Electron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization.

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60-80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

Asynchronous dynamic changes of intracellular free Ca2+ and possible exocytosis in human tracheal gland cells induced by neutrophil elastase.

Measurements of the intracellular free calcium concentration [Ca2+]i in single cells of the human tracheal gland cell line MM 39 demonstrate dynamic changes in [Ca2+]i after their exposure to human neutrophil elastase (HNE). A heterogeneity in [Ca2+]i responses measured cell to cell in monolayer culture is evident: cells generate an initial [Ca2+]i peak rise with or without a delayed time (up to 180 sec) followed either by a rapid return to baseline, asynchronous oscillations or a sustained plateau phase. From basal concentration of 85 +/- 15 nM, HNE (1 microM) produces a [Ca2+]i increase of 91 +/- 66 nM in about 50% of responding cells. At lower concentrations of HNE (0.1 microM, 0.01 microM), the [Ca2+]i rise remains similar, but only 30-40% of the cells are responding. Pretreatment of cells with the recombinant elafin protein, a specific elastase inhibitor, reduces both the [Ca2+]i response to HNE and the number of responding cells. Electron microscopy observations reveal an increased number of secretory granules located beneath the cell plasma membrane after HNE treatment. These results suggest that intracellular [Ca2+]i changes may be associated to the HNE-induced exocytosis in human tracheal gland cells. These findings could have implications with regard to the pathogenesis of increased mucus secretion in human airway diseases.

Three-dimensional co-location of RNA polymerase I and DNA during interphase and mitosis by confocal microscopy.

The relative three-dimensional co-location of RNA polymerase I (RPI) and DNA was studied using confocal laser scanning microscopy during interphase and all the steps of mitosis in human cancerous cells. For each step of the cell cycle, immunolabeled RPI molecules and DNA specifically stained with chromomycin A3 were simultaneously imaged at high resolution through numerous optical sections. Then, all the data obtained were used to generate transverse sections, anaglyphs and volumic representations, which are all prerequisite approaches to a representative study of the three-dimensional organization of the nucleolus and the mitotic chromosomes. Our results indicated that in the interphasic nuclei, in which DNA is organized as a regular 3-D network, RPI was present within numerous irregular spheres arranged as several twisted necklaces. During metaphase, RPI labeling was segregated into pairs of spheres and typical crescent-shaped structures; both were centrally located within the set of chromosomes. During anaphase and telophase, a typical central and symmetric arrangement of labeled structures was systematically seen among the decondensing chromosomes, arranged as a regular cylinder and as a hollow half-sphere, respectively. This typical 3-D organization of structures containing RPI relative to DNA is another strong example of the non-random organization of the genome during interphase and mitosis.

Three-dimensional co-localization of nucleolar argyrophilic components and DNA in cell nuclei by confocal microscopy.

Silver dots deposited specifically on proteins of the nucleolar organizer regions (Ag-NOR proteins) after a one-step silver staining technique were visualized in cells in culture, in cells in smears, and in tissue sections, with a scanning laser confocal microscope working in the reflectance mode. After specific labeling of DNA with the fluorescent dye chromomycin A3, DNA and silver dots could be observed either individually or simultaneously. Therefore, it was possible to study the three-dimensional organization of nucleolar silver-stained structures relative to DNA with a high X, Y, and Z resolution. Our results showed that the argyrophilic components are organized as a twisted necklace structure within interphase nucleoli of cells in culture. We also demonstrated a striking three-dimensional symmetric disposition of NORs within the two sets of chromosomes in telophase cells. Similar results were obtained for cells in smears, although their three-dimensional organization was somewhat disturbed due to air-drying. We also demonstrated that silver dots cannot be visualized in the reflectance mode within sections of paraffin-embedded tissues. However, their simultaneous demonstration in non-confocal transmitted light, together with that of DNA in confocal mode, appeared very useful to study their localization within nuclei and mitotic chromosomes.

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections.

Scanning transmission electron microscopy at 300kV enables the visualization of nucleolar silver-stained structures within thick sections (3-8 microns) of Epon-embedded cells at high tilt angles (-50 degrees; +50 degrees). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-microns-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e-/A2/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-microns-thick sections with a spot-size of 5.6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 microns. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.

Ultrastructural distribution of DNA within the nucleolus of various animal cell lines or tissues revealed by terminal deoxynucleotidyl transferase.

We have used the highly sensitive in situ terminal deoxynucleotidyl transferase method, applied to ultrathin sections, to investigate the location of DNA within nucleoli of various animal cells. In all the nucleoli studied, intense labelling is revealed over the peri- and intranucleolar condensed chromatin. Gold particles are also consistently found over the fibrillar centres, especially at their periphery, namely in the border area between the fibrillar centres and the dense fibrillar component, whereas the dense fibrillar component itself seems to be free of label in nucleoli in which these two compartments can be distinguished. We conclude that, in transcriptionally active nucleoli of this type, DNA is a characteristic constituent of the fibrillar centres, distinguishing them functionally from the dense fibrillar component. Some nucleoli exhibit neither fibrillar centres nor a dense fibrillar component, but have a single, albeit heterogeneous accumulation of fibrillar material; gold particles are consistently seen over some parts of this fibrillar compartment. This suggests that certain parts of the fibrillar material are functionally similar to the fibrillar centres of those nucleoli that possess them.

Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

Digital three-dimensional visualization of cellular organelles studied by medium- and high-voltage electron microscopy.

Tilted thick sections (one-half to several micrometers) of biological specimens observed with medium- to high-voltage electron microscopes are extremely useful for the study of the three-dimensional (3-D) structure of organelles. If high resolution in 3-D visualization and 3-D reconstruction is needed, many images corresponding to various angles of rotation and tilt must be recorded. This necessitates very time-consuming work--including eventual photographic processing--before good positioning of the object is defined. We have developed software which permits very rapid and precise determination of the tilt-axis, the registration of tilted views, 3-D measurements and 3-D visualization. Images are digitized either from negative films or directly with a camera fitted to the microscope. The application of the software is performed in minutes and allows for a rapid check of the quality of the tilt-series and of the features of interest of the object. Application of the software to the study of the 3-D structure of active components of the nucleolus stained with silver is shown.

Three-dimensional imaging of the mucus secretory process in the cryofixed frog respiratory epithelium.

Using the frog palate as a representative model of human mucociliary epithelium, we analyzed, after quick freezing fixation, the three-dimensional (3-D) respiratory mucus secretory release with high voltage (200-300 kV) transmission electron microscopy (TEM). The 3-D vision of the mucus release from the secretory cells was obtained as stereo-pairs and "bas-relief" images after analysis of stereo-pairs using an image analyzer. After standard glutaraldehyde fixation, the secretory cells showed a typical goblet shape with secretory granules heterogeneous in size and electron-density which often fuse together. On the other hand, quick-frozen secretory cells exhibited a columnar shape and their membrane-bound secretory granules contained a homogeneously dark matrix. The expanded gel mucus layer was preserved and its depth never exceeded 2 microns. When the epithelium was immersed in culture medium in presence of cholinergic agonist, a marked discharge of mucus was observed and the granules swelled at the apex of the secretory cell before being discharged in the lumen. In native cryofixed epithelium, the secretory granules exhibited a marked deformability during the process of their extrusion from the secretory cell. Clusters of secretory granules surrounded by cytoplasmic material were observed in the extracellular lumen, suggesting an apocrine-type secretion. These observations indicate that rapid cryofixation and 3-D stereoscopic imaging enable a unique opportunity to analyze, without artifact, the mucous secretory process. We speculate that, apart from the classical merocrine-type secretion mechanism, the respiratory mucus may be released, at least partly by an apocrine-type secretion.

[Silver staining of nucleolus organizer regions (NORs). Application to the study of nucleolar structure and value in pathology]. / Coloration des organisateurs nucléoraires (NORs) par l'argent. Application à l'étude de la structure du nucléole et intérêts en pathologie.

Nucleolus is the morphologic expression of synthesis and maturation of ribosomal RNA (rRNA) from amplified ribosomal DNA (rDNA). Nucleolar Organizer Regions (NORs) are functional subunits of the nucleolus in which actively transcribed rDNA is surrounded with numerous regulatory proteins. Some of them are argyrophilic non-histone proteins (Ag-NOR proteins). By using a cytochemical reaction based on this argyrophilia, active NORs may be stained by the precipitation, at their level, of metallic silver granules whose quantity is directly related to the nucleolar activity. In the present paper, we described various applications of a silver-staining method we developed in our laboratory. The Ag-NOR proteins were ultrastructurally localized within precise nucleolar components. Moreover by viewing thick-sections of silver-stained cells with high-voltage microscopes we were able to describe the three-dimensional structure of nucleoli. Finally, this silver-staining method may be applied at the optical level, to sections of routinely fixed and paraffin-embedded human tissues. With this simple staining method, it is now possible to study the relationships of the number of nucleolar argyrophilic structures with the diagnostic of neoplasms.

Behaviour of nucleolus during mitosis. A comparative ultrastructural study of various cancerous cell lines using the Ag-NOR staining procedure.

The aim of the present work was to study the distribution and the behaviour of the silver-staining nucleolar organizer region (Ag-NOR) proteins at the ultrastructural level during interphase and mitosis in five human and murine cancerous cell lines each characterized by a typical nucleolar morphology. During interphase the Ag-NOR proteins are restricted to the fibrillar centres (F.C.) and/or to the dense fibrillar component (D.F.C.). During prophase the silver-staining components come into close contact with some chromosomes and are arranged with a typical polarity: chromosome, F.C. and D.F.C. Then F.C. and D.F.C. together form roundish silver-stained structures and integrate in part within indentations at the periphery of the metaphase chromosomes. During anaphase and telophase large and small spherical silver-staining structures may be seen. They correspond respectively to the metaphase NORs and to numerous structures which appear de novo within ribonucleoprotein (RNP) material localized between the chromosomes. During late telophase the number of the small silver-staining structures decreases whereas the size of the larger ones increases. Then the interphase nucleoli recover their typical shape. These results suggest that when rRNA synthesis is impaired during mitosis the inactive NORs assume a structure and a localization which are not typical of the cell line. In contrast the F.C. and D.F.C. are probably two aspects of the NORs whose typical distribution, relative to the other nucleolar components, gives the interphasic nucleolus its characteristic morphology.

The three-dimensional ultrastructure of interphasic and metaphasic nucleolar argyrophilic components studied with high-voltage electron microscopy in thick sections.

The three-dimensional structure of the nucleolar argyrophilic components was studied by recording stereo-pairs of tilted thick sections--0.5-2 microns thick--observed with 200 and 300 kV high-voltage electron microscopy (HVEM). Using a very specific silver staining method, the argyrophilic components were stained with a high contrast relatively to the unstained background, thus allowing their study with a high resolution within thick sections. This study was performed on compact nucleoli (of HL60 and K562 cells), on reticulated nucleoli (of human breast cancerous cells) and on metaphasic nucleolar organizer regions (NORs). In compact nucleoli argyrophilic components show a 'knotted rope-like' structure in which knots are constituted of one central fibrillar centre surrounded at some distance by loops of the dense fibrillar component and in which the rope is constituted of dense fibrillar component. In reticulated nucleoli silver deposits are confined to the surface of the nucleolonema as several strands twisted at the periphery of the fibrillar component. During metaphase some NORs get a characteristic crescent-shaped structure disposed at the periphery of some chromosomes.

Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level.

The argyrophilic proteins of the nucleolar organizer region (Ag-NOR proteins) were specifically localized at the optical level with a modified one-step silver technique performed at 20 degrees C. This method was applied to various materials including cells in smears, chromosomes, semi-thin sections of plastic-embedded cells and sections of paraffin-embedded human pathological tissues. In order to improve the visualization of the silver deposits we tested various modes of imaging, including bright-field, Nomarski contrast, reflected light and combined Nomarski contrast with reflected light. The use of Nomarski contrast is useful to define precisely the phases of mitosis. The use of reflected light, which is based on the ability of silver to reflect incident light specifically, gives images with an improved resolution compared to bright-field.

Simultaneous high resolution localization of Ag-NOR proteins and nucleoproteins in interphasic and mitotic nuclei.

Silver stainable proteins of the Nucleolar Organizer Regions (Ag-NOR proteins) of human breast cancer tissues have been localized at the electron microscopical level with a new method which combines a simple and reproducible one step Ag-NOR staining method combined with an acetylation procedure. This new method allows the fine identification of nucleolar components, particularly those which are stained by silver. In order to determine the cytochemical nature of the components associated with Ag-NOR proteins, the EDTA regressive preferential staining procedure for ribonucleoproteins has been applied to sections. By this means the precise localization of the Ag-NOR proteins was studied simultaneously with that of ribonucleoprotein within interphasic nucleoli and mitotic chromosomes. In interphasic nucleoli, stainable Ag-NOR proteins were localized in fibrillar centres and part of the dense fibrillar component. No silver deposits were seen on perichromatin or interchromatin fibrils and granules. In metaphasic nuclei, Ag-NOR proteins were only found on roundish fibrillar ribonucleoprotein structures, which could correspond to secondary constrictions. No silver deposits were seen on the well defined ribonucleoprotein sheet surrounding the chromosomes. In telophasic nuclei, Ag-NOR proteins were seen on the central part of roundish ribonucleoprotein fibrillar structures integrated in decondensing chromosomes. These structures have been interpreted as the nucleolar organizer regions around which rRNA synthesis resumes. In interphasic and mitotic nuclei, Ag-NOR proteins were never found within condensed chromatin but always in association with ribonucleoprotein components. The new method proposed here appears to be a useful tool for the simultaneous study of the localization of ribonucleoprotein and Ag-NOR proteins during the cell cycle.