Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells

A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0°C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.     

THE FINE STRUCTURE OF THE NUCLEOLUS DURING MITOSIS IN THE GRASSHOPPER NEUROBLAST CELL

The behavior of the nucleolus during mitosis was studied by electron microscopy in neuroblast cells of the grasshopper embryo, Chortophaga viridifasciata. Living neuroblast cells were observed in the light microscope, and their mitotic stages were identified and recorded. The cells were fixed and embedded; alternate thick and thin sections were made for light and electron microscopy. The interphase nucleolus consists of two fine structural components arranged in separate zones. Concentrations of 150 A granules form a dense peripheral zone, while the central regions are composed of a homogeneous background substance. Observations show that nucleolar dissolution in prophase occurs in two steps with a preliminary loss of the background substance followed by a dispersal of the granules. Nucleolar material reappears at anaphase as small clumps or layers at the chromosome surfaces. These later form into definite bodies, which disappear as the nucleolus grows in telophase. Evidence suggests both a collecting and a synthesizing role for the nucleolus-associated chromatin. The final, mature nucleolar form is produced by a rearrangement of the fine structural components and an increase in their mass.     

An electron microscopic study of intravascular agglutination in the cerebral cortex due to Babesia argentina infection

The progressive changes in brain capillaries of calves infected with Babesia argentina are described. Initially, the parasitaemia was low (about 5 per cent) and infected red cells had an essentially normal appearance. Terminally, the parasitaemia was > 90 per cent and by this time infected cells were stellate in appearance, connected by fine strands to other infected cells, and to the capillary endothelium which also showed pathological changes. Uninfected cells appeared normal at all stages of the infection. In severe cases most infected cells were haemolysed. The presumed stickiness of infected cells was probably due to changes in the erythrocyte membrane. Parasites were larger than normal and usually possessed two or three nuclei, containing aggregations of chromatin.     

Chemically induced carcinogenesis affecting chromatin structure in rat hepatocarcinoma cells

A new, chemically induced animal tumor cell line (HeDe) was established and characterized by its property of causing aggressively growing tumors in specific strain of rats and changes in the chromatin structure. Results show that (1) the nuclear material in nuclei of normal resting (G0) hepatocytes consists mainly of decondensed veil-like chromatin, chromosomes being clustered in six lobular domains; (2) nuclei of HeDe cells contain primarily supercoiled chromatin; or (3) the nuclear material of tumor cells undergoes apoptosis seen as apoptotic bodies. Heterogeneity of chromatin structures was expressed as contour/area ratio and was nine times higher in apoptotic cells and two times higher in tumor cells compared to resting cells.     

Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO4) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with glutaraldehyde only, but seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO4 increases UAc uptake by a factor of about 3. The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO4; similar levels for final PTA uptake are also observed. An increase in the resin content of embedded chromatin postfixed with OsO4 is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

Structural and ultrastructural aspects of ontogenesis and differentiation of resin secretory cavities in Hymenaea stigonocarpa (Fabaceae‐Caesalpinioideae) leaves

Cell lysis in the formation of secretory cavities in plants has been questioned by some authors and considered as result of technical artifacts. To describe the formation of secretory resin cavities in Hymenaea stigonocarpa leaves, leaflet samples at different stages of differentiation were collected, fixed, and processed for light and electron microscopy as per usual methods. The initial cells of secretory resin cavities are protodermal and grow towards the mesophyll ground meristem; these cells then divide producing cell groups that are distinguished by the shape and arrangement of cytoplasm, and density. At the initial stages of differentiation of the secretory cavities, some central cells in these groups show dark cytoplasm and condensed nuclear chromatin. Later, there is cell wall loosening, tonoplast and plasmalemma rupture resulting in cell death. These cells, however, maintain organelle integrity until lysis, when the cell wall degrades and the plasmalemma ruptures, releasing protoplast residues, marked characteristics of programmed cell death. The secretory epithelium remains active until complete leaf expansion when the cavity is filled with resin and the secretory activity ceases. There are no wall residues between central cells in adult cavities. Our results demonstrate lysigeny and the importance of ontogenetic studies in determining the origin of secretory cavities.     

Observations on the Kinetics of Uranyl Acetate and Phosphotungstic Acid Staining of Chromatin in thin Sections for Electron Microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO₄) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with gutaraldehyde only, but stain uptake is reduced following a long wash with distilled water to a level similar to that seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO₄ increases UAc uptake by a factor of about 3.

The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO₄; similar levels for final PTA uptake are also observed.

An increase in the resin content of embedded chromatin postfixed with OsO₄ is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

RuBP carboxylase determination by enzymic estimation of D-3-PGA formed

RuBPcarboxylase activity was measured in extracts of barley (Hordeum Vulgare L., cv. HOP) seedlings both with the standard radiometric method and by measuring D-3-phosphoglyceric acid formed enzymically in a two stage assay. In the different conditions used, characterized by different NaHCO₃ concentrations, different pH and the presence and absence of oxygen, essentially the same ratio of D-3-PGA formed per ¹⁴CO₂ fixed was obtained. This ratio respected the known stoichiometry of two molecules of D-3-PGA formed per CO₂ fixed. It is suggested that measurement of D-3-PGA enzymically in a two stage assay can be routinely used for the determination of RuBP case activity instead of the radiometric method. The advantages and the validity of the method are discussed.     

Levels of dinoflagellate chromosome-bound metals in conditions of low external ion availability: An X-ray microanalytical study

Cells of the dinoflagellate Glenodinium foliaceum were able to grow and divide in artificial (AE50) liquid culture medium diluted down to 10% of its normal concentration. X-ray microanalysis, with on-line computation of mass fractions, was used to determine the quantitative occurrence of bound (insoluble) elements in the chromosomes of cells grown in normal, 30 and 10 % medium. In normal medium, chromosomes contained high levels of phosphorus (nucleic acid), sulphur (protein) and a variety of bound divalent metals, including Ca, Fe, Ni, Cu and Zn. With a decrease in the level of external ions (30 and 10% dilutions), the level of sulphur showed a significant decrease, but the mass fractions of insoluble phosphorus and total divalent metals did not fall. The presence of these metals in dinoflagellate chromatin does not, therefore, simply involve intracellular deposition in response to high external levels. The occurrence of divalent metals showed variation with medium concentration in terms of the proportions of different metals present, the nature of the metal-nucleoprotein associations, the metal concentrations per unit mass of chromatin and the number of metal atoms per 100 nucleotides. None of the above characteristics is therefore constant in an individual species.     

Apoptotic agents inducing genotoxicity-specific chromatin changes

To visualize characteristic chromatin distortions we have distinguished first among regularly occurring intermediates of chromatin structures in mammalian (Indian muntjac, CHO, murine preB, rat liver, rat myeloid leukemia, K562 human erythroid leukemia) and Drosophila nuclei. Fluorescence microscopy of chromatin structures isolated from nuclei of reversibly permeable cells revealed a common pathway of chromatin condensation in mammalian cells. Different intermediates in mammalian and Drosophila cells indicate alternative mechanisms of chromosome condensation. Genotoxic agents such as irradiation (alpha, gamma, UV-B) and heavy metals (Cd, Pb, Ni, Hg, Ag) caused alterations in chromatin structures leading to apoptosis. Injury-specific chromatin changes manifested at significantly lower concentrations than non-specific signs of cellular toxicity, suggesting that preapoptotic events are useful indicators of genotoxicity.     

Diffuse kinetochores and holokinetic anaphase chromatin movement during mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20

Mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20 was examined by light microscopy of living and fixed cells. During early prometaphase the numerous small (0.30-3.0-microns) chromosomes appear as discrete units that lack a primary constriction. However, by late prometaphase the chromosomes are tightly packed at the spindle equator and are no longer clearly resolvable as individuals. When viewed from the side the metaphase chromatin appears as a 2-3-microns wide band that spans the width of the spindle; when viewed from the pole it appears as a fenestrated disk. The metaphase chromatin splits at anaphase into two sister chromatin plates, each of which exhibits holokinetic poleward movement, i.e., all parts of each plate move as a single unit with the same velocity. In many early-to-mild anaphase cells the separating sister plates are connected by chromatin-containing bridges that break as anaphase progresses. Ultrastructural analyses of serial thick and thin sections from cells fixed by conventional, OsO4/KFeCN, or high pressure rapid freezing methods, reveal that by metaphase all of the chromosomes are interconnected to form a large, irregularly shaped fenestrated disk of chromatin. Similar analyses reveal that adjacent chromatids remain interconnected throughout anaphase. Each disk of metaphase and anaphase chromatin contains numerous kinetochores recessed within its pole-facing surface. Kinetochores consist of a fine, faintly staining fibrillar material arranged along the chromatin surface as thin (0.1-0.3 micron dia.) rods varying considerably (0.15-2.3 microns) in length. From these observations we conclude that the polycentric metaphase chromatin of A. constricta, and its holokinetic behavior during anaphase, arises from the aggregation or cohesion of smaller prometaphase chromosomes, each of which contains a single, diffuse kinetochore.     

Direct Visualization of a Protein Nuclear Architecture

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.     

PRODUCTS OF THE OXIDATION OF THIOSULFATE BY BACTERIA IN MINERAL MEDIA

Various cultures (previously described), which oxidize thiosulfate in mineral media have been studied in an attempt to determine the products of oxidation. The transformation of sodium thiosulfate by Cultures B, T, and K yields sodium tetrathionate and sodium hydroxide; secondary chemical reactions result in the accumulation of some tri- and pentathionates, sulfate, and elemental sulfur. As a result of the initial reaction, the pH increases; the secondary reactions cause a drop in pH after this initial rise. The primary reaction yields much less energy than the reactions effected by autotrophic bacteria. No significant amounts of assimilated organic carbon were detected in media supporting representatives of these cultures. It is concluded that they are heterotrophic bacteria. Th. novellus oxidizes sodium thiosulfate to sodium sulfate and sulfuric acid; the pH drops progressively with growth and oxidation. Carbon assimilation typical of autotrophic bacteria was detected; the ratio of sulfate-sulfur formed to carbon assimilated was 56:1. It is calculated that 5.1 per cent of the energy yielded by the oxidation of thiosulfate is accounted for in the organic cell substance synthesized from inorganic materials. This organism is a facultative autotroph. The products of oxidation of sodium thiosulfate by Th. thioparus are sodium sulfate, sulfuric acid, and elemental sulfur; the ratio of sulfate sulfur to elemental sulfur is 3 to 2. The pH decreases during growth and oxidation. The elemental sulfur is produced by the primary reaction and is not a product of secondary chemical changes. The bacterium synthesizes organic compounds from mineral substances during growth. The ratio of thiosulfate-sulfur oxidized to carbon assimilated was 125:1, with 4.7 per cent of the energy of oxidation recovered as organic cell substance. This bacterium is a strict autotroph.     

Fine Structure of Botrytis fabae Sardina Conidia

The fine structure of Botrytis fabae conidia was studied usinga variety of electron-microscope techniques. The spore walllacks conspicuous ornamentation and consists of microfibrilsembedded in a granular matrix. The two distinct wall layersseen in chemically fixed sections cannot be detected in cross-fracturedreplicas; the two layers are probably structurally similar.The outer surface of the plasmalemma is covered with branchedinvaginations and two kinds of particles. Three distinct typesof particles are present on the inner surface of the plasmalemma.In freeze-etched replicas nuclei, vacuoles, and other organellesalways appear smoothly rounded. Small vesicles pass throughthe plasmalemma into the cell wall. Particles approximately10 nm in diameter occur in compact rows on the cristae of cross-fracturedmitochondria: dense spherical particles, probably of calciumphosphate, are present in chemically fixed mitochondria. Prevacuolesand vesicles with membranous inclusions can be seen in bothcross-fractured replicas and chemically fixed sections. In cross-fracturedreplicas vacuoles and lipid bodies are frequently joined bystrands of endoplasmic reticulum.     

Electron microscopic identification of sex chromatin bodies of tissue culture cells

Summary A method is described for the unequivocal identification of sex chromatin bodies in electron micrographs of thin sections of tissue culture cells derived from human skin. Fibers, rodlets, and circular profiles having a similar diameter of about 200 Å appear to be the only components of the sex chromatin bodies. The fibers and rodlets are sometimes resolvable into two similar subunits and the circular profiles often have a less electron dense center. The overall density of sex chromatin bodies is found to be considerably less than that of metaphase chromosomes. The fibers of the sex chromatin bodies found lying away from the nuclear periphery appear to be organized more compactly than the fibers of the sex chromatin bodies found lying at the nuclear periphery.This paper is based on investigations supported by a research grant No. GM-04738 from the National Institutes of Health, Public Health Service, to Dr. H. Ris, Department of Zoology, University of Wisconsin.     

A Comparison of Low-Temperature and Ambient-Temperature SEM for Viewing Nematode Faces

Faces of lesion nematodes Pratylenchus teres (populations RTB and JK) and P. zeae or the bacterivore Distolabrellus veechi were observed on frozen specimens with low-temperature scanning electron microscopy and as chemically fixed, critical-point dried specimens with conventional scanning electron microscopy. Amphidial secretions were preserved in chemically fixed but not cryofixed lesion nematodes. Overhanging liplets of chemically fixed D. veechi may be artifactual because they appeared as variably filled, mostly empty membranes when cryofixed. The diagnostically useful lips of the frozen lesion nematodes exhibited six sectors of variable prominence that were absent in chemically fixed specimens. This variability may be due to different degrees of muscle contraction captured during cryofixation, which occurs in milliseconds. This is the first evidence that rarely observed lip sectors in Pratylenchus may be something other than an artifact of shrinkage.     

FINE STRUCTURE OF THE PHOTOSYNTHETIC BACTERIUM RHODOMICROBIUM VANNIELII

The fine structure of the photosynthetic bacterium Rhodomicrobium vannielii was studied by the ultra thin sectioning technique. Cells were fixed in buffered osmium tetroxide and embedded in Epoxy resin. The feature most common to nearly all cells was an array of intracellular membranes situated in a concentric manner at the periphery of the cell. The membranes were mostly paired and quite often five pairs were seen aligned together. Calculations from densitometric tracings showed the average width of a "unit" membrane to be 65 A. Sections of material from disrupted cells after passage through a sucrose gradient revealed vesicular forms composed of membranes similar in width to those in the intact cell. Absorption spectra of both intact cells and isolated membranes were very similar in the bacteriochlorophyll regions. Septa and membranes were demonstrated in the filaments that join mature cells. No evidence for chromatophores was obtained although the methods used were adequate for their demonstration in Rhodospirillum rubrum.     

Visualization of chromatin higher-order structures and dynamics in live cells

Chromatin has highly organized structures in the nucleus, and these higher-order structures are proposed to regulate gene activities and cellular processes. Sequencing-based techniques, such as Hi-C, and fluorescent in situ hybridization (FISH) have revealed a spatial segregation of active and inactive compartments of chromatin, as well as the non-random positioning of chromosomes in the nucleus, respectively. However, regardless of their efficiency in capturing target genomic sites, these techniques are limited to fixed cells. Since chromatin has dynamic structures, live cell imaging techniques are highlighted for their ability to detect conformational changes in chromatin at a specific time point, or to track various arrangements of chromatin through long-term imaging. Given that the imaging approaches to study live cells are dramatically advanced, we recapitulate methods that are widely used to visualize the dynamics of higher-order chromatin structures.