Identification of coiled body-like structures in meristematic cells of Pisum sativum cotyledonary buds

This study focused on two types of nuclear bodies visible in plant cells that were previously thought to be similar to the coiled bodies (CBs) of animal cells: the nucleolus-associated body (NAB) and dense body (DB). We show that both NABs and DBs share common features with animal CBs: they consist of ribonucleoproteins, are silver-stainable, and lack DNA. Immunoelectron microscopy shows that only the NABs are rich in snRNAs and fibrillarin, two markers characteristic of animal CBs. This suggests that NABs rather than DBs are the plant counterparts of the CBs of animal cells. These structures appear most frequently in cells blocked in G0–1, their frequency gradually declining with resumption of the cell cycle and nucleolar activity. During this reactivation period, NABs are released from the nucleolus to the nucleoplasm, suggesting that they may act as nuclear transport or sorting structures. Received: 2 November 1998; in revised form: 18 January 1999 / Accepted: 1 February 1999     

Cytochemical characterization of two types of nuclear bodies in Cicer arietinum L.

The present study was carried out to characterize two distinct types of nuclear bodies, the nucleolus-associated bodies (NABs) and the satellites (SATs) using some specific staining, enzyme and immunogold labeling techniques in Cicer arietinum L. These bodies are of interest as the functional components of plant nucleus. DNA-specific staining and labeling with anti-DNA, a monoclonal antibody, were employed to verify the presence of DNA in NABs as well as in SATs. The enzyme-gold labeling technique was used to compare the relative amounts of RNase-gold and protease-gold labeling over the NABs. In NABs, RNase-gold labeling was relatively low compared to the protease-gold labeling. Ag-NOR staining revealed a similar content of NOR-silver proteins in both NABs and granular zone of the nucleolus. The NABs do not contain any DNA as they show negative response to DNA-specific stains and also when incubated with anti-DNA, only few gold particles are found over these structures. The SATs, on the other hand, react positively with DNA-specific stains, and high labeling is recorded with anti-DNA along with the dense chromatin masses.     

Presence of small-nuclear-ribonucleoprotein-containing nuclear bodies in quiescent and early germinatingZea mays embryos

Summary Nucleolus-associated bodies (NABs) occur in interphase nuclei of many plant species. The present work shows that, inZea mays, NABs are present in dry seeds as well as in germinating tissues. The frequency of these nuclear bodies remains more or less constant during the first 24 h of imbibition but decreases significantly during the next 24 h. By the time the nucleolus reaches maturation and contains granular zones, these bodies are still found in close association with the surface of this organelle, as is the case in mature root meristematic cells. Immunocytochemical observations on both dry seeds and germinating tissues further revealed that NABs reacted positively with a monoclonal antibody (mAbK121) recognizing the m3G cap of sn(small nuclear)RNAs. It is, therefore, concluded that the NABs present in such tissues already contain components characterizing snRNPs (small nuclear ribonucleoproteins) in mature tissues. The possible function of NABs as storage deposits of snRNPs in dry seeds and early germinating tissues is discussed. In view of their many similarities with the coiled bodies described in both animal and plant cells, it is most likely that NABs correspond to those structures.Abbreviations BSA bovine serum albumin - DABCO 1,4-diazabicyclo (2.2.2)octane - EDTA ethylenediaminetetraacetic acid - IgM immunoglobulin M - NAB nucleolus-associated body - AO acridine orange - NAC nucleolus-associated chromatin - PBS phosphate-buffered saline - snRNA small nuclear ribonucleic acid - snRNP small nuclear ribonucleoprotein     

Localization of snRNP antigens in nucleolus-associated bodies: study of plant interphase nuclei by confocal and electron microscopy

Using two monoclonal antibodies, mAb Y12 and mAbK121, small nuclear ribonucleoproteins (snRNPs) were localized by immunofluorescence and immunogold electron microscopy in Pisum sativum and Cicer arietinum interphase nuclei. We showed that snRNPs were mostly found in nucleolus associated bodies (NABs) characterizing these two plant species. snRNPs were also found threoughout the nucleoplasm, their distribution being diffuse or speckled depending on the nucleus. Examination of optical sectins furnished by confocal laser microscopy allowed us to obtain more precise information on the number and location of NABs. One, two and rarely three (in green pea) of such structures were observed, their size varied from 0.8 to 1.5 m and they were also systematically observed in intimate contact with the nucleolus. Under electron microscopy, labelling was likewise found to be noticeably higher over NABs than over the nucleoplasm. The possible relationship between NABs and other nuclear bodies found in animal cells and known to be involved in splicing of pre-mRNA is discussed.     

The relationship of ribosomal RNA synthesis to the formation of segregated nucleoli and nucleolus-like bodies

The relationship of ribosomal RNA (rRNA) synthesis to nucleolar ultrastructure was studied in partial nucleolar mutants of Xenopus laevis. These mutations are the result of a partial deletion of rRNA genes and therefore alow studies on nucleolar structure and function without using drugs that inhibit rRNA synthesis. Ultrastructural studies demonstrated that normal embryos have reticulated nucleoli that are composed of a loose meshwork of granules and fibrils and a typical nucleolonema. In contrast, partial nucleolar mutants in which rRNA synthesis is reduced to less than 50% of the normal rate have compact nucleoli and nucleolus-like bodies. The compace nucleoli contain granules and fibrils, but they are segregated into distinct regions, and a nucleolonema is never seen. Since other species of RNA are synthesized normally by partial nucleolar mutants, these results demonstrate that nucleolar segragation is related specifically to a reduction in rRNA synthesis. The nucleolus-like bodies are composed mainly of fibrils,and the number of such bodies are composed mainly of fibrils, and the number of such bodies present in the different nucleolar mutants is inversely related to the relative rate of rRNA synthesis. Although the partial nucleolar organizers produce segregated nucleoli in these mutants, they organize morphologically normal, but smaller, nucleoli in heterozygous embryos. Alternative explanations to account for these results are discussed.     

Composition of nuclear dense bodies and nucleolus-associated bodies in interphase nuclei of the unicellular green alga Chlamydomonas reinhardtii

Summary— The interphase nucleus of the green alga, Chlamydomonas reinhardtii, displayed two types of bodies some of them, the dense bodies, lying apparently free in the nucleoplasm while the others were attached to the nucleolus and were, therefore, referred to as nucleolus-associated bodies (NABs). The presence of DNA, RNA and histones in dense bodies was investigated by means of post-embedding immunocytochemistry and cytochemistry using a monoclonal antibody to single and double stranded DNA, a polyclonal antibody to rye H3 histones and RNase A-gold complexes. The dense bodies were shown to contain significant amounts of RNA but neither DNA nor histones were detected; their composition was thus similar to that of the dense bodies described in higher plant cells. We propose that dense bodies might be implicated in the assembly of the 25 to 45 nm granules observed throughout the nucleoplasm of Chalamydomonas interphase nuclei. The composition of NABs was found to be distinct from that of the dense bodies since they were labeled by the antibody to DNA, specially in cryofixed and cryosubstituted specimens. The presence of DNA in NABs together with their intimate association to the nucleolus suggest that they may correspond to specific segments of chromosomes.     

Occurrence of nucleolar material in the cytoplasm of plant cells.

This paper deals with the induction of cytoplasmic nucleolar bodies in meristematic Allium cepa L. cells after treatment with drugs which interfere with nucleolar functionality. The drugs which interfere with protein synthesis failed to produce these bodies. The ultrastructure origin and physiological significance of these bodies are discussed here, as well as their relation with the mitotic prenucleolar bodies (Moreno-Díaz de la Espina et al., 1976).     

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.     

Nucleolus-like bodies in micronuclei of cultured Xenopus cells

Two of the 36 chromosomes in Xenopus laevis are known to carry nucleolar organizer loci. Partitioning of the chromosomes of cultured, early-passage Xenopus cells among variable numbers of micronuclei could be induced by extended colcemid treatment. A large, obvious nucleolus occurred in a maximum of 4 micronuclei per colcemid-induced tetraploid cell. The large, deeply-stained nucleoli incorporated [³H]uridine and appeared by electron microscopy to have typical nucleolar morphology with fibrillar and granular areas disposed in nucleolonema. In situ hybridization to radioactive ribosomal RNA (rRNA) resulted in heavy labelling of nucleoli in no more than 4 micronuclei per cell. The other micronuclei generally contained small bodies (blobs) which stained for RNA and protein as well as with ammoniacal silver. In the electron microscope, these appeared as round, dense bodies resembling nucleoli segregated by actinomycin D treatment. Nucleoplasmic RNA synthesis occurred in all micronuclei regardless of whether they contained definitive nucleoli. These observations suggest that micronuclei which formed large, typical, RNA-synthesizing nucleoli contained nucleolar organizer chromosomes, while the other micronuclei, which contained nucleolus-like “blobs” probably lacked nucleolar organizer loci. It is possible that the nucleolus-like bodies may have been aggregates of previously synthesized nucleolar RNA and protein trapped in micronuclei after mitosis.     

Peculiar behavior of the nucleolus and appearance of cytoplasmic nucleolus-like bodies in the roottip meristems ofBrodiaea uniflora Engl. Grown at low temperature

Summary Cytoplasmic nucleolus-like bodies, which showed vast variation both in size and in number per cell, were sometimes found in the telophase cells ofBrodiaea uniflora. When the plants were grown at low temperature, the frequency of telophase cells bearing cytoplasmic nucleolus-like bodies increased with the lapse of days. In contrast, growth at moderate temperature reduced this frequency. Prolonged exposure of the plants to low temperature caused peculiar phenomena concerning the behavior of the nucleolus and nucleolar material: 1. retention of nucleolar remnants at high frequency in metaphase, 2. pulverization of the nucleolar remnants into a great number of minute, fluffed fragments during metaphase, 3. appearance of dot-like nucleolar material in anaphase, and 4. appearance of nucleolus-like bodies, sometimes more than 10 m in diameter, in telophase. All these structures were strongly impregnated with silver. Electron microscopy revealed that both the nucleolar remnant and the nucleolus-like body consisted primarily of fibrils. Our observations clearly demonstrate that the nucleolus-like bodies are derived from the fibrillar component of the nucleolus and are formed by fusion of dot-like nucleolar material during anaphase.     

Electronic spectra, excited state structures and interactions of nucleic acid bases and base assemblies: a review

A comprehensive review of recent theoretical and experimental advances in the singlet electronic transitions, excited state structures and dynamics of nucleic acid bases (NABs) and base assemblies are presented. It is well known that NABs absorb ultraviolet radiation, but the absorbed energy is efficiently dissipated in the form of ultrafast internal conversion processes believed to occur in the subpicosecond time scale and, therefore, enabling NABs highly photostable. It is not known how much evolutionary role was played in evolving these molecules and the ultimate selection by nature as genetic materials, but it is well accepted that survival-of-fittest prevails. Recently, significant efforts have been continuously paid to understand the mechanism of electronic excitation deactivation, but universally acceptable mechanism is still elusive. However, recent investigations reveal that electronic excited state geometries of DNA bases are usually nonplanar and this structural nonplanarity may facilitate nonradiative deactivation. Investigation of excited state structures is challenging and, therefore, it is not surprising that despite the impressive theoretical and computational advances, this research area is still hampered by the methodological and computational limitations. Further, stacking has significant influence on the emission properties of molecules. The 2-aminopurine, a fluorescent adenine derivative frequently used in studying DNA dynamics, shows significant attenuations in fluorescence quantum yield when incorporated in the DNA. Theoretical and computational bottlenecks limit a thorough theoretical understanding of effect of stacking interactions on the excited state dynamics of NABs. Despite these limitations the investigations of excited state properties are progressing in the right direction and our better understanding of excited state structure and dynamics of NABs and nucleic acids may help to design preventive strategy for radiation induced illness and photostable materials.     

Basic Domains Target Protein Subunits of the RNase MRP Complex to the Nucleolus Independently of Complex Association

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.     

Identification and definition of nucleolus-related fibrillar bodies in micronucleated cells

Small nucleolus-related bodies which occur in the nucleoplasm of "micronuclei" lacking nucleolar organizers have been studied by immunofluorescence microscopy. These bodies stained specifically with three different antibodies directed against proteins that are normally associated with the dense fibrillar component of functional nucleoli, but not with antibodies specific for certain proteins of the granular component or the fibrillar centers. Our data show that, in the absence of rRNA genes, the various constituent proteins characteristic of the dense fibrillar component spontaneously assemble into spherical entities but that the subsequent fusion of these bodies into larger structures is prevented in these micronuclei. The similarity between these nucleolus-related bodies of micronuclei and the prenucleolar bodies characteristic of early stages of nucleologenesis during mitotic telophase is discussed.     

The nucleolus: a model for the organization of nuclear functions

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. The size and organization of the nucleolus are directly related to ribosome production. The organization of the nucleolus reveals the functional compartmentation of the nucleolar machineries that depends on nucleolar activity. When this activity is blocked, disrupted or impossible, the nucleolar proteins have the capacity to interact independently of the processing activity. In addition, nucleoli are dynamic structures in which nucleolar proteins rapidly associate and dissociate with nucleolar components in continuous exchanges with the nucleoplasm. At the time of nucleolar assembly, the processing machineries are recruited in a regulated manner in time and space, controlled by different kinases and form intermediate structures, the prenucleolar bodies. The participation of stable pre-rRNAs in nucleolar assembly was demonstrated after mitosis and during development but this is an intriguing observation since the role of these pre-rRNAs is presently unknown. A brief report on the nucleolus and diseases is proposed as well as of nucleolar functions different from ribosome biogenesis.Robert Feulgen Lecture presented at the 48th Symposium of the Society for Histochemistry in Stresa, Lake Maggiore, Italy, 7–10 September 2006.     

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.     

M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.