bims-mitdyn Biomed News
on Mitochondrial dynamics: mechanisms
Issue of 2023–12–03
seven papers selected by
Edmond Chan, Queen’s University, School of Medicine
  1. The poxvirus F17 protein counteracts mitochondrially orchestrated antiviral responses.
  2. Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.
  3. Effects of phosphorylation on Drp1 activation by its receptors, actin, and cardiolipin.
  4. Mitochondrial E3 ligase MARCH5 is a safeguard against DNA-PKcs-mediated immune signaling in mitochondria-damaged cells.
  5. Biased placement of Mitochondria fission facilitates asymmetric inheritance of protein aggregates during yeast cell division.
  6. Protocol for evaluating mitochondrial morphology changes in response to CCCP-induced stress through open-source image processing software.
  7. TFAM mislocalization during spermatogenesis.
  1. Nat Commun. 2023 Nov 30. 14(1): 7889
    The poxvirus F17 protein counteracts mitochondrially orchestrated antiviral responses.
    Nathan Meade, Helen K Toreev, Ram P Chakrabarty, Charles R Hesser, Chorong Park, Navdeep S Chandel, Derek Walsh.
      Poxviruses are unusual DNA viruses that replicate in the cytoplasm. To do so, they encode approximately 100 immunomodulatory proteins that counteract cytosolic nucleic acid sensors such as cGAMP synthase (cGAS) along with several other antiviral response pathways. Yet most of these immunomodulators are expressed very early in infection while many are variable host range determinants, and significant gaps remain in our understanding of poxvirus sensing and evasion strategies. Here, we show that after infection is established, subsequent progression of the viral lifecycle is sensed through specific changes to mitochondria that coordinate distinct aspects of the antiviral response. Unlike other viruses that cause extensive mitochondrial damage, poxviruses sustain key mitochondrial functions including membrane potential and respiration while reducing reactive oxygen species that drive inflammation. However, poxvirus replication induces mitochondrial hyperfusion that independently controls the release of mitochondrial DNA (mtDNA) to prime nucleic acid sensors and enables an increase in glycolysis that is necessary to support interferon stimulated gene (ISG) production. To counter this, the poxvirus F17 protein localizes to mitochondria and dysregulates mTOR to simultaneously destabilize cGAS and block increases in glycolysis. Our findings reveal how the poxvirus F17 protein disarms specific mitochondrially orchestrated responses to later stages of poxvirus replication.
    DOI:  https://doi.org/10.1038/s41467-023-43635-y
  2. Nat Metab. 2023 Nov 30.
    Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.
    Wendy K Shoop, Janel Lape, Megan Trum, Alea Powell, Emma Sevigny, Adam Mischler, Sandra R Bacman, Flavia Fontanesi, Jeff Smith, Derek Jantz, Cassandra L Gorsuch, Carlos T Moraes.
      Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.
    DOI:  https://doi.org/10.1038/s42255-023-00932-6
  3. Mol Biol Cell. 2023 Nov 29. mbcE23110427
    Effects of phosphorylation on Drp1 activation by its receptors, actin, and cardiolipin.
    Ao Liu, Anna L Hatch, Henry N Higgs.
      Drp1 is a dynamin family GTPase required for mitochondrial and peroxisomal division. Oligomerization increases Drp1 GTPase activity through interactions between neighboring GTPase domains. In cells, Drp1 is regulated by several factors including Drp1 receptors, actin filaments, cardiolipin, and phosphorylation at two sites: S579 and S600. Commonly, phosphorylation of S579 is considered activating, while S600 phosphorylation is considered inhibiting. However, direct effects of phosphorylation on Drp1 GTPase activity have not been investigated in detail. Here, we compare effects of S579 and S600 phosphorylation on purified Drp1, using phospho-mimetic mutants and in vitro phosphorylation. Both phospho-mimetic mutants are shifted toward smaller oligomers. Both phospho-mimetic mutations maintain basal GTPase activity, but eliminate GTPase stimulation by actin and decrease GTPase stimulation by cardiolipin, Mff, and MiD49. Phosphorylation of S579 by Erk2 produces similar effects. When mixed with wildtype Drp1, both S579D and S600D phospho-mimetic mutants reduce the actin-stimulated GTPase activity of Drp1-WT. Conversely, a Drp1 mutant (K38A) lacking GTPase activity stimulates Drp1-WT GTPase activity under both basal and actin-stimulated conditions. These results suggest that the effect of S579 phosphorylation is not to activate Drp1 directly. In addition, our results suggest that nearest neighbor interactions within the Drp1 oligomer affect catalytic activity.
    DOI:  https://doi.org/10.1091/mbc.E23-11-0427
  4. Cell Death Dis. 2023 Dec 01. 14(12): 788
    Mitochondrial E3 ligase MARCH5 is a safeguard against DNA-PKcs-mediated immune signaling in mitochondria-damaged cells.
    June Heo, Yeon-Ji Park, Yonghyeon Kim, Ho-Soo Lee, Jeongah Kim, Soon-Hwan Kwon, Myeong-Gyun Kang, Hyun-Woo Rhee, Woong Sun, Jae-Ho Lee, Hyeseong Cho.
      Mitochondrial dysfunction is important in various chronic degenerative disorders, and aberrant immune responses elicited by cytoplasmic mitochondrial DNA (mtDNA) may be related. Here, we developed mtDNA-targeted MTERF1-FokI and TFAM-FokI endonuclease systems to induce mitochondrial DNA double-strand breaks (mtDSBs). In these cells, the mtDNA copy number was significantly reduced upon mtDSB induction. Interestingly, in cGAS knockout cells, synthesis of interferon β1 and interferon-stimulated gene was increased upon mtDSB induction. We found that mtDSBs activated DNA-PKcs and HSPA8 in a VDAC1-dependent manner. Importantly, the mitochondrial E3 ligase MARCH5 bound active DNA-PKcs in cells with mtDSBs and reduced the type І interferon response through the degradation of DNA-PKcs. Likewise, mitochondrial damage caused by LPS treatment in RAW264.7 macrophage cells increased phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA in a DNA-PKcs-dependent manner. Accordingly, in March5 knockout macrophages, phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA were prolonged after LPS stimulation. Together, cytoplasmic mtDNA elicits a cellular immune response through DNA-PKcs, and mitochondrial MARCH5 may be a safeguard to prevent persistent inflammatory reactions.
    DOI:  https://doi.org/10.1038/s41419-023-06315-9
  5. PLoS Comput Biol. 2023 Nov 27. 19(11): e1011588
    Biased placement of Mitochondria fission facilitates asymmetric inheritance of protein aggregates during yeast cell division.
    Gordon Sun, Christine Hwang, Tony Jung, Jian Liu, Rong Li.
      Mitochondria are essential and dynamic eukaryotic organelles that must be inherited during cell division. In yeast, mitochondria are inherited asymmetrically based on quality, which is thought to be vital for maintaining a rejuvenated cell population; however, the mechanisms underlying mitochondrial remodeling and segregation during this process are not understood. We used high spatiotemporal imaging to quantify the key aspects of mitochondrial dynamics, including motility, fission, and fusion characteristics, upon aggregation of misfolded proteins in the mitochondrial matrix. Using these measured parameters, we developed an agent-based stochastic model of dynamics of mitochondrial inheritance. Our model predicts that biased mitochondrial fission near the protein aggregates facilitates the clustering of protein aggregates in the mitochondrial matrix, and this process underlies asymmetric mitochondria inheritance. These predictions are supported by live-cell imaging experiments where mitochondrial fission was perturbed. Our findings therefore uncover an unexpected role of mitochondrial dynamics in asymmetric mitochondrial inheritance.
    DOI:  https://doi.org/10.1371/journal.pcbi.1011588
  6. STAR Protoc. 2023 Nov 29. pii: S2666-1667(23)00712-8. [Epub ahead of print]4(4): 102745
    Protocol for evaluating mitochondrial morphology changes in response to CCCP-induced stress through open-source image processing software.
    Sudeshna Nag, Kaitlin Szederkenyi, Christopher M Yip, G Angus McQuibban.
      Mitochondrial morphology is an indicator of cellular health and function; however, its quantification and categorization into different subclasses is a complicated process. Here, we present a protocol for mitochondrial morphology quantification in the presence and absence of carbonyl cyanide m-chlorophenyl hydrazone stress. We describe steps for the preparation of cells for immunofluorescence microscopy, staining, and morphology quantification. The quantification protocol generates an aspect ratio that helps to categorize mitochondria into two clear subclasses. For complete details on the use and execution of this protocol, please refer to Nag et al.1.
    Keywords:  Cell Biology; Cell culture; Metabolism; Microscopy; Molecular/Chemical Probes
    DOI:  https://doi.org/10.1016/j.xpro.2023.102745
  7. Trends Genet. 2023 Nov 29. pii: S0168-9525(23)00257-3. [Epub ahead of print]
    TFAM mislocalization during spermatogenesis.
    Sam Kavoosi, Martin Picard, Brett A Kaufman.
      Mitochondrial DNA (mtDNA) is inherited almost exclusively from the maternal lineage. Paternal destruction of either mtDNA or whole mitochondria has been the dominant model for mtDNA transmission. Recently, Lee et al. provided evidence for mitochondrial transcription factor A (TFAM) import sequence regulation as a potential cause for mtDNA depletion in human sperm before fertilization.
    Keywords:  TFAM; mtDNA; spermatogenesis; uniparental inheritance
    DOI:  https://doi.org/10.1016/j.tig.2023.11.002
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