Immunolocalization of Human p14ARF to the Granular Component of the Interphase Nucleolus

2000 ◽  
Vol 256 (2) ◽  
pp. 400-410 ◽  
Author(s):  
Mikael S. Lindström ◽  
Ulf Klangby ◽  
Rie Inoue ◽  
Pavel Pisa ◽  
Klas G. Wiman ◽  
...  
Keyword(s):  
Granular Component

High-resolution autoradiographic localization of DNA-containing sites and RNA synthesis in developing nucleoli of human preimplantation embryos: a new concept of embryonic nucleologenesis

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 777-791 ◽  
Author(s):  
J. Tesarik ◽  
V. Kopecny ◽  
M. Plachot ◽  
J. Mandelbaum
Keyword(s):  
Rna Synthesis ◽  
Preimplantation Embryos ◽  
Synthetic Activity ◽  
Rrna Synthesis ◽  
Nucleolar Organizer Regions ◽  
Careful Study ◽  
Matrix Elements ◽  
Electron Microscopic Autoradiography ◽  
General Validity ◽  
Granular Component

Human embryos from the 2-cell to the morula stage, obtained by in vitro fertilization, were incubated with [3H]thymidine or [3H]uridine so as to achieve labelling of all replicating nuclear DNA and the newly synthesized RNA, respectively. The label was localized in different structural components of developing nucleoli using electron microscopic autoradiography. Careful study of the relationship between the structural pattern and nucleic acid distribution made it possible to define four stages of embryonic nucleologenesis. Homogeneous nuclear precursors (i) consist of nucleolar matrix elements appearing as filaments of 3 nm thickness, (ii) do not contain recently replicated DNA and (iii) lack RNA synthetic activity. Penetration of DNA into these bodies is a key event leading to their transformation into heterogeneous nucleolar precursors. In addition to the 3 nm matrix filaments, two types of 5 nm fibrillar components can be recognized in them. The denser type contains DNA and is the site of nucleolar RNA synthesis, while the more loosely arranged 5 nm fibrils are not labelled with [3H]thymidine and apparently represent the newly produced pre-rRNA detached from the transcribing rDNA filament. Compact fibrillogranular nucleoli are characterized by the first appearance of the granular component and reduction of the nontranscribing part of the fibrillar component, both indicating the activation of the machinery for rRNA processing. Finally, the granular component is most evident in reticulated nucleoli, occupying mostly the inner parts of their nucleolonema, while the transcription sites tend to be located at the nucleolar periphery. Our findings advocate a unique concept of embryonic nucleologenesis, different from any other nucleolar event during the cell cycle of differentiated cells. This developmental pattern is characterized by a gradual activation of rRNA synthesis and processing, mediated by progressive association of rDNA and, later on, the newly formed pre-rRNA with pre-existing nucleolar matrix elements that are originally topically separated from nucleolar organizer regions. This model may have a general validity in early animal embryos despite some interspecies variability in the timing of individual steps and resulting structural peculiarities.

Distinguishing the sites of pre-rRNA synthesis and accumulation in Ehrlich tumor cell nucleoli

1991 ◽  
Vol 99 (4) ◽  
pp. 759-767
Author(s):  
M. Thiry ◽  
G. Goessens
Keyword(s):  
Tumor Cell ◽  
Condensed Chromatin ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Dense Fibrillar Component ◽  
Ehrlich Tumor ◽  
Granular Component ◽  
Fibrillar Component

The precise location of transcribing rRNA genes within Ehrlich tumor cell nucleoli has been investigated using two approaches: high-resolution autoradiography of cells pulse-labelled with tritiated uridine, varying the exposure time, and in situ-in vitro transcription coupled with an immunogold labelling procedure. When autoradiographic preparations are exposed for a short time, silver grains are found associated almost exclusively with interphasic cell nucleoli. Labelling of extranucleolar areas requires longer exposure. Within the nucleolus, the first sites to be revealed are in the dense fibrillar component. Prolonging exposure increases labelling over the dense fibrillar component, with label becoming more and more apparent over the fibrillar centers. Under these conditions, however, labelling does not extend into the granular component, and no background is observed. Initiation of transcription on ultrathin cell sections occurs preferentially at the borders of condensed chromatin blocks and in their close vicinity. The condensed chromatin areas themselves remain unlabelled. Inside most nucleoli, gold-particle clusters are mainly detected in the fibrillar centers, especially at their periphery, whereas the dense fibrillar component and the granular component remain devoid of label. These results, together with previous observations made on the same cell type, clearly indicate that the fibrillar centers are the sites of rRNA gene transcription in Ehrlich tumor cell nucleoli, while the dense fibrillar component is the site of pre-rRNA accumulation.

scholarly journals Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus.

1989 ◽  
Vol 37 (9) ◽  
pp. 1371-1374 ◽  
Author(s):  
M Biggiogera ◽  
S Fakan ◽  
S H Kaufmann ◽  
A Black ◽  
J H Shaper ◽  
...  
Keyword(s):  
Hela Cell ◽  
Immunoelectron Microscopy ◽  
Silver Staining ◽  
Dense Fibrillar Component ◽  
Specific Antibodies ◽  
Granular Component ◽  
Protein B23 ◽  
Reported Data ◽  
Fibrillar Component

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.

scholarly journals LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HEPATIC CELL NUCLEOLI IN MICE TREATED WITH ACTINOMYCIN D

1967 ◽  
Vol 33 (3) ◽  
pp. 489-496 ◽  
Author(s):  
J. C. H. de Man ◽  
N. J. A. Noorduyn
Keyword(s):  
Orotic Acid ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Electron Microscopic ◽  
Granular Component ◽  
Hepatic Cells ◽  
Progressive Loss ◽  
Nucleolar Rna ◽  
Silver Grains ◽  
Electron Microscopic Radioautography

Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology was studied when actinomycin D treatment preceded labeling with tritiated orotic acid. Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible if a dosage of 25 µg of actinomycin D was used, but nucleolar RNA was still synthesized. After a dosage of 400 µg of actinomycin D, nucleolar RNA synthesis was completely stopped If labeling with tritiated orotic acid preceded treatment with 400 µg of actinomycin D, labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component. At 30 min after actinomicyn D treatment all labeling was lost. Since labeling was associated with the granular component the progressive loss of label as a result of actinomycin D treatment indicated a release of nucleolar granules. The correlation between this release and the loss of 28S RNA from actinomycin D treated nucleoli as described in the literature is discussed.

The Effect of Actinomycin D in Vivo Upon Peripheral Nucleoli and Other Nuclear Organelles in Oocytes of Triturus Cristatus

1972 ◽  
Vol 10 (3) ◽  
pp. 833-855
Author(s):  
M. H. L. SNOW
Keyword(s):  
Phase Contrast ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Triturus Cristatus ◽  
Granular Component ◽  
Azure B ◽  
Final Maturation ◽  
Ribosomal Precursor ◽  
20 Nm

Exposure of the ovaries of Triturus cristatus to actinomycin D at a concentration of 100 µg/ml causes characteristic changes in the peripheral nucleoli and other nuclear organelles in oocytes of 0.6-1.1 mm diameter. Viewed with the light microscope untreated oocytes contain nucleoli that stain uniformly with a variety of dyes. They also appear homogeneous tmder phase-contrast optics. After 2 or 4 h of in vivo incubation with actinomycin D, oocyte sections stained with Haidenhain's haematoxylin or viewed under phase-contrast optics show nucleoli composed of 2 regions. The more heavily stained or contrasted zone is crescent-shaped and directed away from the nuclear membrane. Neither sections stained with azure B bromide nor gallocyanin chrome alum show this feature. Ribonuclease digestion does not eliminate or alter it. Autoradiography with [3H]uridine indicates that all recently synthesized RNA is lost from the nucleolus during actinornycin D treatment. The zonation is not therefore a reflexion of RNA distribution. During recovery from actinomycin D poisoning there is a reduction in the degree of zonation shown by nucleoli which re-establish a normal appearance some 48 h after treatment. Electron microscopy of peripheral nucleoli in oocytes sampled during this treatment indicates that the zonation is not associated with reorganization of ultrastructural components. During incubation with actinomycin D the coarse granules (20 nm diameter) are completely lost from the nucleolus. There is associated shrinkage of the nucleolus which after treatment is found to consist entirely of fibrils (5 nm thick) and small granules. The reappearance of the coarse granules during recovery is completed in about 48 h. It is thought that the loss of the granular component during treatment represents the movement of the 30-S precursor and the 18-s ribosomal unit from the nucleolus. Some 20-30 µm inside the nucleus of untreated oocytes is a region containing many spheroidal bodies, less than 1.0 µm diameter. They have been termed micronucleoli and consist of granules 2.5-5 nm in diameter and fibrils of similar thickness. Actinomycin D treatment causes these components to segregate and eventually (within 24 h of treatment) the granular component is extruded. This component reappears during the second day after treatment. It is postulated that these micronucleoli represent the sites at which the 30-S ribosomal precursor undergoes its final maturation. The segregation of components induced by actinomycin D is probably the morphological manifestation of an abnormal metamorphosis of this precursor. Treatment with actinomycin D also induces the immediate formation within the nucleus of crystalline bodies composed of lamellae 16 nm wide, 4 nm thick and with a centre-to-centre spacing of 8-10 nm. They are not present 24 h after treatment. They are thought to represent a protein fraction normally associated with periods of intense RNA synthesis.

Fine Structure of the Nucleolus in Normal and Mutant Xenopus Embryos

1967 ◽  
Vol 2 (2) ◽  
pp. 151-162
Author(s):  
ELIZABETH D. HAY ◽  
J. B. GURDON
Keyword(s):  
Fine Structure ◽  
Electron Microscope ◽  
Xenopus Laevis ◽  
Fibrous Material ◽  
Osmium Tetroxide ◽  
Wild Type ◽  
Granular Component ◽  
Basic Dyes ◽  
Fibrillar Component ◽  
Xenopus Laevis Embryos

Mutant and normal Xenopus laevis embryos (0-nu, 1-nu, 2-nu) were examined in the electron microscope after glutaraldehyde and/or osmium-tetroxide fixation. During cleavage both 0-nu and wild-type embryos contain multiple small nucleolar bodies, less than 1 µ in diameter, composed mainly of a fibrous material. By the end of cleavage or beginning of gastrulation, granular caps develop on the fibrous nucleolar bodies. In 1-and 2-nu cells, the multiple nucleolar bodies are replaced during gastrula and neurula stages by definitive nucleoli (2-5 µ in diameter) which contain abundant small (150 Å) granules intermingled with fibrous material. In 0-nu cells, one or two pseudonucleoli (1-3 µ in diameter) appear at about the same time that definitive nucleoli develop in wild-type cells. The multiple small nucleolar bodies disappear as the pseudonucleoli enlarge. Pseudonucleoli differ from definitive nucleoli in having a much smaller amount of the granular component, which is located as a cap on the periphery of the fibrous component and not mingled with it. The granular component of the 0-nu pseudonucleoli, however, is not distinguishable in its fine structure from the same component of normal nucleoli. In many 0-nu tadpoles at stage 41, the granular component of the nucleolus is entirely absent and the fibrillar component is very prominent. Both granular and fibrous components of the 0-nu pseudonucleoli contain RNA as judged by RNase sensitivity and staining affinity for basic dyes.

Sites of RNA synthesis and migration in the adrenal cortex cells of the young rat, as shown by radioautography

1983 ◽  
Vol 102 (4) ◽  
pp. 594-602 ◽  
Author(s):  
Maria C. Magalhães ◽  
M. M. Magalhães ◽  
A. Coimbra
Keyword(s):  
Adrenal Cortex ◽  
Rna Synthesis ◽  
Zona Fasciculata ◽  
Time Intervals ◽  
Granular Component ◽  
Sequence Of Events ◽  
Young Rat ◽  
And Migration ◽  
Fibrillar Component ◽  
Silver Grains

Abstract. The sites of RNA synthesis, processing and migration were studied with light and electron microscope radioautography in adrenal zona fasciculata cells of the young rat. Animals were injected iv with [3H]uridine and sacrificed at various time intervals. A marked radioactive peak was found in blood at the 20 min interval, later time intervals being considered as chase periods. Labelling was high over nucleoli of fasciculata cells at 1 h, with the silver grains concentrated over the fibrillar component. Simultaneous though lower peaks occurred over the condensed chromatin and the interchromatin space. Nucleolar and extranucleolar values were decreased at 8 and 24 h, most silver grains overlaying the cytoplasm at this time period. At 8 h, the granular component of the nucleolus was more heavily labelled than the fibrillar component. This sequence of events suggests a relatively slow uptake of the labelled nucleotides and slow formation of pre-rRNA molecules in the fibrillar component of the nucleolus, with the processing into smaller subunits occurring in the granular component during initial chase. Extranucleolar RNAs were synthesized simultaneously but in a smaller rate. Most nucleolar RNA was finally transferred to the cytoplasm. This pattern will serve as a basis for studying the effects of ACTH on the different steps of RNA synthesis and transport in adrenal cortex cells.

scholarly journals Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing

2002 ◽  
Vol 157 (6) ◽  
pp. 941-952 ◽  
Author(s):  
Olivier Gadal ◽  
Daniela Strauss ◽  
Elisabeth Petfalski ◽  
Pierre-Emmanuel Gleizes ◽  
Nicole Gas ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Single Amino Acid ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Particle Assembly ◽  
Granular Component ◽  
Egfp Reporter ◽  
Ribosomal Particle ◽  
Visual Assay

Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.

scholarly journals Compartmentation of the Nucleolar Processing Proteins in the Granular Component Is a CK2-driven Process

2006 ◽  
Vol 17 (6) ◽  
pp. 2537-2546 ◽  
Author(s):  
Emilie Louvet ◽  
Henriette Roberte Junéra ◽  
Isabelle Berthuy ◽  
Danièle Hernandez-Verdun
Keyword(s):  
Binding Sites ◽  
Protein Complexes ◽  
Rrna Processing ◽  
Granular Component ◽  
Permeabilized Cells ◽  
Rrna Transcription ◽  
Transcription Sites ◽  
Protein B23 ◽  
Nucleolar Components

To analyze the compartmentation of nucleolar protein complexes, the mechanisms controlling targeting of nucleolar processing proteins onto rRNA transcription sites has been investigated. We studied the reversible disconnection of transcripts and processing proteins using digitonin-permeabilized cells in assays capable of promoting nucleolar reorganization. The assays show that the dynamics of nucleolar reformation is ATP/GTP-dependent, sensitive to temperature, and CK2-driven. We further demonstrate the role of CK2 on the rRNA-processing protein B23. Mutation of the major CK2 site on B23 induces reorganization of nucleolar components that separate from each other. This was confirmed in assays using extracts containing B23 mutated in the CK2-binding sites. We propose that phosphorylation controls the compartmentation of the rRNA-processing proteins and that CK2 is involved in this process.

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