Endosomal sorting complex required for transport (ESCRT) system drives various cellular processes, including endosome sorting, organelle biogenesis, vesicle transport, maintenance of plasma membrane integrity, membrane fission during cytokinesis, nuclear membrane reformation after mitosis, closure of autophagic vacuoles, and enveloped virus budding. Increasing evidence suggests that the ESCRT system can be hijacked by different family viruses for their proliferation. At different stages of the virus life cycle, viruses can interfere with or exploit ESCRT-mediated physiological processes in various ways to maximize their chance of infecting the host. In addition, many retroviral and RNA viral proteins possess "late domain" motifs, which can recruit host ESCRT subunit proteins to assist in virus endocytosis, transport, replicate, budding and efflux. Therefore, the "late domain" motifs of viruses and ESCRT subunit proteins could serve as promising drug targets in antiviral therapy. This review focuses on the composition and functions of the ESCRT system, the effects of ESCRT subunits and virus "late domain" motifs on viral replication, and the antiviral effects mediated by the ESCRT system, aiming to provide a reference for the development and utilization of antiviral drugs.
Endosomal Sorting Complexes Required for Transport/metabolism* ; Viruses/metabolism* ; Protein Transport ; Virus Replication ; Endosomes/metabolism* ; Virus Release

TRPML1 channel is a non-selective group-2 transient receptor potential (TRP) channel with Ca permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV (MLIV). In the present study, we determined the cryo-electron microscopy (cryo-EM) structures of Mus musculus TRPML1 (mTRPML1) in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain (PMD) may sense the luminal/extracellular stimuli and undergo a "move upward" motion during endocytosis, thus triggering the overall conformational change in TRPML1. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca, and phosphoinositides in a combined manner so as to accommodate the dynamic endocytosis process.
Animals ; Calcium ; metabolism ; Cryoelectron Microscopy ; Endocytosis ; Endosomes ; metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; metabolism ; Mice ; Models, Biological ; Mucolipidoses ; genetics ; metabolism ; pathology ; Nanostructures ; chemistry ; ultrastructure ; Phosphatidylinositols ; metabolism ; Transgenes ; Transient Receptor Potential Channels ; chemistry ; genetics ; metabolism

Parkinson's disease is a multifactorial disorder with several genes linked to the familial types of the disease. ATP13A2 is one of those genes and encode for a transmembrane protein localized in lysosomes and late endosomes. Previous studies suggested the roles of this protein in lysosomal functions and cellular ion homeostasis. Here, we set out to investigate the role of ATP13A2 in lysosomal function and in metabolism of alpha-synuclein, another PD-linked protein whose accumulation is implicated in the pathogenesis. We generated non-sense mutations in both copies of ATP13A2 gene in SH-SY5Y human neuroblastoma cells. We examined lysosomal function of ATP13A2-/- cells by measuring the accumulation of lysosomal substrate proteins, such as p62 and polyubiquitinated proteins, induction of acidic compartments, and degradation of ectopically introduced dextran. None of these measures were altered by ATP13A2 deficiency. The steady-state levels of alpha-synuclein in cells or secretion of this protein were unaltered either in ATP13A2-/- compared to the normal cells. Therefore, the proposed roles of ATP13A2 in lysosomal functions may not be generalized and may depend on the cellular context. The ATP13A2-/- cells generated in the current study may provide a useful control for studies on the roles of PD genes in lysosomal functions.
alpha-Synuclein* ; Dextrans ; Endosomes ; Homeostasis ; Humans ; Lysosomes ; Metabolism* ; Neuroblastoma ; Parkinson Disease ; Polyubiquitin

The intracellular trafficking and subcellular distribution of exogenous gene is very important for gene delivery. A successful gene vehicle should overcome various barriers including endosomal membrane barriers to delivery gene to the target organelle. Traditional nonviral vehicle is unable to avoid endosomal pathway efficiently, so the efficiency of gene delivery is low and the application of gene drugs is limited. In order to achieve efficient nonviral gene delivery, a lot of researches based on endosomal escape have been carried out and some agents with the function of endsomal escape have been found. These agents facilitate the endsomal escape via various mechanisms, such as fusion into the lipid bilayer of endosomes, pore formation in the endosomal membrane, proton sponge effect and photochemical methods to rupture the endosomal membrane. In this review, various reported strategies for endsomal escape are described according to the escape mechanisms, and their applications in intracellular gene delivery are also discussed.
Cell Membrane ; metabolism ; Endosomes ; metabolism ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Humans

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidic acid. PLD is localized in most cellular organelles, where it is likely to play different roles in signal transduction. PLD1 is primarily localized in vesicular structures such as endosomes, lysosomes and autophagosomes. However, the factors defining its localization are less clear. In this study, we found that four hydrophobic residues present in the N-terminal HKD catalytic motif of PLD1, which is involved in intramolecular association, are responsible for vesicular localization. Site-directed mutagenesis of the residues dramatically disrupted vesicular localization of PLD1. Interestingly, the hydrophobic residues of PLD1 are also involved in the interruption of its nuclear localization. Mutation of the residues increased the association of PLD1 with importin-beta, which is known to mediate nuclear importation, and induced the localization of PLD1 from vesicles into the nucleus. Taken together, these data suggest that the hydrophobic amino acids involved in the interdomain association of PLD1 are required for vesicular localization and disturbance of its nuclear localization.
Amino Acid Motifs ; Amino Acid Sequence ; Amino Acids/chemistry ; Cell Nucleus/*enzymology ; Endosomes/enzymology ; HEK293 Cells ; Humans ; Hydrophobic and Hydrophilic Interactions ; Lysosomes/enzymology ; Phagosomes/enzymology ; Phospholipase D/chemistry/*metabolism ; Protein Interaction Domains and Motifs ; Protein Transport ; Transport Vesicles/*enzymology

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a key component of the endosomal sorting complexes required for transport and has been demonstrated to play a regulatory role in endocytosis/exocytosis and the accumulation of internal vesicles in multivesicular bodies. Citron kinase is a Ser/The kinase that we previously reported to enhance human immunodeficiency virus type 1 (HIV-1) virion production. However, the relationship between Hrs and citron kinase in HIV-1 production remains elusive. Here, we report that Hrs interacts with citron kinase via its FYVE domain. Overexpression of Hrs or the FYVE domain resulted in a significant decrease in HIV-1 virion production. Depletion of Hrs by RNA interference in HEK293T cells increased HIV-1 virion production and enhanced the activity of citron kinase. These data suggest that Hrs inhibits HIV-1 production by inhibiting citron kinase-mediated exocytosis.
Down-Regulation ; Endosomal Sorting Complexes Required for Transport ; genetics ; metabolism ; Endosomes ; metabolism ; Exocytosis ; Gene Expression ; Gene Silencing ; drug effects ; HEK293 Cells ; HIV Infections ; genetics ; metabolism ; virology ; HIV-1 ; drug effects ; genetics ; growth & development ; Humans ; Immunoprecipitation ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Microscopy, Fluorescence ; Phosphoproteins ; genetics ; metabolism ; Plasmids ; Protein Binding ; drug effects ; genetics ; Protein Interaction Domains and Motifs ; Protein Structure, Tertiary ; Protein Transport ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA, Small Interfering ; pharmacology ; Transfection ; Virion ; drug effects ; genetics ; growth & development ; Virus Release ; drug effects ; Virus Replication ; drug effects

Exosomes are nanometer sized membrane vesicles, released in the extracellular milieu following the fusion of the external membrane of multivesicular body (MVB) with plasma membrane. They perform a certain function in immune regulation. Exosomes have been shown to be released by cells of hematopoietic and non-hematopoietic origin. Tumour-derived exosomes (TEX) exist in the supernatant of tumour cells, plasma and malignant effusions of tumour patients. They contain native candidate tumour associated antigen and are capable of transferring antigens to T lymphocytes, therefore efficiently promoting cytotoxic T lymphocyte (CTL) activation and producing antitumor immunity. However, recent evidence shows that tumor exosomes may induce immunologic tolerance and even activate immunosuppression which makes tumour escape from the immune surveillance of the host immune system. In addition, tumor exosomes may mediate a growth-promoting effect on tumor cells. These discrepancies are almost certainly due to differences in the phenotype of the exosomes.
Antigen-Presenting Cells ; immunology ; Antigens, Neoplasm ; immunology ; Cytoplasmic Vesicles ; immunology ; Endosomes ; immunology ; metabolism ; Exosomes ; immunology ; Humans ; Neoplasms ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Escape

OBJECTIVETo observe the changes of human trophoblast cells after infected with hepatitis B virus.

METHODSHBV positive serum was used to infect human trophoblast cells in vitro. HBsAg in cell culture medium were detected by ELISA method and HBV DNA in cell culture medium and cells were detected by PCR method. HBV fluorescence polymerase chain reaction diagnose kit were used to detect the HBV DNA concentration. Ultra structure of trophoblast cells were observed with transmission electron microscopy (TEM).

RESULTSHBsAg could be detected in infection group by ELISA. Infection group cell culture medium and infection group cells were HBV DNA positive. HBV DNA concentrations in HBV infection cell culture medium in 0, 12, 36, 60, 84 h after extensively PBS washed were < 10(3), 3 x 10(4), 6 x 10(5), 5 x 10(5), 3 x 10(5) copies/mL. HBV infected trophoblast cells were found many forms of endosomes, some of which contents virus like particle.

CONCLUSIONHBV might take advantage of clathrin-mediated endocytosis to enter trophoblast cell, which might lead to cell infection or across the cell bar by transcytosis.

Animals ; Culture Media, Conditioned ; metabolism ; DNA, Viral ; analysis ; Endosomes ; virology ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; genetics ; isolation & purification ; physiology ; Humans ; Microscopy, Electron, Transmission ; Polymerase Chain Reaction ; Time Factors ; Trophoblasts ; ultrastructure ; virology

Huntington's disease is caused by CAG trinucleotide expansions in the gene encoding huntingtin. N- terminal fragments of huntingtin with polyglutamine produce aggregates in the endosome-lysosomal system, where proteolytic fragments of huntingtin is generated. Heat shock protein 70 (HSP70) prevents the formation of protein aggregates, but the effect of HSP70 on the huntingtin in the endosome-lysosomal system is unknown. This study was to determine whether HSP70 alters the distribution of huntingtin in endosome-lysosomal system. HSP70 expressing stable cells (NIH/3T3 or cerebral hybrid cell line A1) were generated, and mutant [(CAG)100] huntingtin was transiently overexpressed. Analysis of subcellular distribution by immnuocytochemistry or proteolysis cleavage by Western blotting was performed. 18 CAG repeat wild type [WT; (CAG)18] huntingtin was used as a control. Cells with huntingtin showed patterns of endosome- lysosomal accumulation, from a 'dispersed vacuole (DV)' type into a coalescent 'perinuclear vacuole (PV)' type over time. In WT huntingtin, HSP70 increased the cells with the PV types that enhanced the proteolytic cleavage of huntingtin. However, HSP70 reduced cells of the DV and PV types expressing mutant huntingtin, that result in less proteolysis than that of control. In addition, intranuclear inclusions were formed only in mutant cells, which was not affected by HSP70. These results suggest that HSP70 alters the distribution of huntingtin in the endosome-lysosomal system, and that this contributes to huntingtin proteolysis.
Peptide Hydrolases/metabolism ; Nuclear Proteins/genetics/*metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; NIH 3T3 Cells ; Mice ; Lysosomes/*metabolism ; HSP70 Heat-Shock Proteins/genetics/*metabolism ; Endosomes/*metabolism ; Cytoplasm/metabolism ; Cell Survival ; Animals

OBJECTIVETo label rat neural stem cells (NSCs) with the complex of Sinerem, the ultrasmall superparamagnetic iron oxide (USPIO), and poly-L-lysine (PLL), and evaluate the feasibility of tracking the labeled cells with magnetic resonance imaging (MRI) in vitro and in vivo.

METHODSSinerem was incubated with PLL to obtain the complex of Sinerem-PLL. The mesenchymal stem cells (MSCs) isolated from the bone marrow of SD rats were cultured and induced to differentiate into the neural stem cells. The second-passage cells were cultured overnight with the Sinerem-PLL complex, after which Prussian blue staining and transmission electron microscopy were performed to observe the nanoparticles in the cytoplasm. Cell apoptosis assay was performed to assess the cell viability 1 day, 1 week, and 2 weeks after the labeling. Cell tracking with 4.7 MR system was carried out in vivo and in vitro using T(2)WI and T(2)*WI sequences.

RESULTSThe NSCs could be effectively labeled with Sinerem-PLL complex with the labeling efficiency exceeding 95%. Prussian blue staining showed numerous blue iron particles in the cytoplasm, and under transmission electron microscope, these particles accumulated in the endosomes/lysosomes. The labeling did not significantly affect the cell viability and proliferation. Remarkable low signal density changes of the labeled cells was seen on T(2)WI and T(2)*WI in vivo and in vitro.

CONCLUSIONNSCs can be effectively labeled with Sinerem-PLL complex, and MRI can be used to track the labeled cells in vivo and in vitro.

Animals ; Cell Differentiation ; Cells, Cultured ; Dextrans ; metabolism ; Endosomes ; metabolism ; Ferrosoferric Oxide ; metabolism ; Lysosomes ; metabolism ; Magnetic Resonance Imaging ; methods ; Magnetite Nanoparticles ; Male ; Mesenchymal Stromal Cells ; cytology ; Microscopy, Electron, Transmission ; Neurons ; cytology ; metabolism ; ultrastructure ; Polylysine ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; metabolism ; ultrastructure ; Time Factors