bims-mitdyn Biomed News
on Mitochondrial dynamics: mechanisms
Issue of 2022–07–03
29 papers selected by
Edmond Chan, Queen’s University, School of Medicine
  1. Mitochondrial RNA modifications shape metabolic plasticity in metastasis.
  2. Mitochondrial protein import stress regulates the LC3 lipidation step of mitophagy through NLRX1 and RRBP1.
  3. DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos.
  4. Structural basis for mitoguardin-2 mediated lipid transport at ER-mitochondrial membrane contact sites.
  5. Impaired oxygen-sensitive regulation of mitochondrial biogenesis within the von Hippel-Lindau syndrome.
  6. A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery.
  7. RhoA signaling increases mitophagy and protects cardiomyocytes against ischemia by stabilizing PINK1 protein and recruiting Parkin to mitochondria.
  8. Mitochondrial dysfunction in cell senescence and aging.
  9. MICU1 controls spatial membrane potential gradients and guides Ca2+ fluxes within mitochondrial substructures.
  10. Examining Mitochondrial Morphology in Mouse Brains.
  11. In Vivo Analysis of Mitochondrial Protein Synthesis in Saccharomyces cerevisiae Mitochondrial tRNA Mutants.
  12. Fast Determination of Mitochondrial Metabolism and Respiratory Complex Activity in Permeabilized and Intact Cells.
  13. Measuring the Mitochondrial Ubiquinone (Q) Pool Redox State in Isolated Respiring Mitochondria.
  14. Simultaneous Acquisition of Mitochondrial Calcium Retention Capacity and Swelling to Measure Permeability Transition Sensitivity.
  15. Quantifying Regulated Mitochondrial Fission in Macrophages.
  16. Monitoring Mitochondrial Perturbations During Infection.
  17. Monitoring Mitochondrial Respiration in Mouse Cerebellar Granule Neurons.
  18. Measurements of Mitochondrial Respiration in Intact Cells, Permeabilized Cells, and Isolated Tissue Mitochondria Using the Seahorse XF Analyzer.
  19. Investigating Mitochondrial Ca2+ Dynamics in Isolated Mitochondria and Intact Cells: Application of Fluorescent Dyes and Genetic Reporters.
  20. Mitochondrial Calcium Handling in Isolated Mitochondria from a Guinea Pig Heart.
  21. Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE.
  22. Visualizing Mitochondrial Importability of a Protein Using the Yeast Bi-Genomic Mitochondrial-Split-GFP Strain and an Ordinary Fluorescence Microscope.
  23. Monitoring Mitochondrial Morphology and Respiration in Doxorubicin-Induced Cardiomyopathy.
  24. Computational Modeling of Mitochondria to Understand the Dynamics of Oxidative Stress.
  25. Analysis of Mitochondrial Performance in Lymphocytes Using Fluorescent Lifetime Imaging Microscopy.
  26. Monitoring Mitochondrial Membrane Potential in Live Cells Using Time-Lapse Fluorescence Imaging.
  27. Characterizing the Electron Transport Chain: Structural Approach.
  28. Measuring Mitochondrial Function: From Organelle to Organism.
  29. A Plate Reader-Based Measurement of the Cellular ROS Production Using Dihydroethidium and MitoSOX.
  1. Nature. 2022 Jun 29.
    Mitochondrial RNA modifications shape metabolic plasticity in metastasis.
    Sylvain Delaunay, Gloria Pascual, Bohai Feng, Kevin Klann, Mikaela Behm, Agnes Hotz-Wagenblatt, Karsten Richter, Karim Zaoui, Esther Herpel, Christian Münch, Sabine Dietmann, Jochen Hess, Salvador Aznar Benitah, Michaela Frye.
      Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.
    DOI:  https://doi.org/10.1038/s41586-022-04898-5
  2. Mol Cell. 2022 Jun 16. pii: S1097-2765(22)00540-8. [Epub ahead of print]
    Mitochondrial protein import stress regulates the LC3 lipidation step of mitophagy through NLRX1 and RRBP1.
    Samuel A Killackey, Yuntian Bi, Fraser Soares, Ikram Hammi, Nathaniel J Winsor, Ali A Abdul-Sater, Dana J Philpott, Damien Arnoult, Stephen E Girardin.
      Protein import into mitochondria is a highly regulated process, yet how cells clear mitochondria undergoing dysfunctional protein import remains poorly characterized. Here we showed that mitochondrial protein import stress (MPIS) triggers localized LC3 lipidation. This arm of the mitophagy pathway occurs through the Nod-like receptor (NLR) protein NLRX1 while, surprisingly, without the engagement of the canonical mitophagy protein PINK1. Mitochondrial depolarization, which itself induces MPIS, also required NLRX1 for LC3 lipidation. While normally targeted to the mitochondrial matrix, cytosol-retained NLRX1 recruited RRBP1, a ribosome-binding transmembrane protein of the endoplasmic reticulum, which relocated to the mitochondrial vicinity during MPIS, and the NLRX1/RRBP1 complex in turn controlled the recruitment and lipidation of LC3. Furthermore, NLRX1 controlled skeletal muscle mitophagy in vivo and regulated endurance capacity during exercise. Thus, localization and lipidation of LC3 at the site of mitophagosome formation is a regulated step of mitophagy controlled by NLRX1/RRBP1 in response to MPIS.
    Keywords:  NLRX1; Nod-like receptors; mitochondria; mitochondrial protein import; mitophagy
    DOI:  https://doi.org/10.1016/j.molcel.2022.06.004
  3. Nat Commun. 2022 Jul 01. 13(1): 3794
    DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos.
    Yixiong Wang, Lubna Yasmin, Lei Li, Pinzhang Gao, Xia Xu, Xuejun Sun, Roseline Godbout.
      The DEAD box protein DDX1, previously associated with 3'-end RNA processing and DNA repair, forms large aggregates in the cytoplasm of early mouse embryos. Ddx1 knockout causes stalling of embryos at the 2-4 cell stages. Here, we identify a DDX1-containing membrane-bound calcium-containing organelle with a nucleic acid core. We show that aggregates of these organelles form ring-like structures in early-stage embryos which we have named Membrane Associated RNA-containing Vesicles. We present evidence that DDX1 is required for the formation of Membrane Associated RNA-containing Vesicles which in turn regulate the spatial distribution of calcium in embryos. We find that Ddx1 knockout in early embryos disrupts calcium distribution, and increases mitochondria membrane potential, mitochondrial activity, and reactive oxygen species. Sequencing analysis of embryos from Ddx1 heterozygote crosses reveals downregulation of a subset of RNAs involved in developmental and mitochondrial processes in the embryos with low Ddx1 RNA. We propose a role for Membrane Associated RNA-containing Vesicles in calcium-controlled mitochondrial functions that are essential for embryonic development.
    DOI:  https://doi.org/10.1038/s41467-022-31497-9
  4. Nat Commun. 2022 Jun 28. 13(1): 3702
    Structural basis for mitoguardin-2 mediated lipid transport at ER-mitochondrial membrane contact sites.
    Hyunwoo Kim, Seowhang Lee, Youngsoo Jun, Changwook Lee.
      The endoplasmic reticulum (ER)-mitochondria contact site (ERMCS) is crucial for exchanging biological molecules such as phospholipids and Ca2+ ions between these organelles. Mitoguardin-2 (MIGA2), a mitochondrial outer membrane protein, forms the ERMCS in higher eukaryotic cells. Here, we report the crystal structures of the MIGA2 Lipid Droplet (LD) targeting domain and the ER membrane protein VAPB bound to the phosphorylated FFAT motif of MIGA2. These structures reveal that the MIGA2 LD targeting domain has a large internal hydrophobic pocket that accommodates phospholipids and that two phosphorylations of the FFAT motif are required for tight interaction of MIGA2 with VAPB, which enhances the rate of lipid transport. Further biochemical studies show that MIGA2 transports phospholipids between membranes with a strong preference for binding and trafficking phosphatidylserine (PS). These results provide a structural and molecular basis for understanding how MIGA2 mediates the formation of ERMCS and facilitates lipid trafficking at the ERMCS.
    DOI:  https://doi.org/10.1038/s41467-022-31462-6
  5. Nat Metab. 2022 Jun;4(6): 739-758
    Impaired oxygen-sensitive regulation of mitochondrial biogenesis within the von Hippel-Lindau syndrome.
    Shuijie Li, Wenyu Li, Juan Yuan, Petra Bullova, Jieyu Wu, Xuepei Zhang, Yong Liu, Monika Plescher, Javier Rodriguez, Oscar C Bedoya-Reina, Paulo R Jannig, Paula Valente-Silva, Meng Yu, Marie Arsenian Henriksson, Roman A Zubarev, Anna Smed-Sörensen, Carolyn K Suzuki, Jorge L Ruas, Johan Holmberg, Catharina Larsson, C Christofer Juhlin, Alex von Kriegsheim, Yihai Cao, Susanne Schlisio.
      Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel-Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel-Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.
    DOI:  https://doi.org/10.1038/s42255-022-00593-x
  6. Nat Commun. 2022 Jun 28. 13(1): 3720
    A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery.
    Hayden Weng Siong Tan, Guang Lu, Han Dong, Yik-Lam Cho, Auginia Natalia, Liming Wang, Charlene Chan, Dennis Kappei, Reshma Taneja, Shuo-Chien Ling, Huilin Shao, Shih-Yin Tsai, Wen-Xing Ding, Han-Ming Shen.
      PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.
    DOI:  https://doi.org/10.1038/s41467-022-31213-7
  7. Cell Death Differ. 2022 Jun 27.
    RhoA signaling increases mitophagy and protects cardiomyocytes against ischemia by stabilizing PINK1 protein and recruiting Parkin to mitochondria.
    Michelle Tu, Valerie P Tan, Justin D Yu, Raghav Tripathi, Zahna Bigham, Melissa Barlow, Jeffrey M Smith, Joan Heller Brown, Shigeki Miyamoto.
      Mitophagy, a mitochondria-specific form of autophagy, removes dysfunctional mitochondria and is hence an essential process contributing to mitochondrial quality control. PTEN-induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin are critical molecules involved in stress-induced mitophagy, but the intracellular signaling mechanisms by which this pathway is regulated are unclear. We tested the hypothesis that signaling through RhoA, a small GTPase, induces mitophagy via modulation of the PINK1/Parkin pathway as a protective mechanism against ischemic stress. We demonstrate that expression of constitutively active RhoA as well as sphingosine-1-phosphate induced activation of endogenous RhoA in cardiomyocytes result in an accumulation of PINK1 at mitochondria. This is accompanied by translocation of Parkin to mitochondria and ubiquitination of mitochondrial proteins leading to recognition of mitochondria by autophagosomes and their lysosomal degradation. Expression of RhoA in cardiomyocytes confers protection against ischemia, and this cardioprotection is attenuated by siRNA-mediated PINK1 knockdown. In vivo myocardial infarction elicits increases in mitochondrial PINK1, Parkin, and ubiquitinated mitochondrial proteins. AAV9-mediated RhoA expression potentiates these responses and a concurrent decrease in infarct size is observed. Interestingly, induction of mitochondrial PINK1 accumulation in response to RhoA signaling is neither mediated through its transcriptional upregulation nor dependent on depolarization of the mitochondrial membrane, the canonical mechanism for PINK1 accumulation. Instead, our results reveal that RhoA signaling inhibits PINK1 cleavage, thereby stabilizing PINK1 protein at mitochondria. We further show that active RhoA localizes at mitochondria and interacts with PINK1, and that the mitochondrial localization of RhoA is regulated by its downstream effector protein kinase D. These findings demonstrate that RhoA activation engages a unique mechanism to regulate PINK1 accumulation, induce mitophagy and protect against ischemic stress, and implicates regulation of RhoA signaling as a potential strategy to enhance mitophagy and confer protection under stress conditions.
    DOI:  https://doi.org/10.1038/s41418-022-01032-w
  8. J Clin Invest. 2022 Jul 01. pii: e158447. [Epub ahead of print]132(13):
    Mitochondrial dysfunction in cell senescence and aging.
    Satomi Miwa, Sonu Kashyap, Eduardo Chini, Thomas von Zglinicki.
      Mitochondrial dysfunction and cell senescence are hallmarks of aging and are closely interconnected. Mitochondrial dysfunction, operationally defined as a decreased respiratory capacity per mitochondrion together with a decreased mitochondrial membrane potential, typically accompanied by increased production of oxygen free radicals, is a cause and a consequence of cellular senescence and figures prominently in multiple feedback loops that induce and maintain the senescent phenotype. Here, we summarize pathways that cause mitochondrial dysfunction in senescence and aging and discuss the major consequences of mitochondrial dysfunction and how these consequences contribute to senescence and aging. We also highlight the potential of senescence-associated mitochondrial dysfunction as an antiaging and antisenescence intervention target, proposing the combination of multiple interventions converging onto mitochondrial dysfunction as novel, potent senolytics.
    DOI:  https://doi.org/10.1172/JCI158447
  9. Commun Biol. 2022 Jul 01. 5(1): 649
    MICU1 controls spatial membrane potential gradients and guides Ca2+ fluxes within mitochondrial substructures.
    Benjamin Gottschalk, Zhanat Koshenov, Markus Waldeck-Weiermair, Snježana Radulović, Furkan E Oflaz, Martin Hirtl, Olaf A Bachkoenig, Gerd Leitinger, Roland Malli, Wolfgang F Graier.
      Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in mediating Ca2+ control over spatial membrane potential gradients (SMPGs) between CM and IMS was identified. We unveiled alterations of SMPGs by transient CJ openings when Ca2+ binds to MICU1 resulting in spatial cristae depolarization. This Ca2+/MICU1-mediated plasticity of the CJ further provides the mechanistic bedrock of the biphasic mitochondrial Ca2+ uptake kinetics via the mitochondrial Ca2+ uniporter (MCU) during intracellular Ca2+ release: Initially, high Ca2+ opens CJ via Ca2+/MICU1 and allows instant Ca2+ uptake across the CM through constantly active MCU. Second, MCU disseminates into the IBM, thus establishing Ca2+ uptake across the IBM that circumvents the CM. Under the condition of MICU1 methylation by PRMT1 in aging or cancer, UCP2 that binds to methylated MICU1 destabilizes CJ, disrupts SMPGs, and facilitates fast Ca2+ uptake via the CM.
    DOI:  https://doi.org/10.1038/s42003-022-03606-3
  10. Methods Mol Biol. 2022 ;2515 17-28
    Examining Mitochondrial Morphology in Mouse Brains.
    Jessika Royea, Mireille Khacho.
      Mitochondria are dynamic organelles that rely on a balance of opposing fission and fusion events to sustain mitochondrial function and efficiently meet the energy demands of a cell. As high-energy demanding cells, neurons rely heavily on optimally functional mitochondria with balanced mitochondrial dynamics, to ensure a sufficient energy supply required to maintain cell survival, establish membrane excitability and partake in processes of neurotransmission and plasticity. As such, many neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease) and stress conditions (e.g., stroke) leading to neuronal dysfunction or death are often associated with impaired mitochondrial function and dynamics, characterized by excessive mitochondrial fragmentation. For this reason, the assessment of mitochondrial morphology in neurons and within the brain can provide valuable information. The dynamic nature of mitochondria is not only observed in shape changes, but also changes in mitochondrial network connectivity and in cristae architecture. In this chapter, we will describe how mitochondrial morphology can be examined in vitro using hippocampal neuronal cultures and in vivo using mouse brain sections by immunocytochemistry, immunohistochemistry, and electron microscopy techniques.
    Keywords:  Confocal and electron microscopy; Cristae; Hippocampus; Mitochondrial dynamics; Mitochondrial dysfunction; Mitochondrial fission; Mitochondrial fusion; Mitochondrial morphology; Neurodegenerative diseases; Neuronal cultures
    DOI:  https://doi.org/10.1007/978-1-0716-2409-8_2
  11. Methods Mol Biol. 2022 ;2497 243-254
    In Vivo Analysis of Mitochondrial Protein Synthesis in Saccharomyces cerevisiae Mitochondrial tRNA Mutants.
    Arianna Montanari.
      I describe here a protocol for the analysis of mitochondrial protein synthesis as a useful tool to characterize the mitochondrial defects associated with mutations in mitochondrial tRNA genes. The yeast Saccharomyces cerevisiae mutants, bearing human equivalent pathogenic mutations, were used as a simple model for analysis. The mitochondrial proteins were labeled by L[35S]-methionine incorporation in growing cells, extracted from purified mitochondria, and fractionated by SDS-polyacrylamide gel electrophoresis followed by autoradiography. By this method, it is possible to distinguish different protein synthesis profiles in the analyzed mitochondrial tRNA mutants.
    Keywords:  Human equivalent mutations; In vivo L[35S]-methionine labeling; Mitochondria; Mitochondrial protein synthesis; Mitochondrial tRNA mutants; Saccharomyces cerevisiae
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_15
  12. Methods Mol Biol. 2022 ;2497 1-10
    Fast Determination of Mitochondrial Metabolism and Respiratory Complex Activity in Permeabilized and Intact Cells.
    Kareem A Heslop, Amandine Rovini, Monika Gooz, Eduardo N Maldonado.
      Assessment of mitochondrial metabolism is multidimensional and time consuming, usually requiring specific training. Respiration, NADH generation, and mitochondrial membrane potential (ΔΨm) are dynamic readouts of the metabolism and bioenergetics of mitochondria. Methodologies available to determine functional parameters in isolated mitochondria and permeabilized cells are sometimes of limited use or inapplicable to studies in live cells. In particular, the sequential assessment of the activity of each complex in the electron transport chain has not been reported in intact cells. Here, we describe a novel approach to sequentially assess electron flow through all respiratory complexes in permeabilized and intact cells by respirometry. We also describe a highly sensitive and fast method to assess ΔΨm and NADH generation in live cells using plate reader assays. Thus, our combined method allows a relatively inexpensive and fast determination of three major readouts of mitochondrial function in a few hours, using equipment that is frequently available in many laboratories worldwide.
    Keywords:  Electron transport chain; Mitochondria; Mitochondrial membrane potential; Mitochondrial metabolism; NADH; Oxygen consumption; Respiratory complex; TMRM; Warburg Metabolism
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_1
  13. Methods Mol Biol. 2022 ;2497 291-299
    Measuring the Mitochondrial Ubiquinone (Q) Pool Redox State in Isolated Respiring Mitochondria.
    Marten Szibor, Estelle Heyne, Carlo Viscomi, Anthony L Moore.
      The ubiquinone (Q) pool represents a node in the mitochondrial electron transport chain (ETC) onto which the electrons of all respiratory dehydrogenases converge. The redox state of the Q pool correlates closely with the electron flux through the ETC and is thus a parameter of great metabolic value for both the mitochondrial and cellular metabolism. Here, we describe the simultaneous measurement of respiratory rates of isolated mouse heart mitochondria and the redox state of their Q pool using a custom-made combination of a Clark-type oxygen electrode and a Q electrode.
    Keywords:  Mitochondria; Redox state; Respiratory rates; Ubiquinone pool
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_19
  14. Methods Mol Biol. 2022 ;2497 129-140
    Simultaneous Acquisition of Mitochondrial Calcium Retention Capacity and Swelling to Measure Permeability Transition Sensitivity.
    Arielys M Mendoza, Jason Karch.
      The loss of mitochondrial cristae integrity and mitochondrial swelling are hallmarks of multiple forms of necrotic cell death. One of the most well-studied and relevant inducers of mitochondrial swelling is matrix calcium (Ca2+). Respiring mitochondria will intake available Ca2+ into their matrix until a threshold is reached which triggers the opening of the mitochondrial permeability transition pore (MPTP). Upon opening of the pore, mitochondrial membrane potential dissipates and the mitochondria begin to swell, rendering them dysfunctional. The total amount of Ca2+ taken up by a mitochondrion prior to the engagement of the MPTP is referred to as mitochondrial Ca2+ retention capacity (CRC). The CRC/swelling assay is a useful tool for observing the dose-dependent event of mitochondrial dysfunction in real-time. In this technique, isolated mitochondria are treated with specific boluses of Ca2+ until they reach CRC and undergo swelling. A fluorometer is utilized to detect an increase in transmitted light passing through the sample as the mitochondria lose cristae density, and simultaneously measures calcium uptake by way of a Ca2+-specific membrane impermeable fluorescent dye. Here we provide a detailed protocol describing the mitochondrial CRC/swelling assay and we discuss how varying amounts of mitochondria and Ca2+ added to the system affect the dose-dependency of the assay. We also report how to validate the assay by using MPTP and calcium uptake inhibitors and troubleshooting common mistakes that occur with this approach.
    Keywords:  CRC; Calcium Green 5 N; Calcium retention capacity; Cell death; Fluorometry; Mitochondria; Mitochondrial dysfunction; Mitochondrial permeability transition pore (MPTP); Mitochondrial swelling
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_9
  15. Methods Mol Biol. 2022 ;2523 281-301
    Quantifying Regulated Mitochondrial Fission in Macrophages.
    Syeda Farhana Afroz, Nicholas D Condon, Matthew J Sweet, Ronan Kapetanovic.
      Mitochondria have co-evolved with eukaryotic cells for more than a billion years, becoming an important cog in their machinery. They are best known for being tasked with energy generation through the production of adenosine triphosphate, but they also have roles in several other cellular processes, for example, immune and inflammatory responses. Mitochondria have important functions in macrophages, key innate immune cells that detect pathogens and drive inflammation. Mitochondrial activity is influenced by the highly dynamic nature of the mitochondrial network, which alternates between interconnected tubular and fragmented forms. The dynamic balance between this interconnected fused network and fission-mediated mitochondrial fragmentation modulates inflammatory responses such as production of cytokines and mitochondrial reactive oxygen species. Here we describe methods to differentiate mouse bone marrow cells into macrophages and the use of light microscopy, electron microscopy, flow cytometry, and Western blotting to quantify regulated mitochondrial dynamics in these differentiated macrophages.
    Keywords:  Drp1; Fission; Fusion; Inflammation; Macrophages; Microscopy; Mitochondria; Mitochondrial dynamics
    DOI:  https://doi.org/10.1007/978-1-0716-2449-4_18
  16. Methods Mol Biol. 2022 ;2497 281-290
    Monitoring Mitochondrial Perturbations During Infection.
    Varnesh Tiku, Man-Wah Tan.
      Mitochondria are pivotal organelles in the cell that regulate a myriad of cellular functions, which eventually govern cellular physiology and homeostasis. Intriguingly, microbial infection is known to trigger morphological and functional alterations of mitochondria. In fact, a number of bacteria and viruses have been reported to hijack mitochondrial functions including cell death induction and regulation of immune signaling to evade detection, promote their intracellular growth and subsequent dissemination. Here we describe methodologies that can be applied to assess mitochondrial functions upon infection. More specifically, we outline experimental procedures used to evaluate different parameters including mitochondrial morphology, adenosine triphosphate (ATP) levels, reactive oxygen species (ROS) levels, and mitophagy. Together these parameters can help gauge the overall health of mitochondria upon infection.
    Keywords:  ATP levels; Bacterial and viral infection; Mitochondria; Mitochondrial fission and fusion; Mitophagy; ROS levels
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_18
  17. Methods Mol Biol. 2022 ;2515 1-15
    Monitoring Mitochondrial Respiration in Mouse Cerebellar Granule Neurons.
    Ahmad Sharanek, Arezu Jahani-Asl.
      Defects in mitochondrial oxidative phosphorylation have been observed in numerous neurodegenerative disorders and are linked to bioenergetic crises leading to neuronal death. The distinct metabolic profile of neurons is predominantly oxidative, which is characterized by the oxidation of glucose or its metabolites in the mitochondria to produce ATP. This process involves the tricarboxylic acid cycle, electron transfer in the respiratory chain, and oxygen consumption. Therefore, measurement of oxygen consumption rates (OCR) can be accurately applied to assess the rate of mitochondrial respiration. In this chapter, we describe our optimized protocol for the assessment of OCR specifically in primary mouse cerebellar granule neurons (CGN). The protocol includes isolation and manipulation of mouse CGNs followed by real-time assessment of mitochondrial OCR using a Seahorse XFe96 extracellular flux analyzer.
    Keywords:  Cerebellum; Mitochondrion; Oxidative phosphorylation; Primary neurons; Seahorse XFe96 extracellular flux analyzer
    DOI:  https://doi.org/10.1007/978-1-0716-2409-8_1
  18. Methods Mol Biol. 2022 ;2497 185-206
    Measurements of Mitochondrial Respiration in Intact Cells, Permeabilized Cells, and Isolated Tissue Mitochondria Using the Seahorse XF Analyzer.
    Jessica Pfleger.
      Energy homeostasis is critical for cellular function. Significant increases in energy demand or reduced energy supply, however, often result in cellular dysfunction and death. Since mitochondria are the primary cellular energy source, their impairment is often pathogenic. Accordingly, quantitative measurements of cellular and mitochondrial energy utilization and production are crucial for understanding disease development and progression. In the final step of cellular respiration, specifically, oxidative phosphorylation within the mitochondria, oxygen is consumed and drives ATP production. Herein, we provide the complete protocols for measuring oxygen consumption rates and their coupling to ATP production in intact and permeabilized cells, as well as in mitochondria isolated from tissue using the Seahorse XF Extracellular Flux Analyzer (Agilent Technologies).
    Keywords:  Bioenergetics; Cellular respiration; Mitochondrial respiration; Permeabilized cells; Seahorse XF Extracellular Flux Analyzer
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_12
  19. Methods Mol Biol. 2022 ;2497 325-332
    Investigating Mitochondrial Ca2+ Dynamics in Isolated Mitochondria and Intact Cells: Application of Fluorescent Dyes and Genetic Reporters.
    Gauri Bhosale, Michael R Duchen.
      Mitochondrial Ca2+ buffering is a hallmark of eukaryotic cellular physiology, contributing to the spatiotemporal shaping of the cytosolic Ca2+ signals and regulation of mitochondrial bioenergetics. Often, this process is altered in a pathological context; therefore, it can be scrutinized experimentally for therapeutic intervention. In this chapter, we describe fluorescence and bioluminescence measurement of mitochondrial Ca2+ in both isolated mitochondria and intact cells.
    Keywords:  Bioluminescence calcium sensing; Calcium-sensitive genetic probes; Fluorescence calcium imaging; Mitochondrial calcium
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_23
  20. Methods Mol Biol. 2022 ;2497 97-106
    Mitochondrial Calcium Handling in Isolated Mitochondria from a Guinea Pig Heart.
    Jyotsna Mishra, Amadou K S Camara.
      Mitochondrial calcium (Ca2+) plays a key role in regulating normal cardiac function. A physiological increase in mitochondrial matrix calcium [Ca2+]m drives mitochondrial ATP production to meet the high-energy demands during excitation-contraction coupling. However, a pathological increase in [Ca2+]m leads to increased oxidative stress, impaired bioenergetics, and the opening of mitochondrial permeability transition pore (mPTP), a hallmark of the failing heart. Therefore, a better understanding of the [Ca2+]m handling and its role in heart function and dysfunction is of great importance. Here, we describe a detailed protocol for measuring mitochondrial Ca2+ handling in the isolated functionally intact mitochondria from cardiac tissue of the guinea pig.
    Keywords:  Calcium retention capacity; Mitochondrial bioenergetics; Mitochondrial calcium handling; Mitochondrial membrane potential; Mitochondrial oxygen consumption
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_6
  21. Methods Mol Biol. 2022 ;2497 339-348
    Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE.
    Kritarth Singh, Michael R Duchen.
      Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a well-established technique for the isolation and separation of mitochondrial membrane protein complexes in a native conformation with high resolution. In combination with histochemical staining methods, BN-PAGE has been successfully used as clinical diagnostic tool for the detection of oxidative phosphorylation (OXPHOS) defects from small tissue biopsies from patients with primary mitochondrial disease. However, its application to patient-derived primary fibroblasts is difficult due to limited proliferation and high background staining. Here, we describe a rapid and convenient method to analyze the organization and activity of OXPHOS complexes from cultured skin fibroblasts.
    Keywords:  In-gel activity; Mitochondria; Oxidative phosphorylation; Primary fibroblasts; Supercomplex
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_25
  22. Methods Mol Biol. 2022 ;2497 255-267
    Visualizing Mitochondrial Importability of a Protein Using the Yeast Bi-Genomic Mitochondrial-Split-GFP Strain and an Ordinary Fluorescence Microscope.
    Marine Hemmerle, Bruno Senger, Kucharczyk Roza, Hubert D Becker.
      Proving with certainty that a GFP-tagged protein is imported inside mitochondria by visualizing its fluorescence emission with an epifluorescence microscope is currently impossible using regular GFP-tagging. This is particularly true for proteins dual localized in the cytosol and mitochondria, which have been estimated to represent up to one third of the established mitoproteomes. These proteins are usually composed of a surpassingly abundant pool of the cytosolic isoform compared to the mitochondrial isoform. As a consequence, when tagged with a regular GFP, the fluorescence emission of the cytosolic isoform will inevitably eclipse that of the mitochondrial one and prevent the detection of the mitochondrial echoform. To overcome this technical limit, we engineered a yeast strain expressing a new type of GFP called Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). In this strain, one moiety of the GFP is encoded by the mitochondrial DNA while the second moiety of the GFP can be tagged to any nuclear-encoded protein (suspected to be dual localized or bona fide mitochondrial). By doing so, only mitochondrial proteins or echoforms of dual localized proteins, regardless of their organismal origin, trigger GFP reconstitution that can be visualized by regular fluorescence microscopy. The strength of the BiG Mito-Split-GFP system is that proof of the mitochondrial localization of a given protein rests on a simple and effortless microscopy observation.
    Keywords:  BiG Mito-Split-GFP; Dual localized; Epifluorescence microscopy; Living cells; Localization; Mitochondria; Saccharomyces cerevisiae
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_16
  23. Methods Mol Biol. 2022 ;2497 207-220
    Monitoring Mitochondrial Morphology and Respiration in Doxorubicin-Induced Cardiomyopathy.
    Chowdhury S Abdullah, Richa Aishwarya, Mahboob Morshed, Naznin Sultana Remex, Sumitra Miriyala, Manikandan Panchatcharam, Md Shenuarin Bhuiyan.
      Doxorubicin (DOX)-induced cardiomyopathy constitutes dose-dependent cardiac toxicity, culminating in fatal heart failure progression. Cardiac toxicity limits effective and subsequent use of DOX in chemotherapy regimens in pediatric, adult, and recurrent cancer patients. DOX-induced profound alterations in mitochondrial morphology, dynamics, and defects in mitochondrial energy metabolism in the heart comprise key stressors in DOX-induced cardiotoxicity. Hence, the discovery of novel molecular targets and therapeutics to mitigate DOX-induced mitochondrial dysfunctions are imperative. Herein, we provided two laboratory protocols to monitor DOX-induced alterations in mitochondrial morphology and respiration in isolated primary neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes are extensively used to monitor signaling mechanisms regulating cardiomyopathy in vitro. Therefore, these protocols will help researchers study the effects of novel pharmacological and genetic manipulations against DOX-induced alterations in mitochondrial morphology and energy metabolism in cardiomyocytes.
    Keywords:  Doxorubicin-induced cardiomyopathy; Mitochondrial morphology; Mitochondrial respiration; Oxygen consumption rates
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_13
  24. Methods Mol Biol. 2022 ;2497 363-422
    Computational Modeling of Mitochondria to Understand the Dynamics of Oxidative Stress.
    Rashmi Kumar, Mohsin S Jafri.
      Mitochondria are complex organelles that use catabolic metabolism to produce ATP which is the critical energy source for cell function. Oxidative phosphorylation by the electron transport chain, which receives reducing equivalents (NADH and FADH2) from the tricarboxylic acid cycle, also produces reactive oxygen species (ROS) as a by-product at complex I and III. ROS play a significant role in health and disease. In order to better understand this process, a computational model of mitochondrial energy metabolism and the production of ROS has been developed. The model demonstrates the process regulating ROS production and removal and how different energy substrates can affect ROS production.
    Keywords:  Electron transport; Mitochondria; Reactive oxygen species
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_27
  25. Methods Mol Biol. 2022 ;2497 269-280
    Analysis of Mitochondrial Performance in Lymphocytes Using Fluorescent Lifetime Imaging Microscopy.
    Meha Patel, Javier Manzella-Lapeira, Munir Akkaya.
      During lymphocyte maturation and differentiation, cells undergo a series of proliferative stages interrupted with stages of low activity. The rapid proliferation stages are marked by changes in metabolic outputs-adapting to energy demands by either hindering or utilizing metabolic pathways. As such, it is necessary to view these changes in real time; however, current strategies for metabolomics are time consuming and very rarely provide a holistic profile of the cellular metabolism while also characterizing mitochondrial metabolism. Here, we devised a fluorescence lifetime imaging microscopy (FLIM) strategy to image mitochondrial metabolic profiles in lymphocytes as they go through changes in metabolic activity. Our method provides not only a comprehensive view of cellular metabolism but also narrow in mitochondrial contributions while also efficiently excluding non-viable cells with and without the use of a viability dye. Our novel imaging strategy offers a reliable tool to study changes in mitochondrial metabolism.
    Keywords:  FLIM; Immunology; Immunometabolism; Lymphocytes; Microscopy; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_17
  26. Methods Mol Biol. 2022 ;2497 319-324
    Monitoring Mitochondrial Membrane Potential in Live Cells Using Time-Lapse Fluorescence Imaging.
    Gabriel Esteban Valdebenito, Michael R Duchen.
      The mitochondrial membrane potential (ΔΨm) generated by proton pumps (Complexes I, III, and IV) is an essential component in the process of energy generation during oxidative phosphorylation. Tetramethylrhodamine, methyl ester, perchlorate (TMRM) is one of the most commonly used fluorescent reporters of ΔΨm. TMRM is routinely employed in a steady state for the measurement of membrane potential. However, it can also be utilized with time-lapse fluorescence imaging to effectively monitor the changes in membrane potential in response to a given stimulus by analyzing the change in distribution of the dye with time.
    Keywords:  Fluorescence microscopy; Mitochondria membrane potential; Primary skin fibroblasts; TMRM; Uncoupler
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_22
  27. Methods Mol Biol. 2022 ;2497 107-115
    Characterizing the Electron Transport Chain: Structural Approach.
    Ting Liang, Janice Deng, Bijaya Nayak, Xin Zou, Yuji Ikeno, Yidong Bai.
      The mitochondrial respiratory chain which carries out the oxidative phosphorylation (OXPHOS) consists of five multi-subunit protein complexes. Emerging evidences suggest that the supercomplexes which further consist of multiple respiratory complexes play important role in regulating OXPHOS function. Dysfunction of the respiratory chain and its regulation has been implicated in various human diseases including neurodegenerative diseases and muscular disorders. Many mouse models have been established which exhibit mitochondrial defects in brain and muscles. Protocols presented here aim to help to analyze the structures of mitochondrial respiratory chain which include the preparation of the tissue samples, isolation of mitochondrial membrane proteins, and analysis of their respiratory complexes by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) in particular.
    Keywords:  Assembly; Blue Native Gel; Brain; Muscle; Respiratory complex
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_7
  28. Methods Mol Biol. 2022 ;2497 141-172
    Measuring Mitochondrial Function: From Organelle to Organism.
    Matthew T Lewis, Yan Levitsky, Jason N Bazil, Robert W Wiseman.
      Mitochondrial energy production is crucial for normal daily activities and maintenance of life. Herein, the logic and execution of two main classes of measurements are outlined to delineate mitochondrial function: ATP production and oxygen consumption. Aerobic ATP production is quantified by phosphorus magnetic resonance spectroscopy (31PMRS) in vivo in both human subjects and animal models using the same protocols and maintaining the same primary assumptions. Mitochondrial oxygen consumption is quantified by oxygen polarography and applied in isolated mitochondria, cultured cells, and permeabilized fibers derived from human or animal tissue biopsies. Traditionally, mitochondrial functional measures focus on maximal oxidative capacity-a flux rate that is rarely, if ever, observed outside of experimental conditions. Perhaps more physiologically relevant, both measurement classes herein focus on one principal design paradigm; submaximal mitochondrial fluxes generated by graded levels of ADP to map the function for ADP sensitivity. We propose this function defines the bioenergetic role that mitochondria fill within the myoplasm to sense and match ATP demands. Any deficit in this vital role for ATP homeostasis leads to symptoms often seen in cardiovascular and cardiopulmonary diseases, diabetes, and metabolic syndrome.
    Keywords:  ADP sensitivity; Aerobic metabolism; Bioenergetics; Free energy homeostasis; Magnetic resonance; Oxygen consumption
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_10
  29. Methods Mol Biol. 2022 ;2497 333-337
    A Plate Reader-Based Measurement of the Cellular ROS Production Using Dihydroethidium and MitoSOX.
    Chih-Yao Chung, Michael R Duchen.
      Intracellular reactive oxygen species (ROS) act as an important signaling transductor in cells, regulating almost every aspect of cell biology. Measurements of ROS production thus, offer links between oxidative stress and cell pathophysiology. Here, we describe a simple screening assay in intact adherent cells by fluorescence microplate readers, using dihydroethidium (DHE) and MitoSOX to measure cytosolic superoxide and mitochondrial superoxide production, respectively. This assay enables a quick and reliable assessment of ROS generation in a well-controlled environment.
    Keywords:  Dihydroethidium; MitoSOX; Mitochondria; Plate reader; Reactive oxygen species
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_24
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