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     

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.     

Characterization of human myocardial fibroblasts immortalized by HPV16 E6--E7 genes

SMN, the affected protein in spinal muscular atrophy (SMA), is a cytoplasmic protein that also occurs in nuclear structures called "gems" and is involved in snRNP maturation. Coilin-p80 is a marker protein for nuclear Cajal bodies (coiled bodies; CBs) which are also involved in snRNP maturation, storage or transport. We now show that gems and CBs are present in all fetal tissues, even those that lack gems/CBs in the adult. Most gems and CBs occur as separate nuclear structures in fetal tissues, but their colocalization increases with fetal age and is almost complete in the adult. In adult tissues, up to half of all gems/CBs are inside the nucleolus, whereas in cultured cells they are almost exclusively nucleoplasmic. The nucleolar SMN is often more diffusely distributed, compared with nucleoplasmic gems. Up to 30% of cells in fetal tissues have SMN distributed throughout the nucleolus, instead of forming gems in the nucleoplasm. The results suggest a function for gems distinct from Cajal bodies in fetal nuclei and a nucleolar function for SMN. Spinal cord, the affected tissue in SMA, behaves differently in several respects. In both fetal and adult motor neurons, many gems/CBs occur as larger bodies closely associated with the nucleolar perimeter. Uniquely in motor neurons, gems/CBs are more numerous in adult than in fetal stages and colocalization of gems and CBs occurs earlier in development. These unusual features of motor neurons may relate to their special sensitivity to reduced SMN levels in SMA patients.     

Nucleoli undergo structural and molecular modifications during hibernation

The nucleolus is a very dynamic structure able rapidly to adapt its activity to the cellular metabolic state. An interesting physiological model characterized by drastic modifications of cellular metabolism is represented by hibernating animals. In the present study we investigated the hepatocyte nuclei of euthermic and hibernating edible dormice (Glis glis) with the aim of revealing, by means of ultrastructural and immunocytochemical analyses, possible modifications of nucleolar components during hibernation. Our observations demonstrate that, in deep hibernation, nucleoli undergo structural and molecular modifications: (a) they show numerous nucleoplasmic invaginations and clumps of dense fibrillar component extend from the nucleolar surface; (b) they are frequently in contact with coiled bodies and fibro-granular material, two nuclear bodies usually occurring in the nucleoplasm; (c) the dense fibrillar component contains significant amounts of small nuclear ribonucleoproteins, splicing factors usually distributed in the nucleoplasm. Taken together, these results suggest that during hibernation complex relationships are established between the nucleolus and nucleoplasm, probably related to functional activities peculiar to this physiological phase. However, since no evident nucleolar modification was found in early hibernating dormice, it seems likely that the particular structural and molecular arrangement of nucleoli establishes progressively during hibernation, becoming evident only in the deepest phase, and then disappears upon arousal.     

Cytochemical and immunocytochemical study of coiled bodies in different cultured cell lines

We analyzed by different cytochemical and immunocytochemical approaches the biochemical compositon of coiled bodies in three different cultured cell lines. Coiled bodies are stained by the AgNOR staining method and by the EDTA regressive staining method preferential for ribonucleoprotein (RNP). Using the in situ polyadenylate nucleotidyl transferase-immunogold technique or anti-RNA antibodies, we decisively demonstrated the presence of appreciable amounts of RNA in coiled bodies. Neither the in situ terminal deoxynucleotidyl transferase-immunogold technique nor anti-DNA antibodies revealed any DNA in coiled bodies. Coiled bodies thus appear as distinct regions of cell nuclei involved in some steps of RNA metabolism but not directly in RNA synthesis. Their relationships with the dense fibrillar component of the nucleolus and with interchromatin granule clusters are discussed.     

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     

Three structures associated with the nucleolus in male rat germinal cells: round body, coiled body, and "nubecula" and general presence of round body at male meiosis.

In addition to chromosomes and nucleoli, three structures, i.e., round body, coiled body, and nubecula, are encountered in the nucleus during the meiotic prophase in male rats. These structures have been examined by electron microscopy in random and serial sections. The round body is a finely fibrillar, proteinaceous structure closely associated with the granular component of a nucleolus in rat spermatocytes and young spermatids. A similar structure has been observed in man, the monkey Macaca mulatta, the gastropod Achatina fulica, and the insect Locusta migratoria. Together with evidence from the literature, these results support the view that the round body is of general occurrence in the male meiocytes of eukaryotes and may, therefore, play a role in meiosis. The coiled body is a group of electron-dense elements called "coils", which average 35 nm in width, except after mid-pachytene when their size almost doubles. The coils are composed of 2-nm-wide filaments and 8 to 10-nm-wide granules, both of which are ribonucleoprotein. The coiled bodies are interpreted to be groups of "spliceosomes", that is, structures containing heterogeneous RNA and small nuclear RNA. A remarkable feature of the coiled body is its temporary disappearance at early pachytene and its reappearance at late pachytene, possibly due to drastic changes in the turnover rate of its component RNAs. The nubecula is a newly identified nuclear inclusion, composed of weakly staining threads loosely organized into a 560 nm-wide spheroid. It has been observed only in early pachytene nuclei.     

The relationship between SMN, the spinal muscular atrophy protein, and nuclear coiled bodies in differentiated tissues and cultured cells

The spinal muscular atrophy protein, SMN, is a cytoplasmic protein that is also found in distinct nuclear structures called "gems." Gems are closely associated with nuclear coiled bodies and both may have a direct role in snRNP maturation and pre-RNA splicing. There has been some controversy over whether gems and coiled bodies colocalize or form adjacent/independent structures in HeLa and other cultured cells. Using a new panel of antibodies against SMN and antibodies against coilin-p80, a systematic and quantitative study of adult differentiated tissues has shown that gems always colocalize with coiled bodies. In some tissues, a small proportion of coiled bodies (<10%) had no SMN, but independent or adjacent gems were not found. The most striking observation, however, was that many cell types appear to have neither gems nor coiled bodies (e.g., cardiac and smooth muscle, blood vessels, stomach, and spleen) and this expression pattern is conserved across human, rabbit, and pig species. This shows that assembly of distinct nuclear bodies is not essential for RNA splicing and supports the view that they may be storage sites for reserves of essential proteins and snRNPs. Overexpression of SMN in COS-7 cells produced supernumerary nuclear bodies, most of which also contained coilin-p80, confirming the close relationship between gems and coiled bodies. However, when SMN is reduced to very low levels in type I SMA fibroblasts, coiled bodies are still formed. Overall, the data suggest that gem/coiled body formation is not determined by high cytoplasmic SMN concentrations or high metabolic activity alone and that a differentiation-specific factor may control their formation.     

Nuclear domains enriched in RNA 3'-processing factors associate with coiled bodies and histone genes in a cell cycle-dependent manner

Nuclear domains, called cleavage bodies, are enriched in the RNA 3'-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle-dependent expression of histone genes.     

Association between the nucleolus and the coiled body

By means of light and electron microscopic immunocytochemistry, we have localized p80-coilin, a specific protein marker for coiled bodies, in mammalian cell lines as well as in primary rat neuron cultures. p80-coilin-stained nuclear bodies, which also contained fibrillarin, could be subsequently silver stained by a method specific for the visualization of nucleolar organizer regions. In cycling cells, most coiled bodies were not associated with nucleoli, whereas in rat neurons such as association was frequent. The treatment of cycling cells with actinomycin D or 5,6-dichloro-1-beta-D-ribo furanosyl-benzimidazole led to nucleolar segregation and/or disintegration, and to an association of p80-coilin staining structures with nucleoli. p80-coilin-positive structures contained fibrillarin in both untreated and treated cells. These results support the opinion that there might be a special association between coiled bodies and nucleoli, particularly in neuronal cells.     

Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Summary Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance.Nucleolus In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as nucleolar shadow, overlaid by a few small irregular bodies of higher electron density than other nuclear contents.Nucleus The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas.Nissl Substance In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed.The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.This investigation was supported by USPHS Grants NB 03114-04, NB 00690-11 and 5 T 1 GM 495 from the National Institutes of Health, Bethesda, Maryland.Acknowledgement. Mrs. Eleanor W. Morris and Mr. Edwin E. Pitsinger, Jr. gave indispensible aid with the management of the cultures and with photographic procedures.     

Atomic force microscopy of the cell nucleus

In mammals and plants, the cell nucleus is organized in dynamic macromolecular domains involved in DNA and RNA metabolism. These domains can be visualized by light and electron microscopy and their composition analyzed by using several cytochemical approaches. They are composed of chromatin or ribonucleoprotein structures as interchromatin and perichromatin fibers and granules, coiled bodies, and nuclear bodies. In plants, DNA arrangement defines chromocentric and reticulated nuclei. We used atomic force microscopy to study the in situ structure of the plant cell nucleus. Samples of the plants Lacandonia schismatica and Ginkgo biloba were prepared as for electron microscopy and unstained semithin sections were mounted on glass slides. For comparison, we also examined entire normal rat kidney cells using the same approach. Samples were scanned with an atomic force microscope working in contact mode. Recognizable images of the nuclear envelope, pores, chromatin, and nucleolus were observed. Reticulated chromatin was observed in L. schismatica. Different textures in the nucleolus of G. biloba were also observed, suggesting the presence of nucleolar subcompartments. The observation of nuclear structure in situ with the atomic force microscope offers a new approach for the analysis of this organelle at high resolution.     

Localization in the nucleolus and coiled bodies of protein subunits of the ribonucleoprotein ribonuclease P.

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors.     

Intranuclear bodies in neurons of the periaqueductal gray matter in the cat.

The nucleoplasm of neurons in the nucleus lateralis of the periqueductal gray matter in the cat contains fibrillar structures which have no limiting membranes. These intranuclear bodies are associated with neither the nucleolus nor the nuclear membrane and have two characteristic forms. The first, the rodlet, is a compact bundle of fibrils 2 to 8 nm in diameter. It is usually elongated in shape although it appears spherical when sectioned transversely. This rod-like structure appears to correspond to Roncoroni's rodlet or the accessory body of Cajal in light microscopy. The second and more commonly observed form is a long slender bundle of five rows of parallel fibrils. Although similar intranuclear structures have frequently been observed in the highly differentiated neurons of the sympathetic ganglia and the retina, this is the first report of their pbesence in the undifferentiated neurons of the isodendritic core of the brainstem.