bims-mitdyn Biomed News
on Mitochondrial dynamics: mechanisms
Issue of 2024–03–17
nine papers selected by
Edmond Chan, Queen’s University, School of Medicine
  1. Mitochondrial complex I activity in microglia sustains neuroinflammation.
  2. Mitochondrial metabolism sustains CD8+ T cell migration for an efficient infiltration into solid tumors.
  3. NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia.
  4. Genetic ablation of Immt induces a lethal disruption of the MICOS complex.
  5. Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.
  6. MitoLuc: A Luminescence-Based Assay to Study Real-Time Protein Import into Mitochondria.
  7. Tracking the Activity and Position of Mitochondrial β-Barrel Proteins.
  8. Modular Assembly of Mitochondrial β-Barrel Proteins.
  9. Examining Protein Translocation by β-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.
  1. Nature. 2024 Mar 13.
    Mitochondrial complex I activity in microglia sustains neuroinflammation.
    L Peruzzotti-Jametti, C M Willis, G Krzak, R Hamel, L Pirvan, R-B Ionescu, J A Reisz, H A Prag, M E Garcia-Segura, V Wu, Y Xiang, B Barlas, A M Casey, A M R van den Bosch, A M Nicaise, L Roth, G R Bates, H Huang, P Prasad, A E Vincent, C Frezza, C Viscomi, G Balmus, Z Takats, J C Marioni, A D'Alessandro, M P Murphy, I Mohorianu, S Pluchino.
      Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3.
    DOI:  https://doi.org/10.1038/s41586-024-07167-9
  2. Nat Commun. 2024 Mar 11. 15(1): 2203
    Mitochondrial metabolism sustains CD8+ T cell migration for an efficient infiltration into solid tumors.
    Luca Simula, Mattia Fumagalli, Lene Vimeux, Irena Rajnpreht, Philippe Icard, Gary Birsen, Dongjie An, Frédéric Pendino, Adrien Rouault, Nadège Bercovici, Diane Damotte, Audrey Lupo-Mansuet, Marco Alifano, Marie-Clotilde Alves-Guerra, Emmanuel Donnadieu.
      The ability of CD8+ T cells to infiltrate solid tumors and reach cancer cells is associated with improved patient survival and responses to immunotherapy. Thus, identifying the factors controlling T cell migration in tumors is critical, so that strategies to intervene on these targets can be developed. Although interstitial motility is a highly energy-demanding process, the metabolic requirements of CD8+ T cells migrating in a 3D environment remain unclear. Here, we demonstrate that the tricarboxylic acid (TCA) cycle is the main metabolic pathway sustaining human CD8+ T cell motility in 3D collagen gels and tumor slices while glycolysis plays a more minor role. Using pharmacological and genetic approaches, we report that CD8+ T cell migration depends on the mitochondrial oxidation of glucose and glutamine, but not fatty acids, and both ATP and ROS produced by mitochondria are required for T cells to migrate. Pharmacological interventions to increase mitochondrial activity improve CD8+ T cell intratumoral migration and CAR T cell recruitment into tumor islets leading to better control of tumor growth in human xenograft models. Our study highlights the rationale of targeting mitochondrial metabolism to enhance the migration and antitumor efficacy of CAR T cells in treating solid tumors.
    DOI:  https://doi.org/10.1038/s41467-024-46377-7
  3. Nat Commun. 2024 Mar 13. 15(1): 2264
    NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia.
    Chih-Wei Chen, Chi Su, Chang-Yu Huang, Xuan-Rong Huang, Xiaojing Cuili, Tung Chao, Chun-Hsiang Fan, Cheng-Wei Ting, Yi-Wei Tsai, Kai-Chien Yang, Ti-Yen Yeh, Sung-Tsang Hsieh, Yi-Ju Chen, Yuxi Feng, Tony Hunter, Zee-Fen Chang.
      NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.
    DOI:  https://doi.org/10.1038/s41467-024-46385-7
  4. Life Sci Alliance. 2024 Jun;pii: e202302329. [Epub ahead of print]7(6):
    Genetic ablation of Immt induces a lethal disruption of the MICOS complex.
    Stephanie M Rockfield, Meghan E Turnis, Ricardo Rodriguez-Enriquez, Madhavi Bathina, Seng Kah Ng, Nathan Kurtz, Nathalie Becerra Mora, Stephane Pelletier, Camenzind G Robinson, Peter Vogel, Joseph T Opferman.
      The mitochondrial contact site and cristae organizing system (MICOS) is important for crista junction formation and for maintaining inner mitochondrial membrane architecture. A key component of the MICOS complex is MIC60, which has been well studied in yeast and cell culture models. However, only one recent study has demonstrated the embryonic lethality of losing Immt (the gene encoding MIC60) expression. Tamoxifen-inducible ROSA-CreERT2-mediated deletion of Immt in adult mice disrupted the MICOS complex, increased mitochondria size, altered cristae morphology, and was lethal within 12 d. Pathologically, these mice displayed defective intestinal muscle function (paralytic ileus) culminating in dehydration. We also identified bone marrow (BM) hypocellularity in Immt-deleted mice, although BM transplants from wild-type mice did not improve survival. Altogether, this inducible mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex.
    DOI:  https://doi.org/10.26508/lsa.202302329
  5. Cell Death Dis. 2024 Mar 11. 15(3): 203
    Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.
    Emma L Wilson, Yizhou Yu, Nuno S Leal, James A Woodward, Nikolaos Patikas, Jordan L Morris, Sarah F Field, William Plumbly, Vincent Paupe, Suvagata R Chowdhury, Robin Antrobus, Georgina E Lindop, Yusuf M Adia, Samantha H Y Loh, Julien Prudent, L Miguel Martins, Emmanouil Metzakopian.
      Organelles form membrane contact sites between each other, allowing for the transfer of molecules and signals. Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are cellular subdomains characterized by close apposition of mitochondria and ER membranes. They have been implicated in many diseases, including neurodegenerative, metabolic, and cardiac diseases. Although MERCS have been extensively studied, much remains to be explored. To uncover novel regulators of MERCS, we conducted a genome-wide, flow cytometry-based screen using an engineered MERCS reporter cell line. We found 410 genes whose downregulation promotes MERCS and 230 genes whose downregulation decreases MERCS. From these, 29 genes were selected from each population for arrayed screening and 25 were validated from the high population and 13 from the low population. GET4 and BAG6 were highlighted as the top 2 genes that upon suppression increased MERCS from both the pooled and arrayed screens, and these were subjected to further investigation. Multiple microscopy analyses confirmed that loss of GET4 or BAG6 increased MERCS. GET4 and BAG6 were also observed to interact with the known MERCS proteins, inositol 1,4,5-trisphosphate receptors (IP3R) and glucose-regulated protein 75 (GRP75). In addition, we found that loss of GET4 increased mitochondrial calcium uptake upon ER-Ca2+ release and mitochondrial respiration. Finally, we show that loss of GET4 rescues motor ability, improves lifespan and prevents neurodegeneration in a Drosophila model of Alzheimer's disease (Aβ42Arc). Together, these results suggest that GET4 is involved in decreasing MERCS and that its loss is neuroprotective.
    DOI:  https://doi.org/10.1038/s41419-024-06568-y
  6. Methods Mol Biol. 2024 ;2778 185-200
    MitoLuc: A Luminescence-Based Assay to Study Real-Time Protein Import into Mitochondria.
    Holly C Ford, Ian Collinson.
      All but a few mitochondrial proteins are translated into the cytosol and imported in via complicated and varied pathways. These processes occur over short time frames and, as such, are difficult to monitor with classical approaches such as Western blotting or autoradiography that require sample collection at discrete time points. The development of an assay based on a split version of the small luciferase-Mitoluc-has allowed us to monitor the import of proteins into mitochondria in high resolution and real time (Pereira et al., J Mol Biol 431:1689-1699, 2019). Luminescence measurements are acquired using a plate reader in the order of seconds. This allows scores of experiments to be conducted in parallel in a single multi-well plate and permits kinetic analysis yielding information about import mechanisms (Ford et al., Elife 11:e75426, 2022).
    Keywords:  Luminescence; MitoLuc; Mitochondria; NanoLuc; Protein import; Split-luciferase
    DOI:  https://doi.org/10.1007/978-1-0716-3734-0_12
  7. Methods Mol Biol. 2024 ;2778 221-236
    Tracking the Activity and Position of Mitochondrial β-Barrel Proteins.
    Shuo Wang, Stephan Nussberger.
      Total interference reflection fluorescence (TIRF) microscopy of lipid bilayers is an effective technique for studying the lateral movement and ion channel activity of single integral membrane proteins. Here we describe how to integrate the mitochondrial outer membrane preprotein translocase TOM-CC and its β-barrel protein-conducting channel Tom40 into supported lipid bilayers to identify possible relationships between movement and channel activity. We propose that our approach can be readily applied to membrane protein channels where transient tethering to either membrane-proximal or intramembrane structures is accompanied by a change in channel permeation.
    Keywords:  Channels; Mitochondria; Protein translocation; Single molecule; TIRF microscopy; TOM; β-barrel membrane protein
    DOI:  https://doi.org/10.1007/978-1-0716-3734-0_14
  8. Methods Mol Biol. 2024 ;2778 201-220
    Modular Assembly of Mitochondrial β-Barrel Proteins.
    Rituparna Bhowmik, Fabian den Brave, Thomas Becker.
      Mitochondrial β-barrel proteins fulfill crucial roles in the biogenesis and function of the cell organelle. They mediate the import and membrane insertion of proteins and transport of small metabolites and ions. All β-barrel proteins are made as precursors on cytosolic ribosomes and are imported into mitochondria. The β-barrel proteins fold and assemble with partner proteins in the outer membrane. The in vitro import of radiolabelled proteins into isolated mitochondria is a powerful tool to investigate the import of β-barrel proteins, the folding of the β-barrel proteins, and their assembly into protein complexes. Altogether, the in vitro import assay is a versatile and crucial assay to analyze the mechanisms of the biogenesis of mitochondrial β-barrel proteins.
    Keywords:  Blue native electrophoresis; Mitochondria; Protein import assay; SAM complex; TOM complex; β-barrel proteins
    DOI:  https://doi.org/10.1007/978-1-0716-3734-0_13
  9. Methods Mol Biol. 2024 ;2778 83-99
    Examining Protein Translocation by β-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.
    Minh Sang Huynh, Jiaming Caitlyn Xu, Trevor F Moraes.
      β-barrel membrane proteins populate the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts, playing significant roles in multiple key cellular pathways. Characterizing the functions of these membrane proteins in vivo is often challenging due to the complex protein network in the periplasm of Gram-negative bacteria (or intermembrane space in mitochondria and chloroplasts) and the presence of other outer membrane proteins. In vitro reconstitution into lipid-bilayer-like environments such as nanodiscs or proteoliposomes provides an excellent method for examining the specific function and mechanism of these membrane proteins in an isolated system. Here, we describe the methodologies employed to investigate Slam, a 14-stranded β-barrel membrane protein also known as the type XI secretion system that is responsible for translocating proteins across the outer membrane of many bacterial species.
    Keywords:   Denatured protein translocation; Gram-negative bacteria; In vitro translocation; Spheroplast-released translocation; Surface lipoprotein translocation; Type XI Secretion System; outer membrane proteins; β-barrel membrane protein; Proteoliposome reconstitution
    DOI:  https://doi.org/10.1007/978-1-0716-3734-0_6
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