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PubMed2000

Matrix vesicles mediate mineralization of human thyroid cartilage.

Mineralization and ossification of human thyroid cartilage first starts after the end of adolescence when the previously cartilaginous human skeleton has become ossified and the epiphyseal discs are in the process of closing. However, the mechanisms involved in mineralization and ossification of human thyroid cartilage are not well understood. Ultrastructural analysis of human thyroid cartilage revealed that mineralization started close to cartilage canals in a matrix containing gigantic collagen fibers (asbestoid fibers). Matrix vesicles were detected in mineralized areas and were often associated with needle-like crystals. For the first time we were able to isolate matrix vesicles from human thyroid cartilage by mild enzymatic digestions and ultracentrifugation. These particles were oval and varied in size; some were heavily calcified. They were enriched in alkaline phosphatase, calcium, and inorganic phosphate, suggesting that the particles contain Ca2+-Pi complexes. Immunoblot analysis of these vesicles revealed the presence of annexins II, V, and VI, membrane-associated, channel-forming proteins, which allow influx of Ca2+ into the vesicles and intralumenal crystal growth. In addition, the vesicles were associated with types II and X collagen, suggesting that this association not only anchors the vesicles to the extracellular matrix, but, as shown previously, also stimulates Ca2+ influx into these particles. In conclusion, matrix vesicles isolated from human thyroid cartilage contain all the components, enabling them to initiate and mediate the mineralization process in human thyroid cartilage.

Adult↗

PubMed1984

Mineralization by matrix vesicles.

Matrix vesicles are widely regarded as the initial site of calcification in epiphyseal growth plate cartilage, in growing bone and in predentin. This opinion has recently been challenged on grounds that the early aqueous methods used for electron microscopic tissue preparation may have produced an erroneous picture by causing mineral dislocation. However, this argument has now been refuted by multiple investigators throughout the world using a variety of anhydrous methods coupled with electron probe analysis to show convincingly that matrix vesicles are, indeed, associated with initial mineral. Matrix vesicles appear to mineralize by concentrating calcium and phosphate at a protected site close to the inner leaflet of the vesicle membrane. Calcium may be attracted by its affinity for acidic phospholipids of the vesicle membrane, and phosphate may be concentrated by the action of transmembrane phosphatases of the matrix vesicle membrane. Evidence is accumulating to suggest that alkaline phosphatase of the matrix vesicle membrane functions as a phosphotransferase or phosphate vector, transporting PO4 across the vesicle membrane. The mechanism(s) of matrix vesicle biogenesis are discussed including budding from the plasma membrane (for which there is much support), cell degeneration (for which there is gathering support), extrusion of intracytoplasmic vesicles (for which there is weak support), and extracellular subunit self-assembly (for which there is little support). It is suggested that none of these mechanisms is necessarily exclusive, thus more than one mechanism may function in the same tissue. Finally, it is noted that in many calcific diseases, ranging from arthritis to atherosclerosis, mineralization is initiated by extracellular membrane-invested vesicles which are probably analogous to the matrix vesicles of skeletal tissues.

Animals↗

PubMed1986

Horseradish peroxidase uptake of the synaptic vesicles in the superior cervical ganglion of the cat.

The function-dependent changes in synaptic vesicle number and horseradish peroxidase (HRP) labeling were studied in the synapses of cat superior cervical ganglia. It was found that only a low percentage of vesicles contained the extracellular marker. Supra-maximal stimulation (15 min) and subsequent rest (5 min) did not result in significant changes in vesicle number and the percentage of labeled vesicles also remained at about the same level. The low and activity-independent HRP labeling rate does not exclude the possibility of vesicle recycling but indicates that it might be only indirectly connected to the transmitter release itself.

Animals↗

PubMed1987

The basis of antimalarial action: non-weak base effects of chloroquine on acid vesicle pH.

Biologically active concentrations of chloroquine increase the pH of the parasite's acid vesicles within 3-5 min. This increase in pH results from two mechanisms, one of which is markedly reduced in chloroquine-resistant parasites. Because chloroquine is a weak base, it increases vesicle pH by that mechanism in chloroquine-susceptible and resistant parasites and mammalian cells (based on its two pKs and on the delta pH between the acid vesicle and the extracellular environment). In chloroquine-susceptible parasites, but not resistant parasites or mammalian cells, chloroquine increases the pH of acid vesicles 700- to 800-fold more than can be accounted for by its properties as a weak base. The increase in acid vesicle pH caused by these non-weak base effects of nanomolar chloroquine in susceptible parasites suggests that chloroquine acts by interfering with acid vesicle functions in the parasite such as the endocytosis and proteolysis of hemoglobin, and the intracellular targeting of lysosomal enzymes. The non-weak base effects of nanomolar chloroquine on parasite vesicle pH are also responsible for its safety because these chloroquine concentrations do not affect mammalian cells.

Animals↗

PubMedDate not supplied

Matrix vesicle calcification in rat epiphyseal growth plate cartilage prepared anhydrously for electron microscopy.

The preparation of mineralizing tissues for electron microscopy by methods that involve the use of aqueous solvents may result in translocation and/or removal of mineral, leading to the possible erroneous interpretation of initial calcifying loci. The failure of a recent cryoultramicrotomy study to detect a mineral phase associated with matrix vesicles raised doubts about the role of the matrix vesicle in initial mineralization and prompted us to reexamine the ultrastructure of calcifying cartilage using three different anhydrous preparatory techniques. Matrix vesicles or structures similar in profile and location were identified in rat epiphyseal growth plate cartilages that had been subjected to freeze-substitution, cryoultramicrotomy, or ethylene glycol fixation. Furthermore, mineral deposits, as confirmed by energy dispersive x-ray analysis, were observed within or in close association with the matrix vesicles in the extracellular matrix of the growth plate cartilage by all three methods. No evidence of mineral deposition at other sites preceding matrix vesicle calcification was found. Our studies support the conclusion of many investigations that matrix vesicles are the initial site of growth plate calcification.

Animals↗

PubMed1979

Transport of C5 dicarboxylate compounds by Pseudomonas putida.

Induced glutarate and 2-oxoglutarate uptake and transport by Pseudomonas putida were investigated in whole cells and membrane vesicles, respectively. Uptake of 2-oxoglutarate, but not glutarate, was against a concentration gradient to 1.7-fold greater than the initial extracellular concentration. Membrane vesicles transported 2-oxoglutarate and glutarate against gradients to intramembrane concentrations fivefold greater than the initial extravesicle concentrations. The rates of transport of both compounds were greatest in the presence of the artificial electron donor system phenazine methosulfate-ascorbate. Malate and D-lactate were the only naturally occurring compounds that served as electron donors. Uptake and transport were inhibited by KCN, NaN3, and 2,2-dinitrophenol. Kinetic parameters of transport were: glutarate, apparent Km--1.22 mM, Vmax--400 nmol/min per mg of membrane protein; 2-oxoglutarate, apparent Km--131 microM, Vmax--255 nmol/min per mg of membrane protein. Studies of competitive inhibition indicated a common system for transport of five C5 dicarboxylate compounds. The apparent Km and Ki values with 2-oxoglutarate as a substrate placed the substrate affinity for transport in the order 2-oxoglutarate greater than glutarate greater than D-2-hydroxyglutarate and L-2-hydroxyglutarate greater than glutaconate.

Biological Transport↗

PubMed1973

Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.

Animals↗

PubMed1982

Tannic acid-glutaraldehyde fixative and pleomorphic adenomas of the salivary gland: an ultrastructural study.

Four cases of pleomorphic adenomas of parotid glands were fixed by immersion in a mixture of 4% tannic acid and 3% glutaraldehyde followed by OsO4 postfixation and heavy metals staining. This clearly visualized the presence of dense amorphous elastic fibers in cellular and myxochondroid areas. The dense amorphous fibers were associated with varying numbers of peripheral filaments which appeared sometimes as bundles of parallel fibrils without intermingled amorphous material. These intermediate-sized filaments were seen indirect continuity with similar intracellular filaments with or without interposition of basal-membrane-like material and/or with extracellular filament-containing vesicles. It is suggested that these filaments are synthesized intracellularly and released into the extracellular space by neoplastic myoepithelial cells mainly by cytoplasmic vesicle separation. In addition, small round vesicles with a dense core of amorphous material and staining characteristics identical to those of extracellular elastin were frequently seen in the peripheral parts of these myoepithelial-like cells. It is possible that these dense-cored vesicles have a secretory function and contain a precursor of elastin.

Adenoma, Pleomorphic↗

PubMed1983

Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture.

A method is presented for isolating osteoblasts from newborn mouse calvaria without the use of digestive enzymes. The procedure is based on the ability of osteoblasts to migrate from bone onto small glass fragments (Jones, S.J., and A. Boyde, 1977, Cell Tissue Res., 184:179-193). The isolated cells were cultured for up to 14 d in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml of ascorbic acid. 7-d cultures were incubated for 24 h with [3H]proline. High levels of collagen synthesis relative to total protein were found, as measured by collagenase digestion of medium and cell layer proteins. Analysis of pepsin-digested proteins from the same cultures by SDS PAGE showed that type I collagen was predominantly produced with small amounts of type III and V (alpha 1 chains) collagens. Osteoblasts grown in the presence of beta-glycerophosphate were able to initiate mineral deposition in culture. Electron microscopic analysis of the cultures revealed the presence of needle-shaped apatite-like crystals associated with collagen fibrils and vesicles in the extracellular space. Mouse skin fibroblasts cultured under identical conditions failed to initiate mineralization. Electron histochemical studies revealed the presence of alkaline phosphatase activity, associated with osteoblast membranes, matrix vesicles and on or near collagen fibrils. Thus these isolated osteoblasts retained in culture their unique property of initiating mineralization and therefore represent a model of value for studying the mineralization process in vitro.

Animals↗

PubMed1982

Patterns of matrix vesicle calcification in osteomalacia of Gyro mice.

Homozygous Gyro mice manifest widespread abnormalities in the inner ear and other parts of the skeleton. These disorders were attributed to a sex linked dominant gene. In the present study, tissue specimens were obtained from the inner ear and maxillary and tibial bones, and undecalcified samples were processed for light and transmission electron microscopy. Light microscopy revealed osseous changes consistent with osteomalacia. These alterations comprised broad bands of osteoid with partial or complete failure of calcification. Electron micrographs showed longitudinal calcifying fronts composed of hydroxyapatite crystals. Abundance of matrix vesicles could be demonstrated in the extracellular matrix. Many of the vesicles contained crystaline mineral. In addition, calcification alongside collagen fibers was recorded. Occasionally, apatite crystals were attached to plasma membranes. Female and hemizygous male mice showed normal patterns of calcification. The findings in the affected Gyro mice appear to be similar to previously reported changes in human osteomalacia. Further definition of the pathological process in this animal model may be valuable in defining mechanisms operative in the development of osteomalacia in man.

Animals↗

PubMed1994

Use of an oriented transmembrane protein to probe the assembly of a supported phospholipid bilayer.

Planar-supported phospholipid bilayers formed by the adsorption of vesicles are increasingly used in the investigation of lipid-dependent reactions. We have studied the way in which these bilayers are formed with phospholipid vesicles containing the transmembrane protein Tissue Factor (TF). TF complexed with the serine protease, factor VIIa, is the primary initiator of blood coagulation by way of activation of the zymogen factor X. TF has been shown to orient randomly on the inner and outer leaflets of vesicles. We used proteolytic digestion to produce vesicles in which the extracellular domain of TF is located on the inner leaflet. These vesicles show no cofactor activity for factor VIIa as a result of the inability of the extracellular domain of TF to bind VIIa. After freeze/thawing, 50% of the cofactor activity was regained, indicating reorientation of the sequestered, inner leaflet TF. Adsorption of these vesicles to the inner surface of glass microcapillaries results in a continuous phospholipid bilayer. The microcapillaries were perfused with a solution of factors VIIa and X, and the effluent was monitored for factor Xa production, a sensitive measure of the activity of the TF-VIIa complex. For coatings produced with the digested vesicles, minimal TF-VIIa activity was observed, showing that the supported bilayer preserves the orientation of the leaflets in the vesicles, i.e., the outer leaflet of the vesicles forms the outer leaflet of the supported bilayer.

Adsorption↗

PubMed2006

Intracellular localization and trafficking of serine proteinase AhSub and cysteine proteinase AhCP of Acanthamoeba healyi.

Proteinases have been proposed to play important roles in pathogenesis and various biologic actions in Acanthamoeba. Although genetic characteristics of several proteases of Acanthamoeba have been reported, the intracellular localization and trafficking of these enzymes has yet to be studied. In the present study, we analyzed the intracellular localization and trafficking of two proteinases, AhSub and AhCP, of Acanthamoeba healyi by transient transfection. Full-length AhSub-enhanced green fluorescent protein (EGFP) fusion protein was found in intracellular vesicle-like structures of transfected amoebae. Time-lapse photographs confirmed the secretion of the fluorescent material of the vesicle toward the extracellular space. The mutated AhSub, of which the pre or prepro region was deleted, was found to localize diffusely throughout the cytoplasm of the amoeba rather than concentrated in the secretory vesicle. Transfection of the construct containing the pre region only showed the same localization and trafficking of the full-length AhSub. A cysteine proteinase AhCP-EGFP fusion protein showed similar localization in the vesicle-like structure in the amoeba. However, using Lyso Tracker analysis, these vesicular structures of AhCP were confirmed to be lysosomes rather than secretory vesicles. The AhCP construct with a deletion of the prepro region showed a dispersed distribution of fluorescence in the cytoplasm of the cells. These results indicated that AhSub and AhCP would play different roles in Acanthameoba biology and that the pre region of AhSub and pro region of AhCP are important for proper intracellular localization and trafficking of each proteinase.

Acanthamoeba↗

PubMed2005

Exosomes: a bubble ride for prions?

In certain cell types, endosomal multivesicular bodies may fuse with the cell surface in an exocytic manner. During this process, the small 50-90-nm-diameter vesicles contained in their lumen are released into the extracellular environment. The released vesicles are called exosomes. Exosome secretion can be used by cells to eject molecules targeted to intraluminal vesicles of multivesicular bodies, but particular cell types exploit exosomes as intercellular communication devices for transfer of proteins and lipids between cells. The molecular composition of exosomes is determined by sorting events within endosomes that occur concomitantly with the generation of intraluminal vesicles. As other raft-associated components, the glycosylphosphatidylinositol-linked prion protein transits through multivesicular bodies. Recent findings in non-neuronal cell models indicate prion protein association with secreted exosomes. Thus, exosomes could constitute vehicles for transmission of the infectious prion protein, bypassing cell-cell contact in the dissemination of prions.

Animals↗

PubMed1979

Ultrastructure of psammoma bodies of meningioma in tissue culture.

An 8-day-old tissue culture of a human meningioma was studied by electron microscopy. Psammoma bodies were detected in all stages of evolution, affording a unique opportunity for observing the genesis of these structures. Matrix vesicles appeared instrumental in the calcification of a granular extracellular material. Although matrix vesicles are described in both physiologic and pathologic calcification, they have not been previously reported in the very few ultrastructural studies of psammoma bodies in meningiomas.

Cell Membrane↗

PubMed2006

Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane.

Exosomes are secreted, single membrane organelles of approximately 100 nm diameter. Their biogenesis is typically thought to occur in a two-step process involving (1) outward vesicle budding at limiting membranes of endosomes (outward = away from the cytoplasm), which generates intralumenal vesicles, followed by (2) endosome-plasma membrane fusion, which releases these internal vesicles into the extracellular milieu as exosomes. In this study, we present evidence that certain cells, including Jurkat T cells, possess discrete domains of plasma membrane that are enriched for exosomal and endosomal proteins, retain the endosomal property of outward vesicle budding, and serve as sites of immediate exosome biogenesis. It has been hypothesized that retroviruses utilize the exosome biogenesis pathway for the formation of infectious particles. In support of this, we find that Jurkat T cells direct the key budding factor of HIV, HIV Gag, to these endosome-like domains of plasma membrane and secrete HIV Gag from the cell in exosomes.

Antigens, CD↗

PubMed1983

Calcific diseases. A concept.

New observations portray calcification processes as similar whether occurring normally or pathologically. Most forms of calcification are initiated by membranous organelles, ie, extracellular, calcifying "matrix vesicles" or intracellular mitochondria. Matrix vesicles promote calcification through calcium-binding phospholipids and phosphatase activity. Mitochondria use a forceful, inwardly directed Ca and phosphate transport mechanism. After mineral initiation, the proliferation of mineral crystals is dependent on regulatory factors, such as extracellular Ca2+ and PO4(3-) and other mineral inhibitors and promoters. Calcific diseases are defined as those in which (1) Ca uptake is early, (2) calcification is importantly related to dysfunction, and (3) the control of calcification may lead to decreased morbidity or enhanced diagnostic capability. Calcific diseases include such well-known entities as crystal deposition arthritis, atherosclerosis, calcific valvular sclerosis, tumor calcification, dental plaque, and dysfunctional calcification occurring in implanted cardiovascular devices.

Arteriosclerosis↗

PubMed1986

[Dependence of the number of vesicles in vascular smooth muscle cells on the presence of noradrenaline in the extracellular space].

Noradrenaline (NA) effect on the number of vesicles in smooth muscle cells was investigated in small mesenteric arteries of spontaneously hypertensive rats (SHR), aged 8 or 12 weeks, and age-matched normotensive Wistar-Kyoto (WKY) rats. The presence of NA in the incubation medium resulted in an increase in the number of vesicles in SHR of both age groups, but not in WKY. The results are discussed in view of the relationship between the vesicles and Ca transport in smooth muscle cells.

Animals↗

PubMed2003

Matrix vesicles and calcification.

Matrix vesicles (MVs) are extracellular, 100 nM in diameter, membrane-invested particles selectively located at sites of initial calcification in cartilage, bone, and predentin. The first crystals of apatitic bone mineral are formed within MVs close to the inner surfaces of their investing membranes. Matrix vesicle biogenesis occurs by polarized budding and pinching-off of vesicles from specific regions of the outer plasma membranes of differentiating growth plate chondrocytes, osteoblasts, and odontoblasts. Polarized release of MVs into selected areas of developing matrix determines the nonrandom distribution of calcification. Initiation of the first mineral crystals, within MVs (phase 1), is augmented by the activity of MV phosphatases (eg, alkaline phosphatase, adenosine triphosphatase and pyrophosphatase) plus calcium-binding molecules (eg, annexin I and phosphatidyl serine), all of which are concentrated in or near the MV membrane. Phase 2 of biologic mineralization begins with crystal release through the MV membrane, exposing preformed hydroxyapatite crystals to the extracellular fluid. The extracellular fluid normally contains sufficient Ca2+ and PO4(3-) to support continuous crystal proliferation, with preformed crystals serving as nuclei (templates) for the formation of new crystals by a process of homologous nucleation. In diseases such as osteoarthritis, crystal deposition arthritis, and atherosclerosis, MVs initiate pathologic calcification, which, in turn, augments disease progression.

Bone Matrix↗