bims-celmim 2025-02-16 papers

bims-celmim

Biomed News

on Cellular and mitochondrial metabolism

Issue of 2025–02–16
thirty-one papers selected by
Marc Segarra Mondejar

Metabolic signature of renal cell carcinoma tumours and its correlation with the urinary metabolome.
Fluorescence lifetime imaging microscopy of endogenous fluorophores in health and disease.
Emerging Roles of Nanozyme in Tumor Metabolism Regulation: Mechanisms, Applications, and Future Directions.
ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress.
Intercellular mitochondrial transfer contributes to microenvironmental redirection of cancer cell fate.
Look Beyond Plasma Membrane Biophysics: Revealing Considerable Variability of the Dipole Potential Between Plasma and Organelle Membranes of Living Cells.
Subcellular Compartmentalization of Glucose Mediated Insulin Secretion.
A hybrid machine learning framework for functional annotation of mitochondrial glutathione transport and metabolism proteins in cancers.
Metabolic heterogeneity in tumor cells impacts immunology in lung squamous cell carcinoma.
Photothermal imaging of cellular responses to glucose deprivation.
Role of ubiquitination-driven metabolisms in oncogenesis and cancer therapy.
Emerging roles of mitochondrial sirtuin SIRT5 in succinylation modification and cancer development.
Mitofusin 2 displays fusion-independent roles in proteostasis surveillance.
Untargeted urine metabolomics reveals dynamic metabolic differences and key biomarkers across different stages of Alzheimer's disease.
Loss of long-chain acyl-CoA dehydrogenase protects against acute kidney injury.
BBOX1 restrains TBK1-mTORC1 oncogenic signaling in clear cell renal cell carcinoma.
Predicting substrates for orphan solute carrier proteins using multi-omics datasets.
The impact of glycolysis on ischemic stroke: from molecular mechanisms to clinical applications.
Diverse routes to mitophagy governed by ubiquitylation and mitochondrial import.
Metabolic Profiling of Breast Cancer Cell Lines: Unique and Shared Metabolites.
Clear cell renal carcinoma essentially requires CDKL3 for oncogenesis.
Genetically Encoded Metabolic Sensors to Study Retina Metabolism.
Glucokinase activity controls peripherally located subpopulations of β-cells that lead islet Ca2+ oscillations.
Pharmacological Targeting of the NMDAR/TRPM4 Death Signaling Complex with a TwinF Interface Inhibitor Prevents Excitotoxicity-Associated Dendritic Blebbing and Organelle Damage.
Secretory mitophagy: an extracellular vesicle-mediated adaptive mechanism for cancer cell survival under oxidative stress.
Bafilomycin A1 induces colon cancer cell death through impairment of the endolysosome system dependent on iron.
Mitochondrial potassium channels: New properties and functions.
HBimmCue: A Versatile Fluorescent Probe for Multi-Scale Imaging of Lipid Polarity and Membrane Order in Inner Mitochondrial Membrane.
Ratio-based indicators for cytosolic Ca2+ with visible light excitation.
Metabolic control of replisome plasticity in genome surveillance.
Development of selective deconjugases for membrane-anchored LC3A/B in post-mitotic neurons.

Metabolomics. 2025 Feb 13. 21(2): 26

Metabolic signature of renal cell carcinoma tumours and its correlation with the urinary metabolome.

Filipa Amaro, Márcia Carvalho, Carina Carvalho-Maia, Carmen Jerónimo, Rui Henrique, Maria de Lourdes Bastos, Paula Guedes de Pinho, Joana Pinto.

   INTRODUCTION: Despite considerable advances in cancer research, the increasing prevalence and high mortality rate of clear cell renal cell carcinoma (ccRCC) remain a significant challenge. A more detailed comprehension of the distinctive metabolic characteristics of ccRCC is vital to enhance diagnostic, prognostic, and therapeutic strategies.
OBJECTIVES: This study aimed to investigate the metabolic signatures of ccRCC tumours and, for the first time, their correlation with the urinary metabolome of the same patients.
METHODS: We applied a gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach to analyse matched tissue and urine samples from a cohort of 18 ccRCC patients and urine samples from 18 cancer-free controls. Multivariate and univariate statistical methods, as well as pathway and correlation analyses, were performed to assess metabolic dysregulations and correlations between tissue and urine.
RESULTS: The results showed a ccRCC metabolic signature characterized by reprogramming in amino acid, energy, and sugar and inositol phosphate metabolisms. Our study identified, for the first time, significantly decreased levels of asparagine, proline, gluconate, 3-aminoisobutanoate, 4-aminobutanoate and urea in ccRCC tumours, highlighting the involvement of arginine biosynthesis, β-alanine metabolism and purine and pyrimidine metabolism in ccRCC. The correlations between tissue and urine metabolomes provide evidence for the potential usefulness of urinary metabolites in understanding systemic metabolic changes driven by RCC tumours.
CONCLUSIONS: These findings significantly advance our understanding of metabolic reprogramming in ccRCC and the systemic metabolic changes associated with the disease. Future research is needed to validate these findings in larger cohorts and to determine their potential implications for diagnosis and targeted therapies.

Keywords:  Clear cell renal cell carcinoma (ccRCC); Gas-chromatography-mass spectrometry (GC-MS); Metabolomics; Tissue metabolome; Urine metabolome

DOI:  https://doi.org/10.1007/s11306-024-02212-0
J Muscle Res Cell Motil. 2025 Feb 13.

Fluorescence lifetime imaging microscopy of endogenous fluorophores in health and disease.

Barbara Elsnicova.

  Fluorescence Lifetime Imaging Microscopy (FLIM) of endogenous fluorophores has recently emerged as a powerful, marker-free, and non-invasive tool for investigating cellular metabolism. This cutting-edge imaging technique provides valuable insights into cellular energy states by measuring the fluorescence lifetimes of intrinsically fluorescent redox cofactors. The lifetimes of these cofactors reflect their binding states to enzymes, thus indicating enzymatic activity within specific metabolic pathways. As a result, FLIM can help to reveal the overall redox status of the cell and, to some extent, shifts between oxidative phosphorylation and glycolysis. The application of FLIM in metabolic research has shown significant progress across a diverse range of pathological contexts, including cancer, diabetes, neurodegenerative disorders, and various forms of cardiopathology.The aim of this mini-review is to introduce the methodology of NAD(P)H and FAD/FMN FLIM, outline its underlying principles, and demonstrate its ability to reveal changes in cellular metabolism. Additionally, this mini-review highlights FLIM's potential for understanding cellular redox states, detecting metabolic shifts in various disease models, and contributing to the development of therapeutic strategies.

Keywords:  FAD; FLIM; FLIRR; NAD(P)H; Redox state

DOI:  https://doi.org/10.1007/s10974-025-09689-9
ACS Appl Mater Interfaces. 2025 Feb 12.

Emerging Roles of Nanozyme in Tumor Metabolism Regulation: Mechanisms, Applications, and Future Directions.

Yikai Li, Bowen Fu, Wei Jiang.

  Nanozymes, nanomaterials with intrinsic enzyme activity, have garnered significant attention in recent years due to their catalytic abilities comparable to natural enzymes, cost-effectiveness, high catalytic activities, and stability against environmental fluctuations. As functional analogs of natural enzymes, nanozymes participate in various critical metabolic processes, including glucose metabolism, lactate metabolism, and the maintenance of redox homeostasis, all of which are essential for normal cellular functions. However, disruptions in these metabolic pathways frequently promote tumorigenesis and progression, making them potential therapeutic targets. While several therapies targeting tumor metabolism are currently in clinical or preclinical stages, their efficacy requires further enhancement. Consequently, nanozymes that target tumor metabolism are regarded as a promising therapeutic strategy. Despite extensive studies investigating the application of nanozymes in tumor metabolism, relevant reviews are relatively scarce. This article first introduces the physicochemical properties and biological behaviors of nanozymes. Subsequently, we analyze the role of nanozymes in tumor metabolism and explore their potential applications in tumor therapy. In conclusion, this review aims to foster innovative research in related fields and advance the development of nanozyme-based strategies for cancer diagnostics and therapeutics.

Keywords:  Cancer therapy; Metabolism; Nanozymes; Redox homeostasis; Tumor; Tumor catalytic therapy

DOI:  https://doi.org/10.1021/acsami.4c20417
Nat Commun. 2025 Feb 10. 16(1): 1508

ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress.

Isshin Shiiba, Naoki Ito, Hijiri Oshio, Yuto Ishikawa, Takahiro Nagao, Hiroki Shimura, Kyu-Wan Oh, Eiki Takasaki, Fuya Yamaguchi, Ryoan Konagaya, Hisae Kadowaki, Hideki Nishitoh, Takehito Tanzawa, Shun Nagashima, Ayumu Sugiura, Yuuta Fujikawa, Keitaro Umezawa, Yasushi Tamura, Byung Il Lee, Yusuke Hirabayashi, Yasushi Okazaki, Tomohiro Sawa, Ryoko Inatome, Shigeru Yanagi.

The proximal domains of mitochondria and the endoplasmic reticulum (ER) are linked by tethering factors on each membrane, allowing the efficient transport of substances, including lipids and calcium, between them. However, little is known about the regulation and function of mitochondria-ER contacts (MERCs) dynamics under mitochondrial damage. In this study, we apply NanoBiT technology to develop the MERBiT system, which enables the measurement of reversible MERCs formation in living cells. Analysis using this system suggests that induction of mitochondrial ROS increases MERCs formation via RMDN3 (also known as PTPIP51)-VAPB tethering driven by RMDN3 phosphorylation. Disruption of this tethering caused lipid radical accumulation in mitochondria, leading to cell death. The lipid radical transfer activity of the TPR domain in RMDN3, as revealed by an in vitro liposome assay, suggests that RMDN3 transfers lipid radicals from mitochondria to the ER. Our findings suggest a potential role for MERCs in cell survival strategy by facilitating the removal of mitochondrial lipid radicals under mitochondrial damage.

DOI: https://doi.org/10.1038/s41467-025-56666-4
FEBS J. 2025 Feb 11.

Intercellular mitochondrial transfer contributes to microenvironmental redirection of cancer cell fate.

Julie Sofie Bjerring, Yara Khodour, Emilee Anne Peterson, Patrick Christian Sachs, Robert David Bruno.

  The mammary microenvironment has been shown to suppress tumor progression by redirecting cancer cells to adopt a normal mammary epithelial progenitor fate in vivo. However, the mechanism(s) by which this alteration occurs has yet to be defined. Here, we test the hypothesis that mitochondrial transfer from normal mammary epithelial cells to breast cancer cells plays a role in this redirection process. We evaluate mitochondrial transfer in 2D and 3D organoids using our unique 3D bioprinting system to produce chimeric organoids containing normal and cancer cells. We demonstrate that breast cancer tumoroid growth is hindered following interaction with mammary epithelial cells in both 2D and 3D environments. Furthermore, we show mitochondrial transfer occurs between donor mammary epithelial cells and recipient cancer cells primarily through tunneling nanotubes (TNTs) with minimal amounts seen from extracellular transfer of mitochondria, likely via extracellular vesicles (EVs). This organelle exchange results in various cellular and metabolic alterations within cancer cells, reducing their proliferative potential, and making them susceptible to microenvironmental control. Our results demonstrate that mitochondrial transfer contributes to microenvironmental redirection of cancer cells through alteration of metabolic and molecular functions of the recipient cancer cells. To the best of our knowledge, this is the first description of a 3D bioprinter-assisted organoid system for studying mitochondrial transfer. These studies are also the first mechanistic insights into the process of mammary microenvironmental redirection of cancer and provide a framework for new therapeutic strategies to control cancer.

Keywords:  3D bioprinting; breast cancer; cellular redirection; microenvironment; mitochondrial transfer

DOI:  https://doi.org/10.1111/febs.70002
Int J Mol Sci. 2025 Jan 22. pii: 889. [Epub ahead of print]26(3):

Look Beyond Plasma Membrane Biophysics: Revealing Considerable Variability of the Dipole Potential Between Plasma and Organelle Membranes of Living Cells.

Mate Szabo, Bence Cs Szabo, Kitti Kurtan, Zoltan Varga, Gyorgy Panyi, Peter Nagy, Florina Zakany, Tamas Kovacs.

  Due to the lack of measurement techniques suitable for examining compartments of intact, living cells, membrane biophysics is almost exclusively investigated in the plasma membrane despite the fact that its alterations in intracellular organelles may also contribute to disease pathogenesis. Here, we employ a novel, easy-to-use, confocal microscopy-based approach utilizing F66, an environment-sensitive fluorophore in combination with fluorescent organelle markers and quantitative image analysis to determine the magnitude of the molecular order-related dipole potential in the plasma membrane and intracellular organelles of various tumor and neural cell lines. Our comparative analysis demonstrates considerable intracellular variations of the dipole potential that may be large enough to modulate protein functions, with an inward decreasing gradient on the route of the secretory/endocytic pathway (plasma membrane >> lysosome > Golgi > endoplasmic reticulum), whereas mitochondrial membranes are characterized by a dipole potential slightly larger than that of lysosomes. Our approach is suitable and sensitive enough to quantify membrane biophysical properties selectively in intracellular compartments and their comparative analysis in intact, living cells, and, therefore, to identify the affected organelles and potential therapeutic targets in diseases associated with alterations in membrane lipid composition and thus biophysics such as tumors, metabolic, neurodegenerative, or lysosomal storage disorders.

Keywords:  Golgi; endoplasmic reticulum; fluorescence microscopy; intracellular organelles; lysosome; membrane biophysics; membrane dipole potential; mitochondrion

DOI:  https://doi.org/10.3390/ijms26030889
Cells. 2025 Jan 29. pii: 198. [Epub ahead of print]14(3):

Subcellular Compartmentalization of Glucose Mediated Insulin Secretion.

Zhongying Wang, Tatyana Gurlo, Leslie S Satin, Scott E Fraser, Peter C Butler.

  Regulation of blood glucose levels depends on the property of beta cells to couple glucose sensing with insulin secretion. This is accomplished by the concentration-dependent flux of glucose through glycolysis and oxidative phosphorylation, generating ATP. The resulting rise in cytosolic ATP/ADP inhibits KATP channels, inducing membrane depolarization and Ca2+ influx, which prompts insulin secretion. Evidence suggests that this coupling of glucose sensing with insulin secretion may be compartmentalized in the submembrane regions of the beta cell. We investigated the subcellular responses of key components involved in this coupling and found mitochondria in the submembrane zone, some tethered to the cytoskeleton near capillaries. Using Fluorescent Lifetime Imaging Microscopy (FLIM), we observed that submembrane mitochondria were the fastest to respond to glucose. In the most glucose-responsive beta cells, glucose triggers rapid, localized submembrane increases in ATP and Ca2+ as synchronized ~4-min oscillations, consistent with pulsatile insulin release after meals. These findings are consistent with the hypothesis that glucose sensing is coupled with insulin secretion in the submembrane zone of beta cells. This zonal adaptation would enhance both the speed and energy efficiency of beta cell responses to glucose, as only a subset of the most accessible mitochondria would be required to trigger insulin secretion.

Keywords:  ATP; KATP channel; beta cells; calcium; compartmentalization; insulin; mitochondria; oxidative phosphorylation; pulsatile secretion; submembrane

DOI:  https://doi.org/10.3390/cells14030198
BMC Bioinformatics. 2025 Feb 11. 26(1): 48

A hybrid machine learning framework for functional annotation of mitochondrial glutathione transport and metabolism proteins in cancers.

Luke Kennedy, Jagdeep K Sandhu, Mary-Ellen Harper, Miroslava Cuperlovic-Culf.

   BACKGROUND: Alterations of metabolism, including changes in mitochondrial metabolism as well as glutathione (GSH) metabolism are a well appreciated hallmark of many cancers. Mitochondrial GSH (mGSH) transport is a poorly characterized aspect of GSH metabolism, which we investigate in the context of cancer. Existing functional annotation approaches from machine (ML) or deep learning (DL) models based only on protein sequences, were unable to annotate functions in biological contexts.
RESULTS: We develop a flexible ML framework for functional annotation from diverse feature data. This hybrid ML framework leverages cancer cell line multi-omics data and other biological knowledge data as features, to uncover potential genes involved in mGSH metabolism and membrane transport in cancers. This framework achieves strong performance across functional annotation tasks and several cell line and primary tumor cancer samples. For our application, classification models predict the known mGSH transporter SLC25A39 but not SLC25A40 as being highly probably related to mGSH metabolism in cancers. SLC25A10, SLC25A50, and orphan SLC25A24, SLC25A43 are predicted to be associated with mGSH metabolism in multiple biological contexts and structural analysis of these proteins reveal similarities in potential substrate binding regions to the binding residues of SLC25A39.
CONCLUSION: These findings have implications for a better understanding of cancer cell metabolism and novel therapeutic targets with respect to GSH metabolism through potential novel functional annotations of genes. The hybrid ML framework proposed here can be applied to other biological function classifications or multi-omics datasets to generate hypotheses in various biological contexts. Code and a tutorial for generating models and predictions in this framework are available at: https://github.com/lkenn012/mGSH_cancerClassifiers .

Keywords:  Cancer; Gene ontology; Glutathione; Knowledge-based; Machine learning; Mitochondria; Multi-omics; Protein function annotation; SLC25; Transmembrane transport

DOI:  https://doi.org/10.1186/s12859-025-06051-1
Oncoimmunology. 2025 Dec;14(1): 2457797

Metabolic heterogeneity in tumor cells impacts immunology in lung squamous cell carcinoma.

Qian Wang, Na Sun, Chaoyang Zhang, Thomas Kunzke, Philipp Zens, Annette Feuchtinger, Sabina Berezowska, Axel Walch.

  Metabolic processes are crucial in immune regulation, yet the impact of metabolic heterogeneity on immunological functions remains unclear. Integrating metabolomics into immunology allows the exploration of the interactions of multilayered features in the biological system and the molecular regulatory mechanism of these features. To elucidate such insight in lung squamous cell carcinoma (LUSC), we analyzed 106 LUSC tumor tissues. We performed high-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain spatial metabolic profiles, and immunohistochemistry to detect tumor-infiltrating T lymphocytes (TILs). Unsupervised k-means clustering and Simpson's diversity index were employed to assess metabolic heterogeneity, identifying five distinct metabolic tumor subpopulations. Our findings revealed that TILs are specifically associated with metabolite distributions, not randomly distributed. Integrating a validation cohort, we found that heterogeneity-correlated metabolites interact with CD8+ TIL-associated genes, affecting survival. High metabolic heterogeneity was linked to worse survival and lower TIL levels. Pathway enrichment analyses highlighted distinct metabolic pathways in each subpopulation and their potential responses to chemotherapy. This study uncovers the significant impact of metabolic heterogeneity on immune functions in LUSC, providing a foundation for tailoring therapeutic strategies.

Keywords:  Immunology; Spatial metabolomics; lung cancer; metabolic heterogeneity; metabolic tumor subpopulation

DOI:  https://doi.org/10.1080/2162402X.2025.2457797
RSC Chem Biol. 2025 Jan 31.

Photothermal imaging of cellular responses to glucose deprivation.

Jun Miyazaki, Ryotaro Wagatsuma, Koji Okamoto.

In solid tumours, cancer cells modify their metabolic processes to endure environments with nutrient and oxygen scarcity due to inadequate blood flow. A thorough understanding of this adaptive mechanism, which requires reliable microscopic techniques, is crucial for developing effective cancer treatments. In the present study, we used multi-wavelength photothermal (PT) microscopy to visualise the cellular response to glucose deprivation in living cells derived from cervical cancer. We found increased mitochondrial PT signal intensity under glucose deprivation conditions, which is indicative of a correlation between mitochondrial crista density and PT signal intensity. Furthermore, PT microscopy revealed that the activity of the autophagy-lysosome system can be evaluated by detecting substances accumulated in lysosomes. Using this method, we confirmed that ferritin and denatured proteins from the endoplasmic reticulum were present within the lysosomes. The detectability of these substances using PT microscopy at visible wavelengths indicated the presence of iron ions. This method does not require labeling of molecules and provides reliable information and detailed insights into the cellular responses associated with the adaptation of cancer cell metabolism to nutrient stress conditions.

DOI: https://doi.org/10.1039/d4cb00269e
Semin Cancer Biol. 2025 Feb 08. pii: S1044-579X(25)00019-7. [Epub ahead of print]110 17-35

Role of ubiquitination-driven metabolisms in oncogenesis and cancer therapy.

Dongqin Yang, Can Yang, Linlin Huang, Ming Guan, Chunhua Song.

  Ubiquitination represents one of the most critical post-translational modifications, comprising a multi-stage enzyme process that plays a pivotal role in a myriad of cellular biological activities. The deregulation of the processes of ubiquitination and deubiquitination is associated with the development of cancers and other diseases. This typescript reviews the impact of ubiquitination on metabolic processes, elucidating the regulatory functions of ubiquitination on pivotal enzymes within metabolic pathways in pathological contexts. It underscores the role of ubiquitination-driven metabolism disorders in the etiology of cancers, and oncogenesis, and highlights the potential therapeutic efficacy of targeting ubiquitination-driven enzymes in cancer metabolism, their combination with immune checkpoint inhibitors, and their clinical applications.

Keywords:  Cancer Therapy; Deubiquitination; Metabolism; Oncogenesis; Ubiquitination

DOI:  https://doi.org/10.1016/j.semcancer.2025.02.004
Front Immunol. 2025 ;16 1531246

Emerging roles of mitochondrial sirtuin SIRT5 in succinylation modification and cancer development.

Zhangmin Ke, Kaikai Shen, Li Wang, Hao Xu, Xia Pan, Zhenjue Qian, Yuting Wen, Tangfeng Lv, Xiuwei Zhang, Yong Song.

  Succinylation represents an emerging class of post-translational modifications (PTMs), characterized by the enzymatic or non-enzymatic transfer of a negatively charged four-carbon succinyl group to the ϵ-amino group of lysine residues, mediated by succinyl-coenzyme A. Recent studies have highlighted the involvement of succinylation in various diseases, particularly cancer progression. Sirtuin 5 (SIRT5), a member of the sirtuin family, has been extensively studied for its robust desuccinylase activity, alongside its deacetylase function. To date, only a limited number of SIRT5 substrates have been identified. These substrates mediate diverse physiological processes such as glucose oxidation, fatty acid oxidation, ammonia detoxification, reactive oxygen species scavenging, anti-apoptosis, and inflammatory responses. The regulation of these activities can occur through either the same enzymatic activity acting on different substrates or distinct enzymatic activities targeting the same substrate. Aberrant expression of SIRT5 has been closely linked to tumorigenesis and disease progression; however, its role remains controversial. SIRT5 exhibits dual functionalities: it can promote tumor proliferation, metastasis, drug resistance, and metabolic reprogramming, thereby acting as an oncogene; conversely, it can also inhibit tumor cell growth and induce apoptosis, functioning as a tumor suppressor gene. This review aims to provide a comprehensive overview of the current research status of SIRT5. We discuss its structural characteristics and regulatory mechanisms, compare its functions with other sirtuin family members, and elucidate the mechanisms regulating SIRT5 activity. Specifically, we focus on the role of succinylation modification mediated by SIRT5 in tumor progression, highlighting how desuccinylation by SIRT5 modulates tumor development and delineating the underlying mechanisms involved.

Keywords:  SIRT5; cancer; desuccinylase; sirtuin; succinylation

DOI:  https://doi.org/10.3389/fimmu.2025.1531246
Nat Commun. 2025 Feb 10. 16(1): 1501

Mitofusin 2 displays fusion-independent roles in proteostasis surveillance.

Mariana Joaquim, Selver Altin, Maria-Bianca Bulimaga, Tânia Simões, Hendrik Nolte, Verian Bader, Camilla Aurora Franchino, Solenn Plouzennec, Karolina Szczepanowska, Elena Marchesan, Kay Hofmann, Marcus Krüger, Elena Ziviani, Aleksandra Trifunovic, Arnaud Chevrollier, Konstanze F Winklhofer, Elisa Motori, Margarete Odenthal, Mafalda Escobar-Henriques.

Mitochondria are essential organelles and their functional state dictates cellular proteostasis. However, little is known about the molecular gatekeepers involved, especially in absence of external stress. Here we identify a role of MFN2 in quality control independent of its function in organellar shape remodeling. MFN2 ablation alters the cellular proteome, marked for example by decreased levels of the import machinery and accumulation of the kinase PINK1. Moreover, MFN2 interacts with the proteasome and cytosolic chaperones, thereby preventing aggregation of newly translated proteins. Similarly to MFN2-KO cells, patient fibroblasts with MFN2-disease variants recapitulate excessive protein aggregation defects. Restoring MFN2 levels re-establishes proteostasis in MFN2-KO cells and rescues fusion defects of MFN1-KO cells. In contrast, MFN1 loss or mitochondrial shape alterations do not alter protein aggregation, consistent with a fusion-independent role of MFN2 in cellular homeostasis. In sum, our findings open new possibilities for therapeutic strategies by modulation of MFN2 levels.

DOI: https://doi.org/10.1038/s41467-025-56673-5
Front Aging Neurosci. 2025 ;17 1530046

Untargeted urine metabolomics reveals dynamic metabolic differences and key biomarkers across different stages of Alzheimer's disease.

Xiaoya Feng, Shenglan Zhao.

   Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder, with mild cognitive impairment (MCI) often serving as its precursor stage. Early intervention at the MCI stage can significantly delay AD onset.
Methods: This study employed untargeted urine metabolomics, with data obtained from the MetaboLights database (MTBLS8662), combined with orthogonal partial least squares-discriminant analysis (OPLS-DA) to examine metabolic differences across different stages of AD progression. A decision tree approach was used to identify key metabolites within significantly enriched pathways. These key metabolites were then utilized to construct and validate an AD progression prediction model.
Results: The OPLS-DA model effectively distinguished the metabolic characteristics at different stages. Pathway enrichment analysis revealed that Drug metabolism was significantly enriched across all stages, while Retinol metabolism was particularly prominent during the transition stages. Key metabolites such as Theophylline, Vanillylmandelic Acid (VMA), and Adenosine showed significant differencesdifferencesin the early stages of the disease, whereas 1,7-Dimethyluric Acid, Cystathionine, and Indole exhibited strong predictive value during the MCI to AD transition. These metabolites play a crucial role in monitoring AD progression. Predictive models based on these metabolites demonstrated excellent classification and prediction capabilities.
Conclusion: This study systematically analyzed the dynamic metabolic differences during the progression of AD and identified key metabolites and pathways as potential biomarkers for early prediction and intervention. Utilizing urinary metabolomics, the findings provide a theoretical basis for monitoring AD progression and contribute to improving prevention and intervention strategies, thereby potentially delaying disease progression.

Keywords:  Alzheimer’s disease; biomarkers; key biomarkers; mild cognitive impairment; urine metabolomics

DOI:  https://doi.org/10.3389/fnagi.2025.1530046
JCI Insight. 2025 Feb 11. pii: e186073. [Epub ahead of print]

Loss of long-chain acyl-CoA dehydrogenase protects against acute kidney injury.

Takuto Chiba, Akira Oda, Yuxun Zhang, Katherine E Pfister, Joanna Bons, Sivakama S Bharathi, Ayako Kinoshita, Bob B Zhang, Adam C Richert, Birgit Schilling, Eric S Goetzman, Sunder Sims-Lucas.

  The renal tubular epithelial cells (RTECs) are particularly vulnerable to acute kidney injury (AKI). While fatty acids are the preferred energy source for RTECs via fatty acid oxidation (FAO), FAO-mediated H2O2 production in mitochondria has been shown to be a major source of oxidative stress. We have previously shown that a mitochondrial flavoprotein, long-chain acyl-CoA dehydrogenase (LCAD), which catalyzes a key step in mitochondrial FAO, directly produces H2O2 in vitro. Further, we showed that renal LCAD becomes hyposuccinylated during AKI. Here, we demonstrated that succinylation of recombinant LCAD protein suppresses the production of H2O2. Following two distinct models of AKI, cisplatin treatment or renal ischemia/reperfusion injury (IRI), LCAD-/- mice demonstrated renoprotection. Specifically, LCAD-/- kidneys displayed mitigated renal tubular injury, decreased oxidative stress, preserved mitochondrial function, enhanced peroxisomal FAO, and decreased ferroptotic cell death. LCAD deficiency confers protection against two distinct models of AKI. This suggests a therapeutically attractive mechanism whereby preserved mitochondrial respiration as well as enhanced peroxisomal FAO by loss of LCAD mediates renoprotection against AKI.

Keywords:  Cell stress; Fatty acid oxidation; Metabolism; Nephrology

DOI:  https://doi.org/10.1172/jci.insight.186073
Nat Commun. 2025 Feb 11. 16(1): 1543

BBOX1 restrains TBK1-mTORC1 oncogenic signaling in clear cell renal cell carcinoma.

Chengheng Liao, Lianxin Hu, Liwei Jia, Jin Zhou, Tao Wang, Kangsan Kim, Hua Zhong, Hongwei Yao, Lei Dong, Lei Guo, Qian Liang, Cheng Zhang, Fangzhou Zhao, Jun Fang, Hongyi Liu, Shina Li, Lin Xu, Jeremy M Simon, Srinivas Malladi, Payal Kapur, James Brugarolas, Ralph J DeBerardinis, Qing Zhang.

Clear cell renal cell carcinoma (ccRCC), a metabolic disease originating from renal proximal convoluted tubule (PCT) epithelial cells, remains incompletely understood in terms of its initiating signaling events. Here, we identify γ-butyrobetaine hydroxylase 1 (BBOX1), a key enzyme in carnitine synthesis predominantly expressed in PCT cells, as a tumor suppressor in ccRCC. BBOX1 expression is lost during ccRCC malignant transformation, and its restoration reduces cell viability in physiological medium and inhibits xenograft tumor growth. Transcriptomic analyses reveal that BBOX1 suppresses critical metabolic pathways including mTORC1 signaling and glycolysis in ccRCC. Further, we identify TANK-binding kinase 1 (TBK1) as an essential mediator of mTORC1 and glycolysis activation and as a target of BBOX1-mediated tumor suppression. Mechanistically, BBOX1 disrupts TBK1 activation by preventing its interaction with the upstream activator doublecortin-like kinase 2 (DCLK2). This BBOX1-DCLK2-TBK1 axis unveils an important mechanism in ccRCC metabolic dysregulation and highlights potential therapeutic strategies.

DOI: https://doi.org/10.1038/s41467-025-56955-y
BMC Genomics. 2025 Feb 11. 26(1): 130

Predicting substrates for orphan solute carrier proteins using multi-omics datasets.

Y Zhang, S Newstead, P Sarkies.

  Solute carriers (SLC) are integral membrane proteins responsible for transporting a wide variety of metabolites, signaling molecules and drugs across cellular membranes. Despite key roles in metabolism, signaling and pharmacology, around one third of SLC proteins are 'orphans' whose substrates are unknown. Experimental determination of SLC substrates is technically challenging, given the wide range of possible physiological candidates. Here, we develop a predictive algorithm to identify correlations between SLC expression levels and intracellular metabolite concentrations by leveraging existing cancer multi-omics datasets. Our predictions recovered known SLC-substrate pairs with high sensitivity and specificity compared to simulated random pairs. CRISPR-Cas9 dependency screen data and metabolic pathway adjacency data further improved the performance of our algorithm. In parallel, we combined drug sensitivity data with SLC expression profiles to predict new SLC-drug interactions. Together, we provide a novel bioinformatic pipeline to predict new substrate predictions for SLCs, offering new opportunities to de-orphanise SLCs with important implications for understanding their roles in health and disease.

Keywords:  Computational Biology; Deorphanization; Gene expression; Membrane Transport; Metabolomics; Solute Carrier Proteins

DOI:  https://doi.org/10.1186/s12864-025-11330-5
Front Neurol. 2025 ;16 1514394

The impact of glycolysis on ischemic stroke: from molecular mechanisms to clinical applications.

Yingquan Liu, Peijia Hu, Hongliang Cheng, Fangyuan Xu, Yu Ye.

  Ischemic stroke (IS), a leading cause of disability and mortality worldwide, remains a significant challenge due to its complex pathogenesis. Glycolysis, a central metabolic pathway, plays a critical role in bridging the gap between metabolic dysfunction and neurological impairment. During ischemic conditions, glycolysis replaces oxidative phosphorylation as the primary energy source for brain tissue. However, in the ischemia-reperfusion state, neuronal cells show a particular reliance on aerobic glycolysis. Immune cells, such as monocytes, also contribute to atheromatous plaque formation and thrombi through increased aerobic glycolysis. Given glycolysis's involvement in various pathological stages of IS, it offers the potential for improved diagnosis, treatment, and prevention. This review comprehensively explores the role of glycolysis in different phases of IS, addresses existing controversies, and discusses its diagnostic and therapeutic applications. By elucidating the intricate relationship between glycolysis and IS, this review aims to provide novel insights for future research and clinical advancements.

Keywords:  diagnosis; glucose metabolism; glycolysis; ischemic stroke; pathomechanism; treatment

DOI:  https://doi.org/10.3389/fneur.2025.1514394
Trends Cell Biol. 2025 Feb 07. pii: S0962-8924(25)00003-0. [Epub ahead of print]

Diverse routes to mitophagy governed by ubiquitylation and mitochondrial import.

Michael J Clague, Sylvie Urbé.

  The selective removal of mitochondria by mitophagy proceeds via multiple mechanisms and is essential for human well-being. The PINK1/Parkin and NIX/BNIP3 pathways are strongly linked to mitochondrial dysfunction and hypoxia, respectively. Both are regulated by ubiquitylation and mitochondrial import. Recent studies have elucidated how the ubiquitin kinase PINK1 acts as a sensor of mitochondrial import stress through stable interaction with a mitochondrial import supercomplex. The stability of BNIP3 and NIX is regulated by the SCFFBXL4 ubiquitin ligase complex. Substrate recognition requires an adaptor molecule, PPTC7, whose availability is limited by mitochondrial import. Unravelling the functional implications of each mode of mitophagy remains a critical challenge. We propose that mitochondrial import stress prompts a switch between these two pathways.

Keywords:  BNIP3; FBXL4; PINK1; PPTC7; mitophagy; ubiquitin

DOI:  https://doi.org/10.1016/j.tcb.2025.01.003
Int J Mol Sci. 2025 Jan 24. pii: 969. [Epub ahead of print]26(3):

Metabolic Profiling of Breast Cancer Cell Lines: Unique and Shared Metabolites.

Mariana Gallo, Elena Ferrari, Federica Brugnoli, Anna Terrazzan, Pietro Ancona, Stefano Volinia, Valeria Bertagnolo, Carlo M Bergamini, Alberto Spisni, Thelma A Pertinhez, Nicoletta Bianchi.

  Breast Cancer (BrCa) exhibits a high phenotypic heterogeneity, leading to the emergence of aggressive clones and the development of drug resistance. Considering the BrCa heterogeneity and that metabolic reprogramming is a cancer hallmark, we selected seven BrCa cell lines with diverse subtypes to provide their comprehensive metabolome characterization: five lines commonly used (SK-Br-3, T-47D, MCF-7, MDA-MB-436, and MDA-MB-231), and two patient-derived xenografts (Hbcx39 and Hbcx9). We characterized their endometabolomes using 1H-NMR spectroscopy. We found distinct metabolite profiles, with certain metabolites being common but differentially accumulated across the selected BrCa cell lines. High levels of glycine, lactate, glutamate, and formate, metabolites known to promote invasion and metastasis, were detected in all BrCa cells. In our experiment setting were identified unique metabolites to specific cell lines: xanthine and 2-oxoglutarate in SK-Br-3, 2-oxobutyrate in T-47D, cystathionine and glucose-1-phosphate in MCF-7, NAD+ in MDA-MB-436, isocitrate in MDA-MB-231, and NADP+ in Hbcx9. The unique and enriched metabolites enabled us to identify the metabolic pathways modulated in a cell-line-specific manner, which may represent potential candidate targets for therapeutic intervention. We believe this study may contribute to the functional characterization of BrCa cells and assist in selecting appropriate cell lines for drug-response studies.

Keywords:  NMR; breast cancer; metabolic reprogramming; metabolite profiling; metabolome

DOI:  https://doi.org/10.3390/ijms26030969
Proc Natl Acad Sci U S A. 2025 Feb 18. 122(7): e2415244122

Clear cell renal carcinoma essentially requires CDKL3 for oncogenesis.

Lanjing Ma, Zhongqiu Pang, Haijiao Zhang, Xueling Yang, Shaoqin Zheng, Yi Chen, Weijie Ding, Qing Han, Xi Zhang, Liu Cao, Teng Fei, Qiang Wang, Daming Gao, Aina He, Ke-Bang Hu, Xuexin Li, Ren Sheng.

  Clear cell renal cell carcinoma (ccRCC) is the predominant human renal cancer with surging incidence and fatality lately. Hyperactivation of hypoxia-inducible factor (HIF) and mammalian target of rapamycin (mTOR) signaling are the common signatures in ccRCC. Herein, we employed spontaneous ccRCC model to demonstrate the indispensability of an underappreciated Ser/Thr kinase, CDKL3, in the initiation and progression of ccRCC. Ablation of CDKL3 does not affect normal kidney, but abrogates Akt-mTOR hyperactivity and thoroughly prevents the formation and growth of the HIF-agitated ccRCC in vivo. Remarkable clinical correlations also supported the oncogenic role of CDKL3. Mechanism-wise, cytosolic CDKL3 unexpectedly behaves as the adaptor to physically potentiate mTORC2-dependent Akt activation without functioning through kinase activity. And mTORC2 can phosphorylate and stabilize CDKL3 to form a positive feedback loop to sustain the cancer-favored Akt-mTOR overactivation. Together, we revealed the pathological importance and molecular mechanism of CDKL3-mediated Akt-mTOR axis in ccRCC initiation and progression.

Keywords:  Akt; CDKL3; ccRCC; mTOR; oncogene

DOI:  https://doi.org/10.1073/pnas.2415244122
Adv Exp Med Biol. 2025 ;1468 465-469

Genetically Encoded Metabolic Sensors to Study Retina Metabolism.

Gabriele M Wögenstein, Christian Grimm.

  Dysfunctional retinal metabolism has been shown to contribute to retinal diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR) and inherited retinal degeneration (IRD). Data indicates that metabolism in the retina is complex and involves intricate interactions between cell types, including the exchange of metabolites between photoreceptors and retinal pigment epithelium (RPE) cells. To understand these interactions on a single cell level, cell-type specific expression of genetically encoded metabolic sensors can be used to reach a spatial and temporal resolution that is superior to other techniques. These sensors comprise a metabolite binding site and a fluorescent reporter protein. The binding of the metabolite leads to changes in the emission of the fluorophore which can be detected by specialized microscopy. The usage of such sensors together with other techniques in the normal and diseased retina will not only help to resolve metabolic interactions between cells and fluxes of metabolites but also enhance our understanding of pathophysiological changes in the retina.

Keywords:  Energy metabolism; FLIM; FRET; Genetically encoded metabolic sensors; Two-photon microscopy

DOI:  https://doi.org/10.1007/978-3-031-76550-6_76
Elife. 2025 Feb 12. pii: RP103068. [Epub ahead of print]13

Glucokinase activity controls peripherally located subpopulations of β-cells that lead islet Ca2+ oscillations.

Erli Jin, Jennifer K Briggs, Richard K P Benninger, Matthew J Merrins.

  Oscillations in insulin secretion, driven by islet Ca2+ waves, are crucial for glycemic control. Prior studies, performed with single-plane imaging, suggest that subpopulations of electrically coupled β-cells have privileged roles in leading and coordinating the propagation of Ca2+ waves. Here, we used three-dimensional (3D) light-sheet imaging to analyze the location and Ca2+ activity of single β-cells within the entire islet at >2 Hz. In contrast with single-plane studies, 3D network analysis indicates that the most highly synchronized β-cells are located at the islet center, and remain regionally but not cellularly stable between oscillations. This subpopulation, which includes 'hub cells', is insensitive to changes in fuel metabolism induced by glucokinase and pyruvate kinase activation. β-Cells that initiate the Ca2+ wave (leaders) are located at the islet periphery, and strikingly, change their identity over time via rotations in the wave axis. Glucokinase activation, which increased oscillation period, reinforced leader cells and stabilized the wave axis. Pyruvate kinase activation, despite increasing oscillation frequency, had no effect on leader cells, indicating the wave origin is patterned by fuel input. These findings emphasize the stochastic nature of the β-cell subpopulations that control Ca2+ oscillations and identify a role for glucokinase in spatially patterning 'leader' β-cells.

Keywords:  calcium oscillation; cell biology; glucokinase; light-sheet microscope; mouse; pancreatic islets; β-cell

DOI:  https://doi.org/10.7554/eLife.103068
Cells. 2025 Jan 28. pii: 195. [Epub ahead of print]14(3):

Pharmacological Targeting of the NMDAR/TRPM4 Death Signaling Complex with a TwinF Interface Inhibitor Prevents Excitotoxicity-Associated Dendritic Blebbing and Organelle Damage.

Omar A Ramírez, Andrea Hellwig, Zihong Zhang, Hilmar Bading.

  Focal swellings of dendrites ("dendritic blebbing") together with structural damage of mitochondria and the endoplasmic reticulum (ER) are morphological hallmarks of glutamate neurotoxicity, also known as excitotoxicity. These pathological alterations are generally thought to be caused by the so-called "overactivation" of N-methyl-D-aspartate receptors (NMDARs). Here, we demonstrate that the activation of extrasynaptic NMDARs, specifically when forming a protein-protein complex with TRPM4, drives these pathological traits. In contrast, strong activation of synaptic NMDARs fails to induce cell damage despite evoking plateau-type calcium signals that are comparable to those generated by activation of the NMDAR/TRPM4 complex, indicating that high intracellular calcium levels per se are not toxic to neurons. Using confocal laser scanning microscopy and transmission electron microscopy, we show that disrupting the NMDAR/TRPM4 complex using the recently discovered small-molecule TwinF interface inhibitor FP802 inhibits the NMDA-induced neurotoxicity-associated dendritic blebbing and structural damage to mitochondria and the ER. It also prevents, at least in part, the disruption of ER-mitochondria contact sites. These findings establish the NMDAR/TRPM4 complex as the trigger for the structural damage of dendrites and intracellular organelles associated with excitotoxicity. They also suggest that activation of the NMDAR/TRPM4 complex, in addition to inducing high-amplitude, plateau-type calcium signals, generates a second signal required for glutamate neurotoxicity ("two-hit hypothesis"). As structural damage to organelles, particularly mitochondria, is a common feature of many human neurodegenerative diseases, including Alzheimer's disease and amyotrophic lateral sclerosis (ALS), TwinF interface inhibitors have the potential to provide neuroprotection across a broad spectrum of these diseases.

Keywords:  ER–mitochondria contact sites; NMDAR/TRPM4 complex; TwinF interface inhibitor; endoplasmic reticulum; glutamate neurotoxicity; mitochondria; organelle damage

DOI:  https://doi.org/10.3390/cells14030195
Front Cell Dev Biol. 2024 ;12 1490902

Secretory mitophagy: an extracellular vesicle-mediated adaptive mechanism for cancer cell survival under oxidative stress.

Purva V Gade, Angela Victoria Rojas Rivera, Layla Hasanzadah, Sofie Strompf, Thomas Raymond Philipson, Matthew Gadziala, Atharva Tyagi, Arnav Bandam, Rithvik Gabbireddy, Fatah Kashanchi, Amanda Haymond, Lance A Liotta, Marissa A Howard.

  Mitophagy is a critically important survival mechanism in which toxic, aged, or defective mitochondria are segregated into mitophagosomes, which shuttle the damaged mitochondrial segments to the lysosome and proteasome for destruction. Cancer cells rely on mitophagy under conditions of high oxidative stress or increased energy demand. Oxidative stress can generate a large volume of damaged mitochondria, overwhelming lysosomal removal. Accumulated damaged mitochondria are toxic and their proper removal is crucial for maintaining mitochondrial health. We propose a new cancer cell mechanism for survival that is activated when the demand for segregating and eliminating damaged mitochondria exceeds the capacity of the lysosome or proteasome. Specifically, we show that tumor cells subjected to oxidative stress by carbonyl cyanide-3-chlorophenylhdrazone (CCCP) eliminate damaged mitochondria segments by bypassing the lysosome to export them outside the cell via extracellular vesicles (EVs), a process termed "secretory mitophagy". PINK1, the initiator of mitophagy, remains associated with the damaged mitochondria that exported in EVs. Using several types of cancer cells, we show that tumor cells treated with CCCP can be induced to switch over to secretory mitophagy by treatment with Bafilomycin A1, which blocks the fusion of mitophagosomes with lysosomes. Under these conditions, an increased number of PINK1 + EVs are exported. This is associated with greater cell survival by a given CCCP dose, enhanced mitochondrial ATP production, and reduced mitochondrial oxidative damage (membrane depolarization). Our data supports the hypothesis that secretory mitophagy is a previously unexplored process by which cancer cells adapt to survive therapeutic or hypoxic stress. Ultimately, our findings may inform new prevention strategies targeting pre-malignant lesions and therapeutic approaches designed to sensitize tumor cells to oxidative stress-inducing therapies.

Keywords:  PINK1; cancer progression; cell survival; extracellular vesicles; mitophagy; oxidative stress

DOI:  https://doi.org/10.3389/fcell.2024.1490902
Sci Rep. 2025 Feb 12. 15(1): 5148

Bafilomycin A1 induces colon cancer cell death through impairment of the endolysosome system dependent on iron.

Dong Hwa Min, Dasom Kim, Seung Taek Hong, Joohee Kim, Min Jung Kim, Seung-Hae Kwon, Aeree Kim, Ji-Yun Lee.

  The late endolysosomal compartment plays a crucial role in cancer cell metabolism by regulating lysosomal activity, essential for cell proliferation, and the degradation of cellular components during the final stages of autophagy. Modulating late endolysosomal function represents a new target for cancer therapy. In this study, we investigated the effects of bafilomycin A1 (BA1), a vacuolar H+-ATPase inhibitor, on colon cancer and normal colon fibroblasts (CCD-18Co) cells. We found that very low concentrations (~ 2 nM) of BA1 selectively induced cell death in colon cancer cells. This cytotoxicity was associated with lysosomal stress response and dysregulation of iron homeostasis. BA1 treatment resulted in significant alterations to the endolysosomal system, including an increased number and size of lysosomes, lysosomal membrane permeabilization, and autophagy flux blockade. These changes were accompanied by endoplasmic reticulum stress and lipid droplet accumulation. Furthermore, BA1 decreased intracellular Fe2+ levels, as measured using FerroOrange. Notably, iron (III)-citrate supplementation rescued cells from BA1-induced death. These findings suggest that BA1-induced endolysosomal dysfunction impairs iron homeostasis, ultimately leading to colon cancer cell death. Our results highlight the potential of targeting endolysosomal function and iron homeostasis as novel therapeutic strategies for colon cancer, paving the way for more selective and effective treatments.

Keywords:  Autophagy; Bafilomycin A1; Colorectal cancer; Endolysosome; Iron

DOI:  https://doi.org/10.1038/s41598-025-89127-5
Biochim Biophys Acta Bioenerg. 2025 Feb 09. pii: S0005-2728(25)00012-X. [Epub ahead of print] 149546

Mitochondrial potassium channels: New properties and functions.

Adam Szewczyk, Piotr Bednarczyk, Bogusz Kulawiak, Monika Żochowska, Barbara Kalenik, Joanna Lewandowska, Karolina Pytlak, Shur Gałecka, Antoni Wrzosek, Piotr Koprowski.

  Mitochondria are recently implicated in phenomena such as cytoprotection, cellular senescence, tumor metabolism, and inflammation. The basis of these processes relies on biochemical functions of mitochondria such as the synthesis of reactive oxygen species or biophysical properties such as the integrity of the inner mitochondrial membrane. The transport of potassium cations plays an important role in all these events. The K+ influx is mediated by potassium channels present in the inner mitochondrial membrane. In this article, we present an overview of our new findings on the properties of mitochondrial large-conductance calcium-activated and mitochondrial ATP-regulated potassium channels. This concerns the role of mitochondrial potassium channels in cellular senescence, and interactions with other mitochondrial proteins or small molecules such as quercetin, hemin, and hydrogen sulfide. We also discuss the prospects of research on potassium channels present in mitochondria.

Keywords:  Hemin; Hydrogen sulfide; Kinases; Mitochondria; Potassium channels; Quercetin; ROS; Senescence

DOI:  https://doi.org/10.1016/j.bbabio.2025.149546
Adv Sci (Weinh). 2025 Feb 09. e2414343

HBimmCue: A Versatile Fluorescent Probe for Multi-Scale Imaging of Lipid Polarity and Membrane Order in Inner Mitochondrial Membrane.

Shu Gao, Jing Sun, Yiwei Hou, Xichuan Ge, Ming Shi, Hongxi Zheng, Yan Zhang, Meiqi Li, Baoxiang Gao, Peng Xi.

  Mitochondrial membrane environmental dynamics are crucial for understanding function, yet high-resolution observation remains challenging. Here, HBimmCue is introduced as a fluorescent probe localized to inner mitochondrial membrane (IMM) that reports lipid polarity and membrane order changes, which correlate with cellular respiration levels. Using HBimmCue and fluorescence lifetime imaging microscopy (FLIM), IMM lipid heterogeneity is uncovered across scales, from nanoscale structures within individual mitochondria to mouse pre-implantation embryos. At the sub-organelle level, stimulated emission depletion (STED)-FLIM imaging highlights nanoscale polarity variations within the IMM. At the sub-cellular and cellular level, reduced IMM lipid polarity is observed in damaged mitochondria marked for lysosomal degradation and distinct IMM lipid distributions are identified in neurons and disease models. Additionally, metabolic dysfunction associated with oocytes aging and metabolic reprogramming from zygote to blastocyst is detected. Together, the work demonstrates the broad applicability of HBimmCue, offering a new paradigm for investigating lipid polarity and respiration level at multiple scales.

Keywords:  HBimmCue; fluorescence lifetime imaging microscopy; inner mitochondrial membrane; lipid polarity

DOI:  https://doi.org/10.1002/advs.202414343
Proc Natl Acad Sci U S A. 2025 Feb 18. 122(7): e2410436122

Ratio-based indicators for cytosolic Ca2+ with visible light excitation.

Xinqi Zhou, Kayla J Belavek, Marisol X Navarro, Kayli N Martinez, Abigail Hinojosa, Evan W Miller.

  Calcium ions (Ca2+) play central roles in cellular physiology. Fluorescent indicators for Ca2+ ions revolutionized our ability to make rapid, accurate, and highly parallel measurement of Ca2+ concentrations in living cells. The use of ratio-based imaging with one particular indicator, fura-2, allowed practitioners to correct for a number of experimental confounds, including dye bleaching, variations in sample thickness, and fluctuations in illumination intensity. Ratio-based imaging with fura-2 was the most accurate and reliable method for measuring Ca2+ concentrations. Two drawbacks to fura-2 exist. First, it requires ultraviolet (UV) excitation, which is more toxic to living cells than visible light. Second, our ability to use fura-2 for accurate, stable, ratio-based determinations of Ca2+ concentration in living cells is fast becoming a method of the past. This is due, in part, because modern microscopes are phasing out the use of mercury arc lamps that provide the UV excitation needed for fura-2 imaging. To address this problem, we describe the design, synthesis, and cellular application of benzo[b]phosphole-based fluorescent Ca2+ indicators for ratio-based imaging of Ca2+ in living cells that can be used with modern light emitting diode (LED)-equipped fluorescence microscopes. We report isoCaRed-1Me, a Ca2+ indicator that enables ratio-based imaging in immortalized cell lines, primary mammalian hippocampal neurons, and human-induced pluripotent stem cell-derived cardiomyocytes. These data show that isoCaRed-1Me will be useful for ratio-based Ca2+ imaging using modern microscopes.

Keywords:  fluorescence; fluorophore; imaging; physiology

DOI:  https://doi.org/10.1073/pnas.2410436122
Trends Cell Biol. 2025 Feb 12. pii: S0962-8924(25)00028-5. [Epub ahead of print]

Metabolic control of replisome plasticity in genome surveillance.

Mikkel Bo Petersen, Gita Chhetri, Kumar Somyajit.

  Metabolic pathways and DNA replication are both adaptable and essential for early development and cancer progression. While each process is well understood individually, the mechanisms coordinating them are just beginning to emerge. Nucleotide biosynthesis serves as a crucial link, with fluctuating nucleotide pools leading to imbalanced deoxyribonucleotide (dNTP) and increased ribonucleotide (rNTP) levels, impairing DNA synthesis and triggering replication stress; ultimately driving developmental disorders and cancer. To counter these challenges, the replisome - the core machinery of DNA replication - continuously adjusts its architecture and speed in response to physiological changes, including nucleotide fluctuations. This review outlines recent insights into how the replisome aligns its function with metabolic changes in nucleotide levels and explores emerging links between metabolism and genome stability, and their roles in development and disease.

Keywords:  cancer targeting; nucleotide fluctuations; redox signaling; replication fork speed; replisome; ribonucleotide reductase

DOI:  https://doi.org/10.1016/j.tcb.2025.01.006
Mol Brain. 2025 Feb 12. 18(1): 11

Development of selective deconjugases for membrane-anchored LC3A/B in post-mitotic neurons.

Haneul Choi, Sang-Won Park, Deok-Jin Jang, Jin-A Lee.

  Neuronal autophagy is essential for maintaining protein and organelle turnover, thereby safeguarding neuronal health. LC3, a central autophagy protein, exists in lipidated (LC3-II) and non-lipidated (LC3-I) forms, both critical for neurons due to their sensitivity to metabolic and proteostatic stress. To elucidate the specific roles of membrane-anchored LC3A/B in post-mitotic neurons, we engineered deconjugases with enhanced selectivity for lipidated LC3. By modifying LC3-interacting regions (LIRs) at the deconjugase termini, we significantly improved targeting specificity toward LC3A/B. Deconjugases with N-terminal LIR modifications reduced LC3A/B-associated autophagosomes, highlighting the importance of LIR positioning for specificity. Sequential N-terminal LIR arrangements further refined LC3A/B targeting without affecting GABARAP-associated autophagosomes. Moreover, reducing the hydrophobicity of the α3 helix to limit membrane residence time further improved selectivity. These targeted modifications demonstrate the potential of customized deconjugases to dissect and modulate specific autophagic pathways in neurons, paving the way for novel therapeutic strategies against neurodegenerative diseases associated with autophagy dysregulation.

Keywords:  Deconjugases; LC3/GABARAP; Neuronal autophagy; RavZ

DOI:  https://doi.org/10.1186/s13041-025-01184-z