Vesicle distribution in the arterial endothelium determined with ruthenium red as an extracellular marker.
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Animals↗
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Results for “Extracellular vesicles”
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Morphology of interleukin-2-stimulated human peripheral blood mononuclear effector cells killing glioma-derived tumor cells in vitro.
This is the first morphological study of interleukin-2-stimulated human peripheral blood mononuclear (PBM) cells resulting in lymphokine-activated killer (LAK) cell activity against human glioma-derived tumor cells in vitro, in which high-resolution differential interference video light microscopy, scanning electron microscopy, and transmission electron microscopy were used. A subset of cells within the LAK cell population are the effector cells and have an asymmetric cellular architecture characteristic of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Upon binding to target cells, the LAK effector cell nucleus is positioned away from the target cell, whereas the granules, Golgi apparatus, and microtubules orient toward the target cell. These LAK-glioma cell conjugates form very tight plasma membrane bonds with numerous interdigitations, and vesicles were found in the small extracellular spaces between the cells. This morphology was not observed in unstimulated PBM-glioma cell co-cultures. Glioma-derived cells react to LAK effector cells by blebbing, becoming round, and rapidly detaching from the substrate. The injured glioma-derived cells had a highly condensed cytoplasm and chromatin, lobular nucleus, and severe plasma membrane blebs, which are consistent with an apoptotic rather than an osmotic lysis mechanism of cell death. This study provides morphological evidence that supports a common cytotoxic mechanism for CTLs, NK cells, and LAK effector cells. The cytotoxic mechanism is based on the local exocytosis of vesicles by the effector cell into the small extracellular space between the effector-target cell conjugate. Granules found in CTLs, NK cells, and LAK cells contain a pore-forming protein that inserts holes in the target cell's plasma membrane through which a lethal substance(s) not yet identified is thought to enter the cell.
Cell Line↗
Exocytosis of lung surfactant: from the secretory vesicle to the air-liquid interface.
Exocytosis is fundamental in biology and requires an orchestra of proteins and other constituents to fuse a vesicle with the plasma membrane. Although the molecular fusion machinery appears to be well conserved in evolution, the process itself varies considerably with regard to the diversity of physico-chemical and structural factors that govern the delay between stimulus and fusion, the expansion of the fusion pore, the release of vesicle content, and, finally, its extracellular dispersion. Exocytosis of surfactant is unique in many of these aspects. This review deals with the secretory pathway of pulmonary surfactant from the type II cell to the air-liquid interface, with focus on the distinct mechanisms and regulation of lamellar body (LB) fusion and release. We also discuss the fate of secreted material until it is rearranged into units that finally function to reduce the surface tension in the lung.
Animals↗
Epithelial cell-fibroblast interactions: modulation of extracellular matrix proteins in cultured oral cells.
A model system involving co-cultures of human gingival or periodontal ligament fibroblasts with mouse epithelial root sheath cells or human gingival epithelial cells was used to study epithelial cell-fibroblast interactions. Double-labeled immunofluorescence and microfluorometry were used to investigate the expression of extracellular matrix molecules of collagen type I (collagen I), type III (collagen III) and fibronectin in fibroblasts. When fibroblasts from either source were cultured alone, the fluorescence for collagen I and fibronectin ranged from strongly positive to almost negative. Collagen III staining was relatively weak compared with that of collagen I. After 2-3 days of co-culture, gingival fibroblasts and ligament fibroblasts adjacent to the mouse sheath cells exhibited enhanced intracellular fluorescence for collagen I and fibronectin. Very little change was observed for collagen III. Gingival fibroblasts cultured with gingival epithelial cells showed increased fluorescence for collagen I but decreased fluorescence for fibronectin. In contrast, the fluorescence intensity for both collagen I and fibronectin in ligament fibroblasts were reduced after 3 days of co-culture with gingival epithelial cells. Ultrastructural changes in fibroblasts co-cultured with mouse root sheath cells included increased Golgi cisternae and vesicles and an increased abundance of rough endoplasmic reticulum, polyribosomes, secretory vesicles and pinocytotic vesicles. Thus, the expression of extracellular matrix proteins and the metabolic activity of fibroblasts can be modulated by oral epithelial cells.
Animals↗
Calreticulin on the mouse egg surface mediates transmembrane signaling linked to cell cycle resumption.
Calreticulin, a protein best known as an endoplasmic reticulum chaperone, also is found on the extracellular plasma membrane surface of many cell types where it serves as a mediator of adhesion and as a regulator of the immune response. In this report, we demonstrate that calreticulin is present on the extracellular surface of the mouse egg plasma membrane and is increased in the perivitelline space after egg activation. The extracellular calreticulin appears to be secreted by vesicles in the egg cortex that are distinct from cortical granules. An anticalreticulin antibody binds to extracellular calreticulin on live eggs and inhibits sperm-egg binding but not fusion. In addition, engagement of cell surface calreticulin by incubation of mouse eggs in the presence of anticalreticulin antibodies results in alterations in the localization of cortical actin and the resumption of meiosis as indicated by alterations in chromatin configuration, decreases in cdc2/cyclin B1 and MAP kinase activities, and pronuclear formation. These events occur in the absence of any observable alterations in intercellular calcium. These data demonstrate that calreticulin functionally interacts with the egg cytoskeleton and can mediate transmembrane signaling linked to cell cycle resumption. These studies suggest a role for calreticulin as a lectin that may be involved in signal transduction events during or after sperm-egg interactions at fertilization.
Actins↗
Extracellular matrix synthesis in blastula and gastrula stages of normal and hybrid frog embryos. IV. Biochemical and autoradiographic observations on fucose-, glucose-, and mannose-labelled materials.
Normal Rana pipiens gastrulae show more incorporation of isotopically labelled fucose, glucose, and mannose into TCA-insoluble materials than blastulae. Interspecific hybrid embryos which undergo developmental arrest at the onset of gastrulation often synthesize reduced amounts of fucose-, glucose- and mannose-labelled materials. These materials are high-molecular weight and are degraded by Pronase into fragments which are included in Sepharose CL-4B but excluded from Sephadex G-50. Labelled materials contain label predominantly in fucose and galactose with little label in glucose or mannose. Labelled materials migrate slowly on cellulose acetate, bind to DEAE-cellulose and elute at low ionic strength and are precipitated by cetyl pyridinium chloride (CPC) without the addition of carrier compounds. Pulse-chase labelling experiments and light- and electron-microscopic autoradiography were used to examine the site of synthesis, mode of transport, and sites of deposition of fucose-, glucose- and mannose-labelled materials in different developmental stages of normal developing Rana pipiens embryos and interspecific hybrid embryos formed by fertilizing the eggs of R. pipiens with the sperm of R. catesbeiana. In both normal and hybrid embryos, after a 15-30-min pulse, grains are closely associated with juxtanuclear and cytoplasmic collections of membrane-bound vesicles which resemble the Golgi apparatus. In normal embryos following a 15-30-min pulse and a 60-min chase, grains are largely cleared from the cytoplasmic vesicles and deposited in the extracellular spaces or along cell surfaces. In contrast, arrested hybrid embryos given a 15-30-min pulse and a 60-min chase show a marked accumulation of grains over cytoplasmic structures such as the Golgi apparatus and vesicular elements in the cell cortex. Certain interesting features of regional variation in synthetic activity in developing normal embryos are also described.
Animals↗
Location of the carbohydrates present in the HK-ATPase vesicles isolated from hog gastric mucosa.
The glycosylation of H+K(+)-ATPase vesicles isolated from hog gastric mucosa was investigated by various methods. Following protein separation on sodium dodecyl sulfate reducing gels and transfer to poly(vinyl difluoride) membranes, binding of concanavalin A was confined to the 94-kDa band which corresponds to the catalytic subunit. In contrast, wheat germ agglutinin binding occurred in a region below the 94-kDa subunit, corresponding to the 60-85-kDa region, and also to protein just above the catalytic subunit. Treatment with glycopeptidase F removed most of the concanavalin A staining and also the wheat germ agglutinin staining found below the 94-kDa region, but spared the higher molecular weight wheat germ agglutinin reactive material. During the deglycosylation experiments a protein of 35-kDa was produced. Sequencing analysis of V8 protease generated peptide fragments of the 35-kDa protein show at least 30% homology with the Na+K(+)-ATPase beta-subunits. Labeling of the carbohydrates by galactosyltransferase and [3H]uridine diphosphate-galactose showed that the sites of labeling were extracellular and were confined to the wheat germ agglutinin staining regions. Two molecular weight regions, below the 94-kDa region, of 60 and 85 kDa were identified. Electron microscopy using postembedding staining techniques showed that both concanavalin A and wheat germ agglutinin staining occurred on the extracellular face of the gastric vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphatases↗
Maturation of respiratory syncytial virus within HEp-2 cell cytoplasm.
Electron microscopy of HEp-2 cells infected with respiratory syncytial virus (RSV) strain Long revealed the maturation of RSV on an ultrastructural level. The results showed that the virus maturated by two different pathways. In one of them, the virus assembled and matured before reaching the plasma membrane on the internal vesicle membrane within cytoplasm. The mature virus was delivered to the plasma membrane and to the extracellular space most likely by the transport vesicles and exocytosis. In the other pathway, the virus matured on the plasma membrane as described with other members of the family Paramyxoviridae. Using monoclonal antibodies (MoAbs), we localized viral nucleoprotein (NP) and envelope proteins in cytoplasm by immunoelectron microscopy (IEM).
Antibodies, Monoclonal↗
Localization of basic fibroblast growth factor in bovine endothelial cells: immunohistochemical and biochemical studies.
To gain insight into the mechanisms of synthesis, storage, and release of basic fibroblast growth factor (bFGF), we studied the immunohistochemical localization of bFGF in bovine coronary artery, coronary sinus, and adrenal capillary endothelial cells grown in culture. Light and electron microscopic immunohistochemical studies were performed using the ABC immunoperoxidase method on p-formaldehyde-fixed cells. Five different anti-bFGF antibodies gave similar results in all cell types. In subconfluent cells, immunoreactivity was noted in the nuclear chromatin, nucleoli, cytosol, cytoplasmic vesicles (some of which appeared to fuse with the plasma membrane), and extracellular matrix. No reaction was found in endoplasmic reticulum or Golgi zones. Confluent cells demonstrated less immunoreactivity in the nuclei and cytosol but more in the extracellular matrix. Some cells of senescent morphology showed only cytoplasmic staining; however, no cells were found with only nuclear staining. Biochemical studies showed that three forms of bFGF (18, 24, and 26 kDa) were present in endothelial cells and varied with different culture conditions. Protection analysis indicated that bFGF mRNA is less abundant in postconfluent cells than in subconfluent cells. These data suggest that subconfluent cells synthesize bFGF and transport it into the nucleus and exocytotic vesicles, while confluent cells synthesize little bFGF but store it in extracellular matrix, cytoplasmic vesicles, and nuclei.
Animals↗
Macrophage endosomes contain proteases which degrade endocytosed protein ligands.
Rabbit alveolar macrophages rapidly internalize and degrade mannosylated bovine serum albumin (125I-mannose-BSA). Trichloroacetic acid-soluble degradation products appear in the cells as early as 6 min after uptake at 37 degrees C, and in the extracellular medium after 10 min. Incubation of endocytic vesicles containing this ligand in isotonic buffers at pH 7.4 + ATP resulted in intravesicular proteolysis, which was inhibited by monensin, nigericin, or ammonium chloride. At pH 5.0, degradation proceeded rapidly and was abolished by lysis of the vesicles with 0.1% Triton X-100. Readdition of lysosomes to the incubation mixture did not increase the rate of prelysosomal degradation. Proteolysis of 125I-mannose-BSA was optimal at pH 4.5, and inhibited by low concentrations of the cathepsin D inhibitor pepstatin A. After subcellular fractionation of the macrophages on Percoll gradients, 125I-mannose-BSA sedimented with prelysosomal vesicles and was not transported to secondary lysosomes. Addition of pepstatin A to extracellular medium during internalization of prebound 125I-mannose-BSA partially inhibited degradation of ligand, and resulted in transfer of undegraded 125I-mannose-BSA to lysosomes after 20 min. Using 125I-bovine serum albumin as a substrate for the protease in the presence of 0.1% Triton X-100, we have shown that as much as 36% of the total pepstatin A-sensitive activity sediments with nonlysosomal membranes. After intraendosomal iodination using lactoperoxidase, a labeled protease was isolated by affinity chromatography on pepstatin-agarose. The labeled protease, which had a subunit size of 46 kDa, was detected in endocytic vesicles after 5 min of internalization. These results suggest that a cathepsin D-like protease is responsible for the degradation of 125I-mannose-BSA in macrophages, and that this ligand is degraded in a prelysosomal vesicle.
Ammonium Chloride↗
Regulation of matrix vesicle metabolism by vitamin D metabolites.
Matrix vesicles are membrane organelles found in the extracellular matrix of calcifying cells. Vitamin D-responsive alkaline phosphatase specific activity has been localized to matrix vesicles in chondrocyte and osteoblast cultures. The effect of hormone is both metabolite and cell specific. Alkaline phosphatase in matrix vesicles produced by resting zone chondrocytes is stimulated by 24,25(OH)2D3 whereas alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is responsive to 1,25(OH)2D3. However, mesenchymal cell cultures, which exhibit a chondrogenic phenotype when exposed to bone inductive proteins in vitro, produce vesicles with alkaline phosphatase activity that is unaffected by either 1,25(OH)2D3 or 24,25(OH)2D3. Incorporation and release of arachidonic acid into phosphatidylethanolamine is also differentially regulated by 1,25(OH)2D3 and 24,25(OH)2D3 in chondrocytes. These data suggest that vitamin D metabolites may regulate endochondral ossification by altering matrix vesicle enzyme activities, perhaps through changes in membrane phospholipid metabolism.
Alkaline Phosphatase↗
Extracellular trafficking of myocilin in human trabecular meshwork cells.
Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.
Adenoviridae↗
In vitro studies on the regulation of endochondral ossification by vitamin D.
The research described in this article has focused on the complex autocrine, paracrine, and endocrine regulation of endochondral ossification using vitamin D metabolites and TGF-beta as models. By comparing results from a number of laboratories utilizing a diverse array of in vivo and in vitro systems, a coherent picture is beginning to emerge. Vitamin D metabolites influence cell differentiation and maturation and have direct effects on cell function. Differentiation of the mesenchymal cells into chondroblasts is regulated by both 1,25-(OH)2D3 and 24,25-(OH)2D3, as well as by TGF-beta. The resting zone chondrocytes respond primarily to 24,25-(OH)2D3 in terms of matrix synthesis and matrix vesicle biochemistry. They synthesize both metabolites and other factors that stabilize matrix vesicle enzymes like AHSG. In addition to the paracrine role these factors may play in regulating the matrix, it is possible that they may influence the cells in the growth plate itself. Growth zone chondrocytes also synthesize both metabolites, but respond primarily to 1,25-(OH)2D3 for the parameters measured in the studies described. These cells also synthesize TGF-beta which further increases alkaline phosphatase activity, perhaps via an autocrine stimulation of the cell. While cells from the calcified zone have not yet been studied directly in culture, it is likely that they respond to paracrine signals from the avascular cartilage as well as to serum-derived factors. How the signals are transferred among the cells is unknown. Certainly one can postulate information flow in both upward and downward directions. The signal transduction mechanisms for the factors at the cellular level are complex. While it is known that 1,25-(OH)2D3 stimulates gene transcription and stabilization of mRNA for proteins like alkaline phosphatase, its nongenomic effects are only beginning to emerge. Membrane effects of this metabolite have been shown in intestine and kidney in conjunction with studies on Ca flux. It is becoming increasingly evident that other steroid hormones may operate in similar ways. Studies with the rat costochondral chondrocytes are the first to show that there are specific membrane effects for at least two vitamin D metabolites and that membrane enzymes, including those involved in phospholipid metabolism, can be differentially regulated by them. Furthermore, these experiments have provided for the first time a clear hypothesis for how cells can regulate events in the extracellular matrix after the matrix vesicles are produced and incorporated into the matrix.
Animals↗
Proton transport by halorhodopsin.
In halorhodopsin from Natronobacterium pharaonis, a light-driven chloride pump, the chloride binding site also binds azide. When azide is bound at this location the retinal Schiff base transiently deprotonates after photoexcitation with light > 530 nm, like in the light-driven proton pump bacteriorhodopsin. As in the photocycle of bacteriorhodopsin, pyranine detects the release of protons to the bulk. The subsequent reprotonation of the Schiff base is also dependent on azide, but with different kinetics that suggest a shuttling of protons from the surface as described earlier for halorhodopsin from Halobacterium salinarium. This azide-dependent, bacteriorhodopsin-like photocycle results in active electrogenic proton transport in the cytoplasmic to extracellular direction, detected in cell envelope vesicle suspensions both with a potential-sensitive electrode and by measuring light-dependent pH change. We conclude that in halorhodopsin an azide bound to the extracellular side of the Schiff base, and another azide shuttling between the Schiff base and the cytoplasmic surface, fulfill the functions of Asp-85 and Asp-96, respectively, in bacteriorhodopsin. Thus, although halorhodopsin is normally a chloride ion pump, it evidently contains all structural requirements, except an internal proton acceptor and a donor, of a proton pump. This observation complements our earlier finding that when a chloride binding site was created in bacteriorhodopsin through replacement of Asp-85 with a threonine, that protein became a chloride ion pump.
Azides↗
Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor-beta in osteosarcoma cell cultures.
Transforming growth factor-beta (TGF beta) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone-forming cells. TGF beta has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast-like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGF beta plays a role in regulating mineral deposition in the matrix, the effects of TGF beta on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGF beta (0.1-10 ng/ml) to examine the influence of cell maturation on response to TGF beta. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high-density cultures, there was further stimulation at 10 ng/ml. There was a dose-dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low-density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose-dependent increase in phospholipase A2-specific activity in the plasma membranes and matrix vesicles of both high- and low-density cultures. In agreement with previous studies, TGF beta inhibited cellular proliferation 50%. The results show that addition of TGF beta stimulates the activity of enzymes associated with calcification. The effect of TGF beta is dependent on the stage of maturation of the cell. This study indicates that TGF beta may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.
Alkaline Phosphatase↗
Selective internalization of choline-phospholipids in Plasmodium falciparum parasitized human erythrocytes.
We have incubated control and Plasmodium falciparum parasitized human erythrocytes with lipid vesicles containing radiolabeled long-chain phosphatidylcholine and sphingomyelin, in the presence of a nonspecific lipid transfer protein. Most of the radiolabeled phospholipids were, immediately thereafter, available for extracellular phospholipases, suggesting that uptake of vesicles as such did not occur. In time, the amount of phosphatidylcholine inserted in the outer leaflet of the host cell membrane of parasitized erythrocytes decreased, indicating that phosphatidylcholine was being internalized in parasitized erythrocytes. The exclusion of sphingomyelin from the internalization process suggests that the removal of phosphatidylcholine from the outer leaflet of the erythrocyte membrane is caused by transbilayer migration, rather than by endocytosis. The extent of phosphatidylcholine internalization indicates that part of it does not remain in the inner leaflet of the host cell membrane, but is taken up by the intraerythrocytic parasite. Individual phosphatidylcholine species, containing 16:0/18:1-, 16:0/18:2- and 16:0/20:4-fatty acids, showed similar extents of internalization, after being incorporated in parasitized erythrocytes by a phosphatidylcholine specific transfer protein.
Animals↗
Vesicles with lactate dehydrogenase and without alkaline phosphatase present in the resting zone of epiphyseal cartilage.
Matrix vesicles are membrane-invested vesicles that initiate mineralization in the extracellular matrix of calcifying tissues. The epiphyseal cartilages of young-rat rib bones were divided into the growth zone and the resting zone, followed by the isolation of matrix vesicles after collagenase treatment. Matrix vesicles with both alkaline phosphatase and lactate dehydrogenase were detected in the growth cartilage found in the epiphyseal growth plates of young rabbits [Hosokawa, Uchida, Fujiwara & Noguchi (1988) J. Biol. Chem. 263, 10045-10047], but were not detected in the resting zone. By contrast, and surprisingly, lactate dehydrogenase-containing vesicles without alkaline phosphatase were found in the resting zone, but not in the growth zone. In both the growth and resting zones, isoenzyme patterns of lactate dehydrogenase in the two different vesicles were identical with those of cytosolic lactate dehydrogenase of chondrocytes, suggesting the presence of a mechanism for specific uptake of cytosolic lactate dehydrogenase. The same results as for young-rat rib bones were obtained with the resting and growth cartilages of young-dog and monkey rib bones.
Alkaline Phosphatase↗
Morphogenesis of Michaelis-Gutmann bodies in cerebral malacoplakia. An ultrastructural study.
Malacoplakia was found in the brain biopsy specimen from the wall of a cystic lesion in a 4-month-old girl. Ultrastructural study of the Michaelis-Gutmann bodies revealed that the initial stage of calcification appeared to be the deposit of needle-shaped apatite crystals within intracytoplasmic membrane-bound vesicles of histiocytes. Accumulation of apatite crystals and coalescense of calcified vesicles resulted in the formation of large laminated calcospherules. Extracellular Michaelis-Gutmann bodies and apatite crystal-containing matrix vesicles were also noted. Calcified vesicles and the Michaelis-Gutmann bodies were not observed in the phagolysosomes. Bacteria and viral particles were not identified. These findings suggest that matrix and intracytoplasmic membrane-bound vesicles play an important role in the initial stage of the formation of Michaelis-Gutmann bodies and that a mechanism other than bacterial or viral infection may be involved in cerebral malacoplakia.
Apatites↗