A further contribution on nucleoli of human lymphocytes.

Cultured human lymphocytes were investigated by means of light as well as electron microscopic procedures to provide more information on the structural organization of their nucleoli. The transformation of ring shaped nucleoli to nucleoli with less or more distinct nucleolonemata in PHA stimulated cells was characterized by a marked increase of granular RNP components in number indicating the activation of their production. This phenomenon seems to be related not only to the activation of the ribosomal RNA synthesis but also to its processing. The appearance of fibrillar RNP components in the central area of the ring shaped nucleoli apparently represents the first sign of the nucleolar RNA synthesis in these cells. The proportion of fibrillar and granular nucleolar RNP comonents in PHA inresponsive lymphocytes was similar to that in lymphocytes from patients with the usual type of lymphocytic leukemia. The intranucleolar chromatin areas appeared to be larger in PHA stimulated lymphocytes but the proportion of these areas to the nucleolar body did not show substantial difference as compared to the resting cells.     

Structural organization of chromatin in nucleolar organizer regions of nucleoli with a nucleolonema-like and compact ribonucleoprotein distribution

We have studied the relationship between the structural organization of intranucleolar chromatin and fibrillar nucleolar structures, fibrillar centers, and RNP fibrillar component, which are the interphase counterpart of metaphase nucleolar organizer regions (NORs), in regenerating rat hepatocytes and in a human tumor cell line (TG cells). These two cell types were characterized by a nucleolonema-like and compact nucleolar RNP distribution, respectively. We found that, in sections selectively stained for DNA, the intranucleolar chromatin composed of extended, nonnucleosomal DNA filaments formed roundish agglomerates with a spatial distribution which was superimposable on that of the fibrillar centers and the RNP fibrillar component around them and on sites of the silver reaction in samples selectively stained for Ag-NOR proteins. The agglomerates of extended nonnucleosomal DNA filaments were small and numerous in regenerating hepatocyte nucleoli, in which the RNP components had a nucleolonema-like distribution, whereas they were large and few in TG cell nucleoli, in which the RNP components showed a compact organization. Since the pattern of ribosomal RNA synthesis and processing was similar in the two cell types, a model was proposed in which the difference in size and shape of the agglomerates of extended DNA might be responsible for the different structural organization of the RNP components.     

Nucleolus activation in pea cotyledonary buds during 24 hours after decapitation of the main stem: Cytochemical studies

Summary Several cytochemical techniques and routine electron microscopy were used to detect changes in the pea cotyledonary bud nucleolus, from the G_0–1 resting state until the first mitoses (24th hour) following removal of the main stem. In the G_0–1 nucleus the fibrillar RNP nucleolus, shaped like a hollow sphere, possessed clumps of DNA connected to the compact peripheral nuclear chromatin by DNA fibres. The large central space, surrounded by the fibrillar component, showed the characteristics of a heterogeneous fibrillar center. By the 6th hour, this heterogeneous fibrillar center broke up, into small scattered elements in which DNA was detected. Between the 9th and 12th hours, RNA maturation produced a granular zone. Inter- and peri-chromatin granules of RNP were then present. In the bud, which was either in the resting state or in the process of reactivation, Ag-NOR proteins were located throughout the nucleolar mass, but were heavily concentrated in its peripheral region; they constituted a binding network between the various nucleolar components. The role and importance of the G_0–1 nuclei karyosome, present until the 9th hour, is discussed.     

LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HEPATIC CELL NUCLEOLI IN MICE TREATED WITH ACTINOMYCIN D

Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology was studied when actinomycin D treatment preceded labeling with tritiated orotic acid. Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible if a dosage of 25 µg of actinomycin D was used, but nucleolar RNA was still synthesized. After a dosage of 400 µg of actinomycin D, nucleolar RNA synthesis was completely stopped If labeling with tritiated orotic acid preceded treatment with 400 µg of actinomycin D, labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component. At 30 min after actinomicyn D treatment all labeling was lost. Since labeling was associated with the granular component the progressive loss of label as a result of actinomycin D treatment indicated a release of nucleolar granules. The correlation between this release and the loss of 28S RNA from actinomycin D treated nucleoli as described in the literature is discussed.     

EFFECT OF TEMPERATURE OF ALDEHYDE FIXATION ON THE RADIOAUTOGRAPHIC LOCALIZATION OF RIBONUCLEOPROTEIN IN NUCLEOLI OF HELA CELLS : Inhibition by Puromycin and Actinomycin D

In efforts to clarify the role of the nucleolus and substructures thereof in the assembly or synthesis of protein associated with formation of the complete ribosome, the effect of variation of some conditions of aldehyde fixation on the intranuclear distribution of lysine-³H, arginine-³H, and uridine-³H was studied by differential grain count in radioautographs of PPLO-free HeLa cells. It was found that the nucleolus is a site of rapid assembly or synthesis of a protein, the synthesis of which is inhibited equally by puromycin (200 µg/ml) and by actinomycin D under conditions inhibitory for ribosomal precursor RNA synthesis (P < 0.01). This protein is fixed by phosphate-buffered formalin or glutaraldehyde at pH 7.3, but the label is diminished by fixation in customarily employed acetic ethanol or in formalin at acid pH. Elevation of temperature of formalin or glutaraldehyde fixatives to 37°C consistently reduces the nucleolar protein label, but not the RNA label, by a proportion identical with that incurred by puromycin or actinomycin inhibition. This proportional reduction of nucleolar protein label occurs without evident loss of total grain count and is independent of length of fixation between 30 min and 4 hr, but it is not observed at 23°C. The data support the interpretation that the proportion of nucleolar protein not fixed at 37°C is associated with nucleolar ribosomal RNA but that it is dissociated at 37°C in formalin or glutaraldehyde fixatives, probably on the basis of ionic dissociation of a conjugated ribonucleoprotein.     

Intranucleolar visualization of nucleic acids and acidic proteins in inhibited and reactivated pea cotyledonary buds

Summary Nuclease-colloidal gold complexes and silver staining were used to visualize intranucleolar nucleic acids and argyrophilic proteins of the nucleolar organizers in bud cotyledonary cells ofPisum sativum. In the G_0–1 inhibited bud, a few RNA molecules were detected in the fibrillar component and in the unique fibrillar centre, close to the boundary with the fibrillar component of the nucleolus. DNA was present in the fibrillar component, in the fibrillar centre and in a few fibres crossing the perinucleolar halo. The acidic proteins were localized at the periphery of the fibrillar component but they were also present in the unique fibrillar centre. In the reactivated bud, RNA was particularly concentrated in the granular component and along fibres crossing the perinucleolar halo; a few RNA molecules were also detected at the boundary between the small fibrillar centres and the fibrillar component. DNA was localized in the same nucleolar component as in the inhibited bud, but it was distributed between several fibrillar centres. Acidic proteins coated these DNA loci. In the inhibited and reactivated bud connections between nucleolar DNA containing structures were displayed. The data are discussed in relation to the present knowledge of the functional architecture of the nucleolus.Abbreviations DNA deoxyribonucleic acid - DNase deoxyribonuclease - G_0–1 phase G₁ phase of the cell cycle indefinitely prolonged - PEG polyethylene glycol - RNA ribonucleic acid - RNase ribonuclease - S and G₂ phases synthetic and postsynthetic phases of the cell cycle - SPB saline phosphate buffer     

RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells

A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs.     

Signal recognition particle RNA localization within the nucleolus differs from the classical sites of ribosome synthesis

The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I-specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions.     

Localization of chromatin in "persistent" nucleoli in okadaic acid treated HeLa cells.

In okadaic acid treated HeLa cells, the chromosomes sometimes condense without being accompanied by nuclear envelope breakdown. These cells show "persistent" nucleoli. Within these "persistent" nucleoli the intranucleolar chromatin condenses and can be observed in the region of the dense nucleolar component (DNC) of the nucleoli. Other nucleolar components, namely the fibrillar centre (FC) and the granular component (GC) remain unchanged. These observations strongly speak for the localization of nucleolar chromatin (ribosomal cistrons) within the dense nucleolar component of the interphase nucleolus.     

THE ORGANIZATION OF THE NUCLEOLUS IN MERISTEMATIC PLANT CELLS : A Cytochemical Study

The architecture of the nucleolus in Allium porum and Triticum vulgare meristematic cells has been investigated by means of digestions with various enzymes. After staining with azure B at pH4, plant nucleoli exhibit lighter regions which, under electron microscopy, correspond to the fibrillar zones characterizing these organelles. Evidence is presented indicating that these latter zones contain coarse convoluted filaments quite similar to the loops first demonstrated by La Cour (24) and which are assumed to originate from the nucleolar-organizing chromosomes. These coarse, 0.2µ wide filaments are remarkably resistant to the action of deoxyribonuclease, ribonuclease, pepsin, trypsin, or of various combinations of these enzymes and, moreover, they show insignificant incorporation of labeled thymidine even after long exposure to this DNA precursor. The clearing action of pepsin on different regions of the nucleolus lends support to the hypothesis that an amorphous material or matrix pervades the mass of this organelle. This effect is particularly striking within the particulate nucleolar zones themselves. Both ribonuclease and trypsin disorganize the RNP (ribonucleoprotein) nucleolar particles. The effect of the latter enzyme on the RNP particles is taken to indicate that they contain proteins particularly susceptible to trypsin which are essential for maintenance of their morphological integrity. Trypsin also interferes with azure B-staining of the nucleolar mass as a whole and, according to radioautographic data, extracts RNA throughout this organelle. Accordingly, the hypothesis is considered that RNA is complexed with proteins not only within the particulate nucleolar portions, as is already well known, but also in the fibrillar zones.     

Optical control of protein delivery and partitioning in the nucleolus

The nucleolus is a subnuclear membraneless compartment intimately involved in ribosomal RNA synthesis, ribosome biogenesis and stress response. Multiple optogenetic devices have been developed to manipulate nuclear protein import and export, but molecular tools tailored for remote control over selective targeting or partitioning of cargo proteins into subnuclear compartments capable of phase separation are still limited. Here, we report a set of single-component photoinducible nucleolus-targeting tools, designated pNUTs, to enable rapid and reversible nucleoplasm-to-nucleolus shuttling, with the half-lives ranging from milliseconds to minutes. pNUTs allow both global protein infiltration into nucleoli and local delivery of cargoes into the outermost layer of the nucleolus, the granular component. When coupled with the amyotrophic lateral sclerosis (ALS)-associated C9ORF72 proline/arginine-rich dipeptide repeats, pNUTs allow us to photomanipulate poly-proline–arginine nucleolar localization, perturb nucleolar protein nucleophosmin 1 and suppress nascent protein synthesis. pNUTs thus expand the optogenetic toolbox by permitting light-controllable interrogation of nucleolar functions and precise induction of ALS-associated toxicity in cellular models.     

Ultrastructure and behavior of the nucleoli and fibrillar centers during cell differentiation of liver erythroblasts of 12-day-old mouse embryos

The transformation of nucleolus and its structural components in the main groups of erythroid cells (from pronormoblasts to reticulocytes and dividing ones) has been studied. It is shown that during inactivation of the nucleolus, the granular component is reduced, and the degree of chromatin condensation increases. Enlargement and "naking" of fibrillar centres are also observed. At the stage of basophilic and polychromatophilic erythroblasts, the nucleolus has a mushroom-like shape with well developed fibrillar centres, which lie at the border of the nucleolus. Nucleolar RNP components consist predominantly of a fibrillar component and forms "caps" of these mushroom-like structures. Therefore, at this stage "free" fibrillar centres are found on ultrathin sections, if the section plane runs only through the fibrillar centre, or through ring-shaped nucleoli, i.e. the fibrillar centre surrounded by sheet of nucleolar RNP fibrilles, when the mushroom-like nucleolus is cut tangentially. Using serial section technique, small round nucleoli with an extremely weakly developed RNP material or free fibrillar centres, resembling those in telophase nuclei, are shown on the terminal stage of nucleolus transformation. It is noted that the main groups of erythroid cells differ from each other not only in the chromatin condensation degree, but also in the development of nucleolus material and in the size of fibrillar centres. However, such differences exist in either cell group. Consequently, we can distinguish between cell populations being on different stages of maturation. On this basis, we described on intermediate population of cells, which possess signs of pronormoblasts and basophilic erythroblasts. In all the cases, strands of electron opaque material bound with the condensed chromatin are present in fibrillar centres.(ABSTRACT TRUNCATED AT 250 WORDS)     

FINE STRUCTURAL ALTERATIONS OF INTERPHASE NUCLEI OF LYMPHOCYTES STIMULATED TO GROWTH ACTIVITY IN VITRO

This report describes fine structural changes of interphase nuclei of human peripheral blood lymphocytes stimulated to growth by short-term culture with phytohemagglutinin. Chromatin is found highly labile, its changes accompanying the sequential increases of RNA and DNA synthesis which are known to occur in lymphocyte cultures. In "resting" lymphocytes, abundant condensed chromatin appears as a network of large and small aggregates. Early in the response to phytohemagglutinin, small aggregates disappear during increase of diffuse chromatin regions. Small aggregates soon reappear, probably resulting from disaggregation of large masses of condensed chromatin. Loosened and highly dispersed forms then appear prior to the formation of prophase chromosomes. The loosened state is found by radioautography to be most active in DNA synthesis. Small nucleoli of resting lymphocytes have concentric agranular, fibrillar, and granular zones with small amounts of intranucleolar chromatin. Enlarging interphase nucleoli change chiefly (1) by increase in amount of intranucleolar chromatin and alteration of its state of aggregation and (2) by increase in granular components in close association with fibrillar components.     

Relative position of nucleolar chromatin and nucleolar components in ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis> somatic nuclei

According to our computer modeling data obtained earlier, nucleoli in interphase ciliates Didinium nasutum are complex netlike structures, in which the trabeculumor lamella-shaped fibrillar component is located on the periphery, and the granular component in the central part of the nucleolus. Chromatin bodies connected with nucleoli act as the nucleolar organizers in D. nasutum. In the present work, the arrangement of all chromatin bodies, which could correspond to nucleolar organizers by morphological criteria, is studied by means of a 3D-reconstruction. It is shown that all of these chromatin bodies are localized outside the nucleoli, on the fibrillar component’s periphery. Even those chromatin bodies which appeared to be completely surrounded by the fibrillar nucleolar component on single ultrathin sections are actually settled down in nucleolus cavities open to the nucleoplasm. This proves that the RNA processing in D. nasutum nucleoli is directed toward the center of nucleoli, where the granular component is located. The analysis of the nucleolar chromatin distribution made it possible to conclude that different parts of the complex interfase netlike nucleoli of D. nasutum have approximately the same activity.     

小麦核仁在细胞周期中的变化

运用Bernhard染色方法研究了小麦根端分生组织细胞核仁在细胞周期中的变化。结果显示,间期核仁染色很深,能够区分出纤维中心（FC）、致密纤维组分（DFC）和颗粒组分（G）,而染色质被漂白,在染色质间可以观察到细小的RNP颗粒。进入前期,在染色质的边缘有小的RNP颗粒分布。中期,染色体周边分布着类似于间期核仁的深染的大RNP颗粒,形成一个不完全连续的“鞘”状结构;在染色体内部看不到类似核仁的深染颗粒。到了后期时,仍可见RNP“鞘”状结构的存在。进入末期,这些RNP植物逐渐由“鞘”脱离,最后参与新核仁的形成。这些结果表明,核仁解体后的物质直接转移到了中期染色的表面,并形成不连续的表层,没有进入染色体的内部。     

A CORRELATED LIGHT AND ELECTRON MICROSCOPE STUDY OF THE NUCLEOLAR MATERIAL DURING MITOSIS IN VICIA FABA

Root meristematic cells of Vicia faba were examined, with both light and electron microscopes, in order to study the behaviour of the nucleolar material during the mitotic process. Under light microscopy, the preprophase nucleolus is seen to consist of a densely stained material in which are embedded several unstained vacuole-like structures of varying size. The electron microscope reveals that the dense nucleolar material is formed of two structurally distinct components, each segregated into irregularly shaped zones blending with one another. One of these components is represented by 150 A granules which, in places, are arranged into thread-like structures approximately 0.1 µ in diameter; the other component apparently consists of fibrils 60 to 100 A in diameter. The large and medium sized intranucleolar vacuoles contain loosely scattered granules and fibrils similar to those just described. The granular and fibrillar components of the denser portion of the nucleolus persist as such during prophase and disperse throughout the nuclear cavity at the time of nucleolar disintegration. After nuclear membrane breakdown, these granules and fibrils, as well as those of the nucleoplasm, mix freely with similar elements already present within the forming spindle. No evidence has been obtained that, during or after nucleolar disintegration, the structural components of the nucleolus become associated as such with the chromosomes to form an external or internal matrix. Our observations suggest the existence, of a matrix substance within late prophase, metaphase, and anaphase chromosomes, the fine structure of which bears strong resemblance to that of their constituent coiled chromonemata. Data are presented, moreover, that indicate that part of this matrix substance, presumably formed at some time during prophase, is released from the chromosomes during their anaphasic movement. A number of observations indicate that the main bulk of the next nucleolus is derived from a prenucleolar fibrillogranular material, arranged into thread-like structures some 0.1 µ in diameter, which collect in the interchromosomal spaces during early and midtelophase. Finally, our data would seem to favour the view that most of this prenucleolar material results from a resumption of the synthetic activity of the early and midtelophase chromosomes rather than from a mere shedding of a preexisting matrix substance.