bims-mitdyn Biomed News
on Mitochondrial dynamics: mechanisms
Issue of 2023–02–26
twenty-six papers selected by
Edmond Chan, Queen’s University, School of Medicine
  1. Cell lineage-specific mitochondrial resilience during mammalian organogenesis.
  2. TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.
  3. The SLC25A47 locus controls gluconeogenesis and energy expenditure.
  4. Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment.
  5. Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction.
  6. PERK recruits E-Syt1 at ER-mitochondria contacts for mitochondrial lipid transport and respiration.
  7. Mitochondrial membrane biogenesis: A new pathway for lipid transport mediated by PERK/E-Syt1 complex.
  8. Role of human HSPE1 for OPA1 processing independent of HSPD1.
  9. Mitochondrial phosphatidylethanolamine modulates UCP1 to promote brown adipose thermogenesis.
  10. Lipogenesis promotes mitochondrial fusion and maintains cancer stemness in human NSCLC.
  11. The role of programmed mitophagy in germline mitochondrial DNA quality control.
  12. The mitochondrial chaperone TRAP-1 regulates the glutamine metabolism in tumor cells.
  13. Analysis of Mitochondrial DNA Replication by Two-Dimensional Agarose Gel Electrophoresis.
  14. Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels.
  15. Localization of Mitochondrial Nucleoids by Transmission Electron Microscopy Using the Transgenic Expression of the Mitochondrial Helicase Twinkle and APEX2.
  16. Manipulation of Murine Mitochondrial DNA Heteroplasmy with mtZFNs.
  17. Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing.
  18. Isolation of Functional Mitochondria and Pure mtDNA from Murine Tissues.
  19. Concomitant Sequencing of Accessible Chromatin and Mitochondrial Genomes in Single Cells Using mtscATAC-Seq.
  20. Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.
  21. Visualization of mtDNA Using FISH.
  22. Measurement of Nucleoid Size Using STED Microscopy.
  23. Isolating Mitochondria, Mitoplasts, and mtDNA from Cultured Mammalian Cells.
  24. Assessing TFAM Binding to Human Mitochondrial DNA.
  25. Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies.
  26. Studying Mitochondrial Nucleic Acid Synthesis Utilizing Intact Isolated Mitochondria.
  1. Cell. 2023 Feb 17. pii: S0092-8674(23)00093-4. [Epub ahead of print]
    Cell lineage-specific mitochondrial resilience during mammalian organogenesis.
    Stephen P Burr, Florian Klimm, Angelos Glynos, Malwina Prater, Pamella Sendon, Pavel Nash, Christopher A Powell, Marie-Lune Simard, Nina A Bonekamp, Julia Charl, Hector Diaz, Lyuba V Bozhilova, Yu Nie, Haixin Zhang, Michele Frison, Maria Falkenberg, Nick Jones, Michal Minczuk, James B Stewart, Patrick F Chinnery.
      Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments.
    Keywords:  OXPHOS; RNA-seq; SCENIC; mitochondria; mt-Ta; mtDNA; organogenesis; single-cell
    DOI:  https://doi.org/10.1016/j.cell.2023.01.034
  2. Nat Commun. 2023 Feb 23. 14(1): 1009
    TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.
    Lindsey Van Haute, Emily O'Connor, Héctor Díaz-Maldonado, Benjamin Munro, Kiran Polavarapu, Daniella H Hock, Gautham Arunachal, Alkyoni Athanasiou-Fragkouli, Mainak Bardhan, Magalie Barth, Dominique Bonneau, Nicola Brunetti-Pierri, Gerarda Cappuccio, Nikeisha J Caruana, Natalia Dominik, Himanshu Goel, Guy Helman, Henry Houlden, Guy Lenaers, Karine Mention, David Murphy, Bevinahalli Nandeesh, Catarina Olimpio, Christopher A Powell, Veeramani Preethish-Kumar, Vincent Procaccio, Rocio Rius, Pedro Rebelo-Guiomar, Cas Simons, Seena Vengalil, Maha S Zaki, Alban Ziegler, David R Thorburn, David A Stroud, Reza Maroofian, John Christodoulou, Claes Gustafsson, Atchayaram Nalini, Hanns Lochmüller, Michal Minczuk, Rita Horvath.
      Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongation factor responsible for enhancing the processivity of mitochondrial RNA polymerase, POLRMT. We report for the first time that TEFM variants are associated with mitochondrial respiratory chain deficiency and a wide range of clinical presentations including mitochondrial myopathy with a treatable neuromuscular transmission defect. Mechanistically, we show muscle and primary fibroblasts from the affected individuals have reduced levels of promoter distal mitochondrial RNA transcripts. Finally, tefm knockdown in zebrafish embryos resulted in neuromuscular junction abnormalities and abnormal mitochondrial function, strengthening the genotype-phenotype correlation. Our study highlights that TEFM regulates mitochondrial transcription elongation and its defect results in variable, tissue-specific neurological and neuromuscular symptoms.
    DOI:  https://doi.org/10.1038/s41467-023-36277-7
  3. Proc Natl Acad Sci U S A. 2023 Feb 28. 120(9): e2216810120
    The SLC25A47 locus controls gluconeogenesis and energy expenditure.
    Jin-Seon Yook, Zachary H Taxin, Bo Yuan, Satoshi Oikawa, Christopher Auger, Beste Mutlu, Pere Puigserver, Sheng Hui, Shingo Kajimura.
      Mitochondria provide essential metabolites and adenosine triphosphate (ATP) for the regulation of energy homeostasis. For instance, liver mitochondria are a vital source of gluconeogenic precursors under a fasted state. However, the regulatory mechanisms at the level of mitochondrial membrane transport are not fully understood. Here, we report that a liver-specific mitochondrial inner-membrane carrier SLC25A47 is required for hepatic gluconeogenesis and energy homeostasis. Genome-wide association studies found significant associations between SLC25A47 and fasting glucose, HbA1c, and cholesterol levels in humans. In mice, we demonstrated that liver-specific depletion of SLC25A47 impaired hepatic gluconeogenesis selectively from lactate, while significantly enhancing whole-body energy expenditure and the hepatic expression of FGF21. These metabolic changes were not a consequence of general liver dysfunction because acute SLC25A47 depletion in adult mice was sufficient to enhance hepatic FGF21 production, pyruvate tolerance, and insulin tolerance independent of liver damage and mitochondrial dysfunction. Mechanistically, SLC25A47 depletion leads to impaired hepatic pyruvate flux and malate accumulation in the mitochondria, thereby restricting hepatic gluconeogenesis. Together, the present study identified a crucial node in the liver mitochondria that regulates fasting-induced gluconeogenesis and energy homeostasis.
    Keywords:  bioenergetics; metabolism; mitochondria; obesity; type 2 diabetes
    DOI:  https://doi.org/10.1073/pnas.2216810120
  4. Oncogene. 2023 Feb 22.
    Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment.
    Mokryun L Baek, Junegoo Lee, Katherine E Pendleton, Mariah J Berner, Emily B Goff, Lin Tan, Sara A Martinez, Iqbal Mahmud, Tao Wang, Matthew D Meyer, Bora Lim, James P Barrish, Weston Porter, Philip L Lorenzi, Gloria V Echeverria.
      Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in ~45% of patients. Unfortunately, TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. We previously demonstrated mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual TNBC cells surviving NACT. We sought to investigate the mechanism underlying this enhanced reliance on mitochondrial metabolism. Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. The functional impact of mitochondrial structure on metabolic output is highly context dependent. Several chemotherapy agents are conventionally used for neoadjuvant treatment of TNBC patients. Upon comparing mitochondrial effects of conventional chemotherapies, we found that DNA-damaging agents increased mitochondrial elongation, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes instead decreased mitochondrial elongation and OXPHOS. The mitochondrial effects of DNA-damaging chemotherapies were dependent on the mitochondrial inner membrane fusion protein optic atrophy 1 (OPA1). Further, we observed heightened OXPHOS, OPA1 protein levels, and mitochondrial elongation in an orthotopic patient-derived xenograft (PDX) model of residual TNBC. Pharmacologic or genetic disruption of mitochondrial fusion and fission resulted in decreased or increased OXPHOS, respectively, revealing longer mitochondria favor oxphos in TNBC cells. Using TNBC cell lines and an in vivo PDX model of residual TNBC, we found that sequential treatment with DNA-damaging chemotherapy, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22, a specific inhibitor of OPA1, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibit regrowth of residual tumor cells. Our data suggest that TNBC mitochondria can optimize OXPHOS through OPA1-mediated mitochondrial fusion. These findings may provide an opportunity to overcome mitochondrial adaptations of chemoresistant TNBC.
    DOI:  https://doi.org/10.1038/s41388-023-02596-8
  5. Cell Chem Biol. 2023 Feb 17. pii: S2451-9456(23)00033-8. [Epub ahead of print]
    Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction.
    Jun Yang, Peihao Chen, Yu Cao, Shanshan Liu, Wei Wang, Lin Li, Jiaojiao Li, Zhaodi Jiang, Yan Ma, She Chen, Sanduo Zheng, Xiangbing Qi, Hui Jiang.
      Mitochondrial fission is critical for mitochondrial dynamics and homeostasis. The dynamin superfamily GTPase DRP1 is recruited by three functionally redundant receptors, MFF, MiD49, and MiD51, to mitochondria to drive fission. Here, we exploit high-content live-cell imaging to screen for mitochondrial fission inhibitors and have developed a covalent compound, mitochondrial division inhibitor (MIDI). MIDI treatment potently blocks mitochondrial fragmentation induced by mitochondrial toxins and restores mitochondrial morphology in fusion-defective cells carrying pathogenic mitofusin and OPA1 mutations. Mechanistically, MIDI does not affect DRP1 tetramerization nor DRP1 GTPase activity but does block DRP1 recruitment to mitochondria. Subsequent biochemical and cellular characterizations reveal an unexpected mechanism that MIDI targets DRP1 interaction with multiple receptors via covalent interaction with DRP1-C367. Taken together, beyond developing a potent mitochondrial fission inhibitor that profoundly impacts mitochondrial morphogenesis, our study establishes proof of concept for developing protein-protein interaction inhibitors targeting DRP1.
    Keywords:  DRP1 inhibitor; MFF; MIDI; MiD49/51; OPA1; mitochondrial dynamics; mitochondrial fission; mitofusin
    DOI:  https://doi.org/10.1016/j.chembiol.2023.02.002
  6. J Cell Biol. 2023 Mar 06. pii: e202206008. [Epub ahead of print]222(3):
    PERK recruits E-Syt1 at ER-mitochondria contacts for mitochondrial lipid transport and respiration.
    Maria Livia Sassano, Alexander R van Vliet, Ellen Vervoort, Sofie Van Eygen, Chris Van den Haute, Benjamin Pavie, Joris Roels, Johannes V Swinnen, Marco Spinazzi, Leen Moens, Kristina Casteels, Isabelle Meyts, Paolo Pinton, Saverio Marchi, Leila Rochin, Francesca Giordano, Blanca Felipe-Abrio, Patrizia Agostinis.
      The integrity of ER-mitochondria appositions ensures transfer of ions and phospholipids (PLs) between these organelles and exerts crucial effects on mitochondrial bioenergetics. Malfunctions within the ER-mitochondria contacts altering lipid trafficking homeostasis manifest in diverse pathologies, but the molecular effectors governing this process remain ill-defined. Here, we report that PERK promotes lipid trafficking at the ER-mitochondria contact sites (EMCS) through a non-conventional, unfolded protein response-independent, mechanism. PERK operates as an adaptor for the recruitment of the ER-plasma membrane tether and lipid transfer protein (LTP) Extended-Synaptotagmin 1 (E-Syt1), within the EMCS. In resting cells, the heterotypic E-Syt1-PERK interaction endorses transfer of PLs between the ER and mitochondria. Weakening the E-Syt1-PERK interaction or removing the lipid transfer SMP-domain of E-Syt1, compromises mitochondrial respiration. Our findings unravel E-Syt1 as a PERK interacting LTP and molecular component of the lipid trafficking machinery of the EMCS, which critically maintains mitochondrial homeostasis and fitness.
    DOI:  https://doi.org/10.1083/jcb.202206008
  7. J Cell Biol. 2023 Mar 06. pii: e202301132. [Epub ahead of print]222(3):
    Mitochondrial membrane biogenesis: A new pathway for lipid transport mediated by PERK/E-Syt1 complex.
    Sébastien Leterme, Morgane Michaud.
      Despite decades of extensive research, mitochondrial lipid transport is a process far from fully understood. In this issue, Sassano et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202206008) identified a new complex, composed of E-Syt1 and PERK, which mediates lipid transport at ER-mitochondria contact sites and regulates mitochondrial functions in human cells.
    DOI:  https://doi.org/10.1083/jcb.202301132
  8. iScience. 2023 Feb 17. 26(2): 106067
    Role of human HSPE1 for OPA1 processing independent of HSPD1.
    Nelson Yeung, Daisuke Murata, Miho Iijima, Hiromi Sesaki.
      The human mtHSP60/HSPD1-mtHSP10/HSPE1 system prevents protein misfolding and maintains proteostasis in the mitochondrial matrix. Altered activities of this chaperonin system have been implicated in human diseases, such as cancer and neurodegeneration. However, how defects in HSPD1 and HSPE1 affect mitochondrial structure and dynamics remains elusive. In the current study, we address this fundamental question in a human cell line, HEK293T. We found that the depletion of HSPD1 or HSPE1 results in fragmentation of mitochondria, suggesting a decrease in mitochondrial fusion. Supporting this notion, HSPE1 depletion led to proteolytic inactivation of OPA1, a dynamin-related GTPase that fuses the mitochondrial membrane. This OPA1 inactivation was mediated by a stress-activated metalloprotease, OMA1. In contrast, HSPD1 depletion did not induce OMA1 activation or OPA1 cleavage. These data suggest that HSPE1 controls mitochondrial morphology through a mechanism separate from its chaperonin activity.
    Keywords:  Biological sciences; Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2023.106067
  9. Sci Adv. 2023 Feb 24. 9(8): eade7864
    Mitochondrial phosphatidylethanolamine modulates UCP1 to promote brown adipose thermogenesis.
    Jordan M Johnson, Alek D Peterlin, Enrique Balderas, Elahu G Sustarsic, J Alan Maschek, Marisa J Lang, Alejandro Jara-Ramos, Vanja Panic, Jeffrey T Morgan, Claudio J Villanueva, Alejandro Sanchez, Jared Rutter, Irfan J Lodhi, James E Cox, Kelsey H Fisher-Wellman, Dipayan Chaudhuri, Zachary Gerhart-Hines, Katsuhiko Funai.
      Thermogenesis by uncoupling protein 1 (UCP1) is one of the primary mechanisms by which brown adipose tissue (BAT) increases energy expenditure. UCP1 resides in the inner mitochondrial membrane (IMM), where it dissipates membrane potential independent of adenosine triphosphate (ATP) synthase. Here, we provide evidence that phosphatidylethanolamine (PE) modulates UCP1-dependent proton conductance across the IMM to modulate thermogenesis. Mitochondrial lipidomic analyses revealed PE as a signature molecule whose abundance bidirectionally responds to changes in thermogenic burden. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) made mice cold intolerant and insensitive to β3 adrenergic receptor agonist-induced increase in whole-body oxygen consumption. High-resolution respirometry and fluorometry of BAT mitochondria showed that loss of mitochondrial PE specifically lowers UCP1-dependent respiration without compromising electron transfer efficiency or ATP synthesis. These findings were confirmed by a reduction in UCP1 proton current in PE-deficient mitoplasts. Thus, PE performs a previously unknown role as a temperature-responsive rheostat that regulates UCP1-dependent thermogenesis.
    DOI:  https://doi.org/10.1126/sciadv.ade7864
  10. JCI Insight. 2023 Feb 21. pii: e158429. [Epub ahead of print]
    Lipogenesis promotes mitochondrial fusion and maintains cancer stemness in human NSCLC.
    Zhen Liu, Jiaxin Lei, Tong Wu, Weijie Hu, Ming Zheng, Ying Wang, Jingdong Song, Hang Ruan, Lin Xu, Tao Ren, Wei Xu, Zhenke Wen.
      Cancer stem-like cells (CSCs) are critically involved in cancer metastasis and chemoresistance, acting as one major obstacle in clinical practice. While accumulating studies have implicated the metabolic reprogramming of CSCs, mitochondrial dynamics in such cells remain poorly understood. Here we pinpointed OPA1high with mitochondrial fusion as a metabolic feature of human lung CSCs, licensing their stem-like properties. Specifically, human lung CSCs exerted enhanced lipogenesis, inducing OPA1 expression via transcription factor SPDEF. In consequence, OPA1high promoted mitochondrial fusion and stemness of CSCs. Such lipogenesishigh, SPDEFhigh, and OPA1high metabolic adaptions were verified with primary CSCs from lung cancer patients. Accordingly, blocking lipogenesis and mitochondrial fusion efficiently impeded CSC expansion and growth of lung cancer patient-derived organoids. Together, lipogenesis regulates mitochondrial dynamics via OPA1 for controlling CSCs in human lung cancer.
    Keywords:  Lung cancer; Mitochondria; Oncology
    DOI:  https://doi.org/10.1172/jci.insight.158429
  11. Autophagy. 2023 Feb 20.
    The role of programmed mitophagy in germline mitochondrial DNA quality control.
    Jonathan M Palozzi, Thomas R Hurd.
      Mitochondrial DNA (mtDNA) is prone to the accumulation of mutations. To prevent harmful mtDNA mutations from being passed on to the next generation, the female germline, through which mtDNA is exclusively inherited, has evolved extensive mtDNA quality control. To dissect the molecular underpinnings of this process, we recently performed a large RNAi screen in Drosophila and uncovered a programmed germline mitophagy (PGM) that is essential for mtDNA quality control. We found that PGM begins as germ cells enter meiosis, induced, at least in part, by the inhibition of the mTor (mechanistic Target of rapamycin) complex 1 (mTorC1). Interestingly, PGM requires the general macroautophagy/autophagy machinery and the mitophagy adaptor BNIP3, but not the canonical mitophagy genes Pink1 and park (parkin), even though they are critical for germline mtDNA quality control. We also identified the RNA-binding protein Atx2 as a major regulator of PGM. This work is the first to identify and implicate a programmed mitophagy event in germline mtDNA quality control, and it highlights the utility of the Drosophila ovary for studying developmentally regulated mitophagy and autophagy in vivo.
    Keywords:  Drosophila; autophagy; germline; mitochondria; mitochondrial DNA; mitophagy; mtDNA; purifying selection
    DOI:  https://doi.org/10.1080/15548627.2023.2182595
  12. Mitochondrion. 2023 Feb 22. pii: S1567-7249(23)00020-X. [Epub ahead of print]
    The mitochondrial chaperone TRAP-1 regulates the glutamine metabolism in tumor cells.
    Shrikant Purushottam Dharaskar, Amere Subbarao Sreedhar.
      Understanding cancer cell metabolism always provides information on hidden dimensions of tumor adaptations. Warburg's theory that cancer cells opt for aerobic glycolysis over the mitochondrial oxidative phosphorylation (OXPHOS) system is widely accepted. However, the hypothesis does not explain the mitochondrion's role in these cells. Here, we demonstrate that intact mitochondria are used for anaplerotic functions and ATP production by utilizing glutamine with the help of mitochondrial chaperone TRAP-1 (Tumor Necrosis Factor Receptor-associated Protein 1). TRAP-1 otherwise promotes aerobic glycolysis by lowering the mitochondrial OXPHOS in the presence of glucose. Here, we show that TRAP-1 maintains mitochondrial integrity and augments glutamine metabolism upon glucose deprivation to meet the cellular energy demand. The enhanced PER and ECAR correlating with increased ATP production suggest that glutamine fuels mitochondria in the presence of TRAP-1. We also found that TRAP1-dependent glutamine utilization involves the HIF2α-SLC1A5-GLS axis and is independent of hypoxia. Subsequently, we found that the metastatic potential of tumor cells is linked with glucose utilization, whereas the proliferative potential is linked with both glucose and glutamine utilization. Our findings establish that TRAP-1 contributes to enhanced glutamine utilization through the HIF2α-SLC1A5-GLS axis. Our results endow that TRAP-1 inhibitors can be potential drug candidates to combat tumor metabolism. Therefore, their use, either alone or in combination with existing chemotherapeutic agents, may target tumor metabolism and improve anticancer treatment response.
    Keywords:  TRAP-1; cancer; glutamine; metabolism; mitochondria
    DOI:  https://doi.org/10.1016/j.mito.2023.02.011
  13. Methods Mol Biol. 2023 ;2615 241-266
    Analysis of Mitochondrial DNA Replication by Two-Dimensional Agarose Gel Electrophoresis.
    Steffi Goffart, Jaakko Pohjoismäki.
      Two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been employed for nearly two decades in the analysis of replication and maintenance processes of animal mitochondrial DNA, but the method's potential has not been fully exploited. Here, we describe the various steps involved in this technique, from DNA isolation, to two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE), Southern hybridization and interpretation. We also provide examples of the applicability of 2D-AGE to investigate the different features of mtDNA maintenance and regulation.
    Keywords:  2D-AGE; Gel electrophoresis; Mitochondrial DNA; Recombination; Replication intermediates; Replication mechanism; Replication pausing
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_18
  14. Methods Mol Biol. 2023 ;2615 293-314
    Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels.
    Choco Michael Gorospe, Bruno Marçal Repolês, Paulina H Wanrooij.
      Impaired mitochondrial DNA (mtDNA) maintenance, due to, e.g., defects in the replication machinery or an insufficient dNTP supply, underlies a number of mitochondrial disorders. The normal process of mtDNA replication leads to the incorporation of multiple single ribonucleotides (rNMPs) per mtDNA molecule. Given that embedded rNMPs alter the stability and properties of the DNA, they may have consequences for mtDNA maintenance and thereby for mitochondrial disease. They also serve as a readout of the intramitochondrial NTP/dNTP ratios. In this chapter, we describe a method for the determination of mtDNA rNMP content using alkaline gel electrophoresis and Southern blotting. This procedure is suited for the analysis of mtDNA in total genomic DNA preparations as well as in purified form. Moreover, it can be performed using equipment found in most biomedical laboratories, allows the simultaneous analysis of 10-20 samples depending on the gel system employed, and can be modified for the analysis of other mtDNA modifications.
    Keywords:  Alkaline gels; Alkaline hydrolysis; Denaturing gels; Ribonucleotides; Southern blot; rNMPs
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_21
  15. Methods Mol Biol. 2023 ;2615 173-188
    Localization of Mitochondrial Nucleoids by Transmission Electron Microscopy Using the Transgenic Expression of the Mitochondrial Helicase Twinkle and APEX2.
    David Pla-Martín, Felix Babatz, Astrid C Schauss.
      Reminiscent of their evolutionary origin, mitochondria contain their own genome (mtDNA) compacted into the mitochondrial chromosome or nucleoid (mt-nucleoid). Many mitochondrial disorders are characterized by disruption of mt-nucleoids, either by direct mutation of genes involved in mtDNA organization or by interfering with other vital proteins for mitochondrial function. Thus, changes in mt-nucleoid morphology, distribution, and structure are a common feature in many human diseases and can be exploited as an indicator of cellular fitness. Electron microscopy provides the highest possible resolution that can be achieved, delivering spatial and structural information about all cellular structures. Recently, the ascorbate peroxidase APEX2 has been used to increase transmission electron microscopy (TEM) contrast by inducing diaminobenzidine (DAB) precipitation. DAB has the ability to accumulate osmium during classical EM sample preparation and, due to its high electron density, provides strong contrast for TEM. Among the nucleoid proteins, the mitochondrial helicase Twinkle fused with APEX2 has been successfully used to target mt-nucleoids, providing a tool to visualize these subcellular structures with high contrast and with the resolution of an electron microscope. In the presence of H2O2, APEX2 catalyzes the polymerization of DAB, generating a brown precipitate that can be visualized in specific regions of the mitochondrial matrix. Here, we provide a detailed protocol to generate murine cell lines expressing a transgenic variant of Twinkle, suitable to target and visualize mt-nucleoids. We also describe all the necessary steps to validate the cell lines prior to electron microscopy imaging and offer examples of anticipated results.
    Keywords:  APEX2; Mitochondria; Nucleoid; TEM
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_13
  16. Methods Mol Biol. 2023 ;2615 329-344
    Manipulation of Murine Mitochondrial DNA Heteroplasmy with mtZFNs.
    Pavel A Nash, Michal Minczuk.
      Mouse models of mitochondrial DNA mutations hold promise in the development and optimization of mitochondrial gene therapy technology and for gathering pre-clinical data prior to human trials. Their suitability for this purpose stems from the high similarity of human and murine mitochondrial genomes and the increasing availability of rationally designed AAV vectors capable of selectively transducing murine tissues. Our laboratory routinely optimizes mitochondrially targeted zinc finger nucleases (mtZFNs), the compactness of which makes them highly suitable for downstream AAV-based in vivo mitochondrial gene therapy. This chapter discusses the necessary precautions for the robust and precise genotyping of the murine mitochondrial genome as well as the optimization of mtZFNs intended for subsequent use in vivo.
    Keywords:  Gene therapy; Heteroplasmy; MEF; Mitochondria; Mouse; Zinc Finger nuclease; mtDNA; mtZFN
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_23
  17. Methods Mol Biol. 2023 ;2615 381-395
    Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing.
    Andrea Legati, Daniele Ghezzi, Carlo Viscomi.
      Over the last 10 years, next generation sequencing (NGS) became the gold standard for both diagnosis and discovery of new disease genes responsible for heterogeneous disorders, such as mitochondrial encephalomyopathies. The application of this technology to mtDNA mutations poses extra challenges compared to other genetic conditions because of the peculiarities of mitochondrial genetics and the requirement for proper NGS data management and analysis. Here, we describe a detailed, clinically relevant protocol to sequence the whole mtDNA and quantify heteroplasmy levels of mtDNA variants, starting from total DNA through the generation of a single PCR amplicon.
    Keywords:  Heteroplasmy; Mitochondrial DNA; Mitochondrial disease; Mitochondrial haplogroups; Next generation sequencing; Single amplicon
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_26
  18. Methods Mol Biol. 2023 ;2615 3-16
    Isolation of Functional Mitochondria and Pure mtDNA from Murine Tissues.
    Dieu Hien Rozsivalova, Milica Popovic, Harshita Kaul, Aleksandra Trifunovic.
      Detailed analysis of mitochondrial function cannot be achieved without good quality preparations of isolated mitochondria. Ideally, the isolation protocol should be quick, while producing a reasonably pure pool of mitochondria that are still intact and coupled. Here, we describe a fast and simple method for the purification of mammalian mitochondria relying on isopycnic density gradient centrifugation. We describe specific steps that should be taken into consideration when functional mitochondria from different tissues should be isolated. This protocol is suitable for the analysis of many aspects of the organelle's structure and function.
    Keywords:  Blue native PAGE; Electron transport chain; Isopycnic density gradient centrifugation; Mitochondria; Organelle isolation; Oxidative phosphorylation; Respirometry
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_1
  19. Methods Mol Biol. 2023 ;2611 269-282
    Concomitant Sequencing of Accessible Chromatin and Mitochondrial Genomes in Single Cells Using mtscATAC-Seq.
    Leif S Ludwig, Caleb A Lareau.
      Mitochondria are unique organelles of eukaryotic cells that carry their own multicopy number and circular genome. In most mammals, including humans and mice, the size of the chromosome is ~16,000 base pairs and unlike nuclear DNA, mitochondrial DNA (mtDNA) is not densely compacted. This results in mtDNA to be highly accessible for enzymes such as the Tn5 transposase, commonly used for accessible chromatin profiling of nuclear chromatinized DNA. Here, we describe a method for the concomitant sequencing of mtDNA and accessible chromatin in thousands of individual cells via the mitochondrial single-cell assay for transposase accessible chromatin by sequencing (mtscATAC-seq). Our approach extends the utility of existing scATAC-seq products and protocols as we (Nam et al, Nat Rev Genet 22:3-18, 2021) fix cells using formaldehyde to retain mitochondria and its mtDNA within its originating cell, (Buenrostro et al, Nat Methods 10:1213-1218, 2013) modify lysis conditions to permeabilize cells and mitochondria, and (Corces et al, Nat Methods 14:959-962, 2017) optimize bioinformatic processing protocols to collectively increase mitochondrial genome coverage for downstream analysis. Here, we discuss the essentials for the experimental and computational methodologies to generate and analyze thousands of multiomic profiles of single cells over the course of a few days, enabling the profiling of accessible chromatin and mtDNA genotypes to reconstruct clonal relationships and studies of mitochondrial genetics and disease.
    Keywords:  Accessible chromatin profiling; Lineage tracing; Mitochondrial DNA; Mitochondrial disease; Pathogenic mutation; Single cell multiomics; Somatic mutation
    DOI:  https://doi.org/10.1007/978-1-0716-2899-7_14
  20. Methods Mol Biol. 2023 ;2615 427-441
    Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.
    Shilan Wu, Matthew J Longley, Scott A Lujan, Thomas A Kunkel, William C Copeland.
      Mitochondrial DNA (mtDNA) encodes components essential for cellular respiration. Low levels of point mutations and deletions accumulate in mtDNA during normal aging. However, improper maintenance of mtDNA results in mitochondrial diseases, stemming from progressive loss of mitochondrial function through the accelerated formation of deletions and mutations in mtDNA. To better understand the molecular mechanisms underlying the creation and propagation of mtDNA deletions, we developed the LostArc next-generation DNA sequencing pipeline to detect and quantify rare mtDNA species in small tissue samples. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA.
    Keywords:  DNA deletions; Mitochondrial DNA; Mitochondrial DNA Replication; Mitochondrial disease; Next-Generation Sequencing; POLG
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_28
  21. Methods Mol Biol. 2023 ;2615 89-98
    Visualization of mtDNA Using FISH.
    Xie Xie, Xuefeng Zhu.
      Proper mitochondrial DNA (mtDNA) levels are critical for many cellular biological functions and are associated with aging and many mitochondria disorders. Defects in core subunits of the mtDNA replication machinery lead to decreased mtDNA levels. Other indirect mitochondrial contexts including ATP concentration, lipid composition, and nucleotide composition also contribute to mtDNA maintenance. Furthermore, mtDNA molecules are distributed evenly throughout the mitochondrial network. This uniform distribution pattern is required for oxidative phosphorylation and ATP production and has been linked to many diseases when perturbed. Thus, it is important to visualize mtDNA in the cellular context. Here we provide detailed protocols for cellular visualization of mtDNA using fluorescence in situ hybridization (FISH). The fluorescent signals are targeted to the mtDNA sequence directly, ensuring both sensitivity and specificity. This mtDNA FISH method can be combined with immunostaining and used for visualizing mtDNA-protein interactions and dynamics.
    Keywords:  FISH; Imaging; Microscopy; Mitochondria; Visualization; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_7
  22. Methods Mol Biol. 2023 ;2615 107-117
    Measurement of Nucleoid Size Using STED Microscopy.
    Elisa Motori.
      Mitochondria are equipped with their own DNA (mtDNA), which is packed into structures termed nucleoids . While nucleoids can be visualized in situ by fluorescence microscopy , the advent of super-resolution microscopy , and in particular of stimulated emission depletion (STED), has recently enabled the visualization of nucleoids at sub-diffraction resolution. Super-resolution microscopy has proved an invaluable tool for addressing fundamental questions in mitochondrial biology. In this chapter I describe how to achieve efficient labeling of mtDNA and how to quantify nucleoid diameter using an automated approach in fixed cultured cells by STED microscopy .
    Keywords:  Fluorescence microscopy; ImageJ; Immunocytochemistry; Mitochondrial DNA; Nucleoid; STED; Stimulated emission depletion microscopy
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_9
  23. Methods Mol Biol. 2023 ;2615 17-30
    Isolating Mitochondria, Mitoplasts, and mtDNA from Cultured Mammalian Cells.
    Katja E Menger, Thomas J Nicholls.
      Mitochondria are double membrane-bound eukaryotic organelles with roles in a range of cellular activities including energy conversion, apoptosis, cell signalling, and the biosynthesis of enzyme cofactors. Mitochondria contain their own genome, called mtDNA, which encodes subunits of the oxidative phosphorylation machinery as well as the rRNA and tRNA molecules required for their translation within mitochondria. The ability to isolate highly purified mitochondria from cells has been instrumental in a number of studies of mitochondrial function. Differential centrifugation is a long-established method for the isolation of mitochondria. Cells are subjected to osmotic swelling and disruption, followed by centrifugation in isotonic sucrose solutions to separate mitochondria from other cellular components. We present a method using this principle for the isolation of mitochondria from cultured mammalian cell lines. Mitochondria purified by this method can be further fractionated to investigate protein localization, or act as a starting point to purify mtDNA.
    Keywords:  DNA extraction; Mitochondria; Mitochondrial DNA; Mitochondrial isolation; Protein localization
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_2
  24. Methods Mol Biol. 2023 ;2615 139-151
    Assessing TFAM Binding to Human Mitochondrial DNA.
    Takehiro Yasukawa, Dongchon Kang.
      Mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA)-binding protein that plays a crucial dual role in the initiation of mitochondrial transcription initiation and mtDNA maintenance. Because TFAM directly interacts with mtDNA, assessing its DNA-binding property can provide useful information. This chapter describes two in vitro assay methods, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay with recombinant TFAM proteins, which both require simple agarose gel electrophoresis. These are used to investigate the effects of mutations, truncation, and posttranslational modifications on this key mtDNA regulatory protein.
    Keywords:  DNA-unwinding assay; EMSA; Electrophoretic mobility shift assay; Mitochondrial DNA; Mitochondrial nucleoids; Mitochondrial transcription factor A; TFAM; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_11
  25. Methods Mol Biol. 2023 ;2615 79-88
    Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies.
    Xiangjun Di, Jinshan Qin, Yujie Sun, Qian Peter Su.
      Mitochondrial DNA (mtDNA) encodes a variety of rRNAs, tRNAs, and respiratory chain complex proteins. The integrity of mtDNA supports the mitochondrial functions and plays an essential role in numerous physiological and pathological processes. Mutations in mtDNA cause metabolic diseases and aging. The mtDNA within the human cells are packaged into hundreds of nucleoids within the mitochondrial matrix. Knowledge of how the nucleoids are dynamically distributed and organized within mitochondria is key to understanding mtDNA structure and functions. Therefore, visualizing the distribution and dynamics of mtDNA within mitochondria is a powerful approach to gain insights into the regulation of mtDNA replication and transcription. In this chapter, we describe the methods of observing mtDNA and its replication with fluorescence microscopy in both fixed and live cells using different labeling strategies.
    Keywords:  BrdU; EdU; Mitochondrial DNA (mtDNA); POLG2; PdG; TFAM
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_6
  26. Methods Mol Biol. 2023 ;2615 219-228
    Studying Mitochondrial Nucleic Acid Synthesis Utilizing Intact Isolated Mitochondria.
    Jelena Misic, Dusanka Milenkovic.
      Mitochondria are eukaryotic organelles of endosymbiotic origin that contain their own genetic material, mitochondrial DNA (mtDNA), and dedicated systems for mtDNA maintenance and expression. MtDNA molecules encode a limited number of proteins that are nevertheless all essential subunits of the mitochondrial oxidative phosphorylation system. Here, we describe protocols to monitor DNA and RNA synthesis in intact, isolated mitochondria. These in organello synthesis protocols are valuable techniques for studying the mechanisms and regulation of mtDNA maintenance and expression.
    Keywords:  Mitochondria; Radioactive labeling of nucleic acids; in organello replication and transcription; mtDNA; mtDNA maintenance and expression
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_16
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