAUGGAGCAAtt-3′, Drp1 siRNA sense 5′-ACUAUUGAAGGAACUGCAAAAUAUAtt-3′, luciferase siRNA sense 5′-CGUACGCGGAAUACUUCGAdTdT-3′, negative control siRNA sense 5′-UACUAUUCGACACGCGAAGtt-3′, and OPA1 siRNA sense 5′-CACGUUUUAACCUUGAAACtt-3′. Mfn1 and Mfn2 siRNAs (stealth RNA; Invitrogen) have been previously described (24).

Immunoprecipitation and Immunoblotting.

For chemical cross-linking followed by coimmunoprecipitation, cultured cells were subjected to chemical cross-linking with 1% formaldehyde in in culture medium for 10 min at 37 °C, and the cross-linking reaction was stopped by washing in phosphate-buffered saline (PBS) containing 100 mM glycine. The cells were suspended and sonicated in lysis buffer (PBS containing RIPA-B, 1 mM PMSF, and protease inhibitor mixture complete EDTA-free; Roche Diagnostics) and placed on ice for 10 min. Cell lysates were used for coimmunoprecipitation after centrifugation. The lysates were incubated with protein-G–Sepharose, which were washed three times with wash buffer (Tris-B, 150 mM NaCl, 0.1% Nonidet P-40, and 0.2 mM PMSF). The proteins were eluted with 10 μL buffer (0.2 M Tris, 10% glycerol, and 2% SDS) and boiled.

For immunoblotting, proteins were separated on SDS/PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBS-T and incubated with the indicated primary antibodies followed by a mouse or rabbit horseradish peroxidase-conjugated secondary antibody. Blots were detected with ECL Western Blotting Detection Reagents (GE Healthcare) and Immobilon Western Chemiluminescent HRP Substrate (Millipore). Quantification of protein level were performing by ImageJ software (National Institutes of Health) (40).

Immunofluorescence Microscopy.

Cells grown on coverslips were fixed for 15 min with 4% paraformaldehyde, washed twice with PBS, and permeabilized for 5 min with 0.2% Triton X-100 in PBS. After three washes with PBS, the cells were blocked for 30 min with 5% skim milk in PBS, then incubated with primary antibodies for 1 h at room temperature. After four washes with PBS, the cells were incubated with secondary antibodies for 1 h at room temperature. After four washes with PBS, the cells were covered with coverslips (SlowFade Gold antifade reagent; Molecular Probes). Samples were observed under a Zeiss LSM700 confocal microscope with a Plano Apochromat 63×/1.4 numerical aperture oil immersion objective. Confocal images were acquired using Zeiss ZEN 2010LSM software.

Live Imaging.

For time-lapse analysis, HeLa cells expressing mitRFP cultured on glass-bottomed dishes were stained with 100,000-fold diluted SYBR Green I for 5 min at 37 °C, washed four times with growth medium, and changed to fresh growth medium containing 50 mM Hepes buffer (pH 7.4). After 30 min, time-lapse recording was started. The cells were observed under an Olympus IX81 fluorescence microscope, Zeiss LSM700 confocal microscope, or spinning-disk confocal microscope, and analyzed using Zeiss ZEN 2010 LSM software or Metamorph software (Molecular Devices). The data were analyzed and quantified using ImageJ software (NIH) (40).

The mito-PAGFP–based assay was performed as previously described (30). HeLa cells treated with each indicated siRNA were transfected with mito-PAGFP (1 μg). The next day, the cells were stained with 100 nM MitoTracker Red for 20 min, washed four times with growth medium, and changed to fresh growth medium containing 50 mM Hepes buffer (pH 7.4). After 30 min, images were captured on a Zeiss LSM700 confocal microscope. A region was photoactivated by a single-pulse 405-nm laser. Time-lapse images with red and green fluorescent signals were acquired before and immediately following activation every 1 min for 30 min of three z-sections with 0.5 μm of each interval. Images show z-stack reconstructions of representative cells at each time point. Quantitative analysis of changes in fluorescence intensity of photoactivated mito-PAGFP at each time point was carried out using Zeiss ZEN 2010 LSM software and normalized by mito-PAGFP fluorescence after photoactivation.

Quantitative PCR and mtDNA Copy Number Analysis.

DNA from HeLa cells was extracted using a QIAamp DNA Mini Kit (Qiagen). A KAPA SYBR FAST qPCR Kit was used for quantitative PCR with StepOnePlus (Applied Biosystems). To produce a standard curve, 0.5, 1, 2, 4, and 8 ng untreated HeLa cell DNA for mtDNA amplification or 1, 2, 4, 8, and 16 ng untreated HeLa cell DNA for nuclear gene amplification was used. The primer sets for amplification of mtDNA (65 bp) and β2M coding nuclear DNA (95 bp) fragments were as previously described (41).

Protein Expression, Purification, and Analysis.

The bacterial expression vectors encoding ATAD3A or TFAM were transformed into Rosetta strain E. coli. The cells were grown in 50 mL LB medium with 150 μg/mL kanamycin for 10 h at 37 °C. This preculture was used to inoculate 500 mL LB medium containing 150 μg/mL kanamycin, and the culture was allowed to grow at 37 °C until the optical density had reached 1.2 at λ = 600 nm. Expression of recombinant protein was induced by adding 0.1 mM isopropyl-β-d-thiogalactopyranoside for 16 h at 25 °C. The cells were harvested by centrifugation.

For purification of recombinant TFAM protein, the pellet was suspended in 20 mL Buffer-1 (PBS containing 1% Triton X-100, 50 mM imidazole, 10 mM β-mercaptoethanol, and 1 mM PMSF), sonicated (15 s of sonication followed by 15 s of cooling), and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was mixed with 1 mL of a 50% slurry of Ni-NTA resin and 0.5 M NaCl. The sample was rotated for 1.5 h at 4 °C. The resin was washed with 5 mL Buffer-2 (20 mM Tris⋅HCl, pH 8.5, 0.5 M NaCl, 10 mM β-mercaptoethanol, 20 mM imidazole, 10% glycerol, and 0.2 mM PMSF). The resin was washed with 5 mL Buffer-3 (20 mM Tris⋅HCl, pH 8.5, 0.5 M NaCl, 10 mM β-mercaptoethanol, 10% glycerol, 0.2 mM PMSF, and 0.1% Nonidet P-40). The resin was washed with 1 mL Buffer-2. The resin was washed with 5 mL Buffer-4 (20 mM Tris⋅HCl, pH 8.5, 0.5 M NaCl, 10 mM β-mercaptoethanol, 10% glycerol, and 0.2 mM PMSF). The protein was eluted five times using 500 μL Buffer-5 (20 mM Tris⋅HCl, pH 8.5, 0.3 M NaCl, 10 mM β-mercaptoethanol, 250 mM imidazole, and 10% glycerol).

For purification of recombinant ATAD3A protein, the pellet was suspended in 15 mL PBS containing 1% Triton X-100, sonicated, and centrifuged. The supernatant was mixed with 1 mL of a 50% slurry of Glutathione Sepharose 4B resin. The sample was rotated for 1 h at 4 °C. The resin was washed with PBS containing 1% Triton X-100 and 0.5 M NaCl. For transfer to the column, the resin was washed twice with PBS containing 1% Triton X-100 and further washed twice with PBS.

The interaction of ATAD3A and TFAM was analyzed using an on-beads Halo assay. Purified mCherry-fused protein with the TFAM domain (20 ng) was mixed with the GST-fused ATAD3A domains immobilized on Glutathione Sepharose 4B beads. After washing, the mCherry signal (red) on the beads was observed by confocal microscopy.

Data, Materials, and Software Availability

All data are available in the main text and SI Appendix.