bims-mitdyn Biomed News
on Mitochondrial dynamics: mechanisms
Issue of 2022–10–02
eightteen papers selected by
Edmond Chan, Queen’s University, School of Medicine
  1. Oncometabolite d-2HG alters T cell metabolism to impair CD8+ T cell function.
  2. An LKB1-mitochondria axis controls TH17 effector function.
  3. Mitochondrial pyruvate supports lymphoma proliferation by fueling a glutamate pyruvate transaminase 2-dependent glutaminolysis pathway.
  4. Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming.
  5. TraB family proteins are components of ER-mitochondrial contact sites and regulate ER-mitochondrial interactions and mitophagy.
  6. ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.
  7. Unique structural features govern the activity of a human mitochondrial AAA+ disaggregase, Skd3.
  8. Differential requirements for mitochondrial electron transport chain components in the adult murine liver.
  9. Therapeutic implications of mitochondrial stress-induced proteasome inhibitor resistance in multiple myeloma.
  10. Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons.
  11. MICU1 and MICU2 potentiation of Ca2+ uptake by the mitochondrial Ca2+ uniporter of Trypanosoma cruzi and its inhibition by Mg2.
  12. PGC-1β maintains mitochondrial metabolism and restrains inflammatory gene expression.
  13. Mitochondria-ER cooperation: NLRX1 detects mitochondrial protein import stress and promotes mitophagy through the ER protein RRBP1.
  14. Eisosome protein Pil1 regulates mitochondrial morphology, mitophagy, and cell death in Saccharomyces cerevisiae.
  15. MFN2 mediates ER-mitochondrial coupling during ER stress through specialized stable contact sites.
  16. Analysis of Mitochondrial Dynamics in Adult Drosophila Axons.
  17. Clonal Imaging of Mitochondria in the Dissected Fly Wing.
  18. Live Imaging of Mitochondria in the Intact Fly Wing.
  1. Science. 2022 Sep 30. 377(6614): 1519-1529
    Oncometabolite d-2HG alters T cell metabolism to impair CD8+ T cell function.
    Giulia Notarangelo, Jessica B Spinelli, Elizabeth M Perez, Gregory J Baker, Kiran Kurmi, Ilaria Elia, Sylwia A Stopka, Gerard Baquer, Jia-Ren Lin, Alexandra J Golby, Shakchhi Joshi, Heide F Baron, Jefte M Drijvers, Peter Georgiev, Alison E Ringel, Elma Zaganjor, Samuel K McBrayer, Peter K Sorger, Arlene H Sharpe, Kai W Wucherpfennig, Sandro Santagata, Nathalie Y R Agar, Mario L Suvà, Marcia C Haigis.
      Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8+ T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8+ T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.
    DOI:  https://doi.org/10.1126/science.abj5104
  2. Nature. 2022 Sep 28.
    An LKB1-mitochondria axis controls TH17 effector function.
    Francesc Baixauli, Klara Piletic, Daniel J Puleston, Matteo Villa, Cameron S Field, Lea J Flachsmann, Andrea Quintana, Nisha Rana, Joy Edwards-Hicks, Mai Matsushita, Michal A Stanczak, Katarzyna M Grzes, Agnieszka M Kabat, Mario Fabri, George Caputa, Beth Kelly, Mauro Corrado, Yaarub Musa, Katarzyna J Duda, Gerhard Mittler, David O'Sullivan, Hiromi Sesaki, Thomas Jenuwein, Joerg M Buescher, Edward J Pearce, David E Sanin, Erika L Pearce.
      CD4+ T cell differentiation requires metabolic reprogramming to fulfil the bioenergetic demands of proliferation and effector function, and enforce specific transcriptional programmes1-3. Mitochondrial membrane dynamics sustains mitochondrial processes4, including respiration and tricarboxylic acid (TCA) cycle metabolism5, but whether mitochondrial membrane remodelling orchestrates CD4+ T cell differentiation remains unclear. Here we show that unlike other CD4+ T cell subsets, T helper 17 (TH17) cells have fused mitochondria with tight cristae. T cell-specific deletion of optic atrophy 1 (OPA1), which regulates inner mitochondrial membrane fusion and cristae morphology6, revealed that TH17 cells require OPA1 for its control of the TCA cycle, rather than respiration. OPA1 deletion amplifies glutamine oxidation, leading to impaired NADH/NAD+ balance and accumulation of TCA cycle metabolites and 2-hydroxyglutarate-a metabolite that influences the epigenetic landscape5,7. Our multi-omics approach revealed that the serine/threonine kinase liver-associated kinase B1 (LKB1) couples mitochondrial function to cytokine expression in TH17 cells by regulating TCA cycle metabolism and transcriptional remodelling. Mitochondrial membrane disruption activates LKB1, which restrains IL-17 expression. LKB1 deletion restores IL-17 expression in TH17 cells with disrupted mitochondrial membranes, rectifying aberrant TCA cycle glutamine flux, balancing NADH/NAD+ and preventing 2-hydroxyglutarate production from the promiscuous activity of the serine biosynthesis enzyme phosphoglycerate dehydrogenase (PHGDH). These findings identify OPA1 as a major determinant of TH17 cell function, and uncover LKB1 as a sensor linking mitochondrial cues to effector programmes in TH17 cells.
    DOI:  https://doi.org/10.1038/s41586-022-05264-1
  3. Sci Adv. 2022 Sep 30. 8(39): eabq0117
    Mitochondrial pyruvate supports lymphoma proliferation by fueling a glutamate pyruvate transaminase 2-dependent glutaminolysis pathway.
    Peng Wei, Alex J Bott, Ahmad A Cluntun, Jeffrey T Morgan, Corey N Cunningham, John C Schell, Yeyun Ouyang, Scott B Ficarro, Jarrod A Marto, Nika N Danial, Ralph J DeBerardinis, Jared Rutter.
      The fate of pyruvate is a defining feature in many cell types. One major fate is mitochondrial entry via the mitochondrial pyruvate carrier (MPC). We found that diffuse large B cell lymphomas (DLBCLs) consume mitochondrial pyruvate via glutamate-pyruvate transaminase 2 to enable α-ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid cycle in DLBCLs. As a result, MPC inhibition led to decreased glutaminolysis in DLBCLs, opposite to previous observations in other cell types. We also found that MPC inhibition or genetic depletion decreased DLBCL proliferation in an extracellular matrix (ECM)-like environment and xenografts, but not in a suspension environment. Moreover, the metabolic profile of DLBCL cells in ECM is markedly different from cells in a suspension environment. Thus, we conclude that the synergistic consumption and assimilation of glutamine and pyruvate enables DLBCL proliferation in an extracellular environment-dependent manner.
    DOI:  https://doi.org/10.1126/sciadv.abq0117
  4. iScience. 2022 Oct 21. 25(10): 105086
    Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming.
    Ratnakar Tiwari, Prashant V Bommi, Peng Gao, Matthew J Schipma, Yalu Zhou, Susan E Quaggin, Navdeep S Chandel, Pinelopi P Kapitsinou.
      Endothelial cell (EC) metabolism has emerged as a driver of angiogenesis. While hypoxia inactivates the oxygen sensors prolyl-4 hydroxylase domain-containing proteins 1-3 (PHD1-3) and stimulates angiogenesis, the effects of PHDs on EC functions remain poorly defined. Here, we investigated the impact of chemical PHD inhibition by dimethyloxalylglycine (DMOG) on angiogenic competence and metabolism of human vascular ECs. DMOG reduced EC proliferation, migration, and tube formation capacities, responses that were associated with an unfavorable metabolic reprogramming. While glycolytic genes were induced, multiple genes encoding sub-units of mitochondrial complex I were suppressed with concurrent decline in nicotinamide adenine dinucleotide (NAD+) levels. Importantly, the DMOG-induced defects in EC migration could be partially rescued by augmenting NAD+ levels through nicotinamide riboside or citrate supplementation. In summary, by integrating functional assays, transcriptomics, and metabolomics, we provide insights into the effects of PHD inhibition on angiogenic competence and metabolism of human vascular ECs.
    Keywords:  Biological sciences; Metabolomics; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2022.105086
  5. Nat Commun. 2022 Sep 26. 13(1): 5658
    TraB family proteins are components of ER-mitochondrial contact sites and regulate ER-mitochondrial interactions and mitophagy.
    Chengyang Li, Patrick Duckney, Tong Zhang, Yanshu Fu, Xin Li, Johan Kroon, Geert De Jaeger, Yunjiang Cheng, Patrick J Hussey, Pengwei Wang.
      ER-mitochondrial contact sites (EMCSs) are important for mitochondrial function. Here, we have identified a EMCS complex, comprising a family of uncharacterised mitochondrial outer membrane proteins, TRB1, TRB2, and the ER protein, VAP27-1. In Arabidopsis, there are three TraB family isoforms and the trb1/trb2 double mutant exhibits abnormal mitochondrial morphology, strong starch accumulation, and impaired energy metabolism, indicating that these proteins are essential for normal mitochondrial function. Moreover, TRB1 and TRB2 proteins also interact with ATG8 in order to regulate mitochondrial degradation (mitophagy). The turnover of depolarised mitochondria is significantly reduced in both trb1/trb2 and VAP27 mutants (vap27-1,3,4,6) under mitochondrial stress conditions, with an increased population of dysfunctional mitochondria present in the cytoplasm. Consequently, plant recovery after stress is significantly perturbed, suggesting that TRB1-regulated mitophagy and ER-mitochondrial interaction are two closely related processes. Taken together, we ascribe a dual role to TraB family proteins which are component of the EMCS complex in eukaryotes, regulating both interaction of the mitochondria to the ER and mitophagy.
    DOI:  https://doi.org/10.1038/s41467-022-33402-w
  6. Nat Commun. 2022 Sep 30. 13(1): 5750
    ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.
    Paula Clemente, Javier Calvo-Garrido, Sarah F Pearce, Florian A Schober, Megumi Shigematsu, Stefan J Siira, Isabelle Laine, Henrik Spåhr, Christian Steinmetzger, Katja Petzold, Yohei Kirino, Rolf Wibom, Oliver Rackham, Aleksandra Filipovska, Joanna Rorbach, Christoph Freyer, Anna Wredenberg.
      Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3' phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3' phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2.
    DOI:  https://doi.org/10.1038/s41467-022-33368-9
  7. Cell Rep. 2022 Sep 27. pii: S2211-1247(22)01249-9. [Epub ahead of print]40(13): 111408
    Unique structural features govern the activity of a human mitochondrial AAA+ disaggregase, Skd3.
    Ryan R Cupo, Alexandrea N Rizo, Gabriel A Braun, Eric Tse, Edward Chuang, Kushol Gupta, Daniel R Southworth, James Shorter.
      The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with defective protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). How Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. These advances illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.
    Keywords:  AAA+ proteins, protein aggregation; CP: Cell biology; chaperone; disaggregase; mitochondria; mitochondrial disorders; therapeutics
    DOI:  https://doi.org/10.1016/j.celrep.2022.111408
  8. Elife. 2022 Sep 26. pii: e80919. [Epub ahead of print]11
    Differential requirements for mitochondrial electron transport chain components in the adult murine liver.
    Nicholas P Lesner, Xun Wang, Zhenkang Chen, Anderson Frank, Cameron Menezes, Sara House, Spencer D Shelton, Andrew Lemoff, David G McFadden, Janaka Wanaspura, Ralph J DeBerardinis, Prashant Mishra.
      Mitochondrial electron transport chain (ETC) dysfunction due to mutations in the nuclear or mitochondrial genome is a common cause of metabolic disease in humans and displays striking tissue specificity depending on the affected gene. The mechanisms underlying tissue specific phenotypes are not understood. Complex I (cI) is classically considered the entry point for electrons into the ETC, and in vitro experiments indicate that cI is required for basal respiration and maintenance of the NAD+/NADH ratio, an indicator of cellular redox status. This finding has largely not been tested in vivo. Here, we report that mitochondrial complex I is dispensable for homeostasis of the adult mouse liver; animals with hepatocyte-specific loss of cI function display no overt phenotypes or signs of liver damage, and maintain liver function, redox and oxygen status. Further analysis of cI-deficient livers did not reveal significant proteomic or metabolic changes, indicating little to no compensation is required in the setting of complex I loss. In contrast, complex IV (cIV) dysfunction in adult hepatocytes results in decreased liver function, impaired oxygen handling, steatosis, and liver damage, accompanied by significant metabolomic and proteomic perturbations. Our results support a model whereby complex I loss is tolerated in the mouse liver because hepatocytes use alternative electron donors to fuel the mitochondrial ETC.
    Keywords:  cell biology; genetics; genomics; mouse
    DOI:  https://doi.org/10.7554/eLife.80919
  9. Sci Adv. 2022 Sep 30. 8(39): eabq5575
    Therapeutic implications of mitochondrial stress-induced proteasome inhibitor resistance in multiple myeloma.
    Aditi Sharma, Remya Nair, Abhinav Achreja, Anjali Mittal, Pulkit Gupta, Kamakshi Balakrishnan, Claudia L Edgar, Olamide Animasahun, Bhakti Dwivedi, Benjamin G Barwick, Vikas A Gupta, Shannon M Matulis, Manoj Bhasin, Sagar Lonial, Ajay K Nooka, Arun P Wiita, Lawrence H Boise, Deepak Nagrath, Mala Shanmugam.
      The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response-dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11, and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress-induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.
    DOI:  https://doi.org/10.1126/sciadv.abq5575
  10. Sci Adv. 2022 Sep 30. 8(39): eabp8701
    Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons.
    Enrico Zampese, David L Wokosin, Patricia Gonzalez-Rodriguez, Jaime N Guzman, Tatiana Tkatch, Jyothisri Kondapalli, William C Surmeier, Karis B D'Alessandro, Diego De Stefani, Rosario Rizzuto, Masamitsu Iino, Jeffery D Molkentin, Navdeep S Chandel, Paul T Schumacker, D James Surmeier.
      How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.
    DOI:  https://doi.org/10.1126/sciadv.abp8701
  11. Cell Calcium. 2022 Sep 21. pii: S0143-4160(22)00127-0. [Epub ahead of print]107 102654
    MICU1 and MICU2 potentiation of Ca2+ uptake by the mitochondrial Ca2+ uniporter of Trypanosoma cruzi and its inhibition by Mg2.
    Mayara S Bertolini, Roberto Docampo.
      The mitochondrial Ca2+ uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca2+ signaling, is mediated via the calcium uniporter complex (MCUC). In animal cells the MCUC is regulated by the mitochondrial calcium uptake 1 and 2 dimer (MICU1/MICU2), which has been proposed to act as gatekeeper preventing mitochondrial Ca2+ overload at low cytosolic Ca2+ levels. In contrast to animal cells, knockout of either MICU1 or MICU2 in Trypanosoma cruzi, the etiologic agent of Chagas disease, did not allow Ca2+ uptake at low extramitochondrial Ca2+ concentrations ([Ca2+]ext) and it was though that in the absence of one MICU the other would replace its role. However, previous attempts to knockout both genes were unsuccessful. Here, we designed a strategy to generate TcMICU1/TcMICU2 double knockout cell lines using CRISPR/Cas9 genome editing. Ablation of both genes was confirmed by PCR and Southern blot analyses. The absence of both proteins did not allow Ca2+ uptake at low [Ca2+]ext, significantly decreased the mitochondrial Ca2+ uptake at different [Ca2+]ext, without dissipation of the mitochondrial membrane potential, and increased the [Ca2+]ext set point needed for Ca2+ uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg2+ was found to be a negative regulator of MCUC-mediated mitochondrial Ca2+ uptake at different [Ca2+]ext. Occlusion of the MCUC pore by Mg2+ could partially explain the lack of mitochondrial Ca2+ uptake at low [Ca2+]ext in TcMICU1/TcMICU2-KO cells. In addition, TcMICU1/TcMICU2-KO epimastigotes had a lower growth rate, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes.
    Keywords:  CRISPR/Cas9; Gatekeeping; MICU; Magnesium; Mitochondrial calcium uniporter; Trypanosoma cruzi
    DOI:  https://doi.org/10.1016/j.ceca.2022.102654
  12. Sci Rep. 2022 Sep 26. 12(1): 16028
    PGC-1β maintains mitochondrial metabolism and restrains inflammatory gene expression.
    Hannah Guak, Ryan D Sheldon, Ian Beddows, Alexandra Vander Ark, Matthew J Weiland, Hui Shen, Russell G Jones, Julie St-Pierre, Eric H Ma, Connie M Krawczyk.
      Metabolic programming of the innate immune cells known as dendritic cells (DCs) changes in response to different stimuli, influencing their function. While the mechanisms behind increased glycolytic metabolism in response to inflammatory stimuli are well-studied, less is known about the programming of mitochondrial metabolism in DCs. We used lipopolysaccharide (LPS) and interferon-β (IFN-β), which differentially stimulate the use of glycolysis and oxidative phosphorylation (OXPHOS), respectively, to identify factors important for mitochondrial metabolism. We found that the expression of peroxisome proliferator-activated receptor gamma co-activator 1β (PGC-1β), a transcriptional co-activator and known regulator of mitochondrial metabolism, decreases when DCs are activated with LPS, when OXPHOS is diminished, but not with IFN-β, when OXPHOS is maintained. We examined the role of PGC-1β in bioenergetic metabolism of DCs and found that PGC-1β deficiency indeed impairs their mitochondrial respiration. PGC-1β-deficient DCs are more glycolytic compared to controls, likely to compensate for reduced OXPHOS. PGC-1β deficiency also causes decreased capacity for ATP production at steady state and in response to IFN-β treatment. Loss of PGC-1β in DCs leads to increased expression of genes in inflammatory pathways, and reduced expression of genes encoding proteins important for mitochondrial metabolism and function. Collectively, these results demonstrate that PGC-1β is a key regulator of mitochondrial metabolism and negative regulator of inflammatory gene expression in DCs.
    DOI:  https://doi.org/10.1038/s41598-022-20215-6
  13. Autophagy. 2022 Sep 28.
    Mitochondria-ER cooperation: NLRX1 detects mitochondrial protein import stress and promotes mitophagy through the ER protein RRBP1.
    Samuel A Killackey, Yuntian Bi, Dana J Philpott, Damien Arnoult, Stephen E Girardin.
      Mitochondria rely on efficient protein import across their membranes for optimal function. We have shown that numerous mitochondrial stressors all converge on a common pathway disrupting this import efficiency. We identified a novel pathway involving NLRX1 and RRBP1 that responds to this import stress, resulting in LC3 lipidation, mitochondrial targeting and ultimate degradation. Furthermore, we demonstrated the relevance of this mitophagy axis in murine skeletal muscle following acute exercise. We propose that mitochondrial protein import stress is an underlying, common trigger for mitophagy, offering a novel avenue for therapeutic exploration and mechanistic insight.
    Keywords:  Autophagy; NLR; exercise; import; mitochondria; mitophagy; proteostasis
    DOI:  https://doi.org/10.1080/15548627.2022.2129763
  14. J Biol Chem. 2022 Sep 23. pii: S0021-9258(22)00976-0. [Epub ahead of print] 102533
    Eisosome protein Pil1 regulates mitochondrial morphology, mitophagy, and cell death in Saccharomyces cerevisiae.
    Amita Pal, Arunkumar Paripati, Pallavi Deolal, Arpan Chatterjee, Pushpa Rani Prasad, Priyanka Adla, Naresh Babu V Sepuri.
      Mitochondrial morphology and dynamics maintain mitochondrial integrity by regulating its size, shape, distribution, and connectivity, thereby modulating various cellular processes. Several studies have established a functional link between mitochondrial dynamics, mitophagy, and cell death, but further investigation is needed to identify specific proteins involved in mitochondrial dynamics. Any alteration in the integrity of the mitochondria has severe ramifications that include disorders like cancer and neurodegeneration. In this study, we used budding yeast as a model organism and found that Pil1, the major component of the eisosome complex, also localizes to the periphery of mitochondria. Interestingly, the absence of Pil1 causes the branched tubular morphology of mitochondria to be abnormally fused or aggregated, whereas its overexpression leads to mitochondrial fragmentation. Most importantly, pil1Δ cells are defective in mitophagy and bulk autophagy, resulting in elevated levels of ROS and protein aggregates. In addition, we show that pil1Δ cells are more prone to cell death. Yeast two-hybrid analysis and co-immunoprecipitations show the interaction of Pil1 with two major proteins in mitochondrial fission, Fis1 and Dnm1. Additionally, our data suggest that the role of Pil1 in maintaining mitochondrial shape is dependent on Fis1 and Dnm1, but it functions independently in mitophagy and cell death pathways. Together, our data suggest that Pil1, an eisosome protein, is a novel regulator of mitochondrial morphology, mitophagy, and cell death.
    Keywords:  Saccharomyces cerevisiae; autophagy; cell death; mitochondria; mitophagy; protein aggregation; reactive oxygen species (ROS)
    DOI:  https://doi.org/10.1016/j.jbc.2022.102533
  15. Front Cell Dev Biol. 2022 ;10 918691
    MFN2 mediates ER-mitochondrial coupling during ER stress through specialized stable contact sites.
    Benjamin Gottschalk, Zhanat Koshenov, Olaf A Bachkoenig, René Rost, Roland Malli, Wolfgang F Graier.
      Endoplasmic reticulum (ER) functions critically depend on a suitable ATP supply to fuel ER chaperons and protein trafficking. A disruption of the ability of the ER to traffic and fold proteins leads to ER stress and the unfolded protein response (UPR). Using structured illumination super-resolution microscopy, we revealed increased stability and lifetime of mitochondrial associated ER membranes (MAM) during ER stress. The consequent increase of basal mitochondrial Ca2+ leads to increased TCA cycle activity and enhanced mitochondrial membrane potential, OXPHOS, and ATP generation during ER stress. Subsequently, OXPHOS derived ATP trafficking towards the ER was increased. We found that the increased lifetime and stability of MAMs during ER stress depended on the mitochondrial fusion protein Mitofusin2 (MFN2). Knockdown of MFN2 blunted mitochondrial Ca2+ effect during ER stress, switched mitochondrial F1FO-ATPase activity into reverse mode, and strongly reduced the ATP supply for the ER during ER stress. These findings suggest a critical role of MFN2-dependent MAM stability and lifetime during ER stress to compensate UPR by strengthening ER ATP supply by the mitochondria.
    Keywords:  ER stress; mitochondria; mitochondria-associated membranes (MAM); mitochondrial Ca2+; mitofusin 2
    DOI:  https://doi.org/10.3389/fcell.2022.918691
  16. Cold Spring Harb Protoc. 2022 Sep 30.
    Analysis of Mitochondrial Dynamics in Adult Drosophila Axons.
    Daniel C Maddison, Francesca Mattedi, Alessio Vagnoni, Gaynor Ann Smith.
      Neuronal survival depends on the generation of ATP from an ever-changing mitochondrial network. This requires a fine balance between the constant degradation of damaged mitochondria, biogenesis of new mitochondria, movement along microtubules, dynamic processes, and adequate functional capacity to meet firing demands. The distribution of mitochondria needs to be tightly controlled throughout the entire neuron, including its projections. Axons in particular can be enormous structures compared to the size of the cell soma, and how mitochondria are maintained in these compartments is poorly defined. Mitochondrial dysfunction in neurons is associated with aging and neurodegenerative diseases, with the axon being preferentially vulnerable to destruction. Drosophila offer a unique way to study these organelles in fully differentiated adult neurons in vivo. Here, we briefly review the regulation of neuronal mitochondria in health, aging, and disease and introduce two methodological approaches to study mitochondrial dynamics and transport in axons using the Drosophila wing system.
    DOI:  https://doi.org/10.1101/pdb.top107819
  17. Cold Spring Harb Protoc. 2022 Sep 30.
    Clonal Imaging of Mitochondria in the Dissected Fly Wing.
    Daniel C Maddison, Francesca Mattedi, Alessio Vagnoni, Gaynor Ann Smith.
      Mitochondria are essential for long-term neuronal function and survival. They are maintained in neurons, including long axonal stretches, through dynamic processes such as fission, fusion, biogenesis, and mitophagy. Here, we describe a protocol for the in-depth morphological analysis of individual mitochondria in axons in vivo. Most mitochondrial analysis of axons is currently performed in vitro with neurons in a developmental state. Therefore, an understanding of the axonal mitochondrial network during aging in fully differentiated neurons and the long-term consequence of gene knockout is often not developed. By using a clonal system paired with fluorescent genetically encoded markers in the Drosophila wing, we can visualize individual neurons (out of the whole bundle), including their long axons and the mitochondria that they contain, using confocal imaging. The clonal system also allows visualization of neurons with genetic perturbations that would otherwise be lethal if present in the whole organism, allowing investigators to bypass lethality. This protocol can further be adapted to measure the physiological and biochemical state of the mitochondria. Mitochondrial morphology and health in axons are tightly linked to aging, axon injury, and neurodegeneration; therefore, this method can be used to investigate mitochondrial dysfunction associated with novel genes or those linked to neurodegenerative disease and axonopathy.
    DOI:  https://doi.org/10.1101/pdb.prot108051
  18. Cold Spring Harb Protoc. 2022 Sep 30.
    Live Imaging of Mitochondria in the Intact Fly Wing.
    Francesca Mattedi, Daniel C Maddison, Gaynor Ann Smith, Alessio Vagnoni.
      Detailed mechanisms governing the transport of mitochondria in neurons have recently emerged, although it is still poorly understood how the regulation of transport is coordinated in space and time within the physiological context of an organism. Here, we provide a protocol to study the intracellular dynamics of mitochondria in the wing neurons of adult Drosophila in situ. The mounting and imaging procedures that we describe are suitable for use on most microscopes, and they can be easily implemented in any laboratory. Our noninvasive mounting procedures, combined with the translucency of the wing cuticle in adult animals, makes the wing nervous system accessible to advanced microscopy studies in a physiological environment. Combining the powerful genetics of Drosophila with time-lapse live imaging, users of this protocol will be able to analyze mitochondrial dynamics over time in a subset of sensory neurons in the wing. These cells extend long axons with a stereotypical plus-end-out microtubule orientation that represents a unique model to understand the logic of neuronal cargo transport, including the mitochondria. Finally, the neurons in this tissue respond to mechanical and chemical stimulation of the sensory organs of the wing, opening up the possibility of coupling the study of mitochondrial dynamics with the modulation of neuronal activity in aging Drosophila We anticipate that the unique characteristics of this in vivo system will contribute to the discovery of novel mechanisms that regulate mitochondrial dynamics within an organismal context with relevant implications for the pathogenesis of age-dependent neurological disorders.
    DOI:  https://doi.org/10.1101/pdb.prot108052
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