Blood vessels and desmin control the positioning of nuclei in skeletal muscle fibers

Skeletal muscle fibers contain hundreds to thousands of nuclei which lie immediately under the plasmalemma and are spaced out along the fiber, except for a small cluster of specialized nuclei at the neuromuscular junction. How the nuclei attain their positions along the fiber is not understood. Here we show that the nuclei are preferentially localized near blood vessels (BV), particularly in slow-twitch, oxidative fibers. Thus, in rat soleus muscle fibers, 81% of the nuclei appear next to BV. Lack of desmin markedly perturbs the distribution of nuclei along the fibers but does not prevent their close association with BV. Consistent with a role for desmin in the spacing of nuclei, we show that denervation affects the organization of desmin filaments as well as the distribution of nuclei. During chronic stimulation of denervated muscles, new BV form, along which muscle nuclei align themselves. We conclude that the positioning of nuclei along muscle fibers is plastic and that BV and desmin intermediate filaments each play a distinct role in the control of this positioning.     

Possibilities for the histochemical study of the nuclei of normal brain cells and in pathology

Possibilities of application of some histochemical methods to studying cell nuclei of brain are reviewed considering the following techniques: hybridization histochemistry on the light and electron microscope levels, immunohistochemistry and immunoelectron microscopy, absorbtion and fluorescence histochemistry of nucleic acids, histones, non-histone proteins of chromatin, and of cell nucleus lipids, electron histochemistry. Besides, some physicochemical and molecular-biological methods are considered. Data on human brain research in the norm and upon various brain disorders are particularly provided.     

Intracellular localization of beryllium by analytical ion microscopy

After injection in the rat of a soluble beryllium salt, the distribution of this element was studied at the subcellular level by analytical ion microscopy. Beryllium is concentrated inside the nuclei with a particular affinity for the nuclei of the proximal tubule cells of the kidney. The same tissue was studied by electron microscopy and abnormal intranuclear inclusions were observed in the same variety of cells.     

Selective localization of neptunium-237 in nuclei of mammalian cells]

After injection in the rat of soluble neptunium salt, the distribution of this element was studied at the subcellular level by electron microscopy and electron probe microanalysis. Abnormal structures have been observed by electron microscopy in the nuclei of hepatocytes, and the same structures have also been observed in the nuclei of the proximal tubules cells of the kidney. These structures are formed of clusters of very small and dense particles, several nanometers in diameter. The clusters are localized in the central part of the nuclei and they are separate from nucleoli and heterochromatin. Electron probe X-ray analysis of this cluster have shown that they contain neptunium associated with phosphorus. In the cell containing neptunium inclusions, other non specific lesions are also observed (nuclear pycnosis, mitochondrial depletion).     

Distribution of serotonin immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit, and rat

Immunohistochemical techniques were employed to study the distribution of serotonin (5-HT) immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit and rat. 5-HT neurons were principally found to be concentrated in the midline raphe nuclei, particularly, the raphe pallidus, raphe obscurus, raphe magnus, raphe median, raphe pontis and raphe dorsalis nuclei. Characteristically, these cell bodies are displayed in bands or wing-like patterns which extend laterally from the raphe into reticular formations. The formations often appear to blend with the catecholamine system. They are particularly evident in the brainstems of the rabbit and hamster which contain wider and more lateral extensions of the serotonergic (5-HT) neurons than those observed in the brainstems of the rat and guinea pig. The widespread distribution of 5-HT immunoreacted cell bodies in the brainstem shows that there are significant prospects of 5-HT in neuronal activities.     

Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp. PCC 7119.

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.     

Flow-induced hardening of endothelial nucleus as an intracellular stress-bearing organelle

The mechanical contribution of nucleus in adherent cells to bearing intracellular stresses remains unclear. In this paper, the effects of fluid shear stress on morphology and elastic properties of endothelial nuclei were investigated. The morphological observation suggested that the nuclei in the cytoplasm were being vertically compressed under static conditions, whereas they were elongated and more compressed with a fluid shear stress of 2 Pa (20 dyn/cm2) onto the cell. The elongated nuclei remained the shape even after they were isolated from the cells. The micropipette aspiration technique on the isolated nuclei revealed that the elastic modulus of elongated nuclei, 0.62+/-0.15 kPa (n=13, mean+/-SD), was significantly higher than that of control nuclei, 0.42+/-0.12 kPa (n=11), suggesting that the nuclei remodeled their structure due to the shear stress. Based of these results and a transmission electron microscopy, a possibility of the nucleus as an intracellular compression-bearing organelle was proposed, which will impact interpretation of stress distribution in adherent cells.     

ELECTRON MICROSCOPY OF BASOPHILIC STRUCTURES OF SOME INVERTEBRATE OOCYTES : II. FINE STRUCTURE OF THE YOLK NUCLEI

RNA containing yolk nuclei from the surf clam Spisula solidissima have been studied with the light microscope and with the electron microscope. A variety of structures can be seen with both and a correlation can be made between bodies studied with the electron microscope and those studied with the light microscope. The electron microscope shows many of these structures to be composed of double walled lamellae arranged in space, in various ways. The variety of patterns seen with the electron microscope can be satisfactorily explained on the basis of a hypothetical model. The presence of yolk platelets within the yolk nuclei lends support to classical observations on the synthesis of yolk within yolk nuclei or yolk nuclei derivatives. Small granules (about 100 A) are included within the double walled lamellae and their presence probably accounts for the basophilic nature of the entire body. The presence of small granules attached to electron-dense layers relates the yolk nuclei described here to ergastoplasm discussed by others.     

Electron microscopy of basophilic structures of some invertebrate oocytes. II. Fine structure of the yolk nuclei

RNA containing yolk nuclei from the surf clam Spisula solidissima have been studied with the light microscope and with the electron microscope. A variety of structures can be seen with both and a correlation can be made between bodies studied with the electron microscope and those studied with the light microscope. The electron microscope shows many of these structures to be composed of double walled lamellae arranged in space, in various ways. The variety of patterns seen with the electron microscope can be satisfactorily explained on the basis of a hypothetical model. The presence of yolk platelets within the yolk nuclei lends support to classical observations on the synthesis of yolk within yolk nuclei or yolk nuclei derivatives. Small granules (about 100 A) are included within the double walled lamellae and their presence probably accounts for the basophilic nature of the entire body. The presence of small granules attached to electron-dense layers relates the yolk nuclei described here to ergastoplasm discussed by others.     

Endopeptidase 24-16 in Murines: Tissue Distribution, Cerebral Regionalization, and Ontogeny

The tissue distribution, cerebral regionalization, and ontogeny of endopeptidase 24-16 were established in murines by means of its quenched fluorimetric substrate, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp, and its selective dipeptide blocker, Pro-Ile. Endopeptidase 24-16 was particularly abundant in the liver and kidney, and the lowest specific activity was detected in the heart. In the brain, a 16-fold difference in specific activity was observed between the poorest and the richest cerebral areas. Endopeptidase 24-16 appeared in high concentrations in the olfactory bulb and tubercule, cingulate cortex, medial striatum, and globus pallidus, and was particularly weak in the CA1, CA2, and CA3 parts of the hippocampal formation and in the cerebellum. Endopeptidase 24-16 content in thirteen thalamic nuclei indicated a rather homogeneous distribution. This homogeneity was not observed in the hypothalamus, where pronounced variations occurred between enriched zones such as suprachiasmatic and arcuate nuclei and relatively poor areas such as periventricular and supraoptic nuclei. Endopeptidase 24-16 appeared to be developmentally regulated in the mouse brain; it was already detected at the fetal stage, increased transiently after birth, then regularly declined until adulthood.     

Intranuclear silicon detection in a subcutaneous connective tissue cell by energy-dispersive x-ray miscroanalysis using fresh air-dried spread.

Silicon was detected by energy-dispersive x-ray microanalysis in the nucleus of a subcutaneous connective tissue cell of mice fed normally. To eliminate contamination, pieces of connective tissue were spread on copper grids and examined without any treatment by an energy-dispersive spectrometer with a scanning transmission apparatus attached to an electron microscope. Scanning transmission electron microscopy of the spread has demonstrated a well-preserved ultrastructure. Fibrous structures, nuclei and nucleoli of cells and mitochondrial granules were recognized. Electron probe analysis showed peaks for silicon at three spots on the nucleus of a cell in addition to those for phosphorus, sulfur, chlorine, potassium and calcium, whereas no peak of silicon could be detected at the spots on nuclei of other cells, mitochondrial granules and electron-lucent area on the same grid as the above. Silicon appears to play a significant role in the nucleus. Applicability of the technique to know the distribution of easily contaminating elements and diffusible substances is shown.     

HISTOCHEMICAL MAPPING OF LACTATE DEHYDROGENASE AND MONOAMINE OXIDASE IN THEMEDULLA OBLONGATA AND CEREBELLUM OF SQUIRREL MONKEY (SAIMIRI SCIUREUS)

Abstract— —The gross distribution of LDH and MAO was studied in a caudo-cranial series of 50 μ thick sections through the medulla oblongata and cerebellum. In general, LDH exhibited a stronger reaction in the neuropil and in the perikarya, whereas MAO showed moderate activity in the neurons and mild to moderate activity in the neuropil. The axonal processes and nerve fibres showed comparatively stronger MAO activity. The nuclei gracilis, cuneatus medialis and lateralis, cranial nerve nuclei, olivaris inferior, vestibularis and cochlearis nuclei showed particularly strong LDH and equally weak MAO activities. The lateral part of the formato reticularis myelencephali showed much more MAO than did the medial part, whereas the LDH reaction was uniformly strong. The reticularis lateralis showed uniformly strong LDH and very mild MAO activities.
In the cerebellar cortex, the MAO activity was concentrated in the molecular layer and nerve fibre layer, whereas LDH activity was particularly strong in the Purkinje cells and their processes in the molecular layer. The cerebellar nuclei showed strong LDH and weak MAO in the neutrons and stronger MAO and moderate LDH in the neuropil.     

Fluorescent labeling of resin-embedded sections for correlative electron microscopy using tomography-based contrast enhancement

Locating areas of interest by electron microscopy can be laborious. This is particularly true for electron tomography, where the use of thicker sections may obscure relevant details in the projection images. We evaluated the applicability of fluorescent probes to thin plastic sections, in combination with fluorescence microscopy, as an aid in selecting areas for subsequent electron microscopic analysis. We show that pre-embedding labeling of DNA and RNA with acridine orange yielded a predominant nuclear stain. The stain greatly reduced the time needed to scan sections for mitotic cells, or cells with characteristic nuclei such as neutrophils. Post-embedding labeling with SYTOX green yielded a nuclear stain comparable to acridine orange, and wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 labeled mucous granules and the Golgi area in intestinal goblet cells. The fluorescent labels were visualized directly on sections on electron microscope grids. It was therefore possible to establish a coordinate system based on the position of the grid bars, allowing for easy retrieval of selected areas. Because the fluorescent probes were incompatible with osmium tetroxide treatment, contrast in the sections was faint. We propose a simplified electron tomography procedure for the generation of 2D views with enhanced contrast and resolution.     

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated wheter or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M_r 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0° C or 37° C. The matrix fraction retained 20–40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37° C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.     

Morphometric and topologic analysis of freeze-fractured interphase nuclei

Interphase rat liver nuclei were studied by freeze fracturing followed by electron microscopic observations. This method permits information on the native organization of the nuclear components in the hydrated state to be obtained. Morphometric analyses, performed with a Leitz Texture Analysis System, gave precise information on the different nuclear components, based on the histograms of their size distribution in heterochromatin, interchromatin and nucleolar areas. The textural characteristics were analyzed by computer to determine the topologic distribution of the solenoid chromatin fibers, the nucleosome filaments and the ribonucleoproteins in the different nuclear domains.     

Autoradiographic localization of cholecystokinin binding sites in human cerebellar system using a [I]CCK8 probe

Carboxyl-terminal cholecystokinin octapeptide (CCK8) binding sites were studied in the human cerebellar system by autoradiography. High affinity CCK8 binding sites were demonstrated in the main cerebellar afferent nuclei, namely the inferior olivary complex and the pontine nuclei. This localization of CCK8 binding sites was partly correlated with already described CCK containing terminals. In the cerebellar cortex, high affinity CCK8 binding sites were detected with a laminar distribution. Levels were higher in the granular layer (mostly in the superficial part) and lower in the white matter and the Purkinje cell layer. The non-specific binding was homogenous and particularly low (9%) in the cerebellar cortex but a non-specific binding was selectively localized in the deep cerebellar nuclei. Those results illustrate the species variability of CCK binding sites in the cerebellum and are briefly discussed in relation with the low level of CCK immunoreactivity in this structure. The presence of CCK8 binding sites in cerebellar afferent nuclei and cortex suggests a role of CCK in human cerebellar physiology and particularly in the modulation of afferent inputs to the cerebellum.     

Hepatocyte ploidy in normal young rat

The aim of the present study was to examine the relation between hepatocyte size and ploidy in Sprague-Dawley rat liver. Therefore, subpopulations of hepatocytes of various sizes were separated from the isolated crude hepatocyte population either mechanically or by using centrifugal elutriation. Hepatocyte size was determined on scanning electron microscopy photographs. Ploidy of hepatocytes was assessed by flow cytometry. The crude hepatocyte population was very heterogeneous in sizes, with diameters ranging from 8 to 39 microm. Hepatocyte ultrastructure was well preserved as demonstrated by transmission electron microscopy. The distribution of hepatocytes within the ploidy classes was the following: 19.6+/-3.6% diploid, 56.2+/-3.2% tetraploid and 3.4+/-0.6% octoploid mononucleated cells. Thus approximately 79% of hepatocytes appeared mononucleated. The binucleated hepatocytes (21%) had two diploid nuclei (18.7+/-2.9%) or two tetraploid nuclei (2.1+/-0.6%). A similar distribution of hepatocytes into ploidy classes was obtained in subpopulations of hepatocytes of various sizes. Our findings suggest that distribution into ploidy classes is not strictly correlated with hepatocyte size. In accordance with previous observations, our results on hepatocyte ploidy from periportal or perivenous origin using digitonin perfusion, is in favour of the existence of ploidy zonation within the rat hepatic lobule.     

Improved visualization of vertebrate nuclear pore complexes by field emission scanning electron microscopy

Field emission scanning electron microscopy (FESEM) can provide high-resolution three-dimensional surface imaging of many biological structures, including nuclear envelopes and nuclear pore complexes (NPCs). For this purpose, it is important to preserve NPCs as close as possible to their native morphology, embedded in undamaged nuclear membranes. We present optimized methodologies for FESEM imaging in a cell-free reconstitution system and for the direct visualization of mammalian cell nuclei. The use of anchored chromatin templates in the cell-free system is particularly advantageous for imaging fragile intermediates inhibited at early stages of assembly. Our new method for exposing the surface of mammalian nuclei results in an unprecedented quality of NPC images, avoiding detergent-induced and physical damage. These new methodologies pave the way for the combined use of FESEM imaging with biochemical and genetic manipulation, in cell-free systems and in mammalian cells.