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Biomedical subjects

N J Severs

Publications and source records attributed to N J Severs.

At least 109 records · Page 6Linked to original sources
PubMed1991

Development of the distal end and Tomes' processes of ameloblasts observed by freeze-fracture and ultrathin section electron microscopy.

A freeze-fracture examination of distal portions of differentiating and secretory ameloblasts was carried out using upper molar tooth germs from one to four days old rats. Ultrathin sections were prepared for comparison. Freeze-fracture provides a unique opportunity to observe plasma membrane events in distal portions of ameloblasts during differentiation and development. Our results suggest that the distal plasma membrane and Tomes' processes of differentiating and secretory ameloblasts are highly active in endocytosis. In differentiating ameloblasts, endocytic activity is likely to be associated with removal of the basal lamina. During matrix secretion and maturation, ameloblasts are probably removing material from the maturing matrix; in particular amelogenins and water. The present study provides morphological evidence that an endosomal system is present in differentiating and secretory ameloblasts during periods of endocytic activity. This would provide a system whereby ligands, fluid components, membrane receptors and plasma membrane, which enter the cells during endocytosis, may be separated from each other and sorted out for despatch to different destinations.

Ameloblasts↗
PubMed1990

Integrity of the dissociated adult cardiac myocyte: gap junction tearing and the mechanism of plasma membrane resealing.

Dissociation of adult cardiac myocytes by collagenase perfusion techniques requires separation of the junctional contacts that link the cells physically, electrically and metabolically in the intact heart. Gap junctions, one of three types of intercellular junction present at the cardiac intercalated disc, are not split into their component membranes when myocytes are dissociated; they are ripped from the plasma membrane of one cell, to be retained by its neighbour. Partitioning of junctions in this way might be expected to constitute a serious threat to the ionic integrity of dissociated myocytes, but in practice, high yields of functionally intact cells, suitable for experimental studies, are routinely obtained. To explain this apparent paradox, repair mechanisms, operating to seal the membrane lesions caused by gap junction tearing, have been hypothesized, but evidence for their existence has previously been lacking. Using freeze-fracture electron microscopy, the present study identifies repair sites as smooth membrane domains that are continuous with the neighbouring plasma membrane, thus forming intact seals. That these structures are not chemically-induced artefacts is demonstrated by their presence in myocytes that were frozen directly from the living state. Subsarcolemmal vesicle clusters, detected in thin sections and freeze-fracture replicas, are associated with the smooth sealing domains. These structures may represent either rounded-up fragments of mechanically disrupted membrane or structures concerned with the synthesis of new lipid. From their freeze-fracture morphology, the sealing domains appear to be lipid-rich and protein-poor. Cytochemical studies using Ruthenium Red, cationized ferritin and lectins show in addition that they have a lower content of negatively-charged membrane components than the neighbouring plasma membrane, and that the carbohydrate residues normally associated with plasma membrane glycolipids and glycoproteins are absent.

Animals↗
PubMed1990

Arrested exocytosis of atrial secretory granules.

Release of atrial natriuretic peptide (ANP) from atrial muscle cells is thought to occur by exocytosis of secretory granules, as in other secretory systems. However, in the atrial myocyte, exocytosis has previously proved difficult to detect ultrastructurally. In order to study the mechanism of ANP release and related events, we have applied two separate techniques--tannic acid perfusion and ultrarapid freezing--specifically designed to arrest exocytosis. An increased number of fusion sites was found after ultrathin sectioning and freeze-fracture of tannic acid-treated atria enabling a hypothetical release sequence to be constructed. Detail of granule substructure was also obtained, suggesting the presence of a coat to the secretory granule core. Ultrarapid freezing, followed by freeze-fracture and freeze substitution, has confirmed aspects of the proposed exocytotic sequence, suggesting that this release is not due to the application of tannic acid, and these techniques also produced further evidence for the existence of the granule-core coat. Coated pits and vesicles were also found in large numbers in tannic acid-treated atria and interactions between coated vesicles and secretory granules were visualized. The possible role of coated vesicles in an exocytotic/endocytotic membrane retrieval pathway is discussed.

Animals↗
PubMed1990

Cardiac myocyte gap junctions: evidence for a major connexon protein with an apparent relative molecular mass of 70,000.

It is widely accepted that there is a family of gap junction connexon proteins, their distribution appearing to vary with tissue type and species. In cardiac tissues the major junctional channel component identified is a 43K (K = 10(3) Mr) polypeptide. Using a gap junction isolation protocol in which low temperatures are maintained, and which is detergent-free, we have identified a second gap junction-related protein in cardiac tissues with an apparent relative molecular mass of 70,000. Antibodies raised to three synthetic peptides matching portions of the 43K gap junction protein cDNA sequence cross-react with the 70K protein, but biochemical studies indicate that these proteins are distinct from one another. The structures that contain the 70K protein are susceptible to fragmentation at warm temperatures, and by electron microscopy this can be correlated with loss of 'minidomains' within the junctional plaque. Using a gap junction enriched-fraction prepared from purified isolated adult myocytes we show that both the 43K and 70K gap junction proteins are present in ventricular cardiac myocytes. In such preparations, and those from whole heart, the 70K protein appears to be the major gap junction-related protein present.

Amino Acid Sequence↗
PubMed1990

Cardiac gap junctions in rat ventricle: localization using site-directed antibodies and laser scanning confocal microscopy.

Antibodies were raised to three synthetic peptides, each constructed to match a different cytoplasmic region of the 43kD protein of the cardiac gap junction. These antibodies have all been used for immunohistochemical localization of gap junctions in the rat ventricle. Each is shown to have different staining characteristics which provide supporting evidence for the model proposed for the configuration of the gap junction protein within the membrane. Laser scanning confocal microscopy has confirmed that the localization of ventricular myocyte gap junctions is solely within intercalated disks. This technique is demonstrated to provide a greater overview of the three dimensional arrangement of the junctions than other microscopical techniques. Larger gap junctions occur typically at the periphery of the disk, with smaller junctions situated in the central zone. The analysis of the number and distribution of gap junctions over large volumes of myocardial tissue (three-dimensional tomography) is now feasible with this approach, and may be used to delineate conduction pathways.

Animals↗
PubMed1989

Freeze-fracture cytochemistry: review of methods.

"Freeze-fracture cytochemistry" encompasses a diversity of recently developed techniques in which freeze-fracture and cytochemistry are combined. Cytochemical labeling may, in principle, be integrated into one of three basic points in the standard freeze-fracture procedure; 1) before the specimen is frozen, 2) after it has been fractured, or 3) after it has been platinum shadowed and/or carbon coated. Visualization of the labeled cellular structures can be achieved by a variety of different methodologies. For example, the markers (usually colloidal gold particles) may be viewed embedded within a replica, or attached to it via fragments of membrane (or other cellular components). Sectioning is a central strategy in a number of techniques, either in combination with or in place of replication. The different combinations of methods that have been devised are not, for the most part, alternative ways of arriving at the same result; each provides quite distinct information about specific classes of membrane component or other structure in the cell. The purpose of this review is to present, within a single article, a systematic survey of the full range of techniques currently available in freeze-fracture cytochemistry. Emphasis is placed on explaining the principles underlying the methods and on illustrating their applications. With the success recently achieved, freeze-fracture cytochemistry has moved from the phase of experimental development to a position in which it may be expected increasingly to make significant contributions across a wide spectrum of problems in cell and membrane biology.

Animals↗
PubMed1989

Stretch-induced centrifugal movement of atrial specific granules--a preparatory step in atrial natriuretic peptide secretion.

Atrial natriuretic peptide (ANP) is a circulating hormone produced by atrial muscle cells which acts upon renal vascular smooth muscle to raise glomerular filtration pressure, thereby increasing salt and water excretion and reducing blood pressure [2, 5, 8]. As in other peptide hormone-secreting cells, ANP is packaged and stored in dense Golgi-derived secretory granules [4-6]. These atrial specific granules [6] occur predominantly in large clusters deep in the myocyte's interior, associated with Golgi cisternae in myofibril-free cytoplasm at the nuclear poles [5, 6]. Smaller clusters and individual granules are distributed beneath the sarcolemma and between myofibrils [6, 10]. Expansion of the blood volume raises the level of circulating ANP [7], a response mediated by mechanical stretch of the atrial wall [1, 3, 9]. The mechanism by which cell stretch induces release of ANP from atrial myocytes is, however, unknown. We show here that one component of this unusual secretory mechanism involves a sudden centrifugal movement of atrial specific granules toward the cell surface.

Animals↗
PubMed1989

Membrane cytochemistry of the atrial peptide secretory pathway.

The characteristics of the principal membrane systems involved in atrial peptide secretion are explored in rabbit and rat atrial muscle cells using freeze-fracture electron microscopy combined with cytochemistry. Structural features of the Golgi apparatus and secretory granule membrane are described, and two features of the membranes' composition, cholesterol content and terminal glycocomponents, are probed in situ in the cell. Both the secretory granule membrane and the plasma membrane contain morphologically detectable cholesterol, and both are rich in the terminal sugar residues (sialic acid and N-acetyl glucosamine) of membrane glycocomponents. Some cisternal membranes of the Golgi apparatus reveal detectable cholesterol but others do not, and only focal labeling of terminal glycocomponents in Golgi membranes is observed, usually adjacent to the accumulation of secretory product. Nuclear membranes, endoplasmic reticulum, and mitochondria contain virtually no detectable cholesterol nor terminally glycosylated components. The Golgi apparatus, which is exclusively equipped with the enzymes responsible for terminal glycosylation of membrane glycoproteins, appears actively to modify the chemical composition of the membrane segments in which it packages atrial peptide. This ensures that the composition of the atrial granule membrane resembles that of the plasma membrane into which it is ultimately incorporated.

Animals↗
PubMed1989

Fate of gap junctions in isolated adult mammalian cardiomyocytes.

The fate of gap junctions in dissociated adult myocytes, maintained for up to 22 hours in culture medium, was investigated by semiquantitative analysis of thin sections and by freeze-fracture electron microscopy. Gap junctions in the dissociated myocyte are intact bimembranous structures seen either as invaginated surface-located structures or as annular profiles in the cytoplasm. Surface-located junctions are sealed from the exterior by a sheet of nonjunctional membrane originating (together with the "outer" junctional membrane) from the formerly neighboring cell. Serial sectioning was used to establish that at least part of the annular gap junction population in the freshly isolated myocyte represents truly discrete cytoplasmic vesicles; thus, some gap junctions are rapidly endocytosed after myocyte separation. Analysis of the surface-located-to-annular gap junction ratio suggested that no further endocytosis occurred in rabbit and cat myocytes maintained for 22 and 15 hours, respectively. Guinea pig myocytes, by contrast, did appear to continue endocytosis in culture. Analysis of the distance of gap junctional structures from the cell surface suggested that little if any inward migration of gap junction vesicles occurred. Hypoxia had no detectable effect on the internalization or inward movement of gap junctions. The quantity of ultrastructurally detectable gap junction membrane appeared to remain constant over time, as did the incidence of "complex structures" (i.e., annular gap junction profiles with features previously suggested to represent degradation). New gap junction formation was negligible, and a reappraisal of the nature of "complex structures" led to the conclusion that the origin of these structures need not be related to degradation. Taken together, the findings suggest that degradation and disappearance of gap junctional membrane after isolation of the mature myocyte constitute a much slower process than previously believed, and the possibility that the cardiac gap junction protein has a longer half-life than its counterpart in liver remains open.

Animals↗
PubMed1988

Analysis of the rat liver gap junction protein: clarification of anomalies in its molecular size.

The major gap junction polypeptide in most tissues has an apparent molecular mass of 27 kDa with a 47 kDa dimer present in junction-enriched fractions. However, a 54 kDa protein recognized by gap junction-specific antibodies has been reported and a complementary DNA (cDNA) sequence for both human and rat liver gap junctions codes for a 32 kDa protein. In this paper we show that these are all forms of the same gap junction protein that can be observed on SDS-polyacrylamide gels simply by varying the concentration of acrylamide in the gels. A 64 kDa dimer is also obtainable. Antibodies to the gap junction protein or to a synthetic peptide constructed to match the rat liver gap junction amino-terminal sequence recognize all of these forms. Under some conditions a 54 kDa dimer is 'preferred', explaining the presence of this species in whole tissue homogenate Western blots. These results clarify several controversies and indicate that the protein forming the gap junction channel probably undergoes no major post-translational modification as the cDNA sequence codes for a protein of molecular mass 32 kDa and this protein species and its 64 kDa dimer are demonstrable on SDS-polyacrylamide gels under appropriate conditions.

Animals↗
PubMed1987

Lack of cytochemically detectable cholesterol in rabbit vena cava endothelial plasma membrane.

The response of rabbit vascular endothelial plasma membranes to the cholesterol-binding agents filipin and tomatin was investigated by freeze-fracture electron microscopy. In both single and double labelling experiments, the endothelial plasma membranes of the posterior vena cava were remarkably resistant to the agents compared with the endothelial plasma membranes of other vessels (aorta, pulmonary artery, and pulmonary and cardiac capillaries). This suggests that vena cava endothelium differs markedly from other endothelia, either by having exceptionally low cholesterol levels in its plasma membranes, or with respect to other membrane properties that influence the reaction to filipin and tomatin.

Animals↗
PubMed1986

Plasmalemmal vesicles and the effects of sterol-binding agents in rabbit aortic endothelium.

The rims of vesicle openings in capillary endothelium are reported to be highly sensitive to perturbation by the sterol probe filipin when this agent is administered by perfusion at 50 microM concentration in aldehyde fixative for 10 min. If, as supposed, this specific rim response reflects functionally significant aspects of membrane organization at the vesicle-plasmalemma boundary, then it would be expected to be a reproducible and universal feature of endothelial vesicles in general. In the present study, we have investigated in detail the response to filipin of rabbit and rat aortic endothelial plasmalemma. First, the effect of standard filipin treatment (150 microM, 3-20 h) was examined by freeze-fracture. The extensive generalized response observed, taken together with the effects of filipin seen by fluorescence microscopy of stripped aortic endothelial sheets, and the marked corrugation of the plasmalemma observed by freeze-fracture after tomatin treatment, indicate that substantial amounts of cholesterol are present in the membrane. Exposure of rabbit aortic endothelium to 50 microM filipin for 10, 20, 40 and 60 min enabled the progressive appearance of filipin deformations in the plasmalemma to be traced. Even at the very earliest stages of the response, there was no preferential association of filipin deformations with endothelial vesicle rims. Similar experiments in the rat gave the same result. Specific sensitivity of endothelial vesicle rims would, therefore, seem to be either non-universal in occurrence or non-reproducible, and so is unlikely to reflect any membrane property of fundamental significance to endothelial vesicle function.

Animals↗
PubMed1986

Caveolar bands and the effects of sterol-binding agents in vascular smooth muscle plasma membrane. Single and double labeling with filipin and tomatin in the aorta, pulmonary artery, and vena cava.

The effects of single and sequential double treatment with the sterol probes filipin and tomatin were investigated in relation to the distribution of caveolae (flask-shaped membrane invaginations) in the smooth muscle plasma membrane of rabbit aorta, pulmonary artery, and posterior vena cava. The principal structural characteristics of vascular smooth muscle caveolae are documented, and the effects of filipin are quantitatively evaluated and compared in the three vessel types. Aortic and pulmonary artery smooth muscle cells were found to resemble one another with respect to caveolar banding pattern and response to filipin. By comparison, however, the caveolar bands of vena cava smooth muscle were aligned with remarkable precision and regularity, and proved to be significantly (p less than 0.001) more sensitive to filipin than those of the arterial cells. The noncaveolar interband zones in all vessels were resistant to filipin action but sensitive to tomatin. Again, this sensitivity was more marked in vena cava smooth muscle than in arterial smooth muscle. The precision of caveolar banding, combined with the accentuated effects shown by each agent in the vena cava made the result of sequential double filipin-tomatin labeling outstandingly clear-cut in this vessel, with alternate filipin-affected and tomatin-affected zones superimposed, respectively, upon the caveolar bands and interbands. These findings strengthen the view that cholesterol distribution in the smooth muscle plasma membrane is homogeneous, and that the resistance of the smooth muscle interband membrane to filipin is a false-negative cytochemical result due to the presence of underlying membrane associated dense bodies.

Alkaloids↗
PubMed1985

Intercellular junctions and the cardiac intercalated disk.

Cardiac muscle cells are equipped with three distinct types of intercellular junction--gap junctions, "spot" desmosomes, and "sheet" desmosomes (or fasciae adherentes)--located in a specialized portion of the plasma membrane, the intercalated disk. Gap junctions are responsible for electrical coupling and the transfer of small molecules between cells, whereas the desmosomelike junctions (also known as adherens junctions) provide strong intercellular adhesion. The adhesion sites formed by the "spot" desmosome anchor the intermediate-filament cytoskeleton of the cell; those formed by the fascia adherens anchor the contractile apparatus. An understanding of the ultrastructure of these junctions helps explain how they carry out their functions, and new observations in this field have been made through the application of ultrarapid freezing techniques in conjunction with freeze-fracture electron microscopy. With recent findings from biochemical and immunocytochemical studies, this understanding is now being extended to the molecular level.

Animals↗
PubMed1985

Morphometric analysis of the isolated calcium-tolerant cardiac myocyte. Organelle volumes, sarcomere length, plasma membrane surface folds, and intramembrane particle density and distribution.

Using morphometric analysis of thin sections and freeze-fracture replicas, the ultrastructure of isolated rat myocytes prepared by collagenase digestion (Powell et al. 1980) was compared with that of myocytes fixed by perfusion of intact myocardium. The volumes of myofibrils, mitochondria, nuclei, sarcoplasmic reticulum and lipid droplets in the isolated myocytes did not differ from those of their counterparts in the intact heart, but the volume occupied by transverse tubules was apparently reduced. The isolated cells had significantly shorter sarcomeres than did cells in the intact tissue, and this was associated with an altered topography of plasma membrane surface folds at the level of the Z-lines. Plasma membrane intramembrane particles were randomly distributed and showed the same numerical density on the E-faces of both isolated and intact-heart myocytes. However, P-face particle density was slightly reduced in the isolated cells. It is concluded that the few differences detected in the isolated cells do not reflect any fundamental derangement of their properties.

Animals↗