Germline-soma Supply Mitochondria for mtDNA Inheritance in Mouse Oogenesis

Maternal transmission paradigm of mtDNA remains controversial in mammalian oogenesis. Germline-soma-to-oocyte communication by numerous transzonal nanotubes (TZTs) reminds whether intercellular mitochondrial transfer is associated with maternal inheritance. Here, we found that mouse oocytes egocentrically receive mitochondria via TZTs, which projected from germline-soma, to achieve 105 copies, instead of de novo synthesis of mtDNA subpopulation in growing oocytes. De novo assembled TZTs amongst germline-soma and oocytes accumulated mtDNA amounts of the oocytes in vitro. However, mitochondrial supplement from germline-soma gradually diminished along with oocyte growth and was terminated by meiosis resumption, in line with a decrease in the proportion of germline-soma with thriving mtDNA replication and FSH capture capability. Thus, germline-soma-to-oocyte mitochondrial transfer is responsible for mammalian mtDNA inheritance as well as oogenesis and aging.

DRP1 regulates endoplasmic reticulum sheets to control mitochondrial DNA replication and segregation

Mitochondria are multi-faceted organelles crucial for cellular homeostasis that contain their own genome. Mitochondrial DNA (mtDNA) codes for several essential components of the electron transport chain, and mtDNA maintenance defects lead to mitochondrial diseases. mtDNA replication occurs at endoplasmic reticulum (ER)-mitochondria contact sites and is regulated by mitochondrial dynamics. Specifically, mitochondrial fusion is essential for mtDNA maintenance. In contrast, while loss of mitochondrial fission causes the aggregation of nucleoids (mtDNA-protein complexes), its role in nucleoid distribution remains unclear. Here, we show that the mitochondrial fission protein DRP1 regulates nucleoid segregation by altering ER sheets, the ER structure associated with protein synthesis. Specifically, DRP1 loss or mutation leads to altered ER sheets that physically interact with mitobulbs, mitochondrial structures containing aggregated nucleoids. Importantly, nucleoid distribution and mtDNA replication were rescued by expressing the ER sheet protein CLIMP63. Thus, our work identifies a novel mechanism by which DRP1 regulates mtDNA replication and distribution.

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila Mitochondrial Replicative DNA Helicase

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.

Mitochondria Metabolomics Reveals a Role of β-Nicotinamide Mononucleotide Metabolism in Mitochondrial DNA Replication

Summary Mitochondrial DNA (mtDNA) replication is tightly regulated and necessary for cellular homeostasis; however, its relationship with mitochondrial metabolism remains unclear. Advances in metabolomics integrated with the rapid isolation of mitochondria will allow for remarkable progress in analyzing mitochondrial metabolism. Here, we propose a novel methodology for mitochondria-targeted metabolomics, which employs a quick isolation procedure using a hemolytic toxin from Streptococcus pyogenes streptolysin O (SLO). SLO-isolation of mitochondria from cultured HEK293 cells is time- and labor-saving for simultaneous multi-sample processing and has been applied to various other cell lines in this study. Furthermore, our method can detect the time-dependent reduction in mitochondrial ATP in response to a glycolytic inhibitor 2-deoxyglucose, indicating the suitability to prepare metabolite analysis-competent mitochondria. Using this methodology, we searched for specific mitochondrial metabolites associated with mtDNA replication activation, and nucleotides and NAD+ were identified to be prominently altered. Most notably, treatment of β-Nicotinamide Mononucleotide (β-NMN), a precursor of NAD+, to HEK293 cells activated and improved the rate of mtDNA replication by increasing nucleotides in mitochondria and decreasing their degradation products: nucleosides. Our results suggest that β-NMN metabolism play a role in supporting mtDNA replication by maintaining the nucleotide pool balance in the mitochondria.

2-Deoxy-D-glucose couples mitochondrial DNA replication with mitochondrial fitness and promotes the selection of wild-type over mutant mitochondrial DNA

Pathological variants of human mitochondrial DNA (mtDNA) typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood. Because mitochondrial fitness does not favour the propagation of functional mtDNAs in disease states, we sought to create conditions where it would be advantageous. Glucose and glutamine consumption are increased in mtDNA dysfunction, and so we targeted the use of both in cells carrying the pathogenic m.3243A>G variant with 2-Deoxy-D-glucose (2DG), or the related 5-thioglucose. Here, we show that both compounds selected wild-type over mutant mtDNA, restoring mtDNA expression and respiration. Mechanistically, 2DG selectively inhibits the replication of mutant mtDNA; and glutamine is the key target metabolite, as its withdrawal, too, suppresses mtDNA synthesis in mutant cells. Additionally, by restricting glucose utilization, 2DG supports functional mtDNAs, as glucose-fuelled respiration is critical for mtDNA replication in control cells, when glucose and glutamine are scarce. Hence, we demonstrate that mitochondrial fitness dictates metabolite preference for mtDNA replication; consequently, interventions that restrict metabolite availability can suppress pathological mtDNAs, by coupling mitochondrial fitness and replication.

Mitochondrial DNA metabolism is coupled with 20S proteasome function via regulation of deoxyribonucleotide homeostasis in Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) is not coupled with cell cycle. Previous studies have shown that the size of deoxyribonucleoside triphosphate (dNTP) pools plays an important role in regulating mtDNA replication and amplification. In yeast, dNTPs are synthesized by the cytosolic ribonucleotide reductase (RNR). It is currently poorly understood as to how RNR activity is regulated in non-dividing or quiescent cells to finely tune mtDNA metabolism to cope with different metabolic states. Here, we show that defect in the 20S proteasome drastically destabilizes mtDNA. The mtDNA instability phenotype in 20S proteasome mutants is suppressed by overexpression of RNR3 or by the deletion of SML1, encoding a minor catalytic subunit and an intrinsic inhibitor of RNR respectively. We found that Sml1 is stabilized in the 20S proteasomal mutants, suggesting that 20S affects mtDNA stability by stabilizing Sml1. Interestingly, defect in the regulatory 19S proteasomal function has only subtle effect on mtDNA stability, supporting a role of the 20S proteasome in dNTP homeostasis independent of 19S. Finally, we found that when cells are transitioned from glycolytic to oxidative growth, Sml1 level is reduced in a 20S-dependent manner. In summary, our study establishes a link between cellular proteostasis and mtDNA metabolism through the regulation of dNTP homeostasis. We propose that increased degradation of Sml1 by the 20S proteasome under respiratory conditions provides a mechanism to stimulate dNTP synthesis and promote mtDNA amplification.

A new automated tool to quantify nucleoid distribution within mitochondrial networks

Mitochondrial DNA (mtDNA) maintenance is essential to sustain a functionally healthy population of mitochondria within cells. Proper mtDNA replication and distribution within mitochondrial networks are essential to maintain mitochondrial homeostasis. However, the fundamental basis of mtDNA segregation and distribution within mitochondrial networks is still unclear. To address these questions, we developed an algorithm, Mitomate tracker to unravel the global distribution of nucleoids within mitochondria. Using this tool, we decipher the semi-regular spacing of nucleoids across mitochondrial networks. Furthermore, we show that mitochondrial fission actively regulates mtDNA distribution by controlling the distribution of nucleoids within mitochondrial networks. Specifically, we found that primary cells bearing disease-associated mutations in the fission proteins DRP1 and MYH14 show altered nucleoid distribution, and acute enrichment of enlarged nucleoids near the nucleus. Further analysis suggests that the altered nucleoid distribution observed in the fission mutants is the result of both changes in network structure and nucleoid density. Thus, our study provides novel insights into the role of mitochondria fission in nucleoid distribution and the understanding of diseases caused by fission defects.

A genetic bottleneck of mitochondrial DNA during human lymphocyte development

Mitochondria are essential organelles in eukaryotic cells that provide critical support for energetic and metabolic homeostasis. Mutations that accumulate in mitochondrial DNA (mtDNA) in somatic cells have been implicated in cancer, degenerative diseases, and the aging process. However, the mechanisms used by somatic cells to maintain proper functions despite their mtDNA mutation load are poorly understood. Here, we analyzed somatic mtDNA mutations in more than 30,000 human single peripheral and bone marrow mononuclear cells and observed a significant overrepresentation of homoplastic mtDNA mutations in B, T and NK lymphocytes despite their lower mutational burden than other hematopoietic cells. The characteristic mutational landscape of mtDNA in lymphocytes were validated with data from multiple platforms and individuals. Single-cell RNA-seq and computational modeling demonstrated a stringent mitochondrial bottleneck during lymphocyte development likely caused by lagging mtDNA replication relative to cell proliferation. These results illuminate a potential mechanism used by highly metabolically active immune cells for quality control of their mitochondrial genomes.

Controlling the topology of mammalian mitochondrial DNA

The genome of mitochondria, called mtDNA, is a small circular DNA molecule present at thousands of copies per human cell. MtDNA is packaged into nucleoprotein complexes called nucleoids, and the density of mtDNA packaging affects mitochondrial gene expression. Genetic processes such as transcription, DNA replication and DNA packaging alter DNA topology, and these topological problems are solved by a family of enzymes called topoisomerases. Within mitochondria, topoisomerases are involved firstly in the regulation of mtDNA supercoiling and secondly in disentangling interlinked mtDNA molecules following mtDNA replication. The loss of mitochondrial topoisomerase activity leads to defects in mitochondrial function, and variants in the dual-localized type IA topoisomerase TOP3A have also been reported to cause human mitochondrial disease. We review the current knowledge on processes that alter mtDNA topology, how mtDNA topology is modulated by the action of topoisomerases, and the consequences of altered mtDNA topology for mitochondrial function and human health.

Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis

Plant mitochondrial genomes undergo frequent homologous recombination (HR). Ectopic HR activity is inhibited by the HR surveillance pathway, but the underlying regulatory mechanism is unclear. Here, we show that the mitochondrial RNase H1 AtRNH1B impairs the formation of RNA:DNA hybrids (R-loops) and participates in the HR surveillance pathway in Arabidopsis thaliana. AtRNH1B suppresses ectopic HR at intermediate-sized repeats (IRs) and thus maintains mitochondrial DNA (mtDNA) replication. The RNase H1 AtRNH1C is restricted to the chloroplast; however, when cells lack AtRNH1B, transport of chloroplast AtRNH1C into the mitochondria secures HR surveillance, thus ensuring the integrity of the mitochondrial genome and allowing embryogenesis to proceed. HR surveillance is further regulated by the single-stranded DNA-binding protein ORGANELLAR SINGLE-STRANDED DNA BINDING PROTEIN1 (OSB1), which decreases the formation of R-loops. This study uncovers a facultative dual targeting mechanism between organelles and sheds light on the roles of RNase H1 in organellar genome maintenance and embryogenesis.