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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.

Animals↗

Cajal bodies (coiled bodies) in the nuclei of the house cricket (Acheta domesticus) oocytes.

The morphology and fine structure of Cajal bodies (coiled bodies, CB) in the germinal vesicles (oocyte nuclei) of the house cricket, Acheta domesticus have been analyzed. It is shown that in the studied species CBs arise as early as in the youngest previtellogenic oocytes, and are located next to or within aggregations of multiple nucleoli. Surprisingly, two morphological types of CBs have been found in the analyzed specimens. On the basis of EM studies we suggest that they represent subsequent developmental stages of CB morphogenesis.

Animals↗

Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes.

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem-loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

Animals↗

Nopp140 functions as a molecular link between the nucleolus and the coiled bodies.

Coiled bodies are small nuclear organelles that are highly enriched in small nuclear RNAs, and that have long been thought to be associated with the nucleolus. Here we use mutational analysis, transient transfections, and the yeast two-hybrid system to show that the nucleolar phosphoprotein Nopp140 functions as a molecular link between the two prominent nuclear organelles. Exogenous Nopp140 accumulated in the nucleolus rapidly, but only after a lag phase in coiled bodies, suggesting a pathway between the two organelles. The expression of partial Nopp140 constructs exerted dominant negative effects on the endogenous Nopp140 by chasing it and other antigens that were common to both organelles out of the nucleolus. The alternating positively and negatively charged repeat domain of Nopp140 was required for targeting to both organelles. In addition, partial Nopp140 constructs caused formation of novel structures in the nucleoplasm and, in the case of the conserved carboxy terminus, led to the dispersal of coiled bodies. As a final link, we identified the coiled body-specific protein p80 coilin in a yeast two-hybrid screen with Nopp140. The interaction of the two proteins was confirmed by coimmunoprecipitation. Taken together, Nopp140 appeared to shuttle between the nucleolus and the coiled bodies, and to chaperone the transport of other molecules.

3T3 Cells↗

Value of endorectal coil versus body coil MRI for diagnosis of recurrent pelvic malignancies.

BACKGROUND: To compare endorectal coil magnetic resonance imaging (MRI) with body coil MRI in detecting local recurrence of gynecologic tumors and prostate and rectal cancers. METHODS: Forty-six patients with suspected recurrent pelvic malignancies (13 gynecologic, 15 prostatic, and 18 anorectal primaries) were enrolled in the study. Axial T1- and T2-weighted body coil images and T2- and contrast-enhanced T1-weighted axial endorectal coil images were obtained on a 1.5 T system. Results of the MR examinations were compared with histological findings and follow-up examinations with respect to the diagnostic accuracy and diagnostic confidence for assessment or exclusion of local recurrence. RESULTS: Recurrent disease was histologically confirmed in eight patients with primary gynecologic malignancies, seven with suspected prostatic recurrence, and seven with suspected anorectal recurrence. Overall, accuracy of body coil MRI was 67% for gynecologic tumors, 36% for prostatic recurrences, and 59% for rectal recurrences. T2- and contrast-enhanced T1-weighted endorectal sequences yielded similar results, with an accuracy of 73% for depiction of gynecologic recurrence, 77% for prostatic recurrence, and 77% for rectal recurrence. The difference in accuracy between body coil and endorectal coil examinations was statistically significant (p < 0. 05) only for prostatic cancer. Diagnostic confidence was, however, significantly improved (p < 0.05) in all tumors (T2-weighted endorectal coil examination was superior to T2-weighted body coil images in 71% of cases). CONCLUSION: Although the results of endorectal coil MRI are only slightly superior to those of body coil MRI for the detection of recurrent gynecologic and anorectal tumors, diagnosis can be made with greater diagnostic confidence in many cases. For detection of prostatic recurrence, endorectal MRI is highly recommended.

Adult↗

Protein heterogeneity in the coiled body compartment.

Coiled bodies are ubiquitous nuclear inclusions of unknown function. Although a considerable list of coiled body components has been assembled in recent years leading to several functional hypotheses, none have yet been borne out by experimentation. Pinpointing coiled body function is difficult in part because each known component molecule has been shown to be present at other sites in the nucleus. Using probes to individual coiled body molecules is therefore likely to yield ambiguous results. From direct observation of coiled body behavior we know that they are dynamic structures, changing in content, size, and number under different physiological conditions. In our studies, we have found that the number of coiled bodies in mammalian endothelial cells is relatively high. Depending on phenotype, quiescent or angiogenic, endothelial cells can average as few as 4 or as many as 15 coiled bodies per nucleus (as opposed to 2 or 3 for most cell types). This can provide certain advantages in the analysis of their dynamics and composition. Moreover, expression of the coiled body protein, pigpen, is sharply regulated as endothelial cells toggle back and forth between the two phenotypes. Using the endothelial cell system, we present several new observations in this report on the dynamics of coiled bodies and their constituent proteins and reinforce prior observations that we consider important but understated in the literature. With antibodies to p80-coilin, pigpen, and fibrillarin, we show that there may be heterogeneity in the coiled body population of individual cells. We demonstrate that the coiled body marker protein p80-coilin can also be found distributed in the nucleoplasm and in apparent association with the nuclear envelope. This suggests that coilin could play a role in some aspect of nucleocytoplasmic exchange. Finally, we correlate the presence of pigpen in a diffuse nucleoplasmic pool with the expression of a phosphatase-sensitive epitope, indicating that subnuclear localization may depend upon the phosphorylation state of the protein. Our results suggest to us that a viewpoint of coiled bodies as part of a fluid trafficking network may be helpful in discerning their cellular functions.

Animals↗

Nuclear gems and Cajal (coiled) bodies in fetal tissues: nucleolar distribution of the spinal muscular atrophy protein, SMN.

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.

Adult↗

U2 and U1 snRNA gene loci associate with coiled bodies.

The coiled bodies are nuclear structures rich in a variety of nuclear and nucleolar components including snRNAs. We have investigated the possibility that coiled bodies may associate with snRNA genes and report here that there is a high degree of association between U2 and U1 genes with a subset of coiled bodies. As investigated in human HeLa cells grown in monolayer culture, about 75% of nuclei had at least one U2 gene associated with a coiled body, and 45% had at least one U1 locus associated. In another suspension-grown HeLa cell strain, 92% of cells showed associated of one or more U2 genes with coiled bodies. In contrast to the U2 and U1 gene associations, a locus closely linked to the U2 gene cluster appeared associated with a coiled body only in 10% of cells. Associated snRNA gene signals were repeatedly positioned at the edge of the coiled body. Thus, this associated was highly nonrandom and spatially precise. Our analysis revealed a much higher frequency of association for closely spaced "doublet" U2 gene signals, with over 80% of paired signals associated as opposed to 35% for single U2 signals. This finding, coupled with the fact that not all genes were associated in all cells, suggested the possibility of a cell-cycle-dependent, possibly S-phase, association. However, an analysis of S- and non-S-phase cells using BrdU incorporation or cell synchronization did not indicate an increased level of association in S-phase. These and other results suggested that a substantial fraction of paired U2 signals represented association of U2 genes on homologous chromosomes rather than only replicated DNA. Furthermore, triple label analysis showed that in a significant fraction of cells U1 and U2 genes were both associated with the same coiled body. U1 and U2 genes were closely paired in approximately 20% of cells, over 60% of which were associated with a readily identifiable coiled body. This finding raises the possibility that multiple genes of a particular class may be in association with each coiled body. Thus, the coiled body may be a dynamic structure which transiently interacts with or is formed by one or more specific genetic loci, possibly carrying out some function related to their expression.

Alleles↗

Coiled bodies and U2 snRNA genes adjacent to coiled bodies are enriched in factors required for snRNA transcription.

A significant percentage of the gene clusters that contain the human genes for U1 small nuclear RNA (snRNA) or for U2 snRNA have been found associated with small nuclear domains, known as coiled bodies. We show here, by immunofluorescent labeling of human cells, that coiled bodies are enriched in factors required for the transcription of these snRNA genes. The 45-kDa gamma-subunit of the transcription factor, proximal element sequence-binding transcription factor (PTF), which is specific for the snRNA genes, was found in high concentrations in coiled bodies, along with the general transcription factor TATA-box binding protein and a subset of RNA polymerase II. We show that the transcription factors and RNA polymerase II are concentrated in irregularly shaped domains that not only overlap with coiled bodies but also extend to their immediate surroundings. Fluorescent in situ hybridization showed that these domains can overlap with U2 snRNA genes adjacent to coiled bodies. In addition, we found the domains to contain newly synthesized RNA, visualized by 5-bromo-uridine triphosphate labeling. Our data suggest that coiled bodies are involved in the expression of snRNA genes, which leads us to propose the model that coiled bodies are associated with snRNA genes to facilitate and regulate their transcription. These findings point to a general principle of higher order organization of gene expression in the nucleus.

Caco-2 Cells↗

Microinjection of anti-coilin antibodies affects the structure of coiled bodies.

The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.

Antibodies, Monoclonal↗

Identification of a new coiled body component.

Coiled bodies are small, round nuclear inclusions that have been identified in many somatic cell types. Equivalent structures are found in the germinal vesicles of amphibian and insect oocytes, known respectively as sphere organelles and Binnenkörper. Their functions are not known, but their molecular composition is being brought to light. In addition to the nucleolar protein, fibrillarin, coiled bodies contain DNA topoisomerase I and an array of RNA processing molecules characteristic of spliceosomes. One coiled body protein absent from nucleoli and spliceosomes, known as p80-coilin, has also been described. We have now identified pigpen, a new member of the EWS family of proteins, as a second protein enriched in coiled bodies. In an earlier report we found that pigpen's structure and expression pattern were suggestive of a role in endothelial cell proliferation and differentiation. In this brief report we characterize pigpen's nuclear compartment and describe its reorganization during mitosis.

Animals↗

Identification of a new coiled body component.

Coiled bodies are small, round nuclear inclusions that have been identified in many somatic cell types. Equivalent structures are found in the germinal vesicles of amphibian and insect oocytes, known respectively as sphere organelles and Binnenkörper. Their functions are not known, but their molecular composition is being brought to light. In addition to the nucleolar protein, fibrillarin, coiled bodies contain DNA topoisomerase I and an array of RNA processing molecules characteristic of spliceosomes. One coiled body protein absent from nucleoli and spliceosomes, known as p80-coilin, has also been described. We have now identified pigpen, a new member of the EWS family of proteins, as a second protein enriched in coiled bodies. In an earlier report we found that pigpen's structure and expression pattern were suggestive of a role in endothelial cell proliferation and differentiation. In this brief report we characterize pigpen's nuclear compartment and describe its reorganization during mitosis.

Animals↗

Histopathology of uterine cervical carcinoma: diagnostic comparison of endorectal surface coil and standard body coil MRI.

OBJECTIVE: We compared body coil (BC) MR images and endorectal surface coil (ERC) images with respect to the visualization of the cervix and adjacent tissues and the histopathologic correlations of the imaging findings. MATERIALS AND METHODS: Twenty-one patients with cervical carcinoma underwent MRI using both coils for evaluation of the cervix and adjacent tissues. In all patients, surgical confirmation of the diagnosis was obtained after imaging. RESULTS: The ERC images were superior to BC images for visualization of the cervix, parametrium, anterior vaginal wall, and posterior bladder wall (p < 0.005). The accuracy of detecting parametrial invasion by ERC and BC imaging was, respectively, 95 and 79% (p < 0.05), while the differences of accuracy between BC and ERC imaging were not statistically significant in staging or in assessing for invasion to corpus, vagina, bladder, and rectum. All the false-positive sites of parametrial invasion on BC images were diagnosed correctly on ERC images. CONCLUSION: Endorectal surface coil imaging is useful in assessing the extent of cervical carcinoma, especially parametrial invasion, because it can delineate the tumor and the normal tissues in detail.

Adult↗

Assembly of snRNP-containing coiled bodies is regulated in interphase and mitosis--evidence that the coiled body is a kinetic nuclear structure.

Coiled bodies (CBs) are nuclear organelles in which splicing snRNPs concentrate. While CBs are sometimes observed in association with the nucleolar periphery, they are shown not to contain 5S or 28S rRNA or the U3 snoRNA. This argues against CBs playing a role in rRNA maturation or transport as previously suggested. We present evidence here that CBs are kinetic structures and demonstrate that the formation of snRNP-containing CBs is regulated in interphase and mitosis. The coiled body antigen, p80 coilin, was present in all cell types studied, even when CBs were not prominent. Striking changes in the formation of CBs could be induced by changes in cellular growth temperature without a concomitant change in the intracellular p80 coilin level. During mitosis, CBs disassemble, coinciding with a mitotic-specific phosphorylation of p80 coilin. Coilin is shown to be a phosphoprotein that is phosphorylated on at least two additional sites during mitosis. CBs reform in daughter nuclei after a lag period during which they are not detected. CBs are thus, dynamic nuclear organelles and we propose that cycling interactions of splicing snRNPs with CBs may be important for their participation in the processing or transport of pre-mRNA in mammalian cells.

Animals↗

The coiled body.

The coiled body is a nuclear organelle that contains snRNPs involved in splicing, the non-snRNP splicing factor U2AF and the nucleolar protein fibrillarin. It is highly conserved in evolution and is present in both animal and plant cells. The coiled body is a dynamic structure that can undergo regulated cycles of assembly and disassembly during interphase and mitosis and it may represent a distinct metabolic compartment within the nucleus.

Journal Article↗

Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus.

Coiled bodies are conserved subnuclear domains found in both plant and animal cells. They contain a subset of splicing snRNPs and several nucleolar antigens, including Nopp140 and fibrillarin. In addition, autoimmune patient sera have identified a coiled body specific protein, called p80 coilin. In this study we show that p80 coilin is ubiquitously expressed in human tissues. The full-length human p80 coilin protein correctly localizes in coiled bodies when exogenously expressed in HeLa cells using a transient transfection assay. Mutational analysis identifies separate domains in the p80 coilin protein that differentially affect its subnuclear localization. The data show that p80 coilin has a nuclear localization signal, but this is not sufficient to target the protein to coiled bodies. The results indicate that localization in coiled bodies is not determined by a simple motif analogous to the NLS motifs involved in nuclear import. A specific carboxy-terminal deletion in p80 coilin results in the formation of pseudo-coiled bodies that are unable to recruit splicing snRNPs. This causes a loss of endogenous coiled bodies. A separate class of mutant coilin proteins are shown to localize in fibrillar structures that surround nucleoli. These mutants also lead to loss of endogenous coiled bodies, produce a dramatic disruption of nucleolar architecture and cause a specific segregation of nucleolar antigens. The structural change in nucleoli is accompanied by the loss of RNA polymerase I activity. These data indicate that p80 coilin plays an important role in subnuclear organization and suggest that there may be a functional interaction between coiled bodies and nucleoli.

Amino Acid Sequence↗

Preoperative staging of cervical carcinoma: phased array coil fast spin-echo versus body coil spin-echo T2-weighted MR imaging.

OBJECTIVE: This study was performed to compare the diagnostic efficacy of MR imaging in the preoperative evaluation of invasive cervical cancer using the pelvic phased array coil in combination with fast spin-echo T2-weighted imaging and the body coil in combination with conventional spin-echo T2-weighted imaging. MATERIALS AND METHODS: Ninety-four women (22-68 years old) with invasive cervical cancer underwent MR imaging (at 1.5 T) using a body coil conventional spin-echo protocol (n = 62) or a phased array coil fast spin-echo protocol (n = 32). Imaging preceded surgery by no more than 5 weeks. MR images were evaluated for tumor size, local stage, and nodal metastasis using surgical pathology as the standard of reference. RESULTS: Overall staging accuracy for the body coil conventional spin-echo protocol (89%) was not significantly different from that of the phased array coil fast spin-echo protocol (91%). Both techniques also achieved similar accuracy in diagnosing parametrial invasion (95% versus 94%) and lymph node metastases (85% versus 91%) and in tumor sizing (correlation coefficient, .93 versus .94). CONCLUSION: In the preoperative staging of cervical carcinoma by MR imaging, both the newer (phased array coil fast spin-echo protocol) and the older (body coil conventional spin-echo protocol) techniques achieved similarly high accuracies in local staging, assessment of parametrial invasion, and evaluation of tumor size. Decreased imaging time and increased image resolution are advantages of the newer technique, although in our series they did not increase staging accuracy.

Adult↗

Distribution of splicing proteins and putative coiled bodies during pollen development and androgenesis in Brassica napus L.

Small nuclear ribonucleoprotein particles (snRNPs) are subunits of splicing complexes, which show a transcription-dependent localization pattern. We have analyzed the labelling pattern of snRNPs during pollen development and microspore and pollen embryogenesis in Brassica napus with an antibody which recognizes protein D of U1, U2, U4, U5, and U6 snRNPs. It was found that nuclei were labelled almost uniformly for snRNPs in microspores and young bicellular pollen. In the generative nuclei of late-bicellular pollen and in the vegetative nuclei and sperm nuclei of mature pollen no snRNPs could be detected. The snRNP-positive nuclei contained mostly one or two brightly labelled nuclear bodies, most likely coiled bodies, often closely related to the nucleolus. These nuclear bodies increased in size from 0.5 micron in nuclei of young microspores up to 2 microns in nuclei of late microspores and the vegetative nucleus of early-bicellular pollen. Also their number increased during these developmental stages. After induction of embryogenesis the size of the coiled bodies decreased to about 0.5 micron and in several occasions the coiled body was found free in the nucleoplasm, away from the nucleolus. The results support the idea that the size and number of coiled bodies coincide with changes in general nuclear activity. They also indicate that, in nuclei of Brassica napus, at least assembly and disassembly of coiled bodies takes place in the nucleoplasm, whereas mature coiled bodies are located adjacent to the nucleolus.

Brassica napus↗