Structural changes in oocyte nucleoli ofXenopus laevis during oogenesis and meiotic maturation

Immunoelectron microscopy with anti-nucleolin defined substructures within the multiple nucleoli of biosynthetically active stage II–III oocytes and within the nucleoli of relatively quiescent stage VI oocytes of Xenopus laevis. Dense fibrillar components (DFCs) of nucleoli from stage II–III oocytes consisted of nucleolonemas that radiated from a continuous DFC sheath surrounding fibrillar centers (FCs). Discernible granular regions (GRs) were absent in these same nucleoli. Conversely, stage VI oocyte nucleoli displayed compacted DFCs and prominent GRs. Immunofluorescence microscopy then tracked fibrillarin, nucleolin, and condensed DNA through oogenesis and into progesterone-induced meiotic maturation and nuclear breakdown. In stage II–III oocyte nucleoli, fibrillarin was enriched near the FC-DFC boundaries, while nucleolin was distributed throughout these same DFCs. Both proteins were enriched within the compacted DFCs of stage VI oocyte nucleoli. Staining with (DAPI) 4′,6-diamidino-2-phenylindole showed condensed DNA within nucleolar FCs of both stage II–III and stage VI oocyte. Upon nuclear breakdown, we found fibrillarin and nucleolin in small particles and in the surrounding cytoplasm. Although we saw no trace of fibrillarin or nucleolin in nuclear remnants prepared just minutes later, DAPI-stained particles remained within these preparations, thus suggesting that FCs were at least slow to disassemble. Received: 18 March 1996 / Accepted: 16 April 1996     

Nucleolar trafficking of nucleostemin family proteins: common versus protein-specific mechanisms

The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins.     

A novel lysine-rich domain and GTP binding motifs regulate the nucleolar retention of human guanine nucleotide binding protein, GNL3L

A variety of G-proteins and GTPases are known to be involved in nucleolar function. We describe here a new evolutionarily conserved putative human GTPase, guanine nucleotide binding protein-like 3-like (GNL3L). Genes encoding proteins related to GNL3L are present in bacteria and yeast to metazoa and suggests its critical role in development. Conserved domain search analysis revealed that the GNL3L contains a circularly permuted G-motif described by a G5-G4-G1-G2-G3 pattern similar to the HSR1/MMR1 GTP-binding protein subfamily. Highly conserved and critical residues were identified from a three-dimensional structural model obtained for GNL3L using the crystal structure of an Ylqf GTPase from Bacillus subtilis. We demonstrate here that GNL3L is transported into the nucleolus by a novel lysine-rich nucleolar localization signal (NoLS) residing within 1-50 amino acid residues. NoLS identified here is necessary and sufficient to target the heterologous proteins to the nucleolus. We show for the first time that the lysine-rich targeting signal interacts with the nuclear transport receptor, importin-beta and transports GNL3L into the nucleolus. Interestingly, depletion of intracellular GTP blocks GNL3L accumulation into the nucleolar compartment. Furthermore, mutations within the G-domains alter the GTP binding ability of GNL3L and abrogate wild-type nucleolar retention even in the presence of functional NoLS, suggesting that the efficient nucleolar retention of GNL3L involves activities of both basic NoLS and GTP-binding domains. Collectively, these data suggest that GNL3L is composed of distinct modules, each of which plays a specific role in molecular interactions for its nucleolar retention and subsequent function(s) within the nucleolus.     

Yph1p,an ORC-interacting protein: potential links between cell proliferation control,DNA replication,and ribosome biogenesis

Immunoprecipitation of the origin recognition complex (ORC) from yeast extracts identified Yph1p, an essential protein containing a BRCT domain. Two Yph1p complexes were characterized. Besides ORC, MCM proteins, cell-cycle regulatory proteins, checkpoint proteins, 60S ribosomal proteins, and preribosome particle proteins were found to be associated with Yph1p. Yph1p is predominantly nucleolar and is required for 60S ribosomal subunit biogenesis and possibly for translation on polysomes. Proliferating cells depleted of Yph1p arrest in G(1) or G(2), with no cells in S phase, or significantly delay S phase progression after release from a hydroxyurea arrest. Yph1p levels decline as cells commit to exit the cell cycle, and levels vary depending on energy source. Yph1p may link cell proliferation control to DNA replication, ribosome biogenesis, and translation on polysomes.     

Evolutionarily conserved role of nucleostemin: controlling proliferation of stem/progenitor cells during early vertebrate development

Nucleostemin (NS) is a putative GTPase expressed preferentially in the nucleoli of neuronal and embryonic stem cells and several cancer cell lines. Transfection and knockdown studies indicated that NS controls the proliferation of these cells by interacting with the p53 tumor suppressor protein and regulating its activity. To assess the physiological role of NS in vivo, we generated a mutant mouse line with a specific gene trap event that inactivates the NS allele. The corresponding NS(-/-) embryos died around embryonic day 4. Analyses of NS mutant blastocysts indicated that NS is not required to maintain pluripotency, nucleolar integrity, or survival of the embryonic stem cells. However, the homozygous mutant blastocysts failed to enter S phase even in the absence of functional p53. Haploid insufficiency of NS in mouse embryonic fibroblasts leads to decreased cell proliferation. NS also functions in early amphibian development to control cell proliferation of neural progenitor cells. Our results show that NS has a unique ability, derived from an ancestral function, to control the proliferation rate of stem/progenitor cells in vivo independently of p53.     

A nucleostemin-like GTPase required for normal apical and floral meristem development in Arabidopsis

Mammalian nucleostemin (NS) is preferentially expressed in stem cells and acts to promote cell cycle progression. In plants, stem cell activities have to be terminated during flower development, and this process requires the activation of AGAMOUS (AG) gene expression. Here, a nucleostemin-like 1 gene, NSN1, is shown to be required for flower development in Arabidopsis. The NSN1 mRNA was found in the inflorescence meristem and floral primordia, and its protein was localized to the nucleoli. Both heterozygous and homozygous plants developed defective flowers on inflorescences that were eventually terminated by the formation of carpelloid flowers. Overexpression of NSN1 resulted in loss of apical dominance and formation of defective flowers. Expression of the AG gene was found to be up-regulated in nsn1. The carpelloid flower defect of nsn1 was suppressed by the ag mutation in the nsn1 ag double mutant, whereas double mutants of nsn1 apetala2 (ap2) displayed enhanced defective floral phenotypes. These results suggest that in the delicately balanced regulatory network, NSN1 acts to repress AG and plays an additive role with AP2 in floral organ specification. As a midsize nucleolar GTPase, NSN1 represents a new class of regulatory proteins required for flower development in Arabidopsis.     

Nucleolar localization and identification of nuclear/nucleolar localization signals of the calmodulin-binding protein nucleomorphin during growth and mitosis in <Emphasis Type="Italic">Dictyostelium</Emphasis>

The calmodulin-binding protein nucleomorphin isoform NumA1 is a nuclear number regulator in Dictyostelium that localizes to intra-nuclear patches adjacent to the nuclear envelope and to a lesser extent the nucleoplasm. Earlier studies have shown similar patches to be nucleoli but only three nucleolar proteins have been identified in Dictyostelium. Here, actinomycin-D treatment caused the loss of NumA1 localization, while calcium and calmodulin antagonists had no effect. In keeping with a nucleolar function, NumA1 moved out of the presumptive nucleoli during mitosis redistributing to areas within the nucleus, the spindle fibers, and centrosomal region before re-accumulating in the presumptive nucleoli at telophase. Together, these data verify NumA1 as a true nucleolar protein. Prior to this study, the dynamics of specific nucleolar proteins had not been determined during mitosis in Dictyostelium. FITC-conjugated peptides equivalent to presumptive nuclear localization signals within NumA1 localized to nucleoli indicating that they also act as nucleolar localization signals. To our knowledge, these represent the first precisely defined nucleolar localization signals as well as the first nuclear/nucleolar localization signals identified in Dictyostelium. Together, these results reveal that NumA1 is a true nucleolar protein and the only nucleolar calmodulin-binding protein identified in Dictyostelium. The possible use of nuclear/nucleolar localization signal-mediated drug targeting to nucleoli is discussed.     

Inhibiting β-catenin disables nucleolar functions in triple-negative breast cancer

Triple-negative breast cancer (TNBC) patients with upregulated Wnt/β-catenin signaling often have poor clinical prognoses. During pathological examinations of breast cancer sections stained for β-catenin, we made the serendipitous observation that relative to non-TNBC, specimens from TNBC patients have a greater abundance of nucleoli. There was a remarkable direct relationship between nuclear β-catenin and greater numbers of nucleoli in TNBC tissues. These surprising observations spurred our investigations to decipher the differential functional relevance of the nucleolus in TNBC versus non-TNBC cells. Comparative nucleolar proteomics revealed that the majority of the nucleolar proteins in TNBC cells were potential targets of β-catenin signaling. Next, we undertook an analysis of the nucleolar proteome in TNBC cells in response to β-catenin inhibition. This effort revealed that a vital component of pre-rRNA processing, LAS1 like ribosome biogenesis factor (LAS1L) was significantly decreased in the nucleoli of β-catenin inhibited TNBC cells. Here we demonstrate that LAS1L protein expression is significantly elevated in TNBC patients, and it functionally is important for mammary tumor growth in xenograft models and enables invasive attributes. Our observations highlight a novel function for β-catenin in orchestrating nucleolar activity in TNBCs.Subject terms: Breast cancer, Organelles, Breast cancer, Metastasis     

ADP-ribosylation of nucleolar proteins in HeLa tumor cells

ADP-ribosylation reactions in nucleoli of exponentially growing HeLa cells were studied. Isolated nuclei or nucleoli were labeled with ³²P-NAD; then the nucleolar proteins were analyzed by 1-dimensional and 2-dimensional polyacrylamide gel electrophoresis (PAGE) and modified proteins were detected by autoradiography. The labeled nucleolar proteins were also chromatographically fractionated on DEAE-cellulose. Electrophoretic analysis of total nucleolar and chromatographically purified proteins revealed that besides nuclear ADP-ribosyltransferase and histones two characteristic nucleolar phosphoproteins numatrin/B23 and nucleolin/C23 were modified by ADP-ribosylation.     

Cell cycle-dependent migration of the DNA-binding protein Ku80 into nucleoli

The DNA-binding protein Ku (p70/p80) was originally discovered through the use of human autoimmune sera. In attempts to search out nucleolar proteins in relation to nucleolar dynamic changes, we developed monoclonal antibodies against nuclear proteins. One antibody, termed LL1, received particular attention since asynchronous cells exhibited tremendous differences in their nucleolar fluorescence intensities after immunostaining. The LL1 protein was proven to be the Ku subunit p80 (Ku80) by cDNA cloning and sequencing. Possible correlations between the heterogeneous distribution of Ku80 in nucleoli and the cell cycle were examined. HeLa cells were synchronized at M phase by arrest with nocodazole, or at the G1/S boundary by sequential treatments with thymidine and aphidicolin. These cells were then released by culturing in fresh medium to allow the cell cycle to progress synchronously. Immunofluorescent detection of Ku80 revealed that nucleoli of the cells at the G1/S boundary had very small amounts of Ku80, which was mainly present in the nucleoplasm. Ku80 was gradually accumulated in nucleoli during S phase and reached the maximum at late S or G2 phase. Immunoblotting experiments showed that cell extracts prepared from different phases of the cell cycle had virtually identical amounts of Ku80. These results suggest that Ku80 migrates from nucleoplasm to nucleoli in a cell cycle-dependent manner.     

Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D.

Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10(-2) micrograms/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10(-2) micrograms/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10(-1) micrograms/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.     

Morphometry of nuclear and nucleolar structures in a CaCo-2 cell line

The number of the nucleoli in a CaCo-2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo-2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.     

Nucleophosmin is a binding partner of nucleostemin in human osteosarcoma cells

Nucleostemin (NS) is expressed in the nucleoli of adult and embryonic stem cells and in many tumors and tumor-derived cell lines. In coimmunoprecipitation experiments, nucleostemin is recovered with the tumor suppressor p53, and more recently we have demonstrated that nucleostemin exerts its role in cell cycle progression via a p53-dependent pathway. Here, we report that in human osteosarcoma cells, nucleostemin interacts with nucleophosmin, a nucleolar protein believed to possess oncogenic potential. Nucleostemin (NS) and nucleophosmin (NPM) displayed an extremely high degree of colocalization in the granular component of the nucleolus during interphase, and both proteins associated with prenucleolar bodies in late mitosis before the reformation of nucleoli. Coimmunoprecipitation experiments revealed that NS and NPM co-reside in complexes, and yeast two-hybrid experiments confirmed that they are interactive proteins, revealing the NPM-interactive region to be the 46-amino acid N-terminal domain of NS. In bimolecular fluorescence complementation studies, bright nucleolar signals were observed, indicating that these two proteins directly interact in the nucleolus in vivo. These results support the notion that cell cycle regulatory proteins congress and interact in the nucleolus, adding to the emerging concept that this nuclear domain has functions beyond ribosome production.     

Localization of fibrillarin, 53 kDa protein and Ag-NOR proteins in the nuclei of giant antipodal cells of the wheat Triticum aestivum

Distribution of nucleolar argentophylic proteins, fibrillarin and 53 kDa protein, in highly polyploid nuclei of antipodal cells of Triticum aestivum L. was studied at different stages of the embryo sac development. The main results are as follows. 1. Ag-NOR proteins and fibrillarin form clusters are distributed in the giant nucleoli, whereas 53 kDa protein is mainly localized on the nucleolar periphery. Ag-NOR proteins and fibrillarin are accumulated as globular nucleolar-like particles--micronucleoli. 2. Dynamics of Ag-NOR proteins, fibrillarin and 53 kDa protein depends on the proliferative activity of endosperm cells. In embryo sacs with non-dividing endosperm cells at interphase stages, Ag-NOR proteins and fibrillarin were observed only within nucleoli and micronucleoli. In embryo sacs with dividing endosperm cells, fibrillarin and 53 kDa protein formed heterogeneous globular bodies varying in size. Simultaneously, some argentophylic material was observed in giant chromosomes. This may be due, presumably, to a partial or complete disappearance of the nucleoli of antipods and transition of some nucleolar components into the peripheral material of giant polytene chromosomes. We suggest that giant nuclei of antipodal cells may undergo cyclic transformation similar to those in the nuclei of dividing cells.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.