Structure and mode of formation of the nucleolus in meristematic cells of Vicia faba and Allium cepa

Interphase nucleoli from Vicia faba and Allium cepa meristematic cells are roughly classified into two categories: (a) those that commonly show a rather homogeneous texture (except for small light spaces of various sizes) and frequently contain dense particles 140 A in diameter; (b) those found more frequently in Vicia characterized by a very sharp boundary between a dense outer cortex and a much lighter central core. The dense particles are not found in such nucleoli. In Allium the boundary is more irregular and dense particles are sometimes observed in the outer layer. Many nucleoli show a structure intermediate between these two types. They are characterized by a gradient of increasing density from the center to the periphery and occasionally contain dense 140 A granules. During interphase, certain nucleoli are closely associated with segments of chromatin strands which undoubtedly represent nucleolar organizing regions. The dense 140 A granules are followed during the mitotic cycle. In Allium, they are first seen in loose clusters between arms of late anaphase chromosomes where they become more concentrated in early telophase. The substance within which they are scattered slowly increases in density during that time until finally, the particles are limited to small bodies of distinctive character. Evidence is presented suggesting that these small prenucleolar bodies fuse during telophase to give rise to the mature interphase nucleoli. Similar events are described in Vicia material except that a coating of dense substance appears around telophase chromosomes before the formation of prenucleolar bodies.     

Organization of argyrophilic nucleolar material throughout the division cycle of meristematic cells

Summary The silver impregnation of nucleolar material facilitated the study of the morphological changes which take place in the nucleolus throughout the division cycle in root tip cells ofAllium cepa. The nucleolus appears to undergo no morphological changes throughout the interphase. It undergoes disorganization during the prophase, while in the telophase it appears uniformly on the chromatin as condensing into prenucleolar bodies.The appearance of the prenucleolar bodies is unaffected by puromycin, cordycepin, or ethidium bromide. This suggests that the argyrophilic material does not undergo synthesis during the telophase, nor require RNA or protein synthesis to effect the aggregation into prenucleolar bodies. However, the organization of nucleoli from prenucleolar bodies is inhibited by both cordycepin and ethidium bromide, suggesting that RNA synthesis is involved in this proccess.In aneuploid nuclei induced by treatment with colchicine we observed the appearance of prenucleolar bodies during the telophase even in the absence of the nucleolar organizer, but in this case the formation of nucleoli fails to take place. The nucleolar organizers proved to be capable of acting only in the nucleus to which they belong, but not on other nuclei within the same cytoplasm belonging to multinucleate cells.It seems logical to assume that one of the roles of the nucleolar organizer is related with the above-mentioned RNA synthesis, which is required to the aggregation of prenucleolar bodies into nucleoli.The work reported in the paper was undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer.     

Behavior of silver stainable material during mitosis inVicia faba

To reveal the behavior of silver stainable material localized mainly in the nucleoli and nucleolar organizing regions (NORs), the somatic cells ofVicia faba were investigated by silver staining throughout the mitotic cell cycle. Nucleoli of interphase and early prophase nuclei were darkly stained. From late prophase to anaphase the secondary constrictions were discriminated as silver stained NORs and many silver grains appeared throughout the cytoplasm. At late prophase the NOR condensed at the same rate as the chromosome arm. Small spherical bodies and two new nucleoli appeared in telophase nuclei and at the same time the cytoplasmic grains disappeared. On the basis of the above observations on the silver stainable material during each mitotic phase, the behavior of silver stainable material is interpreted.     

BEHAVIOR OF NUCLEOLI AND CONTRACTING NUCLEOLAR VACUOLES IN TOBACCO CELLS GROWING IN MICROCULTURE

The ability to observe for extended periods of time individual tobacco cells growing in microculture has made it possible to describe the behavior of their nucleoli and contracting nucleolar vacuoles. Nucleoli typically disappeared in prophase and reappeared in telophase. If several nucleoli were present in telophase they generally fused to form only one or two during interphase. In one instance a nucleolus was seen to separate into two nucleoli prior to disappearance in late prophase. In aging and senescent cells the number of nucleoli or bodies similar to normal nucleoli often increased, and occasionally fragmentation of nucleoli was noted prior to death of cells. Budding of solid material from the nucleolus was also observed. The amount of nucleolar material decreased rapidly prior to death of tobacco cells. Nucleolar vacuoles were found to be a general and consistent component of tobacco cells in microculture. Nucleolar vacuoles typically formed and contracted repeatedly in interphase nuclei and apparently released a fluid material into the nucleus. Associated with the contraction of the nucleolar vacuoles was a corresponding decrease in diameter of the nucleolus. Nucleolar vacuoles were observed to occur in about 70% of the actively growing cells examined, whereas they were present in only 33% of the senescent or weakened cells. These data indicate a relationship between nucleolar vacuoles and the morphogenic status of the cells. Since it has been shown by others that the nucleolus is an active site of RNA metabolism, it is suggested that the contracting nucleolar vacuoles may be involved in the controlled release of a soluble product associated with RNA metabolism.     

Tomographic distribution of acetylated histone H4 in plant chromosomes,nuclei and nucleoli

Root tip cells of broad bean (Vicia faba L. cv. ’Wase soramame’) and barley (Hordeum vulgare L. cv. ’Minorimugi’) were immunostained with antibodies specific for acetylated histone H4. With an antiserum that recognizes histone H4 acetylated at lysine-5, the nucleolar organizing region (NOR) in mitotic chromosomes was strongly labeled in both species. The broad bean had two signals in the metaphase and telophase chromosome complements and four signals in the prophase and anaphase chromosome complements, while the barley had four signals in the metaphase and telophase chromosome complements and eight signals in the prophase and anaphase complements. Five different patterns of signals were observed at interphase: in type I only nucleoli were wholly stained; in type II perinucleolar knob-like signals and/or fiber-like signals emanated from the nucleus; in type III aggregate signals appeared in the nucleolus; in type IV many small dot-like signals were distributed throughout the nucleus, except nucleoli; and in type V string-like or some granule-like signals appeared in the nucleoli. Type II was very similar to previous results by in situ hybridization with sense rDNA probes. Type III was similar to the patterns of DNA synthesis recognized as chromatin domains by anti-BrdU antibodies. Type V was very similar to the results of in situ hybridization with pTa71, rDNA probes and the appearance of the dense fibrillar components of the nucleolus. Received: 7 August 1996; in revised form: 16 September 1996 / Accepted: 16 September 1996     

Human Y-chromatin : III. The nucleolus

The relative positions of nucleoli and the Y-chromatin body were investigated in human interphase fibroblast nuclei to determine if the reported nucleolar association of the Y-body might be a chance phenomenon. Although nucleolar material was found to be mainly in the central area of the nucleus, the association of the Y-body with a nucleolus was highly significant, irrespective of the morphology or location of the Y-body within the nucleus. The association was corroborated with late interphase and early prophase nuclei in which nucleolar remnants were seen to concentrate around the Y chromosome.     

MITOSIS IN THE ALGA VACUOLARIA VIRESCENS

Details of mitosis in the chloromonadophycean alga Vacuolaria virescens Cienk. have been studied with the light microscope. The chromosomes are relatively large (up to μ in length at metaphase) and so mitotic stages are readily distinguishable. Chromosomes can be recognized in interphase nuclei as fine strands of chromatin. Contraction of these chromosomes marks the beginning of mitosis and continues progressively until the transition from metaphase to anaphase. Disintegration of nucleoli is complete by late prophase and nucleolar reformation begins in telophase. Some chromosomes exhibit less densely stained regions; centromeres are also present as indicated by their differential staining and by the behavior of chromosomes at metaphase and anaphase. At anaphase progeny chromosomes move apart parallel to the division axis of the nucleus. As anaphase progresses the chromosomes fuse at the polar surface of the progeny chromosome groups. This process continues in telophase and the chromosome groups become more spherical. By the end of telophase nucleolar reformation has begun and the chromosomes have relaxed to their interphase condition.     

Study of the positional relationship of nucleolar chromatin and nucleolar compartments in somatic nuclei OPF the ciliate Didinium nasutum

We showed earlier that nucleoli in interphase ciliates Didinium nasutum, appearing on single ultrathin sections as individual structures, actually are parts of more complex network-like structures in which fibrillar component is located on periphery, and granular--in the central part of a nucleolus. It is known, that nucleolar organizers in D. nasutum are represented by chromatin bodies connected with nucleoli. In this work we used 3D reconstruction on the basis of serial ultrathin sections to study localization of chromatin bodies which by morphological criteria might correspond to nucleolar organizers. Our data showed, that all such chromatin bodies settled down outside of nucleoli, near the periphery of fibrillar component. Even those chromatin bodies which on single sections looked completely surrounded by fibrillar nucleolar component, actually settled down in fibrillar component cavities open to nucleoplasm. Analysis of distribution of nucleolar chromatin bodies allowed us to conclude that activity in different parts of interphase complex network-like nucleoli of D. nasutum is approximately the same.     

Whole mount electron microscopy of the nucleolus in salivary gland cells of Drosophila melanogaster.

The nucleolus of Drosophila melanogaster salivary gland cells, examined by whole mount electron microscopy, consists of a fibrillar core region and a peripheral region containing both fibres and granules. These regions appear to correspond to the fibrillar and granular components, respectively, seen in thin sections. Most of the nucleoli were attached to the chromocenter region of the polytene chromosomes, containing the nucleolar organizer. Bundles of relatively straight chromatin fibres, 13 nm in diameter, extended from the chromocenter into the core region of the nucleolus, however it was not possible to trace the path of these chromatin fibres through the nucleolus since they were obscured within the mass of nucleolar fibres. The nucleolar fibres in both the core and peripheral regions were irregular and knobby, with a diameter of about 15 nm. In the core region, the fibres appeared to be of considerable length and were characteristically clustered together to form small interconnected masses. The fibres in the peripheral region were relatively short and some appeared to blend with amorphous, poorly-defined pools of material. Electron dense granules 15-20 nm in diameter were also associated with this amorphous substance. It is hypothesized that the formation and subsequent packaging of the 28s rRNA may be represented by a morphological transition of the peripheral fibres, via an amorphous pool-like intermediate stage, into the nucleolar granules. The results of this study indicate that whole mount electron microscopy may be a useful alternative to thin sectioning in high resolution studies of the nucleolus.     

Ultrastructural Changes of the Nucleolus During Cell Cycle in Triticum aestivum

The ultrastructural changes of the nticleolus during cell cycle in common wheat (Triticum aestivum L. ) were studied by an "en bloc" silver-staining method. It was observed that in interphase, the nucleolus was heavily stained, within which fibrillar centres, dense fibrillar component, granular component and nucleolar vacuoles could be identified. A large quantity of argentine fine granules were distributed in the condensed chromatin. Dur-ing prophase, along with the disintegration of the nucleolus and condensation of the chromatin, the larger heavily-stained granules gradually appeared at the periphery of the chromatin. At late prophase, the materials derived from the nucleolus were spread and deposited on the surface of the chromosomes. The silver-stained, larger granules, deriving from the disintegrated nucleolus, accumulated at the periphery of the metaphase chromosomes and formed an uneven and discontinuous "sheath"-like structure. This "sheath"-like structure was also observed at anaphase. In telophase, the silver-stained nucleolar materials were progressively separated from the "sheath' and fused with each other to form prenucleolar bodies, and at last, participating in the formation of new nucleoli. The results showed that the nucleolar materials were transferred directly to the surface of the chromosomes and formed a discontinuous coat, but not incorporated into the interior of the chromosomes. The silverstained granules inside the chromosomes were neither related to the nucleolus nor to the materials from the disintegrated nucleolus.     

Control of nucleolar growth during interphase in higher plant meristem cells

Summary The evolution of nuclear and nucleolar sizes throughout interphase have been studied in synchronous caffeine-labeled binucleate cells of onion root meristems by using silver impregnation and stereological methods over semithin sections. Nucleus and nucleolus grow independently, since nucleolus enlarges at its fastest rate in G 1, while nucleus grows mostly in two periods: onset of replication and G 2. Nucleolar size in the cycle seems to be a genecontrolled function, hardly affected by protein synthesis inhibition. Hence, there is a biphasic response to cycloheximide (CHM) in the fast growing nucleoli of both early and late G 1 with an initial stimulation later counterbalanced by a depressed rate, so that nucleolar size in S was similar to control shortly afterwards the start of the CHM treatment. The initial enlargement under CHM was due to an increase of all nucleolar structural components, i.e., fibrillar, granular, vacuolar, and lacunar regions. No cycloheximide effect whatsoever was detected in S and G 2 nucleoli.Abbreviations CHM cycloheximide - F fibrillar component - G granular component - L lacunae - V vacuoles - VN nuclear volume - VNu nucleolar volume - VvNu volume density of the nucleoli     

Behaviour of nucleolus during mitosis

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.     

In vivo dynamics and kinetics of pKi-67: transition from a mobile to an immobile form at the onset of anaphase

A cell proliferation marker protein, pKi-67, distributes to the chromosome periphery during mitosis and nucleolar heterochromatin in the interphase. We report here on the structural domains of pKi-67 that are required for its correct distribution. While both the LR domain and the conserved domain were involved in localization to the nucleolar heterochromatin, both the LR domain and the Ki-67 repeat domain were required for its distribution to the mitotic chromosome periphery. Using in vivo time-lapse microscopy, GFP-pKi-67 was dynamically tracked from the mitotic chromosome periphery to reforming nucleoli via prenucleolar bodies (PNBs). The signals in PNBs then moved towards and fused into the reforming nucleoli with a thin string-like fluorescence during early G1 phase. An analysis of the in vivo kinetics of pKi-67 using photobleaching indicated that the association of pKi-67 with chromatin was progressively altered from "loose" to "tight" after the onset of anaphase. These findings indicate that pKi-67 dynamically alters the nature of the interaction with chromatin structure during the cell cycle, which is closely related to the reformation process of the interphase nucleolar chromatin.     

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.     

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.     

CELL DIVISION IN SPIROGYRA. I. MITOSIS

Dividing cells of Spirogyra sp. were examined with both the light and electron microscopes. By preprophase many of the typical transverse wall micro-tubules disappeared while others were seen in the thickened cytoplasmic strands. Microtubules appeared in the polar cytoplasm at prophase and by prometaphase they penetrated the nucleus. They were attached to chromosomes at metaphase and early anaphase, and formed a sheath surrounding the spindle during anaphase; they were seen in the interzonal strands and cytoplasmic strands at telophase. The interphase nucleolus, containing 2 distinct zones and chromatinlike material, fragmented at prophase; at metaphase and anaphase nucleolar material coated the chromosomes, obscuring them by late anaphase. The chromosomes condensed in the nucleoplasm at prophase, moving into the nucleolus at prometaphase. The nuclear envelope was finally disrupted at anaphase during spindle elongation; at telophase membrane profiles coated the reforming nuclei. During anaphase and early telophase the interzonal region contained vacuoles, a few micro-tubules, and sometimes eliminated n ucleolar material; most small organelles, including swollen endoplasmic reticulum and tubular membranes, were concentrated in the polar cytoplasm. Quantitative and qualitative cytological observations strongly suggest movement of intact wall rnicrotubules to the spindle at preprophase and then back again at telophase.     

Localization of histones and their synthesis by ammoniacal silver reaction in meristematic root tip cells of Allium cepa

Summary The ammoniacal silver reaction was used for localization of histones in meristematic root tip cells of Allium cepa. Electron microscopic observations showed that yellow or brown colour of interphase and prophase nuclei and brown nucleolar colour produced in the reaction coincides with the appearance of silver grains, about 400 Å in diameter, in the interphase and prophase chromatin and nucleoli. This together with the complete absence of staining reaction and silver grains in the cytoplasm could mean quite a specific reaction with histones and might suggest also that in these cells the site of histone synthesis is in the nucleolus.     

The dynamics of postmitotic reassembly of the nucleolus

Mammalian cell nucleoli disassemble at the onset of M-phase and reassemble during telophase. Recent studies showed that partially processed preribosomal RNA (pre-rRNA) is preserved in association with processing components in the perichromosomal regions (PRs) and in particles called nucleolus-derived foci (NDF) during mitosis. Here, the dynamics of nucleolar reassembly were examined for the first time in living cells expressing fusions of the processing-related proteins fibrillarin, nucleolin, or B23 with green fluorescent protein (GFP). During telophase the NDF disappeared with a concomitant appearance of material in the reforming nuclei. Prenucleolar bodies (PNBs) appeared in nuclei in early telophase and gradually disappeared as nucleoli formed, strongly suggesting the transfer of PNB components to newly forming nucleoli. Fluorescence recovery after photobleaching (FRAP) showed that fibrillarin-GFP reassociates with the NDF and PNBs at rapid and similar rates. The reentry of processing complexes into telophase nuclei is suggested by the presence of pre-rRNA sequences in PNBs. Entry of specific proteins into the nucleolus approximately correlated with the timing of processing events. The mitotically preserved processing complexes may be essential for regulating the distribution of components to reassembling daughter cell nucleoli.