Structural changes at the myogenic cell surface during the formation of myotendinous junctions

Summary Myotendinous junctions are sites which are morphologically and molecularly specialized for force transmission between intracellular and extracellular structural proteins. In the present investigation, the formation of these specialized junctions is studied in chicken embryos from 9 days following fertilization to 1 day posthatching, using light and electron microscopy. Observations indicate that the first discernible event in myotendinous junction formation is the appearance of basement membrane at the incipient junction at 9–10 days postfertilization, concomitant with the aggregation of fibroblasts at the junctional regions of myogenic cells. Subsequently, subsarcolemmal densities appear at sites opposite basement membrane locations by 13 days postfertilization. Myofibrils insert into subsarcolemmal densities by day 15 and invaginations of the cell membrane are initiated at those insertions. Type I collagen fibers appear at the cell surface at day 17. Junctional structure at day 17 qualitatively resembles that of adult myotendinous junctions. Changes in junctional structure following day 17 are primarily increases in the amount of subsarcolemmal densities, myofibril-membrane associations, and amount of junctional membrane folding.     

Carbonic anhydrase: an ultrastructural study in rat cerebellum

Summary The cellular and intracellular distribution of carbonic anhydrase isozyme II in rat cerebellum has been investigated with the electron microscope by the indirect antibody immunohistochemical technique. Unequivocal evidence is presented supporting the view that this enzyme is exclusively localized in oligodendrocytes. Myelin does not appear to contain detectable amounts of carbonic anhydrase though it is present in oligodendrocyte processes and in the layer of oligodendrocyte cytoplasm frequently seen to coat the external surface of myelinated fibres. The immune precipitate is found to be confined to the cytosol and the cytosolic surfaces of intracellular membranes. The data are discussed in relation to the possible function of the enzyme and the role of oligodendrocytes in the central nervous system.     

Myonexin: an 80-kDa glycoprotein that binds fibronectin and is located at embryonic myotendinous junctions

The distribution and function of an 80-kDa glycoprotein located at the surface of skeletal muscle cells and enriched in gelatin-binding fractions of skeletal muscle extracts are examined in the present study. The glycoprotein was purified by concanavalin A affinity chromatography followed by gel filtration and anion exchange chromatography. The purified protein did not display gelatin-binding although the protein bound to fibronectin in several assays. First, the glycoprotein bound to fibronectin-Sepharose and did not elute in high salt buffers although subsequent basic elutions displaced the 80-kDa protein from the column. Second, gel filtration of the 80-kDa glycoprotein in the presence of fibronectin showed separate peaks corresponding to the mass of the 80-kDa glycoprotein and fibronectin as well as a third, higher mass peak shown in immunoblots to contain both fibronectin and the 80-kDa glycoprotein. Third, immunoprecipitation with affinity-purified anti-80-kDa glycoprotein in the presence of the glycoprotein and radioiodinated fibronectin precipitated labeled fibronectin. The quantity of labeled fibronectin precipitated was reduced by the addition of nonradiolabeled fibronectin. Immunofluorescent microscopy using affinity-purified, anti-80-kDa showed this protein located at the myotendinous junctions of frog tadpoles and embryonic chicks. In chicks, it was discernible by immunofluorescence only during the morphogenetic stages that myotendinous junctions were being assembled. Amino acid analysis shows that the 80-kDa glycoprotein has a high concentration of acidic residues. There is only one cysteine per molecule in the 80-kDa glycoprotein and comparisons of reducing and nonreducing gels show that no disulfides are present, indicating that this is not an integrin protein. Amino terminal sequencing reveals that the protein contains marked similarity to the amino terminal of calsequestrin although the protein is distinct from calsequestrin in lacking Ca2(+)-dependent phenyl sepharose affinity and in its molecular weight and distribution. The observations indicate that the 80-kDa glycoprotein is a fibronectin receptor present at chick myotendinous junctions during junction morphogenesis. This apparently novel protein is named "myonexin" to reflect its location and likely function in attaching fibronectin to the surface of muscle cells.     

Isolated pancreatic islets of the rat: an ultrastructural study.

Pancreatic islets from adult Wistar rats were isolated by an improved collagenase digestion technique. Examination of the preparations showed that they contained B cells possessing secretory granules, each having an eccentric electron-dense core surrounded by an electron-lucent halo; the Golgi bodies with their characteristic features were located in a juxtanuclear position. Roughly surfaced endoplasmic reticulum and mitochondria were present and were mostly normal in appearance. The cell possessed all the ultrastructural attributes indicating that they were fully functional and structurally intact.     

Plasma protein changes in horse after prolonged physical exercise: a proteomic study

Physical exercise induces various stress responses and metabolic adaptations that have not yet been completely elucidated. Novel biomarkers are needed in sport veterinary medicine to monitor training levels and to detect subclinical conditions that can develop into exercise-related diseases. In this study, protein modifications in horse plasma induced by prolonged, aerobic physical exercise were investigated by using a proteomic approach based on 2-DE and combined mass spectrometry procedures. Thirty-eight protein spots, associated with expression products of 13 genes, showed significant quantitative changes; spots identified as membrane Cu amine oxidase, α-1 antitrypsin, α-1 antitrypsin-related protein, caeruloplasmin, α-2 macroglobulin and complement factor C4 were augmented in relative abundance after the race, while haptoglobin β chain, apolipoprotein A-I, transthyretin, retinol binding protein 4, fibrinogen γ chain, complement factor B and albumin fragments were reduced. These results indicate that prolonged physical exercise affects plasma proteins involved in pathways related to inflammation, coagulation, immune modulation, oxidant/antioxidant activity and cellular and vascular damage, with consequent effects on whole horse metabolism.     

Physiological activity-dependent ultrastructural plasticity in normal adult rat neuromuscular junctions

The major finding of the present study is that the ultrastructural organization of the neuromuscular synapse can be modified by a small, 4-week-long, physiological increase in the locomotor activity of the extensor digitorum longus muscle of normal adult rats trained to walk. This study measures these plastic adaptations using several synaptic morphological parameters. The observed changes in neuromuscular junctions affect both pre- and postsynaptic membranes. In particular, the presynaptic membrane densities in the active zones and the postsynaptic adaxonal membrane densities become larger, which shows that in the normal adult mammal neuromuscular junction, there is an activity-dependent modulation of the neurotransmission-related structures in response to slight physiologic functional demands. The nature and magnitude of these changes are discussed.     

Myomuscular junctions in the gill-sac muscle of the Atlantic hagfish,Myxine glutinosa: analogy with myotendinous junctions

Summary Myomuscular junctions between muscle fibers in the gill sacs of the Atlantic hagfish, Myxine glutinosa, were examined by electron microscopy. According to the presence of sarcolemmal differentiations typical of myotendinous junctions, the myomuscular junctions can be described as a symmetric myotendinous junction     

Postnatal development of the vomeronasal epithelium in the rat: an ultrastructural study

Three basic types of cells are distinguished in the rat vomeronasal epithelium at birth: bipolar neurons, supporting cells, and basal cells. Neurons at this time include both immature and differentiated cells. By the end of the first postnatal week, all neurons show morphological signs of maturity in their cytoplasm, including abundant granular and smooth endoplasmic reticulum, neurotubules, dense lamellar bodies, apical centrioles, and tufts of microvilli. During the third week microvilli are more frequently encountered and appear to be longer and more branched. Supporting cells appear well-developed by the second day after birth. During the first ten days of life, supporting cells lose their centrioles and all of the complex associated with ciliary generation in the apical zone. Basal cells appear to be more numerous in newborns than in older animals. Protrusions projecting into the lumen are frequently observed in the epithelium of newborn animals, both on the dendrites of neurons and on supporting cells. After the third week, such protrusions are only observed in the transitional zone between the sensory and the non-sensory epithelia of the vomeronasal tubes. In this transitional zone, a fourth cell type showing apical protrusions with microvilli differentiates. Cytoplasm in this type resembles that of neighboring ciliated cells but has no cilia or centrioles. These transitional cells are considered to be cells in an intermediate state of differentiation, between that of the differentiated neurons and supporting cells of the sensory epithelium and that of the predominate ciliated cells of the non-sensory epithelium. The results suggest that by the end of the third week the vomeronasal epithelium is morphologically mature.     

Ultrastructural characteristics of rat neuromuscular junctions after physical load

It is interesting to ascertain the adaptive reaction of rat neuromuscular junctions (NMJ) of muscle fibers of different types to a chronic physical load. We examined ultrastructural changes in NMJ following both static load (pre- and postnatal ontogenesis of Wistar rats till a 2 month age took place under a constant rotation on the centrifuge at hypergravity conditions 2G), and after three kinds of dynamic loads (1/run on treadmill with a speed 35 m/min for 6 wks, 10-60 min/day; 2/swimmings, each 10 hrs/day for 10 days; 3/strength exercises on a vertical treadmill with load for 6 wks). Differences in NMJ reaction of muscle fibers of the same type to various loads were established. A low secretory activity of axonal terminals of type I muscle fibers of m. soleus was shown after the static load. The dynamic load (run) is accompanied with a high secretory activity of axonal terminals in m. soleus type I muscle fibers and of some axonal terminals of m. quadriceps femoris IIB type muscle fibers after strength exercises; the secretory activity of axonal terminals of m. quadriceps femoris IIA and IIB types muscle fibers is expressed in a lesser degree after swimming. The NMJ ultrastructure remodelling (terminal renewal) of type I muscle fibers of the 2 month old control rats increases after static and dynamic (run) loads. Some correlations between different kinds of physical load, muscle fiber type and the degree of NMJ ultrastructure transformation have been shown.     

Effects of physical training on rat myocardium. An enzymatic and ultrastructural morphometric study

Rats subjected to physical training through swimming increased their weight at a slower rate than controls, which initially had the same characteristics. The ratio heart weight/body weight was 23% greater in the trained rats. However, the absolute weights of the hearts were only 7% greater. The ultrastructural morphometric study, backed up by and analysis of the hierarchical variance, did not reveal significant changes neither in the myofibrillar and mitochondrial volume nor in the number of mitochondria per surface unit of myocardium. Furthermore, no variations were recorded, due to training, in the amount of mitochondrial protein nor in the specific mitochondrial activities of malate dehydrogenase, cytochrome c oxidase and ATPase. It is therefore suggested that the increase in the measured parameters, due to training, is proportional to the increase in weight and size of the heart. On the other hand, the specific activity of LDH increased by 15% after the first weeks of training.     

Corneal glycoconjugates: an ultrastructural lectin--gold study

The oligosaccharide chains of cell surface and extracellular matrix glycoconjugates are essential for the biological properties of these molecules. We have, therefore, investigated carbohydrate residues in the rat cornea using biotinylated lectin--gold probes. Fixed corneas were removed and embedded in Lowicryl HM20 or LR White. Ultrathin sections were incubated in one of the lectins: Triticum vulgare (WGA), Canavalia ensiformis (Con A), Griffonia simplicifolia (GS-1), Limax flavus (LFA) and Allomyrina dichotoma (Allo A), followed by streptavidin--gold, or the sections were incubated in cationic colloidal gold. Semi-quantification of gold labelling was determined for corneal endothelium, Descemet's membrane, stroma and epithelium from electron micrographs. WGA and Con A binding sites were expressed either moderately or strongly through out the cornea, suggesting a preponderance of alpha-mannose and N-acetylglucosamine residues. A particular concentration of these sugars was found in Descemet's membrane. In contrast, GS-1 (specific for alpha-galactose) and Allo A (specific for beta-galactose) labelled all regions weakly. Sialic acid residues, as defined by LFA labelling and the expression of neuraminidase-sensitive cationic colloidal gold binding sites, were sparsely distributed throughout the stroma, Descemet's membrane and endothelium. In contrast, sialoglycoconjugates were found in significant concentrations in the epithelium. Electron microscopy proved useful in providing new information on the cellular and subcellular localization of these lectin binding sites. © Chapman & Hall     

Uptake of plasma triacylglycerol by a muscular artery in the rat: an ultrastructural study

The uptake of plasma triacylglycerol by the dorsalis pedis artery in the rat was studied using intravenous infusion of an emulsion of triacylglycerol at a rate of 2.3 mumol per min for 1.5 or 5 h. Electron microscopy revealed lipid droplets in the arterial lumen near the endothelium and in the medial smooth muscle cells (SMC), but not in the endothelial cells or in the extracellular space. Lamellar structures with a periodicity of 40 A developed in the arterial tissue when glutaraldehyde-fixed specimens were incubated at +25 degrees C before postfixation in osmium. Lamellae were present at the luminal and basal surfaces and within endothelial cells, and also in the medial extracellular space associated with the plasma membrane of SMC, in the intracellular channels and near and inside the mitochondria of the medial SMC. No lipid droplets or lamellae were found in the arterial tissue of the control rats. The findings indicate that plasma triacylglycerol is not taken up by the arterial tissue as intact lipid particles, but that these are hydrolyzed at the luminal surface of the endothelium, the lipolytic products then being transferred to the medial SMC for re-esterification and storage in the form of triacylglycerol. The lamellar structures found in the fixed and incubated arterial tissue are thought to represent fatty acids produced by the lipolysis of triacylglycerol during incubation, and we suggest that the transport of fatty acids from the arterial lumen to the medial SMC occurs by lateral movement in a continuum of cell membranes.     

Localization of the loosely bound nuclear proteins of rat brain: an immunocytochemical ultrastructural study

Antibodies against the loosely bound subnuclear protein fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the ultrastructural distribution of the antigenic determinants in rat cerebellum was studied using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. A localization restricted to the nucleus was observed. Both neuronal and neuroglial nuclei exhibited antigens, whereas nuclei of pericytes and endothelial cells did not. The immunoreaction product was homogeneously distributed in dispersed chromatin and was absent from condensed chromatin, suggesting that the antigens were confined to the active regions of the genoma. The outer nuclear membrane and the nucleolus appeared to be free of the antigens, while a perinucleolar ring of immunoreaction was detectable. Liver preparations showed a nuclear reaction markedly weaker than the one for brain nuclei. Adsorption of the serum with isolated liver nuclei nullified the reactivity in liver tissue, whereas a sharp reaction was still observed in the cerebellum, indicating the subcellular reaction under examination to contain antigens specifically concentrated in the nervous system or unique to the brain.     

Innervation of abdominal paraganglia: an ultrastructural study

Abdominal extra-adrenal chromaffin tissue, or paraganglia, was examined at the ultrastructural level to elucidate the innervation of this adrenal medullary homologue. Paraganglia display unmyelinated nerve fibers surrounded by Schwann cell cytoplasm. These nerves are separated from the paraganglion Type I (granule-containing) cells by cytoplasmic projections of paraganglion Type II (satellite) cells. However, serial sections show that the nerves eventually make synaptic contact with the Type I cell. At the axon-chromaffin cell junction, only the outer aspect of the nerve is covered by the satellite cell. The presynaptic endings contain numerous synaptic vesicles, mitochondria and glycogen particles. The vesicles are predominantly of the clear-cored variety, but a few possess centers which are elecron opaque. The pre- and postsynaptic membranes are separated bya subsynaptic space and occasionally exhibit the membranal densities usually associated with synaptic areas. These ultrastructural studies establish definite evidence that abdominal paraganglion cells are innervated.     

Dynamics of DNA replication: an ultrastructural study

DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.     

An ultrastructural and electrophysiological study of the development of neuro-muscular junctions between previously dissociated foetal rat cells in vitro

Summary The development of neuro-muscular junctions between previously dissociated foetal rat spinal cord and somatic muscle has been investigated. The first indications of junction formation, both ultrastructurally and electrophysiologically, were observed after circa 18 days in vitro. The junctions contained numerous vesicles, but no secondary folds were developed even after 6 weeks in culture, and synaptic densities were not well marked. Functional endplates were found, and action potentials, endplate potentials and miniature endplate potentials recorded.The authors wish to thank Mr. D. Fraser, B. Sc., for valued technical help, and Mr. S. Waterman for photographic printing.