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Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Azheimer's disease (AD). Sequential cleavage of APP by β and γ secretases leads to generation of Aβ40 (non-amyloidogenic) and Aβ42 (amyloidogenic) peptides. Despite intense studies, the biological function of these peptides and the mechanism of Aβ42 toxicity is poorly understood. In the previous publications we proposed that association of Aβ peptides with the endosomal membranes may have important implications for pathogenesis of AD (Kim and Bezprozvanny, IJMS, 2021, vol 22, 13600; Kim and Bezprozvanny, IJMS, 2023, vol 24, 2092). To understand potential biological importance of such interaction, we focused on the region of Aβ peptides involved in peri-membrane association (E682 to N698). We discovered that association of this region with the membranes is reminiscent of several known anti-microbial peptides (AMP) such as PA13, Aurein1.2 and BP100. Our analysis further revealed that energy of peri-membrane association of Aβ40 is significantly weaker than for Aβ42 or AMP peptides, but it can be increased in the presence of non-amyloidogenic FAD mutations or in the presence of cholesterol in the membrane. Based on similarity with established mechanism of action of AMP peptides, we propose that Aβ peptides affect the curvature of endosomal membranes and shift the balance between endosomal recycling to plasma membrane and late endosomal/lysosomal pathway. We further propose that these effects are enhanced as a result of non-amyloidogenic FAD mutations in the sequence of Aβ peptides or in the presence of cholesterol in the membrane. The proposed model provides potential mechanistic explanation to synaptic defects induced by increased levels of Aβ42, by non-amyloidogenic FAD mutations in APP and by age-related increase in the levels of cholesterol in the brain., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:I.B. is a member of Neurodon SAB and holds Neurodon stock or stock options., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mapping the endocytic vesicular acidification process is of prior importance to better understand the health and pathological processes of cells. Herein, by integrating a pH-sensitive i-motif and a pair of fluorescence resonance energy transfer (FRET) into a tetrahedral DNA framework (TDF), we develop a pH-responsive DNA nanomachine, allowing for efficient sensing of pH from 7.0 to 5.5 via the pH-triggered spatial proximity modulation of FRET. The inheriting endo-lysosome-targeting ability of TDF enables spatiotemporal tracking of endocytic vesicle acidification during the endosomal maturation process. Analysis of pH-dependent FRET response at single fluorescent spot level reveals the significant difference of endocytic vesicular acidification between normal and cancer cells. The performance of pH-responsive DNA nanomachine underlines its potential for studies on vesicle acidification-related pathologies as a universal platform., (© 2024 Wiley-VCH GmbH.)

Endosomal acid base balance functions as a master orchestrator within the cell, engaging with many cellular pathways to maintain homeostasis. Mutations in the endosomal pH regulator Na + /H + exchanger NHE6 may disrupt this delicate balancing act and cause monogenic Parkinsonism. Here, gene expression studies in post-mortem substantia nigra of Parkinson's disease (PD) patients and normal controls were performed to investigate whether NHE6 represents a pathophysiological link between monogenic and sporadic PD. The substantia nigra in PD displayed down-regulation of NHE6, coincident with a loss of expression of several SNARE signalling pathway members, suggesting impaired membrane fusion and vesicle-recycling. Increased abundance of related NHE9 was also identified in the parkinsonian nigra that could reflect compensatory changes or be a consequence of neuronal dysfunction. The current model suggests the possibility that neurons expressing low levels of NHE6 are more susceptible to injury in PD, potentially directly contributing to the loss of nigral dopaminergic neurons and the genesis of the disease. These results have important implications for disease-modifying therapies as they suggest that endosomal pH correctors, including epigenetic modifiers that regulate NHE6 expression, may be beneficial for PD. Thus, aberrant endosomal acidification in the nigrostriatal pathway is a possible unifying pathomechanism in both monogenic and sporadic PD, with implications for understanding and treating this disorder. Replication of these observations in the post-mortem brains of Alzheimer's disease and frontotemporal dementia patients supports a model of conserved mechanisms underlying injury and death of neurons., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The endomembrane system is essential for healthy cell function, with the various compartments carrying out a large number of specific biochemical reactions. To date, almost all of our understanding of the endomembrane system has come from the study of cultured cells growing as monolayers. However, monolayer-grown cells only poorly represent the environment encountered by cells in the human body. As a first step to address this disparity, we have developed a platform that allows us to investigate and quantify changes to the endomembrane system in three-dimensional (3D) cell models, in an automated and highly systematic manner. HeLa Kyoto cells were grown on custom-designed micropatterned 96-well plates to facilitate spheroid assembly in the form of highly uniform arrays. Fully automated high-content confocal imaging and analysis were then carried out, allowing us to measure various spheroid-, cellular- and subcellular-level parameters relating to size and morphology. Using two drugs known to perturb endomembrane function, we demonstrate that cell-based assays can be carried out in these spheroids, and that changes to the Golgi apparatus and endosomes can be quantified from individual cells within the spheroids. We also show that image texture measurements are useful tools to discriminate cellular phenotypes. The automated platform that we show here has the potential to be scaled up, thereby allowing large-scale robust screening to be carried out in 3D cell models., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mysior, Simpson. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of host-pathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially regarding the early dynamics of downstream TLR pathways. Here, we present a label-free optical biosensor-based assay as a method for detecting TLR activation in a native and label-free environment and defining the dynamics of TLR pathway activation. This technology is sufficiently sensitive to detect TLR signaling and readily discriminates between different TLR signaling pathways. We define pharmacological modulators of cell surface and endosomal TLRs and downstream signaling molecules and uncover TLR signaling signatures, including potential biased receptor signaling. These findings highlight that optical biosensor assays complement traditional assays that use a single endpoint and have the potential to facilitate the future design of selective drugs targeting TLRs and their downstream effector cascades., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Microtubule-dependent endosomal transport is crucial for polar growth, ensuring the precise distribution of cellular cargos such as proteins and mRNAs. However, the molecular mechanism linking mRNAs to the endosomal surface remains poorly understood. Here, we present a structural analysis of the key RNA-binding protein Rrm4 from Ustilago maydis . Our findings reveal a different type of MademoiseLLE domain (MLLE) featuring a seven-helical bundle that provides a distinct binding interface. A comparative analysis with the canonical MademoiseLLE domain of the poly(A)-binding protein Pab1 disclosed unique characteristics of both domains. Deciphering the MLLE binding code enabled prediction and verification of previously unknown Rrm4 interactors containing short linear motifs. Importantly, we demonstrated that the human MLLE domains, such as those of PABPC1 and UBR5, employed a similar principle to distinguish among interaction partners. Thus, our study provides detailed mechanistic insights into how structural variations in the widely distributed MLLE domain facilitate mRNA attachment during endosomal transport., Competing Interests: Competing interests statement:The authors declare no competing interest.

The therapeutic options for Alzheimer's disease (AD) are limited, underscoring the critical need for finding an effective regulator of Aβ42 production. In this study, with 489 human postmortem brains, we revealed that homotrimer G protein subunit gamma 5 (GNG5) expression is upregulated in the hippocampal-entorhinal region of pathological AD compared with normal controls, and is positively correlated with Aβ pathology. In vivo and in vitro experiments confirm that increased GNG5 significantly promotes Aβ pathology and Aβ42 production. Mechanically, GNG5 regulates the cleavage preference of γ-secretase towards Aβ42 by directly interacting with the γ-secretase catalytic subunit presenilin 1 (PS1). Moreover, excessive GNG5 increases the protein levels and the activation of Rab5, leading to the increased number of early endosomes, the major cellular organelle for production of Aβ42. Furthermore, immunoprecipitation and immunofluorescence revealed co-interaction of Aβ42 with GPCR family CXCR2, which is known as the receptor for IL-8, thus facilitating the dissociation of G-proteins βγ from α subunits. Treatment of Aβ42 in neurons combined with structure prediction indicated Aβ42 oligomers as a new ligand of CXCR2, upregulating γ subunit GNG5 protein levels. The co-localizations of GNG5 and PS1, CXCR2 and Aβ42 were verified in eight human brain regions. Besides, GNG5 is significantly reduced in extracellular vesicles (EVs) derived from cerebral cortex or serum of AD patients compared with healthy cognition controls. In brief, GNG5 is a novel regulator of Aβ42 production, suggesting its clinical potential as a diagnosis biomarker and the therapeutic target for AD. The GNG5 content in EVs derived from serum and brain tissue of patients with AD significantly reduced. The GNG5 expression in the hippocampal-entorhinal neurons of donors with pathological AD significantly increased, and can exist in homotrimer subtypes. GNG5 expression positively correlates with Aβ pathology and Aβ42 production. Homotrimer-GNG5 binds to the γ-secretase catalytic subunit PS1 and preferentially generates Aβ42 in early endosome. GNG5 leads to enhanced Rab5 protein and activation levels, increased number of early endosome, promoting Aβ42 production. Further, Aβ42 binds to CXCR2 to upregulate GNG5 levels in a feedback loop., Competing Interests: Competing interests The authors declare no competing interests. Ethical approval and consent to participate Human material study has been approved by the Ethics Committee of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The ethics approval for animal work was provided by the Ethics Committee of Chinese Academy of Medical Sciences., (© 2024. The Author(s).)

Membrane penetration by non-enveloped viruses is diverse and generally not well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. Enteroviruses enter cells by receptor-mediated endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing enteroviruses deform, rupture, and release the virus particles into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of particles that released their genomes, but did not prevent them from reaching the cytoplasm. Inhibiting post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. The rupture of endosomes also occurs in control cells and after the endocytosis of very low-density lipoprotein. In summary, our results show that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus particles into the cytoplasm to initiate infection. Since the studied enteroviruses employ different receptors for cell entry but are delivered into the cytoplasm by cell-mediated endosome disruption, it is likely that most if not all enteroviruses, and probably numerous other viruses from the family Picornaviridae, can utilize endosome rupture to infect cells., (© 2024. The Author(s).)

Classical G-protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap, we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial-trans Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments., Competing Interests: WJ, KS, GF, SL, NL No competing interests declared, (© 2024, Jang et al.)

RIG-I-like receptors (RLRs) are cytosolic RNA sensors critical for antiviral immunity. RLR activation is regulated by polyubiquitination and oligomerization following RNA binding. Yet, little is known about how RLRs exploit subcellular organelles to facilitate their posttranslational modifications and activation. Endosomal adaptor TAPE regulates the endosomal TLR and cytosolic RLR pathways. The potential interplay between RIG-I signaling and endosomes has been explored. Here, we report that endosomes act as platforms for facilitating RIG-I polyubiquitination and complex formation. RIG-I was translocated onto endosomes to form signaling complexes upon activation. Ablation of endosomes impaired RIG-I signaling to type I IFN activation. TAPE mediates the interaction and polyubiquitination of RIG-I and TRIM25. TAPE-deficient myeloid cells were defective in type I IFN activation upon RNA ligand and virus challenges. Myeloid TAPE deficiency increased the susceptibility to RNA virus infection in vivo. Our work reveals endosomes as signaling platforms for RIG-I activation and antiviral immunity.

We have previously shown that Golgi stacks and recycling endosomes (REs) exist as Golgi/RE units in sea urchin embryos. In this study, we showed that Golgi/RE units were scattered throughout the cytoplasm at early developmental stages but gathered to form a "Golgi ring" surrounding the centric REs at the blastula stage. This change in the cell-wide arrangement of Golgi/RE units coincided with a dramatic change in microtubule organization from a randomly oriented cortical pattern to radial arrays under the apical plasma membrane. A single gigantic Golgi apparatus surrounding centric RE is clearly associated with the center of the radial microtubule arrays. Furthermore, we found that in some animal species belonging to different clades, Golgi stacks lack lateral connections but are likely centralized by microtubule motors. These results suggest that Golgi centralization depends on the organization of the microtubule array in addition to the lateral linking between Golgi stacks.Key words: Golgi stack, recycling endosome, Golgi-ribbon, microtubule, cilium, sea urchin, ascidian.

Deleterious mutations in the lipopolysaccharide responsive beige-like anchor protein (LRBA) gene cause severe childhood immune dysregulation. The complexity of the symptoms involving multiple organs and the broad range of unpredictable clinical manifestations of LRBA deficiency complicate the choice of therapeutic interventions. Although LRBA has been linked to Rab11-dependent trafficking of the immune checkpoint protein CTLA-4, its precise cellular role remains elusive. We show that LRBA, however, only slightly colocalizes with Rab11. Instead, LRBA is recruited by members of the small GTPase Arf protein family to the TGN and to Rab4+ endosomes, where it controls intracellular traffic. In patient-derived fibroblasts, loss of LRBA led to defects in the endosomal pathway promoting the accumulation of enlarged endolysosomes and lysosome secretion. Thus, LRBA appears to regulate flow through the endosomal system on Rab4+ endosomes. Our data strongly suggest functions of LRBA beyond CTLA-4 trafficking and provide a conceptual framework to develop new therapies for LRBA deficiency., (© 2024 Szentgyörgyi et al.)

Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homologue of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Because MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Cellular membrane homoeostasis is maintained via a tightly regulated membrane and cargo flow between organelles of the endocytic and secretory pathways. Adaptor protein complexes (APs), which are recruited to membranes by the small GTPase ARF1, facilitate cargo selection and incorporation into trafficking intermediates. According to the classical model, small vesicles would facilitate bi-directional long-range transport between the Golgi, endosomes and plasma membrane. Here we revisit the intracellular organization of the vesicular transport machinery using a combination of CRISPR-Cas9 gene editing, live-cell high temporal (fast confocal) or spatial (stimulated emission depletion) microscopy as well as correlative light and electron microscopy. We characterize tubulo-vesicular ARF1 compartments that harbour clathrin and different APs. Our findings reveal two functionally different classes of ARF1 compartments, each decorated by a different combination of APs. Perinuclear ARF1 compartments facilitate Golgi export of secretory cargo, while peripheral ARF1 compartments are involved in endocytic recycling downstream of early endosomes. Contrary to the classical model of long-range vesicle shuttling, we observe that ARF1 compartments shed ARF1 and mature into recycling endosomes. This maturation process is impaired in the absence of AP-1 and results in trafficking defects. Collectively, these data highlight a crucial role for ARF1 compartments in post-Golgi sorting., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis., Competing Interests: Declarations Competing interest The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Bismuth compounds, particularly colloidal bismuth subcitrate (CBS), have been widely used in the treatment of gastrointestinal diseases. However, overdose of CBS has been linked to cases of acute renal failure, primarily due to the intracellular accumulation of bismuth in the kidney. To date, the detailed mechanisms of CBS internalization and its metabolic fate remain unclear. In this study, CBS was characterized as a type of nano-object using transmission electron microscopy and dynamic light scattering. Renal cells internalized CBS primarily via clathrin-mediated endocytosis in an active transport manner. Gene knockdown techniques revealed that CBS binds to the transferrin receptor likely through complexing with transferrin before cellular uptake. Once internalized, CBS was sorted into early endosomes, late endosomes, and lysosomes, mediated by microtubules and the Golgi apparatus. Additionally, differentially expressed genes analysis revealed that CBS endocytosis stimulated oxidative stress, significantly affecting the metabolism of glutathione and cysteine within cells. This led to the formation of black bismuth sulfide particles as a result of CBS conjugating with intracellular glutathione. These findings provide crucial insights into the cellular mechanisms underlying excessive CBS exposure, which is essential for understanding and potentially mitigating the risks associated with the use of bismuth compounds in medical treatments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In the past decade, nucleic acid therapies have seen a boon in development and clinical translation largely due to advances in nanotechnology that have enabled their safe and targeted delivery. Nanoparticles can protect nucleic acids from degradation by serum enzymes and can facilitate entry into cells. Still, achieving endosomal escape to allow nucleic acids to enter the cytoplasm has remained a significant barrier, where less than 5% of nanoparticles within the endo-lysosomal pathway are able to transfer their cargo to the cytosol. Lipid-based drug delivery vehicles, particularly lipid nanoparticles (LNPs), have been optimized to achieve potent endosomal escape, and thus have been the vector of choice in the clinic as demonstrated by their utilization in the COVID-19 mRNA vaccines. The success of LNPs is in large part due to the rational design of lipids that can specifically overcome endosomal barriers. In this review, we chart the evolution of lipid structure from cationic lipids to ionizable lipids, focusing on structure-function relationships, with a focus on how they relate to endosomal escape. Additionally, we examine recent advancements in ionizable lipid structure as well as discuss the future of lipid design., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sortilin-related receptor 1 (SorL1) deficiency is a genetic predisposition to familial Alzheimer's disease (AD), but its pathology is poorly understood. In SorL1-null rats, a disorder of the global endosome-lysosome network (ELN) is found in hippocampal neurons. Deletion of amyloid precursor protein (APP) in SorL1-null rats could not completely rescue the neuronal abnormalities in the ELN of the hippocampus and the impairment of spatial memory in SorL1-null young rats. These in vivo observations indicated that APP is one of the cargoes of SorL1 in the regulation of the ELN, which affects hippocampal-dependent memory. When SorL1 is depleted, the endolysosome takes up more of the lysosome flux and damages lysosomal digestion, leading to pathological lysosomal storage and disturbance of cholesterol and iron homeostasis in the hippocampus. These disturbances disrupt the original homeostasis of the material-energy-subcellular structure and reprogram energy metabolism based on fatty acids in the SorL1-null hippocampus, instead of glucose. Although fatty acid oxidation increases ATP supply, it cannot reduce the levels of the harmful byproduct ROS during oxidative phosphorylation, as it does in glucose catabolism. Therefore, the SorL1-null rats exhibit hippocampal degeneration, and their spatial memory is impaired. Our research sheds light on the pathology of SorL1 deficiency in AD., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Cells utilize diverse organelles to maintain homeostasis and to respond to extracellular stimuli. Recently, multifaceted aspects of organelle stress caused by various factors have been emerging. The endosome is an essential organelle, functioning as the central hub for membrane trafficking in cooperation with the ubiquitin system. However, knowledge regarding endosomal stress, which refers to organelle stress of the endosome, is currently limited. We recently revealed ubiquitin-mediated endosomal stress of early endosomes (EEs) and its responsive signaling pathways. These findings shed light on the relevance of ubiquitin-mediated endosomal stress to physiological and pathological processes. Here, we present a hypothesis that ubiquitin-mediated endosomal stress may have significant roles in biological contexts and that ubiquitin-specific protease 8 is a key regulator of ubiquitin clearance from EEs., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

G protein-coupled receptors (GPCRs) have historically been associated with signalling events driven from the plasma membrane. More recently, signalling from endosomes has been recognized as a feature of internalizing receptors. However, there was little consideration given to the notion that GPCRs can be targeted to distinct subcellular locations that did not involve an initial trafficking to the cell surface. Here, we focus on the evidence for and the potential impact of GPCR signalling specifically initiated from the nuclear membrane. We also discuss the possibilities for selectively targeting this and other internal pools of receptors as novel venues for drug discovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2023 Wiley Periodicals LLC.)

Lipid nanoparticle (LNP)-mRNA complexes are transforming medicine. However, the medical applications of LNPs are limited by their low endosomal disruption rates, high toxicity and long tissue persistence times. LNPs that rapidly hydrolyse in endosomes (RD-LNPs) could solve the problems limiting LNP-based therapeutics and dramatically expand their applications but have been challenging to synthesize. Here we present an acid-degradable linker termed 'azido-acetal' that hydrolyses in endosomes within minutes and enables the production of RD-LNPs. Acid-degradable lipids composed of polyethylene glycol lipids, anionic lipids and cationic lipids were synthesized with the azido-acetal linker and used to generate RD-LNPs, which significantly improved the performance of LNP-mRNA complexes in vitro and in vivo. Collectively, RD-LNPs delivered mRNA more efficiently to the liver, lung, spleen and brains of mice and to haematopoietic stem and progenitor cells in vitro than conventional LNPs. These experiments demonstrate that engineering LNP hydrolysis rates in vivo has great potential for expanding the medical applications of LNPs., Competing Interests: Competing interests The authors declare the following competing interests: H.H., K.G. and N.M. own equity in Opus Biosciences. All the other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Multivesicular endosomes (MVEs) sequester membrane proteins destined for degradation within intralumenal vesicles (ILVs), a process mediated by the membrane-remodeling action of Endosomal Sorting Complex Required for Transport (ESCRT) proteins. In Arabidopsis , endosomal membrane constriction and scission are uncoupled, resulting in the formation of extensive concatenated ILV networks and enhancing cargo sequestration efficiency. Here, we used a combination of electron tomography, computer simulations, and mathematical modeling to address the questions of when concatenated ILV networks evolved in plants and what drives their formation. Through morphometric analyses of tomographic reconstructions of endosomes across yeast, algae, and various land plants, we have found that ILV concatenation is widespread within plant species, but only prevalent in seed plants, especially in flowering plants. Multiple budding sites that require the formation of pores in the limiting membrane were only identified in hornworts and seed plants, suggesting that this mechanism has evolved independently in both plant lineages. To identify the conditions under which these multiple budding sites can arise, we used particle-based molecular dynamics simulations and found that changes in ESCRT filament properties, such as filament curvature and membrane binding energy, can generate the membrane shapes observed in multiple budding sites. To understand the relationship between membrane budding activity and ILV network topology, we performed computational simulations and identified a set of membrane remodeling parameters that can recapitulate our tomographic datasets., Competing Interests: Competing interests statement:The authors declare no competing interest.

Nutrient-sensing pathways undergo deregulation in aged animals, exerting a pivotal role in regulating the cell cycle and subsequent stem cell division. Nevertheless, their precise functions in governing pluripotent stem cell differentiation remain largely elusive. Here, we uncovered a significant alteration in the cellular constituents of the intestinal epithelium in aged humans and mice. Employing Drosophila midgut and mouse organoid culture models, we made an observation regarding the altered trajectory of differentiation in intestinal stem cells (ISC) during overnutrition or aging, which stems from the erroneous activation of the insulin receptor signaling pathway. Through genetic analyses, we ascertained that the nutrient-sensing pathway regulated the direction of ISC differentiation by modulating the maturation of endosomes and SOX21A transcription factor. This study elucidates a nutrient-sensing pathway-mediated mechanism underlying stem cell differentiation, offering insights into the etiology of stem cell dysfunction in aged animals, including humans., (© 2024. The Author(s).)

Atlantic cod has a peculiar immune system, characterized by the loss of Major Histocompatibility Complex (MHC) class II pathway, and an extreme expansion of the MHC class I gene repertoire. This has led to the hypothesis that some of the MHC I variants have replaced MHC II by presenting exogenous-peptides in a process similar to cross-presentation. In mammals, MHC I loads endogenous antigens in the endoplasmic reticulum, but we recently found that different Atlantic cod MHC I gene variants traffic to endolysosomes. There, they colocalize with Tapasin and other components of the peptide-loading complex, indicating a plausible peptide-loading system outside the endoplasmic reticulum. In this study, we further characterize the identity of the Atlantic cod MHC I compartment (cMIC). We found that, similarly to mammalian MHC II compartment, cMIC contains late endosomal markers such as Rab7, LAMP1 and CD63. Furthermore, we identified Hsp90b1 (also known as grp94) and LRP1 (also known as CD91) as interactors of MHC I by mass spectrometry. As these two proteins are involved in cross-presentation in mammals, this further suggests that Atlantic cod MHC I might use a similar mechanism to present exogenous peptides, thus, compensating for the absence of MHC II., (© 2024. The Author(s).)

B cells rapidly adapt their endocytic pathway to promote the uptake and processing of extracellular antigens recognized through the B-cell receptor (BCR). The mechanisms coupling changes in endomembrane trafficking to the capacity of B cells to screen for antigens within lymphoid tissues remain unaddressed. We investigated the role of SNX5, a member of the sorting nexin family, which interacts with endocytic membranes to regulate vesicular trafficking and macropinocytosis. Our results show that in steady state, B cells form SNX5-rich protrusions at the plasma membrane, which dissipate upon interaction with soluble antigens, whereas B cells activated with immobilized antigens accumulate SNX5 at the immune synapse where it regulates actin-dependent spreading responses. B cells silenced for SNX5 exhibit enlarged lysosomes, which are not recruited to the synaptic membrane, decreasing their capacity to extract immobilized antigens. Overall, our findings reveal that SNX5 is critical for actin-dependent plasma membrane remodeling in B cells involved in antigen screening and immune synapse formation, as well as endolysosomal trafficking required to promote antigen extraction and presentation., (© 2024 Cabrera-Reyes et al.)

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells., Competing Interests: SK, MT, MT, TO, TN, KK, MM No competing interests declared, (© 2024, Koike et al.)

The major organelles of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) (~1.5 billion years ago). Their acquisitions were defining milestones during eukaryogenesis. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct interorganelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of additional, distinct cellular compartments, infer ancestral states predating LECA, and explore the process of eukaryogenesis itself. Here, we undertake a molecular evolutionary analysis (including providing a transcriptome of the jakobid flagellate Reclinomonas americana ), exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles (LROs), the Biogenesis of LRO Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery is present across the eukaryotic tree of life and was likely in place prior to LECA, making it an underappreciated facet of eukaryotic cellular organisation. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1 and MON1 proteins, and an unexpected relationship with the "homotypic fusion and vacuole protein sorting" (HOPS) and "Class C core vacuole/endosomal tethering" (CORVET) complexes), offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LROs biogenesis machinery from before the LECA to current eukaryotic diversity, integrating it into the larger mechanistic framework describing endomembrane evolution., Competing Interests: Competing interests statement:The authors declare no competing interest.

Fungal biosynthetic gene clusters often include genes encoding transmembrane proteins, which have been mostly thought to be transporters exporting the products. However, there is little knowledge about subcellular compartmentalization of transmembrane proteins essential for biosynthesis. Fungal mycotoxin cyclochlorotine is synthesized by non-ribosomal peptide synthetase, which is followed by modifications with three transmembrane UstYa-family proteins. Heterologous expression in Aspergillus oryzae revealed that total biosynthesis of cyclochlorotine requires additional two transporter proteins. Here, we investigated subcellular localizations of the five transmembrane proteins under heterologous expression in A. oryzae. Enhanced green fluorescent protein (EGFP) fusions to the transmembrane proteins, which were confirmed to normally function in cyclochlorotine production, were expressed together with organellar markers. All the transmembrane proteins exhibited localizations commonly in line of the trans-Golgi, endosomes, and vacuoles. This study suggests that subcellular compartmentalization of UstYa family proteins and transporters allows corporative functions of delivering intermediates and subsequent modifications, completing cyclochlorotine biosynthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Polyphosphate (polyP) is a ubiquitous polyanion present throughout the tree of life. While polyP's widely varied functions have been interrogated in single-celled organisms, little is known about the cellular distribution and function of polyP in multicellular organisms. To study polyP in metazoans, we developed the nematode Caenorhabditis elegans as a model system. We designed a high-throughput, longitudinal-orientation cryosectioning method that allowed us to scrutinize the intracellular localization of polyP in fixed C. elegans using fluorescent polyP probes and co-immunostaining targeting appropriate marker proteins. We discovered that the vast majority of polyP is localized within the endo-lysosomal compartments of the intestinal cells and is highly sensitive toward the disruption of endo-lysosomal compartment generation and food availability. This study lays the groundwork for further mechanistic research of polyPs in multicellular organisms and provides a reliable method for immunostaining hundreds of fixed worms in a single experiment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) or programmed cell death-ligand 1 (PD-L1) have achieved impressive antitumor clinical outcomes. However, the limited response rates suggest the incomplete understanding of PD-L1 regulation. Here, we demonstrate that vacuole protein sorting 11 and 18 (VPS11/18), two key players in vesicular trafficking, positively regulate PD-L1 and confer resistance to immune checkpoint blockade therapy. VPS11/18 interact with PD-L1 in endosome recycling accompanied by promoting PD-L1 glycosylation and protein stability. VPS18 deficiency enhances antitumor immune response. Pharmacological inhibition by VPS18 inhibitor RDN impaired PD-L1 member trafficking and protein stability. Combination treatment of RDN and anti-cytotoxic T lymphocyte-associated antigen 4 synergistically enhances antitumor efficacy in aggressive and drug-resistant tumors. RDN exerted lung-preferred distribution and good bioavailability, suggesting a favorable drug efficacy. Together, our study links VPS18/11-mediated trans-Golgi network recycling of PD-L1 and points to a promising treatment strategy for the enhancement of antitumor immunity.

Extracellular vesicles (EVs) are attracting growing attention for therapeutic use and as diagnostic markers, particularly for cancer. Although therapies based on small interfering RNAs are under intensive research, other therapeutic molecules, especially proteins, have not been sufficiently investigated. One of the major method for loading proteins into EVs is electroporation; however, it damages membrane integrity and requires repeated purification, precluding clinical applications. Thus, natural and efficient protein transfer is a prerequisite for the clinical application of protein-based EV therapy. Another prerequisite is an efficient endosomal escape, as most EVs incorporated into receptor cells result in endosomal degradation. Therefore, we generated a short CD9 (sCD9)-INF/TAT tag for efficiently transfers fused proteins to the EV and enhances endosomal escape to address the abovementioned problems. Interestingly, protein transfer via EVs drastically improved when the EV producer and receptor cells were cocultured, strongly indicating bystander effects of cells producing therapeutic proteins fused with a sCD9-INF/TAT tag. This method can be applied to a wide range of therapeutic technologies, including cellular transplantation or viral therapy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Inano, Kitano. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Small interfering RNAs (siRNAs), comprising 21-23 nucleotides, function by complementary binding to specific mRNA sequences, thereby suppressing target protein expression. Despite their vast potential in disease therapy, siRNAs face challenges due to their susceptibility to degradation and high electronegativity, rendering them unstable in the bloodstream and impeding their passage across endothelial barriers. Moreover, successful intracellular delivery necessitates overcoming endosomal entrapment, posing a significant hurdle for carrier material development. In this study, leveraging the strong affinity of histidine oligomers (His6) for metal ions, we engineered nanoparticles (HmA) by gentle assembly with divalent zinc ions under pH = 8 conditions. We designed the RNA-binding functional peptide L2-NTD to enhance siRNA stability and delivery efficiency when complexed with HmA. The resulting siRNA+L2-NTD@HmA nanoparticles were formed via in situ encapsulation, ensuring efficient siRNA delivery into cells with minimal cytotoxicity and degradation. This approach presents a novel strategy for the design and artificial fabrication of carriers for effective RNA delivery.

Many disease-causing proteins have multiple pathogenic mechanisms, and conventional inhibitors struggle to reliably disrupt more than one. Targeted protein degradation (TPD) can eliminate the protein, and thus all its functions, by directing a cell's protein turnover machinery towards it. Two established strategies either engage catalytic E3 ligases or drive uptake towards the endolysosomal pathway. Here we describe CYpHER (CatalYtic pH-dependent Endolysosomal delivery with Recycling) technology with potency and durability from a catalytic mechanism that shares the specificity and straightforward modular design of endolysosomal uptake. By bestowing pH-dependent release on the target engager and using the rapid-cycling transferrin receptor as the uptake receptor, CYpHER induces endolysosomal delivery of surface and extracellular targets while re-using drug, potentially yielding increased potency and reduced off-target tissue exposure risks. The TfR-based approach allows targeting to tumors that overexpress this receptor and offers the potential for transport to the CNS. CYpHER function was demonstrated in vitro with EGFR and PD-L1, and in vivo with EGFR in a model of EGFR-driven non-small cell lung cancer., (© 2024. The Author(s).)

Kinesin and dynein-dynactin motors move endosomes and other vesicles bidirectionally along microtubules, a process mainly studied under in vitro conditions. Here, we provide a physiological bidirectional transport model following color-coded, endogenously tagged transport-related proteins as they move through a crowded cellular environment. Late endosomes (LEs) surf bidirectionally on Protrudin-enriched endoplasmic reticulum (ER) membrane contact sites, while hopping and gliding along microtubules and bypassing cellular obstacles, such as mitochondria. During bidirectional transport, late endosomes do not switch between opposing Rab7 GTPase effectors, RILP and FYCO1, or their associated dynein and KIF5B motor proteins, respectively. In the endogenous setting, far fewer motors associate with endosomal membranes relative to effectors, implying coordination of transport with other aspects of endosome physiology through GTPase-regulated mechanisms. We find that directionality of transport is provided in part by various microtubule-associated proteins (MAPs), including MID1, EB1, and CEP169, which recruit Lis1-activated dynein motors to microtubule plus ends for transport of early and late endosomal populations. At these microtubule plus ends, activated dynein motors encounter the dynactin subunit p150 glued and become competent for endosomal capture and minus-end movement in collaboration with membrane-associated Rab7-RILP. We show that endosomes surf over the ER through the crowded cell and move bidirectionally under the control of MAPs for motor activation and through motor replacement and capture by endosomal anchors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The endosomal sorting complex required for transport (ESCRT)-III is involved in membrane remodeling and abscission during intraluminal vesicle (ILV) formation at endosomes. Our data now suggest that ESCRT-III function could be connected to lipid remodeling of the endosomal membrane. This notion is based on our finding that ESCRT-III proteins bind to the yeast serine incorporator (SERINC) homolog Tms1. Human SERINC3 and SERINC5 are HIV-1 restriction factors and have been shown to act as scramblases, flipping phospholipids between membrane leaflets. Due to the extraordinarily high sequence conservation between Tms1 and human SERINCs, it is likely that Tms1 is also a scramblase. While deletion of TMS1 had only a moderate effect on the sorting of multivesicular body (MVB) cargo proteins, the simultaneous deletion of a component of the Vps55/Vps68 complex led to a strong synergistic phenotype. This pronounced synergism suggests that Tms1 and Vps55/Vps68 perform a parallel function at endosomes. Vps55/Vps68 loosely resembles Tms1 in its overall structure. Thus, it is possible that Vps55/Vps68 is also a scramblase. Since both Vps55 and Tms1 physically interact with ESCRT-III proteins, we propose that the recruitment of a scramblase plays a crucial role in ESCRT-III-dependent membrane remodeling at endosomes., Competing Interests: Conflicts of interest The author has no competing interests to declare that are relevant to the content of this article., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Target of Myb1 (TOM1) facilitates the transport of endosomal ubiquitinated proteins destined for lysosomal degradation; however, the mechanisms regulating TOM1 during this process remain unknown. Here, we identified an adjacent DXXLL motif-containing region to the TOM1 VHS domain, which enhances its affinity for ubiquitin and can be modulated by phosphorylation. TOM1 is an endosomal phosphatidylinositol 5-phosphate (PtdIns5P) effector under Shigella flexneri infection. We pinpointed a consensus PtdIns5P-binding motif in the VHS domain. We show that PtdIns5P binding by TOM1 is pH-dependent, similarly observed in its binding partner TOLLIP. Under acidic conditions, TOM1 retained its complex formation with TOLLIP, but was unable to bind ubiquitin. S. flexneri infection inhibits pH-dependent endosomal maturation, leading to reduced protein degradation. We propose a model wherein pumping of H + to the cytosolic side of endosomes contributes to the accumulation of TOM1, and possibly TOLLIP, at these sites, thereby promoting PtdIns5P- and pH-dependent signaling, facilitating bacterial survival., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Multivesicular bodies are key endosomal compartments implicated in cellular quality control through their degradation of membrane-bound cargo proteins 1-3 . The ATP-consuming ESCRT protein machinery mediates the capture and engulfment of membrane-bound cargo proteins through invagination and scission of multivesicular-body membranes to form intraluminal vesicles 4,5 . Here we report that the plant ESCRT component FREE1 6 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation. We use a minimal physical model, reconstitution experiments and in silico simulations to identify the dynamics of this process and describe intermediate morphologies of nascent intraluminal vesicles. Furthermore, we find that condensate-wetting-induced line tension forces and membrane asymmetries are sufficient to mediate scission of the membrane neck without the ESCRT protein machinery or ATP consumption. Genetic manipulation of the ESCRT pathway in several eukaryotes provides additional evidence for condensate-mediated membrane scission in vivo. We find that the interplay between condensate and machinery-mediated scission mechanisms is indispensable for osmotic stress tolerance in plants. We propose that condensate-mediated scission represents a previously undescribed scission mechanism that depends on the physicomolecular properties of the condensate and is involved in a range of trafficking processes. More generally, FREE1 condensate-mediated membrane scission in multivesicular-body biogenesis highlights the fundamental role of wetting in intracellular dynamics and organization., (© 2024. The Author(s).)

The majority of viruses classified as pandemic threats are enveloped viruses which enter the cell through receptor-mediated endocytosis and take advantage of endosomal acidification to activate their fusion machinery. Here we report that the endosomal fusion of low pH-requiring viruses is highly dependent on TRPM7, a widely expressed TRP channel that is located on the plasma membrane and in intracellular vesicles. Using several viral infection systems expressing the envelope glycoproteins of various viruses, we find that loss of TRPM7 protects cells from infection by Lassa, LCMV, Ebola, Influenza, MERS, SARS-CoV-1, and SARS-CoV-2. TRPM7 ion channel activity is intrinsically necessary to acidify virus-laden endosomes but is expendable for several other endosomal acidification pathways. We propose a model wherein TRPM7 ion channel activity provides a countercurrent of cations from endosomal lumen to cytosol necessary to sustain the pumping of protons into these virus-laden endosomes. This study demonstrates the possibility of developing a broad-spectrum, TRPM7-targeting antiviral drug to subvert the endosomal fusion of low pH-dependent enveloped viruses., (© 2024. The Author(s).)

Endocytosis and recycling control the uptake and retrieval of various materials, including membrane proteins and lipids, in all eukaryotic cells. These processes are crucial for cell growth, organization, function and environmental communication. However, the mechanisms underlying efficient, fast endocytic recycling remain poorly understood. Here, by utilizing a biosensor and imaging-based screening, we uncover a recycling mechanism that couples endocytosis and fast recycling, which we name the clathrin-associated fast endosomal recycling pathway (CARP). Clathrin-associated tubulovesicular carriers containing clathrin, AP1, Arf1, Rab1 and Rab11, while lacking the multimeric retrieval complexes, are generated at subdomains of early endosomes and then transported along actin to cell surfaces. Unexpectedly, the clathrin-associated recycling carriers undergo partial fusion with the plasma membrane. Subsequently, they are released from the membrane by dynamin and re-enter cells. Multiple receptors utilize and modulate CARP for fast recycling following endocytosis. Thus, CARP represents a previously unrecognized endocytic recycling mechanism with kiss-and-run membrane fusion., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Trace elemental iron is an essential nutrient that participates in diverse metabolic processes. Dysregulation of cellular iron homeostasis, both iron deficiency and iron overload, is detrimental and tightly associated with disease pathogenesis. IRPs-IREs system is located at the center for iron homeostasis regulation. Additionally, ferritinophagy, the autophagy-dependent ferritin catabolism for iron recycling, is emerging as a novel mechanism for iron homeostasis regulation. It is still unclear whether IRPs-IREs system and ferritinophagy are synergistic or redundant in determining iron homeostasis. Here we report that IRP2, but not IRP1, is indispensable for ferritinophagy in response to iron depletion. Mechanistically, IRP2 ablation results in compromised AMPK activation and defective ATG9A endosomal trafficking, leading to the decreased engulfment of NCOA4-ferritin complex by endosomes and the subsequent dysregulated endosomal microferritinophagy. Moreover, this defective endosomal microferritinophagy exacerbates DNA damage and reduces colony formation in IRP2-depleted cells. Collectively, this study expands the physiological function of IRP2 in endosomal microferritinophagy and highlights potential crosstalk between IRPs-IREs and ferritinophagy in manipulating iron homeostasis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Presentation of metabolites by the major histocompatibility complex class I-related protein 1 (MR1) molecule to mucosal-associated invariant T cells is impaired during herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections. This is surprising given these viruses do not directly synthesise MR1 ligands. We have previously identified several HSV proteins responsible for rapidly downregulating the intracellular pool of immature MR1, effectively inhibiting new surface antigen presentation, while preexisting ligand-bound mature MR1 is unexpectedly upregulated by HSV-1. Using flow cytometry, immunoblotting, and high-throughput fluorescence microscopy, we demonstrate that the endocytosis of surface MR1 is impaired during HSV infection and that internalized molecules accumulate in EEA1-labeled early endosomes, avoiding degradation. We establish that the short MR1 cytoplasmic tail is not required for HSV-1-mediated downregulation of immature molecules; however it may play a role in the retention of mature molecules on the surface and in early endosomes. We also determine that the HSV-1 US3 protein, the shorter US3.5 kinase and the full-length HSV-2 homolog, all predominantly target mature surface rather than total MR1 levels. We propose that the downregulation of intracellular and cell surface MR1 molecules by US3 and other HSV proteins is an immune-evasive countermeasure to minimize the effect of impaired MR1 endocytosis, which might otherwise render infected cells susceptible to MR1-mediated killing by mucosal-associated invariant T cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinomas (HCCs) are characterized by a vast spectrum of somatic copy number alterations (CNAs); however, their functional relevance is largely unknown. By performing a genome-wide survey on prognosis-associated focal CNAs in 814 HCC patients by an integrative computational framework based on transcriptomic data, genomic amplification is identified at 8q24.13 as a promising candidate. Further evidence is provided that the 8q24.13 amplification-driven overexpression of Rab GTPase activating protein TBC1D31 exacerbates HCC growth and metastasis both in vitro and in vivo through activating Epidermal growth factor receptor (EGFR) signaling. Mechanistically, TBC1D31 acts as a Rab GTPase activating protein to catalyze GTP hydrolysis for Rab22A and then reduces the Rab22A-mediated endolysosomal trafficking and degradation of EGFR. Notably, overexpression of TBC1D31 markedly increases the resistance of HCC cells to lenvatinib, whereas inhibition of the TBC1D31-EGFR axis can reverse this resistance phenotype. This study highlights that TBC1D31 at 8q24.13 is a new critical oncogene, uncovers a novel mechanism of EGFR activation in HCC, and proposes the potential strategies for treating HCC patients with TBC1D31 amplification or overexpression., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Growth arrest-specific 6 (GAS6) is a secreted protein that acts as a ligand for TAM receptors (TYRO3, AXL, and MERTK). In humans, GAS6 circulating levels and genetic variations in GAS6 are associated with hyperglycemia and increased risk of type 2 diabetes. However, the mechanisms by which GAS6 influences glucose metabolism are not understood. Here, we show that Gas6 deficiency in mice increases insulin sensitivity and protects from diet-induced insulin resistance. Conversely, increasing GAS6 circulating levels is sufficient to reduce insulin sensitivity in vivo. GAS6 inhibits the activation of the insulin receptor (IR) and reduces insulin response in muscle cells in vitro and in vivo. Mechanistically, AXL and IR form a complex, while GAS6 reprograms signaling pathways downstream of IR. This results in increased IR endocytosis following insulin treatment. This study contributes to a better understanding of the cellular and molecular mechanisms by which GAS6 and AXL influence insulin sensitivity., (© 2024 by the American Diabetes Association.)

The early diagnosis of hepatocellular carcinomas (HCCs) remains challenging in the clinic. Primovist-enhanced magnetic resonance imaging (MRI) aids HCC diagnosis but loses sensitivity for tumors <2 cm. Therefore, developing advanced MRI contrast agents is imperative for improving the diagnostic accuracy of HCCs in very-early-stage. To address this challenge, PEGylated ultra-small iron oxide nanoparticles (PUSIONPs) are synthesized and employed as liver-specific T1 MRI contrast agents. Intravenous delivery produces simultaneous hyperintense HCC and hypointense hepatic parenchyma signals on T1 imaging, creating an extraordinarily high tumor-to-liver contrast. Systematic studies uncover PUSIONP distribution in hepatic parenchyma, HCC lesions at the organ, tissue, cellular, and subcellular levels, revealing endosomal confinement of PUSIONP without aggregation. By mimicking such situations, the dependency of relaxometric properties on local PUSIONP concentration is investigated, emphasizing the key role of different endosomal concentrations in liver and tumor cells for high tumor-to-liver contrast and clear tumor boundaries. These findings offer exceptional imaging capabilities for early HCC diagnosis, potentially benefiting real HCC patients., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Type IV P-type ATPases (P4-ATPases) are lipid flippases that generate an asymmetric membrane organization essential for cell viability. The five budding yeast P4-ATPases traffic between the Golgi complex, plasma membrane, and endosomes but how they are recycled from the endolysosomal system to the Golgi complex is poorly understood. In this study, we find that P4-ATPase endosomal recycling is primarily driven by the retromer complex and the F-box protein Rcy1. Defects in P4-ATPase recycling result in their mislocalization to the vacuole and a substantial loss of membrane asymmetry. The P4-ATPases contain multiple predicted retromer sorting signals, and the characterization of these signals in Dnf1 and Dnf2 led to the identification of a novel retromer-dependent signal, IPM[ST] that acts redundantly with predicted motifs. Together, these results emphasize the importance of endosomal recycling for the functional localization of P4-ATPases and membrane organization., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Background: Endolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of proinflammatory cytokines/chemokines. However, excessive proinflammatory responses can lead to a potentially lethal cytokine storm., Objectives: Here we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants., Methods: HSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release., Results: We show that Toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust proinflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein., Conclusions: These findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs., Competing Interests: Disclosure statement This study was funded by grants from the Swedish Society for Medical Research (SSMF; grant number PG-22-0423-H-01), Magnus Bergvall Foundation, the Swedish Research Council, the Swedish Cancer Foundation, the Swedish Heart and Lung Foundation, the Knut and Alice Wallenberg Foundation, the Erling-Persson Foundation, and the Consul Berg Foundation. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Endosomes are central protein-sorting stations at the crossroads of numerous membrane trafficking pathways in all eukaryotes. They have a key role in protein homeostasis and cellular signalling and are involved in the pathogenesis of numerous diseases. Endosome-associated protein assemblies or coats collect transmembrane cargo proteins and concentrate them into retrieval domains. These domains can extend into tubular carriers, which then pinch off from the endosomal membrane and deliver the cargoes to appropriate subcellular compartments. Here we discuss novel insights into the structure of a number of tubular membrane coats that mediate the recruitment of cargoes into these carriers, focusing on sorting nexin-based coats such as Retromer, Commander and ESCPE-1. We summarize current and emerging views of how selective tubular endosomal carriers form and detach from endosomes by fission, highlighting structural aspects, conceptual challenges and open questions., (© 2024. Springer Nature Limited.)

Treatments for cancer that incorporate small interfering RNA (siRNA) to target iron-dependent ferroptosis are thought to be highly promising. However, creating a reliable and clinically feasible siRNA delivery system continues to be a major obstacle in the field of cancer treatment. Here, three imidazole-based ionizable lipid nanoparticles (LNPs) with pH-sensitive effects are rationally designed and synthesized for siRNA delivery. LNPs formulated with the top-performing lipid (O12-D3-I3) encapsulating FVII siRNA (FVII@O-LNP) elicited greater gene silencing than those with the benchmark Onpattro lipid DLin-MC3-DMA (MC3) due to its stronger endosomal escape. Moreover, Fc-siRNA@O-LNPs encapsulated with ferrocene (Fc) and SLC7A11/Nrf2-targeted siRNA is formulated. The outcomes demonstrate optimal safety profiles and a significant anti-tumor effect by inducing long-lasting and efficient ferroptosis through a synergistic action in vivo. In summary, this work shows that imidazolyl lipid-prepared LNPs are efficient delivery vehicles for cancer therapy and ferroptosis-targeting siRNA administration, both of which have extensive clinical application potential., (© 2024 Wiley‐VCH GmbH.)

In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport. During retrograde transport these autophagosomes mature through repetitive fusion events. Complete lysosomal cargo degradation occurs largely in the cell body. The precipitating events to stimulate retrograde autophagosome transport have been debated but their importance is clear: disrupting neuronal autophagy or autophagosome transport is detrimental to neuronal health and function. We have identified the HOPS complex as essential for early autophagosome maturation and consequent initiation of retrograde transport from the axon terminal. In yeast and mammalian cells, HOPS controls fusion between autophagosomes and late endosomes with lysosomes. Using zebrafish strains with loss-of-function mutations in vps18 and vps41 , core components of the HOPS complex, we found that disruption of HOPS eliminates autophagosome maturation and disrupts retrograde autophagosome transport initiation from the axon terminal. We confirmed this phenotype was due to loss of HOPS complex formation using an endogenous deletion of the HOPS binding domain in Vps18. Finally, using pharmacological inhibition of lysosomal proteases, we show that initiation of autophagosome retrograde transport requires autophagosome maturation. Together, our data demonstrate that HOPS-mediated fusion events are critical for retrograde autophagosome transport initiation through promoting autophagosome maturation. This reveals critical roles for the HOPS complex in neuronal autophagy which deepens our understanding of the cellular pathology of HOPS-complex linked neurodegenerative diseases. Abbreviations : CORVET: Class C core vacuole/endosome tethering; gRNA: guide RNA; HOPS: homotypic fusion and protein sorting; pLL: posterior lateral line; Vps18: VPS18 core subunit of CORVET and HOPS complexes; Vps41: VPS41 subunit of HOPS complex.