Search Results (114)

Exosomes, a major subpopulation of small extracellular vesicles (sEV), are conserved mediators of intercellular communication, yet the properties of their endosomal precursors, intraluminal vesicles (ILV), have not been systematically quantified across species or imaging modalities. This study systematically evaluates ILV sizes across diverse eukaryotic species and modalities while assessing their relationship to secreted sEV sizes. We carried out two complementary meta-analyses of ILV sizes based on transmission electron microscopy (TEM) and cryogenic electron microscopy (cryo-EM) data across species. This was followed by in situ assessment of sEVs secreted by HEK293T cells with TEM, nanoparticle tracking analysis and super-resolution microscopy characterization. Across species, imaging modalities, and cellular contexts, ILV sizes were under approximately 200 nm, with a mean diameter of 100.5 nm, overlapping with the size range of sEVs. This study addresses an existing knowledge gap by systematically evaluating ILV size across species and revealing an upper size limit of approximately 200 nm. Full article

Background and Objectives: The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a key innate immune complex, and its aberrant activation contributes to metabolic and neurodegenerative diseases. Brain-derived neurotrophic factor (BDNF) is a neurotrophin with anti-inflammatory and metabolic regulatory functions, but its role in NLRP3 inflammasome activation and gasdermin D (GSDMD)-mediated pyroptosis remains unclear. The aim of this study was to investigate the effects of BDNF deficiency on LPS- and nigericin-induced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis in vivo, and to elucidate the involvement of NF-κB signaling, autophagy, and ESCRT-III-dependent plasma membrane repair in this process. Materials and Methods: In this in vivo study, male Bdnf ^+/+ and Bdnf ^+/− mice were subjected to lipopolysaccharide (LPS) plus nigericin-induced NLRP3 inflammasome activation. Serum and hippocampus, cortex, liver, epididymal adipose, and muscle tissues were collected 24 h after stimulation for analysis of inflammasome-related, autophagy-related, and membrane repair-related proteins by Western blotting and of serum BDNF, interleukin-1β (IL-1β), and interleukin-18 (IL-18) by ELISA. Results: Bdnf ^+/− mice displayed significantly reduced circulating BDNF levels and exhibited exaggerated LPS plus nigericin-induced increases in IL-1β and IL-18 compared with Bdnf ^+/+ mice. Across all tissues, BDNF deficiency enhanced NF-κB p65, NLRP3, active caspase-1 p20, and GSDMD expression, indicating amplified inflammasome activation and pyroptosis. Conversely, LC3B and SQSTM1/p62 levels were decreased, and VPS4A expression, a key component of the ESCRT-III membrane repair machinery, was suppressed in Bdnf ^+/− mice, suggesting impaired selective autophagy, autophagosome formation, and plasma membrane repair. Conclusions: Together, these findings indicate that BDNF restrains NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through inhibition of NF-κB signaling and coordinated activation of autophagy and ESCRT-III-dependent membrane repair. BDNF thus emerges as an endogenous negative regulator of inflammasome activity and a potential therapeutic target for conditions characterized by aberrant NLRP3-driven inflammation. Full article

The epigenome and nuclear architectural mechanisms that regulate neuronal activity-induced transcriptional responses in cortical neurons remain incompletely understood. Previously, we have shown that the chromatin organizer SATB2 and the inner nuclear membrane protein LEMD2 form a chromatin tether at the nuclear lamina, and that activity-induced transcription is impaired in both Satb2 and Lemd2 loss-of-function models. Interaction of SATB2 and LEMD2 with subunits of the ESCRT-III complex indicates that the ESCRT-III complex could serve as an activity-dependent, dynamic component of this tether. Here, we study the activity-dependent subcellular localization and function of the ESCRT-III components CHMP7 and CHMP4B in primary cortical neurons. We find that increased neuronal activity correlates with the accumulation of co-localized CHMP7 and CHMP4B foci at the nuclear envelope. shRNA-mediated Chmp7 knockdown causes a reduction in the expression of activity-regulated genes and genes with highly specialized functions in synaptic organization and trans-synaptic signaling. Furthermore, the observed similarity in the global transcriptome responses in Satb2, Lemd2, and Chmp7 loss-of-function models points toward a previously unrecognized role of the SATB2–LEMD2–CHMP7 tether in linking chromatin architecture and nuclear envelope plasticity to activity-dependent gene regulation. Full article

During open mitosis, reassembly of the nuclear envelope requires the coordinated recruitment of the ESCRT machinery, initiated by the chromatin-associated factor BAF1 and the nuclear-envelope-associated factor LEM2. Because telomeres are enriched at the reforming envelope, we investigated whether ESCRT factors contribute to telomere integrity. Reduction in the pivotal nuclear ESCRT factor CHMP7 caused DNA damage, heterochromatin disorganization, and telomere defects, including sister telomere associations and telomere free ends. Extending this analysis, we found that additional ESCRT components, including TSG101, VPS28, CHMP4B, and the ESCRT-associated factor AKTIP/Ft1, also contribute to telomere integrity, although with different strengths. Genetic interaction analyses suggest that CHMP7 converges in a common pathway with CHMP4B and AKTIP/Ft1, while it functions in parallel routes to TNKS1, a telomere-specific regulator of the shelterin TRF1. More genetic analyses indicated that BAF1 and LEM2 contribute to safeguarding of telomeres during nuclear envelope reassembly. Because defects in nuclear envelope dynamics and chromatin–membrane coupling are hallmarks of disorders associated with nuclear deformation and fragility, including aging and cancer, our findings contribute a new angle into these conditions and suggest potential targets for selectively modulating telomere maintenance pathways. Full article

Glioblastoma multiforme (GBM) is characterized by aggressive growth, extensive vascularization, high metabolic malleability, and a striking capacity for therapy resistance. Current treatments involve surgical resection and concomitant radiation therapy and chemotherapy, prolonging survival times marginally due to the therapy resistance that is built up by the tumor cells. A growing body of research has identified exosomes as critical enablers of therapy resistance. These nanoscale vesicles enable GBM cells to disseminate oncogenic proteins, nucleic acids, and lipids that collectively promote angiogenesis, maintain autophagy under metabolic pressure, and suppress apoptosis. As interest grows in targeting tumor communication networks, exosome-based therapeutic strategies have emerged as promising avenues for improving therapeutic outcomes in GBM. This review integrates current insights into two complementary therapeutic strategies: inhibiting exosome biogenesis and secretion, and engineering exosomes as precision vehicles for the delivery of anti-tumor molecular cargo. Key molecular regulators of exosome formation—including the endosomal sorting complex required for transport (ESCRT) machinery, tumor susceptibility gene 101 (TSG101) protein, ceramide-driven pathways, and Rab GTPases—govern the sorting and release of factors that enhance GBM survival. Targeting these pathways through pharmacological or genetic means has shown promise in suppressing angiogenic signaling, disrupting autophagic flux via modulation of autophagy-related gene (ATG) proteins, and sensitizing tumor cells to apoptosis by destabilizing mitochondria and associated survival networks. In parallel, advances in exosome engineering—encompassing siRNA loading, miRNA enrichment, and small-molecule drug packaging—offer new routes for delivering therapeutic agents across the blood–brain barrier with high cellular specificity. Engineered exosomes carrying anti-angiogenic, autophagy-inhibiting, or pro-apoptotic molecules can reprogram the tumor microenvironment and activate both the intrinsic mitochondrial and extrinsic ligand-mediated apoptotic pathways. Collectively, current evidence underscores the potential of strategically modulating endogenous exosome biogenesis and harnessing exogenous engineered therapeutic exosomes to interrupt the angiogenic and autophagic circuits that underpin therapy resistance, ultimately leading to the induction of apoptotic cell death in GBM. Full article

Exosomes are nanoscale extracellular vesicles that mediate intercellular communication by transporting microRNAs, proteins, and lipids. Generated through Endosomal Sorting Complex Required for Transport (ESCRT)-dependent mechanisms or ESCRT-independent pathways, exosomes are released when multivesicular bodies fuse with the plasma membrane. The ESCRT-dependent pathway involves sequential protein complexes (ESCRT-0, I, II, III) that recognize and sort ubiquitinated cargo, induce membrane budding, and facilitate vesicle scission. In contrast, the ESCRT-independent pathway relies on membrane lipids such as ceramide and proteins like tetraspanins (CD9, CD63, CD81) to promote vesicle formation without ESCRT machinery. Furthermore, post-translational modifications, including ubiquitination, sumoylation, and phosphorylation, further serve as molecular switches, modulating the affinity of ESCRT complexes or cargo proteins for membrane domains and affecting ILV formation rates. In reproductive medicine, exosomes regulate oocyte maturation, embryo–endometrial crosstalk, placental development, and maternal–fetal communication. Altered exosomal signaling contributes to obstetric complications, including preeclampsia, gestational diabetes mellitus, and preterm birth, whereas distinct exosomal miRNA signatures serve as potential diagnostic biomarkers. In gynecology, dysregulated exosomes are implicated in endometriosis, polycystic ovary syndrome, premature ovarian insufficiency, and gynecological malignancies. In contrast, mesenchymal stem cell-derived exosomes show therapeutic promise in restoring ovarian function and enhancing fertility outcomes. The distinctive molecular profiles of circulating exosomes enable minimally invasive diagnosis, while their biocompatibility and ability to cross biological barriers position them as vehicles for targeted drug delivery. Characterization of accessible data provides non-invasive opportunities for disease monitoring. However, clinical translation faces challenges, including standardization of isolation protocols, establishment of reference ranges for biomarkers, and optimization of therapeutic dosing. This review summarizes exosome biogenesis, characterization methods, physiological functions, and clinical applications in obstetrics and gynecology, with an emphasis on their diagnostic and therapeutic potential. Future directions include large-scale biomarker validation studies, engineering approaches to enhance exosome targeting, and integration with precision medicine platforms to advance personalized reproductive healthcare. Full article

The Endosomal Sorting Complex Required for Transport (ESCRT) is a highly conserved machinery best known for its role in endosomal trafficking and membrane remodeling. Increasing evidence shows that ESCRT components are also key regulators during open mitosis, where precise membrane dynamics are essential for nuclear envelope reformation and spindle disassembly. In this review, we explore how the ESCRT machinery coordinates mitotic processes under physiological conditions and how their dysregulation contributes to genomic instability, altered cell division, and disease. We highlight recent findings on the spatiotemporal control of ESCRT recruitment at mitotic membranes, the interplay with chromatin and nuclear envelope-associated factors, and the consequences of defective ESCRT function in pathological contexts such as cancer and neurodegeneration. By connecting molecular mechanisms with cellular outcomes, we provide an integrated view of how the ESCRT machinery acts as critical guardian of mitotic fidelity and offer some routes for the identification of potential therapeutic targets in human disease. Full article

Drug resistance is still one of the main challenges for the treatment of colorectal cancer (CRC). Whilst some resistance mechanisms are well known, from the static therapy success rate, clearly, still much is undiscovered. Intracellular transport mechanisms have attracted attention as having a possible role in drug resistance, and here, the Endosomal Sorting Complex Required for Transport (ESCRT) protein family is studied as a source of drug resistance modulation using human CRC cell lines and clinical material. From an initial screening of ESCRT proteins in a panel of 10 CRC wild-type cell lines using immunoblotting, Vacuolar Protein Sorting-Associated Protein A4 (VPS4A) was identified as being consistently highly expressed, and it was selected for further investigation. Immunohistopathological evaluation in a small panel of CRC patient samples demonstrated high expression in the tumor epithelium compared to normal intestinal epithelium. The knockdown of VPS4A resulted in enhanced sensitivity of cells to oxaliplatin, and it was subsequently seen that oxaliplatin-resistant sublines had significantly higher VPS4A expression than their wild-type variants. In addition, it was demonstrated that a small molecule inhibitor of VPS4A, aloperine, could interact synergistically with oxaliplatin to enhance its sensitivity in an oxaliplatin-resistant cell line. We hypothesize from initial RNA sequencing analysis that the mechanism of action of VPS4A modulation is through depleting levels of the drug efflux transporter MRP2 in the cell, preventing oxaliplatin egress and increasing cell exposure to the drug. The evidence presented here thus indicates that ESCRT machinery, specifically VPS4A, may act as a modulator of oxaliplatin resistance in CRC. Full article

Endosomal dysfunction is one of the earliest cellular signs in Alzheimer’s disease. Tumor susceptibility gene 101 protein (TSG101) is a component of the endosomal sorting complex required for transport (ESCRT)-I, which plays a key role in sorting ubiquitinated cell surface proteins and lipids onto intraluminal vesicles of multivesicular bodies for trafficking to lysosomes or autophagosomes for degradation, or to the plasma membrane for exosomal secretion. TSG101-dependent trafficking has been implicated in the propagation and spread of misfolded proteins associated with neurodegenerative diseases. We used transgenesis mice to study the in vivo consequences of disrupting TSG101-dependent trafficking in adult neurons. Mice lacking Tsg101 in forebrain neurons (Tsg101^ck2-null) showed rapid loss of hippocampal neurons and progressive forebrain atrophy. Astrogliosis was apparent in the dentate gyrus within 1 week of deleting Tsg101, followed by apoptosis of hippocampal CA3 neurons and accumulation of the autophagy adapter P62/SQSTM1 and ubiquitinated proteins. Failure to detect lipidated LC3 indicated autophagy was impaired rather than upregulated. Endosomal markers (RAB5 and RAB7) and amyloid protein also accumulated in hippocampal neurons of Tsg101^ck2-null mice. Our data establish a critical role for TSG101 in neuronal survival and demonstrate the importance of the in vivo assessment of gene and protein functions. Full article

The endosomal sorting complexes required for transport (ESCRT) comprise a fundamental cellular machinery with remarkable versatility in membrane remodeling. It is multifunctional in the multivesicular body (MVB) biogenesis, exosome formation and secretion, virus budding, cytokinesis, plasma membrane repair, neuron pruning, and autophagy. ESCRT’s involvement in cellular mechanisms extends beyond basic membrane trafficking. By directly interacting with autophagy-related (ATG) proteins and facilitating autophagosome-lysosome fusion, ESCRT ensures cellular homeostasis. Dysregulation in ESCRT function has been implicated in cancer, neurodegenerative disorders, and infectious diseases, underscoring its critical role in numerous pathologies. Hepatitis B virus (HBV) is an enveloped virus that exploits ESCRT and autophagy pathways for viral replication, assembly, and secretion. This review synthesizes recent mechanistic insights into ESCRT’s multifaceted roles, particularly focusing on its interactions with autophagy formation and the HBV lifecycle. Full article

Chitin synthase 3 complex assembly begins at the endoplasmic reticulum where the formation of a Chs3/Chs7 complex facilitates its exit from the ER and its transport along the secretory route. In the present study, our work shows that orphan molecules of Chs7 can exit the ER and are later recycled from the early Golgi by coat protein I (COPI) machinery via the adaptor complex Erv41/Erv46. Moreover, an eventual excess of the protein in the Golgi is recognized by the GGA complex and targeted to the vacuole for degradation through the ESCRT machinery. Non-oligomerizable versions of Chs3 can also exit the ER individually and follow a similar route to that of Chs7. We therefore demonstrate the traffic of unassembled CS3 subunits and describe the cellular mechanisms that guarantee the correct assembly of this protein complex at the ER while providing a default traffic route to the vacuole in case of its failure. This traffic route is shared with canonical ER adaptors, such as Erv29 and Erv14, and other components of protein complexes. The comparative analysis of their traffic allows us to discern a cellular program that combines COPI recycling, proteasomal degradation, and vacuolar disposal for maintaining protein homeostasis at the ER. Full article

The ESCRT (endosomal sorting complex required for transport) machinery is essential for various cellular processes, yet its role in head and neck squamous cell carcinoma (HNSCC) is poorly understood. We utilized The Cancer Genome Atlas (TCGA) datasets to analyze the expression of ESCRT genes. Bulk RNA-sequencing data and HNSCC tissue microarrays (TMAs) were used to evaluate VPS25 expression and its clinical significance. Single-cell RNA sequencing of tumor tissues and VPS25 knockdown experiments in CAL27 cells were used to investigate its biological functions. Immunohistochemistry, spatial transcriptomics, and immunotherapy datasets highlighted the involvement of VPS25 in immune suppression and its potential as a predictive biomarker. The results demonstrated significant VPS25 overexpression in HNSCC tissues, which correlated with poor clinical outcomes. It promoted tumor cell proliferation and migration while reducing immune cell infiltration in the tumor microenvironment (TME). Additionally, by upregulating PVR expression in tumor cells, VPS25 activated the immunosuppressive PVR-TIGIT signaling axis, thereby facilitating immune evasion. Furthermore, VPS25 emerged as a potential biomarker for predicting immunotherapy response. These findings highlight VPS25 as a pivotal regulator of tumor progression and immune evasion in HNSCC and a promising target for therapeutic strategies. Full article

We previously isolated a cDNA clone for galactosylceramide expression factor 1, which is the rat homologue of hepatocyte-growth-factor-regulated tyrosine kinase substrate (HGS) and induces galactosylceramide expression and morphological changes in COS-7 cells, and reported that overexpression of HGS induced morphological changes in canine kidney epithelial MDCK cells. HGS is a component of the endosomal sorting complexes required for transport machinery that mediates endosomal multivesicle body formation. In this study, the overexpression of HGS induced epithelial–mesenchymal transition and caused transformation in MDCK cells, whereas the overexpression of a coiled-coil domain of HGS inhibited induction of epithelial–mesenchymal transition by HGF stimulation. The overexpression of HGS in mouse melanoma B16 cells and human colorectal cancer COLO205 cells promoted cancer characteristic anchorage-independent cell growth ability and tumor growth, whereas the overexpression of the coiled-coil domain of HGS in these cells suppressed them. The oligopeptide OP12-462 constituting the coiled-coil domain suppressed the anchorage-independent cell growth ability and tumor growth of COLO205 cells. The coiled-coil domain of HGS and OP12-462 are novel tumor growth inhibitors that do not directly destroy cancer cells but rather inhibit only the anchorage-independent cell growth ability of cancer cells. Full article

The HIV-1 protease is a critical enzyme for viral replication. Because protease activity is necessary to generate mature infectious virions, it is a primary target of antiretroviral treatment. Here, we provide an overview of the mechanisms regulating protease activation and the methods available to assess protease activity. Finally, we will highlight some of the key discoveries regarding the kinetics of protease activation from the last decade, including how the manipulation of activation kinetics may provide novel HIV-1 treatment strategies. Full article

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101–Ub–Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding. Full article