Lycopene is a carotenoid with strong antioxidant properties. Treatment with tripropylamine (TPA) greatly increases lycopene production by Blakeslea trispora, but the mechanism of this effect remains unclear. In this study, a reference-free transcriptome was used to analyze changes in gene expression in B. trispora after treatment with TPA. Downregulated genes were mostly involved in primary and secondary metabolic pathways, whereas upregulated genes were mostly related to autophagy and nucleotide excision repair. In addition, after TPA treatment, the mycelium swelling and the number of spores decreased. Our study suggests that TPA causes DNA damage, which upregulates the expression of nucleotide excision repair genes Xpb, Xpg, Csb, Polr2, and genome stability regulator gene Rmi1, as well as downregulation of Lsm2 and Dars2 and upregulation of G10, which are related to mRNA spliceosomes, leaving the cell in a state of starvation and triggering lysosomal-, peroxisomal-, and vacuole-autophagy. The autophagosome recycles material from spores, reduces spore production, and induces mycelial swelling, which, in turn, increases lycopene production and storage.

Improving treatment options for head and neck squamous cell carcinoma (HNSCC) requires a deeper understanding of the tumor microenvironment, particularly cancer-associated fibroblasts (CAFs). We previously reported that HNSCC-derived FGF2/bFGF (fibroblast growth factor 2) triggers cytokine release from CAFs via secretory autophagy. Here, using transmission electron microscopy, live-cell imaging, and immunofluorescence, we show that CAF autophagosomes transport cargo, including IL6, to the plasma membrane for secretion. Autophagy in CAFs is constitutive and independent of STAT3, MAPK1/ERK2-MAPK3/ERK1 and phosphoinositide 3-kinase (PI3K) signaling. Despite the significant role of secretory autophagy in CAFs, its molecular machinery has remained elusive. Using both a literature based, and an unbiased approach, we studied the molecular machinery involved in autophagosome trafficking in CAFs. We identified TRIM16, a protein previously reported to traffic to autophagosomes, upregulated in CAFs compared to normal oral fibroblasts. Immunohistochemistry of patient HNSCC stroma revealed co-expression of TRIM16 and LC3B, linking TRIM16 to autophagosome function. An unbiased proteomics profiling of immunoprecipitated LC3B+ vesicles in primary HNSCC CAFs revealed enrichment in trafficking proteins, focal adhesion, and mitochondrial proteins. We demonstrate that SEC22B, SNAP23, VAMP3, and STX4 colocalize with LC3B, IL6, and TRIM16 in CAFs. TRIM16 knockdown reduced autophagosomes at the plasma membrane and decreased IL6 secretion from CAFs. These findings uncover key molecular components involved in autophagy-mediated IL6 secretion in CAFs and suggest potential therapeutic targets for HNSCC.Abbreviations: ACTA2/αSMA: actin alpha 2, smooth muscle; CAF: cancer-associated fibroblasts; CM: conditioned media; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethylsulfoxide; EGFP: enhanced green fluorescent protein; ELISA: enzyme-linked immunosorbent assay; ER: endoplasmic reticulum; FGF2/bFGF: fibroblast growth factor 2; FGFR: fibroblast growth factor receptor; GO: gene ontology; GORASP2/GRASP55: golgi reassembly stacking protein 2; HMGB1: high mobility group box 1; HNSCC: head and neck squamous cell carcinoma; HPV: human papillomavirus; IL6: interleukin 6; IP: immunoprecipitation; LC-MS/MS: liquid chromatography-mass spectrometry/mass spectrometry; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; NFs: normal oral fibroblasts; NSCLC: non-small cell lung cancer; PLA: proximity ligation assay; SQSTM1/p62: sequestosome 1; STAT3: signal transducer and activator of transcription 3; SNAP23: synaptosome associated protein 23; SNARE: soluble N-ethyl-maleimide-sensitive factor attachment protein receptor; STX4: syntaxin 4; TEM: transmission electron microscopy; TGFB1: transforming growth factor beta 1; TMA: tissue microarray; TRIM: tri-partite motif; VAMP: vesicle associated membrane protein; VC: vehicle control.

Efferocytosis, the process by which apoptotic cells (ACs) are recognized and cleared by phagocytes, is a critical mechanism in maintaining intestinal immune homeostasis and promoting the resolution of inflammation. Inflammatory bowel disease (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC), is characterized by chronic intestinal inflammation, wherein defective efferocytosis contributes to the accumulation of ACs, secondary necrosis, and sustained mucosal damage. This review delineates the molecular mechanisms underlying efferocytosis and systematically examines its functional roles across five key intestinal phagocytic cell types: macrophages, dendritic cells (DCs), neutrophils, intestinal epithelial cells (IECs), and Paneth cells (PCs). Particular emphasis is placed on the dysregulation of efferocytosis capacity in IBD pathogenesis and the consequences of impaired apoptotic cell clearance in both professional and non-professional phagocytes. Furthermore, we evaluate emerging therapeutic strategies designed to restore or enhance efferocytosis, including modulation of macrophage polarization, LC3-associated phagocytosis pathways, nanotechnology-enabled delivery systems, and stem cell-based interventions. A comprehensive understanding of cell-type-specific efferocytosis in the intestinal microenvironment offers promising directions for the development of targeted, inflammation-resolving therapies for IBD.

Cell-autonomous immunity protects cells by utilizing membrane trafficking to detect and counteract diverse microbial pathogens, including selective autophagy and extracellular expulsion. However, the mechanisms underlying the mutual regulation among these systems has remained unknown. Here, we demonstrate that Rab GTPase-activating protein 1-like (RabGAP1L) modulates cell-autonomous immune responses via inactivation of two distinct Rab GTPases during group A Streptococcus (GAS) infection. Confocal microscopy analyses revealed that Rab7A positively regulates selective autophagy induction against GAS by facilitating endolysosomal trafficking and that Rab7A and Rab10 negatively regulate GAS expulsion from infected cells by inhibiting Rab11A-positive recycling endosome formation. RabGAP1L suppressed these pathways via inactivation of Rab7A and Rab10. By contrast, ATG7 and ATG5 knockout, resulting in autophagy deficiency, increased RabGAP1L-dependent bacterial expulsion from infected cells via the endocytic recycling pathway. Our findings suggest a regulatory mechanism of cell-autonomous immunity mediated by RabGAP1L, which contributes to the efficient elimination of intracellular pathogens.

Lysosomes, as crucial organelles within cells, carry out diverse biological functions such as waste degradation, regulation of the cellular environment, and precise control of cell signaling. This paper reviews the core functions and structural characteristics of lysosomes, and delves into the current research status of lysosomes damage repair mechanisms. Subsequently, we explore in depth the close association between lysosomes and various diseases, including but not limited to age-related chronic diseases, neuro-degenerative diseases, tumors, inflammation, and immune imbalance. Additionally, we also provide a detailed discussion of the application of lysosome-targeted substances in the field of regenerative medicine, especially the enormous potential demonstrated in key areas such as stem cell regulation and therapy, and myocardial cell repair. Though the integration of multidisciplinary research efforts, we believe that lysosomes damage repair mechanisms will demonstrate even greater application value in disease treatment and regenerative medicine.

Mouse oocytes undergo drastic changes in organellar composition and their activities during maturation from the germinal vesicle (GV) to metaphase II (MII) stage. After fertilization, the embryo degrades parts of the maternal components via lysosomal degradation systems, including autophagy and endocytosis, as zygotic gene expression begins during embryogenesis. Here, we demonstrate that endosomal-lysosomal organelles form large spherical assembly structures, termed endosomal-lysosomal organellar assemblies (ELYSAs), in mouse oocytes. ELYSAs are observed in GV oocytes, attaining sizes up to 7-8 μm in diameter in MII oocytes. ELYSAs comprise tubular-vesicular structures containing endosomes and lysosomes along with cytosolic components. Most ELYSAs are also positive for an autophagy regulator, LC3. These characteristics of ELYSA resemble those of ELVA (endolysosomal vesicular assemblies) identified independently. The signals of V1-subunit of vacuolar ATPase tends to be detected on the periphery of ELYSAs in MII oocytes. After fertilization, the localization of the V1-subunit on endosomes and lysosomes increase as ELYSAs gradually disassemble at the 2-cell stage, leading to further acidification of endosomal-lysosomal organelles. These findings suggest that the ELYSA/ELVA maintain endosomal-lysosomal activity in a static state in oocytes for timely activation during early development.

While dysregulated autophagy has been linked to acute respiratory distress syndrome (ARDS) development in sepsis, the exact regulatory mechanisms driving this process remain unclear. This study systematically investigated autophagy-related genes in sepsis-induced ARDS using integrative bioinformatics, including weighted gene coexpression network analysis (WGCNA), differential gene expression analysis (DEGs), receiver operating characteristic (ROC) curve analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, protein‒protein interaction (PPI) network analysis, and immune infiltration analysis. Hub genes were further validated by qPCR in Beas-2B cells receiving lipopolysaccharide (LPS) stimulation. We identified 18 autophagy-related DEGs with diagnostic potential for sepsis-induced ARDS. These DEGs were linked to endocytosis, protein kinase inhibition, and enigmatic Ficolin-1-rich granules. The downregulated hallmark signaling pathways involved apoptosis, complement, IL-2/STAT5, and KRAS signaling. Immune infiltration analysis revealed alterations in 7 immune cell subsets, including CD8 + T-cell exhaustion, natural killer cell reduction, and the type 1 helper T-cell response. When Beas-2B cells were treated with LPS, we discovered that 6 out of the 18 hub genes were significantly downregulated. Our findings provide novel insights into autophagy-mediated ARDS pathogenesis in sepsis. The hub genes represent promising candidates for clinical biomarker development and therapeutic targeting, which necessitates further validation.

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, a leading cause of irreversible blindness worldwide. IOP homeostasis is maintained through a balance between aqueous humor production and its drainage through the trabecular meshwork (TM)/Schlemm's Canal (SC) outflow pathway. Prior studies by our laboratory reported a key role of mechanical forces and primary cilia (PC)-dependent stretch-induced autophagy in IOP homeostasis. However, the precise mechanism regulating this process remains elusive. In this study, we investigated the upstream signaling pathway orchestrating autophagy activation during cyclic mechanical stretch (CMS) in primary cultured human TM cells, using biochemical and cell biological analyses. Our results indicate that TM cells express catalytic subunits of class IA PI3Ks (PIK3CA, B, and D), and that inhibition of class IA isoforms, but not class II and III, significantly prevent CMS-induced autophagy. Importantly, PIK3CA was found to localize in the PC. Furthermore, we identified a coordinated action of Class IA PI3Ks along INPP4A/B, a 4' inositol phosphatase, responsible for the formation of PI(3,4)P2 and PI(3)P and stretch-induced autophagy in TM cells. These findings contribute to a deeper understanding of the molecular mechanisms underlying IOP homeostasis.

Previous studies have discussed the association between serum metabolism and lactation performance among Sanhe and Holstein cows of different parities and found that the metabolic profiles of these two breeds vary differently with parity. Since the rumen is the central organ for nutrient absorption and production transformation in dairy cows, it remains unknown whether the differences observed under the same dietary conditions are related to the structure of the rumen microbiome. This study measured the apparent digestibility and rumen fermentation parameters of Sanhe cows (S1/S2/S3/S4) and Holstein cows (H1/H2/H3/H4) across four parities and generated a comprehensive rumen microbiome dataset using high-throughput sequencing technology. Significant differences in dry matter digestibility (p = 0.001) and ammonia nitrogen (p = 0.024) were observed among the S groups, with higher trends of various VFA contents in S1 (0.05 < p < 0.1). The H group showed significant differences in crude protein digestibility (p = 0.001), higher isovaleric acid content in H1 (p = 0.002), and the lowest acetate to propionate ratio (p = 0.002) in H3. Metagenomic sequencing results indicated consistency between rumen microbiome patterns and metabolic changes, with S1 distinctly different from S2/S3/S4, and H1 and H2 different from H3 and H4. The species composition of the rumen microbiome was similar between Sanhe and Holstein cows, but differences in abundance were noted. Rhizophagus , Neocallimastix, and Piromyces were more abundant in S1, H1, and H2, and pathways such as autophagy-animal, plant-pathogen interaction, and endocytosis were significantly enriched in these parities. Multiparous Sanhe cows had higher abundances of ATP-binding cassette transporters pathways. Additionally, CAZymes such as GH84 and GH37 were significantly associated with differential physiological indicators and milk traits. In conclusion, this study reveals the complex relationship between rumen microbiota and metabolic characteristics in Sanhe and Holstein cows of different parities, indicating that changes in the structure of the rumen microbiome may be key factors affecting lactation performance and metabolic differences in dairy cows.

The villous trophoblast cells are of fundamental importance because they fulfill a variety of functions that are vital for the growth of the fetus and the maintenance of pregnancy. A simple in vitro villous trophoblast cell model that grows on standard tissue culture plates has been utilized for various functional studies on villous trophoblast cells. Despite the potential value of incorporating electron microscopy analysis in reports on functional analysis of primary human trophoblast cells, electron microscopy analysis is exclusively ancillary to functional analysis in previous publications. In the context of autophagy research of villous trophoblast cells using primary trophoblast cells, a detailed ultrastructural analysis of autophagy flux using electron microscopy is imperative; however, it has not been conducted to date. In this study, we isolated term villous trophoblast cells (i.e., cytotrophoblast cells, CTB cells) using the most up-to-date isolation method for isolating pure CTB cells from human term placenta and investigated the ultrastructural dynamic process of autophagy of cultured CTB cells by means of transmission electron microscopy. The initial 6 h culture resulted in CTB cell aggregation; however, the majority of CTB cells did not differentiate into syncytial cells. In contrast, after 72 h, CTB cells exhibited a promotion of differentiation into syncytial cells. The electron microscopy analysis revealed the upregulation of autophagy and visualized unique autophagic profiles during differentiation into syncytial cells, which exhibited perinuclear accumulation of extremely large autophagosomes/autolysosomes. This study provides novel insights into the reproductive biology of primary trophoblast cells, thereby demonstrating the substantial value of primary trophoblast cells as research resources.

The evolution of insect metamorphosis has profoundly influenced their successful adaptation and diversification. Two key physiological processes during insect metamorphosis are notable: wing maturation and prothoracic gland (PG) histolysis. The ecdysone-induced protein 93 (E93) is a transcription factor indispensable for metamorphosis. While it has been established that both wing maturation and PG histolysis are dependent on E93, the molecular mechanisms through which E93 regulates these seemingly 'opposing' events remain poorly understood. In this study, time-course transcriptome profiles were generated for wing pads and PGs during metamorphosis in Blattella germanica, a hemimetabolous model insect. Comparative transcriptomic analyses demonstrated that E93 exerts predominant control over extensive gene transcription during wing morphogenesis and PG histolysis. During wing morphogenesis, E93 selectively enhances the expression of genes associated with cell proliferation, energy supply, signal transduction, actin cytoskeleton organization, and cell adhesion, etc. Additionally, E93 activates the transcription of the majority of genes within the wing gene network that are crucial for wing development in B. germanica. During PG histolysis, E93 preferentially promotes the expression of genes related to endocytosis, focal adhesion, the AMPK signaling pathway, adipocytokine signaling pathway, Toll and Imd signaling pathways, and autophagy, etc. The key genes involved in the aforementioned pathways were subsequently confirmed to contribute to the E93-dependent degeneration of the PG in B. germanica. In summary, our results reveal that E93 functions as a master transcriptional regulator orchestrating both tissue morphogenesis and histolysis during insect metamorphosis. These findings contribute to a deeper understanding of the genetic underpinnings of insect metamorphosis.

Cell death mechanisms are broadly classified into accidental cell death (ACD) and regulated cell death (RCD). ACD such as necrosis, is an uncontrolled, accidental process, while RCD is tightly regulated by specific signaling pathways and molecular mechanisms. Tumor cells are characterized by their ability to evade cell death and sustain uncontrolled proliferation. The failure of programmed cell death is a key contributor to tumor initiation, progression, and resistance to cancer therapies. Traditionally, research has focused primarily on apoptosis as the dominant form of RCD in cancer. However, emerging evidence highlights the importance of other non-apoptotic forms of RCD, such as pyroptosis, ferroptosis, necroptosis, and parthanatos, in tumorigenesis and treatment response. These pathways are gaining attention for their potential roles in overcoming therapy resistance. In this review, we will discuss the recent advances in the study of non-apoptotic cell death pathways in malignant tumors and explore their therapeutic implications, offering insights into new targets for cancer treatment strategies.

Protein homeostasis (proteostasis) refers to the balance of the cellular protein environment, tightly regulated by pathways governing protein synthesis, folding, trafficking, and degradation. Growing evidence supports the interconnection of these pathways to ensure the robustness of the proteo-stasis network. A recent study by Park et al. showed that, in macroautophagy/autophagy-deficient cells, the loss of proteasome or nuclear pore components causes synthetic lethality, as cytoplasmic proteins that accumulate under impaired autophagy are transported to the nucleus and degraded by nuclear proteasomes. The authors illustrated the mechanistic basis for why cells with conditions such as Huntington disease, where both autophagy and cytoplasm-to-nuclear shuttling are compromised, are more vulnerable to proteostasis perturbation.Abbreviation: UPR: unfolded protein response; UPS: ubiquitin-proteasome system.

Sodium taurocholate co-transporting polypeptide (NTCP) has been identified as an entry receptor for hepatitis B virus (HBV), but the molecular events of the viral post-endocytosis steps remain obscure. In this study, we discovered that manganese (Mn) could strongly inhibit HBV infection in NTCP-reconstituted HepG2 cells without affecting viral replication. We therefore profiled the antiviral effects of Mn2+ in an attempt to elucidate the regulatory mechanisms involved in early HBV infection. Intriguingly, Mn2+ conspicuously stimulated lysosomal activity, as evidenced by hyperactivation of mTORC1 and increased endo/lysosomal acidity. After HBV-triggered internalization, the NTCP receptor was sorted to late endosomal compartments by the ESCRT machinery in concert with the invading virion. The establishment of HBV infection was found to be independent of lysosomal fusion-driven late endosome maturation; Mn2+-induced lysosomal hyperfunction virtually impaired infection, suggesting that virions may gain cytosolic access directly from late endosomes. In contrast, suppression of lysosomal activity substantially enhanced HBV infection. Prolonged mTORC1 inactivation facilitated viral infection by depleting lysosomes and accelerating endocytic transport of virions. Notably, treatment with the natural steroidal alkaloid tomatidine recapitulated the effects of Mn2+ in stimulating lysosomal activity and exhibited potent anti-HBV activity in HepG2-NTCP cells and in proliferating human hepatocyte organoids. These findings provide new insights into the post-endocytosis events of HBV infection. The negative regulation of early HBV infection by endo/lysosomal activity makes it a promising target for antiviral therapies.

Antigen-presenting cells (APCs) are readily activated after phagocytosing infected or DNA-damaged cells but not normal apoptotic cells for reasons that are not well understood. Here, we demonstrate that after DNA damage events, cytosolic dsDNA species trigger intrinsic STING signaling and the production of key immunogenic proteins, including CCL5, which renders such cells capable of APC activation upon phagocytosis. These events involve the generation of immunogenic STING-inducible endosomal vesicles (SIEVEs) additionally comprising critical autophagy-associated proteins associated with cytosolic DNA species. After phagocytosis, extrinsic cGAS-STING signaling is triggered via engulfed, immunogenic transactivating DNA vesicles resulting in APC stimulation. These results help explain how APCs are predominantly activated by DNA-damaged or infected cells in contrast with normal apoptotic cells and suggest that reconstitution of STING signaling or key inducible genes in cGAS-STING-defective malignancies could substantially augment cancer immunotherapies.

Porcine epidemic diarrhea virus (PEDV) causes enormous economic losses to the pork industry, and its extensive cell tropism poses a substantial challenge to public health and safety. However, the invasion mechanisms and relevant host factors of PEDV remain poorly understood. In this study, we identified 422 differentially expressed genes related to PEDV infection through transcriptome analysis. Among these, Annexin A2 (ANXA2), Prohibitin-2 (PHB2), and Caveolin-2 (CAV2) were identified through screening and verifying as having a specific interaction with the PEDV S protein, and positive regulation of PEDV internalization was validated by siRNA and overexpression tests. Subsequently, using host membrane protein interaction networks and co-immunoprecipitation analysis, we found that ANXA2 PHB2 or CAV2 directly interact with Rab11a. Next, we constructed a pseudovirus model (LV-PEDV S-GFP) to further confirm that the downregulation of Rab11a could promote PEDV invasion. In detail, ANXA2, PHB2, or CAV2 promoted PEDV invasion via downregulating Rab11a. Furthermore, we showed that the S-protein fusion peptide (FP) was sufficient for S-protein interaction with ANXA2, PHB2, CAV2, and Rab11a, and the addition of exogenous GTP could regulate the efficiency of PEDV invasion. Collectively, ANXA2, PHB2, or CAV2 influenced the membrane fusion of PEDV with host cells through the host restriction factor Rab11a. This study could be targeted for future research to develop strategies for the control of PEDV.

The endogenous ecto-enzyme and exogenously administered alkaline phosphatase (ALP) have been evidenced to significantly attenuate inflammatory conditions, including Toll-like receptor 4 (TLR4)-related signaling and cytokine overexpression, barrier tissue dysfunction and oxidative stress, and metabolic syndrome and insulin resistance, in experimental models of colitis, liver failure, and renal and cardiac ischemia-reperfusion injury. This suggests multiple mechanisms of ALP anti-inflammatory action that remain to be fully elucidated.

Recent studies have contributed to a deeper comprehension of the role played by ALP in immune metabolism. This review outlines the established effects of ALP on lipopolysaccharide (LPS)-induced inflammation, including the neutralization of LPS and the modulation of purinergic signaling.

The additional mechanisms of anti-inflammatory activity of ALP observed in different pathologies are proposed.

The anti-inflammatory pathways of ALP may include a scavenger receptor (CD36)-mediated activation of β-oxidation and oxidative phosphorylation, caveolin-dependent endocytosis, and selective autophagy-dependent degradation.

The human Atg8 family member GABARAP is involved in numerous autophagy-related and -unrelated processes. We recently observed that specifically the deficiency of GABARAP enhances epidermal growth factor receptor (EGFR) degradation upon ligand stimulation. Here, we report on two putative LC3-interacting regions (LIRs) within EGFR, the first of which (LIR1) is selected as a GABARAP binding site in silico. Indeed, in vitro interaction studies reveal preferential binding of LIR1 to GABARAP and GABARAPL1. Our X-ray data demonstrate interaction of core LIR1 residues FLPV with both hydrophobic pockets of GABARAP suggesting canonical binding. Although LIR1 occupies the LIR docking site, GABARAP Y49 and L50 appear dispensable in this case. Our data support the hypothesis that GABARAP affects the fate of EGFR at least in part through direct binding.

The coordinated interaction between mitochondria and lysosomes, mainly manifested by mitophagy, mitochondria-derived vesicles, and direct physical contact, is essential for maintaining cellular life activities. The VPS39 subunit of the homotypic fusion and protein sorting complex could play a key role in the regulation of organelle dynamics, such as endolysosomal trafficking and mitochondria-vacuole/lysosome crosstalk, thus contributing to a variety of physiological functions. The abnormalities of VPS39 and related subunits have been reported to be involved in the pathological process of some diseases. Here, we analyze the potential mechanisms and the existing problems of VPS39 in regulating organelle dynamics, which, in turn, regulate physiological functions and disease pathogenesis, so as to provide new clues for facilitating the discovery of therapeutic targets for mitochondrial and lysosomal diseases.

Porcine deltacoronavirus (PDCoV) poses a serious threat to pork industry and has the potential for cross-species transmission. Yet, the invasion mechanisms and host factors involved are still unknown. In the present work, using siRNA interference and co-immunoprecipitation, we identified Annexin A2 (ANXA2), Prohibitin-2 (PHB2), or Caveolin-2 (CAV2) as host factors positively regulating the internalization of PDCoV. We further found that Rab11a co-localized with PDCoV S and inhibited PDCoV internalization. Subsequently, a pseudoviral infection model (LV-PDCoV S-GFP) was constructed, and ANXA2 or CAV2 promoted PDCoV invasion by downregulating Rab11a. Our results also indicated that ANXA2, CAV2, and Rab11a interact with the S protein via S-FP, thereby regulating virus-host membrane fusion. Through LV-PDCoV S-GFP infection, we found that Rab11a may act as a host restriction factor, and it could regulate the invasion efficiency of PDCoV by adding of exogenous GTP. These findings revealed that Rab11a was an exciting target to restrict fusion of PDCoV with host cell membranes. AVAILABILITY OF DATA AND MATERIAL: Not applicable.

Giardia duodenalis, an important zoonotic protozoan parasite, adheres to host intestinal epithelial cells (IECs) via the ventral disc and causes giardiasis characterized mainly by diarrhea. To date, it remains elusive how excretory-secretory products (ESPs) of Giardia enter IECs and how the cells respond to the entry. Herein, we initially demonstrated that ESPs evoked IEC endocytosis in vitro. We indicated that ESPs contributed vitally in triggering intrinsic apoptosis, pro-inflammatory responses, tight junction (TJ) protein expressional changes, and autophagy in IECs. Endocytosis was further proven to be implicated in those ESPs-triggered IEC responses. Ten predicted virulent excretory-secretory proteins of G. duodenalis were investigated for their capability to activate clathrin/caveolin-mediated endocytosis (CME/CavME) in IECs. Pyridoxamine 5'-phosphate oxidase (PNPO) was confirmed to be an important contributor. PNPO was subsequently verified as a vital promoter in the induction of giardiasis-related IEC apoptosis, inflammation, and TJ protein downregulation. Most importantly, this process seemed to be involved majorly in PNPO-evoked CME pathway, rather than CavME. Collectively, this study identified Giardia ESPs, notably PNPO, as potentially important pathogenic factors during noninvasive infection. It was also noteworthy that ESPs-evoked endocytosis might play a role in triggering giardiasis-inducing cellular regulation. These findings would deepen our understanding about the role of ESPs, notably PNPO, in the pathogenesis of giardiasis and the potential attributed endocytosis mechanism.

Lysosomes, crucial cellular organelles, undergo bidirectional transport along microtubules, mediated by motor proteins such as cytoplasmic dynein-1 (dynein) and various kinesins. While the kinesin-3 family member KIF1C is established in mediating anterograde vesicle transport, its role in lysosomal transport remains unclear. Our study reveals that KIF1C unexpectedly supports the retrograde transport of lysosomes, driven by dynein, and contributes to their perinuclear localization. Notably, while KIF1C facilitates this perinuclear positioning, its motor activity is not required and, instead, exerts an inhibitory effect on this process. Mechanistically, KIF1C facilitates this process by interacting with the dynein-activating adaptor Hook3, which associates with the lysosome-anchored protein RUFY3. This regulatory mechanism is critical for the efficient degradation of cargo in autophagic and endocytic pathways. Our findings identify an unconventional, non-motor role for KIF1C in activating dynein-driven lysosomal transport, expanding our understanding of its functional diversity in cellular trafficking.

Acinetobacter baumannii, an opportunistic pathogen has shown an upsurge in its multi-drug resistant isolates. OmpA of A. baumannii induces incomplete autophagy and apoptosis in host cells. Various therapeutic alternatives are under investigation against A. baumannii. Here, the major emphasis has been laid on comparing the efficacy of AgNP with different capping agents. OmpA targeted lead, Ivermectin capped AgNP (IVM-AgNP) has been compared with the antibacterial polyvinylpyrrolidone capped AgNP (PVP-AgNP) for their role in the modulations of host autophagy. Upregulation of p62 and LC3B confirmed by real-time PCR analysis indicated an increased autophagic flux upon the treatment with AgNPs. The elongation and closure of autophagic vacuoles was also supported by upregulated Atg genes (Atg4, Atg3, Atg5) in A. baumannii infected cells after treatment with AgNP. Autophagic flux increased on treatment with PVP-AgNP as suggested by the rise in mcherryLC3B fluorescence in A549 cells treated with PVP-AgNP as compared to the GFP-LC3B of IVM-AgNP. This suggests that PVP-AgNP treatment more effectively promotes the elongation and maturation stages of autophagy by increasing autophagic flux. These results indicate that capped AgNPs have the efficiency to revert the incomplete autophagy induced by A. baumannii back to normal autophagic levels.

The incorporation of different molecules by eukaryotic cells occurs through endocytosis, which is critical to the cell's survival and ability to reproduce. Although this process has been studied in greater detail in mammalian and yeast cells, several groups working with pathogenic protists have made relevant contributions. This review analysed the most relevant data on the endocytic process in anaerobic protists (Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, and Tritrichomonas foetus). Many protozoa can exert endocytic activity across their entire surface and do so with great intensity, as with E. histolytica. The available data on the endocytic pathway and the participation of PI-3 kinase, Rab, and Rho molecular complexes is reviewed from a historical perspective.

Autophagy is a complex physiological pathway mediating homeostasis and survival of cells degrading damaged organelles and regulating their recycling. Physiologic autophagy can maintain normal lung function, decrease lung cellular senescence, and inhibit myofibroblast differentiation. It is well known that autophagy is activated in several chronic inflammatory diseases; however, its role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and the expression of autophagy-related genes (ATGs) in lower airways of COPD patients is still controversial. The expression and localization of all ATG proteins that represented key components of the autophagic machinery modulating elongation, closure, and maturation of autophagosome membranes were retrospectively measured in peripheral lungs of patients with stable COPD (n = 10), control smokers with normal lung function (n = 10), and control nonsmoking subjects (n = 8) using immunohistochemical analysis. These results show an increased expression of ATG4 protein in alveolar septa and bronchiolar epithelium of stable COPD patients compared to smokers with normal lung function and non-smoker subjects. In particular, the genes in the ATG4 protein family (including ATG4A, ATG4B, ATG4C, and ATG4D) that have a key role in the modulation of the physiological autophagic machinery are the most important ATGs increased in the compartment of lower airways of stable COPD patients, suggesting that the alteration shown in COPD patients can be also correlated to impaired modulation of autophagic machinery modulating elongation, closure, and maturation of autophagosomes membranes. Statistical analysis was performed by the Kruskal-Wallis test and the Mann-Whitney U test for comparison between groups. A statistically significant increased expression of ATG4A (p = 0.0047), ATG4D (p = 0.018), and ATG5 (p = 0.019) was documented in the bronchiolar epithelium as well in alveolar lining for ATG4A (p = 0.0036), ATG4B (p = 0.0054), ATG4C (p = 0.0064), ATG4D (p = 0.0084), ATG5 (p = 0.0088), and ATG7 (p = 0.018) in patients with stable COPD compared to control groups. The ATG4 isoforms may be considered as additional potential targets for the development of new drugs in COPD.

Mitochondria are crucial for cellular ATP production. They are highly dynamic organelles, whose morphology and function are controlled through mitochondrial fusion and fission. The specific roles of mitochondria in podocytes, the highly specialized cells of the kidney glomerulus, remain less understood. Given the significant structural, functional, and molecular similarities between mammalian podocytes and Drosophila nephrocytes, we employed fly nephrocytes to explore the roles of mitochondria in cellular function. Our study revealed that alterations in the Pink1-Park (mammalian PINK1-PRKN) pathway can disrupt mitochondrial dynamics in Drosophila nephrocytes. This disruption led to either fragmented or enlarged mitochondria, both of which impaired mitochondrial function. The mitochondrial dysfunction subsequently triggered defective intracellular endocytosis, protein aggregation, and cellular damage. These findings underscore the critical roles of mitochondria in nephrocyte functionality.

Endolysosomes perform a wide range of cellular functions, including nutrient sensing, macromolecule digestion and recycling, as well as plasma membrane repair. Because of their high activity in cancerous cells, endolysosomes are attractive targets for the development of novel cancer treatments. Light-activated compounds termed photosensitizers (PS) can catalyze the oxidation of specific biomolecules and intracellular organelles. To selectively damage endosomes and lysosomes, HT-29 colorectal cancer cells were incubated with nanomolar concentrations of meso-tetraphenylporphine disulfonate (TPPS2a), an amphiphilic PS taken up via endocytosis and activated by green light (522 nm, 2.1 J.cm-1). Several cellular responses were characterized by a combination of immunofluorescence and immunoblotting assays. We showed that TPPS2a photosensitization blocked autophagic flux without extensive endolysosomal membrane rupture. Nevertheless, there was a severe functional failure of endolysosomes due to a decrease in CTSD (cathepsin D, 55%) and CTSB (cathepsin B, 52%) maturation. PSAP (prosaposin) processing (into saposins) was also considerably impaired, a fact that could be detrimental to glycosphingolipid homeostasis. Therefore, photosensitization of HT-29 cells previously incubated with a low concentration of TPPS2a promotes endolysosomal dysfunction, an effect that can be used to improve cancer therapies.

Mammalian autophagy-related protein Atg8, including the LC3 subfamily and GABARAP subfamily. Atg8 proteins play a vital role in autophagy initiation, autophagosome formation and transport, and autophagy-lysosome fusion. GABARAP subfamily proteins (GABARAPs) share a high degree of homology with LC3 family proteins, and their unique roles are often overlooked. GABARAPs are as indispensable as LC3 in autophagy. Deletion of GABARAPs fails autophagy flux induction and autophagy lysosomal fusion, which leads to the failure of autophagy. GABARAPs are also involved in the transport of selective autophagy receptors. They are engaged in various particular autophagy processes, including mitochondrial autophagy, endoplasmic reticulum autophagy, Golgi autophagy, centrosome autophagy, and dorphagy. Furthermore, GABARAPs are closely related to the transport and delivery of the inhibitory neurotransmitter γ-GABAA and the angiotensin II AT1 receptor (AT1R), tumor growth, metastasis, and prognosis. GABARAPs also have been confirmed to be involved in various diseases, such as cancer, cardiovascular disease, and neurodegenerative diseases. In order to better understand the role and therapeutic potential of GABARAPs, this article comprehensively reviews the autophagic and non-autophagic functions of GABARAPs, as well as the research progress of the role and mechanism of GABARAPs in cancer, cardiovascular diseases and neurodegenerative diseases. It emphasizes the significance of GABARAPs in the clinical prevention and treatment of diseases, and may provide new therapeutic ideas and targets for human diseases. GABARAP and GABARAPL1 in the serum of cancer patients are positively correlated with the prognosis of patients, which can be used as a clinical biomarker, predictor and potential therapeutic target.

Biallelic loss-of-function variants in TBC1D2B have been reported in five subjects with cognitive impairment and seizures with or without gingival overgrowth. TBC1D2B belongs to the family of Tre2-Bub2-Cdc16 (TBC)-domain containing RAB-specific GTPase activating proteins (TBC/RABGAPs). Here, we report five new subjects with biallelic TBC1D2B variants, including two siblings, and delineate the molecular and clinical features in the ten subjects known to date. One of the newly reported subjects was compound heterozygous for the TBC1D2B variants c.2584C>T; p.(Arg862Cys) and c.2758C>T; p.(Arg920*). In subject-derived fibroblasts, TBC1D2B mRNA level was similar to control cells, while the TBC1D2B protein amount was reduced by about half. In one of two siblings with a novel c.360+1G>T splice site variant, TBC1D2B transcript analysis revealed aberrantly spliced mRNAs and a drastically reduced TBC1D2B mRNA level in leukocytes. The molecular spectrum included 12 different TBC1D2B variants: seven nonsense, three frameshifts, one splice site, and one missense variant. Out of ten subjects, three had fibrous dysplasia of the mandible, two of which were diagnosed as cherubism. Most subjects developed gingival overgrowth. Half of the subjects had developmental delay. Seizures occurred in 80% of the subjects. Six subjects showed a progressive disease with mental deterioration. Brain imaging revealed cerebral and/or cerebellar atrophy with or without lateral ventricle dilatation. The TBC1D2B disorder is a progressive neurological disease with gingival overgrowth and abnormal mandible morphology. As TBC1D2B has been shown to positively regulate autophagy, defects in autophagy and the endolysosomal system could be associated with neuronal dysfunction and the neurodegenerative disease in the affected individuals.

Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, PKD1) and Polycystin-2 (PC2, PKD2) contain mutations. PC1 is a large membrane receptor that can interact and form a complex with the calcium-permeable cation channel PC2. This complex localizes to the plasma membrane, primary cilia and ER. Dysregulated calcium signalling and consequential alterations in downstream signalling pathways in ADPKD are linked to cyst formation and expansion; however, it is not completely understood how PC1 and PC2 regulate calcium signalling. We have studied Polycystin-2 mediated calcium signalling in the model organism Dictyostelium discoideum by overexpressing and knocking down the expression of the endogenous Polycystin-2 homologue, Polycystin-2. Chemoattractant-stimulated cytosolic calcium response magnitudes increased and decreased in overexpression and knockdown strains, respectively, and analysis of the response kinetics indicates that Polycystin-2 is a significant contributor to the control of Ca2+ responses. Furthermore, basal cytosolic calcium levels were reduced in Polycystin-2 knockdown transformants. These alterations in Ca2+ signalling also impacted other downstream Ca2+-sensitive processes including growth rates, endocytosis, stalk cell differentiation and spore viability, indicating that Dictyostelium is a useful model to study Polycystin-2 mediated calcium signalling.

Metastasis remains the most critical factor limiting patient survival and the most challenging part of cancer-targeted therapy. Identifying the causal drivers of metastasis and characterizing their properties in various key aspects of cancer biology is essential for the development of novel metastasis-targeting approaches. Metastasis-associated in colon cancer 1 (MACC1) is a prognostic and predictive biomarker that is now recognized in more than 20 cancer entities. Although MACC1 can already be linked with many hallmarks of cancer, one key process-the facilitation of immune evasion-remains poorly understood. In this review, we explore the direct and indirect links between MACC1 and the mechanisms of immune escape. Therein, we highlight the signaling pathways and secreted factors influenced by MACC1 as well as their effects on the infiltration and anti-tumor function of immune cells.

The primary objective of this study was to examine the effect of acute ammonia stress on hepatic physiological alterations in yellow catfish by performing a comprehensive analysis of the metabolome and transcriptome. The present study showed that ammonia stress led to liver metabolic disruption, functional incapacitation, and oxidative damage. Transcriptomic and metabolomic analyses revealed transcriptional and metabolic differences in the liver of yellow catfish under control and high ammonia stress conditions. After 96 h of acute exposure to ammonia, the mRNA levels of 596 liver genes were upregulated, whereas those of 603 genes were downregulated. Enrichment analysis of the differentially expressed genes identified multiple signalling pathways associated with autophagy, including the endocytosis, autophagy-animal, and mammalian target of rapamycin signalling pathways. A total of 186 upregulated and 117 downregulated metabolites, primarily associated with amino acid biosynthesis pathways, were identified. Multi-omics integration revealed the solute carrier family 38 member 9 (SLC38A9)-mammalian target of rapamycin axis as a signalling nexus for amino acid-mediated modulation of autophagy flux, and q-PCR was used to assess the expression of autophagy-related genes (LC3a and sqstm1), revealing an initial inhibition followed by the restoration of autophagic flux during ammonia stress. Subsequent utilisation of arginine as a specific SLC38A9 activator during ammonia stress demonstrated that augmented SLC38A9 expression hindered autophagy, exacerbated ammonia toxicity, and caused a physiological decline (total cholesterol, total triglyceride, acid phosphatase, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase levels were significantly increased), oxidative stress, and apoptosis. Autophagy activation may be an adaptive mechanism to resist ammonia stress.

Sensorineural hearing loss (SNHL) poses a major threat to both physical and mental health; however, there is still a lack of effective drugs to treat the disease. Recently, novel biological therapies, such as mesenchymal stem cells (MSCs) and their products, namely, exosomes, are showing promising therapeutic potential due to their low immunogenicity, few ethical concerns, and easy accessibility. Nevertheless, the precise mechanisms underlying the therapeutic effects of MSC-derived exosomes remain unclear.

Exosomes derived from MSCs reduced hearing and hair cell loss caused by neomycin-induced damage in models in vivo and in vitro. In addition, MSC-derived exosomes modulated autophagy in hair cells to exert a protective effect. Mechanistically, exogenously administered exosomes were internalized by hair cells and subsequently upregulated endocytic gene expression and endosome formation, ultimately leading to autophagy activation. This increased autophagic activity promoted cell survival, decreased the mitochondrial oxidative stress level and the apoptosis rate in hair cells, and ameliorated neomycin-induced ototoxicity.

In summary, our findings reveal the otoprotective capacity of exogenous exosome-mediated autophagy activation in hair cells in an endocytosis-dependent manner, suggesting possibilities for deafness treatment.

Brain plays a central role in adapting to environmental changes and is highly sensitive to the oxygen level. Although previous studies investigated the molecular response of brain exposure to acute hypoxia in fish, the lack of studies at the translational level hinders further understanding of the regulatory mechanism response to hypoxia from multi-omics levels. Yellow catfish (Pelteobagrus fulvidraco) is an important freshwater aquaculture species; however, hypoxia severely restricts the sustainable development of its breeding industry. In the present study, the transcriptome, translatome, and proteome were integrated to study the global landscapes of yellow catfish brain response to hypoxia. The evidently increased amount of cerebral cortical cells with oedema and pyknotic nuclei has been observed in hypoxia group of yellow catfish. A total of 2750 genes were significantly changed at the translational level. Comparative transcriptional and translational analysis suggested the HIF-1 signaling pathway, autophagy and glycolysis/gluconeogenesis were up-regulated after hypoxia exposure. KEGG enrichment of translational efficiency (TE) differential genes suggested that the lysosome and autophagy were highly enriched. Our result showed that yellow catfish tends to inhibit the TE of genes by increasing the translation of uORFs to adapt to hypoxia. Correlation analysis showed that transcriptome and translatome exhibit higher correlation. In summary, this study demonstrated that hypoxia dysregulated the cerebral function of yellow catfish at the transcriptome, translatome, and proteome, which provides a better understanding of hypoxia adaptation in teleost.

Uveal melanoma is an ocular tumor with a high risk of developing metastases. The endo-lysosomal system can affect the melanoma progression by accelerating and facilitating invasion or metastasis. This study aims to conduct comparative analysis of normal choroidal melanocytes and uveal melanoma cells ultrastructure with a focus on intracellular transport system, and to examine the patterns of autophagy- and vesicular trafficking-related proteins expression in a case series of uveal melanomas. Transmission electron microscopy was used to assess the ultrastructure of normal choroidal melanocytes and uveal melanoma cells. The expression levels of autophagy- and vesicular trafficking-related proteins in three histological types of uveal melanoma were analyzed by immunofluorescence staining. Electron microscopy results showed that the autophagic vacuoles were more abundant in normal choroidal melanocytes, than in uveal melanoma cells. The normal choroidal melanocytes were characterized by active intracellular vesicular trafficking; however, the proportion of caveolae was higher in uveal melanoma cells. The spindle type of tumor was characterized by a high expression levels of LC3 beta, while Rab7 and Rab11 proteins expression was significantly up-regulated in the mixed-type tumor cells. The results indicate that uveal melanoma cells probably have lower basal levels of autophagy and higher receptor-mediated endocytic trafficking-associated with caveolae than normal choroidal melanocytes. RESEARCH HIGHLIGHTS: The autophagic vacuoles are abundant in normal choroidal melanocytes. Uveal melanoma cells are characterized by a high proportion of caveolae. The high expression levels of LC3 beta were revealed in a spindle type of tumor, while Rab7 and Rab11 proteins expression was up-regulated in the mixed-type tumor cells.

Glaucoma is a heterogeneous group of progressive diseases that leads to irreversible blindness. Secondary glaucoma refers to glaucoma caused by a known underlying condition. Pseudoexfoliation and pigment dispersion syndromes are common causes of secondary glaucoma. Their respective deposits may obstruct the trabecular meshwork, leading to aqueous humor outflow resistance, ocular hypertension, and optic neuropathy. There are no disease-specific interventions available for either. Pseudoexfoliation syndrome is characterized by fibrillar deposits (pseudoexfoliative material) on anterior segment structures. Over a decade of multiomics analyses taken together with the current knowledge on pseudoexfoliative glaucoma warrant a re-think of mechanistic possibilities. We propose that the presence of nucleation centers (e.g., vitamin D binding protein), crosslinking enzymes (e.g., transglutaminase 2), aberrant extracellular matrix, flawed endocytosis, and abnormal aqueous-blood barrier contribute to the formation of proteolytically resistant pseudoexfoliative material. Pigment dispersion syndrome is characterized by abnormal iridolenticular contact that disrupts iris pigment epithelium and liberates melanin granules. Iris melanogenesis is aberrant in this condition. Cytotoxic melanogenesis intermediates leak out of melanosomes and cause iris melanocyte and pigment epithelium cell death. Targeting melanogenesis can likely decrease the risk of pigmentary glaucoma. Skin and melanoma research provides insights into potential therapeutics. We propose that specific prostanoid agonists and fenofibrates may reduce melanogenesis by inhibiting cholesterol internalization and de novo synthesis. Additionally, melatonin is a potent melanogenesis suppressor, antioxidant, and hypotensive agent, rendering it a valuable agent for pigmentary glaucoma. In pseudoexfoliative glaucoma, where environmental insults drive pseudoexfoliative material formation, melatonin's antioxidant and hypotensive properties may offer adjunct therapeutic benefits. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology.

Spinocerebellar ataxia type 6 (SCA6) is a rare disease that is characterized by cerebellar dysfunction. Patients have progressive motor coordination impairment, and postmortem brain tissue reveals degeneration of cerebellar Purkinje cells and a reduced level of cerebellar brain-derived neurotrophic factor (BDNF). However, the pathophysiological changes underlying SCA6 are not fully understood. We carried out RNA-sequencing of cerebellar vermis tissue in a mouse model of SCA6, which revealed widespread dysregulation of genes associated with the endo-lysosomal system. Since disruption to endosomes or lysosomes could contribute to cellular deficits, we examined the endo-lysosomal system in SCA6. We identified alterations in multiple endosomal compartments in the Purkinje cells of SCA6 mice. Early endosomes were enlarged, while the size of the late endosome compartment was reduced. We also found evidence for impaired trafficking of cargo to the lysosomes. As the proper functioning of the endo-lysosomal system is crucial for the sorting and trafficking of signaling molecules, we wondered whether these changes could contribute to previously identified deficits in signaling by BDNF and its receptor tropomyosin kinase B (TrkB) in SCA6. Indeed, we found that the enlarged early endosomes in SCA6 mice accumulated both BDNF and TrkB. Furthermore, TrkB recycling to the cell membrane in recycling endosomes was reduced, and the late endosome transport of BDNF for degradation was impaired. Therefore, mis-trafficking due to aberrant endo-lysosomal transport and function could contribute to SCA6 pathophysiology through alterations to BDNF-TrkB signaling, as well as mishandling of other signaling molecules. Deficits in early endosomes and BDNF localization were rescued by chronic administration of a TrkB agonist, 7,8-dihydroxyflavone, that we have previously shown restores motor coordination and cerebellar TrkB expression. The endo-lysosomal system is thus both a novel locus of pathophysiology in SCA6 and a promising therapeutic target.

As an integral organelle in the eukaryote, the lysosome is the degradation center and metabolic signal center in living cells, and partakes in significant physiological processes such as autophagy, cell death and cellular senescence. Fluorescent probe has become a favorite tool for studying organelles and their chemical microenvironments because of its high specificity and non-destructive merits. Over recent years, it has been reported that increasingly new lysosome-targeted probes play a major role in the diagnosis and monitor of diseases, in particular cancer and neurodegenerative diseases. In order to deepen the relevant research on lysosome, it is challenging and inevitability to design novel lysosomal targeting probes. This review first introduces the concepts of lysosome and its closely related biological activities, and then introduces the fluorescent probes for lysosome in detail according to different detection targets, including targeting mechanism, biological imaging, and application in diseases. Finally, we summarize the specific challenges and discuss the future development direction facing the current lysosome-targeted fluorescent probes. We hope that this review can help biologists grasp the application of fluorescent probes and broaden the research ideas of researchers targeting fluorescent probes so as to design more accurate and functional probes for application in diseases.

Endocytosis and autophagy are the main pathways to deliver cargoes in vesicles and autophagosomes, respectively, to vacuoles/lysosomes in eukaryotes. Multiple positive regulators but few negative ones are reported to regulate the entry of vesicles and autophagosomes into vacuoles/lysosomes. In yeast, the Rab5 GTPase Vps21 and the ESCRT (endosomal sorting complex required for transport) are positive regulators in endocytosis and autophagy. During autophagy, Vps21 regulates the ESCRT to phagophores (unclosed autophagosomes) to close them. Phagophores accumulate on vacuolar membranes in both vps21∆ and ESCRT mutant cells under a short duration of nitrogen starvation. The vacuolar transport chaperon (VTC) complex proteins are recently found to be negative regulators in endocytosis and autophagy. Phagophores in vps21∆ cells are promoted to enter vacuoles when the VTC complex proteins are absent. Phagophores are easily observed inside vacuoles when any of these VTC complex proteins (Vtc1, 2, 4, 5) are removed. However, it is unknown whether the removal of VTC complex proteins will also promote the entry of phagophores into vacuoles in ESCRT mutant cells under the same conditions. Snf7 is a core subunit of ESCRT subcomplex III (ESCRT-III), and phagophores accumulate in snf7∆ cells under a short duration of nitrogen starvation. We used green fluorescence protein (GFP) labeled Atg8 to display phagophores and FM4-64-stained or Vph1-GFP-labeled membrane structures to show vacuoles, then examined fluorescence localization and GFP-Atg8 degradation in snf7∆ and snf7∆vtc4∆ cells. Results showed that Vtc4 depletion promoted the entry of phagophores in snf7∆ cells into vacuoles as it did for vps21∆ cells, although the promotion level was more obvious in vps21∆ cells. This observation indicates that the VTC complex proteins may have a widespread role in negatively regulating cargos to enter vacuoles in yeast.

The asexual sporulation of filamentous fungi is an important mechanism for their reproduction, survival, and pathogenicity. In Aspergillus and several filamentous fungi, BrlA, AbaA, and WetA are the key elements of a central regulatory pathway controlling conidiation, and MedA is a developmental modifier that regulates temporal expression of central regulatory genes; however, their roles are largely unknown in nematode-trapping (NT) fungi. Arthrobotrys oligospora is a representative NT fungus, which can capture nematodes by producing adhesive networks (traps). Here, we characterized the function of AoMedA and three central developmental regulators (AoBrlA, AoAbaA, and AoWetA) in A. oligospora by gene disruption, phenotypic comparison, and multi-omics analyses, as these regulators are required for conidiation and play divergent roles in mycelial development, trap formation, lipid droplet accumulation, vacuole assembly, and secondary metabolism. A combined analysis of phenotypic traits and transcriptome showed that AoMedA and AoWetA are involved in the regulation of peroxisome, endocytosis, and autophagy. Moreover, yeast one-hybrid analysis showed that AoBrlA can regulate AoMedA, AoAbaA, and AoWetA, whereas AoMedA and AoAbaA can regulate AoWetA. Our results highlight the important roles of AoMedA, AoBrlA, AoAbaA, and AoWetA in conidiation, mycelia development, trap formation, and pathogenicity of A. oligospora and provide a basis for elucidating the relationship between conidiation and trap formation of NT fungi. IMPORTANCE Conidiation is the most common reproductive mode for many filamentous fungi and plays an essential role in the pathogenicity of fungal pathogens. Nematode-trapping (NT) fungi are a special group of filamentous fungi owing to their innate abilities to capture and digest nematodes by producing traps (trapping devices). Sporulation plays an important role in the growth and reproduction of NT fungi, and conidia are the basic components of biocontrol reagents for controlling diseases caused by plant-parasitic nematodes. Arthrobotrys oligospora is a well-known NT fungus and is a routinely used model fungus for probing the interaction between fungi and nematodes. In this study, the functions of four key regulators (AoMedA, AoBrlA, AoAbaA, and AoWetA) involved in conidiation were characterized in A. oligospora. A complex interaction between AoMedA and three central regulators was noted; these regulators are required for conidiation and trap formation and play a pleiotropic role in multiple intracellular activities. Our study first revealed the role of AoMedA and three central regulators in conidiation, trap formation, and pathogenicity of A. oligospora, which contributed to elucidating the regulatory mechanism of conidiation in NT fungi and helped in developing effective reagents for biocontrol of nematodes.