Cytochemical observations of the coiled bodies in neurons of rat sympathetic ganglia

Summary Coiled bodies were investigated by means of ultrastructural cytochemistry. Preferential staining methods for localization of various proteins (ribonucleoproteins, basic proteins, phosphoproteins and glycoproteins) and DNA were applied. The results of cytochemical tests revealed that coiled bodies have a proteinaceous nature. They are composed of ribonucleoproteins, probably of nucleolar origin. They also contain phosphoproteins and glycoproteins but lack cytochemically detectable DNA. Coiled bodies present ultrastructural and cytochemical characteristics similar to the fibrillar part of the nucleous and to the interchromatin granules. The origin and possible functional role of coiled bodies are briefly discussed.     

Ultrastructural and cytochemical studies of nucleolus-like bodies in neurons of rat sympathetic ganglia

Intracytoplasmic fibrillar inclusions, generally referred to as nucleolus-like bodies (NLBs) were studied by means of ultrastructural cytochemistry. The structure of these bodies was visualized by several different staining procedures: conventional electron microscopy and preferential staining methods for localization of various proteins including ribonucleoproteins, basic proteins, glycoproteins and phosphorylated proteins. The results of the cytochemical tests indicate that NLBs have an essentially proteinaceous nature. They consist of ribonucleoproteins, basic proteins and glycoproteins but do not contain phosphorylated proteins. These findings suggest that NLBs are, at least partially, of the same nature as nucleoli and coiled bodies. The origin of NLBs and their possible functional role is briefly discussed.     

Ultrastructural and cytochemical studies of nucleolus-like bodies in neurons of rat sympathetic ganglia

Summary Intracytoplasmic fibrillar inclusions, generally referred to as nucleolus-like bodies (NLBs) were studied by means of ultrastructural cytochemistry. The structure of these bodies was visualized by several different staining procedures: conventional electron microscopy and preferential staining methods for localization of various proteins including ribonucleoproteins, basic proteins, glycoproteins and phosphorylated proteins. The results of the cytochemical tests indicate that NLBs have an essentially proteinaceous nature. They consist of ribonucleoproteins, basic proteins and glycoproteins but do not contain phosphorylated proteins. These findings suggest that NLBs are, at least partially, of the same nature as nucleoli and coiled bodies. The origin of NLBs and their possible functional role is briefly discussed.     

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.     

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.     

Structural components of the nuclear body in nuclei of Allium cepa cells

INTRODUCTIONSmall spherical nucleax bodies have long beenobserved in both hamal and plain interphasenuclei. In the case of animal cells, these nuclear bodies are generally called coiled bodies[1].As for plant cells, they have been vaxiously described as coiled bodies, ~somes, micronucleolior nucloolus-associated bodies because they sometimes appeared in the vicinity of nucleolusl2-4].Eaxly cytologists noted that nuclear bodies in platcells appeared as a tangle of coiled threads forming a …     

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.     

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.     

Coiled‐coils: The long and short of it

Coiled‐coils are found in proteins throughout all three kingdoms of life. Coiled‐coil domains of some proteins are almost invariant in sequence and length, betraying a structural and functional role for amino acids along the entire length of the coiled‐coil. Other coiled‐coils are divergent in sequence, but conserved in length, thereby functioning as molecular spacers. In this capacity, coiled‐coil proteins influence the architecture of organelles such as centrioles and the Golgi, as well as permit the tethering of transport vesicles. Specialized coiled‐coils, such as those found in motor proteins, are capable of propagating conformational changes along their length that regulate cargo binding and motor processivity. Coiled‐coil domains have also been identified in enzymes, where they function as molecular rulers, positioning catalytic activities at fixed distances. Finally, while coiled‐coils have been extensively discussed for their potential to nucleate and scaffold large macromolecular complexes, structural evidence to substantiate this claim is relatively scarce.     

Further ultrastructural characterization of the intranuclear ring-shaped bodies of the plant Lacandonia schismatica

Ring-shaped bodies are found in the nucleus of Lacandonia schismatica, a rare plant with the sexual organs inverted. They are 0.5-microm-diameter structures that present an electron-dense external ring surrounding a central core. Ultrastructural studies indicate that these bodies contain RNA. The external ring is labeled with antibodies against small nuclear ribonucleoproteins, suggesting that they may be involved in pre-mRNA metabolism. In the present work we further characterized these intranuclear ring-shaped structures by serial-sectioning analysis. Moreover, we tested the presence of additional molecular elements related to pre-mRNA metabolism, such as SR proteins and poly(A)(+) RNA, using immunoelectron microscopy and ultrastructural in situ hybridization. Our results show that these nuclear bodies are spherical. They contain SR proteins involved in splicing and postsplicing events and little to no poly(A)(+) RNA. We also found similar nuclear bodies in other plant and animal species. Therefore, ring-shaped bodies in L. schismatica are spherical, highly compartmentalized nuclear structures that may be involved in pre-mRNA metabolism.     

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.     

The nucleolus: a site of ribonucleoprotein maturation

The nucleolus is the site of ribosomal RNA synthesis, processing and ribosome maturation. Various small ribonucleoproteins also undergo maturation in the nucleolus, involving RNA modification and RNA-protein assembly. Such steps and other activities of small ribonucleoproteins also take place in Cajal (coiled) bodies. Events of ribosome biogenesis are found solely in the nucleolus, which is the final destination of small nucleolar RNAs after their traffic through Cajal bodies. However, nucleoli are just a stopping point in the intricate cellular traffic for small nuclear RNAs and other ribonucleoproteins.     

The Evolution and Structure Prediction of Coiled Coils across All Genomes

Coiled coils are α-helical interactions found in many natural proteins. Various sequence-based coiled-coil predictors are available, but key issues remain: oligomeric state and protein-protein interface prediction and extension to all genomes. We present SpiriCoil (http://supfam.org/SUPERFAMILY/spiricoil), which is based on a novel approach to the coiled-coil prediction problem for coiled coils that fall into known superfamilies: hundreds of hidden Markov models representing coiled-coil-containing domain families. Using whole domains gives the advantage that sequences flanking the coiled coils help. SpiriCoil performs at least as well as existing methods at detecting coiled coils and significantly advances the state of the art for oligomer state prediction. SpiriCoil has been run on over 16 million sequences, including all completely sequenced genomes (more than 1200), and a resulting Web interface supplies data downloads, alignments, scores, oligomeric state classifications, three-dimensional homology models and visualisation. This has allowed, for the first time, a genomewide analysis of coiled-coil evolution. We found that coiled coils have arisen independently de novo well over a hundred times, and these are observed in 16 different oligomeric states. Coiled coils in almost all oligomeric states were present in the last universal common ancestor of life. The vast majority of occasions that individual coiled coils have arisen de novo were before the last universal common ancestor of life; we do, however, observe scattered instances throughout subsequent evolutionary history, mostly in the formation of the eukaryote superkingdom. Coiled coils do not change their oligomeric state over evolution and did not evolve from the rearrangement of existing helices in proteins; coiled coils were forged in unison with the fold of the whole protein.     

Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway.

BACKGROUND: Small nuclear ribonucleoproteins (snRNPs), which are essential components of the mRNA splicing machinery, comprise small nuclear RNAs, each complexed with a set of proteins. An early event in the maturation of snRNPs is the binding of the core proteins - the Sm proteins - to snRNAs in the cytoplasm followed by nuclear import. Immunolabelling with antibodies against Sm proteins shows that splicing snRNPs have a complex steady-state localisation within the nucleus, the result of the association of snRNPs with several distinct subnuclear structures. These include speckles, coiled bodies and nucleoli, in addition to a diffuse nucleoplasmic compartment. The reasons for snRNP accumulation in these different structures are unclear. RESULTS: When mammalian cells were microinjected with plasmids encoding the Sm proteins B, D1 and E, each tagged with either the green fluorescent protein (GFP) or yellow-shifted GFP (YFP), a pulse of expression of the tagged proteins was observed. In each case, the newly synthesised GFP/YFP-labelled snRNPs accumulated first in coiled bodies and nucleoli, and later in nuclear speckles. Mature snRNPs localised immediately to speckles upon entering the nucleus after cell division. CONCLUSIONS: The complex nuclear localisation of splicing snRNPs results, at least in part, from a specific pathway for newly assembled snRNPs. The data demonstrate that the distribution of snRNPs between coiled bodies and speckles is directed and not random.     

Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50-100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2-3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen-antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.