bims-auttor 2025-06-29 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2025–06–29
thirty papers selected by
Viktor Korolchuk, Newcastle University

MAPK Signaling in the Interplay Between Oxidative Stress and Autophagy.
Acidosis attenuates the hypoxic stabilization of HIF-1α by activating lysosomal degradation.
Autophagy and Alzheimer's Disease: Mechanisms and Impact Beyond the Brain.
Sestrin2 improves intestinal ischemia-reperfusion injury through the TFEB-mediated autophagy/lysosomal pathway.
Analysis of Autophagy Under Abiotic Stress in Arabidopsis Seedlings Expressing the GFP-ATG8 Autophagosome Marker.
Rag GTPases control lysosomal acidification by regulating v-ATPase assembly in Drosophila.
Inhibition of lysosomal LAMTOR1 increases autophagy by suppressing the MTORC1 pathway to ameliorate lipid accumulations in MAFLD.
The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy.
NAD+ repletion restores cardioprotective autophagy and mitophagy in obesity-associated heart failure by suppressing excessive trophic signaling.
Retinal Autophagy for Sustaining Retinal Integrity as a Proof of Concept for Age-Related Macular Degeneration.
Cooperative Function of Atg8- and TORC1-Mediated Activities in Yeast.
TNIP1 and Autophagy Receptors regulate STING Signaling.
Transferrin receptor controls both autophagosome formation and closure via phosphatidylinositol 3-phosphate synthesis.
A substrate-interacting region of Parkin directs ubiquitination of the mitochondrial GTPase Miro1.
Microaggrephagy: an ESCRT-I-PTPN23-dependent pathway for MAPT/tau aggregate clearance.
Dynamic Interplay Between Autophagy and Oxidative Stress in Stem Cells: Implications for Regenerative Medicine.
Enhancing autophagy by redox regulation extends lifespan in Drosophila.
Autophagy-targeted NBR1-p62/SQSTM1 complexes promote breast cancer metastasis by sequestering ITCH.
Liver Metabolism at the Crossroads: The Reciprocal Control of Nutrient-Sensing Nuclear Receptors and Autophagy.
Mitochondrial protein nmd regulates lipophagy and general autophagy during development.
The transcription factors Tfeb and Tfe3 are required for survival and embryonic development of pancreas and liver in zebrafish.
Rapamycin Alleviates Heart Failure Caused by Mitochondrial Dysfunction and SERCA Hypoactivity in Syntaxin 12/13 Deficient Models.
Advances in Autophagy-Lysosomal Pathway and Neurodegeneration via Brain-Gut Axis.
A lysosomal surveillance response to stress extends healthspan.
Secondary accumulation of lyso-platelet activating factors in lysosomal storage diseases.
The human autophagy-initiating complexes ULK1C and PI3KC3-C1.
Calcium at the crossroads: TPC2's role in LRRK2-linked Parkinson's disease.
Exploration of Bromodomain Proteins as Drug Targets for Niemann-Pick Type C Disease.
Flow Cytometric Quantification of Mitochondrial Properties: A High-Throughput Approach for Single Organelle Analysis.
The expanding repertoire of ESCRT functions in cell biology and disease.

Antioxidants (Basel). 2025 May 30. pii: 662. [Epub ahead of print]14(6):

MAPK Signaling in the Interplay Between Oxidative Stress and Autophagy.

Enrico Desideri, Serena Castelli, Maria Rosa Ciriolo.

  The term autophagy identifies several mechanisms that mediate the degradation of intracellular and extracellular components via the lysosomal pathway. Three main forms of autophagy exist, namely macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy, which have distinct mechanisms but share lysosomes as the final destination of their cargo. A basal autophagic flux is crucial for the maintenance of cellular homeostasis, being involved in the physiological turnover of proteins and organelles. Several stressors, including nutrient shortage and genotoxic and oxidative stress, increase the autophagic rate, which prevents the accumulation of damaged and potentially harmful cell components, thus preserving cell viability. In this context, several studies have highlighted the role of MAPKs, serine-threonine kinases activated by several stimuli, in linking oxidative stress and autophagy. Indeed, several oxidative stressors activate autophagy by converging on MAPKs, directly or indirectly. In this regard, the different transcription factors that bridge MAPKs and autophagic activation are here described. In this review, we summarize the current knowledge regarding the regulation of autophagy by MAPK, including the atypical ones, with a particular focus on the regulation of autophagy by oxidative stress.

Keywords:  CMA; MAPK; atypical MAPK; autophagy; eMI; oxidative stress

DOI:  https://doi.org/10.3390/antiox14060662
J Cell Biol. 2025 Aug 04. pii: e202409103. [Epub ahead of print]224(8):

Acidosis attenuates the hypoxic stabilization of HIF-1α by activating lysosomal degradation.

Bobby White, Zhenyi Wang, Matthew Dean, Johanna Michl, Natalia Nieora, Sarah Flannery, Iolanda Vendrell, Roman Fischer, Alzbeta Hulikova, Pawel Swietach.

Hypoxia-inducible factors (HIFs) mediate cellular responses to low oxygen, notably enhanced fermentation that acidifies poorly perfused tissues and may eventually become more damaging than adaptive. How pH feeds back on hypoxic signaling is unclear but critical to investigate because acidosis and hypoxia are mechanistically coupled in diffusion-limited settings, such as tumors. Here, we examined the pH sensitivity of hypoxic signaling in colorectal cancer cells that can survive acidosis. HIF-1α stabilization under acidotic hypoxia was transient, declining over 48 h. Proteomic analyses identified responses that followed HIF-1α, including canonical HIF targets (e.g., CA9, PDK1), but these did not reflect a proteome-wide downregulation. Enrichment analyses suggested a role for lysosomal degradation. Indeed, HIF-1α destabilization was blocked by inactivating lysosomes, but not proteasome inhibitors. Acidotic hypoxia stimulated lysosomal activity and autophagy via mammalian target of rapamycin complex I (mTORC1), resulting in HIF-1α degradation. This response protects cells from excessive acidification by unchecked fermentation. Thus, alkaline conditions are permissive for at least some aspects of HIF-1α signaling.

DOI: https://doi.org/10.1083/jcb.202409103
Cells. 2025 Jun 16. pii: 911. [Epub ahead of print]14(12):

Autophagy and Alzheimer's Disease: Mechanisms and Impact Beyond the Brain.

Zaw Myo Hein, Thirupathirao Vishnumukkala, Barani Karikalan, Aisyah Alkatiri, Farida Hussan, Saravanan Jagadeesan, Mohd Amir Kamaruzzaman, Muhammad Danial Che Ramli, Che Mohd Nasril Che Mohd Nassir, Prarthana Kalerammana Gopalakrishna.

  Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by neuronal loss, cognitive decline, and pathological hallmarks such as amyloid-beta (Aβ) plaques and tau neurofibrillary tangles. Recent evidence highlights autophagy as a pivotal mechanism in cellular homeostasis, mediating the clearance of misfolded proteins and damaged organelles. However, impaired autophagy contributes significantly to AD pathogenesis by disrupting proteostasis, exacerbating neuroinflammation, and promoting synaptic dysfunction. This review aims to scrutinize the intricate relationship between autophagy dysfunction and AD progression, explaining key pathways including macroautophagy, chaperone-mediated autophagy (CMA), and selective autophagy processes such as mitophagy and aggrephagy. This further extends the discussion beyond the central nervous system, evaluating the role of hepatic autophagy in Aβ clearance and systemic metabolic regulation. An understanding of autophagy's involvement in AD pathology via various mechanisms could give rise to a novel therapeutic strategy targeting autophagic modulation to mitigate disease progression in the future.

Keywords:  Alzheimer’s disease; amyloid-beta clearance; autophagy; neurodegeneration; tau pathology

DOI:  https://doi.org/10.3390/cells14120911
Free Radic Biol Med. 2025 Jun 24. pii: S0891-5849(25)00787-7. [Epub ahead of print]

Sestrin2 improves intestinal ischemia-reperfusion injury through the TFEB-mediated autophagy/lysosomal pathway.

Jing-Yuan Xie, Man Ye, Jie Luo, Ning Hu, Yi-Guo Zhang, Le-le Zhang, Zi-Yi Wen, Xiang-Yun Fu, Rong Chen, Qing-Tao Meng.

  Sestrin2 is a stress-inducible protein that exhibits protective effects against ischemia-reperfusion injury in various organs. However, the specific roles and mechanisms of Sestrin2 in intestinal ischemia-reperfusion (IIR) injury have yet to be fully elucidated. The present study aims to investigate the role of Sestrin2 in intestinal IIR injury and its underlying mechanisms. We found that in the IIR model of C57BL/6J mice, Sestrin2 expression increased following IIR injury, accompanied by enhanced lysosomal activity and autophagy activation. Further cellular experiments demonstrated that overexpression of Sestrin2 increased autophagic flux, enhanced lysosomal activity, and mitigated cellular injury. These effects were abrogated by Sestrin2 knockdown. Additionally, we discovered that Sestrin2 interacts with transcription factor EB (TFEB), and that knockdown of Sestrin2 resulted in decreased nuclear translocation of TFEB, leading to a reduction in autophagic flux due to impaired lysosomal function. The TFEB activator (TFEB A1) promoted TFEB nuclear translocation and reversed autophagy/lysosomal pathway (ALP) dysfunction and cellular damage caused by Sestrin2 knockdown. In conclusion, Sestrin2 protects against IIR injury by promoting TFEB nuclear translocation, enhancing lysosomal activity, accelerating autophagosome turnover and substrate degradation, and increasing autophagic flux. These findings provide novel insights and potential targets for the treatment of IIR injury.

Keywords:  Intestinal ischemia-reperfusion; Lysosomal activation; Sestrin2; TFEB; autophagy

DOI:  https://doi.org/10.1016/j.freeradbiomed.2025.06.037
Curr Protoc. 2025 Jun;5(6): e70166

Analysis of Autophagy Under Abiotic Stress in Arabidopsis Seedlings Expressing the GFP-ATG8 Autophagosome Marker.

William Agbemafle, Vishadinie Jayasinghe, Diane C Bassham.

  Plant autophagy is a catabolic process where cellular components such as protein aggregates and dysfunctional organelles are degraded and recycled to maintain homeostasis and facilitate stress resilience. Autophagy relies on a double-membrane vesicle called the autophagosome, which delivers cellular cargo to the vacuole for degradation. The Arabidopsis GFP-ATG8 reporter line is a valuable tool widely used to visualize and quantify autophagosomes via microscopy and monitor autophagic degradation via immunoblotting. Consistent assessment of autophagic activity requires standardized protocols for sample preparation, imaging, and data analysis. Here, we present protocols for monitoring autophagy in Arabidopsis seedlings expressing GFP-ATG8, including treatments to induce or inhibit autophagic flux, as well as imaging and image analysis procedures. These methods enable reliable evaluation of autophagic activity and can be adapted for diverse experimental conditions and genotypes. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth of Arabidopsis seedlings Basic Protocol 2: Activation of autophagy in Arabidopsis seedlings by abiotic stresses Basic Protocol 3: Inhibition of vacuolar degradation by concanamycin A treatment Basic Protocol 4: Quantification of GFP-ATG8-labeled autophagosomes in Arabidopsis seedlings via microscopy Basic Protocol 5: Analysis of autophagic degradation of GFP-ATG8 via immunoblotting.

Keywords:  Arabidopsis; GFP‐ATG8; autophagic bodies; autophagosomes; autophagy; immunoblotting; microscopy

DOI:  https://doi.org/10.1002/cpz1.70166
J Biol Chem. 2025 Jun 19. pii: S0021-9258(25)02250-1. [Epub ahead of print] 110400

Rag GTPases control lysosomal acidification by regulating v-ATPase assembly in Drosophila.

Ying Zhou, Xiaodie Yang, Wenyu Xu, Sulin Shen, Weikang Fan, Guoqiang Meng, Yang Cheng, Yingying Lu, Youheng Wei.

  The Rag GTPases play an important role in sensing amino acids and activating the target of rapamycin complex 1 (TORC1), a master regulator of cell metabolism. Previously, we have shown that GDP-bound RagA stimulates lysosome acidification and autophagic degradation, which are essential for young egg chamber survival under starvation in Drosophila. However, the underlying mechanism is unclear. Here we demonstrate that the GDP-bound RagA breaks the physical interaction between chaperonin containing tailless complex polypeptide 1 (CCT) and Vacuolar H+-ATPase (v-ATPase) subunit V1, and thus promotes the assembly of active v-ATPase and increases the lysosomal acidification. Consistently, knockdown of CCT complex components rescued the accumulation of defective autolysosomes in RagA RNAi. Moreover, the knockdown of Lamtor4, a component of lysosomal adaptor and MAPK and mTOR activator (LAMTOR) that anchors Rag GTPases to the lysosome, resulted in autolysosome accumulation, suggesting that RagGTPases regulate lysosomal acidification depend on their lysosomal localization. Knockdown of the CCT complex components attenuated the autophagic defects in Lamtor 4 RNAi. Our work highlights the interaction between CCT and v-ATPase in regulating lysosomal acidification.

Keywords:  Drosophila melanogaster; Rag GTPases; V-ATPase assembly; autophagy; chaperonin containing tailless complex polypeptide 1

DOI:  https://doi.org/10.1016/j.jbc.2025.110400
Autophagy. 2025 Jun 23.

Inhibition of lysosomal LAMTOR1 increases autophagy by suppressing the MTORC1 pathway to ameliorate lipid accumulations in MAFLD.

Yunyeong Jang, Minjeong Ko, Ju Yeon Lee, Jin Young Kim, Eun-Woo Lee, Ho Jeong Kwon.

  Metabolic dysfunction-associated fatty liver disease (MAFLD) is a serious metabolic disorder characterized by fat accumulation in the liver, which can trigger liver inflammation and fibrosis, potentially leading to cirrhosis or liver cancer. Despite many studies, effective treatments for MAFLD remain elusive due to its complex etiology. In this study, we have focused on the discovery of therapeutic agents and molecular targets for MAFLD treatment. We demonstrated that the natural compound acacetin (ACA) alleviates MAFLD by regulating macroautophagy/autophagy in a CDAHFD mouse model of rapidly induced steatohepatitis. In addition, ACA inhibits lipid accumulation in 3T3-L1 adipocytes through autophagy induction. To identify the target responsible for the autophagy activity induced by ACA, we performed drug affinity responsive target stability (DARTS) combined with LC-MS/MS proteomic analysis. This led to the identification of LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and MTOR activator 1), a lysosomal membrane adaptor protein. We found that binding of ACA to LAMTOR1 induces its release from the LAMTOR complex, leading to inhibition of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1), thereby increasing autophagy. This process helps ameliorate metabolic disorders by modulating the MTORC1-AMPK axis. Genetic knockdown of LAMTOR1 phenocopies the effects of ACA treatment, further supporting the role of LAMTOR1 as a target of ACA. These findings suggest LAMTOR1 plays a crucial role in ACA's therapeutic effects on MAFLD. In summary, our study identifies LAMTOR1 as a key protein target of ACA, revealing a potential therapeutic avenue for MAFLD by modulating autophagy via the LAMTOR1-MTORC1-AMPK signaling pathway.

Keywords:  Acacetin; DARTS; LAMTOR1; MAFLD; MTORC1; autophagy

DOI:  https://doi.org/10.1080/15548627.2025.2519054
Elife. 2025 06 24. pii: RP101531. [Epub ahead of print]13

The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy.

Yutaro Hama, Yuko Fujioka, Hayashi Yamamoto, Noboru Mizushima, Nobuo N Noda.

  In mammals, autophagosome formation, a central event in autophagy, is initiated by the ULK complex comprising ULK1/2, FIP200, ATG13, and ATG101. However, the structural basis and mechanism underlying the ULK complex assembly have yet to be fully clarified. Here, we predicted the core interactions organizing the ULK complex using AlphaFold, which proposed that the intrinsically disordered region of ATG13 engages the bases of the two UBL domains in the FIP200 dimer via two phenylalanines and also binds the tandem microtubule-interacting and transport domain of ULK1, thereby yielding the 1:1:2 stoichiometry of the ULK1-ATG13-FIP200 complex. We validated the predicted interactions by point mutations and demonstrated direct triad interactions among ULK1, ATG13, and FIP200 in vitro and in cells, wherein each interaction was additively important for autophagic flux. These results indicate that the ULK1-ATG13-FIP200 triadic interaction is crucial for autophagosome formation and provides a structural basis and insights into the regulation mechanism of autophagy initiation in mammals.

Keywords:  ATG13; AlphaFold; FIP200; ULK complex; autophagy; cell biology; human

DOI:  https://doi.org/10.7554/eLife.101531
Autophagy. 2025 Jun 24.

NAD+ repletion restores cardioprotective autophagy and mitophagy in obesity-associated heart failure by suppressing excessive trophic signaling.

Mahmoud Abdellatif, Francisco Vasques-Nóvoa, João Pedro Ferreira, Junichi Sadoshima, Abhinav Diwan, Wolfgang A Linke, Guido Kroemer, Simon Sedej.

  Macroautophagy/autophagy is markedly inhibited in the hearts of elderly obese patients with heart failure and preserved ejection fraction (HFpEF). However, the therapeutic relevance and underlying signaling mechanisms of the decline of autophagy in HFpEF remain unclear. We observed that therapeutic nicotinamide adenine dinucleotide (NAD+) repletion via nicotinamide supplementation restores cardioprotective autophagy and mitophagy in preclinical models of obesity-related HFpEF. Targeted and untargeted cardiac acetylome profiling revealed no significant deacetylation of essential autophagy-related proteins, including ATG5, ATG7 and mammalian Atg8-family members (ATG8s), suggesting a SIRT (sirtuin)-independent mechanism of autophagy induction by nicotinamide. Instead, cardiac transcriptomic analysis revealed major shifts in insulin-IGF1 (insulin-like growth factor 1) signaling, a known autophagy inhibitory pathway. Nicotinamide supplementation reversed the HFpEF-associated increase in insulin-IGF1 signaling, whereas exogenous IGF1 counteracts nicotinamide-induced autophagy. Importantly, nicotinamide fails to exert cardioprotective effects in mice lacking the autophagy-related protein ATG5 in cardiomyocytes, implicating autophagy as essential for the therapeutic response. In patients with HFpEF, a metabolic shift diverting nicotinamide away from NAD+ biosynthesis toward catabolism strongly correlates with worsening heart failure and increased cardiovascular mortality, even after adjusting for traditional risk factors. In sum, we demonstrate that NAD+ replenishment improves cardiometabolic HFpEF by restoring cardiac autophagy through suppression of excessive IGF1 signaling.

Keywords:  Acetylation; HFpEF; IGF1; insulin; nutrient signaling; sirtuins

DOI:  https://doi.org/10.1080/15548627.2025.2522127
Int J Mol Sci. 2025 Jun 16. pii: 5773. [Epub ahead of print]26(12):

Retinal Autophagy for Sustaining Retinal Integrity as a Proof of Concept for Age-Related Macular Degeneration.

Roberto Pinelli, Gloria Lazzeri, Caterina Berti, Francesca Biagioni, Elena Scaffidi, Michela Ferrucci, Violet Vakunseh Bumah, Francesco Fornai.

  Current evidence indicates that most types of autophagy represent a pivot in promoting retinal integrity. In healthy conditions, autophagy acts on multiple pathways, which are fundamental for the biochemistry and the fine structure of the retina. Autophagy is essential in granting visual processes. On the other hand, autophagy dysfunction characterizes several retinal disorders. This is mostly evident in age-related macular degeneration (AMD), which represents the most common degenerative disease leading to blindness. The involvement of autophagy in AMD is documented in vitro and in vivo experiments, and it is strongly suggested by clinical findings in humans. The present manuscript provides an overview of the specific types of autophagy, which prevail in the retina and their alterations in retinal degeneration with an emphasis on AMD. The dysfunction of specific autophagy steps was analyzed in relation to hallmarks of AMD pathology and symptoms. An extended session of the manuscript analyzes the connection between altered autophagy and cell pathology within retinal pigment epithelium, as well as the site and structure of extracellular aggregates named drusen. The significance of the drusen in relation to visual function is discussed in the light of the role of autophagy in regulating key steps of phototransduction.

Keywords:  autophagoproteasome; drusen; lipophagy; lysosome; mitophagy; proteasome; pseudodrusen; retinal degeneration; retinal neurovascular unit; retinal pigment epithelium

DOI:  https://doi.org/10.3390/ijms26125773
Yeast. 2025 Jun 26.

Cooperative Function of Atg8- and TORC1-Mediated Activities in Yeast.

Yumiko Oba, Miyuki Higuchi, Naoka Takahashi, Haruko Katsuta, Naoki Koike, Takashi Ushimaru, Yoko Kimura.

  The target of rapamycin complex 1 (TORC1) protein kinase plays an important role in regulating various cellular activities in response to nutrient availability. In this study, an autophagy-related protein 8 (atg8) mutant of Saccharomyces cerevisiae was highly sensitive to cellular processes in which TORC1 activity was inhibited by rapamycin treatment or by a mutated allele of KOG1 which encodes a subunit of TORC1. Atg8 exhibits both lipidation-dependent and -independent activities, each involving distinct factors. Lipidation of Atg8 is necessary for autophagy and functions with autophagy-related proteins like Atg7, whereas the lipidation-independent activities of Atg8 require Hfl1. The atg7Δhfl1Δ double mutant exhibited defects for the impaired TORC1 activities, suggesting that both lipidation-dependent and -independent functions of Atg8 are required for survival during impaired TORC1 activity. Moreover, atg8Δ and atg7Δhfl1Δ mutants exhibited sensitivity to metal ion Zn2+ during low-dose rapamycin treatment. The results suggest that Atg8-mediated functions and TORC1 signaling events play an important role in cell growth, possibly by maintaining vacuole integrity.

Keywords:  Atg8; Hfl1; TORC1; rapamycin; vacuole; yeast

DOI:  https://doi.org/10.1002/yea.4003
bioRxiv. 2025 Apr 23. pii: 2025.04.21.649822. [Epub ahead of print]

TNIP1 and Autophagy Receptors regulate STING Signaling.

Eric N Bunker, Tara D Fischer, Peng-Peng Zhu, François Le Guerroué, Richard J Youle.

Activation of the cGAS-STING pathway stimulates innate immune signaling as well as LC3B lipidation and ubiquitylation at Golgi-related vesicles upon STING trafficking. Although ubiquitylation at these subcellular sites has been associated with regulating NF-κB-related innate immune signaling, the mechanisms of Golgi-localized polyubiquitin chain regulation of immune signaling is not well understood. We report here that the ubiquitin- and LC3B-binding proteins, TNIP1 and autophagy receptors p62, NBR1, NDP52, TAX1BP1, and OPTN associate with STING-induced ubiquitin and LC3B-labeled vesicles, and that p62 and NBR1 act redundantly in spatial clustering of the LC3B-labeled vesicles in the perinuclear region. We also find that while TBK1 kinase activity is not required for the recruitment of TNIP1 and the autophagy receptors, it also plays a role in sequestration of the LC3B-labeled vesicles. The ubiquitin binding domains, rather than the LC3B-interacting regions, of TNIP1 and OPTN are specifically important for their recruitment to Ub/LC3B-associated perinuclear vesicles, while OPTN is also recruited through a TBK1-dependent mechanism. Functionally, we find that TNIP1 and OPTN play a role in STING-mediated innate immune signaling, with TNIP1 acting as a significant negative regulator of both NF-κB- and Interferon-mediated gene expression. Together, these results highlight autophagy-independent mechanisms of autophagy receptors and TNIP1 with unanticipated roles in regulating STING-mediated innate immunity.

DOI: https://doi.org/10.1101/2025.04.21.649822
Dev Cell. 2025 Jun 13. pii: S1534-5807(25)00327-2. [Epub ahead of print]

Transferrin receptor controls both autophagosome formation and closure via phosphatidylinositol 3-phosphate synthesis.

Claudia Puri, So Jung Park, Lidia Wrobel, David C Rubinsztein.

  Autophagosome formation involves multiple sequential steps that need to be coordinated and linked. Here, we describe in mammalian cells that the transferrin receptor (TfR) links LC3 family conjugation to phagophore membranes, an early step in autophagosome biogenesis, with subsequent autophagosome closure. TfR depletion impairs autophagic flux and its overexpression stimulates this catabolic process in an iron-independent manner. TfR is ubiquitinated by the ubiquitin ligase MARCH8 in the RAB11A-LC3B-positive membranes that are conjugated by LC3 family members from which phagophores emanate. Ubiquitinated TfR recruits the VPS34 component VPS15, enabling phosphatidylinositol 3-phosphate (PI(3)P) synthesis on nascent autophagosome membranes. This PI(3)P is not only important for LC3-lipid conjugation but also for subsequent phagophore closure, where TfR-dependent PI(3)P recruits the endosomal sorting complexes required for transport (ESCRT) complex. This TfR activity occurs after endocytosis of iron-containing transferrin, its canonical function, as TfR only binds VPS15 after iron detachment from transferrin that is enabled by pH lowering in the endocytic compartment.

Keywords:  ESCRT complex; PI(3)P; autophagosome closure; autophagy; transferrin receptor

DOI:  https://doi.org/10.1016/j.devcel.2025.05.016
J Cell Biol. 2025 Aug 04. pii: e202408025. [Epub ahead of print]224(8):

A substrate-interacting region of Parkin directs ubiquitination of the mitochondrial GTPase Miro1.

Joanna Koszela, Anne Rintala-Dempsey, Giulia Salzano, Viveka Pimenta, Outi Kamarainen, Mads Gabrielsen, Aasna L Parui, Gary S Shaw, Helen Walden.

Mutations in the E3 ubiquitin ligase Parkin gene have been linked to early onset Parkinson's disease. Besides many other roles, Parkin is involved in clearance of damaged mitochondria via mitophagy-a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognizes its substrates for ubiquitination. Here, we characterize a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results may explain fast kinetics of Miro1 ubiquitination by Parkin in recombinant assays and provide a biochemical explanation for Miro1-dependent Parkin recruitment to the mitochondrial membrane observed in cells. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinson's disease.

DOI: https://doi.org/10.1083/jcb.202408025
Autophagy. 2025 Jun 26.

Microaggrephagy: an ESCRT-I-PTPN23-dependent pathway for MAPT/tau aggregate clearance.

Shoshiro Hirayama, Shigeo Murata.

  The clearance mechanisms for ubiquitinated protein aggregates, such as MAPT/tau in neurodegenerative diseases, remain incompletely understood, particularly regarding the role of microautophagy. To identify mediators of this process, we performed an unbiased genome-wide CRISPR knockout screen using cells propagating MAPT/tau repeat domain (MAPT/tauRD) aggregates. This screen identified the ESCRT-I complex and the accessory protein PTPN23 as essential for the clearance of ubiquitinated MAPT/tauRD aggregates via a microautophagy-dependent pathway, operating independently of macroautophagy and chaperone-mediated autophagy. We designate this pathway "microaggrephagy". Mechanistically, microaggrephagy involves the recognition of polyubiquitinated aggregates by the ESCRT-I subunit TSG101, with PTPN23 acting as an adaptor bridging ESCRT-I and ESCRT-III to facilitate microautophagic engulfment. Furthermore, a disease-associated mutation in the ESCRT-I component UBAP1 disrupts its interaction with PTPN23 and impairs MAPT/tau clearance, implicating dysfunction of this pathway in neurodegenerative pathogenesis. These findings establish microaggrephagy as a distinct cellular mechanism for degrading pathological protein aggregates, provide a molecular basis for its function, and suggest potential therapeutic targets for proteinopathies.

Keywords:  ESCRT-I complex; MAPT/tau repeat domain (mapt/taurd); PTPN23; microaggrephagy; microautophagy; protein aggregates

DOI:  https://doi.org/10.1080/15548627.2025.2525866
Antioxidants (Basel). 2025 Jun 06. pii: 691. [Epub ahead of print]14(6):

Dynamic Interplay Between Autophagy and Oxidative Stress in Stem Cells: Implications for Regenerative Medicine.

Daniela Rossin, Maria-Giulia Perrelli, Marco Lo Iacono, Raffaella Rastaldo, Claudia Giachino.

  The crosstalk between autophagy and oxidative stress is a cornerstone of stem cell biology. These processes are tightly interwoven, forming a regulatory network that impacts stem cell survival, self-renewal, and differentiation. Autophagy, a cellular recycling mechanism, ensures the removal of damaged organelles and proteins, thereby maintaining cellular integrity and metabolic balance. Oxidative stress, driven by the accumulation of reactive oxygen species (ROS), can act as both a signalling molecule and a source of cellular damage, depending on its levels and context. The interplay between autophagy and oxidative stress shapes stem cell fate by either promoting survival under stress conditions or triggering senescence and apoptosis when dysregulated. Recent evidence underscores the bidirectional relationship between these processes, where autophagy mitigates oxidative damage by degrading ROS-generating organelles, and oxidative stress can induce autophagy as a protective response. This crosstalk is critical not only for preserving stem cell function but also for addressing age-related decline and enhancing regenerative potential. Understanding the molecular mechanisms that govern this interplay offers novel insights into stem cell biology and therapeutic strategies. This review delves into the intricate molecular dynamics of autophagy and oxidative stress in stem cells, emphasizing their synergistic roles in health, disease, and regenerative medicine applications.

Keywords:  ROS; ageing; antioxidant; autophagy; mitophagy; regenerative medicine; stem cell

DOI:  https://doi.org/10.3390/antiox14060691
Nat Commun. 2025 Jun 25. 16(1): 5379

Enhancing autophagy by redox regulation extends lifespan in Drosophila.

Claudia Lennicke, Ivana Bjedov, Sebastian Grönke, Katja E Menger, Andrew M James, Jorge Iván Castillo-Quan, Lucie A G van Leeuwen, Andrea Foley, Marcela Buricova, Jennifer Adcott, Alex Montoya, Holger B Kramer, Pavel V Shliaha, Angela Logan, Filipe Cabreiro, Michael P Murphy, Linda Partridge, Helena M Cochemé.

Dysregulation of redox homeostasis is implicated in the ageing process and the pathology of age-related diseases. To study redox signalling by H2O2 in vivo, we established a redox-shifted model by manipulating levels of the H2O2-degrading enzyme catalase in Drosophila. Here we report that ubiquitous over-expression of catalase robustly extends lifespan in females. As anticipated, these flies are strongly resistant to a range of oxidative stress challenges, but interestingly are sensitive to starvation, which could not be explained by differences in levels of energy reserves. This led us to explore the contribution of autophagy, which is an important mechanism for organismal survival in response to starvation. We show that autophagy is essential for the increased lifespan by catalase upregulation, as the survival benefits are completely abolished upon global autophagy knock-down. Furthermore, using a specific redox-inactive knock-in mutant, we highlight the in vivo role of a key regulatory cysteine residue in Atg4a, which is required for the lifespan extension in our catalase model. Altogether, these findings confirm the redox regulation of autophagy in vivo as an important modulator of longevity.

DOI: https://doi.org/10.1038/s41467-025-60603-w
Nat Cell Biol. 2025 Jun 27.

Autophagy-targeted NBR1-p62/SQSTM1 complexes promote breast cancer metastasis by sequestering ITCH.

Gourish Mondal, Hugo Gonzalez, Timothy Marsh, Andrew M Leidal, Ariadne Vlahakis, Pravin R Phadatare, Sofı́a Bustamante Eguiguren, Michael Bruck, Akul Naik, Mark Jesus M Magbanua, Laura A Huppert, Arun P Wiita, Jeroen P Roose, Jennifer M Rosenbluth, Jayanta Debnath.

Autophagy deficiency in breast cancer promotes metastasis through the accumulation of the autophagy cargo receptor NBR1. Here we show that autophagy normally suppresses breast cancer metastasis by enabling the clearance of NBR1-p62/SQSTM1 complexes that instruct p63-mediated pro-metastatic basal differentiation programmes. When autophagy is inhibited, the autophagy cargo receptors NBR1 and p62/SQSTM1 accumulate within biomolecular condensates in cells, which drives basal differentiation in both mouse and human breast cancer models. Mechanistically, these NBR1-p62/SQSTM1 complexes sequester ITCH, a ubiquitin ligase that degrades and negatively regulates p63 in breast cancer cells, thereby stabilizing and activating p63. Accordingly, mutant forms of NBR1 unable to sequester ITCH into NBR1-p62/SQSTM1 complexes do not promote basal differentiation and metastasis in vivo. Overall, our findings illuminate how proteostatic defects arising in the setting of therapeutic autophagy inhibition modulate epithelial lineage fidelity and metastatic progression.

DOI: https://doi.org/10.1038/s41556-025-01689-8
Int J Mol Sci. 2025 Jun 18. pii: 5825. [Epub ahead of print]26(12):

Liver Metabolism at the Crossroads: The Reciprocal Control of Nutrient-Sensing Nuclear Receptors and Autophagy.

Eun Young Kim, Jae Man Lee.

  Peroxisome proliferator-activated receptor α (PPARα, encoded by NR1C1) and farnesoid X receptor (FXR, encoded by NR1H4) are the two prominent nutrient-sensing nuclear receptors essential for maintaining hepatic metabolism during fasting and fed states, respectively. These nuclear receptors comprehensively regulate the transcription of numerous genes involved in fatty acid oxidation (FAO), ketogenesis, bile acid (BA) biosynthesis, and other metabolic processes critical for liver energy homeostasis. These receptors have been shown to have opposite impacts on autophagy, which is triggered by PPARα activation but inhibited by FXR activation. Recent studies have further revealed that liver-specific genetic ablation of key autophagic genes tremendously impairs the activation of these nuclear receptors, thereby profoundly affecting hepatic metabolism in both fasting and feeding states. This review explores the roles and mechanisms of PPARα and FXR in regulating liver metabolism and autophagy, highlighting the necessity of basal autophagic activity in ensuring the proper signaling of these nutrient-sensing nuclear receptors. Finally, we examine the potential therapeutic strategies that leverage the interplay between PPARα, FXR, and autophagy for the treatment of metabolic liver disorders. We also delve into the clinical implications of this complex relationship, emphasizing its significance for translational medicine and future therapeutic interventions.

Keywords:  FXR; PPARα; autophagy; liver; nuclear receptor

DOI:  https://doi.org/10.3390/ijms26125825
Autophagy. 2025 Jun 26.

Mitochondrial protein nmd regulates lipophagy and general autophagy during development.

Wei Wang, Xufeng Wang, Xiaoqi Zhou, Lu Jiang, Weina Shang, Liquan Wang, Chao Tong.

  Lipophagy engulfs lipid droplets and delivers them to lysosomes for degradation. We found that lipophagy levels were low in most fly tissues, except for the prothoracic gland (PG) during larval development. Therefore, we performed a small-scale screening in the PG to identify regulators of lipophagy. We discovered that the loss of nmd, a gene encoding a mitochondrial AAA-ATPase, led to developmental failure and reduced lipophagy in the PG. Further studies indicated that nmd was not only required for lipophagy but also essential for general macroautophagy/autophagy in both PG and fat body tissues. Autophagy was induced but blocked at the autophagosome-lysosome fusion stage upon nmd reduction. Additionally, nmd interacted with mitochondrial protein import machinery, such as Tom20, Tom40, and the import cargo, such as Idh. Loss of nmd decreased protein import into mitochondria. Similar to the loss of nmd, reduction of Tom20 or Tom40 also resulted in reduced lipophagy in the PG. In adult flies, reducing nmd expression in the eyes caused lipid droplet accumulation and severe degeneration during aging. Overexpression of bmm, a triglyceride lipase, reduced lipid droplets in the eye but did not rescue the eye degeneration caused by the reduction of nmd.

Keywords:  Drosophila; lipophagy; mitochondrial protein import; neuronal homeostasis; nmd; prothoracic gland

DOI:  https://doi.org/10.1080/15548627.2025.2522124
PLoS Genet. 2025 Jun 27. 21(6): e1011754

The transcription factors Tfeb and Tfe3 are required for survival and embryonic development of pancreas and liver in zebrafish.

Alberto Rissone, Martina La Spina, Erica Bresciani, Zulfeqhar A Syed, Christian A Combs, Martha Kirby, Abdel Elkahloun, Vicky Chen, Raman Sood, Shawn M Burgess, Rosa Puertollano.

The transcription factors TFEB and TFE3 modulate expression of lysosomal, autophagic, and metabolic genes to restore energy and cellular homeostasis in response to a variety of stress conditions. Since their role during vertebrate development is less characterized, we used CRISPR/Cas9 to deplete tfeb, tfe3a, and tfe3b in zebrafish. The simultaneous lack of these genes compromised embryo survival during early development, with an almost complete lethality of the larvae by 8-10 dpf. The knockout animals showed apoptosis in brain and retina and alterations in pancreas, liver, and gut. Exocrine pancreas presented the most severe defects, with accumulation of abnormal zymogen granules leading to acinar atrophy in embryos and pancreatitis-like phenotypes in adults; likely due to a block of the autophagy machinery implicated in removal of damaged granules. Knockout animals displayed increased susceptibility to oxidative and heat-shock stress. Our work reveals an essential role of Tfeb and Tfe3 in maintaining cellular and tissue homeostasis during development.

DOI: https://doi.org/10.1371/journal.pgen.1011754
Adv Sci (Weinh). 2025 Jun 26. e07210

Rapamycin Alleviates Heart Failure Caused by Mitochondrial Dysfunction and SERCA Hypoactivity in Syntaxin 12/13 Deficient Models.

Run-Zhou Yang, Fang Li, Jiao Liu, Shu-Ang Li, Dan-Hua Liu, Zhuanbin Wu, Pei-Pei Liu, Wenju Liu, Bin Zhou, Cizhong Jiang, Haibing Zhang, Ying Yu, Jian-Sheng Kang.

  SYNTAXIN 12/13 (STX12), a member of the syntaxin protein family enriched in the brain and heart, plays important roles in vesicle recycling. Currently, the role of STX12 in cardiovascular physiology remains unclear. Using zebrafish and mice, it is shown that STX12 loss leads to pericardial edema, cardiac malformations, and heart failure. Stx12 depletion disrupts mitochondrial morphology, reduces iron and zinc levels, and impairs ATP production. Stx12-deficient cardiomyocytes exhibit prolonged repolarization due to decreased sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Treatment with rapamycin, an mTOR inhibitor, restores mitochondrial protein expression and function by prompting the TFEB-PGC1α axis, enhances SERCA activity via the CAMKII-phospholamban pathway, and reduces the expression of stress markers. These findings suggest that STX12 plays an important role in the energy metabolism and metal homeostasis of cardiomyocytes. Enhancing mitochondrial function, autophagy, and SERCA activity through the administration of rapamycin may provide a potential therapeutic approach for cardiomyopathies associated with STX12 deficiency and hypometabolism.

Keywords:  SERCA; Syntaxin 12/13; heart failure; mitochondria; rapamycin

DOI:  https://doi.org/10.1002/advs.202507210
Biomedicines. 2025 Jun 05. pii: 1390. [Epub ahead of print]13(6):

Advances in Autophagy-Lysosomal Pathway and Neurodegeneration via Brain-Gut Axis.

Ping Yao, Hailong Han.

  Background/Objectives: The autophagy-lysosomal pathway (ALP) is crucial for neuronal health by clearing misfolded proteins and damaged organelles. While much research has focused on ALP dysfunction in the central nervous system, new evidence shows its importance in the gut, where it affects neurodegeneration via the gut-brain axis. Past reviews have mainly studied the ALP's direct neuroprotective effects or the gut microbiota's role in neurodegeneration separately. However, the two-way relationship between the ALP and the gut microbiota in neurodegenerative diseases is not well understood. We combine the latest findings on the ALP's role in gut health, microbial imbalance, and neuroinflammation, providing a comprehensive view of their combined effects in Alzheimer's, Parkinson's, and Huntington's diseases. Methods: This narrative review synthesizes evidence from preclinical, clinical, and translational studies (2014-2025) to explore the interplay between the autophagy-lysosomal pathway (ALP) and the gut-brain axis in neurodegeneration. The literature was identified via PubMed and Web of Science using search terms including autophagy, lysosome, gut microbiota, neurodegeneration, and gut-brain axis, with additional manual screening of reference lists. The inclusion criteria prioritized studies elucidating molecular mechanisms (e.g., ALP-microbiota crosstalk), while excluding case reports or non-peer-reviewed sources. Results: The gut-brain axis facilitates bidirectional communication between the gut and the brain through neural, immune, and metabolic pathways. Autophagy dysfunction may disrupt intestinal homeostasis, promote gut microbiota dysbiosis, and trigger chronic neuroinflammation, ultimately accelerating neurodegeneration. Notably, strategies targeting the gut microbiota and restoring intestinal barrier function via the ALP have demonstrated promising potential in delaying the progression of neurodegenerative diseases. Conclusions: This review establishes the ALP as a dynamic regulator of gut-brain communication, highlighting microbiota-targeted therapies as promising strategies for neurodegeneration.

Keywords:  autophagy; gut microbiota; gut–brain axis; lysosome; neurodegenerative diseases

DOI:  https://doi.org/10.3390/biomedicines13061390
Nat Cell Biol. 2025 Jun 26.

A lysosomal surveillance response to stress extends healthspan.

Terytty Yang Li, Arwen W Gao, Rendan Yang, Yu Sun, Yuxuan Lei, Xiaoxu Li, Lin Chen, Yasmine J Liu, Rachel N Arey, Kimberly Morales, Raya B Liu, Wenzheng Wang, Ang Zhou, Tong-Jin Zhao, Weisha Li, Amélia Lalou, Qi Wang, Tanes Lima, Riekelt H Houtkooper, Johan Auwerx.

Lysosomes are cytoplasmic organelles central for the degradation of macromolecules to maintain cellular homoeostasis and health. However, how lysosomal activity can be boosted to counteract ageing and ageing-related diseases remains elusive. Here we reveal that silencing specific vacuolar H+-ATPase subunits (for example, vha-6), which are essential for intestinal lumen acidification in Caenorhabditis elegans, extends lifespan by ~60%. This longevity phenotype can be explained by an adaptive transcriptional response typified by induction of a set of transcripts involved in lysosomal function and proteolysis, which we termed the lysosomal surveillance response (LySR). LySR activation is characterized by boosted lysosomal activity and enhanced clearance of protein aggregates in worm models of Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis, thereby improving fitness. The GATA transcription factor ELT-2 governs the LySR programme and its associated beneficial effects. Activating the LySR pathway may therefore represent an attractive mechanism to reduce proteotoxicity and, as such, potentially extend healthspan.

DOI: https://doi.org/10.1038/s41556-025-01693-y
Mol Genet Metab. 2025 Jun 17. pii: S1096-7192(25)00171-4. [Epub ahead of print]145(4): 109180

Secondary accumulation of lyso-platelet activating factors in lysosomal storage diseases.

Pamela Kell, Sonali Mishra, Heather L Gray-Edwards, Douglas R Martin, Angel Gaudioso Guirado, María Dolores Ledesma, Ernesto R Bongarzone, Mark S Sands, Ellen Sidransky, Daniel S Ory, Xuntian Jiang.

  Lysosomal storage diseases (LSDs) are a group of inherited disorders caused by defects in genes that encode lysosomal enzymes, transmembrane proteins, or transport proteins. These defects typically lead to the accumulation of undegraded substrates or obstructed substances in lysosomes, serving as primary storage materials. However, in certain LSDs, secondary storage products-such as glycosphingolipids, phospholipids, and cholesterol-can also accumulate in tissues, independent of the primary enzyme or protein defect. In our recent studies, we identified lyso-platelet activating factors (lyso-PAFs) as secondary storage compounds in multiple LSDs, including Niemann-Pick disease type C1 (NPC1), GM2 activator deficiency, and GM1 gangliosidosis (GM1). Our ongoing work suggests that lyso-PAFs are also prevalent secondary storage products in Niemann-Pick disease type A (NPA), Sandhoff disease (SD), Tay-Sachs disease (TSD), and Krabbe disease (KD). We observed that elevated lyso-PAF levels were significantly correlated with the accumulation of primary storage substances in these disorders, indicating their potential as biomarkers for disease progression in these LSDs. Moreover, treatment with adeno-associated virus (AAV)-based gene therapies led to a reduction in lyso-PAF levels in the central nervous systems of TSD sheep and GM1 cats, further supporting their potential as biomarkers for therapeutic efficacy. While it remains unclear whether changes in lyso-PAFs contribute directly to disease pathology or simply reflect disease progression, further research into the enzymes involved in their synthesis and degradation is essential for uncovering their functional role in the cellular physiology and pathology of LSDs. Thus, further exploration of lyso-PAF in biofluids as prognostic and pharmacodynamic biomarkers is warranted.

Keywords:  Krabbe disease; Lyso-platelet activating factors; Niemann-Pick disease type A; Sandhoff disease; Tay-Sachs disease

DOI:  https://doi.org/10.1016/j.ymgme.2025.109180
J Biol Chem. 2025 Jun 19. pii: S0021-9258(25)02241-0. [Epub ahead of print] 110391

The human autophagy-initiating complexes ULK1C and PI3KC3-C1.

Minghao Chen, James Hurley.

The unc-51-like kinase complex (ULK1C) and the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) are the key regulators of macroautophagy initiation. Understanding the assembly and coordination of these two complexes is essential for deciphering their cellular regulation and targeting them for therapeutic enhancement. This review highlights recent advances in our understanding of the structural organization and activation mechanisms of ULK1C and PI3KC3-C1 at the molecular level and discusses their roles within the protein interaction network governing autophagy initiation.

DOI: https://doi.org/10.1016/j.jbc.2025.110391
Cell Calcium. 2025 Jun 17. pii: S0143-4160(25)00058-2. [Epub ahead of print]130 103049

Calcium at the crossroads: TPC2's role in LRRK2-linked Parkinson's disease.

Federica De Lazzari, Simone Wanderoy, Alexander J Whitworth.

  The pathogenic mechanisms of LRRK2 are hotly debated but regulation of lysosomal homeostasis has emerged as a leading focus area. In recent work, Gregori et al. show that Ca2+ release through the lysosomal Two-Pore Channel 2 (TPC2) could be a significant contributor to dopaminergic neuron vulnerability.

Keywords:  LRRK2; Lysosomal calcium; Parkinson's disease; TPC2

DOI:  https://doi.org/10.1016/j.ceca.2025.103049
Int J Mol Sci. 2025 Jun 16. pii: 5769. [Epub ahead of print]26(12):

Exploration of Bromodomain Proteins as Drug Targets for Niemann-Pick Type C Disease.

Martina Parente, Amélie Barthelemy, Claudia Tonini, Sara Caputo, Alessandra Sacchi, Stefano Leone, Marco Segatto, Frank W Pfrieger, Valentina Pallottini.

  Defects in lysosomal cholesterol handling provoke fatal disorders presenting neurovisceral symptoms with variable onset and life spans. A prime example is Niemann-Pick type C disease (NPCD), where cholesterol export from the endosomal-lysosomal system is impaired due to variants of either NPC intracellular cholesterol transporter 1 (NPC1) or NPC intracellular cholesterol transporter 2 (NPC2). Therapeutic options for NPCD are limited to palliative care and disease-modifying drugs, and there is a need for new treatments. Here, we explored bromodomain and extra-terminal domain (BET) proteins as new drug targets for NPCD using patient-derived skin fibroblasts. Treatment with JQ1, a prototype BET protein inhibitor, raised the level of NPC1 protein, diminished lysosomal expansion and cholesterol accumulation, and induced extracellular release of lysosomal components in a dose-, time-, and patient-dependent manner. Lastly, JQ1 enhanced and reduced cholesterol accumulation induced by pharmacologic inhibition of NPC1 and of histone deacetylase (HDAC) activity, respectively. Taken together, bromodomain proteins should be further explored as therapeutic drug targets for lysosomal diseases like NPCD, and as new components regulating lysosomal function and cholesterol metabolism.

Keywords:  BET inhibitor; cholesterol; drug therapy; epigenetic regulation; lysosome; rare disease

DOI:  https://doi.org/10.3390/ijms26125769
Int J Mol Sci. 2025 Jun 07. pii: 5481. [Epub ahead of print]26(12):

Flow Cytometric Quantification of Mitochondrial Properties: A High-Throughput Approach for Single Organelle Analysis.

Andrew J Piasecki, Hannah C Sheehan, Jonathan L Tilly, Dori C Woods.

  Recent advances in flow cytometry facilitate the detection of subcellular components, such as organelles and vesicles. Fluorescence-activated mitochondria sorting (FAMS) is a flow cytometry-based technique that allows for quantitative analysis and sorting of mitochondria as individual organelles from various tissues and in vitro cell culture. This manuscript details three novel applications of this technique to study mitochondrial function on an organelle-specific level, which is not possible with other approaches. Specifically, we detail the further development and versatility of this nanoscaled flow cytometry approach, including assays to quantitatively assess mitochondrial subpopulations, mitochondrial protein translocation, and both free-floating and EV-encapsulated secreted mitochondria. We demonstrate a multi-parameter quantitative assay for the analysis of mitochondrial autophagy using antibodies targeting the proteins PINK1 and Parkin corresponding to ΔΨM and further show how these can be assessed for mtDNA content on a single organelle level. Further, we establish parameters for the size and surface marker-based analysis of EVs, many of which contain identifiable and respiring mitochondria, as well as free-floating respiratory-competent mitochondria. These results display the versatility of nanoscaled flow cytometry in terms of both sample input and target organelle and provide an important methodological means for the quantitative assessment of mitochondrial features.

Keywords:  extracellular vesicle sorting; flow cytometry; fluorescence-activated mitochondria sorting; mitochondria; organelle sorting

DOI:  https://doi.org/10.3390/ijms26125481
Nature. 2025 Jun 25.

The expanding repertoire of ESCRT functions in cell biology and disease.

James H Hurley, Alyssa N Coyne, Marta Miączyńska, Harald Stenmark.

The endosomal sorting complex required for transport (ESCRT) is a multicomplex machinery comprising proteins that are conserved from bacteria to humans and has diverse roles in regulating the dynamics of cellular membranes. ESCRT functions have far-reaching consequences for cell biological processes such as intracellular traffic, membrane repair, cell signalling, metabolic regulation, cell division and genome maintenance. Here we review recent insights that emphasize the pathophysiological consequences of ESCRT dysfunctions, including infections, immune disorders, cancers and neurological diseases. We highlight the possibilities of using our knowledge about ESCRT structures and functions for drug discovery.

DOI: https://doi.org/10.1038/s41586-025-08950-y