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PubMed2003

Imaging zinc: old and new tools.

As recently as 20 years ago, all zinc in biological systems was believed to be tightly bound to proteins, and the idea of imaging zinc was considered heretical. Beginning with Maske's research with dithizonate staining of the hippocampus in the 1950s, however, zinc-sensitive dyes have indicated that, in mammalian cells, free zinc can exist in at least three separate pools. These pools include vesicular zinc sequestered in presynaptic vesicles and secretory granules, zinc released from these vesicles into the extracellular space after physiological stimulation, and transient increases in zinc in cells in the regions where extracellular release of zinc has occurred. This Perspective covers the zinc-imaging tools, from dithizonate to the newest FRET-based sensors, that have galvanized biomedical science.

Animals↗

PubMed2002

Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyphaga: an electron micrograph study.

Parachlamydiaceae are endosymbionts of free-living amoeba first identified in 1997. Two developmental stages, elementary and reticulate bodies, were observed; however, their localization and proportions according to culture condition and duration remain unknown. The life cycle of Parachlamydia acanthamoeba within Acanthamoeba polyphaga was studied by transmission electron microscopy of 8-, 36-, and 144-h coculture. Morphometry and quantification were performed using SAMBA software. The elementary body, the predominant stage within the amoebae, was located mainly within their vacuoles. The multiplication of Parachlamydia bacteria by binary fission of reticulate bodies was independently associated with culture in PYG broth (odds ratio [OR] = 4.4; 95% confidence interval [CI], 1.55 to 12.46) and with the presence of reticulate bodies within the amoebae (OR = 2.10; 95% CI, 1.53 to 2.89). A third developmental stage was observed, the crescent body. Its presence outside and inside the amoebae was associated mainly with prolonged incubation time (OR = 3.98; 95% CI, 1.49 to 10.68, and OR = 5.98; 95% CI, 1.75 to 20.4, respectively). Elementary and crescent bodies were released into the extracellular medium within vesicles or after amoebal lysis. For both, phagocytosis was their mode of entry. This electron micrograph study revealed another infective developmental stage, the crescent body, and provided quantitative analysis of the life cycle of P. acanthamoeba within A. polyphaga.

Acanthamoeba↗

PubMed1999

Structural maturation of the transmissible gastroenteritis coronavirus.

During the life cycle of the transmissible gastroenteritis coronavirus (TGEV), two types of virus-related particles are detected in infected swine testis cells: large annular viruses and small dense viruses. We have studied the relationships between these two types of particles. Immunoelectron microscopy showed that they are closely related, since both large and small particles reacted equally with polyclonal and monoclonal antibodies specific for TGEV proteins. Monensin, a drug that selectively affects the Golgi complex, caused an accumulation of large annular viral particles in perinuclear elements of the endoplasmic reticulum-Golgi intermediate compartment. A partial reversion of the monensin blockade was obtained in both the absence and presence of cycloheximide, a drug that prevented the formation of new viral particles. After removal of monensin, the Golgi complex recovered its perinuclear location, and a decrease in the number of perinuclear large viral particles was observed. The release of small dense viral particles into secretory vesicles and the extracellular medium was also observed, as was a partial recovery of infectivity in culture supernatants. Small viral particles started to be seen between the third and the fourth Golgi cisternae of normally infected cells. All of these data strongly indicate that the large annular particles are the immature precursors of the small dense viruses, which are the infectious TGEV virions. The immature viral particles need to reach a particular location at the trans side of the Golgi stack to complete their morphological maturation.

Animals↗

PubMed1988

Ultrastructural localization of calcium in matrix vesicles and preodontoblasts of developing rat molar tooth germs during initial dentinogenesis.

We investigated the ultrastructural localization of calcium in progenitor predentine and preodontoblasts of developing rat molar tooth germs using the potassium pyroantimonate technique. At the precalcification stage, antimonate reaction product was sparsely, randomly distributed in the preodontoblasts and in the progenitor predentine but no significant reaction could be noticed associated with matrix vesicles. At the matrix vesicle calcification stage, large amounts of antimonate reaction product tended to be localized in the region adjacent to the distal, outer surface membrane of preodontoblasts in which moderate antimonate reaction activity could be observed in mitochondria. Strong antimonate reaction was detected preferentially on the outer surface membrane of some matrix vesicles at this stage. At the subsequent collagen calcification stage, definite antimonate reaction was no longer seen within mitochondria of the late preodontoblasts, instead precipitate was mainly distributed in Golgi area, secretory granules and lateral intercellular spaces. It is suggested that although matrix vesicles contain few calcium capable of reacting to antimonate immediately after their biogenesis, subsequently, large amounts of calcium are accumulated associated with the outer surface membrane of matrix vesicles in the extracellular matrix.

Animals↗

PubMed1995

Characteristics of Actinobacillus actinomycetemcomitans invasion of and adhesion to cultured epithelial cells.

Actinobacillus actinomycetemcomitans (A.a.) is highly implicated in periodontitis. We have developed several in vitro models using the KB oral cell line to examine A.a.-epithelial cell interactions. In support of the use of KB cell line model systems is our finding that A.a. invaded KB and primary gingival cells to the same extent. Invasion is an active event which requires new protein synthesis by both KB and A.a. Like many other intracellular parasites, A.a. invade by receptor-mediated endocytosis. We observed that internalized A.a. were surrounded by foci of actin which had been transported from the periphery of the KB cell. Adhesion of A.a. to KB cells occurred rapidly and stimulated the formation of microvilli. Adhesion is affected by both host factors (saliva, serum, [NaCl]) and culture conditions. Multiple determinants [fimbriae, outer membrane proteins, vesicles, and/or an extracellular amorphous material (ExAmMat)] which are either associated with the A.a. surface or are released into the milieu are involved. We determined that ExAmMat can convey adhesiveness to weakly adherent A.a. and to at least one other oral species (Streptococcus parasanguis).

Adhesins, Bacterial↗

PubMedDate not supplied

The exocytotic fusion pore interface: a model of the site of neurotransmitter release.

Ultrastructural techniques have shown that an early event in the exocytotic fusion of a secretory vesicle is the formation of a narrow, water-filled pore spanning both the vesicle and plasma membranes and connecting the lumen of the secretory vesicle to the extracellular environment. Smaller precursors of the exocytotic fusion pore have been detected using electrophysiological techniques, which reveal a dynamic fusion pore that quickly expands to the size of the pores seen with electron microscopy. While it is clear that in the latter stages of expansion, when the size of the fusion pore is several orders of magnitude bigger than any known macromolecule, the fusion pore must be mainly made of lipids, the structure of the smaller precursors is unknown. Patch-clamp measurements of the activity of individual fusion pores in mast cells have shown that the fusion pore has some unusual and unexpected properties, namely that there is a large flux of lipid through the pore and the rate of pore closure has a discontinuous temperature dependency, suggesting a purely lipidic fusion pore. Moreover, comparisons of experimental data with theoretical fusion pores and with breakdown pores support the view that the fusion pore is initially a pore through a single bilayer, as would be expected for membrane fusion proceeding through a hemifusion mechanism. Based on these observations we present a model where the fusion pore is initially a pore through a single bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1975

The effect of glucose-1 phosphate calcium on the epiphyseal cartilage of the rat.

Electron microscopic investigations were carried out on the proximal tibial epiphyseal cartilage of albino rats treated with 2 mg G-1-P-Ca daily for one and three weeks. The treatment--enhanced the secretory activity of the upper cells of the zone of maturing cartilage with a consequential enlargement of the Golgi region, depletion of secretory products, and an increase in the number of microtubuli. --In addition, the treatment increased the number of matrix vesicles in the extracellular space showing incipient calcification. This findings confirm the earlier light micriscopic observation of the calcification stimulating effect of G-1-P-Ca.

Animals↗

PubMed1990

Ultrastructural in vitro characterization of a porous hydroxyapatite/bone cell interface.

In order for the hydroxyapatite implant material interface to new bone to be characterized, osteogenic mouse calvarial mesenchymal cells were grown in vitro in contact with a porous hydroxyapatite (PHA). After the mesenchymal cell culture was incubated for 12 to 13 days, the resulting tissue-containing bone colonies were fixed, embedded, sectioned, and stained for microscopic evaluation. Light and transmission electron microscopy (with conventional staining) and phosphotungstic acid cytochemistry were used to explore and record the optical microscopic and ultrastructural interfaces at the hydroxyapatite surface. Osteoblasts, fibroblasts, bone, and cartilage were observed and photographed at the implant surface. Osteoblasts found in conjunction with well-developed collagen, matrix vesicles in the extracellular matrix, and newly formed hydroxyapatite crystals on the PHA surface confirmed the beginning of woven bone formation. Collagen fibers were observed directly in contact with the PHA when osteoblasts were present. Polysaccharides were localized among the collagen fibers in the implant-cell extracellular space, indicating a rich complex carbohydrate layer in relation to the collagen of immature bone. Fibroblasts and chondroblasts at the implant surface secreted no collagen, but an amorphous layer was visible between the fibroblasts and the implant surface. When polysaccharides were stained, an electron-dense film appeared where the amorphous layer came into contact with the implant material. Collagen was secreted from the cell surface furthest from the implant. Osteoblasts and fibroblasts/chondroblasts, when surrounding PHA, seem to take on two different interfacial functions: Osteoblasts secrete collagen in a bone-initiating extracellular matrix with carbohydrates at the implant surface, whereas fibroblasts/chondroblasts appear to attach to the implant with a carbohydrate-rich attachment substance, but with no collagen at the interface. This study confirms in vivo data that PHA is a viable implant material because it is biocompatible and, unlike several other materials, appears to stimulate, or at least to permit, osteogenesis.

Animals↗

PubMed1981

[Cholesteatoma, oto- and tympanosclerosis: their general mechanism of tissue destruction (author's transl)].

The connective tissue contains extracellular enzymes for the turnover of the ground substance. If this enzyme system gets out of control there is development of a complex metabolic disturbance of all tissue components. The lysosomal dysfunction is the basis of the general mechanism of tissue destruction in cholesteatoma, oto- and tympanosclerosis. This process seems most enhanced in cholesteatoma. Emphasis is placed on the role of extracellular lysosomes (= matrix vesicles).

Cholesteatoma↗

PubMed1976

[Oocyte maturation in Xenopus laevis. Arguments in favor of the role of calcium ions].

Influx and efflux of 45Ca++ ions in Xenopus leavis isolated full-grown oocytes were measured. Calcium influx was from 0.5 to 1 pmole/oocyte/h. Progesterone, which induces maturation does not influence Ca++ fluxes while PCMB caused a significant increase of Ca++ influx during the course of PCMB (p-Cl-HgBzO-) induced maturation. Our results show that the presence of Ca++ is not necessary in extracellular medium for germinal vesicle breakdown (GVBD) induced by progesterone. In contrast, the kinetic and the percent of germinal vesicle breakdown observed when maturation was induced by PCMB depend on the extracellular Ca++ concentration in Ca-EGTA buffers.

Animals↗

PubMed1976

Intracellular membrane junctions during the exocytosis of insulin.

In the process of insulin release by exocytosis, a fusion between the membranes of secretory vesicles and the plasma membrane occurs which is followed by the rupture of these membranes and the formation of an exocytotic stoma. As revealed by freeze-fracturing, the membrane structure is altered in the region of membrane interaction. An accumulation of membrane-associated particles can be observed in membranes of secretory vesicles contacting the plasma membrane. At the exocytotic stoma the boundary between granule limiting membrane and plasma membrane is occasionally outlined by a ring-like aggregation of membrane-associated particles. The effects of hypertonic solutions on tissue structure indicate that the membrane junctions during exocytosis are permeable to ions and small molecules, forming a communication between the interior of the secretory vesicle and the extracellular space.

Animals↗

PubMed2004

Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD.

Patients with ESRD have a high circulating calcium (Ca) x phosphate (P) product and develop extensive vascular calcification that may contribute to their high cardiovascular morbidity. However, the cellular mechanisms underlying vascular calcification in this context are poorly understood. In an in vitro model, elevated Ca or P induced human vascular smooth muscle cell (VSMC) calcification independently and synergistically, a process that was potently inhibited by serum. Calcification was initiated by release from living VSMC of membrane-bound matrix vesicles (MV) and also by apoptotic bodies from dying cells. Vesicles released by VSMC after prolonged exposure to Ca and P contained preformed basic calcium phosphate and calcified extensively. However, vesicles released in the presence of serum did not contain basic calcium phosphate, co-purified with the mineralization inhibitor fetuin-A and calcified minimally. Importantly, MV released under normal physiologic conditions did not calcify, and VSMC were also able to inhibit the spontaneous precipitation of Ca and P in solution. The potent mineralization inhibitor matrix Gla protein was found to be present in MV, and pretreatment of VSMC with warfarin markedly enhanced vesicle calcification. These data suggest that in the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release. These vesicles contain mineralization inhibitors derived from VSMC and serum, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcification.

Adolescent↗

PubMed2006

A cell-semiconductor synapse: transistor recording of vesicle release in chromaffin cells.

The release of dense-core vesicles in bovine chromaffin cells is a model for the presynaptic process in neurons. It is usually studied by microamperometry of catecholamines with carbon fibers. Here we introduce transistor recording as a tool to study vesicle release. When we stimulate a chromaffin cell placed on a field-effect transistor, the gate voltage exhibits peaks that correlate with a simultaneously performed amperometric recording. We attribute the transistor signal to a release of protons from the extruded matrix of vesicles that lowers the extracellular pH and changes the electrical surface potential of the gate oxide. The rise time of the transistor signals is similar to that of amperometric responses, whereas their duration is distinctly longer. In a model computation, the rise time is identified with the extrusion of vesicle matrix into the narrow extracellular space between cell and gate oxide, and the decay time is attributed to pH equilibration through slow diffusion in the extruded matrix. Because the transistor recording relies on protons, it can be applied to acidic vesicles with electrochemically inactive hormones or transmitters.

Animals↗

PubMed2001

Expression of cathepsin K mRNA and protein in odontoclasts after experimental tooth movement in the mouse maxilla by in situ hybridization and immunoelectron microscopy.

This study demonstrated the simultaneous expression of cathepsin K (CK) mRNA by in situ hybridization and CK protein by immunoelectron microscopy in odontoclasts in mouse maxillae after experimental tooth movement. On the pressure side (the area under pressure during tooth movement), CK mRNA was detected in odontoclasts in resorption lacunae in the tooth root, in osteoclasts in bone resorption lacuane, and in fibroblasts in the periodontal ligament. Using electron microscopy, CK protein was detected at the apex of odontoclasts, intracellularly in vesicles and granules, and extracellularly in irregularly shaped vacuoles (extracellular spaces), on the plasma membrane of the ruffled border, and on and between typical striated type I collagen fibrils in the lacunae. These vesicles and granules appeared to fuse with irregular vacuoles containing CK-positive fragmented fibril-like structures close to the ruffled border. In the basolateral portion of odontoclasts, small amounts of CK-positive rough endoplasmic reticulum (ER) were found. CK-positive intracellular vacuoles (not extracellular spaces) also appeared to fuse with the vesicles and granules. However, these fused organelles rarely contained fragmented fibril-like structures. They are probably endolysosomes. The distribution of CK in odontoclasts was similar to that previously seen in osteoclasts. Furthermore, CK-positive fibril-like structures were found in the vacuoles of fibroblasts. These results indicated that during tooth movement CK is synthesized in odontoclasts on the pressure side and secreted into the tooth resorption lacunae. Therefore, CK may take part in the degradation of the dentin matrix (type I collagen fibrils and non-collagenous protein) of the tooth root, and in the subsequent intracellular degradation of endocytosed fragmented fibril-like structures in endolysosomes.

Animals↗

PubMed1994

Characterization of very acidic phagosomes in breast cancer cells and their association with invasion.

Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5-10 microns) in which endocytosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcourrier et al. (1990). Cancer Res. 50, 6045-6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epifluorescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 microns diameter latex beads and fluorescence Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are heterophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H(+)-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (< 4), lower than the lysosomal value (pH approximately 5) in the same cells and in specialized phagocytotic cells such as macrophages. We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.

Anti-Bacterial Agents↗

PubMed2006

Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells.

Exocytosis of secretory vesicles begins with a fusion pore connecting the vesicle lumen to the extracellular space. This pore may then expand or it may close to recapture the vesicle intact. The contribution of the latter, termed kiss-and-run, to exocytosis of pancreatic beta cell large dense-core vesicles (LDCVs) is controversial. Examination of single vesicle fusion pores demonstrated that rat beta cell LDCVs can undergo exocytosis by rapid pore expansion, by the formation of stable pores, or via small transient kiss-and-run fusion pores. Elevation of cAMP shifted LDCV fusion pore openings to the transient mode. Under this condition, the small fusion pores were sufficient for release of ATP, stored within LDCVs together with insulin. Individual ATP release events occurred coincident with amperometric "stand alone feet" representing kiss-and-run. Therefore, the LDCV kiss-and-run fusion pores allow small transmitter release but likely retain the larger insulin peptide. This may represent a mechanism for selective intraislet signaling.

Adenosine Triphosphate↗

PubMed2000

Interplay between membrane dynamics, diffusion and swelling pressure governs individual vesicular exocytotic events during release of adrenaline by chromaffin cells.

Release of adrenaline by chromaffin cells occurs through a process involving docking and then fusion of a secretory vesicle to the cytoplasmic membrane of the cell. Fusion proceeds in two main stages. The first one leads to the creation of a stable fusion pore passing through the two membranes and which gives a constant release flux of neurotransmitter (pore-release stage). After a few milliseconds, this initial stage which is not investigated here proceeds through a sudden enlargement of the initial pore (full-fusion stage) up to the complete incorporation of the vesicle membrane into that of the cell and total exposure of the initial matrix vesicle core to the extracellular fluid. The precise time-resolved dynamics of the release and of the vesicle membrane during the full-fusion phase can be extracted with a precision never achieved so far by de-convolution of experimental chronoamperometric currents monitored during individual exocytotic secretion events. The peculiar dynamics of the vesicle membrane proves that exocytotic events are powered by the swelling of the matrix polyelectrolyte core of the vesicle, although they are kinetically regulated by diffusion in the matrix and by the dynamics of the vesicle and cell membranes. Two simple theoretical models based on the dynamics of pores are developed to account for these dynamics and are shown to predict behaviors which are essentially identical to the experimental ones. This offers a new view of the kinetic grounds which control the full-fusion stage, and therefore provides a new interpretation of the sudden transition between the pore-release and the full-fusion stages. This transition occurs when the increasing membrane surface tension energy due to the refrained internal swelling pressure overcomes the edge energy of the pore, so that the initial fusion pore becomes unstable and is disrupted. This new view predicts that secretory vesicles which contain matrixes energetically similar to those of the adrenal cells investigated here can be separated into two classes according to their radius and catecholamine content. Small vesicles (less than ca. 25 nm radius, and containing less than ca. 20000 molecules) should always release through pores. Larger vesicles should always end into fusing except if another mechanism closes the pore before ca. 10000 molecules of catecholamines have been released.

Animals↗

PubMed1981

Normal and abnormal mineralization in mammals.

It is widely accepted that cells play a crucial role in the mineralization of dentin, cartilage and bone. The initial locus of calcification appears to be the matrix vesicle, a 200 nm submicroscopic extracellular, membrane-invested particle which is shed from the osteoblast, chondrocyte, or odontoblast and carries into the matrix calcifiable lipids and phosphatases. During Phase 1 of mineralization, the initiation phase, apatite appears within matrix vesicles, probably preceded by amorphous calcium phosphate. Initially, acidic phospholipids of matrix vesicles may attract calcium to form complexes with phosphate and protein. In vitro studies of cartilage slices and isolated matrix vesicles indicate the essentiality of vesicle phosphatases for mineral initiation. In pathological calcification is self-sustaining. Given physiological amounts of Ca2+ and PO43(-), calcification will spread by crystal proliferation into the extracellular matrix surrounding vesicles (Phase 2). During Phase 2 the rate of crystal proliferation is controlled by matrical factors: Collagen can orient and apparently promote apatite formation. Proteoglycans, pyrophosphate, gamma-carboxyglutamic acid-containing proteins and phosphoproteins in calcifying matrix bind Ca2+ by their anionic subgroups, and all have been shown to impede hydroxyapatite formation in vitro. The latter substances are visualized as regulating Phase 2 by inhibiting mineral growth. The calcification process involves an interaction of all of the above. When viewed as a 2-phase phenomenon, it is possible to integrate each of these factors into a comprehensive concept od biological mineralization.

Calcification, Physiologic↗