bims-mitdyn Biomed News
on Mitochondrial dynamics: mechanisms
Issue of 2024–08–11
fiveteen papers selected by
Edmond Chan, Queen’s University, School of Medicine
  1. PERK-ATAD3A interaction provides a subcellular safe haven for protein synthesis during ER stress.
  2. SLC25A48 controls mitochondrial choline import and metabolism.
  3. Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.
  4. Heterozygous de novo dominant negative mutation of REXO2 results in interferonopathy.
  5. Preserving mitochondrial homeostasis protects against drug-induced liver injury via inducing OPTN (optineurin)-dependent Mitophagy.
  6. Purinosomes spatially co-localize with mitochondrial transporters.
  7. Naturally occurring hyperactive variants of human parkin.
  8. Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level.
  9. Hif1α-dependent mitochondrial acute O2 sensing and signaling to myocyte Ca2+ channels mediate arterial hypoxic vasodilation.
  10. Neuro-intestinal acetylcholine signalling regulates the mitochondrial stress response in Caenorhabditis elegans.
  11. Mitochondrial Transfer Between Mesenchymal Stem Cells and Cancer Cells.
  12. Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.
  13. Methods for Monitoring Mitophagy Using mt-Keima.
  14. Analysis of Mitophagy Reporters in Drosophila.
  15. Assessing Neuronal Mitophagy During Development in the Nematode Caenorhabditis elegans.
  1. Science. 2024 Aug 08. eadp7114
    PERK-ATAD3A interaction provides a subcellular safe haven for protein synthesis during ER stress.
    Karinder K Brar, Daniel T Hughes, Jordan L Morris, Kelly Subramanian, Shivaani Krishna, Fei Gao, Lara-Sophie Rieder, Sebastian Uhrig, Joshua Freeman, Heather L Smith, Rebekkah Jukes-Jones, Edward Avezov, Jodi Nunnari, Julien Prudent, Adrian J Butcher, Giovanna R Mallucci.
      Endoplasmic Reticulum (ER) stress induces repression of protein synthesis throughout the cell. Attempts to understand how localized stress leads to widespread repression have been limited by difficulties in resolving translation rates at the subcellular level. Here, using live-cell imaging of reporter mRNA translation, we unexpectedly found that during ER stress active translation at mitochondria was significantly protected. The mitochondrial protein, ATAD3A, interacted with PERK and mediated this effect on localized translation by competing for binding with PERK's target, eIF2. PERK-ATAD3A interactions increased during ER stress, forming mitochondria-ER contact sites. Furthermore, ATAD3A binding attenuated local PERK signaling and rescued the expression of some mitochondrial proteins. Thus, PERK-ATAD3A interactions can control translational repression at a subcellular level, mitigating the impact of ER stress on the cell.
    DOI:  https://doi.org/10.1126/science.adp7114
  2. Cell Metab. 2024 Aug 01. pii: S1550-4131(24)00281-X. [Epub ahead of print]
    SLC25A48 controls mitochondrial choline import and metabolism.
    Anthony R P Verkerke, Xu Shi, Mark Li, Yusuke Higuchi, Tadashi Yamamuro, Daisuke Katoh, Hiroshi Nishida, Christopher Auger, Ichitaro Abe, Robert E Gerszten, Shingo Kajimura.
      Choline is an essential nutrient for the biosynthesis of phospholipids, neurotransmitters, and one-carbon metabolism with a critical step being its import into mitochondria. However, the underlying mechanisms and biological significance remain poorly understood. Here, we report that SLC25A48, a previously uncharacterized mitochondrial inner-membrane carrier protein, controls mitochondrial choline transport and the synthesis of choline-derived methyl donors. We found that SLC25A48 was required for brown fat thermogenesis, mitochondrial respiration, and mitochondrial membrane integrity. Choline uptake into the mitochondrial matrix via SLC25A48 facilitated the synthesis of betaine and purine nucleotides, whereas loss of SLC25A48 resulted in increased production of mitochondrial reactive oxygen species and imbalanced mitochondrial lipids. Notably, human cells carrying a single nucleotide polymorphism on the SLC25A48 gene and cancer cells lacking SLC25A48 exhibited decreased mitochondrial choline import, increased oxidative stress, and impaired cell proliferation. Together, this study demonstrates that SLC25A48 regulates mitochondrial choline catabolism, bioenergetics, and cell survival.
    Keywords:  bioenergetics; brown adipose tissue; cancer metabolism; choline; mitochondria; purine nucleotides
    DOI:  https://doi.org/10.1016/j.cmet.2024.07.010
  3. Nat Commun. 2024 Aug 08. 15(1): 6777
    Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.
    Soeun Kang, Maciek R Antoniewicz, Nissim Hay.
      Metabolic rewiring during the proliferation-to-quiescence transition is poorly understood. Here, using a model of contact inhibition-induced quiescence, we conducted 13C-metabolic flux analysis in proliferating (P) and quiescent (Q) mouse embryonic fibroblasts (MEFs) to investigate this process. Q cells exhibit reduced glycolysis but increased TCA cycle flux and mitochondrial respiration. Reduced glycolytic flux in Q cells correlates with reduced glycolytic enzyme expression mediated by yes-associated protein (YAP) inhibition. The increased TCA cycle activity and respiration in Q cells is mediated by induced mitochondrial pyruvate carrier (MPC) expression, rendering them vulnerable to MPC inhibition. The malate-to-pyruvate flux, which generates NADPH, is markedly reduced by modulating malic enzyme 1 (ME1) dimerization in Q cells. Conversely, the malate dehydrogenase 1 (MDH1)-mediated oxaloacetate-to-malate flux is reversed and elevated in Q cells, driven by high mitochondrial-derived malate levels, reduced cytosolic oxaloacetate, elevated MDH1 levels, and a high cytoplasmic NAD+/NADH ratio. Transcriptomic analysis revealed large number of genes are induced in Q cells, many of which are associated with the extracellular matrix (ECM), while YAP-dependent and cell cycle-related genes are repressed. The results suggest that high TCA cycle flux and respiration in Q cells are required to generate ATP and amino acids to maintain de-novo ECM protein synthesis and secretion.
    DOI:  https://doi.org/10.1038/s41467-024-51117-y
  4. Nat Commun. 2024 Aug 06. 15(1): 6685
    Heterozygous de novo dominant negative mutation of REXO2 results in interferonopathy.
    Elina Idiiatullina, Mahmoud Al-Azab, Meng Lin, Katja Hrovat-Schaale, Ziyang Liu, Xiaotian Li, Caiqin Guo, Xixi Chen, Yaoying Li, Song Gao, Jun Cui, Wenhao Zhou, Li Liu, Yuxia Zhang, Seth L Masters.
      Mitochondrial RNA (mtRNA) in the cytosol can trigger the innate immune sensor MDA5, and autoinflammatory disease due to type I IFN. Here, we show that a dominant negative mutation in the gene encoding the mitochondrial exonuclease REXO2 may cause interferonopathy by triggering the MDA5 pathway. A patient characterized by this heterozygous de novo mutation (p.T132A) presented with persistent skin rash featuring hyperkeratosis, parakeratosis and acanthosis, with infiltration of lymphocytes and eosinophils around small blood vessels. In addition, circulating IgE levels and inflammatory cytokines, including IFNα, are found consistently elevated. Transcriptional analysis highlights a type I IFN gene signature in PBMC. Mechanistically, REXO2 (T132A) lacks the ability to cleave RNA and inhibits the activity of wild-type REXO2. This leads to an accumulation of mitochondrial dsRNA in the cytosol, which is recognized by MDA5, leading to the associated type I IFN gene signature. These results demonstrate that in the absence of appropriate regulation by REXO2, aberrant cellular nucleic acids may accumulate and continuously trigger innate sensors, resulting in an inborn error of immunity.
    DOI:  https://doi.org/10.1038/s41467-024-50878-w
  5. Autophagy. 2024 Aug 04. 1-20
    Preserving mitochondrial homeostasis protects against drug-induced liver injury via inducing OPTN (optineurin)-dependent Mitophagy.
    Jiajia Wang, Yueping Qiu, Lijun Yang, Jincheng Wang, Jie He, Chengwu Tang, Zhaoxu Yang, Wenxiang Hong, Bo Yang, Qiaojun He, Qinjie Weng.
      Disruption of mitochondrial function is observed in multiple drug-induced liver injuries (DILIs), a significant global health threat. However, how the mitochondrial dysfunction occurs and whether maintain mitochondrial homeostasis is beneficial for DILIs remains unclear. Here, we show that defective mitophagy by OPTN (optineurin) ablation causes disrupted mitochondrial homeostasis and aggravates hepatocytes necrosis in DILIs, while OPTN overexpression protects against DILI depending on its mitophagic function. Notably, mass spectrometry analysis identifies a new mitochondrial substrate, GCDH (glutaryl-CoA dehydrogenase), which can be selectively recruited by OPTN for mitophagic degradation, and a new cofactor, VCP (valosin containing protein) that interacts with OPTN to stabilize BECN1 during phagophore assembly, thus boosting OPTN-mediated mitophagy initiation to clear damaged mitochondria and preserve mitochondrial homeostasis in DILIs. Then, the accumulation of OPTN in different DILIs is further validated with a protective effect, and pyridoxine is screened and established to alleviate DILIs by inducing OPTN-mediated mitophagy. Collectively, our findings uncover a dual role of OPTN in mitophagy initiation and implicate the preservation of mitochondrial homeostasis via inducing OPTN-mediated mitophagy as a potential therapeutic approach for DILIs.Abbreviation: AILI: acetaminophen-induced liver injury; ALS: amyotrophic lateral sclerosis; APAP: acetaminophen; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DILI: drug-induced liver injury; FL: full length; GCDH: glutaryl-CoA dehydrogenase; GOT1/AST: glutamic-oxaloacetic transaminase 1; GO: gene ontology; GSEA: gene set enrichment analysis; GPT/ALT: glutamic - pyruvic transaminase; INH: isoniazid; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMP: mitochondrial membrane potential; MST: microscale thermophoresis; MT-CO2/COX-II: mitochondrially encoded cytochrome c oxidase II; OPTN: optineurin; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; TSN: toosendanin; VCP: valosin containing protein, WIPI2: WD repeat domain, phosphoinositide interacting 2.
    Keywords:  Drug-induced liver injury; mitochondrial homeostasis; mitophagy; optineurin; phagophore formation
    DOI:  https://doi.org/10.1080/15548627.2024.2384348
  6. J Biol Chem. 2024 Aug 02. pii: S0021-9258(24)02121-5. [Epub ahead of print] 107620
    Purinosomes spatially co-localize with mitochondrial transporters.
    Zhou Sha, Stephen J Benkovic.
      In this study, we advance our understanding of the spatial relationship between the purinosome, a liquid condensate consisting of six enzymes involved in de novo purine biosynthesis, and mitochondria. Previous research has shown that purinosomes move along tubulin toward mitochondria, suggesting a direct uptake of glycine from mitochondria. Here, we propose that the purinosome is located proximally to the mitochondrial transporters SLC25A13 and SLC25A38, facilitating the uptake of glycine, aspartate, and glutamate, essential factors for purine synthesis. We utilized the proximity ligation assay (PLA) and APEX proximity labeling to investigate the association between purinosome proteins and mitochondrial transporters. Our results indicate that purinosome assembly occurs close to the mitochondrial membrane under purine-deficient conditions, with the transporters migrating to be adjacent to the purinosome. Furthermore, both targeted and non-targeted analyses suggest that the SLC25A13-APEX2-V5 probe accurately reflects endogenous cellular status. These findings provide insights into the spatial organization of purine biosynthesis and lay the groundwork for further investigations into additional proteins involved in this pathway.
    Keywords:  APEX; Purinosomes; de novo purine biosynthesis; mitochondrial transporter; proximity ligation assay (PLA)
    DOI:  https://doi.org/10.1016/j.jbc.2024.107620
  7. Commun Biol. 2024 Aug 08. 7(1): 961
    Naturally occurring hyperactive variants of human parkin.
    Tahrima Saiha Huq, Jean Luo, Rayan Fakih, Véronique Sauvé, Kalle Gehring.
      Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. Although most cases are sporadic and occur later in life, 10-15% of cases are genetic. Loss-of-function mutations in the ring-between-ring E3 ubiquitin ligase parkin, encoded by the PRKN gene, cause autosomal recessive forms of early onset PD. Together with the kinase PINK1, parkin forms a mitochondrial quality control pathway that tags damaged mitochondria for clearance. Under basal conditions, parkin is inhibited and compounds that increase its activity have been proposed as a therapy for PD. Recently, several naturally occurring hyperactive parkin variants were identified, which increased mitophagy in cultured cells. Here, we validate the hyperactivities of these variants in vitro and compare the levels of activity of the variants to those of the wild-type and the well-characterized hyperactive variant, W403A. We also study the effects of mutating the parkin ACT (activating element) on parkin activity in vitro. This work advances our understanding of the pathogenicity of parkin variants and is an important first step in the design of molecules to increase parkin activity.
    DOI:  https://doi.org/10.1038/s42003-024-06656-x
  8. EMBO J. 2024 Aug 05.
    Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level.
    Rodaria Roussou, Dirk Metzler, Francesco Padovani, Felix Thoma, Rebecca Schwarz, Boris Shraiman, Kurt M Schmoller, Christof Osman.
      Mitochondrial DNA (mtDNA) is present in multiple copies within cells and is required for mitochondrial ATP generation. Even within individual cells, mtDNA copies can differ in their sequence, a state known as heteroplasmy. The principles underlying dynamic changes in the degree of heteroplasmy remain incompletely understood, due to the inability to monitor this phenomenon in real time. Here, we employ mtDNA-based fluorescent markers, microfluidics, and automated cell tracking, to follow mtDNA variants in live heteroplasmic yeast populations at the single-cell level. This approach, in combination with direct mtDNA tracking and data-driven mathematical modeling reveals asymmetric partitioning of mtDNA copies during cell division, as well as limited mitochondrial fusion and fission frequencies, as critical driving forces for mtDNA variant segregation. Given that our approach also facilitates assessment of segregation between intact and mutant mtDNA, we anticipate that it will be instrumental in elucidating the mechanisms underlying the purifying selection of mtDNA.
    Keywords:  Heteroplasmy; Mathematical Modeling; Mitochondria; Mitochondrial Fission; mtDNA
    DOI:  https://doi.org/10.1038/s44318-024-00183-5
  9. Nat Commun. 2024 Aug 05. 15(1): 6649
    Hif1α-dependent mitochondrial acute O2 sensing and signaling to myocyte Ca2+ channels mediate arterial hypoxic vasodilation.
    Alejandro Moreno-Domínguez, Olalla Colinas, Ignacio Arias-Mayenco, José M Cabeza, Juan L López-Ogayar, Navdeep S Chandel, Norbert Weissmann, Natascha Sommer, Alberto Pascual, José López-Barneo.
      Vasodilation in response to low oxygen (O2) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O2 supply to tissues according to demand. However, how blood vessels react to O2 deficiency is not well understood. A common belief is that arterial myocytes are O2-sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Ca2+channels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O2 modulation of Ca2+ channels and hypoxic vasodilation. Mitochondria function as O2 sensors and effectors that signal myocyte Ca2+ channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O2-sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology.
    DOI:  https://doi.org/10.1038/s41467-024-51023-3
  10. Nat Commun. 2024 Aug 03. 15(1): 6594
    Neuro-intestinal acetylcholine signalling regulates the mitochondrial stress response in Caenorhabditis elegans.
    Rebecca Cornell, Wei Cao, Bernie Harradine, Rasoul Godini, Ava Handley, Roger Pocock.
      Neurons coordinate inter-tissue protein homeostasis to systemically manage cytotoxic stress. In response to neuronal mitochondrial stress, specific neuronal signals coordinate the systemic mitochondrial unfolded protein response (UPRmt) to promote organismal survival. Yet, whether chemical neurotransmitters are sufficient to control the UPRmt in physiological conditions is not well understood. Here, we show that gamma-aminobutyric acid (GABA) inhibits, and acetylcholine (ACh) promotes the UPRmt in the Caenorhabditis elegans intestine. GABA controls the UPRmt by regulating extra-synaptic ACh release through metabotropic GABAB receptors GBB-1/2. We find that elevated ACh levels in animals that are GABA-deficient or lack ACh-degradative enzymes induce the UPRmt through ACR-11, an intestinal nicotinic α7 receptor. This neuro-intestinal circuit is critical for non-autonomously regulating organismal survival of oxidative stress. These findings establish chemical neurotransmission as a crucial regulatory layer for nervous system control of systemic protein homeostasis and stress responses.
    DOI:  https://doi.org/10.1038/s41467-024-50973-y
  11. Methods Mol Biol. 2024 ;2835 39-48
    Mitochondrial Transfer Between Mesenchymal Stem Cells and Cancer Cells.
    S Kumar Panda, M Torsiello, A Rehman, Vincenzo Desiderio, V Del Vecchio.
      Mitochondrial transfer (MT) is a biological process that allows a donor cell to horizontally share its own mitochondria with a recipient cell. Mitochondria are highly dynamic membrane-bound sub-cellular organelles prominently involved in the regulation of the cell energy balance, calcium homeostasis, and apoptotic machinery activation. They physiologically undergo fusion and fission processes in response to the cell requirement, with a continuous morphological re-arrangement. This structural and functional plasticity is at the basis of the MT, described in tissue regeneration, cardiac and neurological diseases, as well as in cancer. Here, the MT has been observed in the tumor micro-environment (TME) from the adipose-derived stem cells (ASCs) to the cancer cells, eventually reverting the lack of the mitochondria respiration function, or enhancing their motility and drug resistance. In this chapter, we outline some key protocols for evaluating this exciting phenomenon of MT. These methodological and technical approaches are very important, considering all the limitations that scientists constantly face, especially in this field of the research.
    Keywords:  Mitochondrial transfer; Stromal cells; Tumor microenvironment (TME)
    DOI:  https://doi.org/10.1007/978-1-0716-3995-5_4
  12. Methods Mol Biol. 2024 ;2845 1-14
    Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.
    Yuki Nakayama, Koji Okamoto.
      Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.
    Keywords:  Atg32; Budding yeast; Fluorescence microscopy; Immunoblotting; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_1
  13. Methods Mol Biol. 2024 ;2845 151-160
    Methods for Monitoring Mitophagy Using mt-Keima.
    Alexandra J Gilsrud, Derek P Narendra.
      Mitochondria-targeted Keima (mt-Keima) is a pH-sensitive, acid-stable fluorescent protein used for the quantification of mitophagy. Mt-Keima contains a mitochondrial matrix targeting sequence and has bimodal excitation with peaks at 440 nM in neutral environments and 586 nM in acidic environments. From this bimodal excitation, a ratiometric signal may be calculated to quantify mitophagy in live cells. This chapter describes procedures for measuring mitophagy by flow cytometry and live cell confocal microscopy with mt-Keima.
    Keywords:  Mitochondria; Mitophagy; PINK1; Parkin; Selective autophagy
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_12
  14. Methods Mol Biol. 2024 ;2845 79-93
    Analysis of Mitophagy Reporters in Drosophila.
    Alvaro Sanchez-Martinez, Aitor Martinez, Alexander J Whitworth.
      Mitophagy is the degradation of mitochondria via the autophagy-lysosome system, disruption of which has been linked to multiple neurodegenerative diseases. As a flux process involving the identification, tagging, and degradation of subcellular components, the analysis of mitophagy benefits from the microscopy analysis of fluorescent reporters. Studying the pathogenic mechanisms of disease also benefits from analysis in animal models in order to capture the complex interplay of molecular and cell biological phenomena. Here, we describe protocols to analyze mitophagy reporters in Drosophila by light microscopy.
    Keywords:  Brain; Drosophila; Light microscopy; Mitochondria; Mitophagy; Muscle; Neurodegeneration; Reporter; mito-QC; mtx-QC
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_7
  15. Methods Mol Biol. 2024 ;2845 55-66
    Assessing Neuronal Mitophagy During Development in the Nematode Caenorhabditis elegans.
    Christina Ploumi, Antonis Roussos, Konstantinos Palikaras.
      Preserving mitochondrial homeostasis is vital, particularly for the energetically demanding and metabolically active nerve cells. Mitophagy, the selective autophagic removal of mitochondria, stands out as a prominent mechanism for efficient mitochondrial turnover, which is crucial for proper neuronal development and function. Dysfunctional mitochondria and disrupted mitophagy pathways have been linked to a diverse array of neurological disorders. The nematode Caenorhabditis elegans, with its well-defined nervous system, serves as an excellent model to unravel the intricate involvement of mitophagy in developing neurons. This chapter describes the use of Rosella biosensor in C. elegans to monitor neuronal mitophagy, providing a user-friendly platform for screening genes and drugs affecting mitophagic pathways under physiological conditions or in the context of neurodevelopmental pathologies.
    Keywords:  Caenorhabditis elegans; Development; Mitochondria; Mitophagy; Neurodevelopmental diseases; Neurons; Rosella biosensor
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_5
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