bims-midmar Biomed News
on Mitochondrial DNA maintenance and replication
Issue of 2022–04–17
ten papers selected by
Flavia Söllner, Ludwig-Maximilians University



  1. Methods Mol Biol. 2022 ;2431 409-416
      Mitochondria are essential organelles that generate energy and play vital roles in cellular metabolism. The small circular mitochondrial genome encodes key components of the mitochondrial respiratory apparatus. Depletion of, or mutations in mitochondrial DNA (mtDNA) cause mitochondrial dysfunction and disease. mtDNA is packaged into nucleoids, which are transported throughout the cell within mitochondria. Efficient transport of nucleoids is essential in neurons, where mitochondrial function is required locally at synapses. Here I describe methods for visualization of nucleoids in Drosophila neurons using a GFP fusion of the mitochondrial transcription factor TFAM. TFAM-GFP, together with mCherry-labeled mitochondria, was used to visualize nucleoids in fixed larval segmental nerves. I also describe how these tools can be used for live imaging of nucleoid dynamics. Using Drosophila as a model system, these methods will enable further characterization and analysis of nucleoid dynamics in neurons.
    Keywords:  Drosophila; Live imaging; Mitochondrial DNA; Nucleoid; TFAM
    DOI:  https://doi.org/10.1007/978-1-0716-1990-2_21
  2. Methods Mol Biol. 2022 ;2431 291-310
      Mitochondria are highly dynamic organelles which form intricate networks with complex dynamics. Mitochondrial transport and distribution are essential to ensure proper cell function, especially in cells with an extremely polarised morphology such as neurons. A layer of complexity is added when considering mitochondria have their own genome, packaged into nucleoids. Major mitochondrial morphological transitions, for example mitochondrial division, often occur in conjunction with mitochondrial DNA (mtDNA) replication and changes in the dynamic behaviour of the nucleoids. However, the relationship between mtDNA dynamics and mitochondrial motility in the processes of neurons has been largely overlooked. In this chapter, we describe a method for live imaging of mitochondria and nucleoids in differentiated SH-SY5Y cells by instant structured illumination microscopy (iSIM). We also include a detailed protocol for the differentiation of SH-SY5Y cells into cells with a pronounced neuronal-like morphology and show examples of coordinated mitochondrial and nucleoid motility in the long processes of these cells.
    Keywords:  Axonal transport; Instant structured illumination microscopy (iSIM); Mitochondria; Mitochondrial DNA; Mitochondrial fission; Neuronal differentiation; Nucleoids; SH-SY5Y cells; Superresolution
    DOI:  https://doi.org/10.1007/978-1-0716-1990-2_15
  3. Bioinformatics. 2022 Apr 12. pii: btac216. [Epub ahead of print]
       SUMMARY: We present MitoVisualize, a new tool for analysis of the human mitochondrial DNA (mtDNA). MitoVisualize enables visualization of: (1) the position and effect of variants in mitochondrial transfer RNA (tRNA) and ribosomal RNA (rRNA) secondary structures alongside curated variant annotations, (2) data across RNA structures, such as to show all positions with disease-associated variants or with post-transcriptional modifications, and (3) the position of a base, gene or region in the circular mtDNA map, such as to show the location of a large deletion. All visualizations can be easily downloaded as figures for reuse. MitoVisualize can be useful for anyone interested in exploring mtDNA variation, though is designed to facilitate mtDNA variant interpretation in particular.
    AVAILABILITY AND IMPLEMENTATION: MitoVisualize can be accessed via https://www.mitovisualize.org/. The source code is available at https://github.com/leklab/mito_visualize/.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btac216
  4. Elife. 2022 Apr 11. pii: e76557. [Epub ahead of print]11
      High frequencies of mutant mitochondrial DNA (mtDNA) in human cells lead to cellular defects that are associated with aging and disease. Yet much remains to be understood about the dynamics of the generation of mutant mtDNAs and their relative replicative fitness that informs their fate within cells and tissues. To address this, we utilize long-read single-molecule sequencing to track mutational trajectories of mtDNA in the model organism Saccharomyces cerevisiae. This model has numerous advantages over mammalian systems due to its much larger mtDNA and ease of artificially competing mutant and wild-type mtDNA copies in cells. We show a previously unseen pattern that constrains subsequent excision events in mtDNA fragmentation in yeast. We also provide evidence for the generation of rare and contentious non-periodic mtDNA structures that lead to persistent diversity within individual cells. Finally, we show that measurements of relative fitness of mtDNA fit a phenomenological model that highlights important biophysical parameters governing mtDNA fitness. Altogether, our study provides techniques and insights into the dynamics of large structural changes in genomes that we show are applicable to more complex organisms like humans.
    Keywords:  S. cerevisiae; computational biology; genetics; genomics; systems biology
    DOI:  https://doi.org/10.7554/eLife.76557
  5. Int J Mol Sci. 2022 Mar 23. pii: 3474. [Epub ahead of print]23(7):
      Mitochondria are key organelles that combine features inherited from their bacterial endosymbiotic ancestor with traits that arose during eukaryote evolution. These energy producing organelles have retained a genome and fully functional gene expression machineries including specific ribosomes. Recent advances in cryo-electron microscopy have enabled the characterization of a fast-growing number of the low abundant membrane-bound mitochondrial ribosomes. Surprisingly, mitoribosomes were found to be extremely diverse both in terms of structure and composition. Still, all of them drastically increased their number of ribosomal proteins. Interestingly, among the more than 130 novel ribosomal proteins identified to date in mitochondria, most of them are composed of a-helices. Many of them belong to the nuclear encoded super family of helical repeat proteins. Here we review the diversity of functions and the mode of action held by the novel mitoribosome proteins and discuss why these proteins that share similar helical folds were independently recruited by mitoribosomes during evolution in independent eukaryote clades.
    Keywords:  helical repeat proteins; mitochondrial gene expression; pentatricopeptide repeat proteins; ribosomes; single particle cryo-EM; translation
    DOI:  https://doi.org/10.3390/ijms23073474
  6. STAR Protoc. 2022 Jun 17. 3(2): 101278
      The Golgi apparatus is subjected to fragmentation under several cellular processes such as mitosis. Here we describe two complementary approaches to analyze different Golgi morphological changes during its mitotic fragmentation, using classical immunofluorescence and imaging flow cytometry. Although fluorescent microscopy provides information on the exact Golgi architecture in distinct cells, the imaging flow cytometry combines the morphological data with the high-throughput quantification of flow cytometry. Taken together, both approaches provide robust and significant unbiased data analysis. For complete details on the use and execution of this protocol, please refer to Wortzel et al. (2021).
    Keywords:  Cell Biology; Flow Cytometry/Mass Cytometry; Microscopy
    DOI:  https://doi.org/10.1016/j.xpro.2022.101278
  7. RSC Adv. 2022 Jan 18. 12(5): 2668-2674
      Glutathione (GSH), an abundant non-protein thiol, plays a crucial role in numerous biotic processes. Herein, a mitochondria-targeted near-infrared GSH probe (JGP) was synthesized, which displayed desired properties with high specificity and sensitivity, appreciable water solubility, and rapid response time. In the presence of GSH, nearly a 13-fold fluorescence emission growth appeared at 730 nm and the solvent color changed from blue to cyan. The sensing mechanism of JGP and GSH was confirmed by a high-resolution mass spectroscopy analysis. Moreover, good cell penetration enabled JGP to be successfully used for imaging biological samples such as HeLa cells, C. elegans, and especially rat brain slices. Imaging experiments showed that JGP could monitor the GSH concentration changes with a dose-dependent direct ratio in all the tested samples. The successful application of JGP in brain imaging indicates that JGP is a suitable GSH optical probe, which may have wide application value in fields of brain imaging. It also lays a theoretical and practical foundation for the further application of fluorescent probes in brain sciences.
    DOI:  https://doi.org/10.1039/d1ra08917j
  8. J Agric Food Chem. 2022 Apr 14.
      Bisulfite (HSO3-)/Sulfite (SO32-) is widely used as a food additive, but excessive use often leads to serious consequences, so the detection of HSO3-/SO32- is of great importance. In this paper, a novel 1,4-diethylpiperazine-modified coumarin-benzopyran derivative (probe QLP) has been synthesized and characterized. In PBS (10 mM, pH = 7.4), QLP displays good selectivity and is sensitive for HSO3-/SO32- over various analytes with fluorescent "OFF-ON" rapid responding (2 min), long-wavelength emission (600 nm), and a detection limit of 177 nM. With the treatment of HSO3-/SO32-, the color of the QLP solution obviously changes from blue-green to yellow, and the fluorescent color of QLP changes from colorless to amaranth. The fluorescence-enhanced mechanism is qualitatively evaluated by density functional theory calculations using the CAM-B3LYP/6-31G (d) method, which reveals that the photoinduced electron transfer leads to the fluorescence emission of the QLP-SO3H adduct. Importantly, nontoxic QLP can be used to detect HSO3-/SO32- in sugar, natural water samples, and living cells and localized to the mitochondria and monitor the mitochondrial HSO3-/SO32- level.
    Keywords:  bisulfite/sulfite; fluorescent probe; food samples; mitochondria-targeted; rapid detection; turn-on responding
    DOI:  https://doi.org/10.1021/acs.jafc.2c00820
  9. J Cell Sci. 2022 Apr 01. pii: jcs258986. [Epub ahead of print]135(7):
      For the past century, the nucleus has been the focus of extensive investigations in cell biology. However, many questions remain about how its shape and size are regulated during development, in different tissues, or during disease and aging. To track these changes, microscopy has long been the tool of choice. Image analysis has revolutionized this field of research by providing computational tools that can be used to translate qualitative images into quantitative parameters. Many tools have been designed to delimit objects in 2D and, eventually, in 3D in order to define their shapes, their number or their position in nuclear space. Today, the field is driven by deep-learning methods, most of which take advantage of convolutional neural networks. These techniques are remarkably adapted to biomedical images when trained using large datasets and powerful computer graphics cards. To promote these innovative and promising methods to cell biologists, this Review summarizes the main concepts and terminologies of deep learning. Special emphasis is placed on the availability of these methods. We highlight why the quality and characteristics of training image datasets are important and where to find them, as well as how to create, store and share image datasets. Finally, we describe deep-learning methods well-suited for 3D analysis of nuclei and classify them according to their level of usability for biologists. Out of more than 150 published methods, we identify fewer than 12 that biologists can use, and we explain why this is the case. Based on this experience, we propose best practices to share deep-learning methods with biologists.
    Keywords:  3D microscopy images; 3D nucleus; 3D segmentation; Deep learning; Image dataset; Open source
    DOI:  https://doi.org/10.1242/jcs.258986
  10. Cells. 2022 Apr 06. pii: 1240. [Epub ahead of print]11(7):
      As a fast-growing, woody grass plant, Moso bamboo (Phyllostachys edulis) can supply edible shoots, building materials, fibrous raw material, raw materials for crafts and furniture and so on within a relatively short time. Rapid growth of Moso bamboo occurs after the young bamboo shoots are covered with a shell and emerge from the ground. However, the molecular reactions of bioenergetic processes essential for fast growth remain undefined. Herein, total and mitochondrial transcriptomes and proteomes were compared between spring and winter shoots. Numerous key genes and proteins responsible for energy metabolism were significantly upregulated in spring shoots, including those involved in starch and sucrose catabolism, glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and oxidative phosphorylation. Accordingly, significant decreases in starch and soluble sugar, higher ATP content and higher rates of respiration and glycolysis were identified in spring shoots. Further, the upregulated genes and proteins related to mitochondrial fission significantly increased the number of mitochondria, indirectly promoting intracellular energy metabolism. Moreover, enhanced alternate-oxidase and uncoupled-protein pathways in winter shoots showed that an efficient energy-dissipating system was important for winter shoots to adapt to the low-temperature environment. Heterologous expression of PeAOX1b in Arabidopsis significantly affected seedling growth and enhanced cold-stress tolerance. Overall, this study highlights the power of comparing total and mitochondrial omics and integrating physiochemical data to understand how bamboo initiates fast growth through modulating bioenergetic processes.
    Keywords:  Phyllostachys edulis; RNA sequencing; cold tolerance; energy metabolism; fast growth; mitochondrial fission; proteomics
    DOI:  https://doi.org/10.3390/cells11071240