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.     

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     

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     

Spermatogenesis in animals as revealed by electron microscopy

Summary The early spermatid nuclei of the grasshopper, Acrida lata, have been observed electron microscopically. The irregularly compact chromatin mass appears closely attached to the nuclear envelope. This mass migrates subsequently into a more central portion. It seems to participate in the formation of the nucleolus as a nucleolar organizer. At the time when the chromatin mass and frequently the nucleolus undergo involution, clusters of peculiar granular bodies 130 m in average diameter and 200 A wide filamentous elements among the bodies make their appearance in the nucleoplasm. The particles constituting the granular bodies are composed of DNA, but their matrix consists of RNA. The term microkaryosome is proposed for such granular body, because it is similar in chemical components to karyosome, but the former is smaller in size than the latter. It is suggested that the microkaryosome may be related with the paracrystalline formation of nucleoprotein.     

Nucleosphaeridies in early pig embryos

Summary Spherical fibrillogranular nuclear structures, here called nucleosphaeridies, were observed in pig embryos ranging between the two-cell-stage and the early blastocyststage. Up to four nucleosphaeridies, averaging 2 to 4 m in diameter and different from the common nucleoplasmic structures, were found in a single thin section. As a rule the nucleosphaeridies are situated at random in the nucleoplasm, sometimes in contiguity with the nuclear envelope. Occasionally, they are located within the nucleolus. There is morphological similarity between the nucleosphaeridies situated within the nucleolus and those situated in the nucleoplasm. Based on these morphological observations, considerations are given as to whether these nucleosphaeridies are synthesized by the nucleoli, or inversely, these structures are precursors in the development and maturation of the nucleoli.After Büttner et al. (1967).This work was supported by the Agricultural Research Council of Norway.     

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.     

Unusual prophase structures and multiple nucleoli in male meiosis of Drosophila species of the virilis group

With silver nitrate (Ag-NOR) staining, unusual fibrillar structures, apparently coupled to the nucleolus, were found is several species of the D. virilis group. In D. littoralis, beaded strings appear in connection with these structures, whereas the late prophase is characterized by the appearance of multiple nucleoli in the nucleoplasm. In D. virilis, the nucleus has a prominent pointed protrusion in the region of the nucleolus and often a fibril protrudes from this point. Small nucleoli are budding from the nucleolus during prophase. The multiple nucleoli at late prophase are smaller and fewer. A nucleolar body with black spots appears at prometaphase and persists through metaphase and anaphase. In D. lummei, the nucleolus becomes surrounded by fibrils, which are released into the nucleoplasm and on which multiple nucleoli are synthesized.These phenomena are similar to the events described in oocyte meiosis of many animals, where rDNA amplification, coupled to the synthesis of multiple nucleoli in late prophase, has been established.     

Ultrastructural studies of late meiotic prophase nuclei of spermatocytes in Ascaris suum

Post pachytene stages of meiotic prophase in males of Ascaris suum have been analyzed with the electron microscope. No synaptonemal-like polycomplexes (PCs) have been observed in the nucleoplasm or cytoplasm during the period from pachytene to diakinesis. From Serially sectioned diplotene nuclei it was found that the bivalents are located near the periphery of the nuclei, the central part of the nuclei being vacant. Each nucleus contains one nucleolus. Up to 1 m long stretches of unpaired lateral elements (LEs) are found in some of the diplotene bivalents. These LEs are morphologically similar to unpaired LEs in early zygotene nuclei. Partial 3-dimensional reconstruction of two nuclei shows that the bivalents contain some small stretches of synaptonemal complex (SC) up to 1.9 m long. Some bivalents at diakinesis show remnants of SCs. At this stage chromosomes are fibrous, condensed, attached to the nuclear envelope and mostly with a rounded profile in cross section. The synchronous development of the spermatocytes and small bivalents at diplotene in A. suum make this system a good object for the study of localization of SC remnants.     

Identification of coiled bodies inBrassica napus nuclei during embryogenesis and early germination

Summary Nucleolus-associated bodies characterize interphase nuclei of many plant species. The recent demonstration that such bodies contain small nuclear ribonucleoproteins as well as coilin clearly indicates that they belong to a larger family of nuclear structures, known as coiled bodies, that have been intensively studied in a variety of animal cell types. In a previous work, we have shown that coiled bodies were present in close association with the nucleolus inZea mays dry seeds as well as during subsequent stages of germination. This study reveals that similar nuclear structures were also present duringBrassica napus embryogenesis starting at the torpedo stage and that they were, likewise, generally located on the nucleolar surface. As in the case ofZ. mays, coiled bodies were observed in cells of dry seeds as well as in those of early germinating tissues. These bodies were labelled with monoclonal antibody K121, an antibody reacting with the unique 5-terminal cap structure containing 2,2,7-trimethylguanosine that characterizes small nuclear RNAs. Owing to their intimate association with the nucleolus in all stages studied, the possibility is considered that, in these plant cells, coiled bodies are assembled on an organizer element located within this organelle.Abbreviations BSA bovine serum albumin - IgM immunoglobulin M - NAB nucleolus-associated body - NAC nucleolus-associated chromatin - PBS phosphate-buffered saline - snRNA small nuclear ribonucleic acid - snRNP small nuclear ribonucleoprotein     

Fine structural cytochemical and immunocytochemical analysis of nucleic acids and ribonucleoprotein distribution in nuclei of pig oocytes and early preimplantation embryos

The fine structure of pig oocytes at the germinal vesicle (GV) stage and early preimplantation embryos (one to four blastomeres) isolated at slaughter was investigated by cytochemical and immunocytochemical methods. The distribution of nucleic acids and ribonucleoproteins (RNPs) in compact nucleoli [denominated nucleolus-like bodies (NLB) in oocytes and nucleolus precursor bodies (NPB) in early embryos] and in intranuclear bodies or granules was investigated by staining methods preferential for nuclear RNPs or using the osmium ammine or ethidium bromide-phosphotungstic acid (EB-PTA) reactions for nucleic acids. The distributions of the Sm antigen of nucleoplasmic small nuclear RNPs (snRNPs), the methyl-3 guanosine (m₃G) cap of snRNAs and the splicing factor SC-35 were detected by immunoelectron microscopy using specific antibodies. The RNP nature of both NLBs and NPBs, and of nuclear granules in oocytes and embryos, and of fibrillar strands radially projecting from NLBs was revealed. Cytochemical evidence for RNA as a component of NLBs was further provided by EB-PTA staining in combination with the enzymatic removal of RNA, or by osmium-ammine staining without previous acid hydrolysis, while the absence of DNA in NLBs was established by Feulgen-like osmium-ammine staining. In addition, autoradiography demonstrated the absence of [6-³H]thymidine incorporation into NPBs. Other autoradiographic evidence attested the accumulation of RNA in NLBs of oocytes after a 60 min in vitro pulse of [5-³H]uridine. Immunoelectron microscopy using specific antibodies revealed the occurrence of nucleoplasmic snRNPs in both NLBs and NPBs. The presence of snRNA in NLB was confirmed by means of an antibody recognizing the m₃G-cap structure. Another spliceosomal component, the protein SC-35 was also detected in NLBs. Among the numerous and variable intranuclear granules occurring mostly in aggregates, the Sm antigen was clearly detected only in the interchromatin granule-type component. Some Sm labeling was occasionally seen in other categories of larger granules. No reaction was detected over any granules when using the anti-m₃G-cap antibody. The aggregates consisting of large granules and a finely fibrillar component were intensely immunolabeled by the anti-SC-35 splicing factor probe. Our observations suggest that the compact nucleoli, known to be present before and after fertilization in mammals (NLBs of oocytes and NPBs of early embryos), represent nuclear structural elements containing nonnucleolar, spliceosomal components.     

Coiled bodies in supraoptic nuclei of the rat hypothalamus during the postnatal period

In electron microscopic studies of the supraoptic nuclei of the rat hypothalamus, structures identified as "coiled bodies" were found in magnocellular neurons. Although they could be seen elsewhere in mature neurosecretory cells, coiled bodies were commonly encountered in developing neurons during the postnatal period in both sexes. They appeared as distinctive nuclear inclusions consisting of round-to-oval networks of short electron-dense strands embedded in a less dense, fibrillar matrix, and lacking a limiting membrane. In fine structure and stain-affinity, they bore a resemblance to the fibrillar component of the nucleolus. Coiled bodies were located either in close association with the nucleolus or free within the nucleoplasm, showing no specific relationships with the perinucleolar chromatin or with the nuclear envelope. Their origin and functional meaning is discussed in the light of recent ultrastructural and biochemical data on cellular differentiation and nucleolar behavior.     

Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants

We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM). Cajal bodies (CBs) were observed in quiescent embryos and germinating cells in both species. The number and distribution of CBs were investigated. To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy. Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced. AtRSp31 was detected in quiescent nuclei and in germinating cells. This study revealed an unexpected speckled nuclear organization of atRSp31 in root epidermal cells where micro-clusters of interchromatin granules were also observed by EM. Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium -mediated transient expression. When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells. Treatments with -amanitin, okadaic acid, staurosporine or heat shock induced the speckles to reorganize. Furthermore, we generated stable Arabidopsis transgenics expressing atRSp31-GFP. The distribution of the fusion protein was identical to that of endogenous atRSp31. Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.Communicated by P. ShawS. Docquier and P. Motte contributed equally to this work     

Coiled Bodies in the Meristematic Cells of the Root of Lupinus luteus L.

The nature of nucleolar associate bodies in the meristematic cells of the root of Lupinus luteus L. was investigated using immunocytochemical methods, in situ hybridisation with light, confocal, and electron microscopy. The nuclear bodies of lupin proved to be structures containing fibrillarin and coilin, but devoid of rRNA and DNA, like animal coiled bodies (CBs). In lupin cells we have observed the occurrence of small nuclear ribonucleoprotein (snRNP) in the cytoplasm, in nucleoplasm, CBs and in nucleoli. This type of snRNP localisation pattern is in agreement with recently presented models of the small nuclear RNA cycle. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Movement of Coiled Bodies Visualized in Living Plant Cells by the Green Fluorescent Protein

Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3'- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3'-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"-green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2B" gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body.     

In vivo analysis of Cajal body movement, separation, and joining in live human cells

Cajal bodies (also known as coiled bodies) are subnuclear organelles that contain specific nuclear antigens, including splicing small nuclear ribonucleoproteins (snRNPs) and a subset of nucleolar proteins. Cajal bodies are localized in the nucleoplasm and are often found at the nucleolar periphery. We have constructed a stable HeLa cell line, HeLa(GFP-coilin), that expresses the Cajal body marker protein, p80 coilin, fused to the green fluorescent protein (GFP-coilin). The localization pattern and biochemical properties of the GFP-coilin fusion protein are identical to the endogenous p80 coilin. Time-lapse recordings on 63 nuclei of HeLa(GFP-coilin) cells showed that all Cajal bodies move within the nucleoplasm. Movements included translocations through the nucleoplasm, joining of bodies to form larger structures, and separation of smaller bodies from larger Cajal bodies. Also, we observed Cajal bodies moving to and from nucleoli. The data suggest that there may be at least two classes of Cajal bodies that differ in their size, antigen composition, and dynamic behavior. The smaller size class shows more frequent and faster rates of movement, up to 0.9 microm/min. The GFP-coilin protein is dynamically associated with Cajal bodies as shown by changes in their fluorescence intensity over time. This study reveals an unexpectedly high level of movement and interactions of nuclear bodies in human cells and suggests that these movements may be driven, at least in part, by regulated mechanisms.     

Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are required for pre-mRNA splicing throughout the nucleoplasm, yet snRNPs also concentrate in Cajal bodies (CBs). To address a proposed role of CBs in snRNP assembly, we have used fluorescence resonance energy transfer (FRET) microscopy to investigate the subnuclear distribution of specific snRNP intermediates. Two distinct complexes containing the protein SART3 (p110), required for U4/U6 snRNP assembly, were localized: SART3.U6 snRNP and SART3.U4/U6 snRNP. These complexes segregated to different nuclear compartments, with SART3.U6 snRNPs exclusively in the nucleoplasm and SART3.U4/U6 snRNPs preferentially in CBs. Mutant cells lacking the CB-specific protein coilin and consequently lacking CBs exhibited increased nucleoplasmic levels of SART3.U4/U6 snRNP complexes. Reconstitution of CBs in these cells by expression of exogenous coilin restored accumulation of SART3.U4/U6 snRNP in CBs. Thus, while some U4/U6 snRNP assembly can occur in the nucleoplasm, these data provide evidence that SART3.U6 snRNPs form in the nucleoplasm and translocate to CBs where U4/U6 snRNP assembly occurs.