Nucleolar Dense Fibrillar Component Research Articles

DEAD-box RNA helicase 10 is required for 18S rRNA maturation by controlling the release of U3 snoRNA from pre-rRNA in embryonic stem cells

Ribosome biogenesis plays a pivotal role in maintaining stem cell homeostasis, yet the precise regulatory mechanisms governing this process in mouse embryonic stem cells (mESCs) remain largely unknown. In this investigation, we ascertain that DEAD-box RNA helicase 10 (DDX10) is indispensable for upholding cellular homeostasis and the viability of mESCs. Positioned predominantly at the nucleolar dense fibrillar component (DFC) and granular component (GC), DDX10 predominantly binds to 45S ribosomal RNA (rRNA) and orchestrates ribosome biogenesis. Degradation of DDX10 prevents the release of U3 snoRNA from pre-rRNA, leading to perturbed pre-rRNA processing and compromised maturation of the 18S rRNA, thereby disrupting the biogenesis of the small ribosomal subunit. Moreover, DDX10 participates in the process of liquid-liquid phase separation (LLPS), which is necessary for efficient ribosome biogenesis. Notably, the NUP98-DDX10 fusion associated with acute myelocytic leukemia (AML) alters the cellular localization of DDX10 and results in loss of ability to regulate pre-rRNA processing. Collectively, this study reveals the critical role of DDX10 as a pivotal regulator of ribosome biogenesis in mESCs.

Unexpected distribution of KRIT1 inside the nucleus: new insight in a complex molecular pathway

KRIT1 is an 84kDa protein that lacks any relevant catalytic domains, associated with the cerebral cavernous malformation disease. We have investigated by means of ultrastructural immunocytochemistry the nuclear distribution of KRIT1 in different cell lines, revealing its unexpected localization on actively transcribing nuclear domains such as the perichromatin fibrils and the nucleolar dense fibrillar component. These preliminary data indicate a still undescribed and unknown role for KRIT1 inside the nucleus.

Proteolytic Processing of cGMP-Dependent Protein Kinase I Mediates Nuclear cGMP Signaling in Vascular Smooth Muscle Cells

Cyclic GMP modulates gene expression in vascular smooth muscle cells (SMCs) in part by stimulating cGMP-dependent protein kinase I (PKGI) and the phosphorylation of transcription factors. In some cells, cGMP increases nuclear translocation of PKGI and PKGI-dependent phosphorylation of transcription regulators; however, these observations have been variable, and the mechanisms mediating nuclear PKGI translocation are incompletely understood. We tested the hypothesis that proteolytic cleavage of PKGI is required for cGMP-stimulated nuclear compartmentation of PKGI and phosphorylation of transcription factors. We detected an NH(2)-terminal PKGI fragment with leucine zipper domain immunoreactivity in the cytosol and endoplasmic reticulum of SMCs, but only a COOH-terminal PKGI fragment containing the catalytic region (now termed PKGIgamma) was observed in the Golgi apparatus (GA) and nucleoplasm. Posttranslational PKGI processing in the GA was critical for nuclear compartmentation of PKGIgamma because GA disruption with nocodazol or brefeldin A inhibited PKGIgamma nuclear localization. PKGIgamma immunoreactivity was particularly abundant in the nucleolus of interphase SMCs where its colocalization with the nucleolar dense fibrillar component protein fibrillarin closely matched the level of nucleolar assembly. Purified nucleolar PKGIgamma enzyme activity was insensitive to cGMP stimulation, which is consistent with its lack of the NH(2)-terminal autoinhibitory domain. Mutation of a putative proteolytic cleavage region in PKGI inhibited cGMP-mediated phosphorylation of cAMP response element-binding protein, cAMP response element-dependent transcription, and nuclear localization of PKGIgamma. These observations suggest that posttranslational modification of PKGI critically influences the nuclear translocation of PKGI and activities of cGMP in SMCs.

Revealing the unseen: the organizer region of the nucleolus.

We carried out a high-resolution ultrastructural analysis of the nucleolus in mouse P815 cells by combining specific DNA and RNA staining, anti-fibrillarin immunolabeling, contrast enhancement by energy filtering TEM and phosphorus mapping by ESI to visualize nucleic acids. We demonstrated that specifically contrasted DNA, fibrillarin and phosphorus overlap within the nucleolar dense fibrillar component. Moreover, we describe a 'DNA cloud' consisting of an inner core of DNA fibers (fibrillar center) and a periphery made of extremely thin fibrils overlapping the anti-fibrillarin immunolabeling (dense fibrillar component). This highly sensitive approach has allowed us to demonstrate, for the first time, the exact distribution of DNA within the decondensed interphase counterpart of the NOR, which includes both the fibrillar center and the dense fibrillar component.

Non-isotopic detection of nucleolar transcription in pre-implantation mouse embryos.

Nucleolar transcription was analysed in permeabilized pre-implantation mouse embryos at the four-cell, eight-cell, morula and early blastocyst stages using confocal microscopy to detect incorporated 5-bromouridine. The results demonstrated that the patterns of nucleolar transcription sites were common for all embryonic stages studied. They consisted most frequently of tightly associated groups of transcription foci similar to those encountered in somatic interphase cells. In addition, the nucleologenesis accompanying each cell cycle apparently gave rise to a different fluorescent pattern, that is to spatially separated fluorescent foci in the cells just after the resumption of rRNA synthesis. An immunoelectron microscopic analysis of the nucleolar transcription was also performed in the eight-cell embryos. A signal, usually consisting of clustered gold particles, was found specifically within nucleolar dense fibrillar components. This result was in agreement with established findings, which identify dense fibrillar component as the major site of nucleolar transcription in somatic cells.

Does the Synthesis of Ribosomal RNA Take Place within Nucleolar Fibrillar Centers or Dense Fibrillar Components? A Critical Appraisal

The localization of transcribing rRNA genes within nucleoli of mammalian cells, although intensively studied, has not been established. Most published papers on this topic situate transcribing ribosomal genes either to nucleolar fibrillar centers or to nucleolar dense fibrillar components. To clarify this point, we have generated the electron microscopic affinity cytochemistry picture of the nucleolus of cultured mammalian cells. Three kinds of affinity probes have been used: (1) probes to nucleolar chromatin, including rDNA sequences; (2) probes to a number of macromolecules (such as RNA polymerase I) which are directly, or indirectly, involved in the synthesis and processing of rRNA and formation of preribosomes; (3) antibodies to bromouridine for a recently standardized nonisotopical method depicting incorporated bromouridine within RNA. The results suggest the localization of transcription sites not only to dense fibrillar components but also to the border region between these components and fibrillar centers. Our data support a hypothesis that in metabolically active mammalian nucleoli, fibrillar centers and dense fibrillar components form a single functional domain for the transcription of rRNA genes, with nascent transcripts generating "automatically" dense fibrillar components. Through the active process of transcription, individual rRNA genes thus become engulfed within dense fibrillar components.

Distribution of B-36 nucleolar protein in relation to transcriptional activity in plant cells

The Mr 34000 nucleolar protein B-36 was immunolocalized using a mouse monoclonal antibody in different plant cells by light and electron microscopy. Specific immunofluorescence was observed in nucleoli of cryofixed and cryosectioned material. Immunoelectron microscopy was performed on ultrathin cryosections and Lowicryl sections. Immunogold labelling revealed that B-36 is mainly localized in the nucleolar dense fibrillar component (DFC). The fibrillar centres and the granular component were practically devoid of gold particles. Nucleoli with different rates of transcriptional activity and, as a consequence, with different morphologies showed the same immunolocalization. This was true for both somatic and germinal tissues of the different plant species studied: Allium cepa root meristematic cells and Scilla peruviana and Capsicum annuum pollen grains. The presence of the B-36 antigen in the DFC of nucleoli with very different rates of transcriptional activity indicates that it is a constitutive protein. Moreover, the localization suggests that it could play a role in the transcription and/or early processing of rRNA. The presence of B-36 in the different plant tissues and species studied, confirms once more its high level of conservation throughout evolution. Another Mr 34000 protein called fibrillarin was also found in the DFC. Taking these and other published data together, we think that the antibodies used in the different studies are probably binding different epitopes of identical or very similar Mr 34000 nucleolar proteins.

Ultrastructural, cytochemical and immunocytochemical investigation of the interphase nucleus in the unicellular green alga Chlamydomonas reinhardtii

The ultrastructural organization of the interphase nucleus of the green alga Chlamydomonas reinhardtii was investigated and found to be largely dependent on the fixation conditions. In specimens stained with bismuth, densely contrasted granules ranging from 25 to 45 nm in diameter were localized throughout the interchromatin space and often formed clusters. These granules were labeled by RNase A-gold complexes and may represent the counterparts of animal and higher plant cll interchromatin granules. Within the nucleolus the Ag-NOR and pyroantimonate stains and, to a lesser extent, the bismuth stain reacted with the nucleolar dense fibrillar component (DFC). When cells were subjected to a heat shock at 42°C, the nucleolar DFC was found to progressively separate from the nucleolus and, after 3 h, appeared as a continuous meandering thread about 0.1 μm in width. Within the nucleolus, labeling on conventional preparations occurred as small clusters with antibodies to H3 histones or to DNA whereas RNase A-gold complexes labeled most of it including fibrillar centers. Improved ultrastructural preservation in cryofixed, cryosubstituted specimens gently fixed in glutaraldehyde permitted to localize nucleolar DNA predominantly at the outer edge of fibrillar centers and to a lesser extent within the neighbouring DFC. Our results indicate that the structure and composition of Chlamydomonas interphase nuclei are comparable, despite particularities, to those of animal and higher plant nuclei.

Nucleoli, nucleolar chromosomes and ribosomal genes in the human spermatocyte

The formation and development of nucleoli and their connections with the nucleolar chromosomes were studied in human spermatocytes using electron microscopy, silver staining of nucleolus organizer regions (NORs), high resolution autoradiography and in situ hybridization in order to localize rRNA genes and their transcription in the different stages of meiotic prophase I. At leptotene, new nucleoli were formed, consisting of a fibrillar centre surrounded by a cap of dense fibrillar component. Following [3H]uridine uptake, label was found only over the dense fibrillar component. In situ hybridization revealed rDNA mainly in the dense fibrillar component and in the chromatin. During zygotene, nucleoli increased in size. The fibrillar centre was connected with the secondary constriction region of the nucleolar bivalent and was partially surrounded by dense fibrillar component. This shell of dense fibrillar component merged into a fibrillo-granular mesh that extended away from the fibrillar centre. Autoradiography following [3H]uridine uptake again showed the label overlaying the dense fibrillar component and the proximal part of the fibrillo-granular strands. With in situ hybridization in both the light and electron microscope, signal was mainly found in the dense fibrillar component. A small quantity of label was observed in the peripheral region of the fibrillar centre and in the adjacent chromatin. From early to late pachytene segregation of nucleolar components occurred, with a reduction in the dense fibrillar component that formed a narrow rim around the fibrillar centre with small extensions along the granular component. [3H]uridine incorporation progressively decreased. In situ hybridization showed signal located mainly in the dense fibrillar component and in the chromatin corresponding to the condensed short arm of the nucleolar bivalent. Our results indicate that the majority of rDNA is located and transcribed in the dense fibrillar component; only a small amount is present in the peripheral part of the fibrillar centre and may be transcribed there. Moreover, from leptotene to zygotene, rDNA unravels from the nucleolar chromosome into the nucleolar dense fibrillar component. From zygotene to late pachytene a progressive return to the condensed acrocentric short arm is observed.

Ethanol-phosphotungstic acid and bismuth staining of spermatid nucleoli in mouse spermiogenesis

The nucleoli of developing mouse spermatids were examined with ethanol-phosphotungstic acid (E-PTA) staining, and also with bismuth staining following formaldehyde fixation (FA-Bi staining) and glutaraldehyde fixation (GA-Bi staining). Only the cortical zone of the nucleolar dense fibrillar component (DFC) in the round spermatids was stained with E-PTA, while the inner area remained either faintly (early Golgi-phase spermatids) or completely unstained (cap-phase spermatids). Incubation of the fixed testis with dithiothreitol before E-PTA staining resulted in homogeneously intense staining of the DFC. The facts suggest that numerous E-PTA-positive basic proteins were present in the DFC, but disulfide crosslinks formed in the DFC proteins prevent penetration of PTA into the DFC interior. The DFC was stained with bismuth after FA-Bi and GA-Bi staining until the disappearance of the nucleoli occurring in acrosomephase spermatids. The fibrillar center, homogeneously stained using E-PTA, FA-Bi, and GA-Bi methods was present in the nucleoli of Golgi-phase and early cap-phase spermatids, but disappeared in the nucleoli of late capphase spermatids. These results are discussed based on the previous studies dealing with the ribosomal RNA synthesis in mouse spermiogenesis.

Fine structural ribonucleoprotein components of the cell nucleus visualized after spreading and high resolution autoradiography

The fine structure of the nuclear components was studied following mild lysis of mouse or Drosophila tissue culture cells and spreading of nuclear material. Particular attention was paid to nuclear ribonucleoprotein (RNP) constituents, which were analysed by high resolution autoradiography after [3H]uridine pulse labelling of cells. Comparison with the labelling kinetics of various in situ nuclear RNP constituents described previously suggests strong similarities between in situ constituents and structures observed within spread nuclear components. The present observations suggest that the nucleolar dense fibrillar component, shown previously in ultrathin sections of [3H]uridine-labelled intact cells as carrying rapidly labelled pre-rRNA, in fact consists of highly compacted transcribing ribosomal genes. The growing RNP fibrils appearing in transcription complexes of extranucleolar active genes and the in situ observed perichromatin fibrils also show the same labelling properties. This confirms that the two structures indeed represent the same nucleoplasmic constituents. As for the nuclear structures involved in post-transcriptional events, our observations demonstrate the occurrence of a rapidly labelled RNP fibro-granular network. Its granular elements correspond, in size and perichromatin location, to the perichromatin granules seen in the situ preparations and suggest similarities between the two constituents. The results are discussed in the light of other data providing information on the role of various nuclear structural constituents.

Does the synthesis of ribosomal RNA take place‐within nucleolar fibrillar centers or dense fibrillar components?

By means of immunocytochemistry performed on cryosections of cultured cells, RNA polymerase I was localized mainly to nucleolar fibrillar centers. The labelling of nucleolar dense fibrillar components was low and depended on the cell type. In contrast, DNA topoisomerase I and RNP complexes containing U3 snRNA were enriched in dense fibrillar components, their occurrence in fibrillar centers being usually much less.