An EM Study on the Nucleolar Residual in Meristematic Cells of Vicia faba

A special structure which originates from the nucleolus and persists during mitosis is observed in meristematic cells of Vicia faba, and is called nucleolar residual in this paper. The result of conventional staining showed that the nucleolar residual (NR) was a medium electron-dense structure that could be distinguished from chromosomes. At the end of prophase, the NR was observed in the dispersing nucleolar material, which consisted principally of granules and fibrils about 15 to 20 nm in diameter. In prometaphase, the NR, composed mainly of the similar granules and fibrils, was found attached to chromosomes. Results of Bernhard's technique for RNP (ribonucleoprotein) preferential staining indicated that the main chemical component of the NR was RNP, and sometimes bleached regions showing the same reaction as chromatin in Bernhard's staining appeared in the structure. In metaphase and anaphase some of the NRs were attached to chromosomes while others existed randomly in the cytoplasm. The NR is supposed, according to its cytochemical features and its behavi0ur during the nucleolar disintegration, to be a structure composed mainly of nucleolar matrix material or nucleolar skeleton.     

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.     

蚕豆核仁残体的电镜研究

本文报道了有丝分裂过程中蚕豆(Vicia faba)根端分生组织细胞内一种由核仁物质组成的特殊结构,我们将其称作核仁残体。经常规染色后,可在前期末正在分散的核仁物质中看到由直径15—20nm 的颗粒和纤维组成的核仁残体;前中期时,核仁残体附着在染色体上,其电子密度低于染色体。Bernhard 染色结果表明,核仁残体的主要成分是核糖核蛋白(RNP)颗粒和纤维,一些核仁残体中存在着与染色质染色反应相似的被漂白区。有的核仁残体附着在中、后期染色体上,有的游离存在于细胞质中。本文讨论了核仁残体的成分及其本质等问题。     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

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.     

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.     

Mitotic Segregation of the Nucleolar Ribosomal RNA in Physarum polycephalum

In the naturally synchronous mitosis of the syncytial plasmodium of Physarum polycephalum , the nucleolus disintegrates in prophase, releasing a large amount of ribosomal RNA. Using biotinylated rDNA probes, we studied by high-resolution in situ hybridization the behavior of this nucleolar RNA throughout mitosis. Our results demonstrate that this rRNA is stable and maintained within the mitotic nucleus mainly, but not exclusively, associated with fibrillar nucleolar remnants. The distribution of these rRNA molecules on both sides of the cleavage plane in telophase is indicative of a precise mechanism of mitotic partition of the nucleolar components, supporting our recent findings concerning the rDNA minichromosomes (Puvion-Dutilleul and Pierron, 1992, Exp. Cell Res. 203, 354-364). Taking advantage of the stability of this RNA component in mitosis, we unambiguously demonstrate that the nucleolar remnants are the precursors of the prenucleolar bodies appearing in the newly divided nuclei which, by fusion, reconstitute a single nucleolus. Our data exemplify the persistence of the nucleolar rRNA in mitosis and demonstrate that in Physarum, following its disintegration, the nucleolus is segregated and inherited.     

Differences in the fine structure and chemical constitution of nucleolar bodies and the true nucleolus in Xenopus laevis embryos

Numerous bodies resembling nucleoli, named “prenucleolar bodies”, were seen in the interphase nucleus of Xenopus laevis embryos between stages 7 and 11 of Nieuwkoop and Faber (1956) but not at stage 12. These bodies are composed of thick strands, 200 A in diameter, and apparently differ from the fibrillar component of the true nucleolus which consists of thin fibrils, 50 A in diameter. The granular component of the true nucleolus consists of fibers and granules which are both also 150–200 A in diameter, but which differ in chemical nature from the prenucleolar bodies. The granular component and fibrillar component are readily digested by RNase with or without pretreatment with trypsin, while the prenucleolar body is only digested with RNase after pretreatment with trypsin. This suggests that the prenucleolar body consists of strands of RNA coated with protein. At stage 9, another type of nucleolus-like body is formed, which is larger (2–2.6 μ in diameter) than the prenucleolar body (0.2–1 μ) and consists of thin fibrils of 50 A. This body resembles the fibrillar component of the true nucleolus in the size of the elemental fibrils as well as in its susceptibility to actinomycin D, RNase and trypsin. It seems to be a precursor of the true nucleolus and for this reason was named the “primary nucleolus.” From stage 9 to stage 10, each nucleus in the presumptive ectodermal and mesodermal areas contains 2 primary nucleoli together with multiple prenucleolar bodies. At stage 12, the prenucleolar body is not seen at all, but a new type of nucleolus-like body appears. There are usually 2 of these bodies in each nucleus, and they consist of 2 components: a network of 50 A fibrils, and a group of strands, 150–200 A in diameter, containing some granule-like elements. The former has the same susceptibility to actinomycin D, RNase and/or trypsin as the fibrillar component of the definitive nucleolus and the primary nucleolus, while the latter has the same susceptibility as the granular component of the definitive nucleolus. Thus, this body may     

蚕豆染色体周边RNP形成过程的电镜研究

本文运用Bernhard染色方法研究了蚕豆根端分生组织细胞中染色体周边RNP的超微结构以及这种周边RNP在有丝分裂前期到中期的形成过程。我们观察到,在前期核仁解体过程中,来自核仁的RNP物质结合于染色体表面,形成染色体周边RNP。前期末时,大量核仁RNP颗粒向周围扩散并进一步结合于染色体表面,使染色体周边RNP有所增加。中期染色体的周边RNP明显多于前期,由直径15-20 nm的RNP颗粒构成。RNP物质在染色体周边的分布是不均匀的。姊妹染色单体之间往往有较多的RNP物质存在。本文观察结果表明染色体周边RNP来源于核仁RNP。     

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.     

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.     

Alteration of nucleolar dispersion in prophase by 5-FUdR treatment.

The action of 5-Fluorodeoxyuridine (FUdR) used as an inhibitor of RNA synthesis on the nucleolar evolution during mitosis, has been studied in meristematic cells. Under FUdR treatment the nucleolar dispersion appears as a continuous process, but generally it is not completed and nucleolar remnants remain throughout the whole mitosis. The nucleolar material which was dispersed is transported by the mitotic chromosomes, and in telophase contributed to the formation of the new nucleolus. The non-dispersed part persisted in the cytoplasm during telophase, coexisting with both the prenucleolar bodies and the new nucleolus which was being formed. Our results suggest the necessity of some kind of RNA synthesis, preferentially blocked by FUdR, for nucleolar dispersion to take place.     

An autoimmune antibody from scleroderma patients recognizes a component of the plant cell nucleolus

Summary An auto-antibody from human serum of patients with the autoimmune disease scleroderma was used to localize the nucleolus in meristematic cells of onion and soybean roots using indirect immunofluorescence microscopy. Similar lots of antiserum recognized a single 34 kD, nucleolar protein, fibrillarin, in a variety of animal cells (Ochs, et al. 1984, 1985). In both plants, antibody linked fluorescence is associated with the one to several nucleoli present in the interphase nucleus. The fluorescence becomes diffuse around condensing prophase chromosomes and becomes more diffused at metaphase with slightly more intense fluorescence surrounding the chromosomes. At anaphase-telophase the fluorescence is localized in dense areas within the chromosomes, presumably representing prenucleolar bodies which will form the interphase nucleoli of the daughter nuclei. This antiserum provides a new, valuable tool for the study of the nucleolus and the highly conversed nucleolar antigen(s) that it recognizes.     

Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells

The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed during mitosis in Chinese hamster ovary CHO cells using specific antibodies to this protein and immunocytochemical techniques. The 100 kDa protein was visualized at discrete sites on metaphase chromosomes, corresponding to nucleolus organizer regions, and in large, immunostained nucleolar remnants that are discarded in the cytoplasm after nucleolar disintegration. After mitosis, the 100 kDa protein was shown to play an early role in nucleolar reformation. It was first detected in small deposits around the anaphase chromosomes. In telophase, the protein accumulated simultaneously in prenucleolar bodies and in the reforming nucleoli. The early presence of the 100 kDa protein in the telophase nucleus suggests that it is essential for the reestablishment of nucleolar function after mitosis. Thus this protein is present throughout the CHO cell cycle, an observation which supports the hypothesis that it plays a fundamental role in cell organization.     

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。     

Electron Microscopic Studies on the Silver-stained Nucleolar Cycle of Physarum polycephalum

The dynamic changes of nucleolar ultrastructure in the cell cycle of Physarum polycephalum Schw. were studied by an en bloc silver-staining method. The results showed that the nucleolus was large in size and situated in the center of the nucleus in late G2-phase, and the fibrillar centers, dense fibrillar components and granular components could be observed in the nucleolus. During prophase, the nucleolus moved towards the periphery of the nucleus and in late prophase disintegrated near the nuclear envelope. In metaphase, the disintegrated nucleolar components were dispersed in masses and located at the periphery of the chromosomal region of the nucleus. No specifically silver-stained area and argentophilic protein sheath were observed on the chromosomes, but there were some big dispersed silver particles within the chromosomes. During telophase the nucleolar components moved towards the two poles along with the chromosomes and co-existed with the decondensing chromatin in daughter nuclei. The nucleolar components then gradually converged with one another and separated from the chromatin. A big nucleolus was formed in the nucleus about 120 min after the completion of mitosis.     

Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.     

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.