bims-mitdyn Biomed News
on Mitochondrial dynamics: mechanisms
Issue of 2021‒06‒13
twenty-five papers selected by
Edmond Chan
Queen’s University, School of Medicine
  1. An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial membrane.
  2. Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes.
  3. ALS- and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy.
  4. MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking.
  5. The role of Mfn2 in the structure and function of ER-mitochondrial tethering in vivo.
  6. Spermidine-induced hypusination preserves mitochondrial and cognitive function during aging.
  7. Maintenance of complex I and its super-complexes by NDUF-11 is essential for mitochondrial structure, function and health.
  8. The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.
  9. Antipsychotic drugs counteract autophagy and mitophagy in multiple sclerosis.
  10. Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes.
  11. A hitchhiker's guide to mitochondrial quantification.
  12. Imaging Mitochondrial Hydrogen Peroxide in Living Cells.
  13. Norbormide-Based Probes and Their Application for Mitochondrial Imaging in Drosophila Melanogaster.
  14. Mitochondrial Functional Assessment in Mammalian Gametes Using Fluorescent Probes.
  15. An Update on Isolation of Functional Mitochondria from Cells for Bioenergetics Studies.
  16. Computer-Aided Prediction of Protein Mitochondrial Localization.
  17. Mitochondrial Regulation Assessment by 13C-NMR Isotopomer Analysis.
  18. mTRIP, an Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Resolution.
  19. Measuring PGC-1α and Its Acetylation Status in Mouse Primary Myotubes.
  20. Following the Dynamism of the Mitochondrial Network in T Cells.
  21. Methods to Monitor Mitophagy and Mitochondrial Quality: Implications in Cancer, Neurodegeneration, and Cardiovascular Diseases.
  22. Split Green Fluorescent Protein-Based Contact Site Sensor (SPLICS ) for Heterotypic Organelle Juxtaposition as Applied to ER -Mitochondria Proximities.
  23. Sequence-Specific Control of Mitochondrial Gene Transcription Using Programmable Synthetic Gene Switches Called MITO-PIPs.
  24. Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.
  25. Mito-SinCe2 Approach to Analyze Mitochondrial Structure-Function Relationship in Single Cells.
  1. EMBO J. 2021 Jun 08. e106438
    An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial membrane.
    Fujiao Lv, Fei Qi, Zhi Zhang, Maorong Wen, Justin Kale, Alessandro Piai, Lingyu Du, Shuqing Wang, Liujuan Zhou, Yaqing Yang, Bin Wu, Zhijun Liu, Juan Del Rosario, Justin Pogmore, James J Chou, David W Andrews, Jialing Lin, Bo OuYang.
      Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.
    Keywords:  NMR structure; bax core dimer; functional mutagenesis; membrane lipid bilayer; pore formation
    DOI:  https://doi.org/10.15252/embj.2020106438
  2. EMBO Rep. 2021 Jun 04. 22(6): e51323
    Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes.
    Hyun Ku Yeo, Tae Hyun Park, Hee Yeon Kim, Hyonchol Jang, Jueun Lee, Geum-Sook Hwang, Seong Eon Ryu, Si Hoon Park, Hyun Kyu Song, Hyun Seung Ban, Hye-Jin Yoon, Byung Il Lee.
      In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51-VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function.
    Keywords:  MAM; PTPIP51; endoplasmic reticulum; mitochondria; phospholipid
    DOI:  https://doi.org/10.15252/embr.202051323
  3. Proc Natl Acad Sci U S A. 2021 Jun 15. pii: e2025053118. [Epub ahead of print]118(24):
    ALS- and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy.
    Olivia Harding, Chantell S Evans, Junqiang Ye, Jonah Cheung, Tom Maniatis, Erika L F Holzbaur.
      TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia, including missense mutations that disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type-specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals.
    Keywords:  OPTN; Parkin; TBK1; mitophagy; neurodegeneration
    DOI:  https://doi.org/10.1073/pnas.2025053118
  4. EMBO Rep. 2021 Jun 06. e52006
    MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking.
    Fei Wang, Xiaoping Chen, Haipeng Cheng, Lu Song, Jianghong Liu, Steve Caplan, Li Zhu, Jane Y Wu.
      Tunneling nanotubes (TNTs) are actin-rich structures that connect two or more cells and mediate cargo exchange between spatially separated cells. TNTs transport signaling molecules, vesicles, organelles, and even pathogens. However, the molecular mechanisms regulating TNT formation remain unclear and little is known about the endogenous mechanisms suppressing TNT formation in lung cancer cells. Here, we report that MICAL2PV, a splicing isoform of the neuronal guidance gene MICAL2, is a novel TNT regulator that suppresses TNT formation and modulates mitochondrial distribution. MICAL2PV interacts with mitochondrial Rho GTPase Miro2 and regulates subcellular mitochondrial trafficking. Moreover, down-regulation of MICAL2PV enhances survival of cells treated with chemotherapeutical drugs. The monooxygenase (MO) domain of MICAL2PV is required for its activity to inhibit TNT formation by depolymerizing F-actin. Our data demonstrate a previously unrecognized function of MICAL2 in TNT formation and mitochondrial trafficking. Furthermore, our study uncovers a role of the MICAL2PV-Miro2 axis in mitochondrial trafficking, providing a mechanistic explanation for MICAL2PV activity in suppressing TNT formation and in modulating mitochondrial subcellular distribution.
    Keywords:  F-actin; Miro2; lung cancer; mitochondria; tunneling nanotubes
    DOI:  https://doi.org/10.15252/embr.202052006
  5. J Cell Sci. 2021 Jun 10. pii: jcs.253443. [Epub ahead of print]
    The role of Mfn2 in the structure and function of ER-mitochondrial tethering in vivo.
    Song Han, Fanpeng Zhao, Jeffrey Hsia, Xiaopin Ma, Yi Liu, Sandy Torres, Hisashi Fujioka, Xiongwei Zhu.
      The mitochondria-ER contacts (MERCs) plays an essential role in multiple cell physiological process. While Mfn2 was the first protein implicated in the formation of MERCs, it is debated whether it acts as a tether or antagonizer, largely based on in vitro studies. To understand the role of Mfn2 in MERCs in vivo, we characterized ultrastructural and biochemical changes of MERCs in pyramidal neurons of hippocampus in Mfn2 conditional knockout (KO) mice and in Mfn2 overexpression (OE) mice and found Mfn2 ablation caused reduced close contacts while Mfn2 OE caused increased close contacts between ER and mitochondria in vivo. Functional studies on SH-SY5Y cells with Mfn2 KO or overexpression demonstrating similar biochemical changes found that mitochondrial calcium uptake along with IP3R3-Grp75 interaction was decreased in Mfn2 KO cells but increased in the Mfn2 OE cells. Lastly, we found Mfn2 KO decreased and Mfn2 OE increased the interaction between the ER-mitochondria tethering pair of VAPB-PTPIP51. In conclusion, our study supports the notion that Mfn2 plays a critical role in ER-mitochondrial tethering and the formation of close contacts in neuronal cells in vivo.
    Keywords:  ER-mitochondria tethering; Mfn2; Mitochondria-ER contact; Mitochondria-associated membrane; Mitochondrial calcium uptake; VAPB
    DOI:  https://doi.org/10.1242/jcs.253443
  6. Autophagy. 2021 Jun 09. 1-3
    Spermidine-induced hypusination preserves mitochondrial and cognitive function during aging.
    Sebastian J Hofer, YongTian Liang, Andreas Zimmermann, Sabrina Schroeder, Jörn Dengjel, Guido Kroemer, Tobias Eisenberg, Stephan J Sigrist, Frank Madeo.
      Spermidine is a natural polyamine, central to cellular homeostasis and growth, that promotes macroautophagy/autophagy. The polyamine pathway is highly conserved from bacteria to mammals and spermidine (prominently found in some kinds of aged cheese, wheat germs, nuts, soybeans, and fermented products thereof, among others) is an intrinsic part of the human diet. Apart from nutrition, spermidine is available to mammalian organisms from intracellular biosynthesis and microbial production in the gut. Importantly, externally supplied spermidine (via drinking water or food) prolongs lifespan, activates autophagy, improves mitochondrial function, and refills polyamine pools that decline during aging in various tissues of model organisms, including mice. In two adjacent studies, we explored how dietary spermidine supplementation enhances eEF5/EIF5A hypusination, cerebral mitochondrial function and cognition in aging Drosophila melanogaster and mice.
    Keywords:  Autophagy; Drosophila; Pink1; hypusination; learning; memory; mitophagy; polyamines; spermidine
    DOI:  https://doi.org/10.1080/15548627.2021.1933299
  7. J Cell Sci. 2021 Jun 09. pii: jcs.258399. [Epub ahead of print]
    Maintenance of complex I and its super-complexes by NDUF-11 is essential for mitochondrial structure, function and health.
    Amber Knapp-Wilson, Gonçalo C Pereira, Emma Buzzard, Holly C Ford, Andrew Richardson, Robin A Corey, Chris Neal, Paul Verkade, Andrew P Halestrap, Vicki A M Gold, Patricia Kuwabara, Ian Collinson.
      Mitochondrial super-complexes form around a conserved core of monomeric complex I and dimeric complex III; wherein subunit NDUFA11, of the former, is conspicuously situated at the interface. We identified B0491.5 (NDUF-11) as the C. elegans homologue, of which animals homozygous for a CRISPR-Cas9 generated knockout allele arrested at the L2 development stage. Reducing (but not eliminating) expression by RNAi allowed development to adulthood, enabling characterisation of the consequences: destabilisation of complex I and its super-complexes, and perturbation of respiratory function. The loss of NADH-dehydrogenase activity is compensated by enhanced complex II activity, with the potential for detrimental ROS-production. Electron cryo-tomography highlight aberrant cristae morphology and inter-membrane-space widening and cristae-junctions. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development presumably arises due to its involvement in complex I/ super-complex maintenance. This highlights the importance of respiratory complex integrity for health and the potential of its perturbation for mitochondrial disease.
    Keywords:  Caenorhabditis elegans; Electron-transfer chain; Mitochondria; Mitochondrial ultrastructure; electron cryo-tomography; NDUF-11; Respirasome; Respiration; Super-complexes; Worm
    DOI:  https://doi.org/10.1242/jcs.258399
  8. J Biol Chem. 2021 Apr 29. pii: S0021-9258(21)00525-1. [Epub ahead of print] 100736
    The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.
    Victor Vitvitsky, Roshan Kumar, Marouane Libiad, Allison Maebius, Aaron Landry, Ruma Banerjee.
      Hydrogen sulfide is synthesized by enzymes involved in sulfur metabolism and oxidized via a dedicated mitochondrial pathway that intersects with the electron transport chain (ETC) at the level of complex III. Studies with H2S are challenging since it is volatile and also reacts with oxidized thiols in the culture medium, forming sulfane sulfur species. The half-life of exogenously added H2S to cultured cells is unknown. In this study, we first examined the half-life of exogenously added H2S to human colonic epithelial cells. In plate cultures, H2S disappeared with a t1/2 of 3-4 min at 37°C with a small fraction being trapped as sulfane sulfur species. In suspension cultures, the rate of abiotic loss of H2S was slower, and we demonstrated that sulfide stimulated aerobic glycolysis, which was sensitive to the mitochondrial but not the cytoplasmic NADH pool. Oxidation of mitochondrial NADH using the genetically encoded mito-LbNOX tool, blunted the cellular sensitivity to sulfide-stimulated aerobic glycolysis and enhanced its oxidation to thiosulfate. In contrast, sulfide did not affect flux through the oxidative pentose phosphate pathway or the TCA cycle. Knockdown of sulfide quinone oxidoreductase, which commits H2S to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of H2S to activate oxidative phosphorylation at low concentrations, but inhibit it at high concentrations, suggests that it might play a role in tuning electron flux and therefore, cellular energy metabolism, particularly during cell proliferation.
    Keywords:  Hydrogen sulfide; aerobic glycolysis; electron transport chain; sulfide quinone oxidoreductase
    DOI:  https://doi.org/10.1016/j.jbc.2021.100736
  9. Proc Natl Acad Sci U S A. 2021 Jun 15. pii: e2020078118. [Epub ahead of print]118(24):
    Antipsychotic drugs counteract autophagy and mitophagy in multiple sclerosis.
    Simone Patergnani, Massimo Bonora, Selene Ingusci, Maurizio Previati, Saverio Marchi, Silvia Zucchini, Mariasole Perrone, Mariusz R Wieckowski, Massimiliano Castellazzi, Maura Pugliatti, Carlotta Giorgi, Michele Simonato, Paolo Pinton.
      Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by myelin damage followed by axonal and ultimately neuronal loss. The etiology and physiopathology of MS are still elusive, and no fully effective therapy is yet available. We investigated the role in MS of autophagy (physiologically, a controlled intracellular pathway regulating the degradation of cellular components) and of mitophagy (a specific form of autophagy that removes dysfunctional mitochondria). We found that the levels of autophagy and mitophagy markers are significantly increased in the biofluids of MS patients during the active phase of the disease, indicating activation of these processes. In keeping with this idea, in vitro and in vivo MS models (induced by proinflammatory cytokines, lysolecithin, and cuprizone) are associated with strongly impaired mitochondrial activity, inducing a lactic acid metabolism and prompting an increase in the autophagic flux and in mitophagy. Multiple structurally and mechanistically unrelated inhibitors of autophagy improved myelin production and normalized axonal myelination, and two such inhibitors, the widely used antipsychotic drugs haloperidol and clozapine, also significantly improved cuprizone-induced motor impairment. These data suggest that autophagy has a causal role in MS; its inhibition strongly attenuates behavioral signs in an experimental model of the disease. Therefore, haloperidol and clozapine may represent additional therapeutic tools against MS.
    Keywords:  antipsychotic drugs; autophagy; mitochondria; multiple sclerosis; remyelination
    DOI:  https://doi.org/10.1073/pnas.2020078118
  10. Redox Biol. 2021 Jun 01. pii: S2213-2317(21)00179-8. [Epub ahead of print]45 102021
    Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes.
    Sehwan Jang, Xavier R Chapa-Dubocq, Yulia Y Tyurina, Claudette M St Croix, Alexandr A Kapralov, Vladimir A Tyurin, Hülya Bayır, Valerian E Kagan, Sabzali Javadov.
      Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10) and the oxoglutarate carrier (OGC, SLC25A11) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro. In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.
    Keywords:  Ferroptosis; Glutathione; Heart; Ischemia-reperfusion; Mitochondria; Oxidized phosphatidylethanolamine
    DOI:  https://doi.org/10.1016/j.redox.2021.102021
  11. Mitochondrion. 2021 Jun 05. pii: S1567-7249(21)00078-7. [Epub ahead of print]
    A hitchhiker's guide to mitochondrial quantification.
    Irene Mgm Hemel, Bob Ph Engelen, Nicole Luber, Mike Gerards.
      The variety of available mitochondrial quantification tools makes it difficult to select the most reliable and accurate quantification tool. Here, we performed elaborate analyses on five open source ImageJ tools. Excessive clustering of mitochondrial structures was observed in four tools, caused by the global thresholding applied by these tools. The Mitochondrial Analyzer, which uses adaptive thresholding, outperformed the other examined tools, with accurate structural segregation and identification. Additionally, we showed that the Mitochondrial Analyzer successfully identifies mitochondrial morphology differences. Based on the observed performance, we consider the Mitochondrial Analyzer the best open source tool for mitochondrial network morphology quantification.
    Keywords:  Image analysis; ImageJ; Mitochondria; Mitochondrial Dynamics; Mitochondrial quantification
    DOI:  https://doi.org/10.1016/j.mito.2021.06.005
  12. Methods Mol Biol. 2021 ;2275 127-140
    Imaging Mitochondrial Hydrogen Peroxide in Living Cells.
    Alexander R Lippert, Bryan C Dickinson, Elizabeth J New.
      Hydrogen peroxide (H2O2) produced from mitochondria is intimately involved in human health and disease, but is challenging to selectively monitor inside living systems. The fluorescent probe MitoPY1 provides a practical tool for imaging mitochondrial H2O2 and has been demonstrated to function in a variety of diverse cell types. In this chapter, we describe the synthetic preparation of the small molecule probe MitoPY1 , methods for validating this probe in vitro and in live cells, and an example procedure for measuring mitochondrial H2O2 in a cell culture model of Parkinson's disease.
    Keywords:  Boronate; Fluorescence microscopy; Fluorescent probes; Hydrogen peroxide; Mitochondria; Triphenylphosphonium cations
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_8
  13. Methods Mol Biol. 2021 ;2275 279-289
    Norbormide-Based Probes and Their Application for Mitochondrial Imaging in Drosophila Melanogaster.
    Alessia Forgiarini, Zifei Wang, Sergio Bova, Margaret Anne Brimble, Brian Hopkins, David Rennison, Genny Orso.
      Fluorescent live imaging on Drosophila melanogaster is a microscopy technique in rapid expansion. The growing number of probes available to detect cellular components and the relatively easy genetic manipulation of fruit fly make this model one of the most used for in vivo analysis of several physiological and/or pathological processes. Here we describe the chemical synthesis of two norbormide-derived BODIPY-conjugated fluorescent probes (NRBMC009 and NRBZLW0047). Moreover, we describe the larval dissection method, and subsequent live imaging acquisition. Both probes are able to label mitochondria in different Drosophila larval tissues, which allows for the characterization of mitochondrial morphological alterations by using a simple and quick method that avoids the fixation artefacts that often occur in immunofluorescence studies.
    Keywords:  Confocal microscopy; Drosophila melanogaster; Fluorescent probes; Live imaging; Norbormide
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_17
  14. Methods Mol Biol. 2021 ;2310 57-68
    Mitochondrial Functional Assessment in Mammalian Gametes Using Fluorescent Probes.
    Renata S Tavares, Sara Escada-Rebelo, Maria M Soares, Andreia F Silva, Teresa Almeida-Santos, Sandra Amaral, Ana Paula Sousa, João Ramalho-Santos.
      A positive relationship between mitochondrial functionality and gamete quality, ultimately contributing to fertilization success and normal embryo development has been established for some years now. Both mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production are major indicators of mitochondrial function, and the need for accurate biomarkers mirroring gamete quality highlights the importance of a precise assessment of mitochondrial quality and function. In this chapter, we discuss the use of some mitochondrial fluorescent probes coupled to flow cytometry and/or fluorescence microscopy to specifically assess mitochondrial ROS production and MMP in both sperm and oocytes. Furthermore, as the distribution/aggregation of mitochondria in the oocyte is of interest to determine its quality, a detailed protocol is also given. These methodologies are easy, accurate and can be safely applied in research- and/or clinical-based contexts.
    Keywords:  Fluorescent probes; Mitochondrial aggregation; Mitochondrial distribution; Mitochondrial membrane potential; Oocytes; Reactive oxygen species; Sperm
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_5
  15. Methods Mol Biol. 2021 ;2310 79-89
    An Update on Isolation of Functional Mitochondria from Cells for Bioenergetics Studies.
    Magdalena Lebiedzinska-Arciszewska, Lech Wojtczak, Mariusz R Wieckowski.
      Mitochondria are the organelles where the most fundamental processes of energy transformation within the cell are located. They are also involved in several processes like apoptosis and autophagy, reactive oxygen species formation, and calcium signaling, which are crucial for proper cell functioning. In addition, mitochondrial genome hosts genes encoding important proteins incorporated in respiratory chain complexes and indispensable for the oxidative phosphorylation. Studying isolated mitochondria is, therefore, crucial for better understanding of cell physiology. The presented protocol describes a relatively simple and handy method for crude mitochondrial fraction isolation from different mammalian cell lines. It includes mechanical cells disruption (homogenization) and differential centrifugation. In addition, this chapter presents two basic ways to assess mitochondrial functionality: by measuring mitochondrial inner membrane potential and coupled respiration.
    Keywords:  Cell cultures; Mitochondria isolation; Mitochondrial membrane potential; Oxidative phosphorylation; Oxygen consumption
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_7
  16. Methods Mol Biol. 2021 ;2275 433-452
    Computer-Aided Prediction of Protein Mitochondrial Localization.
    Pier Luigi Martelli, Castrense Savojardo, Piero Fariselli, Giacomo Tartari, Rita Casadio.
      Protein sequences, directly translated from genomic data, need functional and structural annotation. Together with molecular function and biological process, subcellular localization is an important feature necessary for understanding the protein role and the compartment where the mature protein is active. In the case of mitochondrial proteins, their precursor sequences translated by the ribosome machinery include specific patterns from which it is possible not only to recognize their final destination within the organelle but also which of the mitochondrial subcompartments the protein is intended for. Four compartments are routinely discriminated, including the inner and the outer membranes, the intermembrane space, and the matrix. Here we discuss to which extent it is feasible to develop computational methods for detecting mitochondrial targeting peptides in the precursor sequence and to discriminate their final destination in the organelle. We benchmark two of our methods on the general task of recognizing human mitochondrial proteins endowed with an experimentally characterized targeting peptide (TPpred3) and predicting which submitochondrial compartment is the final destination (DeepMito). We describe how to adopt our web servers in order to discriminate which human proteins are endowed with mitochondrial targeting peptides, the position of cleavage sites, and which submitochondrial compartment are intended for. By this, we add some other 1788 human proteins to the 450 ones already manually annotated in UniProt with a mitochondrial targeting peptide, providing for each of them also the characterization of the suborganellar localization.
    Keywords:  Arginine motifs; Cleavage site; Machine and deep learning; Prediction of subcellular localization; Targeting peptide
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_28
  17. Methods Mol Biol. 2021 ;2310 259-270
    Mitochondrial Regulation Assessment by 13C-NMR Isotopomer Analysis.
    Francisco X Carvalho, Bárbara Guerra-Carvalho, Ivana Jarak, Rui A Carvalho.
      Mitochondria play a central role in metabolic reprograming that occurs in numerous disease conditions. A precise evaluation of the extent of mitochondrial involvement in the metabolic alterations is essential for a better definition of metabolically based therapeutic strategies. In this chapter, some simple protocols are presented, using carbon 13 tracers and nuclear magnetic resonance isotopomer analysis, for the evaluation of mitochondrial contributions to intermediary metabolism and the metabolic effects of the implementation of some mitochondrial regulatory mechanisms.
    Keywords:  13C isotope tracers; Intermediary metabolism; Metabolic reprograming; Mitochondria; Mitochondrial regulation; NMR isotopomer analysis; TCA cycle
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_14
  18. Methods Mol Biol. 2021 ;2275 247-263
    mTRIP, an Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Resolution.
    Laurent Chatre, Miria Ricchetti.
      Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling of mtDNA, mtRNA, and proteins or mitochondria, respectively. Altogether, qualitative and quantitative alterations of the dynamics of mtDNA processing are detected by mTRIP in human cells undergoing physiological changes, as well as stress and dysfunction. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.
    Keywords:  DNA replication; FISH; Imaging; Mitochondrial DNA; Mitochondrial disease; Single-cell; Transcription; mTRIP
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_15
  19. Methods Mol Biol. 2021 ;2310 301-309
    Measuring PGC-1α and Its Acetylation Status in Mouse Primary Myotubes.
    João A Amorim, David A Sinclair.
      Metabolic flexibility is vital for organisms to respond to and survive changes in energy availability. A critical metabolic flexibility regulator is peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), which regulates various transcription factors and nuclear receptors that, in turn, regulate mitochondrial homeostasis and fatty acid oxidation. PGC-1α is itself regulated, with one of the significant modes of regulation being acetylation. Thus, measuring the acetylation status of PGC-1α is a critical indicator of cells' metabolic flexibility. In this chapter, we describe a method of evaluating PGC-1α acetylation in primary mouse myotubes. This method can also be used with other cell types and tissues.
    Keywords:  Acetylation; Aging; Cachexia; Diabetes; GCN5; Mitochondrial biogenesis; PGC-1α; Respiration; SIRT1; Sarcopenia; Sirtuin deacetylase
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_17
  20. Methods Mol Biol. 2021 ;2310 287-299
    Following the Dynamism of the Mitochondrial Network in T Cells.
    Arianna Di Daniele, Luca Simula, Silvia Campello.
      The dynamism of mitochondria, considered as complex and motile organelles, is brought about by mitochondria ability to undergo cycles of fission and fusion events, whose fine balance determines their morphology in a specific physiological context. A huge body of evidence makes it possible to associate mitochondrial organization to regulation of an increasing number of key cellular processes, such as biosynthetic pathways, oxidative phosphorylation and ATP production, calcium buffering, mtDNA homeostasis, autophagy, and cell death. Here, we review the recently developed imaging methods for studying mitochondrial dynamics, including live-cell imaging, by using mitochondrial-targeted fluorescent proteins. In more details, we focus our attention on two different protocols in the T cell model, an example of nonadherent cells, which present some particularities and difficulties in the analysis of mitochondrial shape. Also, we discuss some examples of mouse models carrying mitochondria-targeted fluorescent proteins, which allow to investigate the mitochondrial morphology in vivo.
    Keywords:  Dynamic network; Fission; Fusion; Imaging; Mitochondria; Morphology
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_16
  21. Methods Mol Biol. 2021 ;2310 113-159
    Methods to Monitor Mitophagy and Mitochondrial Quality: Implications in Cancer, Neurodegeneration, and Cardiovascular Diseases.
    Simone Patergnani, Massimo Bonora, Esmaa Bouhamida, Alberto Danese, Saverio Marchi, Giampaolo Morciano, Maurizio Previati, Gaia Pedriali, Alessandro Rimessi, Gabriele Anania, Carlotta Giorgi, Paolo Pinton.
      Mitochondria are dynamic organelles that participate in a broad array of molecular functions within the cell. They are responsible for maintaining the appropriate energetic levels and control the cellular homeostasis throughout the generation of intermediary metabolites. Preserving a healthy and functional mitochondrial population is of fundamental importance throughout the life of the cells under pathophysiological conditions. Hence, cells have evolved fine-tuned mechanisms of quality control that help to preserve the right amount of functional mitochondria to meet the demand of the cell. The specific recycling of mitochondria by autophagy, termed mitophagy, represents the primary contributor to mitochondrial quality control. During this process, damaged or unnecessary mitochondria are recognized and selectively degraded. In the past few years, the knowledge in mitophagy has seen rapid progress, and a growing body of evidence confirms that mitophagy holds a central role in controlling cellular functions and the progression of various human diseases.In this chapter, we will discuss the pathophysiological roles of mitophagy and provide a general overview of the current methods used to monitor and quantify mitophagy. We will also outline the main established approaches to investigate the mitochondrial function, metabolism, morphology, and protein damage.
    Keywords:  Cardiovascular diseases (CVD); Homeostasis; Metabolism; Mitochondrial morphology; Mitochondrial quality control; Pathology
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_9
  22. Methods Mol Biol. 2021 ;2275 363-378
    Split Green Fluorescent Protein-Based Contact Site Sensor (SPLICS ) for Heterotypic Organelle Juxtaposition as Applied to ER -Mitochondria Proximities.
    Tito Calì, Marisa Brini.
      In the last decades, membrane contact sites (MCSs) have been the object of intense investigation in different fields of cell physiology and pathology and their importance for the correct functioning of the cell is now widely recognized. MCS between any known intercellular organelles, including endoplasmic reticulum (ER), mitochondria, Golgi, endosomes, peroxisomes, lysosomes, lipid droplets, and the plasma membrane (PM), have been largely documented and in some cases the molecules responsible for the tethering also identified. They represent specific membrane hubs where a tightly coordinated exchange of ions, lipids, nutrients, and factors required to maintain proper cellular homeostasis takes place. Their delicate, dynamic, and sometimes elusive nature prevented and/or delayed the development of tools to easily image interorganelle proximity under physiological conditions and in living organisms. Nowadays, this aspect received great momentum due to the finding that MCSs' dysregulation is involved in several pathological conditions. We have recently developed modular, split-GFP-based contact site sensors (SPLICS) engineered to fluoresce when homo- and heterotypic juxtapositions between ER and mitochondria occur over a range of specific distances. Here we describe in detail, by highlighting strengths and weaknesses, the use and the application of these novel genetically encoded SPLICS sensors and how to properly quantify short- and long-range ER-mitochondria interactions.
    Keywords:  ER–Mitochondria tethering; Endoplasmic reticulum; Mitochondria; Organelle contact sites; SPLICS; Split GFP
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_23
  23. Methods Mol Biol. 2021 ;2275 217-225
    Sequence-Specific Control of Mitochondrial Gene Transcription Using Programmable Synthetic Gene Switches Called MITO-PIPs.
    Takuya Hidaka, Hiroshi Sugiyama, Ganesh N Pandian.
      Mitochondria possess multiple copies of mitochondrial DNA (mtDNA) that encode 37 genes and their transcription and replication get controlled by unique molecular codes different from that in the nuclear DNA. The mtDNA has been gaining increased attention as one of the critical therapeutic targets as mutations in them impair the function of mitochondria and cause mitochondrial diseases like MELAS. In this chapter, we describe artificial control of mitochondrial transcription based on mtDNA sequence information with a new type of compounds termed MITO-PIPs, which encompasses two domains: pyrrole-imidazole polyamide as DNA recognition domain and mitochondrial penetrating peptide as the mitochondria-targeting domain. Because MITO-PIPs are amenable to tunability, they can be expanded as a synthetic strategy to modulate mitochondrial gene(s) on demand.
    Keywords:  DNA binding ligand; Fmoc solid-phase synthesis; MITO-PIP; Mitochondrial DNA; Pyrrole–imidazole polyamide; Transcription
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_13
  24. Methods Mol Biol. 2021 ;2310 271-285
    Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.
    Yue Yang, Anthony A Sauve.
      NAD+ is a redox cofactor essential to the proper functioning of a variety of important metabolic pathways, including key steps in mitochondrial energy metabolism. In addition, it serves as a signaling substrate for enzymes such as sirtuins and the poly-ADP ribosyl-polymerase family of enzymes. Sirtuins, which are NAD+-dependent protein deacylases, harness changes in cellular NAD+ concentrations to produce changes in protein acylation status, thereby affecting downstream functions including energy metabolism, stress resistance, and cell survival. Thus, the availability of NAD+ in cells, or in specific organelles such as the mitochondrion, regulates downstream signaling and key biological processes. This concept has driven a need for researchers to easily and precisely measure NAD+ concentrations in biological samples. We herein describe several protocols for the measurement of NAD+ and NADH concentrations in tissues, cells, or subcellular compartments such as mitochondria. These protocols include a cycling assay that can quickly measure NAD+ or NADH levels using a plate reader equipped with fluorescence measurement capabilities. This plate assay relies only upon commercially available materials in addition to the biological samples of interest. In addition, we describe a protocol employing stable isotope-labeled NAD+ as an internal standard to determine biological NAD+ content by isotope-dilution methods. This method requires mass spectrometry to ratio endogenous NAD+ with exogenous isotope-labeled NAD+ to obtain quantification using HPLC and mass spectrometry.
    Keywords:  18O-NAD+; Diaphorase; HPLC; Isotopes; LC-MS; Lactate; Lactate dehydrogenase (LDH); Mitochondria isolation; NAD+; NAD+/NADH cycling assay; Resazurin; Resorufin
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_15
  25. Methods Mol Biol. 2021 ;2275 415-432
    Mito-SinCe2 Approach to Analyze Mitochondrial Structure-Function Relationship in Single Cells.
    B Spurlock, K Mitra.
      The cross talk between mitochondrial dynamic structure, determined primarily by mitochondrial fission and fusion events, and mitochondrial function of energetics, primarily ATP and ROS production, is widely appreciated. Understanding the mechanistic details of such cross talk between mitochondrial structure and function needs integrated quantitative analyses between mitochondrial dynamics and energetics. Here we describe our recently designed approach of mito-SinCe2 that involves high resolution confocal microscopy of genetically expressed ratiometric fluorescent probes targeted to mitochondria, and its quantitative analyses. Mito-SinCe2 analyses allows for quantitative analyses of mitochondrial structure-function relationship in single cells toward understanding the role of mitochondria and their heterogeneity in various physiological and pathological conditions.
    Keywords:  ATP; Confocal Microscopy; Fission; Fusion; Mitochondrial Dynamics; Mitochondrial Energetics; Quantitative analyses; Ratiometric Probes; Redox State; Single Cell; Structure–Function Relationships
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_27
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