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Results for “Extracellular vesicles”

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PubMed2004

Association of hepatitis C virus envelope proteins with exosomes.

As the human tetraspanin CD81 binds hepatitis C virus (HCV) envelope glycoprotein E2, we addressed the role CD81 may play in cellular trafficking of HCV envelope proteins. Studies on HCV life cycle are complicated by the lack of a robust cell culture system; we therefore transfected mammalian cells with HCV E1-E2 cDNA, with or without human CD81 (huCD81) cDNA. In the absence of huCD81, HCV envelope proteins are almost completely retained in the endoplasmic reticulum. Instead, when huCD81 is present, a fraction of HCV envelope proteins passes through the Golgi apparatus, matures acquiring complex sugars and is found extracellularly associated with exosomes. These are 60-100-nm membrane vesicles enriched in tetraspanins, released into the extracellular milieu by many cell types and having fusogenic activity. We also report that human plasma contains exosomes and that in HCV patients, viral RNA is associated with these circulating vesicles. We propose that the HCV-CD81 complex leaves cells in the form of exosomes, circulates in this form and exploits the fusogenic capabilities of these vesicles to infect cells even in the presence of neutralizing antibodies.

Animals↗

PubMed1991

A contribution with review to the description of mineralization of bone and other calcified tissues in vivo.

This manuscript considers certain aspects of mineral deposition in bone and other vertebrate calcifying tissues in order to examine physical, chemical, and biological factors important in the mineralization process. The paper in a discussion format principally presents a new data and the formulation of concepts based on such data as well as a summary of background material as necessary review. Mineralization is found to occur at spatially independent sites throughout the organic extracellular tissue matrices. Matrix vesicles and collagen fibrils each may serve as independent nucleation centers for mineral with vesicle mineralization being local and collagen mineralization dominating the tissues as a whole. Collagen fibril organization is suggested to be such that hole zones are aligned in three dimensions, creating extensive channels for mineral accommodation. Nucleation occurs initially in hole zones and crystal growth leads to the development of plate-like mineral particles whose orientation, disposition, and sizes within fibrils are detailed. Effects of diffusion, crystallinity, and critical nucleation and growth events are described with respect to their influence on mineral deposition in bulk and local regions of tissue matrices.

Animals↗

PubMed1978

Effects of acute cold exposure on the ultrastructure of the mouse pinealocyte.

Ultrastructural changes in pinealocytes were investigated qualitatively and quantitatively in mice exposed to low temperatures at 0 degree C or 5 degree C for 30 or 60 min. A quantitative estimation of the granulated vesicles of about 100 nm diameter in pinealocytes demonstrated that the number of these vesicles per pinealocyte in mice exposed to cold at 0 degree C or 5 degree C increased remarkably as compared with that of the controls. This finding indicates that cold stimuli may activate the formation of the granulated vesicles in pinealocytes. In mice exposed to cold at 0 degree C for 60 min, granulated vesicles were often found in gourps close to the plasma membrane of the pinealocyte cell bodies or processes, indicating the release of the vesicle content into the extracellular spaces. Additionally, the pinealocytes of the mice exposed to cold at 0 degree C for 60 min were characterized by the frequent occurrence of lysosome-like bodies and filaments. The development of these structures in response to severe cold may represent a depressed functional state of pienalocytes.

Animals↗

PubMed1986

Ultrastructural localization of calcium in the CNS of vertebrates.

The ultrastructural localization of calcium in synaptic areas of the CNS of fish was investigated. Prefixation with phosphate-buffered glutaraldehyde followed by post-fixation with osmium/potassium-bichromate was used to precipitate and visualize endogenous calcium without the addition of external calcium. The presence of calcium in the electron-dense precipitates produced using this method was demonstrated by electron spectroscopic imaging using a Zeiss EM-902 transmission electron microscope, and in various control experiments using the calcium chelator EGTA. In the optic tectum of fish, electron dense precipitates containing calcium were found not only in intracellular compartments, e.g. the smooth endoplasmic reticulum, mitochondria and synaptic vesicles, but also at extracellular locations, particularly in synaptic clefts. In the extracellular sites, only chelate complexes of ionic calcium were found. This would seem to be in agreement with electrophysiological and biochemical data reported in earlier studies. Thus, using the present method, it should be possible to obtain further ultrastructural information concerning the mechanisms of synaptic transmission.

Animals↗

PubMed1988

Molecular mechanisms of exocytosis: the adrenal chromaffin cell as a model system.

1. The release of neurotransmitters, hormones, and enzymes involves exquisitely regulated events which ultimately result in the fusion of the secretory vesicle with the cell's plasma membrane, releasing the vesicle contents into the extracellular space. 2. The biochemical and cellular mechanisms mediating exocytosis have been extensively studied in a model system of primary cultured adrenal chromaffin cells. 3. This paper briefly reviews current understanding, and directions of future studies in exocytosis using this model system.

Adrenal Medulla↗

PubMed1991

Subcellular localization of acidic and basic PR proteins in tobacco mosaic virus-infected tobacco.

Infection of Samsum NN tobacco with tobacco mosaic virus (TMV) results in the induction of the synthesis of acidic and basic isoforms of many pathogenesis-related (PR) proteins. By immunogold-electromicroscopy we have shown that PR proteins accumulate mainly in cells around the necrotic spots of TMV-induced lesions. The acidic chitinases, beta-(1,3)-glucanases and thaumatin-like proteins were found to accumulate in extracellular "pocket-like" vesicles while the basic chitinases were found in electron dense inclusion bodies in the vacuoles. These structures were not detectable in PR-containing leaves devoid of virus nor in healthy plants.

Antibodies, Viral↗

PubMed1999

Developmental regulation and ultrastructure of glycogen deposits during murine tooth morphogenesis.

The distribution and ultrastructure of glycogen deposits were investigated in the murine tooth germ by histochemical periodic acid-Schiff (PAS) staining and transmission electron microscopy. Lower and upper first molars were examined in mouse embryos at embryonic days 11.5-17 (E11.5-E17) and in 2-day-old postnatal (P2) mice. The oral and dental epithelia and the mesenchymal cells were generally PAS-positive during tooth morphogenesis. PAS-negative cells were present at E13 in the distal tip of the tooth bud epithelium and in the contacting mesenchyme, and this complete lack of PAS reactivity continued in the dental papilla mesenchyme and inner enamel epithelium during the cap and bell stages. The lack of glycogen deposits in the interacting epithelium and mesenchyme during early morphogenesis may be associated with their demonstrated high signaling activities. Mesenchymal cells in the dental follicle consistently possessed small clusters or large pools of glycogen, which disappeared by P2. Since an intense PAS reaction was seen in mesenchymal cells at future bone sites, the glycogen in the dental follicle cells may be associated with their development into hard-tissue-forming cells. Ultrastructural observation of the enamel organ cells from the cap to early bell stages (E14-E15) revealed the occurrence of glycogen pools, which were associated with the Golgi apparatus and with vesicles having amorphous contents. Glycogen particles were also occasionally present inside vesicles or in the extracellular matrix. These may be associated with the exocytosis of glycosaminoglycan components into extracellular spaces and the formation of the stellate reticulum.

Animals↗

PubMed1989

Calcification of in vitro developed hypertrophic cartilage.

We have recently reported that dedifferentiated cells derived from stage 28-30 chick embryo tibiae, when transferred in suspension culture in the presence of ascorbic acid, develop in a tissue closely resembling hypertrophic cartilage. Ultrastructural examination of this in vitro formed cartilage showed numerous matrix vesicles associated with the extracellular matrix (C. Tacchetti, R. Quarto, L. Nitsch, D. J. Hartmann, and R. Cancedda, 1987, J. Cell Biol. 105, 999-1006). In the present article we report that the in vitro developed hypertrophic cartilage undergoes calcification. We indicate a correlation between the levels of alkaline phosphatase activity and calcium deposition at different times of development. Following the transfer of cells into suspension culture and an initial lag phase, the level of alkaline phosphatase activity rapidly increased. In most experiments the maximum of activity was reached after 5 days of culture. When alkaline phosphatase activity and 45Ca deposition were measured in the same experiment, we observed that the increase in alkaline phosphatase preceded the deposition of nonwashable calcium deposits in the cartilage.

Alkaline Phosphatase↗

PubMed1976

Quantitative and comparative ultrastructure of the vertebrate cornea. I. Urodele Amphibia.

The cornea of the urodele amphibian Triturus c. cristatus was studied ultrastructurally in order to provide the basis for a comparison among corneas throughout the vertebrate phylum. The cornea of this salamander consists of relatively thick epithelium and basement membrane and thin Descemet's membrane, unlike the mammalian corneas. The outermost epithelial cells contain Ruthenium Red stainable extracellular filaments and intracellular vesicles which are thought to play a role in the process of lubricating the corneal surface. Occluding junctions have been observed in the apical region of the superficial epithelial cells and are considered as barriers to the intercellular passage of material. A thin substantia propria (stroma) consists of about 40 collagenous highly organized lamellae. The thicknesses of the basement membrane, Descemet's membrane and the epithelium are believed to represent the primitive situation in the process of corneal evolution.

Animals↗

PubMed1992

Demonstration of the capacity of nacre to induce bone formation by human osteoblasts maintained in vitro.

Nacre implanted in vivo in bone is osteogenic suggesting that it may possess factor(s) which stimulate bone formation. The present study was undertaken to test the hypothesis that nacre can induce mineralization by human osteoblasts in vitro. Nacre chips were placed on a layer of first passage human osteoblasts. None of the chemical inducers generally required to obtain bone formation in vitro was added to the cultures. Osteoblasts proliferated and were clearly attracted by nacre chips to which they attached. Induction of mineralization appeared preferentially in bundles of osteoblasts surrounding the nacre chips. Three-dimensional nodules were formed by a dense osteoid matrix with cuboidal osteoblasts at the periphery and osteocytic-like cells in the center. These nodules contained foci with features of mineralized structures and bone-like structures, both radiodense to X-ray. Active osteoblasts (e.m.) with abundant rough endoplasmic reticulum, extrusion of collagen fibrils and budding of vesicles were observed. Matrix vesicles induced mineral deposition. Extracellular collagen fibrils appeared cross-banded and electrodense indicating mineralization. These results demonstrate that a complete sequence of bone formation is reproduced when human osteoblasts are cultured in the presence of nacre. This model provides a new approach to study the steps of osteoblastic differentiation and the mechanisms of induction of mineralization.

Animals↗

PubMed1993

In vitro differentiation and mineralization of cartilaginous nodules from enzymatically released rat nasal cartilage cells.

Nasal cartilage cells from 21-day-old rat fetuses were cultured at high density in the presence of ascorbic acid and beta-glycerophosphate over a 12-day period. Immediately after plating, the cells exhibited a fibroblastic morphology, lost their chondrocyte phenotype and expressed type I collagen. On day 3, clusters of enlarged polygonal cells were found. These cell clusters synthetized type II collagen and formed an alcian-blue-positive matrix. The following days, a progressive increase in the number of cells positive for type II collagen was noted and, on day 8, typical cartilaginous nodules were formed. These nodules increased in size and number, spreading outward, laying down a dense matrix which mineralized. Light and electron microscopy observations of cross-sections of nodules confirmed the cartilaginous nature of this tissue formed in vitro with typical chondrocytes embedded in a hyaline matrix. Furthermore, at the electron microscopic level, matrix vesicles were seen in extracellular matrix associated with the initiation of mineralization. Typical rod-like crystals were present in the intercellular spaces along the collagen fibers. These results indicated that in a specific environment, dedifferentiated chondrocytes were able to redifferentiate and to form nodular structures with morphological ultrastructure of calcified cartilage observed in vivo.

Alcian Blue↗

PubMed1994

Expression of the galactose binding protein Mac-2 by human melanoma cell-lines.

The Mac-2 protein is a lectin specific for galactose-containing glycoconjugates. Present in some normal cells, it was also associated with the metastatic potential of some carcinoma cells. We studied Mac-2 expression in three human melanoma cell lines and in five variants and clones from one of them. By using the M3/38 rat monoclonal antibody, Mac-2 was demonstrated on cell surface by flow cytometry as well as in the cytoplasm and in the nucleus by confocal microscopy. The expression of Mac-2 was not correlated with that of terminal unsialylated Gal beta 1-3 GalNac structures on metastatic melanoma cell lines. However, the presence of extracellular Mac-2 containing vesicles was observed in cell lines with metastatic potency. Western blot analysis of cell lysates, in reducing or non-reducing conditions, revealed two bands of 34-36 and 93-98 kDa apparent M(r), also found in HL60 and P388.D1 cell lines used as positive controls.

Antigens, Differentiation↗

PubMedDate not supplied

Exosome secretion and red cell maturation: Exploring molecular components involved in the docking and fusion of multivesicular bodies in K562 cells.

During reticulocyte maturation, some membrane proteins and organelles that are not required in the mature red cell are lost. These proteins are released into the extracellular medium associated with vesicles present in multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles, termed exosomes. By studying MVBs fusion and exosome release in K562 cells, a human erythroleukemic cell line, we have determined the functional significance of Rab11 and calcium in these events. Additionally, in the transformation process that occurs during erythrocyte maturation, intracellular organelles are likely removed as a consequence of autophagic sequestration and degradation. We propose K562 cells as a useful tool to analyze, at the molecular level, the role of autophagy in the terminal differentiation of red cells.

Autophagy↗

PubMed2006

Zinc-transporter genes in human visceral and subcutaneous adipocytes: lean versus obese.

Zinc ions influence adipose tissue metabolism by regulating leptin secretion and by promoting free fatty acid release and glucose uptake. The mechanisms controlling zinc metabolism in adipose tissue are unknown. We therefore examined the gene-expression levels of a number of zinc-transporting proteins in adipose tissue, comparing subcutaneous fat with visceral fat from lean and obese humans. Both ZnT-proteins responsible for zinc transport from cytosol to extracellular compartments and intracellular vesicles and Zip-proteins responsible for zinc transport to the cytoplasm were expressed in all samples. This suggests that zinc metabolism in adipocytes is actively controlled by zinc-transporters. The expression levels were different in lean and obese subjects suggesting a role for these proteins in obesity. Furthermore, the expression levels were different from subcutaneous fat to intra-abdominal fat suggesting that the metabolic activity in adipocytes is to some extent dependent upon zinc and the activity of zinc-transporting proteins or vice versa.

Adipocytes↗

PubMed2006

Frequency-dependent kinetics and prevalence of kiss-and-run and reuse at hippocampal synapses studied with novel quenching methods.

The kinetics of exo-endocytotic recycling could restrict information transfer at central synapses if neurotransmission were entirely reliant on classical full-collapse fusion. Nonclassical fusion retrieval by kiss-and-run would be kinetically advantageous but remains controversial. We used a hydrophilic quencher, bromophenol blue (BPB), to help detect nonclassical events. Upon stimulation, extracellular BPB entered synaptic vesicles and quenched FM1-43 fluorescence, indicating retention of FM dye beyond first fusion. BPB also quenched fluorescence of VAMP (synaptobrevin-2)-EGFP, thus indicating the timing of first fusion of vesicles in the total recycling pool. Comparison with FM dye destaining revealed that kiss-and-run strongly prevailed over full-collapse fusion at low frequency, giving way to a near-even balance at high frequency. Quickening of kiss-and-run vesicle reuse was also observed at higher frequency in the average single vesicle fluorescence response. Kiss-and-run and reuse could enable hippocampal nerve terminals to conserve scarce vesicular resources when responding to widely varying input patterns.

Animals↗

PubMed2006

Intra-endosomal membrane traffic.

Following endocytosis, ubiquitinated signaling receptors are incorporated within intraluminal vesicles of forming multivesicular endosomes. These vesicles then follow the pathway from early to late endosomes, remaining within the endosomal lumen, and are eventually delivered to lysosomes, where they are degraded together with their protein cargo. However, intraluminal vesicles do not always end up in lysosomes for degradation; they can also fuse back with the limiting membrane of late endosomes. This route, which might be regulated by lyso-bisphosphatidic acid and its putative effector Alix, can be hijacked by the anthrax toxin and vesicular stomatitis virus and is presumably exploited by proteins and lipids that transit through intraluminal vesicles. Alternatively, these vesicles can be released extracellularly, like HIV in macrophages, upon fusion of endosomes or lysosomes with the plasma membrane.

Animals↗

PubMed2004

Proteomic analysis of exosomes secreted by human mesothelioma cells.

Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins present in exosomes secreted by tumor cells have been poorly defined. This study describes the protein composition of mesothelioma cell-derived exosomes in more detail. After electrophoresis of exosome preparations, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) was used to characterize the protein spots. MHC class I was found to be present together with the heat shock proteins HSC70 and HSP90. In addition, we found annexins and PV-1, proteins involved in membrane transport and function. Cytoskeleton proteins and their associated proteins ezrin, moesin, actinin-4, desmoplakin, and fascin were also detected. Besides the molecular motor kinesin-like protein, many enzymes were detected revealing the cytoplasmic orientation of exosomes. Most interesting was the detection of developmental endothelial locus-1 (DEL-1), which can act as a strong angiogenic factor and can increase the vascular development in the neighborhood of the tumor. In conclusion, mesothelioma cells release exosomes that express a discrete set of proteins involved in antigen presentation, signal transduction, migration, and adhesion. Exosomes may play an important role in the interaction between tumor cells and their environment.

Antigen-Presenting Cells↗

PubMed2003

Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin L.

Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine protease, cathepsin L, with a Ki of 0.7 nM, but it does not inhibit the structurally related lysosomal cysteine protease cathepsin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and 7.2. At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease to remain active longer, although the rate of proteolysis is reduced. These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu.

Amino Acid Sequence↗