bims-celmim Biomed News
on Cellular and mitochondrial metabolism
Issue of 2024–10–27
fourteen papers selected by
Marc Segarra Mondejar
  1. Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry.
  2. Mitostasis in age-associated neurodegeneration.
  3. Glycolysis: A multifaceted metabolic pathway and signalling hub.
  4. CENCAT enables immunometabolic profiling by measuring protein synthesis via bioorthogonal noncanonical amino acid tagging.
  5. Mitoception, or transfer of normal cell mitochondria to cancer cells, reverses remodeling of store-operated Ca2+ entry in tumor cells.
  6. Glucose impacts onto the reciprocal reprogramming between mammary adipocytes and cancer cells.
  7. Synthetic syntrophy for adenine nucleotide cross-feeding between metabolically active nanoreactors.
  8. Mitochondrial Transfer Via Tunneling Nanotubes Between Mesenchymal Stem Cells and Retinal Pigment Epithelium In Vitro.
  9. Interplay between de novo and salvage pathways of GDP-fucose synthesis.
  10. High-throughput identification of calcium-regulated proteins across diverse proteomes.
  11. Fluorescent Reporters, Imaging, and Artificial Intelligence Toolkits to Monitor and Quantify Autophagy, Heterophagy, and Lysosomal Trafficking Fluxes.
  12. Recent Developments in Single-Cell Metabolomics by Mass Spectrometry─A Perspective.
  13. Golgiphagy: a novel selective autophagy to the fore.
  14. Imaging interorganelle contacts at a glance.
  1. J Vis Exp. 2024 Oct 04.
    Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry.
    Terri A Pietka, Rita T Brookheart.
      Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.
    DOI:  https://doi.org/10.3791/66834
  2. Biochim Biophys Acta Mol Basis Dis. 2024 Oct 20. pii: S0925-4439(24)00541-6. [Epub ahead of print] 167547
    Mitostasis in age-associated neurodegeneration.
    Mrutyunjaya Panda, Maria Markaki, Nektarios Tavernarakis.
      Mitochondria are essential organelles that play crucial roles in various metabolic and signalling pathways. Proper neuronal function is highly dependent on the health of these organelles. Of note, the intricate structure of neurons poses a critical challenge for the transport and distribution of mitochondria to specific energy-intensive domains, such as synapses and dendritic appendages. When faced with chronic metabolic challenges and bioenergetic deficits, neurons undergo degeneration. Unsurprisingly, disruption of mitostasis, the process of maintaining cellular mitochondrial content and function within physiological limits, has been implicated in the pathogenesis of several age-associated neurodegenerative disorders. Indeed, compromised integrity and metabolic activity of mitochondria is a principal hallmark of neurodegeneration. In this review, we survey recent findings elucidating the role of impaired mitochondrial homeostasis and metabolism in the onset and progression of age-related neurodegenerative disorders. We also discuss the importance of neuronal mitostasis, with an emphasis on the major mitochondrial homeostatic and metabolic pathways that contribute to the proper functioning of neurons. A comprehensive delineation of these pathways is crucial for the development of early diagnostic and intervention approaches against neurodegeneration.
    Keywords:  Age-associated neurodegenerative disorders; Metabolism; Mitochondria; Mitostasis; Neurodegeneration; Neurodegenerative diseases
    DOI:  https://doi.org/10.1016/j.bbadis.2024.167547
  3. J Biol Chem. 2024 Oct 21. pii: S0021-9258(24)02408-6. [Epub ahead of print] 107906
    Glycolysis: A multifaceted metabolic pathway and signalling hub.
    Sarah J Kierans, Cormac T Taylor.
      Glycolysis is a highly conserved metabolic pathway responsible for the anaerobic production of adenosine triphosphate (ATP) from the breakdown of glucose molecules. While serving as a primary metabolic pathway in prokaryotes, glycolysis is also utilised by respiring eukaryotic cells, providing pyruvate to fuel oxidative metabolism. Furthermore, glycolysis is the primary source of ATP production in multiple cellular states (e.g. hypoxia) and is particularly important in maintaining bioenergetic homeostasis in the most abundant cell type in the human body, the erythrocyte. Beyond its role in ATP production, glycolysis also functions as a signalling hub, producing several metabolic intermediates which serve roles in both signalling and metabolic processes. These signals emanating from the glycolytic pathway can profoundly impact cell function, phenotype and fate, and have previously been overlooked. In this review, we will discuss the role of the glycolytic pathway as a source of signalling molecules in eukaryotic cells, emphasising the newfound recognition of glycolysis' multifaceted nature and its importance in maintaining cellular homeostasis, beyond its traditional role in ATP synthesis.
    DOI:  https://doi.org/10.1016/j.jbc.2024.107906
  4. Cell Rep Methods. 2024 Oct 21. pii: S2667-2375(24)00266-2. [Epub ahead of print]4(10): 100883
    CENCAT enables immunometabolic profiling by measuring protein synthesis via bioorthogonal noncanonical amino acid tagging.
    Frank Vrieling, Hendrik J P van der Zande, Britta Naus, Lisa Smeehuijzen, Julia I P van Heck, Bob J Ignacio, Kimberly M Bonger, Jan Van den Bossche, Sander Kersten, Rinke Stienstra.
      Cellular energy metabolism significantly contributes to immune cell function. To further advance immunometabolic research, novel methods to study the metabolism of immune cells in complex samples are required. Here, we introduce CENCAT (cellular energetics through noncanonical amino acid tagging). This technique utilizes click labeling of alkyne-bearing noncanonical amino acids to measure protein synthesis inhibition as a proxy for metabolic activity. CENCAT successfully reproduced known metabolic signatures of lipopolysaccharide (LPS)/interferon (IFN)γ and interleukin (IL)-4 activation in human primary macrophages. Application of CENCAT in peripheral blood mononuclear cells revealed diverse metabolic rewiring upon stimulation with different activators. Finally, CENCAT was used to analyze the cellular metabolism of murine tissue-resident immune cells from various organs. Tissue-specific clustering was observed based on metabolic profiles, likely driven by microenvironmental priming. In conclusion, CENCAT offers valuable insights into immune cell metabolic responses, presenting a powerful platform for studying cellular metabolism in complex samples and tissues in both humans and mice.
    Keywords:  CP: Immunology; CP: Metabolism; OXPHOS; SCENITH; energy metabolism; glycolysis; immunometabolism
    DOI:  https://doi.org/10.1016/j.crmeth.2024.100883
  5. Biochim Biophys Acta Mol Cell Res. 2024 Oct 20. pii: S0167-4889(24)00205-2. [Epub ahead of print]1872(1): 119862
    Mitoception, or transfer of normal cell mitochondria to cancer cells, reverses remodeling of store-operated Ca2+ entry in tumor cells.
    Verónica Feijóo, Sendoa Tajada, Alejandra Méndez-Mena, Lucía Núñez, Carlos Villalobos.
      Most cancer cells show the Warburg effect, the rewiring of aerobic metabolism to glycolysis due to defective mitochondrial ATP synthesis. As a consequence, tumor cells display enhanced mitochondrial potential (∆Ψ), the driving force for mitochondrial Ca2+ uptake. Mitochondria control the Ca2+-dependent inactivation of store-operated channels (SOCs), leading to enhanced and sustained store-operated Ca2+ entry (SOCE) involved in cancer hallmarks. We asked here whether the transfer of mitochondria (mitoception) from normal cells to tumor cells may reverse SOCE remodeling in cancer cells. For this end, we labeled mitochondria in normal NCM460 human colonic cells, isolated them and transferred them to tumor HT29 cells. We tested the viability and efficiency of mitoception using flow cytometry and confocal microscopy, as well as calcium imaging to investigate the effects of mitoception on SOCE. Our results show that mitoception of tumor HT29 cells with normal mitochondria restores a low ∆Ψ and SOCE. Conversely, self-mitoception of tumor HT29 cells with tumor cell mitochondria increases further ∆Ψ and SOCE, thus excluding the possibility that effects of mitoception are due to increased mitochondrial mass. Strikingly, mitoception of normal NCM460 cells with tumor cell mitochondria has no effects on either ∆Ψ or SOCE. These results are consistent with the previous proposal that transformed mitochondria may modulate SOC channels involved in SOCE. Further research is warranted to test whether mitoception of cancer cells with normal mitochondria may reverse Ca2+ remodeling associated to cancer.
    Keywords:  Colon cancer; Mitoception; Mitochondria; Store-operated Ca(2+) entry
    DOI:  https://doi.org/10.1016/j.bbamcr.2024.119862
  6. Sci Rep. 2024 10 21. 14(1): 24674
    Glucose impacts onto the reciprocal reprogramming between mammary adipocytes and cancer cells.
    Maria Rosaria Ambrosio, Michiel Adriaens, Kasper Derks, Teresa Migliaccio, Valerio Costa, Domenico Liguoro, Simona Cataldi, Vittoria D'Esposito, Giovanni Maneli, Rita Bassolino, Simone Di Paola, Marinella Pirozzi, Fabrizio Schonauer, Francesco D'Andrea, Francesco Beguinot, Ilja Arts, Pietro Formisano.
      An established hallmark of cancer cells is metabolic reprogramming, largely consisting in the exacerbated glucose uptake. Adipocytes in the tumor microenvironment contribute toward breast cancer (BC) progression and are highly responsive to metabolic fluctuations. Metabolic conditions characterizing obesity and/or diabetes associate with increased BC incidence and mortality. To explore BC-adipocytes interaction and define the impact of glucose in such dialogue, Mammary Adipose-derived Mesenchymal Stem Cells (MAd-MSCs) were differentiated into adipocytes and co-cultured with ER+ BC cells while exposed to glucose concentration resembling hyperglycemia or normoglycemia in humans (25mM or 5.5mM). The transcriptome of both cell types in co-culture as in mono-culture was profiled by RNA-Seq to define the impact of adipocytes on BC cells and viceversa (i), the action of glucose on BC cells, adipocytes (ii) and their crosstalk (iii). Noteworthy, we provided evidence that co-culture with adipocytes in a glucose-rich environment determined a re-program of BC cell transcriptome driving lipid accumulation, a hallmark of BC aggressiveness, promoting stem-like properties and reducing Tamoxifen responsiveness. Moreover, our data point out to a transcriptional effect through which BC cells induce adipocytes de-lipidation, paralleled by pluripotency gain, as source of lipids when glucose lowering occurs. Thus, modulating plasticity of peri-tumoral adipocytes may represent a key point for halting BC progression in metabolically unbalanced patients.
    Keywords:  Adipogenesis; Breast Cancer; Glucose; Mammary Adipocytes; Transcriptional signatures; Tumor Microenvironment
    DOI:  https://doi.org/10.1038/s41598-024-76522-7
  7. Nat Nanotechnol. 2024 Oct 21.
    Synthetic syntrophy for adenine nucleotide cross-feeding between metabolically active nanoreactors.
    Laura Heinen, Marco van den Noort, Martin S King, Edmund R S Kunji, Bert Poolman.
      Living systems depend on continuous energy input for growth, replication and information processing. Cells use membrane proteins as nanomachines to convert light or chemical energy of nutrients into other forms of energy, such as ion gradients or adenosine triphosphate (ATP). However, engineering sustained fuel supply and metabolic energy conversion in synthetic systems is challenging. Here, inspired by endosymbionts that rely on the host cell for their nutrients, we introduce the concept of cross-feeding to exchange ATP and ADP between lipid-based compartments hundreds of nanometres in size. One population of vesicles enzymatically produces ATP in the mM concentration range and exports it. A second population of vesicles takes up this ATP to fuel internal reactions. The produced ADP feeds back to the first vesicles, and ATP-dependent reactions can be fuelled sustainably for up to at least 24 h. The vesicles are a platform technology to fuel ATP-dependent processes in a sustained fashion, with potential applications in synthetic cells and nanoreactors. Fundamentally, the vesicles enable studying non-equilibrium processes in an energy-controlled environment and promote the development and understanding of constructing life-like metabolic systems on the nanoscale.
    DOI:  https://doi.org/10.1038/s41565-024-01811-1
  8. J Vis Exp. 2024 Oct 04.
    Mitochondrial Transfer Via Tunneling Nanotubes Between Mesenchymal Stem Cells and Retinal Pigment Epithelium In Vitro.
    Jing Yuan, Zi-Bing Jin.
      Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods. We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.
    DOI:  https://doi.org/10.3791/66917
  9. PLoS One. 2024 ;19(10): e0309450
    Interplay between de novo and salvage pathways of GDP-fucose synthesis.
    Edyta Skurska, Mariusz Olczak.
      GDP-fucose is synthesised via two pathways: de novo and salvage. The first uses GDP-mannose as a substrate, and the second uses free fucose. To date, these pathways have been considered to work separately and not to have an influence on each other. We report the mutual response of the de novo and salvage pathways to the lack of enzymes from a particular route of GDP-fucose synthesis. We detected different efficiencies of GDP-fucose and fucosylated structure synthesis after a single inactivation of enzymes of the de novo pathway. Our study demonstrated the unequal influence of the salvage enzymes on the production of GDP-fucose by enzymes of the de novo biosynthesis pathway. Simultaneously, we detected an elevated level of one of the enzymes of the de novo pathway in the cell line lacking the enzyme of the salvage biosynthesis pathway. Additionally, we identified dissimilarities in fucose uptake between cells lacking TSTA3 and GMDS proteins.
    DOI:  https://doi.org/10.1371/journal.pone.0309450
  10. Cell Rep. 2024 Oct 18. pii: S2211-1247(24)01230-0. [Epub ahead of print]43(11): 114879
    High-throughput identification of calcium-regulated proteins across diverse proteomes.
    Timothy M Locke, Rose Fields, Hayden Gizinski, George M Otto, Melissa J S MacEwen, Domnita-Valeria Rusnac, Peixian He, David M Shechner, Chris D McGann, Matthew D Berg, Judit Villen, Yasemin Sancak, Devin K Schweppe.
      Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by a lack of high-throughput, unbiased, and quantitative methods to identify protein-calcium engagement. To address this, we adapted protein thermostability assays in budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2,884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-coenzyme A (CoA) reductase DECR1 binds calcium at physiologically relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These discovery-based proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.
    Keywords:  CP: Cell biology; CP: Metabolism; TMTpro; calcium engagement; calcium ion engagement; calcium regulated proteins; cell signaling; magnesium engagement; oxidation of polyunsaturated fatty acids; sample multiplexed quantitation; thermal stability proteomics
    DOI:  https://doi.org/10.1016/j.celrep.2024.114879
  11. Traffic. 2024 Oct;25(10): e12957
    Fluorescent Reporters, Imaging, and Artificial Intelligence Toolkits to Monitor and Quantify Autophagy, Heterophagy, and Lysosomal Trafficking Fluxes.
    Mikhail Rudinskiy, Diego Morone, Maurizio Molinari.
      Lysosomal compartments control the clearance of cell-own material (autophagy) or of material that cells endocytose from the external environment (heterophagy) to warrant supply of nutrients, to eliminate macromolecules or parts of organelles present in excess, aged, or containing toxic material. Inherited or sporadic mutations in lysosomal proteins and enzymes may hamper their folding in the endoplasmic reticulum (ER) and their lysosomal transport via the Golgi compartment, resulting in lysosomal dysfunction and storage disorders. Defective cargo delivery to lysosomal compartments is harmful to cells and organs since it causes accumulation of toxic compounds and defective organellar homeostasis. Assessment of resident proteins and cargo fluxes to the lysosomal compartments is crucial for the mechanistic dissection of intracellular transport and catabolic events. It might be combined with high-throughput screenings to identify cellular, chemical, or pharmacological modulators of these events that may find therapeutic use for autophagy-related and lysosomal storage disorders. Here, discuss qualitative, quantitative and chronologic monitoring of autophagic, heterophagic and lysosomal protein trafficking in fixed and live cells, which relies on fluorescent single and tandem reporters used in combination with biochemical, flow cytometry, light and electron microscopy approaches implemented by artificial intelligence-based technology.
    Keywords:  ER‐phagy; ER‐to‐lysosome‐associated degradation (ERLAD); artificial intelligence; autophagy; autophagy flux; endolysosomes (EL); heterophagy; lysosomal storage disorders (LSD); lysosomes; tandem fluorescent reporters
    DOI:  https://doi.org/10.1111/tra.12957
  12. J Proteome Res. 2024 Oct 22.
    Recent Developments in Single-Cell Metabolomics by Mass Spectrometry─A Perspective.
    Boryana Petrova, Arzu Tugce Guler.
      Recent advancements in single-cell (sc) resolution analyses, particularly in sc transcriptomics and sc proteomics, have revolutionized our ability to probe and understand cellular heterogeneity. The study of metabolism through small molecules, metabolomics, provides an additional level of information otherwise unattainable by transcriptomics or proteomics by shedding light on the metabolic pathways that translate gene expression into functional outcomes. Metabolic heterogeneity, critical in health and disease, impacts developmental outcomes, disease progression, and treatment responses. However, dedicated approaches probing the sc metabolome have not reached the maturity of other sc omics technologies. Over the past decade, innovations in sc metabolomics have addressed some of the practical limitations, including cell isolation, signal sensitivity, and throughput. To fully exploit their potential in biological research, however, remaining challenges must be thoroughly addressed. Additionally, integrating sc metabolomics with orthogonal sc techniques will be required to validate relevant results and gain systems-level understanding. This perspective offers a broad-stroke overview of recent mass spectrometry (MS)-based sc metabolomics advancements, focusing on ongoing challenges from a biologist's viewpoint, aimed at addressing pertinent and innovative biological questions. Additionally, we emphasize the use of orthogonal approaches and showcase biological systems that these sophisticated methodologies are apt to explore.
    Keywords:  cellular heterogeneity; mass spectrometry; metabolic imaging; metabolomics; multiomics; single-cell
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00646
  13. Cell Biosci. 2024 Oct 22. 14(1): 130
    Golgiphagy: a novel selective autophagy to the fore.
    Yifei Chen, Yihui Wu, Xianyan Tian, Genbao Shao, Qiong Lin, Aiqin Sun.
      The Golgi apparatus is the central hub of the cellular endocrine pathway and plays a crucial role in processing, transporting, and sorting proteins and lipids. Simultaneously, it is a highly dynamic organelle susceptible to degradation or fragmentation under various physiological or pathological conditions, potentially contributing to the development of numerous human diseases. Autophagy serves as a vital pathway for eukaryotes to manage intracellular and extracellular stress and maintain homeostasis by targeting damaged or redundant organelles for removal. Recent research has revealed that autophagy mechanisms can specifically degrade Golgi components, known as Golgiphagy. This review summarizes recent findings on Golgiphagy while also addressing unanswered questions regarding its mechanisms and regulation, aiming to advance our understanding of the role of Golgiphagy in human disease.
    Keywords:  Autophagy; Golgi apparatus; Golgi fragmentation; Golgiphagy; Receptor
    DOI:  https://doi.org/10.1186/s13578-024-01311-8
  14. J Cell Sci. 2024 Oct 15. pii: jcs262020. [Epub ahead of print]137(20):
    Imaging interorganelle contacts at a glance.
    Maria Clara Zanellati, Chih-Hsuan Hsu, Sarah Cohen.
      Eukaryotic cells are compartmentalized into membrane-bound organelles that must coordinate their responses to stimuli. One way that organelles communicate is via membrane contact sites (MCSs), sites of close apposition between organelles used for the exchange of ions, lipids and information. In this Cell Science at a Glance article and the accompanying poster, we describe an explosion of new methods that have led to exciting progress in this area and discuss key examples of how these methods have advanced our understanding of MCSs. We discuss how diffraction-limited and super-resolution fluorescence imaging approaches have provided important insight into the biology of interorganelle communication. We also describe how the development of multiple proximity-based methods has enabled the detection of MCSs with high accuracy and precision. Finally, we assess how recent advances in electron microscopy (EM), considered the gold standard for detecting MCSs, have allowed the visualization of MCSs and associated proteins in 3D at ever greater resolution.
    Keywords:  Biosensors; Electron microscopy; Light microscopy; Membrane contact sites; Organelles; Super-resolution microscopy
    DOI:  https://doi.org/10.1242/jcs.262020
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