bims-celmim Biomed News
on Cellular and mitochondrial metabolism
Issue of 2025–10–19
twenty-six papers selected by
Marc Segarra Mondejar, AINA
  1. Analyzing Endolysosome-Mitochondria Membrane Contact Sites by Thin Section TEM of Human Fibroblasts.
  2. Glioma neuron symbiosis: a hypothesis.
  3. SLC25A45 is required for mitochondrial uptake of methylated amino acids and de novo carnitine biosynthesis.
  4. Nutrient allocation fuels T cell-mediated immunity.
  5. Power and Poison: The intersections of H2S and O2 metabolism.
  6. The role of fatty acid oxidation in the tumor microenvironment: Implications for cancer progression and therapeutic strategies.
  7. Differential bioenergetic profile of human glioblastoma following transplantation of myocyte-derived mitochondria.
  8. Visualization of oxygen profile in reconstructed human epidermis by phosphorescence-lifetime imaging microscopy using Ir(III) complex.
  9. Monitoring Autophagosome Biogenesis and Degradation in Mammalian Cells Using LC3B Blotting.
  10. Metabolic Dependency on De Novo Pyrimidine Synthesis is a Targetable Vulnerability in Platinum Resistant Ovarian Cancer.
  11. Aged mice exhibit widespread metabolic changes but preserved major fluxes.
  12. Metabolites in the extracellular tumor microenvironment and the shaping of macrophage function.
  13. Machine-learning-guided discovery of SLC25A45 as a mediator of mitochondrial methylated amino acid import and carnitine synthesis.
  14. Exploiting metabolic cell death for cancer therapy.
  15. Succinate in innate immunity: linking metabolic reprogramming to immune modulation.
  16. Label-Free Imaging of Lysosomes in Living Cells by Photothermal Microscopy.
  17. Global conformation of the Rag GTPase heterodimer governs eukaryotic amino acid sensing.
  18. Lysosomal Immunoprecipitation (LysoIP) from Cells and Tissues for Metabolomic and Proteomic Analyses.
  19. Backtracking metabolic dynamics in single cells predicts bacterial replication in human macrophages.
  20. Visualization of lysosomal membrane proteins by cryo electron tomography.
  21. Detection methods for mitochondrial DNA heteroplasmy and their potential single-cell applications in cancer.
  22. HIFα isoform specific activities drive cell-type specificity of VHL-associated oncogenesis.
  23. MAPL regulates gasdermin-mediated release of mtDNA from lysosomes to drive pyroptotic cell death.
  24. Spatial metabolic gradients in the liver and small intestine.
  25. Determining Lysosomal Enzyme Activity Using Fluorogenic Probes.
  26. Methods for Assessing MAGL Enzymatic Activity: An Extensive Review of Past and Emerging Approaches.
  1. Methods Mol Biol. 2026 ;2976 119-134
    Analyzing Endolysosome-Mitochondria Membrane Contact Sites by Thin Section TEM of Human Fibroblasts.
    Claudia Parra-Ruiz, Carlos Enrich, Silvana Zanlungo.
      Membrane contact sites (MCS) are dynamic nanoregions of close apposition between two different organelles, functioning as discrete lipid or ion transfer sites. This new concept in cell biology involves unique proteins at both membrane sites, named tethers, and emerges in early observations by transmission electron microscopy (TEM). Currently, this technique still constitutes a valuable tool for MCS visualization and quantification. In the last decade, Lysosomal Storage Diseases (LSD) have been instrumental in studying the MCS between lysosomes (Ly), or endolysosomes (EL), and other organelles in close proximity such as mitochondria or the endoplasmic reticulum (ER). At present, the analysis of composition, functioning, and alterations/rewiring of MCS in health and disease represents an innovative area of research for designing therapeutic strategies in a variety of pathologies. Here, we describe procedures for chemical fixation using the Flat Embedding technique to characterize and quantify the MCS between LE/Lys and mitochondria in human fibroblasts by thin-section TEM.
    Keywords:  Flat embedding; Image analysis; Lysosomal storage diseases; Lysosomes; Membrane contact sites; Mitochondria; Transmission electron microscopy-chemical fixation
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_10
  2. Front Neurosci. 2025 ;19 1646148
    Glioma neuron symbiosis: a hypothesis.
    Avital Schurr.
      Glioma cells, just like all cancerous cells, consume substantial amounts of glucose for their energy needs, using glycolysis, an inefficient metabolic pathway (Warburg effect) to produce only two moles of adenosine triphosphate and two moles of lactate for each mole of glucose consumed. By contrast, neurons consume glucose via glycolysis and utilize its end-product lactate as the substrate of the mitochondrial tricarboxylic acid cycle and its coupled oxidative phosphorylation, a process eighteen times more efficient at adenosine triphosphate than glycolysis alone. It hypothesizes here that glioma-produced lactate is the preferred oxidative energy substrate of their surrounding neurons. Consequently, by using lactate, neurons bypass glycolysis, sparing their glucose and making it readily available for the glucose-craving cancer cells. Moreover, glioma cells' ability to secrete glutamate, which excites glutamatergic neurons, could drive the latter to consume even more lactate, sparing more glucose. Such symbiotic exchange, especially at the initial stages of malignancy, assures the budding cancer cells an ample glucose supply ahead of the development of additional vasculature. While this hypothesis focuses on gliomas, it may also apply to other cancer types.
    Keywords:  cancer; energy metabolism; glioma; glucose; glycolysis; lactate; neuron; symbiosis
    DOI:  https://doi.org/10.3389/fnins.2025.1646148
  3. Mol Cell. 2025 Oct 10. pii: S1097-2765(25)00703-8. [Epub ahead of print]
    SLC25A45 is required for mitochondrial uptake of methylated amino acids and de novo carnitine biosynthesis.
    Marilia M Dias, Martin S King, Engy Shokry, Sergio Lilla, Nikki Paul, Peter Thomason, Sara Zanivan, David Sumpton, Edmund R S Kunji, Thomas MacVicar.
      Methylated amino acids accumulate upon the degradation of methylated proteins and are implicated in diverse metabolic and signaling pathways. Disturbed methylated amino acid homeostasis is associated with cardiovascular disease and renal failure. Mitochondria are core processing hubs in conventional amino acid metabolism, but how they interact with methylated amino acids is unclear. Here, we reveal that the orphan mitochondrial solute carrier 25A45 (SLC25A45) is required for the mitochondrial uptake of methylated amino acids. SLC25A45 binds with dimethylarginine and trimethyllysine but has no affinity for unmethylated arginine and lysine. A non-synonymous mutation of human SLC25A45 (R285C) stabilizes the carrier by limiting its proteolytic degradation and associates with altered methylated amino acids in human plasma. Metabolic tracing of trimethyllysine in cancer cells demonstrates that SLC25A45 drives the biosynthesis of the key amino acid derivative, carnitine. SLC25A45 is therefore an essential mediator of compartmentalized methylated amino acid metabolism.
    Keywords:  SLC25; carnitine; metabolism; metabolite transport; methylated amino acids; mitochondria; solute carriers
    DOI:  https://doi.org/10.1016/j.molcel.2025.08.018
  4. Cell Metab. 2025 Oct 15. pii: S1550-4131(25)00393-6. [Epub ahead of print]
    Nutrient allocation fuels T cell-mediated immunity.
    Joseph Longo, McLane J Watson, Kelsey S Williams, Ryan D Sheldon, Russell G Jones.
      T cell activation and function are intricately linked to metabolic reprogramming. The classic view of T cell metabolic reprogramming centers on glucose as the dominant bioenergetic fuel, where T cell receptor (TCR) stimulation promotes a metabolic switch from relying primarily on oxidative phosphorylation (OXPHOS) for energy production to aerobic glycolysis (i.e., the Warburg effect). More recently, studies have revealed this classic model to be overly simplistic. Activated T cells run both glycolysis and OXPHOS programs concurrently, allocating diverse nutrient sources toward distinct biosynthetic and bioenergetic fates. Moreover, studies of T cell metabolism in vivo and ex vivo highlight that physiologic nutrient availability influences how glucose is allocated by T cells to fuel both optimal proliferation and effector function. Here, we summarize recent advancements that support a revised model of effector T cell metabolism, where strategic nutrient allocation fuels optimal T cell-mediated immunity.
    Keywords:  T cells; adaptive immunity; effector function; glucose; immunometabolism; nutrient allocation
    DOI:  https://doi.org/10.1016/j.cmet.2025.09.008
  5. J Biol Chem. 2025 Oct 13. pii: S0021-9258(25)02662-6. [Epub ahead of print] 110810
    Power and Poison: The intersections of H2S and O2 metabolism.
    Joseph Brake, Ruma Banerjee.
      The metabolic interaction between hydrogen sulfide (H2S) and O2 exemplifies the interplay between chemical power and poison at the electron transport chain as these gases influence the conversion of nutrient energy to cellular currency. H2S is a product of mammalian and microbial metabolism and is both an inorganic nutrient and a respiratory poison. In its former role, H2S transfers its reducing power to coenzyme Q as it is oxidized by sulfide quinone oxidoreductase in the inner mitochondrial membrane. As a respiratory poison, H2S inhibits complex IV and profoundly influences intracellular O2 levels with pleiotropic effects on hypoxia sensing and signaling, and on cellular metabolism, glimpses of which are only just beginning to emerge. The high concentration of luminal sulfide in the lower gut combined with the steep radial O2 gradient, ranging from a virtually anoxic lumen to a highly vascular lamina propria, raises many questions about how the interaction between these gases plays out with local and long-range impacts on biology. Their interaction is equally germane in other hypoxic tissues where endogenous H2S production and/or constitutively low sulfide oxidation capacity could potentially dial up O2 availability. Importantly, H2S oxidation can prevail even when its concentration rises to levels that poison complex IV, and is enabled by rerouting electrons through complex II, using fumarate as a terminal electron acceptor. Methodological advancements that support the quantitative analysis of in vivo models will be critical for broadening our understanding of the metabolic and physiological import of the O2-H2S interplay.
    Keywords:  Hydrogen sulfide; butyrate; fumarate; gut dysbiosis; gut microbiome; hypoxia; hypoxia inducible factor; oxygen metabolism
    DOI:  https://doi.org/10.1016/j.jbc.2025.110810
  6. Biochim Biophys Acta Rev Cancer. 2025 Oct 10. pii: S0304-419X(25)00216-1. [Epub ahead of print]1880(6): 189474
    The role of fatty acid oxidation in the tumor microenvironment: Implications for cancer progression and therapeutic strategies.
    Nasot Rashed, Wenbin Liu, Xiangjian Luo.
      Fatty acid oxidation (FAO), or β-oxidation, is a catabolic process that breaks down fatty acids into acetyl-CoA. FAO plays a pivotal role in the metabolic reprogramming of cancer cells and the tumor microenvironment (TME), serving as a crucial energy source that sustains cellular functions under conditions of nutrient deprivation and metabolic stress. This process significantly influences cancer cell survival, proliferation, metastasis, and therapeutic resistance. In this review, we discuss the biological functions of FAO in cancer cells, immune cells, and stromal cells, with a particular focus on its regulatory role in tumor progression and therapy resistance. Furthermore, we explore FAO inhibitors and emerging therapeutic strategies targeting FAO as a potential approach to disrupting tumor metabolism and enhancing cancer treatment efficacy.
    Keywords:  Cancer progression; Fatty acid oxidation; Immune cells; Stromal cells; Therapeutic resistance; Tumor microenvironment
    DOI:  https://doi.org/10.1016/j.bbcan.2025.189474
  7. PLoS One. 2025 ;20(10): e0330322
    Differential bioenergetic profile of human glioblastoma following transplantation of myocyte-derived mitochondria.
    Kent L Marshall, Ethan Meadows, Alan Mizener, John M Hollander, Christopher P Cifarelli.
      Glioblastoma (GBM) exhibits profound plasticity, enabling adaptation to fluctuating microenvironmental stressors such as hypoxia and nutrient deprivation. However, this metabolic rewiring also creates subtype-specific vulnerabilities that may be exploited therapeutically. Here, we investigate whether mitochondrial transplantation using non-neoplastic, human myocyte-derived mitochondria alters the metabolic architecture of GBM cells and modulates their response to ionizing radiation. Using a cell-penetrating peptide-mediated delivery system, we successfully introduced mitochondria into two mesenchymal-subtype GBM cell lines, U3035 and U3046. Transplanted cells exhibited enhanced mitochondrial polarization and respiratory function, particularly in the metabolically flexible U3035 line. Bioenergetic profiling revealed significant increases in basal respiration, spare respiratory capacity, and glycolytic reserve in U3035 cells post-transplantation, whereas U3046 cells showed minimal bioenergetic augmentation. Transcriptomic analyses using oxidative phosphorylation (OXPHOS) and glycolysis gene sets confirmed these functional findings. At baseline, U3035 cells expressed high levels of both glycolytic and OXPHOS genes, while U3046 cells were metabolically suppressed. Following radiation, U3035 cells downregulated key OXPHOS and glycolysis genes, suggesting metabolic collapse. In contrast, U3046 cells transcriptionally upregulated both pathways, indicating compensatory adaptation. These results identify and establish mitochondrial transplantation as a metabolic priming strategy that sensitizes adaptable GBM subtypes like U3035 to therapeutic stress by inducing bioenergetic overextension. Conversely, rigid subtypes like U3046 may require inhibition of post-radiation metabolic compensation for effective targeting. Our findings support a novel stratified approach to GBM treatment which integrates metabolic subtype profiling with bioenergetic modulation.
    DOI:  https://doi.org/10.1371/journal.pone.0330322
  8. Sci Rep. 2025 Oct 15. 15(1): 36008
    Visualization of oxygen profile in reconstructed human epidermis by phosphorescence-lifetime imaging microscopy using Ir(III) complex.
    Misaki Ochiai-Noguchi, Aoi Horikoshi, Seina Hirakata, Toshitada Yoshihara.
      Reconstructed human epidermis (RHE) is a useful experimental tool for evaluating the effects of various stimuli on the skin. Here, we present a method to visualize the partial pressure of oxygen in RHE with cellular-level spatial resolution by means of confocal phosphorescence-lifetime imaging microscopy (PLIM) with a cell-permeable phosphorescent probe, BTPDM1 (an iridium-based cationic lipophilic dye). Z-stack PLIM images of RHE revealed an oxygen partial pressure gradient in the direction of differentiation, with a decrease in oxygen levels from the basal layer to the cornified layer. FCCP (carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone), an uncoupler of mitochondrial oxidative phosphorylation, significantly decreased pO₂, suggesting that changes in mitochondrial respiration may contribute to the O₂ concentration gradient in RHE. Antimycin A (Ant A), an inhibitor of mitochondrial respiration, decreased expression of the differentiation markers filaggrin and loricrin, indicating that mitochondrial respiration is essential for normal epidermal maturation. In 2D keratinocyte cultures, addition of calcium as a differentiation inducer led to an increase in mitochondrial oxygen consumption and oxidative phosphorylation. These results indicate that oxygen imaging is an effective method for evaluating not only mitochondrial respiration status, but also the differentiation state of reconstructed human epidermis.
    Keywords:  Differentiation; Iridium(III) complex; Keratinocyte; Oxygen; Phosphorescence lifetime; Skin
    DOI:  https://doi.org/10.1038/s41598-025-19891-x
  9. Methods Mol Biol. 2026 ;2976 47-60
    Monitoring Autophagosome Biogenesis and Degradation in Mammalian Cells Using LC3B Blotting.
    Jennifer E Palmer, David C Rubinsztein.
      Autophagy is a conserved lysosomal degradation pathway that recycles protein aggregates and damaged organelles to maintain cytoplasmic quality control. Measuring the amount of the lipid-conjugated autophagic protein LC3B-II is a useful way to test whether a particular perturbation affects autophagy. However, the level of LC3B-II is affected by factors that alter either the rate of autophagosome biogenesis or degradation. Consequently, the same steady-state LC3B-II level can be reached by opposing autophagic fluxes. It is thus essential when measuring LC3B-II to perform the assay both in the absence and presence of a lysosomal inhibitor, enabling measurement of the rate of synthesis independent of its degradation. LC3B-II is also a small protein that can be challenging to detect by western blotting. In this chapter, we will provide a method for the efficient western blotting of LC3B-II and guidance as to the interpretation of the results.
    Keywords:  ATG8; Autophagy; Bafilomycin A1; LC3B; SDS-PAGE; Western blotting
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_5
  10. Cancer Res. 2025 Oct 15.
    Metabolic Dependency on De Novo Pyrimidine Synthesis is a Targetable Vulnerability in Platinum Resistant Ovarian Cancer.
    Horacio Cardenas, Yinu Wang, Guangyuan Zhao, Delan Xingyue Hao, Ana Maria Isac, Vanessa Hernandez, Ujin Kim, Wenan Qiang, Hao F Zhang, Daniela Matei.
      Ovarian cancer (OC) is lethal due to near universal development of resistance to platinum-based chemotherapy. Metabolic adaptations can play a pivotal role in therapy resistance. Here, we aimed to identify key metabolic pathways that regulate platinum response and represent potential therapeutic targets. Transcriptomic and metabolomic analyses in cisplatin sensitive and resistant ovarian cancer cells identified enrichment of pyrimidine metabolism related to upregulated de novo pyrimidine synthesis. 15N-glutamine flux analysis confirmed increased de novo pyrimidine synthesis in cisplatin resistant cells. Targeting this pathway using brequinar (BRQ), an inhibitor of the key enzyme dihydroorotate dehydrogenase (DHODH), decreased cell viability, delayed G2/M cell cycle progression, and altered expression of genes related to mitochondrial electron transport in resistant cells. Under basal conditions, cisplatin resistant cells had a lower oxygen consumption rate (OCR) and spare respiratory capacity (SRC) than sensitive cells. BRQ suppressed OCR in both sensitive and resistant but only inhibited SRC in resistant cells. In cell line-derived and patient-derived xenograft models, BRQ attenuated the growth of cisplatin resistant ovarian tumors and enhanced the inhibitory effects of carboplatin. Together, these results identify metabolic reprogramming in cisplatin resistant ovarian cancer that induces an acquired dependency on de novo pyrimidine synthesis, which can be targeted to sensitize tumors to chemotherapy.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-25-0043
  11. Cell Metab. 2025 Oct 16. pii: S1550-4131(25)00394-8. [Epub ahead of print]
    Aged mice exhibit widespread metabolic changes but preserved major fluxes.
    Connor S R Jankowski, Laith Z Samarah, Michael R MacArthur, Sarah J Mitchell, Daniel R Weilandt, Craig J Hunter, Xianfeng Zeng, Melanie R McReynolds, Joshua D Rabinowitz.
      Metabolic dysregulation is a hallmark of aging. Here, we investigate in mice age-induced metabolic alterations using metabolomics and stable isotope tracing. Circulating metabolite fluxes and serum and tissue concentrations were measured in young and old (20-30 months) C57BL/6J mice, with young obese (ob/ob) mice as a comparator. For major circulating metabolites, concentrations changed more with age than fluxes, and fluxes changed more with obesity than with aging. Specifically, glucose, lactate, 3-hydroxybutryate, and many amino acids (but notably not taurine) change significantly in concentration with age. Only glutamine circulatory flux does so. The fluxes of major circulating metabolites remain stable despite underlying metabolic changes. For example, lysine catabolism shifts from the saccharopine toward the pipecolic acid pathway, and both pipecolic acid concentration and flux increase with aging. Other less-abundant metabolites also show coherent, age-induced concentration and flux changes. Thus, while aging leads to widespread metabolic changes, major metabolic fluxes are largely preserved.
    Keywords:  aging; fluxomics; glutamine; metabolic flux; metabolism; metabolomics; obesity; stable isotope tracing; systemic metabolism
    DOI:  https://doi.org/10.1016/j.cmet.2025.09.009
  12. Mol Cell. 2025 Oct 16. pii: S1097-2765(25)00776-2. [Epub ahead of print]85(20): 3779-3792
    Metabolites in the extracellular tumor microenvironment and the shaping of macrophage function.
    Sara Lamorte, Matthew Bianca, Zhe Qi Liu, Tracy L McGaha.
      It has been a century since it was discovered that cancer cells have a distorted metabolism compared to healthy cells and tissues. It is now universally accepted that the abnormal metabolic state of cancers is essential for proliferation and survival in the harsh environment of most solid tumors. However, the impact of the altered metabolite pools generated from this rewiring is complex and has been challenging to functionally disentangle. Macrophages are innate immune cells and a major cellular constituent of the tumor microenvironment (TME). Macrophages are functionally plastic and highly sensitive to changes in metabolite exposure, with the potential to change the TME in a profound, disease-altering fashion. However, it was not until the recent advent of sensitive, high-dimensional analysis that the impact of metabolites on tumor macrophage diversity and function was fully appreciated. In this review, we discuss recent developments in our knowledge of how altered metabolites, resulting from metabolic reprogramming in the TME, influence macrophage phenotype and the implications for tumor development and progression. Furthermore, we examine emerging therapeutic strategies aimed at targeting tumor-metabolite crosstalk to improve disease outcomes.
    Keywords:  immunity; macrophage; metabolism; metabolites; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.molcel.2025.09.016
  13. Cell Metab. 2025 Oct 10. pii: S1550-4131(25)00434-6. [Epub ahead of print]
    Machine-learning-guided discovery of SLC25A45 as a mediator of mitochondrial methylated amino acid import and carnitine synthesis.
    Artem Khan, Frederick S Yen, Gokhan Unlu, Nicole L DelGaudio, Ranya Erdal, Michael Xiao, Khando Wangdu, Kevin Cho, Eric R Gamazon, Gary J Patti, Kıvanç Birsoy.
      Solute carriers (SLCs) regulate cellular and organismal metabolism by transporting small molecules and ions across membranes, yet the physiological substrates of ∼20% remain elusive. To address this, we developed a machine-learning platform to predict gene-metabolite associations. This approach identifies UNC93A and SLC45A4 as candidate plasma membrane transporters for acetylglucosamine and polyamines, respectively. Additionally, we uncover SLC25A45 as a mitochondrial transporter linked to serum levels of methylated basic amino acids, products of protein catabolism. Mechanistically, SLC25A45 is necessary for the mitochondrial import of methylated basic amino acids, including ADMA and TML, the latter serving as a precursor for carnitine synthesis. In line with this observation, SLC25A45 loss impairs carnitine synthesis and blunts upregulation of carnitine-containing metabolites under fasted conditions. By facilitating mitochondrial TML import, SLC25A45 connects protein catabolism to carnitine production, sustaining β-oxidation during fasting. Altogether, our study identifies putative substrates for three SLCs and provides a resource for transporter deorphanization.
    Keywords:  SLC25A45; SLC45A4; UNC93A; acetylglucosamine; carnitine synthesis; fasting; metabolomic GWAS; mitochondrial metabolism; polyamines; solute carrier transporters
    DOI:  https://doi.org/10.1016/j.cmet.2025.09.015
  14. Nat Rev Cancer. 2025 Oct 15.
    Exploiting metabolic cell death for cancer therapy.
    Chao Mao, Dadi Jiang, Albert C Koong, Boyi Gan.
      Resistance to cell death is a hallmark of cancer, driving tumour progression and limiting therapeutic efficacy. Metabolic cell death pathways have been identified as unique vulnerabilities in cancer, with ferroptosis being the most extensively studied, alongside the more recently discovered pathways of cuproptosis and disulfidptosis - each triggered by distinct metabolic perturbations. In this Review, we examine the molecular mechanisms and regulatory networks that govern these forms of metabolic cell death in cancer cells. We further examine the potential crosstalk between these pathways and discuss how insights gained and challenges encountered from extensive studies on ferroptosis can guide future research and therapeutic strategies targeting cuproptosis and disulfidptosis in cancer treatment. We highlight the complexity and dual roles of metabolic cell death in cancer and offer our perspective on how to leverage these cell death processes to develop innovative, targeted cancer therapies.
    DOI:  https://doi.org/10.1038/s41568-025-00879-8
  15. Front Immunol. 2025 ;16 1661948
    Succinate in innate immunity: linking metabolic reprogramming to immune modulation.
    Reham Atallah, Juergen Gindlhuber, Akos Heinemann.
      Succinate is an essential metabolite in the tricarboxylic acid (TCA) cycle. In mitochondria, succinate holds a unique position connecting the TCA cycle and the electron transport chain (ETC), thereby providing a shortcut path for adenosine triphosphate (ATP) production. Beyond this fundamental role in cellular metabolism, succinate is increasingly acknowledged as a key modulator of immune cell function. Production of reactive oxygen species (ROS), hypoxia-inducible factor-1α (HIF-1α) stabilization, protein succinylation and cell-cell communication mediated by succinate receptor 1 (SUCNR1) are traits induced by succinate. During inflammation, succinate plays key dual roles, culminating in either pro- or anti-inflammatory effects that are tissue- and context-dependent. In this review, we provide a succinct overview focusing on the regulatory role of succinate in innate immune cells, highlighting involved mechanisms and research gaps that represent promising targets for future study.
    Keywords:  hypoxia-inducible factor-1α (HIF-1α); inflammation; innate immune cells; reactive oxygen species (ROS); succinate; succinate dehydrogenase (SDH); succinate receptor 1 (SUCNR1); succinylation
    DOI:  https://doi.org/10.3389/fimmu.2025.1661948
  16. Methods Mol Biol. 2026 ;2976 35-45
    Label-Free Imaging of Lysosomes in Living Cells by Photothermal Microscopy.
    Jun Miyazaki, Ryotaro Wagatsuma.
      Photothermal microscopy is an optical imaging technique used to visualize the distribution of trace amounts of endogenous dyes in living cells. This technique facilitates lysosomal observation and functional analysis by detecting and identifying accumulated substances within them, eliminating the need for molecular labeling. Here, we describe the fundamental principles and system configuration of a photothermal microscope. We also present a detailed procedure for performing photothermal measurements in living cells.
    Keywords:  Autophagy; Label-free; Live-cell imaging; Optical absorption; Photothermal microscope
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_4
  17. Proc Natl Acad Sci U S A. 2025 Oct 21. 122(42): e2517050122
    Global conformation of the Rag GTPase heterodimer governs eukaryotic amino acid sensing.
    Dylan D Doxsey, Kuang Shen.
      The Rag GTPase heterodimer is a central mediator of amino acid sensing in eukaryotic cells. When amino acids are abundant, it binds to the mechanistic target of rapamycin complex 1 to activate cellular programs for growth and proliferation. In its functional cycle, besides local conformational changes near the nucleotides that are commonly observed in monomeric signaling GTPases, the relative positioning of the two Rag subunits, i.e., the global conformation, is unique due to the heterodimeric architecture. Although various global conformations have been captured in static structural models, dynamic transitions between these conformations and their biological relevance remain unclear. Here, we visualize the global conformation of the Rag GTPase heterodimer using single-molecule Förster resonance energy transfer. By tracking the movement of individual protein molecules, we found that the two subunits explore a wide conformational space, which is strictly dictated by the bound nucleotides, regulators, and mutations. Additionally, we demonstrate that proper modulation of the global conformation is crucial for correctly interpreting amino acid signals. Our results defined a checkpoint of amino acid sensing in eukaryotic cells.
    Keywords:  Rag GTPase; amino acid sensing; mTORC1; protein conformation; single-molecule FRET
    DOI:  https://doi.org/10.1073/pnas.2517050122
  18. Methods Mol Biol. 2026 ;2976 85-102
    Lysosomal Immunoprecipitation (LysoIP) from Cells and Tissues for Metabolomic and Proteomic Analyses.
    Nouf N Laqtom, Monther Abu-Remaileh.
      Lysosomes, known for degrading biomolecules and damaged cellular components, are now recognized as signaling hubs for nutrient sensing and metabolic adaptation, and their dysfunction is implicated in diseases including cancer and neurodegeneration. To understand the composition of the lysosome, the dynamic behavior of its contents, and its specific roles in health and disease, we describe a lysosomal immunoprecipitation method, termed "LysoIP," that enables the isolation of intact lysosomes from cultured cells and mouse tissues. This method utilizes a lysosome-localized 3xHA epitope tag (LysoTag) and a simple, yet robust organelle immunoprecipitation workflow. Isolated lysosomes are extracted with optimized buffers to allow the efficient retrieval of lysosomal proteins, polar metabolites, and lipids, maintaining compatibility with downstream liquid chromatography and mass spectrometry (LC-MS) analyses.
    Keywords:  LC-MS analyses; LysoIP; LysoTag; LysoTag mouse; Lysosomes; Metabolomics; Proteomics; TMEM192
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_8
  19. Nat Commun. 2025 Oct 16. 16(1): 9189
    Backtracking metabolic dynamics in single cells predicts bacterial replication in human macrophages.
    Mariatou Dramé, Francisco-Javier Garcia-Rodriguez, Dmitry Ershov, Jessica E Martyn, Jean-Yves Tinevez, Carmen Buchrieser, Pedro Escoll.
      Accurately tracking dynamic state transitions is crucial for modeling and predicting biological outcomes, as it captures heterogeneity of cellular responses. To build a model to predict bacterial infection in single cells, we have monitored in parallel infection progression and metabolic parameters in thousands of human primary macrophages infected with the intracellular pathogen Legionella pneumophila. By combining live-cell imaging with a tool for classifying cells based on infection outcomes, we were able to trace the specific evolution of metabolic parameters linked to distinct outcomes, such as bacterial replication or cell death. Our findings revealed that early changes in mitochondrial membrane potential (Δψm) and in the production of mitochondrial Reactive Oxygen Species (mROS) are associated with macrophages that will later support bacterial growth. We used these data to train an explainable machine-learning model and achieved 83% accuracy in predicting L. pneumophila replication in single, infected cells before bacterial replication starts. Our results highlight backtracking as a valuable tool to gain new insights in host-pathogen interactions and identify early mitochondrial alterations as key predictive markers of success of bacterial infection.
    DOI:  https://doi.org/10.1038/s41467-025-64225-0
  20. Nat Commun. 2025 Oct 17. 16(1): 9234
    Visualization of lysosomal membrane proteins by cryo electron tomography.
    Bridget M McVeigh, José J De Jesús-Pérez, Dirk H Siepe, Prerana Gogoi, Shrawan Kumar Mageswaran, Marian Kalocsay, Elaine M Mihelc, Vera Y Moiseenkova-Bell.
      Lysosomes are essential organelles for cellular homeostasis and signaling, with dysfunction linked to neurological disorders, lysosomal storage diseases, and cancer. While proteomics has advanced our understanding of lysosomal composition, the structural characterization of lysosomal membrane proteins in their native environment remains a significant challenge. Here, we developed a cryo electron tomography workflow to visualize lysosomal membrane proteins within intact, native lysosomal membranes. We isolated endolysosomes by independently targeting two lysosomal membrane proteins, transient receptor potential mucolipin 1 and transmembrane protein 192, enriching organelles that exhibited the expected morphology and proteomic composition of the endolysosomal system. Sub-tomogram averaging enabled the structural refinement of key membrane and membrane-associated proteins, including V-ATPase, Flotillin, and Clathrin, directly within the lysosomal membrane, revealing their heterogeneous distribution across endolysosomal organelles. By integrating proteomics with structural biology, our workflow establishes a powerful platform for studying lysosomal membrane protein function in health and disease, paving the way for future discoveries in membrane-associated lysosomal mechanisms.
    DOI:  https://doi.org/10.1038/s41467-025-64314-0
  21. Biochim Biophys Acta Rev Cancer. 2025 Oct 10. pii: S0304-419X(25)00214-8. [Epub ahead of print] 189472
    Detection methods for mitochondrial DNA heteroplasmy and their potential single-cell applications in cancer.
    Tianguang Cheng, Min Pan, Yi Qiao, Jing Tu.
      Mitochondrial DNA (mtDNA) is crucial for cellular metabolism, oxidative stress responses, and genomic stability, with mutations linked to cancer progression and therapeutic resistance. Mitochondrial heteroplasmy, the coexistence of wild-type and mutant mtDNA within a cell or across populations, plays a key role in mitochondrial dysfunction, tumor heterogeneity, and disease pathogenesis. Advances in single-cell technologies like quantitative PCR (qPCR), digital droplet PCR (ddPCR), next-generation sequencing (NGS), and long-read sequencing (TGS) have enabled precise mapping of heteroplasmic variants, providing insights into their role in cancer. This review evaluates current detection methods, discussing their strengths, limitations, and relevance to cancer research. We also explore the biological implications of heteroplasmy in cellular dynamics, nuclear mitochondrial DNA segments (NUMTs), and cancer pathogenesis, highlighting emerging technologies and future directions for studying mtDNA mutations at single-cell resolution in cancer. Ultimately, this review provides a critical synthesis of how single-cell mtDNA heteroplasmy analysis is reshaping our understanding of tumorigenesis and identifies key methodological and challenges that must be addressed to realize its full potential in precision oncology.
    Keywords:  Cancer metabolism; Heteroplasmy; Mitochondrial DNA; Sequencing; Single cell
    DOI:  https://doi.org/10.1016/j.bbcan.2025.189472
  22. Nat Commun. 2025 Oct 16. 16(1): 9185
    HIFα isoform specific activities drive cell-type specificity of VHL-associated oncogenesis.
    Joanna D C C Lima, Madeleine Hooker, Ran Li, Ayslan B Barros, Norma Masson, Christopher W Pugh, David R Mole, Julie Adam, Peter J Ratcliffe, Samvid Kurlekar.
      Cancers arising from dysregulation of generally operative signaling pathways are often tissue specific, but the mechanisms underlying this paradox are poorly understood. Based on striking cell-type specificity, we postulated that these mechanisms must operate early in cancer development and set out to study them in a model of von Hippel Lindau (VHL) disease. Biallelic mutation of the VHL ubiquitin ligase leads to constitutive activation of hypoxia inducible factors HIF1A and HIF2A and is generally a truncal event in clear cell renal carcinoma. We used an oncogenic tagging strategy in which VHL-mutant cells are marked by tdTomato, enabling their observation, retrieval, and analysis early after VHL-inactivation. Here, we reveal markedly different consequences of HIF1A and HIF2A activation, but that both contribute to renal cell-type specific consequences of VHL-inactivation in the kidney. Early involvement of HIF2A in promoting proliferation within the proximal tubular epithelium supports therapeutic targeting of HIF2A early in VHL disease.
    DOI:  https://doi.org/10.1038/s41467-025-64214-3
  23. Nat Cell Biol. 2025 Oct;27(10): 1708-1724
    MAPL regulates gasdermin-mediated release of mtDNA from lysosomes to drive pyroptotic cell death.
    Mai Nguyen, Jack J Collier, Olesia Ignatenko, Genevieve Morin, Vanessa Goyon, Alexandre Janer, Camila Tiefensee Ribeiro, Austen J Milnerwood, Sidong Huang, Michel Desjardins, Heidi M McBride.
      Mitochondrial control of cell death is of central importance to disease mechanisms from cancer to neurodegeneration. Mitochondrial anchored protein ligase (MAPL) is an outer mitochondrial membrane small ubiquitin-like modifier ligase that is a key determinant of cell survival, yet how MAPL controls the fate of this process remains unclear. Combining genome-wide functional genetic screening and cell biological approaches, we found that MAPL induces pyroptosis through an inflammatory pathway involving mitochondria and lysosomes. MAPL overexpression promotes mitochondrial DNA trafficking in mitochondrial-derived vesicles to lysosomes, which are permeabilized in a process requiring gasdermin pores. This triggers the release of mtDNA into the cytosol, activating the DNA sensor cGAS, required for cell death. Additionally, multiple Parkinson's disease-related genes, including VPS35 and LRRK2, also regulate MAPL-induced pyroptosis. Notably, depletion of MAPL, LRRK2 or VPS35 inhibited inflammatory cell death in primary macrophages, placing MAPL and the mitochondria-lysosome pathway at the nexus of immune signalling and cell death.
    DOI:  https://doi.org/10.1038/s41556-025-01774-y
  24. Nature. 2025 Oct 15.
    Spatial metabolic gradients in the liver and small intestine.
    Laith Z Samarah, Clover Zheng, Xi Xing, Won Dong Lee, Amichay Afriat, Uthsav Chitra, Michael R MacArthur, Wenyun Lu, Connor S R Jankowski, Cong Ma, Craig J Hunter, Michael Neinast, Daniel R Weilandt, Benjamin J Raphael, Joshua D Rabinowitz.
      The properties of mammalian cells depend on their location within organs. Gene expression in the liver varies between periportal and pericentral hepatocytes1-3, and in the intestine from crypts to villus tips4,5. A key element of tissue spatial organization is probably metabolic, but direct assessments of spatial metabolism remain limited. Here we map spatial metabolic gradients in the mouse liver and intestine. We develop an integrated experimental-computational workflow using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS), isotope tracing and deep-learning artificial intelligence. Most measured metabolites (>90%) showed significant spatial concentration gradients in the liver lobules and intestinal villi. In the liver, tricarboxylic acid (TCA)-cycle metabolites and their isotope labelling from both glutamine and lactate localized periportally. Energy-stress metabolites, including adenosine monophosphate (AMP), also localized periportally, consistent with a high periportal energy demand. In the intestine, the TCA intermediates malate (tip) and citrate (crypt) showed opposite spatial patterns, aligning with higher glutamine catabolism in tips and lactate oxidation in crypts based on isotope tracing. Finally, we mapped the fate of the obesogenic dietary sugar fructose. In the intestine, oral fructose was catabolized faster in the villus bottom than in the tips. In the liver, fructose-derived carbon accumulated pericentrally as fructose-1-phosphate and triggered pericentral adenosine triphosphate (ATP) depletion. Thus, we both provide foundational knowledge regarding intestine and liver metabolic organization and identify fructose-induced focal derangements in liver metabolism.
    DOI:  https://doi.org/10.1038/s41586-025-09616-5
  25. Methods Mol Biol. 2026 ;2976 1-10
    Determining Lysosomal Enzyme Activity Using Fluorogenic Probes.
    Etienne Sauvageau, Stephane Lefrancois.
      Lysosomes are responsible for a number of cellular functions, including the degradation of various biological molecules. Soluble enzymes within the lysosomal lumen are required to perform this function. Lysosomal activity can be disrupted in a variety of diseases, and measuring the activity of specific enzymes can be performed. In this chapter, we detail how lysosomal enzyme activity can be measured either in cell lysates or intact cells. This can be used to study fundamental cell biology or the effect of therapeutics targeting lysosomal function.
    Keywords:  Batten disease; Fluorogenic substrates; Lysosomal enzyme activity; Lysosomes
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_1
  26. Int J Mol Sci. 2025 Oct 09. pii: 9829. [Epub ahead of print]26(19):
    Methods for Assessing MAGL Enzymatic Activity: An Extensive Review of Past and Emerging Approaches.
    Giulia Bononi, Eva Landucci, Miriana Di Stefano, Lisa Piazza, Simone Bertini, Marco Macchia, Carlotta Granchi.
      Monoacylglycerol lipase (MAGL) is a key serine hydrolase involved in lipid metabolism, catalyzing the hydrolysis of monoacylglycerols into free fatty acids and glycerol. MAGL plays a central role in regulating endocannabinoid signaling and lipid homeostasis, processes often dysregulated in cancer and other pathological conditions. In recent years, MAGL has emerged as a promising therapeutic target, particularly in oncology, where its inhibition has shown potential to impair tumor growth, metastasis, and inflammation-driven processes. Alongside the development of selective MAGL inhibitors, several biochemical methods have been established to measure MAGL enzymatic activity, providing essential tools for target validation and inhibitor characterization. In this review, we provide a comprehensive and critical overview of the main approaches developed for MAGL activity evaluation, including radiometric, chromatographic, colorimetric, fluorescence-based, bioluminescence-based, and activity-based protein profiling (ABPP) assays. For each method, we discuss principles, advantages, and limitations. This review aims to support researchers in the selection of the most appropriate assay strategy for their experimental needs, ultimately fostering the rapid and accurate development of novel MAGL inhibitors with potential applications in cancer therapy and metabolic disease management.
    Keywords:  MAGL; biochemical methods; enzymatic activity; enzymatic assays; monoacylglycerol lipase
    DOI:  https://doi.org/10.3390/ijms26199829
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