G-patch domain-containing protein 4 localizes to both the nucleoli and Cajal bodies and regulates cell growth and nucleolar structure.

Ribosome formation occurs in the nucleolus through interaction with various trans-acting factors. Therefore, hundreds of nucleolar proteins have a function in ribosome formation, although the precise function of each nucleolar protein in ribosome formation is largely unclear. We have previously identified an uncharacterized protein, G-patch domain-containing protein 4 (GPATCH4 or G4), as a component of the pre-ribosomes purified with either nucleolin (NCL) or NPM1. In this present study, we sought to clarify the localization and function of G4. We identified that G4 localizes to both the nucleolus and the Cajal body. Although knockdown of G4 did not have a significant effect on pre-ribosomal RNA processing, cell growth did decrease. Interestingly, G4 knockdown also decreased the number of fibrillar center and dense fibrillar component regions inside the nucleolus. This data has identified G4 as a novel nucleolar protein involved in the regulation of cell growth and nucleolar structure.

Low-voltage scanning electron microscopy study of lampbrush chromosomes and nuclear bodies in avian and amphibian oocytes.

Nucleus is a highly compartmentalized part of the cell where the key processes of genome functionality are realized through the formation of non-membranous nuclear domains. Physically nuclear domains appear as liquid droplets with different viscosity stably maintained throughout the interphase or during the long diplotene stage of meiosis. Since nuclear body surface represents boundary between two liquid phases, the ultrastructural surface topography of nuclear domains is of an outstanding interest. The aim of this study was to examine ultrathin surface topography of the amphibian and avian oocyte nuclear structures such as lampbrush chromosomes, nucleoli, histone-locus bodies, Cajal body-like bodies, and the interchromatin granule clusters via low-voltage scanning electron microscopy. Our results demonstrate that nuclear bodies with similar molecular composition may differ dramatically in the surface topography and vice versa, nuclear bodies that do not share common molecular components may possess similar topographical characteristics. We also have analyzed surface distribution of particular nuclear antigens (double stranded DNA, coilin and splicing snRNA) using indirect immunogold labeling with subsequent secondary electron detection of gold nanoparticles. We suggest that ultrastructural surface morphology reflects functional status of a nuclear body.

Fluorescent In Situ Hybridization of Nuclear Bodies in Drosophila melanogaster Ovaries.

Fluorescent in situ hybridization (FISH) is a technique for determining the cytological localization of RNA or DNA molecules. There are many approaches available for generating in situ hybridization probes and conducting the subsequent hybridization steps. Here, we describe a simple and reliable FISH method to label small RNAs (200-500 nucleotides in length) that are enriched in nuclear bodies in Drosophila melanogaster ovaries, such as Cajal bodies (CBs) and histone locus bodies (HLBs). This technique can also be applied to other Drosophila tissues, and to abundant mRNAs such as histone transcripts.

Localization of α-Dystrobrevin in Cajal Bodies and Nucleoli: A New Role for α-Dystrobrevin in the Structure/Stability of the Nucleolus.

α-Dystrobrevin (α-DB) is a cytoplasmic component of the dystrophin-associated complex involved in cell signaling; however, its recently revealed nuclear localization implies a role for this protein in the nucleus. Consistent with this, we demonstrated, in a previous work that α-DB1 isoform associates with the nuclear lamin to maintain nuclei morphology. In this study, we show the distribution of the α-DB2 isoform in different subnuclear compartments of N1E115 neuronal cells, including nucleoli and Cajal bodies, where it colocalizes with B23/nucleophosmin and Nopp140 and with coilin, respectively. Recovery in a pure nucleoli fraction undoubtedly confirms the presence of α-DB2 in the nucleolus. α-DB2 redistributes in a similar fashion to that of fibrillarin and Nopp140 upon actinomycin-mediated disruption of nucleoli and to that of coilin after disorganization of Cajal bodies through ultraviolet-irradiation, with relocalization of the proteins to the corresponding reassembled structures after cessation of the insults, which implies α-DB2 in the plasticity of these nuclear bodies. That localization of α-DB2 in the nucleolus is physiologically relevant is demonstrated by the fact that downregulation of α-DB2 resulted in both altered nucleoli structure and decreased levels of B23/nucleophosmin, fibrillarin, and Nopp140. Since α-DB2 interacts with B23/nucleophosmin and overexpression of the latter protein favors nucleolar accumulation of α-DB2, it appears that targeting of α-DB2 to the nucleolus is dependent on B23/nucleophosmin. In conclusion, we show for the first time localization of α-DB2 in nucleoli and Cajal bodies and provide evidence that α-DB2 is involved in the structure of nucleoli and might modulate nucleolar functions.

Nuclear signs of pre-neurodegeneration.

Nuclear architecture is highly concerted including the organization of chromosome territories and distinct nuclear bodies, such as nucleoli, Cajal bodies, nuclear speckles of splicing factors, and promyelocytic leukemia nuclear bodies, among others. The organization of such nuclear compartments is very dynamic and may represent a sensitive indicator of the functional status of the cell. Here, we describe methodologies that allow isolating discrete cell populations from the brain and the fine observation of nuclear signs that could be insightful predictors of an early neuronal injury in a wide range of neurodegenerative disorders. The tools here described may be of use for the early detection of pre-degenerative processes in neurodegenerative diseases and for validating novel rescue strategies.

Poly(A) RNAs including coding proteins RNAs occur in plant Cajal bodies.

The localisation of poly(A) RNA in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus, Arabidopsis thaliana) nuclei was studied through in situ hybridisation. In both types of nuclei, the amount of poly(A) RNA was much greater in the nucleus than in the cytoplasm. In the nuclei, poly(A) RNA was present in structures resembling nuclear bodies. The molecular composition as well as the characteristic ultrastructure of the bodies containing poly(A) RNA demonstrated that they were Cajal bodies. We showed that some poly(A) RNAs in Cajal bodies code for proteins. However, examination of the localisation of active RNA polymerase II and in situ run-on transcription assays both demonstrated that CBs are not sites of transcription and that BrU-containing RNA accumulates in these structures long after synthesis. In addition, it was demonstrated that accumulation of poly(A) RNA occurs in the nuclei and CBs of hypoxia-treated cells. Our findings indicated that CBs may be involved in the later stages of poly(A) RNA metabolism, playing a role storage or retention.

Immunocytochemical study of YB-1 nuclear distribution in different cell types.

In the present work we studied the distribution of YB-1 in the nuclei of mouse hepatocytes, early embryos and human skin fibroblasts with the use of light and electron microscopy. To reveal YB-1, we applied rat polyclonal antibody against the C-terminal fragment of YB-1 molecule and rabbit polyclonal antibody against full-length YB-1 molecule. YB-1 distribution patterns varied significantly in different cell types. YB-1 was found to be colocalized with RNA polymerase I in mouse hepatocytes and embryos. Besides, YB-1 was revealed in a population of Cajal bodies in 2-cell mouse embryos but not in other cells studied.

Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies.

Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.

Pearls are novel Cajal body-like structures in the Xenopus germinal vesicle that are dependent on RNA pol III transcription.

We have identified novel nuclear bodies, which we call pearls, in the giant oocyte nuclei of Xenopus laevis and Xenopus tropicalis. Pearls are attached to the lampbrush chromosomes at specific loci that are transcribed by RNA polymerase III, and they disappear after inhibition of polymerase III activity. Pearls are enriched for small Cajal body-specific RNAs (scaRNAs), which are guide RNAs that modify specific nucleotides on splicing snRNAs. Surprisingly, snRNAs themselves are not present in pearls, suggesting that pearls are not functionally equivalent to Cajal bodies in other systems, which contain both snRNAs and scaRNAs. We suggest that pearls may function in the processing of RNA polymerase III transcripts, such as tRNA, 5S rRNA, and other short non-coding RNAs.

Progressive supranuclear palsy presenting as primary lateral sclerosis but lacking parkinsonism, gaze palsy, aphasia, or dementia.

We report an autopsy case of progressive supranuclear palsy (PSP) that clinically showed only slowly progressive and symmetric upper motor neuron syndrome over a disease course of 12 years. A female patient initially exhibited dysarthria at the age of 65, followed by gait disturbance and dysphagia. Neurological examination at age 67 disclosed pseudobulbar palsy, spastic gait, hyperreflexia, and presence of bilateral Hoffmann and Babinski signs. However, muscle atrophy, weakness, evidence of denervation on electromyography, vertical gaze palsy, parkinsonism, gait freezing, aphasia, speech apraxia, or dementia was not noted throughout the course. She was clinically diagnosed as having motor neuron disease consistent with so-called primary lateral sclerosis. Pathological examination disclosed histopathological features of PSP, including argyrophilic and tau-positive tufted astrocytes, neurofibrillary tangles, coiled bodies, and thread-like processes in the motor cortex and superior frontal gyrus, and to a lesser degree, in the basal ganglia and brain stem nuclei. In addition, severe fibrillary gliosis was noted in the precentral gyrus and corticospinal tract, being consistent with upper motor neuron syndrome observed in this case. No TAR-DNA binding protein 43-positive lesion, FUS pathology, Bunina body, or Lewy body-like hyaline inclusion was noted in the motor cortex or lower motor neurons. These findings suggest that when tau pathology is prominent in the motor cortex but is minimal in the basal ganglia and brain stem nuclei, a PSP case can lack all classic clinical features of PSP and show only slowly progressive upper motor syndrome, consistent with clinical picture of primary lateral sclerosis.

[Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

Arrangement of nuclear structures is not transmitted through mitosis but is identical in sister cells.

Although it is well known that chromosomes are non-randomly organized during interphase, it is not completely clear whether higher-order chromatin structure is transmitted from mother to daughter cells. Therefore, we addressed the question of how chromatin is rearranged during interphase and whether heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during interphase, especially when histone hyperacetylation was induced or transcription was inhibited. This natural phenomenon can influence the analysis of nuclear arrangement. Using photoconversion of Dendra2-tagged core histone H4 we showed that the distribution of chromatin in daughter interphase nuclei differed from that in mother cells. Similarly, the nuclear distribution of heterochromatin protein 1ß (HP1ß) was not completely identical in mother and daughter cells. However, identity between mother and daughter cells was in many cases evidenced by nucleolar composition. Moreover, morphology of nucleoli, HP1ß protein, Cajal bodies, chromosome territories, and gene transcripts were identical in sister cell nuclei. We conclude that the arrangement of interphase chromatin is not transmitted through mitosis, but the nuclear pattern is identical in naturally synchronized sister cells. It is also necessary to take into account the possibility that cell rotation and the degree of chromatin condensation during functionally specific cell cycle phases might influence our view of nuclear architecture.

[Cajal body in the neuroendocrine neurons of the paleoamygdala].

The article demonstrates the ultrastructure of Cajal body (CB) that was detected during the electron microscopic study of nucleoplasm of the neuroendocrine neurons of the paleoamygdala of the adult Wistar rats in the study of the dynamics of their functional states throughout the estrous cycle. CB is located in the nucleoplasm close to the nucleolus and appears as a polygon structure, having the size of 0.4 x 0.5 microm, consisting of twisted strands of 40 to 60 nm thickness, which are separated from each other by the material of low electron density, obviously, a continuation of the nucleoplasm. Structural association of CB with other nuclear domains--nucleoli, interchromatin granule clusters were not noticed. CB was found in neurons only at the stage of "return to the initial state", which characterizes the completion of the functional activity of neurons. The number of these neurons was increased at the stage of metestrus. They are characterized by a segregation of nucleolar components, indicating the blockade of the protein synthesis. This fact is associated with the restructuring of CB modular organization, caused by the functional state of neurons.

A change of developmental program induces the remodeling of the interchromatin domain during microspore embryogenesis in Brassica napus L.

After a stress treatment, in vitro-cultured pollen changes its normal gametophytic developmental pathway towards embryogenesis producing multicellular embryos from which, finally, haploid and double haploid plants develop. The architecture of the well-organized nuclear functional domains changes in response to DNA replication, RNA transcription, processing and transport dynamics. A number of subnuclear structures present in the interchromatin region (IR, the nuclear domain between chromosome territories) have been shown as involved, either directly or indirectly, in transcriptional regulation. These structures include the interchromatin granule clusters (IGCs), perichromatin fibrils (PFs), Cajal bodies (CBs) and perichromatin granules (PGs). In this work, we present a cytochemical, immunocytochemical, quantitative and morphometric analysis at the light, confocal and electron microscopy levels to characterize the changes in the functional architecture of the nuclear interchromatin domain during two developmental programs followed by the microspore: differentiation to mature pollen grains (transcriptionally inactive), and microspore embryogenesis involving proliferation in the first stages (highly engaged in transcription). Our results revealed characteristic changes in size, shape and distribution of the different interchromatin structures as a consequence of the reprogramming of the microspore, allowing us to relate the remodeling of the interchromatin domain to the variations in transcriptional activities during proliferation and differentiation events, and suggesting that RNA-associated structures could be a regulatory mechanism in the process. In addition, we document the presence of two structurally different types of CBs, and of IGC and CB-associated regions, similar to those present in animal cells, and not yet described in plants.

The dynamics of RNA participation in the vitellogenesis of Rhipicephalus sanguineus ticks Latreille 1806 (Acari:Ixodidae). I. Nucleoli or Cajal bodies?

To understand the morphological and histological aspects of internal systems of ticks has become important matter since these arthropods have an impact in the areas of the economy and public health. In this context, this study has provided morphological data on female germinative cells of Rhipicephalus sanguineus ticks, ectoparasites of dogs that maintain a close relationship with human on a daily basis. Oocytes of engorged females were analyzed, through the PAS reaction (detection of polysaccharides) counterstained by methyl green (detection of RNA) revealing information that allowed to infer for the first time the presence of Cajal bodies, in the germinal vesicles (nuclei) of developing oocytes, as well as showing how the RNA and the polysaccharides are involved in the dynamics of the vitellogenesis in this species.

A conserved WD40 protein binds the Cajal body localization signal of scaRNP particles.

Small Cajal body (CB)-specific RNPs (scaRNPs) function in posttranscriptional modification of small nuclear (sn)RNAs. An RNA element, the CAB box, facilitates CB localization of H/ACA scaRNPs. Using a related element in Drosophila C/D scaRNAs, we purified a fly WD40 repeat protein that UV crosslinks to RNA in a C/D CAB box-dependent manner and associates with C/D and mixed domain C/D-H/ACA scaRNAs. Its human homolog, WDR79, associates with C/D, H/ACA, and mixed domain scaRNAs, as well as with telomerase RNA. WDR79's binding to human H/ACA and mixed domain scaRNAs is CAB box dependent, and its association with mixed domain RNAs also requires the ACA motif, arguing for additional interactions of WDR79 with H/ACA core proteins. We demonstrate a requirement for WDR79 binding in the CB localization of a scaRNA. This and other recent reports establish WDR79 as a central player in the localization and processing of nuclear RNPs.

Universal nuclear domains of somatic and germ cells: some lessons from oocyte interchromatin granule cluster and Cajal body structure and molecular composition.

It is now clear that two prominent nuclear domains, interchromatin granule clusters (IGCs) and Cajal bodies (CBs), contribute to the highly ordered organization of the extrachromosomal space of the cell nucleus. These functional domains represent structurally stable but highly dynamic nuclear organelles enriched in factors that are required for different nuclear activities, especially RNA biogenesis. IGCs are considered to be the main sites for storage, assembly, and/or recycling of the essential spliceosome components. CBs are involved in the biogenesis of several classes of small RNPs as well as the modification of newly assembled small nuclear RNA. We have summarized data on the molecular composition, structure, and functional roles of IGCs and CBs in the nuclei of mammalian somatic cells and oocytes of some animals with a special focus on insects. We have focused on similarities and differences between the IGCs and CBs of oocytes and the well-studied CBs and IGCs of cultured mammalian somatic cells. We have shown the heterogeneous character of oocyte IGCs and CBs, both in structure and molecular content. We have also demonstrated the unique capacity of oocytes to form close structural interactions between IGC and CB components. We proposed to consider these joint structures as integrated entities, sharing the features of both IGCs and CBs.

De novo formation of a subnuclear body.

The mammalian cell nucleus contains structurally stable functional compartments. We show here that one of them, the Cajal body (CB), can be formed de novo. Immobilization on chromatin of both CB structural components, such as coilin, and functional components of the CB, such as the SMN complex, spliceosomal small nuclear ribonucleoproteins (RNPs), small nucleolar RNPs, and small Cajal body-specific RNPs, is sufficient for the formation of a morphologically normal and apparently functional CB. Biogenesis of the CB does not follow a hierarchical assembly pathway and exhibits hallmarks of a self-organizing structure.

Nuclear bodies in Douglas fir (Pseudotsuga menziesii Mirb.) microspores.

The identification of nucleolar proteins and immunocytochemical localization of small nuclear ribonucleoprotein (snRNP) elements revealed the presence of three types of nuclear bodies in Douglas fir microspore nuclei. One type consists of structures resembling Cajal bodies (CBs) and contains nucleolar proteins as well as snRNPs and U2 snRNA. The second type is bizonal bodies, which are nuclear bodies also linked with the splicing system. The bizonal body comprises two parts: the first contains Sm proteins and stains strongly with silver stain, and the second resembles CBs in terms of the degree of silver staining and molecular composition. Douglas fir is the second species after larch where the presence of bizonal bodies has been demonstrated. Pseudotsuga menziesii Mirb and Larix decidua Mill are species with one of the longest microsporogenesis processes known in plants. The presence of bizonal bodies in both species may be linked to the intensification of the splicing processes in microspores with an exceptionally long cell cycle. The third type of structure is dense bodies, whose morphology and degree of silver staining strongly indicate their functional and spatial relationship to the dense part of bizonal bodies.

Structure of the insect oocyte nucleus with special reference to interchromatin granule clusters and cajal bodies.

The nuclear structure of insect oocyte is described with reference to interchromatin granule clusters and Cajal bodies. It is suggested that the intranuclear structure is determined by the ovary organization and reflects the sources of oocyte RNA. Inactivated oocyte chromosomes in meroistic ovaries form a karyosphere, and their features are discussed. In insects with panoistic ovaries, the organization and molecular composition of complicated oocyte Cajal bodies are discussed, and the homology between a structure located inside Cajal bodies and interchromatin granule clusters is proved. Along with revealing some essential Cajal body components and factors involved in RNA polymerase II transcription, we analyze the absence of RNAP II itself from oocyte Cajal bodies of Acheta. In insects with meroistic ovaries, Cajal bodies of Drosophila and homology of Panorpa nuclear bodies to Cajal bodies and their peculiarities are described, because the nuclear bodies may share some features of both Cajal bodies and interchromatin granule clusters to represent a single nuclear domain.