The mechanisms by which pancreatic β cells sense and communicate environmental cues to mitochondria to maintain insulin secretion remain poorly understood. We identified that β cells transduce metabolic stress, such as during obesity or glucolipotoxicity, through the innate immune signaling adaptor TRAF6 to regulate mitochondrial quality control. TRAF6 was dispensable for β-cell function at baseline yet was essential for glucose-stimulated insulin secretion in mouse or human β cells following nutrient excess. TRAF6 was required for the recruitment of components of both ubiquitin- and receptor-mediated mitophagy pathways to clear dysfunctional mitochondria following metabolic stress. Interestingly, concomitant loss of the ubiquitin-mediated mitophagy effector Parkin restored glucose homeostasis and mitophagy flux in TRAF6-deficient β cells by relieving impediments in receptor-mediated mitophagy. Together, we found that TRAF6 forms a molecular relay linking environmental signals to multiple mitophagy pathways to equip β cells with the means to adapt to metabolic demand and prevent diabetes.Abbreviations: ADP: adenosine diphosphate; ATP: adenosine triphosphate; BNIP3: BCL2 interacting protein 3; GSIS: glucose-stimulated insulin secretion; HFD: high-fat diet; MFN2: mitofusin 2; PRKN: parkin RBR E3 ubiquitin protein ligase; RNF41/NRDP1: ring finger protein 41; SQSTM1/p62: sequestosome 1; T2D: type 2 diabetes; TAX1BP1: Tax1 binding protein 1; TLR: Toll-like receptor; TRAF6: TNF receptor associated factor 6.

Mitochondria of embryos in most plants and animals are inherited from the egg cell rather than the sperm cell, a phenomenon known as mitochondrial maternal inheritance. Nevertheless, how plants regulate paternal mitochondrial elimination (PME) has long remained a mystery. We discovered micromitophagy in higher plants and revealed that micromitophagy could serve as a specific mechanism underlying PME, which is triggered by fate determination of male germline (MG) cells. This process underpins maternal mitochondrial inheritance and normal early embryogenesis. Mechanistically, we demonstrate that the vacuole-localized ATG5 (autophagy-related protein 5) undergoes neofunctionalization and interacts with the mitochondrion-localized HSP90.2 (heat shock protein 90.2) to mediate paternal micromitophagy. Collectively, through interdisciplinary research on spermatogenesis, micromitophagy and mitochondrial inheritance, our work fills a long-standing gap in plant PME, and offers new insights into the functional divergence of autophagy-related genes and the adaptive evolution of autophagy.Abbreviations: PME, paternal mitochondrial elimination; 3-MA, 3-Methyladenine; ATG5, autophagy-related protein 5; HSP90.2, heat shock protein 90.2; MG, male germline; TEM, transmission electron microscopy; MVB, Multivesicular body.

Although the Golgi apparatus undergoes extensive disassembly during mitosis to ensure its equitable partitioning into two daughter cells, the extreme asymmetry of oocyte meiosis obviates this need, raising fundamental questions about the existence and necessity of Golgi disassembly in oocytes. Herein, we demonstrated the existence of a Golgi disassembly process during oocyte meiosis I, which peaked at germinal vesicle breakdown (GVBD) and was largely completed by metaphase II (MII). Strikingly, the vesiculation stage of Golgi disassembly exhibited a function divergent from mitosis by governing spindle cortical migration during metaphase I (MI), thereby ensuring oocyte polarity establishment. Moreover, we found that macroautophagy/autophagy negatively regulated the vesiculation stage of Golgi disassembly: its hyperactivation suppressed vesiculation, whereas its inhibition conversely accelerated this process. Mechanistically, MIA2 and SFT2D2 were identified as key downstream effectors through which autophagy controlled Golgi vesiculation. MIA2 mediated autophagy-dependent regulation of Golgi dynamics through its two LIR motifs, thereby linking autophagic signaling to Golgi remodeling. Specifically, autophagy activation downregulated MIA2, thus inhibiting the ubiquitin-dependent degradation of SFT2D2, consequently suppressing Golgi vesiculation and spindle migration during oocyte meiosis I; conversely, autophagy inhibition accelerated these processes by increasing MIA2 to promote SFT2D2 degradation. These findings elucidate the key role of autophagy in regulating Golgi disassembly, thereby providing a new organelle-based perspective on how autophagy contributes to oocyte spindle migration. Furthermore, our work identifies a novel Golgi-dependent mechanism for oocyte polarity establishment and uncovers the divergent functions of Golgi remodeling across distinct division modes.

Xenophagy, a selective autophagy pathway, is a critical innate immune defense mechanism that targets pathogens for lysosomal degradation. However, the molecular mechanisms enabling autophagosomes to specifically recognize and engulf bacteria remain incompletely understood. Here, we identify WIPI2, a core component that drives autophagosome biogenesis, as a novel phosphorylation substrate of TBK1 during Salmonella Typhimurium infection. We demonstrate that TBK1 phosphorylates WIPI2 at Ser96, which enhances its interaction with ATG16L1 and its binding to PtdIns3P. Crucially, the recruitment of WIPI2 to intracellular bacteria is dependent on its interaction with ATG16L1, which is localized to the Salmonella-containing vacuole. Furthermore, TBK1-mediated phosphorylation of WIPI2 is required for efficient bacterial clearance. Collectively, our findings reveal a molecular mechanism whereby TBK1-mediated phosphorylation of WIPI2 directs localized phagophore expansion around invading bacteria, thereby bridging bacterial recognition with autophagosome assembly.

We developed a computationally enhanced intravital imaging framework for high-resolution analysis of macroautophagy/autophagy dynamics in vivo. By integrating two-photon microscopy with an optimized tissue stabilization method, motion artifacts were minimized, enabling real-time imaging of LC3-positive structures in the liver and skeletal muscle under physiological stress conditions, including starvation, ischemia, and ethanol exposure. Image quality was further improved using a trained denoising model and enhanced Super-Resolution Radial Fluctuations (eSRRF), enabling nanoscale visualization of autophagy-related compartments. To differentiate increased autophagosome formation from impaired degradation, autophagic flux was assessed via chloroquine-mediated lysosomal inhibition, enabling functional interpretation beyond static structural measurements. Together, this pipeline overcomes major technical limitations of intravital imaging and provides a robust framework for integrated structural and functional investigation of autophagy and related intracellular processes in vivo.

Alphaviruses encompass a variety of human and animal pathogens, such as getah virus (GETV), chikungunya virus (CHIKV), and Sindbis virus (SINV). Increasing evidence suggests that innate immunity plays an important role in host resistance to alphavirus infection, while alphaviruses have evolved multiple strategies to evade host antiviral innate immunity. Mitochondria, as key immune hubs, play a central role in the initiation of innate antiviral responses. It remains unclear whether alphaviruses inhibit innate immune responses by manipulating mitochondria. In this study, we found that GETV infection damages mitochondrial structure and function, as shown by mitochondrial membrane potential depolarization, reduction in mitochondrial numbers, and an imbalance in mitochondrial dynamics. GETV infection induces mitophagy in host cells and promotes viral replication. Nsp3 protein of alphaviruses (CHIKV, SINV, GETV) can directly trigger receptor-mediated mitophagy through its interaction with BNIP3L/NIX. Knockdown of BNIP3L could alleviate mitophagy caused by GETV infection. Mitophagy induced by alphavirus Nsp3 protein leads to degradation of MAVS which inhibits the IFN response and promotes viral replication. Collectively, our results demonstrated that alphaviruses Nsp3 serves as a novel virulence factor, inducing BNIP3L-mediated mitophagy to degrade MAVS and evade the host immune response.

Selective autophagy is a regulated process that ensures the specific recognition and lysosomal degradation of defined intracellular substrates, including damaged organelles, protein aggregates, and invading pathogens. Despite intense research interest, several mechanistic aspects of selective autophagy remain incompletely understood. Comparative analysis across different models offers a valuable framework for identifying evolutionarily conserved molecular components and regulatory principles. In this review, we provide a detailed overview of seven major evolutionarily conserved selective autophagy pathways: mitophagy, xenophagy, pexophagy, lysophagy, ER-phagy, Golgiphagy, and ribophagy, building on data from four widely used model organisms (mammals, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae). We focus on the core molecular machinery including the modes of cargo recognition via already characterized and predicted selective receptors, and orchestration of cargo capture into forming autophagosomes.

Intracerebral hemorrhage (ICH) is a devastating neurological disorder characterized by extensive neuronal death, in which ferroptosis has emerged as a central pathological mechanism. Yet, the role of mitophagy in regulating ferroptosis during ICH remains elusive. Herein, we identified a lactylation-mediated HSPA5 (heat shock protein family A (Hsp70) member 5)-VGLL3 (vestigial like family member 3) signaling axis that confers neuroprotection following ICH. We observed a marked upregulation of VGLL3 expression in ICH models, and loss-of-function experiments revealed that VGLL3 silencing exacerbated BNIP3 (BCL2/adenovirus E1B interacting protein 3)-BNIP3L/NIX (BCL2/adenovirus E1B interacting protein 3-like)-mediated, mitophagy-dependent ferroptosis. Mechanistically, VGLL3 enhanced EGLN3 (egl-9 family hypoxia-inducible factor 3) expression and reduced HIF1A/HIF-1α (hypoxia inducible factor 1, alpha subunit) stability, thereby suppressing BNIP3-BNIP3L-driven mitophagy. Upstream, HSPA5 activated VGLL3, and HSPA5 knockdown phenocopied the effects of VGLL3 silencing. Pharmacological activation of HSPA5 with the selective agonist BIP inducer X (BIX) suppressed mitophagy-dependent ferroptosis in cultured neurons, while in vivo BIX administration alleviated neuronal death and improved motor function in ICH mice. Epigenetic analyses further demonstrated that HSPA5 expression was transcriptionally modulated by histone H3-Lys18 (H3K18) lactylation, while its non-histone lactylation was catalyzed by the acetyltransferase EP300 (E1A binding protein p300) and removed by the NAD-dependent protein deacetylase SIRT1 (sirtuin 1). Collectively, our findings delineate a lactylation-driven HSPA5-VGLL3-EGLN3-HIF1A/HIF1-1α axis that represses BNIP3-BNIP3L-mediated, mitophagy-dependent ferroptosis. Targeting this pathway may provide a mechanistic rationale and therapeutic strategy for mitigating neuronal injury and functional deficits following ICH.Abbreviations: BNIP3: BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; EGLN3: egl-9 family hypoxia-inducible factor 3; GPX4: glutathione peroxidase 4; GSH: glutathione; GSSG: oxidized glutathione; HIF1A/HIF-1α: hypoxia inducible factor 1, alpha subunit; HSPA5: heat shock protein family A (Hsp70) member 5; IB: immunoblot; ICH: intracerebral hemorrhage; MDA: malondialdehyde; ROS: reactive oxygen species; SLC7A11: solute carrier family 7 member 11; VGLL3: vestigial like family member 3.

Endoplasmic reticulum (ER)-phagy is an important ER quality-control pathway that selectively removes misfolded or unfolded proteins and damaged ER membranes through autophagic degradation in the vacuole. Recent studies have identified small guanosine triphosphatases (GTPases) as important regulators of the autophagy pathway. However, how they couple stress signals to ER-phagy remains poorly understood in plants. Our recent work demonstrated that RABC1 regulates ER-phagy under dithiothreitol (DTT)-, tunicamycin (TM)-, and heat-induced ER stress through interaction with the exocyst complex component SEC5A. Upon ER stress, green fluorescent protein (GFP)-RABC1 was recruited to the ATG8e-positive autophagosomes from the ER and Golgi in root cells, whereas autophagic flux and calnexin 1 (CNX1)-mRFP-tagged ER membranes turnover were impaired in the rabc1 mutant. Together, our findings support a model in which RABC1 acts as a molecular switch that interacts with distinct effectors to regulate autophagy under different stress conditions.Abbreviations: RAB, ras-related in brain; ER, endoplasmic reticulum; GTPases, guanosine triphosphatases; GFP, green fluorescent protein; CNX1, calnexin 1; GDP, guanosine diphosphate; GTP, guanosine triphosphate; SAR1D, secretion associated ras related GTPase 1D; COPII, coat protein complex II; LDs, lipid droplets; DTT, dithiothreitol; TM, tunicamycin; TOR, target of rapamycin; ATG, autophagy-related; Rho, ras homology.

At Autophagy we request that authors express centrifugal values as relative centrifugal force (RCF) or g-force as opposed to revolutions per minute (RPM). This is not an arbitrary decision but to understand the reason we need to provide a brief explanation of the difference between these two terms and, yes, there is a difference. Hence, it is annoying when during the editing process we request that an author convert a stated value such as "12,000 RPM" to "×g" and they simply return the paper with "12,000 ×g." The problem is that "12,000 RPM" is not equivalent to "12,000 ×g."

Effector-triggered immunity (ETI) safeguards intestinal epithelial cells (IECs) by surveilling bacterial virulence effectors or the damage they cause, such as pore-forming toxins (PFTs) that breach plasma membrane integrity (PMI). However, how metazoan ETI regulators guard IEC homeostasis compromised by PFT in vivo remains elusive. Moreover, while the molecular architectures of ETI are well-defined in plants, functionally analogous systems in animals are only beginning to emerge. Here, we unveil a conserved ETI guard system - the intestinal ETI regulator methyl-HLH-30/TFEB upregulates a novel intrinsic cellular defense (INCED) orchestrator Y54G11A.4/TTC38 - in Caenorhabditis elegans and human IECs. This conserved ETI system coordinates the restoration of PMI, microvilli architecture, and nutrient absorption concurrently by promoting phase condensation of the PTRN-1/CAMSAP-dependent guard program through an HSPA/HSP70-HSP90 chaperone system. Together, our findings reveal an intestinal ETI guard system that couples PFT-damage sensing to comprehensive epithelial recuperation, underscoring its physiological relevance for mucosal homeostasis across metazoans.Abbreviations and acronyms B. thuringiensis (Bt): Bacillus thuringiensis; CAMSAP: calmodulin regulated spectrin associated protein; C. elegans: Caenorhabditis elegans; Cry toxins: crystal toxins; ETI: effector-triggered immunity; FRAP: fluorescence recovery after photobleaching; FRET: Förster/fluorescence resonance energy transfer; GFP: green fluorescent protein; hlh/HLH: helix loop helix; HSP: heat shock protein; IDRs: intrinsically disordered regions; IECs: intestinal epithelial cells; INCED: intrinsic cellular defense; IPTG: isopropyl-β-D-1-thiogalactopyranoside; lgg/LGG: LC3 and GABARAP family; ncMTOC: non-centrosomal microtubule-organizing center; LLPS: liquid-liquid phase separation; PFTs: pore-forming toxins; PI: propidium iodide; PMI: plasma membrane integrity; PTMs: post-translational modifications; qRT-PCR: quantitative real-time PCR; RNAi: ribonucleic acid interference; SEM: scanning electron microscopy; SLO: streptolysin O; SQST-1/SQSTM1/p62: sequestosome related 1; TFEB: transcription factor EB; TPR: tetratricopeptide repeat motifs; TTC38: tetratricopeptide repeat domain 38.

Mitochondrial ubiquitination is a central component of mitochondrial quality control. The PINK1 (PTEN induced kinase 1)-PRKN (parkin RBR E3 ubiquitin protein ligase) pathway established how loss of mitochondrial membrane potential can trigger a phospho-ubiquitin feed-forward cascade on the outer mitochondrial membrane (OMM). It remains less clear how mitochondrial ubiquitination is achieved when PRKN is absent or inactivated. In our recent work, we identify a recruitment platform organized by AMBRA1 (autophagy and beclin 1 regulator 1), in which RMC1 (regulator of MON1-CCZ1) positions HUWE1 (HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1) at mitochondria. This spatial arrangement promotes HUWE1-dependent ubiquitination and turnover of OMM proteins, including MFN2 (mitofusin 2), VDAC1 (voltage-dependent anion channel 1), and VDAC2 (voltage-dependent anion channel 2). Our findings raise the question of how cells select among distinct mitochondrial ubiquitination pathways and whether these pathways function independently, sequentially, or cooperatively.

Metabolism and autophagy are closely interconnected, but whether metabolic alterations can chemically modify proteins and influence their selective autophagic degradation remains poorly understood. Our recent findings show that increased S-nitrosylation resulting from the loss of the denitrosylase AKR1A1/SCoR impairs glycolysis and promotes methylglyoxal accumulation, leading to carbonyl modification and selective autophagic degradation of KEAP1. These findings suggest that metabolic rewiring can influence the fate of specific proteins. In particular, we discuss whether glycation may act as a metabolic "mark" for selective autophagy and how the removal of metabolically modified proteins may, in turn, sustain signaling pathways involved in tumor progression and therapy resistance.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, for which radiotherapy constitutes the key component of standard comprehensive treatment; however, tumor relapse could inevitably arise from intrinsically radioresistant GBM subclones. Radiotherapy exerts biphasic regulatory effects on the tumor immune microenvironment (TIME), with transient activation followed by sustained immunosuppression. Nevertheless, it remains elusive how radioresistant GBM cells remodel such an immunosuppressive TIME to evade immune surveillance. Herein, integrative analyses encompassing clinical specimens, public single-cell RNA-seq datasets, orthotopic glioma models, primary CD8[+] T cell co-culture systems, and tandem mass tag (TMT) proteomics indicated that CD81 was highly expressed in radioresistant GBM and governed GBM immune evasion. CD81-high tumor cells were surrounded by functionally exhausted CD8[+] T cells, alongside immunosuppressive signature within neighboring myeloid and NK cells. CD81 depletion promoted selective macroautophagic/autophagic degradation of CD274/PD-L1, increased CD8[+] T cell infiltration and cytotoxic activity, reduced M2-like tumor-associated macrophages, and suppressed intracranial tumor growth. Moreover, CD81-knockdown augmented the antitumor efficacy of anti-PDCD1/PD-1, yielding a pronounced survival benefit when combined with radiotherapy. Mechanistically, CD81 interacted with CD274 via its large extracellular loop (LEL) and recruited the deubiquitinase USP14 through its cytoplasmic C-terminal tail (CCT). This resultant ternary complex erased K63-linked ubiquitination at the K280 residue of CD274, thereby abolishing SQSTM1/p62-dependent recognition and subsequent autophagy-lysosomal degradation of CD274 to maintain its protein stability. Collectively, our work establishes CD81 as a pivotal bridge connecting radioresistance to immune escape via sustaining CD274 abundance in GBM, highlighting CD81 as a promising therapeutic target to optimize radioimmunotherapy.Abbreviations: BBB: blood-brain barrier; CTLs: cytotoxic T lymphocytes; CCK-8: cell counting kit-8; ELISA: enzyme-linked immunosorbent assay; GBM: glioblastoma; IHC: Immunohistochemistry; GZMB: granzyme B; MDSCs: myeloid-derived suppressor cells; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OS: overall survival; PFA: paraformaldehyde; PLA: proximity ligation assay; PRF1: Perforin1; STR: short tandem repeat; SQSTM1: sequestosome 1; TMT: tandem mass tag; TMZ: temozolomide; TME: tumor microenvironment; TDEs: tumor-derived exosomes; Tregs: regulatory T cells; WT: wild type.

Lactylation is an emerging post-translational modification that is well established for its involvement in epigenetic regulation. However, its functional significance and regulatory mechanisms in non-small cell lung cancer (NSCLC) remain poorly understood. In this study, integrated proteomic and lactylomic analyses of clinical NSCLC specimens and matched adjacent normal tissues showed that USP7 (ubiquitin specific peptidase 7) was upregulated and USP7 K1084 lactylation was increased in NSCLC. Knockout of USP7 or inhibition of USP7 lactylation impaired cellular mitophagy, resulting in mitochondrial damage and attenuated NSCLC tumorigenicity. We identified CREBBP/CBP (CREB binding lysine acetyltransferase) as the key lactyltransferase responsible for USP7 K1084 lactylation. USP7 lactylation induced its localization to mitochondria and increased its interaction with PINK1 (PTEN induced kinase 1), promoting PINK1 deubiquitination and stabilization. Pharmacological inhibition of CREBBP reduced USP7 lactylation levels and promoted PINK1 ubiquitination and degradation, exerting antitumor effects in vitro and in vivo. Analysis of NSCLC clinical specimens showed that the protein levels of USP7, CREBBP, and PINK1 were positively correlated, and their high expression was associated with poor patient prognosis. Collectively, our findings establish the CREBBP-USP7-PINK1 axis as a promising therapeutic target for NSCLC treatment.Abbreviations: CHX: cycloheximide; CREBBP/CBP: CREB binding lysine acetyltransferase; DUB: deubiquitinating enzyme; IHC: immunohistochemistry; IP: immunoprecipitation; KO: knockout; LDHA: lactate dehydrogenase A; MS: mass spectrometry; NALA: L-sodium lactate; NSCLC: non-small cell lung cancer; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PINK1: PTEN induced kinase 1; PLA: proximity ligation assay; ROS: reactive oxygen species; shRNA: short hairpin RNA; TEM: transmission electron microscopy; TUBE: tandem ubiquitin binding entity; UPS: ubiquitin-proteasome system; USP7: ubiquitin specific peptidase 7; WT: wild type.