Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the 3D organisation of the A. thaliana genome

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Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Saez-Vasquez, Julio

2017-01-01

The nucleolus is the site of ribosomal RNA (rRNA) gene transcription, rRNA processing and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli by Fluorescence Activated Cell Sorting (FACS) and identified Nucleolus-Associated Chromatin Domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein, NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions and mostly inactive protein-coding genes. However, NADs also include active ribosomal RNA genes, and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. PMID:27477271

Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome.

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Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Sáez-Vásquez, Julio

2016-08-09

The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Plant cell nucleolus as a hot spot for iron.

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Roschzttardtz, Hannetz; Grillet, Louis; Isaure, Marie-Pierre; Conéjéro, Geneviève; Ortega, Richard; Curie, Catherine; Mari, Stéphane

2011-08-12

Many central metabolic processes require iron as a cofactor and take place in specific subcellular compartments such as the mitochondrion or the chloroplast. Proper iron allocation in the different organelles is thus critical to maintain cell function and integrity. To study the dynamics of iron distribution in plant cells, we have sought to identify the different intracellular iron pools by combining three complementary imaging approaches, histochemistry, micro particle-induced x-ray emission, and synchrotron radiation micro X-ray fluorescence. Pea (Pisum sativum) embryo was used as a model in this study because of its large cell size and high iron content. Histochemical staining with ferrocyanide and diaminobenzidine (Perls/diaminobenzidine) strongly labeled a unique structure in each cell, which co-labeled with the DNA fluorescent stain DAPI, thus corresponding to the nucleus. The unexpected presence of iron in the nucleus was confirmed by elemental imaging using micro particle-induced x-ray emission. X-ray fluorescence on cryo-sectioned embryos further established that, quantitatively, the iron concentration found in the nucleus was higher than in the expected iron-rich organelles such as plastids or vacuoles. Moreover, within the nucleus, iron was particularly accumulated in a subcompartment that was identified as the nucleolus as it was shown to transiently disassemble during cell division. Taken together, our data uncover an as yet unidentified although abundant iron pool in the cell, which is located in the nuclei of healthy, actively dividing plant tissues. This result paves the way for the discovery of a novel cellular function for iron related to nucleus/nucleolus-associated processes.

[Advances in the study of the nucleolus].

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Feng, Jin-Mei; Sun, Jun; Wen, Jian-Fan

2012-12-01

As the most prominent sub-nuclear compartment in the interphase nucleus and the site of ribosome biogenesis, the nucleolus synthesizes and processes rRNA and also assembles ribosomal subunits. Though several lines of research in recent years have indicated that the nucleolus might have additional functions-such as the assembling of signal recognition particles, the processing of mRNA, tRNA and telomerase activities, and regulating the cell cycle-proteomic analyses of the nucleolus in three representative eukaryotic species has shown that a plethora of proteins either have no association with ribosome biogenesis or are of presently unknown function. This phenomenon further indicates that the composition and function of the nucleolus is far more complicated than previously thought. Meanwhile, the available nucleolar proteome databases has provided new approaches and led to remarkable progress in understanding the nucleolus. Here, we have summarized recent advances in the study of the nucleolus, including new discoveries of its structure, function, genomics/proteomics as well as its origin and evolution. Moreover, we highlight several of the important unresolved issues in this field.

Empirical Derivation of Correction Factors for Human Spiral Ganglion Cell Nucleus and Nucleolus Count Units.

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Robert, Mark E; Linthicum, Fred H

2016-01-01

Profile count method for estimating cell number in sectioned tissue applies a correction factor for double count (resulting from transection during sectioning) of count units selected to represent the cell. For human spiral ganglion cell counts, we attempted to address apparent confusion between published correction factors for nucleus and nucleolus count units that are identical despite the role of count unit diameter in a commonly used correction factor formula. We examined a portion of human cochlea to empirically derive correction factors for the 2 count units, using 3-dimensional reconstruction software to identify double counts. The Neurotology and House Histological Temporal Bone Laboratory at University of California at Los Angeles. Using a fully sectioned and stained human temporal bone, we identified and generated digital images of sections of the modiolar region of the lower first turn of cochlea, identified count units with a light microscope, labeled them on corresponding digital sections, and used 3-dimensional reconstruction software to identify double-counted count units. For 25 consecutive sections, we determined that double-count correction factors for nucleus count unit (0.91) and nucleolus count unit (0.92) matched the published factors. We discovered that nuclei and, therefore, spiral ganglion cells were undercounted by 6.3% when using nucleolus count units. We determined that correction factors for count units must include an element for undercounting spiral ganglion cells as well as the double-count element. We recommend a correction factor of 0.91 for the nucleus count unit and 0.98 for the nucleolus count unit when using 20-µm sections. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

High-Throughput Live-Cell Microscopy Analysis of Association Between Chromosome Domains and the Nucleolus in S. cerevisiae.

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Wang, Renjie; Normand, Christophe; Gadal, Olivier

2016-01-01

Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell's nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.

Regulation of nucleolus assembly by non-coding RNA polymerase II transcripts.

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Caudron-Herger, Maïwen; Pankert, Teresa; Rippe, Karsten

2016-05-03

The nucleolus is a nuclear subcompartment for tightly regulated rRNA production and ribosome subunit biogenesis. It also acts as a cellular stress sensor and can release enriched factors in response to cellular stimuli. Accordingly, the content and structure of the nucleolus change dynamically, which is particularly evident during cell cycle progression: the nucleolus completely disassembles during mitosis and reassembles in interphase. Although the mechanisms that drive nucleolar (re)organization have been the subject of a number of studies, they are only partly understood. Recently, we identified Alu element-containing RNA polymerase II transcripts (aluRNAs) as important for nucleolar structure and rRNA synthesis. Integrating these findings with studies on the liquid droplet-like nature of the nucleolus leads us to propose a model on how RNA polymerase II transcripts could regulate the assembly of the nucleolus in response to external stimuli and during cell cycle progression.

NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle

International Nuclear Information System (INIS)

Zhang, Heyu; Ma, Xi; Shi, Taiping; Song, Quansheng; Zhao, Hongshan; Ma, Dalong

2010-01-01

NSA2 (Nop seven-associated 2) was previously identified in a high throughput screen of novel human genes associated with cell proliferation, and the NSA2 protein is evolutionarily conserved across different species. In this study, we revealed that NSA2 is broadly expressed in human tissues and cultured cell lines, and located in the nucleolus of the cell. Both of the putative nuclear localization signals (NLSs) of NSA2, also overlapped with nucleolar localization signals (NoLSs), are capable of directing nucleolar accumulation. Moreover, over-expression of the NSA2 protein promoted cell growth in different cell lines and regulated the G1/S transition in the cell cycle. SiRNA silencing of the NSA2 transcript attenuated the cell growth and dramatically blocked the cell cycle in G1/S transition. Our results demonstrated that NSA2 is a nucleolar protein involved in cell proliferation and cell cycle regulation.

Stressing on the nucleolus in cardiovascular disease.

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Hariharan, Nirmala; Sussman, Mark A

2014-06-01

The nucleolus is a multifunctional organelle with multiple roles involving cell proliferation, growth, survival, ribosome biogenesis and stress response signaling. Alteration of nucleolar morphology and architecture signifies an early response to increased cellular stress. This review briefly summarizes nucleolar response to cardiac stress signals and details the role played by nucleolar proteins in cardiovascular pathophysiology. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease. © 2013.

[PIWI protein as a nucleolus visitor in Drosophila melanogaster].

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Mikhaleva, E A; Iakushev, E Iu; Stoliarenko, A D; Klenov, M S; Pozovskiĭ, Ia M; Gvozdev, V A

2015-01-01

The evolutionarily conserved nuclear Piwi protein of Drosophila melanogaster is a representative of the Argonaute small RNA binding protein family. Guided by small piRNAs, Piwi functions in transposon silencing in somatic and germ cells of the gonad. We found that in ovarian somatic and germ cells, as well as in the established ovarian somatic cell line, Piwi is concentrated predominantly in the nucleolus--the main nuclear compartment, participating not only in rRNA synthesis, but also in various cell stress responses. We demonstrated the colocalization of Piwi with nucleolar marker proteins--fibrillarin and Nopp140. A mutation preventing Piwi transport to the nucleus and disturbing transposon silencing (piwi(Nt)) leads to 6-8-fold upregulation of rRNA genes expression, as evaluated by the level of transcripts of transposon insertions in 28S rRNA genes. RNase treatment of live cultured ovarian somatic cells depletes Piwi from the nucleolus. The same effect is observed upon inhibiting RNA polymerase I which transcribes rRNA, but not RNA polymerase II. In contrast, upon heat shock Piwi is concentrated in the nucleolus and is depleted from the nucleoplasm. These results implicate Piwi in RNA polymerase activity modulation and stress response in the nucleolus. We discuss possible noncanonical Piwi functions along with its canonical role in transposon silencing by piRNAs.

Initial genomics of the human nucleolus.

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Attila Németh

2010-03-01

Full Text Available We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD-localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD-specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture.

Initial Genomics of the Human Nucleolus

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Németh, Attila; Conesa, Ana; Santoyo-Lopez, Javier; Medina, Ignacio; Montaner, David; Péterfia, Bálint; Solovei, Irina; Cremer, Thomas; Dopazo, Joaquin; Längst, Gernot

2010-01-01

We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs) in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD–localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD–specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture. PMID:20361057

A phosphorescent silver(I)-gold (I) cluster complex that specifically lights up the nucleolus of living cells with FLIM imaging.

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Chen, Min; Lei, Zhen; Feng, Wei; Li, Chunyan; Wang, Quan-Ming; Li, Fuyou

2013-06-01

The phosphorescent silver(I)-gold(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1) selectively stains the nucleolus, with a much lower uptake in the nucleus and cytoplasm, and exhibits excellent photostability. This Ag-Au cluster, which has a photoluminescent lifetime of microseconds, is particularly attractive as a probe in applications of time-gated microscopy. Investigation of the pathway of cellular entry indicated that N1 permeates the outer membrane and nuclear membrane of living cells through an energy-dependent and non-endocytic route within 10 min. High concentrations of N1 in the nucleolus have been quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy coupled with an energy dispersive X-ray analysis (TEM-EDXA), which also helped to elucidate the mechanism of the specific staining. Intracellular selective staining may be correlated with the microenvironment of the nucleolus, which is consistent with experiments conducted at different phases of the cell cycle. These results prove that N1 is a very attractive phosphorescent staining reagent for visualizing the nucleolus of living cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nucleolus disassembly and distribution of segregated nucleolar material in prophase of root-tip meristematic cells in Triticum aestivum L.

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Wang Jianyue

2015-01-01

Full Text Available This paper presents details of the process of nucleolar disassembly, studied by conventional transmission electron microscopy (TEM in wheat root cells. In early prophase, chromatin condensation and irregular nucleolar morphology are observed, with many small particles appearing around the nucleolus. In middle prophase, the nucleolus radiates outwards; in late prophase, the fine structure of the nucleolus disappears and nucleolar material diffuses away. Using “en bloc” silver-staining to distinguish between nucleoli and chromatin, we observed that the dispersed nucleolar material aggregates around the chromatin, forming a sheath-like perichromosomal structure that coats the chromosomes in late prophase.

Apoptosis and expression of argyrophilic nucleolus organizer regions in epithelial neoplasms of the larynx

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Christiana Vargas Ribeiro

2015-04-01

Full Text Available INTRODUCTION: Occurrence of apoptosis and expression of proliferative markers are powerful tools to establish a prognosis in the follow-up of cancer.OBJECTIVE: To evaluate the growth fraction in papillomas and laryngeal squamous cell carcinomas with three degrees of differentiation through apoptosis and the expression of nucleolus organizer regions.METHODS: Retrospective study from which paraffin material was submitted to microtomy and hematoxylin-eosin and silver staining. Stained slides were used to quantify the apoptotic index and the number of nucleolus organizer regions by morphometry.RESULTS: Apoptosis was significantly more frequent in well differentiated carcinomas and in papillomas, and a higher growth fraction of expressed nucleolus organizer regions and cells that expressed a greater than average number of nucleolus organizer regions were more frequently noted in undifferentiated carcinomas.CONCLUSIONS: Thus, it was possible to verify that a high apoptotic index was associated with a lower chance of tumor differentiation in carcinomas, while a greater number of total nucleolus organizer regions, cells expressing nucleolus organizer regions above average and a higher growth fraction were associated with greater likelihood of abnormal cell proliferation and increased tumor differentiation.

Enhanced NOLC1 promotes cell senescence and represses hepatocellular carcinoma cell proliferation by disturbing the organization of nucleolus.

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Yuan, Fuwen; Zhang, Yu; Ma, Liwei; Cheng, Qian; Li, Guodong; Tong, Tanjun

2017-08-01

The nucleolus is a key organelle that is responsible for the synthesis of rRNA and assembly of ribosomal subunits, which is also the center of metabolic control because of the critical role of ribosomes in protein synthesis. Perturbations of rRNA biogenesis are closely related to cell senescence and tumor progression; however, the underlying molecular mechanisms are not well understood. Here, we report that cellular senescence-inhibited gene (CSIG) knockdown up-regulated NOLC1 by stabilizing the 5'UTR of NOLC1 mRNA, and elevated NOLC1 induced the retention of NOG1 in the nucleolus, which is responsible for rRNA processing. Besides, the expression of NOLC1 was negatively correlated with CSIG in the aged mouse tissue and replicative senescent 2BS cells, and the down-regulation of NOLC1 could rescue CSIG knockdown-induced 2BS senescence. Additionally, NOLC1 expression was decreased in human hepatocellular carcinoma (HCC) tissue, and the ectopic expression of NOLC1 repressed the proliferation of HCC cells and tumor growth in a HCC xenograft model. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

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Qin, Rong; Zhang, Huaning; Li, Shaoshan; Jiang, Wusheng; Liu, Donghua

2014-09-01

Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.

NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

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de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

2013-01-01

NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1–33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species. PMID:23516598

Assembly and disassembly of the nucleolus during the cell cycle.

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Hernandez-Verdun, Danièle

2011-01-01

The nucleolus is a large nuclear domain in which transcription, maturation and assembly of ribosomes take place. In higher eukaryotes, nucleolar organization in three sub-domains reflects the compartmentation of the machineries related to active or inactive transcription of the ribosomal DNA, ribosomal RNA processing and assembly with ribosomal proteins of the two (40S and 60S) ribosomal subunits. The assembly of the nucleoli during telophase/early G(1) depends on pre-existing machineries inactivated during prophase (the transcription machinery and RNP processing complexes) and on partially processed 45S rRNAs inherited throughout mitosis. In telophase, the 45S rRNAs nucleate the prenucleolar bodies and order the dynamics of nucleolar assembly. The assembly/disassembly processes of the nucleolus depend on the equilibrium between phosphorylation/dephosphorylation of the transcription machinery and on the RNP processing complexes under the control of the CDK1-cyclin B kinase and PP1 phosphatases. The dynamics of assembly/disassembly of the nucleolus is time and space regulated.

Roles of the nucleolus in the CAG RNA-mediated toxicity.

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Tsoi, Ho; Chan, Ho Yin Edwin

2014-06-01

The nucleolus is a subnuclear compartment within the cell nucleus that serves as the site for ribosomal RNA (rRNA) transcription and the assembly of ribosome subunits. Apart from its classical role in ribosomal biogenesis, a number of cellular regulatory roles have recently been assigned to the nucleolus, including governing the induction of apoptosis. "Nucleolar stress" is a term that is used to describe a signaling pathway through which the nucleolus communicates with other subcellular compartments, including the mitochondria, to induce apoptosis. It is an effective mechanism for eliminating cells that are incapable of performing protein synthesis efficiently due to ribosome biogenesis defects. The down-regulation of rRNA transcription is a common cause of nucleolar function disruption that subsequently triggers nucleolar stress, and has been associated with the pathogenesis of neurological disorders such as spinocerebellar ataxias (SCAs) and Huntington's diseases (HD). This article discusses recent advances in mechanistic studies of how expanded CAG trinucleotide repeat RNA transcripts trigger nucleolar stress in SCAs, HD and other trinucleotide repeat disorders. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease. Copyright © 2013 Elsevier B.V. All rights reserved.

Recent studies implicate the nucleolus as the major site of nuclear translation.

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McLeod, Tina; Abdullahi, Akilu; Li, Min; Brogna, Saverio

2014-08-01

The nucleolus is the most prominent morphological feature within the nucleus of eukaryotic cells and is best known for its role in ribosome biogenesis. It forms around highly transcribed ribosomal RNA gene repeats which yield precursor rRNAs that are co-transcriptionally processed, folded and, while still within the nucleolus, associate with most of the ribosomal proteins. The nucleolus is therefore often thought of as a factory for making ribosomal subunits, which are exported as inactive precursors to the cytoplasm where late maturation makes them capable of mRNA binding and translation initiation. However, recent studies have shown substantial evidence for the presence of functional, translation competent ribosomal subunits within the nucleus, particularly in the nucleolus. These observations raise the intriguing possibility that the nucleolus, as well as being a ribosome factory, is also an important nuclear protein-synthesis plant.

A Nucleolus for Stochastic Cooperative Games

OpenAIRE

Suijs, J.P.M.

1996-01-01

This paper extends the definition of the nucleolus to stochastic cooperative games, that is, to cooperative games with random payoffs to the coalitions. It is shown that the nucleolus is nonempty and that it belongs to the core whenever the core is nonempty. Furthermore, it is shown for a particular class of stochastic cooperative games that the nucleolus can be determined by calculating the traditional nucleolus introduced by Schmeidler (1969) of a specific deterministic cooperative game.

Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

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Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

2016-08-25

Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

Sequential recovery of macromolecular components of the nucleolus.

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Bai, Baoyan; Laiho, Marikki

2015-01-01

The nucleolus is involved in a number of cellular processes of importance to cell physiology and pathology, including cell stress responses and malignancies. Studies of macromolecular composition of the nucleolus depend critically on the efficient extraction and accurate quantification of all macromolecular components (e.g., DNA, RNA, and protein). We have developed a TRIzol-based method that efficiently and simultaneously isolates these three macromolecular constituents from the same sample of purified nucleoli. The recovered and solubilized protein can be accurately quantified by the bicinchoninic acid assay and assessed by polyacrylamide gel electrophoresis or by mass spectrometry. We have successfully applied this approach to extract and quantify the responses of all three macromolecular components in nucleoli after drug treatments of HeLa cells, and conducted RNA-Seq analysis of the nucleolar RNA.

The Multiple Functions of the Nucleolus in Plant Development, Disease and Stress Responses

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Natalia O. Kalinina

2018-02-01

Full Text Available The nucleolus is the most conspicuous domain in the eukaryotic cell nucleus, whose main function is ribosomal RNA (rRNA synthesis and ribosome biogenesis. However, there is growing evidence that the nucleolus is also implicated in many other aspects of cell biology, such as regulation of cell cycle, growth and development, senescence, telomerase activity, gene silencing, responses to biotic and abiotic stresses. In the first part of the review, we briefly assess the traditional roles of the plant nucleolus in rRNA synthesis and ribosome biogenesis as well as possible functions in other RNA regulatory pathways such as splicing, nonsense-mediated mRNA decay and RNA silencing. In the second part of the review we summarize recent progress and discuss already known and new hypothetical roles of the nucleolus in plant growth and development. In addition, this part will highlight studies showing new nucleolar functions involved in responses to pathogen attack and abiotic stress. Cross-talk between the nucleolus and Cajal bodies is also discussed in the context of their association with poly(ADP ribosepolymerase (PARP, which is known to play a crucial role in various physiological processes including growth, development and responses to biotic and abiotic stresses.

The Multiple Functions of the Nucleolus in Plant Development, Disease and Stress Responses

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Kalinina, Natalia O.; Makarova, Svetlana; Makhotenko, Antonida; Love, Andrew J.; Taliansky, Michael

2018-01-01

The nucleolus is the most conspicuous domain in the eukaryotic cell nucleus, whose main function is ribosomal RNA (rRNA) synthesis and ribosome biogenesis. However, there is growing evidence that the nucleolus is also implicated in many other aspects of cell biology, such as regulation of cell cycle, growth and development, senescence, telomerase activity, gene silencing, responses to biotic and abiotic stresses. In the first part of the review, we briefly assess the traditional roles of the plant nucleolus in rRNA synthesis and ribosome biogenesis as well as possible functions in other RNA regulatory pathways such as splicing, nonsense-mediated mRNA decay and RNA silencing. In the second part of the review we summarize recent progress and discuss already known and new hypothetical roles of the nucleolus in plant growth and development. In addition, this part will highlight studies showing new nucleolar functions involved in responses to pathogen attack and abiotic stress. Cross-talk between the nucleolus and Cajal bodies is also discussed in the context of their association with poly(ADP ribose)polymerase (PARP), which is known to play a crucial role in various physiological processes including growth, development and responses to biotic and abiotic stresses. PMID:29479362

The maternal nucleolus plays a key role in centromere satellite maintenance during the oocyte to embryo transition.

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Fulka, Helena; Langerova, Alena

2014-04-01

The oocyte (maternal) nucleolus is essential for early embryonic development and embryos originating from enucleolated oocytes arrest at the 2-cell stage. The reason for this is unclear. Surprisingly, RNA polymerase I activity in nucleolus-less mouse embryos, as manifested by pre-rRNA synthesis, and pre-rRNA processing are not affected, indicating an unusual role of the nucleolus. We report here that the maternal nucleolus is indispensable for the regulation of major and minor satellite repeats soon after fertilisation. During the first embryonic cell cycle, absence of the nucleolus causes a significant reduction in major and minor satellite DNA by 12% and 18%, respectively. The expression of satellite transcripts is also affected, being reduced by more than half. Moreover, extensive chromosome bridging of the major and minor satellite sequences was observed during the first mitosis. Finally, we show that the absence of the maternal nucleolus alters S-phase dynamics and causes abnormal deposition of the H3.3 histone chaperone DAXX in pronuclei of nucleolus-less zygotes.

The nucleolus: a raft adrift in the nuclear sea or the keystone in nuclear structure?

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O'Sullivan, Justin M; Pai, Dave A; Cridge, Andrew G; Engelke, David R; Ganley, Austen R D

2013-06-01

The nucleolus is a prominent nuclear structure that is the site of ribosomal RNA (rRNA) transcription, and hence ribosome biogenesis. Cellular demand for ribosomes, and hence rRNA, is tightly linked to cell growth and the rRNA makes up the majority of all the RNA within a cell. To fulfill the cellular demand for rRNA, the ribosomal RNA (rDNA) genes are amplified to high copy number and transcribed at very high rates. As such, understanding the rDNA has profound consequences for our comprehension of genome and transcriptional organization in cells. In this review, we address the question of whether the nucleolus is a raft adrift the sea of nuclear DNA, or actively contributes to genome organization. We present evidence supporting the idea that the nucleolus, and the rDNA contained therein, play more roles in the biology of the cell than simply ribosome biogenesis. We propose that the nucleolus and the rDNA are central factors in the spatial organization of the genome, and that rapid alterations in nucleolar structure in response to changing conditions manifest themselves in altered genomic structures that have functional consequences. Finally, we discuss some predictions that result from the nucleolus having a central role in nuclear organization.

Curcumin homing to the nucleolus: mechanism for initiation of an apoptotic program.

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Ghosh, Mistuni; Ryan, Robert O

2014-11-01

Curcumin is a plant-derived polyphenol that displays antitumor properties. Incubation of cultured SF-767 glioma cells with curcumin gave rise to intense intranuclear foci of curcumin fluorescence. In vitro studies revealed that nuclear homing by curcumin is not a result of DNA/chromatin binding. On the other hand, curcumin fluorescence colocalized with nucleophosmin, a nucleolus marker protein. To determine the temporal relationship between curcumin-induced apoptosis and nucleolar homing, confocal live cell imaging was performed. The data show that curcumin localization to the nucleolus occurs prior to cell surface exposure of phosphatidylserine. In studies of the mechanism of curcumin-induced apoptosis in SF-767 cells, its effect on the subcellular location of p14(ARF) was determined. Whereas p14(ARF) was confined to the nucleolus in untreated cells, 2 h following incubation with curcumin, it displayed a diffuse nuclear distribution. Given the role of nuclear p14(ARF) in binding the E3 ubiquitin ligase, mouse double minute 2 homolog (MDM2), the effect of curcumin treatment on cellular levels of the tumor suppressor protein, p53, was examined. Between 2 and 4 h following curcumin treatment, p53 levels increased with maximum levels reached by 8 h. Thus, curcumin homing to the nucleolus induces redistribution of p14(ARF) to the nucleoplasm where interaction with MDM2 leads to stabilization of p53, with subsequent initiation of apoptosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Directed proteomic analysis of the human nucleolus

DEFF Research Database (Denmark)

Andersen, Jens S; Lyon, Carol E; Fox, Archa H

2002-01-01

BACKGROUND: The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspec...

Nucleolar protein trafficking in response to HIV-1 Tat: rewiring the nucleolus.

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Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W; Gautier, Virginie W

2012-01-01

The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these

A mRNA and cognate microRNAs localize in the nucleolus.

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Reyes-Gutierrez, Pablo; Ritland Politz, Joan C; Pederson, Thoru

2014-01-01

We previously discovered that a set of 5 microRNAs are concentrated in the nucleolus of rat myoblasts. We now report that several mRNAs are also localized in the nucleoli of these cells as determined by microarray analysis of RNA from purified nucleoli. Among the most abundant of these nucleolus-localized mRNAs is that encoding insulin-like growth factor 2 (IGF2), a regulator of myoblast proliferation and differentiation. The presence of IGF2 mRNA in nucleoli was confirmed by fluorescence in situ hybridization, and RT-PCR experiments demonstrated that these nucleolar transcripts are spliced, thus arriving from the nucleoplasm. Bioinformatics analysis predicted canonically structured, highly thermodynamically stable interactions between IGF2 mRNA and all 5 of the nucleolus-localized microRNAs. These results raise the possibility that the nucleolus is a staging site for setting up particular mRNA-microRNA interactions prior to export to the cytoplasm.

Focal accumulation of preribosomes outside the nucleolus during metaphase-anaphase in budding yeast.

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Moriggi, Giulia; Gaspar, Sonia G; Nieto, Blanca; Bustelo, Xosé R; Dosil, Mercedes

2017-09-01

Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud. © 2017 Moriggi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Focal accumulation of preribosomes outside the nucleolus during metaphase–anaphase in budding yeast

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Moriggi, Giulia; Gaspar, Sonia G.; Nieto, Blanca; Bustelo, Xosé R.

2017-01-01

Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud. PMID:28588079

Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent.

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Zhang, Duo; Chen, Hui-Peng; Duan, Hai-Feng; Gao, Li-Hua; Shao, Yong; Chen, Ke-Yan; Wang, You-Liang; Lan, Feng-Hua; Hu, Xian-Wen

2016-07-01

Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Viral Impact in Autoimmune Diseases: Expanding the “X Chromosome–Nucleolus Nexus” Hypothesis

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Brooks, Wesley H.

2017-01-01

Viruses are suspected of significant roles in autoimmune diseases but the mechanisms are unclear. We get some insight by considering demands a virus places on host cells. Viruses not only require production of their own proteins, RNA and/or DNA, but also production of additional cellular machinery, such as ribosomes, to handle the increased demands. Since the nucleolus is a major site of RNA processing and ribonucleoprotein assembly, nucleoli are targeted by viruses, directly when viral RNA and proteins enter the nucleolus and indirectly when viruses induce increased expression of cellular polyamine genes. Polyamines are at high levels in nucleoli to assist in RNA folding. The size and activity of nucleoli increase directly with increases in polyamines. Nucleolar expansion due to abnormal increases in polyamines could disrupt nearby chromatin, such as the inactive X chromosome, leading to expression of previously sequestered DNA. Sudden expression of a large concentration of Alu elements from the disrupted inactive X can compete with RNA transcripts containing intronic Alu sequences that normally maintain nucleolar structural integrity. Such disruption of nucleolar activity can lead to misfolded RNAs, misassembled ribonucleoprotein complexes, and fragmentation of the nucleolus. Many autoantigens in lupus are, at least transiently, components of the nucleolus. Considering these effects of viruses, the “X chromosome–nucleolus nexus” hypothesis, which proposed disruption of the inactive X by the nucleolus during stress, is now expanded here to propose subsequent disruption of the nucleolus by previously sequestered Alu elements, which can fragment the nucleolus, leading to generation of autoantigens. PMID:29234321

Kinetics of the association of dengue virus capsid protein with the granular component of nucleolus.

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Tiwary, Ashish Kumar; Cecilia, D

2017-02-01

Dengue virus (DENV) replicates in the cytoplasm but translocation of the capsid protein (C) to the nucleoli of infected cells has been shown to facilitate virus multiplication for DENV-2. This study demonstrates that the nucleolar localization of C occurs with all four serotypes of DENV. The interaction of C with the nucleolus was found to be dynamic with a mobile fraction of 66% by FRAP. That the C shuttled between the nucleus and cytoplasm was suggested by FLIP and translation inhibition experiments. Colocalization with B23 indicated that DENV C targeted the granular component (GC) of the nucleolus. Presence of DENV C in the nucleolus affected the recovery kinetics of B23 in infected and transfected cells. Sub-nucleolar localization of DENV C of all serotypes to the GC, its mobility in and out of the nucleolus and its affect on the dynamics of B23 is being shown for the first time. Copyright © 2016 Elsevier Inc. All rights reserved.

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria.

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Walsh, Charles J

2012-01-01

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.

A Method to Identify Nucleolus-Associated Chromatin Domains (NADs).

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Carpentier, Marie-Christine; Picart-Picolo, Ariadna; Pontvianne, Frédéric

2018-01-01

The nuclear context needs to be taken into consideration to better understand the mechanisms shaping the epigenome and its organization, and therefore its impact on gene expression. For example, in Arabidopsis, heterochromatin is preferentially localized at the nuclear and the nucleolar periphery. Although chromatin domains associating with the nuclear periphery remain to be identified in plant cells, Nucleolus Associated chromatin Domains (NADs) can be identified thanks to a protocol allowing the isolation of pure nucleoli. We describe here the protocol enabling the identification of NADs in Arabidopsis. Providing the transfer of a nucleolus marker as described here in other crop species, this protocol is broadly applicable.

A Nucleolus for Stochastic Cooperative Games

NARCIS (Netherlands)

Suijs, J.P.M.

1996-01-01

This paper extends the definition of the nucleolus to stochastic cooperative games, that is, to cooperative games with random payoffs to the coalitions. It is shown that the nucleolus is nonempty and that it belongs to the core whenever the core is nonempty. Furthermore, it is shown for a particular

Cytoplasmic movement profiles of mouse surrounding nucleolus and not-surrounding nucleolus antral oocytes during meiotic resumption.

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Bui, Thi Thu Hien; Belli, Martina; Fassina, Lorenzo; Vigone, Giulia; Merico, Valeria; Garagna, Silvia; Zuccotti, Maurizio

2017-05-01

Full-grown mouse antral oocytes are classified as surrounding nucleolus (SN) or not-surrounding nucleolus (NSN), depending on the respective presence or absence of a ring of Hoechst-positive chromatin surrounding the nucleolus. In culture, both types of oocytes resume meiosis and reach the metaphase II (MII) stage, but following insemination, NSN oocytes arrest at the two-cell stage whereas SN oocytes may develop to term. By coupling time-lapse bright-field microscopy with image analysis based on particle image velocimetry, we provide the first systematic measure of the changes to the cytoplasmic movement velocity (CMV) occurring during the germinal vesicle-to-MII (GV-to-MII) transition of these two types of oocytes. Compared to SN oocytes, NSN oocytes display a delayed GV-to-MII transition, which can be mostly explained by retarded germinal vesicle break down and first polar body extrusion. SN and NSN oocytes also exhibit significantly different CMV profiles at four main time-lapse intervals, although this difference was not predictive of SN or NSN oocyte origin because of the high variability in CMV. When CMV profile was analyzed through a trained artificial neural network, however, each single SN or NSN oocyte was blindly identified with a probability of 92.2% and 88.7%, respectively. Thus, the CMV profile recorded during meiotic resumption may be exploited as a cytological signature for the non-invasive assessment of the oocyte developmental potential, and could be informative for the analysis of the GV-to-MII transition of oocytes of other species. © 2017 Wiley Periodicals, Inc.

New roles for Dicer in the nucleolus and its relevance to cancer.

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Roche, Benjamin; Arcangioli, Benoît; Martienssen, Rob

2017-09-17

The nucleolus is a distinct compartment of the nucleus responsible for ribosome biogenesis. Mis-regulation of nucleolar functions and of the cellular translation machinery has been associated with disease, in particular with many types of cancer. Indeed, many tumor suppressors (p53, Rb, PTEN, PICT1, BRCA1) and proto-oncogenes (MYC, NPM) play a direct role in the nucleolus, and interact with the RNA polymerase I transcription machinery and the nucleolar stress response. We have identified Dicer and the RNA interference pathway as having an essential role in the nucleolus of quiescent Schizosaccharomyces pombe cells, distinct from pericentromeric silencing, by controlling RNA polymerase I release. We propose that this novel function is evolutionarily conserved and may contribute to the tumorigenic pre-disposition of DICER1 mutations in mammals.

Changes of the nucleolus architecture in absence of the nuclear factor CTCF.

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Hernández-Hernández, A; Soto-Reyes, E; Ortiz, R; Arriaga-Canon, C; Echeverría-Martinez, O M; Vázquez-Nin, G H; Recillas-Targa, F

2012-01-01

CTCF is a multifunctional nuclear factor involved in many cellular processes like gene regulation, chromatin insulation and genomic organization. Recently, CTCF has been shown to be involved in the transcriptional regulation of ribosomal genes and nucleolar organization in Drosophila cells and different murine cell types, including embryonic stem cells. Moreover, it has been suggested that CTCF could be associated to the nucleolus of human erythroleukemic K562 cells. In the present work, we took advantage of efficient small hairpin RNA interference against human CTCF to analyze nucleolar organization in HeLa cells. We have found that key components of the nucleolar architecture are altered. As a consequence of such alterations, an upregulation of ribosomal gene transcription was observed. We propose that CTCF contributes to the structural organization of the nucleolus and, through epigenetic mechanisms, to the regulation of the ribosomal gene expression. Copyright © 2012 S. Karger AG, Basel.

Human nucleolus organizers on nonhomologous chromosomes can share the same ribosomal gene variants.

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Krystal, M; D'Eustachio, P; Ruddle, F H; Arnheim, N

1981-01-01

The distributions of three human ribosomal gene polymorphisms among individual chromosomes containing nucleolus organizers were analyzed by using mouse--human hybrid cells. Different nucleolus organizers can contain the same variant, suggesting the occurrence of genetic exchanges among ribosomal gene clusters on nonhomologous chromosomes. Such exchanges appear to occur less frequently in mice. This difference is discussed in terms of the nucleolar organization and chromosomal location of ribosomal gene clusters in humans and mice. Images PMID:6272316

The nucleolus in the mouse oocyte is required for the early step of both female and male pronucleus organization.

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OGUSHI, Sugako; SAITOU, Mitinori

2010-10-01

During oocyte growth in the ovary, the nucleolus is mainly responsible for ribosome biogenesis. However, in the fully-grown oocyte, all transcription ceases, including ribosomal RNA synthesis, and the nucleolus adopts a specific monotonous fibrillar morphology without chromatin. The function of this inactive nucleolus in oocytes and embryos is still unknown. We previously reported that the embryo lacking an inactive nucleolus failed to develop past the first few cleavages, indicating the requirement of a nucleolus for preimplantation development. Here, we reinjected the nucleolus into oocytes and zygotes without nucleoli at various time points to examine the timing of the nucleolus requirement during meiosis and early embryonic development. When we put the nucleolus back into oocytes lacking a nucleolus at the germinal vesicle (GV) stage and at second metaphase (MII), these oocytes were fertilized, formed pronuclei with nucleoli and developed to full term. When the nucleolus was reinjected at the pronucleus (PN) stage, most of the reconstructed zygotes cleaved and formed nuclei with nucleoli at the 2-cell stage, but the rate of blastocyst formation and the numbers of surviving pups were profoundly reduced. Moreover, the zygotes without nucleoli showed a disorder of higher chromatin organization not only in the female pronucleus but also, interestingly, in the male pronucleus. Thus, the critical time point when the nucleolus is required for progression of early embryonic development appears to be at the point of the early step of pronucleus organization.

The relationship between the nucleolus and cancer: Current evidence and emerging paradigms.

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Orsolic, Ines; Jurada, Deana; Pullen, Nick; Oren, Moshe; Eliopoulos, Aristides G; Volarevic, Sinisa

2016-06-01

The nucleolus is the most prominent nuclear substructure assigned to produce ribosomes; molecular machines that are responsible for carrying out protein synthesis. To meet the increased demand for proteins during cell growth and proliferation the cell must increase protein synthetic capacity by upregulating ribosome biogenesis. While larger nucleolar size and number have been recognized as hallmark features of many tumor types, recent evidence has suggested that, in addition to overproduction of ribosomes, decreased ribosome biogenesis as well as qualitative changes in this process could also contribute to tumor initiation and cancer progression. Furthermore, the nucleolus has become the focus of intense attention for its involvement in processes that are clearly unrelated to ribosome biogenesis such as sensing and responding to endogenous and exogenous stressors, maintenance of genome stability, regulation of cell-cycle progression, cellular senescence, telomere function, chromatin structure, establishment of nuclear architecture, global regulation of gene expression and biogenesis of multiple ribonucleoprotein particles. The fact that dysregulation of many of these fundamental cellular processes may contribute to the malignant phenotype suggests that normal functioning of the nucleolus safeguards against the development of cancer and indicates its potential as a therapeutic approach. Here we review the recent advances made toward understanding these newly-recognized nucleolar functions and their roles in normal and cancer cells, and discuss possible future research directions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interchanges in popcorn (Zea mays L. involving the nucleolus organizer chromosome

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Maria Suely Pagliarini

2006-01-01

Full Text Available The analysis of microsporogenesis in endogamous plants of popcorn (S5 to S7 showed several and distinctinterchanges which involve the nucleolus organizer (chromosome 6. The detection of cells with interchanges was facilitatedby the presence of two nucleoli of different sizes in contrast to normal ones with a single big nucleolus. Interchange points donot always seem to be at the same place. Whereas in several situations the interchange point clearly involved more than twochromosome pairs, a simple terminal translocation seemed to occur in others. During diplotene, a cross-shaped configurationconnected with the nucleoli was observed in some meiocytes. Some heteromorphic bivalents were found during diakinesis,after which meiosis progressed normally to the end and gave rise to apparently normal tetrads with one normal nucleolus ineach microspore. Tests of pollen viability in fixed pollen grains showed 100% stainability in normal and in affected plants.This is the first report on chromosome interchanges in popcorn.

Electron Microscopy Analysis of the Nucleolus of Trypanosoma cruzi

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López-Velázquez, Gabriel; Hernández, Roberto; López-Villaseñor, Imelda; Reyes-Vivas, Horacio; Segura-Valdez, María De L.; Jiménez-García, Luis F.

2005-08-01

The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical techniques as well as three-dimensional reconstruction to analyze the nucleolus of Trypanosoma cruzi, which is an early divergent eukaryote of medical importance. In T. cruzi epimastigotes the nucleolus is a spherical intranuclear ribonucleoprotein organelle localized in a relatively central position within the nucleus. Dense fibrillar and granular components but not fibrillar centers were observed. In addition, nuclear bodies resembling Cajal bodies were observed associated to the nucleolus in the surrounding nucleoplasm. Our results provide additional morphological data to better understand the synthesis and processing of the ribosomal RNA in kinetoplastids.

The nucleolus: an emerging target for cancer therapy.

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Hein, Nadine; Hannan, Katherine M; George, Amee J; Sanij, Elaine; Hannan, Ross D

2013-11-01

For over 100 years, pathologists have utilised an increase in size and number of nucleoli, the subnuclear site of ribosome synthesis, as a marker of aggressive tumours. Despite this, the contribution of the nucleolus and ribosomal RNA synthesis to cancer has been largely overlooked. This concept has recently changed with the demonstration that the nucleolus indirectly controls numerous other cellular functions, in particular, the cellular activity of the critical tumour suppressor protein, p53. Moreover, selective inhibition of ribosomal gene transcription in the nucleolus has been shown to be an effective therapeutic strategy to promote cancer-specific activation of p53. This article reviews the largely untapped potential of the nucleolus and ribosomal gene transcription as exciting new targets for cancer therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nucleolar Proteome Analysis and Proteasomal Activity Assays Reveal a Link between Nucleolus and 26S Proteasome in A. thaliana

Science.gov (United States)

Montacié, Charlotte; Durut, Nathalie; Opsomer, Alison; Palm, Denise; Comella, Pascale; Picart, Claire; Carpentier, Marie-Christine; Pontvianne, Frederic; Carapito, Christine; Schleiff, Enrico; Sáez-Vásquez, Julio

2017-01-01

In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity. PMID:29104584

Nepro is localized in the nucleolus and essential for preimplantation development in mice.

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Hashimoto, Masakazu; Sato, Tatsuya; Muroyama, Yuko; Fujimura, Lisa; Hatano, Masahiko; Saito, Tetsuichiro

2015-09-01

We generated knockout (KO) mice of Nepro, which has been shown to be necessary to maintain neural progenitor cells downstream of Notch in the mouse developing neocortex by using knockdown experiments, to explore its function in embryogenesis. Nepro KO embryos were morphologically indistinguishable from wild type (WT) embryos until the morula stage but failed in blastocyst formation, and many cells of the KO embryos resulted in apoptosis. We found that Nepro was localized in the nucleolus at the blastocyst stage. The number of nucleolus precursor bodies (NPBs) and nucleoli per nucleus was significantly higher in Nepro KO embryos compared with WT embryos later than the 2-cell stage. Furthermore, at the morula stage, whereas 18S rRNA and ribosomal protein S6 (rpS6), which are components of the ribosome, were distributed to the cytoplasm in WT embryos, they were mainly localized in the nucleoli in Nepro KO embryos. In addition, in Nepro KO embryos, the amount of the mitochondria-associated p53 protein increased, and Cytochrome c was distributed in the cytoplasm. These findings indicate that Nepro is a nucleolus-associated protein, and its loss leads to the apoptosis before blastocyst formation in mice. © 2015 Japanese Society of Developmental Biologists.

The Nucleolus of Caenorhabditis elegans

Directory of Open Access Journals (Sweden)

Li-Wei Lee

2012-01-01

Full Text Available Nucleolar size and appearance correlate with ribosome biogenesis and cellular activity. The mechanisms underlying changes in nucleolar appearance and regulation of nucleolar size that occur during differentiation and cell cycle progression are not well understood. Caenorhabditis elegans provides a good model for studying these processes because of its small size and transparent body, well-characterized cell types and lineages, and because its cells display various sizes of nucleoli. This paper details the advantages of using C. elegans to investigate features of the nucleolus during the organism's development by following dynamic changes in fibrillarin (FIB-1 in the cells of early embryos and aged worms. This paper also illustrates the involvement of the ncl-1 gene and other possible candidate genes in nucleolar-size control. Lastly, we summarize the ribosomal proteins involved in life span and innate immunity, and those homologous genes that correspond to human disorders of ribosomopathy.

Environmental cues induce a long noncoding RNA–dependent remodeling of the nucleolus

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Jacob, Mathieu D.; Audas, Timothy E.; Uniacke, James; Trinkle-Mulcahy, Laura; Lee, Stephen

2013-01-01

The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Its structural plasticity has long been appreciated, particularly in response to transcriptional inhibition and other cellular stresses, although the mechanism and physiological relevance of these phenomena are unclear. Using MCF-7 and other mammalian cell lines, we describe a structural and functional adaptation of the nucleolus, triggered by heat shock or physiological acidosis, that depends on the expression of ribosomal intergenic spacer long noncoding RNA (IGS lncRNA). At the heart of this process is the de novo formation of a large subnucleolar structure, termed the detention center (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate–positive hydrophobic signature. Its formation is accompanied by redistribution of nucleolar factors and arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA by environmental signals operates as a molecular switch that regulates the structure and function of the nucleolus. PMID:23904269

Environmental cues induce a long noncoding RNA-dependent remodeling of the nucleolus.

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Jacob, Mathieu D; Audas, Timothy E; Uniacke, James; Trinkle-Mulcahy, Laura; Lee, Stephen

2013-09-01

The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Its structural plasticity has long been appreciated, particularly in response to transcriptional inhibition and other cellular stresses, although the mechanism and physiological relevance of these phenomena are unclear. Using MCF-7 and other mammalian cell lines, we describe a structural and functional adaptation of the nucleolus, triggered by heat shock or physiological acidosis, that depends on the expression of ribosomal intergenic spacer long noncoding RNA (IGS lncRNA). At the heart of this process is the de novo formation of a large subnucleolar structure, termed the detention center (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate-positive hydrophobic signature. Its formation is accompanied by redistribution of nucleolar factors and arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA by environmental signals operates as a molecular switch that regulates the structure and function of the nucleolus.

Proteomic Analysis of Bovine Nucleolus

Institute of Scientific and Technical Information of China (English)

Amrutlal K.Patel; Doug Olson; Suresh K. Tikoo

2010-01-01

Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eu-karyotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells,we analyzed the proteomie composition of the bovine nueleoli. The nucleoli were isolated from Madin Darby bo-vine kidney cells and subjected to proteomie analysis by LC-MS/MS after fractionation by SDS-PAGE and strongcation exchange chromatography. Analysis of the data using the Mascot database search and the GPM databasesearch identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in theproteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggestedthat the bovine nueleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional,translational and post-translational regulation, transport, and structural organization.

Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro.

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Arcangeletti, Maria-Cristina; Rodighiero, Isabella; Mirandola, Prisco; De Conto, Flora; Covan, Silvia; Germini, Diego; Razin, Sergey; Dettori, Giuseppe; Chezzi, Carlo

2011-01-01

The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.

MXD1 localizes in the nucleolus, binds UBF and impairs rRNA synthesis.

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Lafita-Navarro, Maria Del Carmen; Blanco, Rosa; Mata-Garrido, Jorge; Liaño-Pons, Judit; Tapia, Olga; García-Gutiérrez, Lucía; García-Alegría, Eva; Berciano, María T; Lafarga, Miguel; León, Javier

2016-10-25

MXD1 is a protein that interacts with MAX, to form a repressive transcription factor. MXD1-MAX binds E-boxes. MXD1-MAX antagonizes the transcriptional activity of the MYC oncoprotein in most models. It has been reported that MYC overexpression leads to augmented RNA synthesis and ribosome biogenesis, which is a relevant activity in MYC-mediated tumorigenesis. Here we describe that MXD1, but not MYC or MNT, localizes to the nucleolus in a wide array of cell lines derived from different tissues (carcinoma, leukemia) as well as in embryonic stem cells. MXD1 also localizes in the nucleolus of primary tissue cells as neurons and Sertoli cells. The nucleolar localization of MXD1 was confirmed by co-localization with UBF. Co-immunoprecipitation experiments showed that MXD1 interacted with UBF and proximity ligase assays revealed that this interaction takes place in the nucleolus. Furthermore, chromatin immunoprecipitation assays showed that MXD1 was bound in the transcribed rDNA chromatin, where it co-localizes with UBF, but also in the ribosomal intergenic regions. The MXD1 involvement in rRNA synthesis was also suggested by the nucleolar segregation upon rRNA synthesis inhibition by actinomycin D. Silencing of MXD1 with siRNAs resulted in increased synthesis of pre-rRNA while enforced MXD1 expression reduces it. The results suggest a new role for MXD1, which is the control of ribosome biogenesis. This new MXD1 function would be important to curb MYC activity in tumor cells.

Principles of protein targeting to the nucleolus.

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Martin, Robert M; Ter-Avetisyan, Gohar; Herce, Henry D; Ludwig, Anne K; Lättig-Tünnemann, Gisela; Cardoso, M Cristina

2015-01-01

The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.

Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development.

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Vogt, Edgar J; Meglicki, Maciej; Hartung, Kristina Ilka; Borsuk, Ewa; Behr, Rüdiger

2012-12-01

The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus.

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Hult, Caitlin; Adalsteinsson, David; Vasquez, Paula A; Lawrimore, Josh; Bennett, Maggie; York, Alyssa; Cook, Diana; Yeh, Elaine; Forest, Mark Gregory; Bloom, Kerry

2017-11-02

Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells.

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Hong, Jin-Bon; Chou, Fu-Ju; Ku, Amy T; Fan, Hsiang-Hsuan; Lee, Tung-Lung; Huang, Yung-Hsin; Yang, Tsung-Lin; Su, I-Chang; Yu, I-Shing; Lin, Shu-Wha; Chien, Chung-Liang; Ho, Hong-Nerng; Chen, You-Tzung

2014-01-01

PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.

Inverse size scaling of the nucleolus by a concentration-dependent phase transition.

Science.gov (United States)

Weber, Stephanie C; Brangwynne, Clifford P

2015-03-02

Just as organ size typically increases with body size, the size of intracellular structures changes as cells grow and divide. Indeed, many organelles, such as the nucleus [1, 2], mitochondria [3], mitotic spindle [4, 5], and centrosome [6], exhibit size scaling, a phenomenon in which organelle size depends linearly on cell size. However, the mechanisms of organelle size scaling remain unclear. Here, we show that the size of the nucleolus, a membraneless organelle important for cell-size homeostasis [7], is coupled to cell size by an intracellular phase transition. We find that nucleolar size directly scales with cell size in early C. elegans embryos. Surprisingly, however, when embryo size is altered, we observe inverse scaling: nucleolar size increases in small cells and decreases in large cells. We demonstrate that this seemingly contradictory result arises from maternal loading of a fixed number rather than a fixed concentration of nucleolar components, which condense into nucleoli only above a threshold concentration. Our results suggest that the physics of phase transitions can dictate whether an organelle assembles, and, if so, its size, providing a mechanistic link between organelle assembly and cell size. Since the nucleolus is known to play a key role in cell growth, this biophysical readout of cell size could provide a novel feedback mechanism for growth control. Copyright © 2015 Elsevier Ltd. All rights reserved.

The transcription factor EGR1 localizes to the nucleolus and is linked to suppression of ribosomal precursor synthesis.

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Ponti, Donatella; Bellenchi, Gian Carlo; Puca, Rosa; Bastianelli, Daniela; Maroder, Marella; Ragona, Giuseppe; Roussel, Pascal; Thiry, Marc; Mercola, Dan; Calogero, Antonella

2014-01-01

EGR1 is an immediate early gene with a wide range of activities as transcription factor, spanning from regulation of cell growth to differentiation. Numerous studies show that EGR1 either promotes the proliferation of stimulated cells or suppresses the tumorigenic growth of transformed cells. Upon interaction with ARF, EGR1 is sumoylated and acquires the ability to bind to specific targets such as PTEN and in turn to regulate cell growth. ARF is mainly localized to the periphery of nucleolus where is able to negatively regulate ribosome biogenesis. Since EGR1 colocalizes with ARF under IGF-1 stimulation we asked the question of whether EGR1 also relocate to the nucleolus to interact with ARF. Here we show that EGR1 colocalizes with nucleolar markers such as fibrillarin and B23 in the presence of ARF. Western analysis of nucleolar extracts from HeLa cells was used to confirm the presence of EGR1 in the nucleolus mainly as the 100 kDa sumoylated form. We also show that the level of the ribosomal RNA precursor 47S is inversely correlated to the level of EGR1 transcripts. The EGR1 iseffective to regulate the synthesis of the 47S rRNA precursor. Then we demonstrated that EGR1 binds to the Upstream Binding Factor (UBF) leading us to hypothesize that the regulating activity of EGR1 is mediated by its interaction within the transcriptional complex of RNA polymerase I. These results confirm the presence of EGR1 in the nucleolus and point to a role for EGR1 in the control of nucleolar metabolism.

The transcription factor EGR1 localizes to the nucleolus and is linked to suppression of ribosomal precursor synthesis.

Directory of Open Access Journals (Sweden)

Donatella Ponti

Full Text Available EGR1 is an immediate early gene with a wide range of activities as transcription factor, spanning from regulation of cell growth to differentiation. Numerous studies show that EGR1 either promotes the proliferation of stimulated cells or suppresses the tumorigenic growth of transformed cells. Upon interaction with ARF, EGR1 is sumoylated and acquires the ability to bind to specific targets such as PTEN and in turn to regulate cell growth. ARF is mainly localized to the periphery of nucleolus where is able to negatively regulate ribosome biogenesis. Since EGR1 colocalizes with ARF under IGF-1 stimulation we asked the question of whether EGR1 also relocate to the nucleolus to interact with ARF. Here we show that EGR1 colocalizes with nucleolar markers such as fibrillarin and B23 in the presence of ARF. Western analysis of nucleolar extracts from HeLa cells was used to confirm the presence of EGR1 in the nucleolus mainly as the 100 kDa sumoylated form. We also show that the level of the ribosomal RNA precursor 47S is inversely correlated to the level of EGR1 transcripts. The EGR1 iseffective to regulate the synthesis of the 47S rRNA precursor. Then we demonstrated that EGR1 binds to the Upstream Binding Factor (UBF leading us to hypothesize that the regulating activity of EGR1 is mediated by its interaction within the transcriptional complex of RNA polymerase I. These results confirm the presence of EGR1 in the nucleolus and point to a role for EGR1 in the control of nucleolar metabolism.

The Nucleolus: In Genome Maintenance and Repair.

Science.gov (United States)

Tsekrekou, Maria; Stratigi, Kalliopi; Chatzinikolaou, Georgia

2017-07-01

The nucleolus is the subnuclear membrane-less organelle where rRNA is transcribed and processed and ribosomal assembly occurs. During the last 20 years, however, the nucleolus has emerged as a multifunctional organelle, regulating processes that go well beyond its traditional role. Moreover, the unique organization of rDNA in tandem arrays and its unusually high transcription rates make it prone to unscheduled DNA recombination events and frequent RNA:DNA hybrids leading to DNA double strand breaks (DSBs). If not properly repaired, rDNA damage may contribute to premature disease onset and aging. Deregulation of ribosomal synthesis at any level from transcription and processing to ribosomal subunit assembly elicits a stress response and is also associated with disease onset. Here, we discuss how genome integrity is maintained within nucleoli and how such structures are functionally linked to nuclear DNA damage response and repair giving an emphasis on the newly emerging roles of the nucleolus in mammalian physiology and disease.

The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B.

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Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Bögler, Oliver; Duchrow, Michael

2003-01-01

The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase. Copyright 2002 John Wiley & Sons, Ltd.

Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation.

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Dillinger, Stefan; Straub, Tobias; Németh, Attila

2017-01-01

Mammalian chromosomes are organized in structural and functional domains of 0.1-10 Mb, which are characterized by high self-association frequencies in the nuclear space and different contact probabilities with nuclear sub-compartments. They exhibit distinct chromatin modification patterns, gene expression levels and replication timing. Recently, nucleolus-associated chromosomal domains (NADs) have been discovered, yet their precise genomic organization and dynamics are still largely unknown. Here, we use nucleolus genomics and single-cell experiments to address these questions in human embryonic fibroblasts during replicative senescence. Genome-wide mapping reveals 1,646 NADs in proliferating cells, which cover about 38% of the annotated human genome. They are mainly heterochromatic and correlate with late replicating loci. Using Hi-C data analysis, we show that interactions of NADs dominate interphase chromosome contacts in the 10-50 Mb distance range. Interestingly, only minute changes in nucleolar association are observed upon senescence. These spatial rearrangements in subdomains smaller than 100 kb are accompanied with local transcriptional changes. In contrast, large centromeric and pericentromeric satellite repeat clusters extensively dissociate from nucleoli in senescent cells. Accordingly, H3K9me3-marked heterochromatin gets remodelled at the perinucleolar space as revealed by immunofluorescence analyses. Collectively, this study identifies connections between the nucleolus, 3D genome structure, and cellular aging at the level of interphase chromosome organization.

Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation.

Directory of Open Access Journals (Sweden)

Stefan Dillinger

Full Text Available Mammalian chromosomes are organized in structural and functional domains of 0.1-10 Mb, which are characterized by high self-association frequencies in the nuclear space and different contact probabilities with nuclear sub-compartments. They exhibit distinct chromatin modification patterns, gene expression levels and replication timing. Recently, nucleolus-associated chromosomal domains (NADs have been discovered, yet their precise genomic organization and dynamics are still largely unknown. Here, we use nucleolus genomics and single-cell experiments to address these questions in human embryonic fibroblasts during replicative senescence. Genome-wide mapping reveals 1,646 NADs in proliferating cells, which cover about 38% of the annotated human genome. They are mainly heterochromatic and correlate with late replicating loci. Using Hi-C data analysis, we show that interactions of NADs dominate interphase chromosome contacts in the 10-50 Mb distance range. Interestingly, only minute changes in nucleolar association are observed upon senescence. These spatial rearrangements in subdomains smaller than 100 kb are accompanied with local transcriptional changes. In contrast, large centromeric and pericentromeric satellite repeat clusters extensively dissociate from nucleoli in senescent cells. Accordingly, H3K9me3-marked heterochromatin gets remodelled at the perinucleolar space as revealed by immunofluorescence analyses. Collectively, this study identifies connections between the nucleolus, 3D genome structure, and cellular aging at the level of interphase chromosome organization.

Immunoelectron microscopic characterization of nucleolus-associated domains during hibernation.

Science.gov (United States)

Malatesta, Manuela; Zancanaro, Carlo; Biggiogera, Marco

2011-01-01

The nucleolus represents a highly dynamic nuclear compartment involved in multiple functions and able to promptly respond to variations of metabolic needs. In the hibernator dormouse, which drastically modifies its metabolic activity during the seasonal cycle, the nucleolus undergoes structural and molecular changes during the torpor bouts; in particular, it shows many nucleoplasmic invaginations containing weakly contrasted areas of unknown nature. To analyze the molecular composition of these nucleolus-associated domains (NADs) and to understand their functional significance, the fine nucleolar composition has been investigated by means of ultrastructural immunocytochemistry in different tissues of euthermic, hibernating, and arousing hazel dormice (Muscardinus avellanarius): in particular, the intranucleolar location of several protein factors involved in the transcription and processing of either pre-rRNA or pre-mRNA has been considered. NADs proved to form during hibernation and disappear upon arousal and were found to contain m₃-G-capped snRNAs, snRNPs, hnRNPs, and the survival motor neuron protein; they were, on the contrary, devoid of the nucleolar factors tested (polymerase I, fibrillarin, nucleolin, and the ribosomal phosphoproteins P₀, P₁, and P₂). We hypothesize that NADs may represent a transient storage site for those molecules involved in the pre-mRNA splicing, which usually transit through the nucleolus; upon arousal, this would facilitate the resumption of RNA maturation by promoting the rapid reactivation of the molecular trafficking from the nucleolus. © 2010 Wiley-Liss, Inc.

The actin family protein ARP6 contributes to the structure and the function of the nucleolus

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Kitamura, Hiroshi [Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoka-ku, Sendai 981-8555 (Japan); Matsumori, Haruka [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811 (Japan); Kalendova, Alzbeta; Hozak, Pavel [Department of Biology of the Cell Nucleus, Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Prague (Czech Republic); Goldberg, Ilya G. [Image Informatics and Computational Biology Unit, Laboratory of Genetics, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224 (United States); Nakao, Mitsuyoshi [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811 (Japan); Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo 102-0076 (Japan); Saitoh, Noriko [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811 (Japan); Harata, Masahiko, E-mail: mharata@biochem.tohoku.ac.jp [Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoka-ku, Sendai 981-8555 (Japan)

2015-08-21

The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. - Highlights: • ARP6, an actin related protein, is important for nucleolar function and structure. • A population of ARP6 is localized in the center of nucleolus. • Depletion of ARP6 resulted in aberrant shape of the nucleolus. • ARP6 maintains the active rDNA transcription under high glucose. • ARP6 is required for the repression of rDNA transcription under starvation.

The actin family protein ARP6 contributes to the structure and the function of the nucleolus

International Nuclear Information System (INIS)

Kitamura, Hiroshi; Matsumori, Haruka; Kalendova, Alzbeta; Hozak, Pavel; Goldberg, Ilya G.; Nakao, Mitsuyoshi; Saitoh, Noriko; Harata, Masahiko

2015-01-01

The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. - Highlights: • ARP6, an actin related protein, is important for nucleolar function and structure. • A population of ARP6 is localized in the center of nucleolus. • Depletion of ARP6 resulted in aberrant shape of the nucleolus. • ARP6 maintains the active rDNA transcription under high glucose. • ARP6 is required for the repression of rDNA transcription under starvation

Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions

DEFF Research Database (Denmark)

Pendle, Alison F; Clark, Gillian P; Boon, Reinier

2005-01-01

The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic ...

Stalled RNAP-II molecules bound to non-coding rDNA spacers are required for normal nucleolus architecture.

Science.gov (United States)

Freire-Picos, M A; Landeira-Ameijeiras, V; Mayán, María D

2013-07-01

The correct distribution of nuclear domains is critical for the maintenance of normal cellular processes such as transcription and replication, which are regulated depending on their location and surroundings. The most well-characterized nuclear domain, the nucleolus, is essential for cell survival and metabolism. Alterations in nucleolar structure affect nuclear dynamics; however, how the nucleolus and the rest of the nuclear domains are interconnected is largely unknown. In this report, we demonstrate that RNAP-II is vital for the maintenance of the typical crescent-shaped structure of the nucleolar rDNA repeats and rRNA transcription. When stalled RNAP-II molecules are not bound to the chromatin, the nucleolus loses its typical crescent-shaped structure. However, the RNAP-II interaction with Seh1p, or cryptic transcription by RNAP-II, is not critical for morphological changes. Copyright © 2013 John Wiley & Sons, Ltd.

Strongly Essential Coalitions and the Nucleolus of Peer Group Games

NARCIS (Netherlands)

Brânzei, R.; Solymosi, T.; Tijs, S.H.

2003-01-01

Most of the known efficient algorithms designed to compute the nucleolus for special classes of balanced games are based on two facts: (i) in any balanced game, the coalitions which actually determine the nucleolus are essential; and (ii) all essential coalitions in any of the games in the class

Structural and functional organization of ribosomal genes within the mammalian cell nucleolus.

Science.gov (United States)

Derenzini, Massimo; Pasquinelli, Gianandrea; O'Donohue, Marie-Françoise; Ploton, Dominique; Thiry, Marc

2006-02-01

Data on the in situ structural-functional organization of ribosomal genes in the mammalian cell nucleolus are reviewed here. Major findings on chromatin structure in situ come from investigations carried out using the Feulgen-like osmium ammine reaction as a highly specific electron-opaque DNA tracer. Intranucleolar chromatin shows three different levels of organization: compact clumps, fibers ranging from 11 to 30 nm, and loose agglomerates of extended DNA filaments. Both clumps and fibers of chromatin exhibit a nucleosomal organization that is lacking in the loose agglomerates of extended DNA filaments. In fact, these filaments constantly show a thickness of 2-3 nm, the same as a DNA double-helix molecule. The loose agglomerates of DNA filaments are located in the fibrillar centers, the interphase counterpart of metaphase NORs, therefore being constituted by ribosomal DNA. The extended, non-nucleosomal configuration of this rDNA has been shown to be independent of transcriptional activity and characterizes ribosome genes that are either transcribed or transcriptionally silent. Data reviewed are consistent with a model of control for ribosome gene activity that is not mediated by changes in chromatin structure. The presence of rDNA in mammalian cells always structurally ready for transcription might facilitate a more rapid adjustment of the ribosome production in response to the metabolic needs of the cell.

Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation.

Science.gov (United States)

Moore, Henna M; Bai, Baoyan; Boisvert, François-Michel; Latonen, Leena; Rantanen, Ville; Simpson, Jeremy C; Pepperkok, Rainer; Lamond, Angus I; Laiho, Marikki

2011-10-01

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

Structural aspects of the nucleolus and its associated heterochromatin in neurons

International Nuclear Information System (INIS)

Pessacq, T.P.; Vidal Rioja, L.A.

1975-01-01

The study of the structure of the nucleolus has received considerable attention in recent years. Most data have been provided by a number of electromicroscopical studies which to some extent are confirmatory of the previous opticomicroscopical findings of Estable and Sotelo (1951, 1952, 1955), Davis (1960), Altmann, Stoecker, and Thoenes (1963), O'Donell (1961), Lettre, and Siebs (1961), Bernhard, Bauer, Gropp, Haguenau and Oberling (1955) among others. In a recent work Melker (1970) points out the existence in neurons of a mammal of a particular arrangement of the nucleolus and its associated chromatin very similar to that already established in avian neurons (Pessacq 1969). The research reported here adds some new structural facts on nucleolar-heterochromatinic rapports which although recognizable in a great variety of cellular types appears with schematic clearness only in a limited number of highly specialized cells of some species. (auth.)

A new role of GCN2 in the nucleolus.

Science.gov (United States)

Nakamura, Akito; Kimura, Hiromichi

2017-04-01

General control nonderepressible 2 (GCN2) is activated by the accumulation of uncharged tRNA in response to amino acid shortage and regulates amino acid starvation response in the cytosol. Here we report the nucleolar localization of GCN2 and the association between GCN2 and small RNA transcripts. Immunofluorescence analysis revealed that GCN2 was constitutively localized to the nucleolus or recruited to the nucleolus by amino acid starvation stress. The nucleolus is the largest structure in the nucleus, where it primarily serves as the site of ribosome and RNA synthesis in addition to acting as a stress sensor through the regulation of p53 function. We found that siRNA-mediated depletion of GCN2 increases small RNA transcripts such as tRNA and 5S rRNA, and induces the p53 pathway activation. Derepression of these transcripts and p53 pathway activation by GCN2 depletion was restored by depletion of B-related factor 1 (BRF1), a primary subunit of RNA polymerase III (pol III) components. These data suggest that the excess amount of small RNA transcripts following GCN2 depletion was responsible for the p53 activation. Our findings reveal a role of GCN2 in the nucleolus that is involved in the expression of small RNA transcripts and serves as alternative stress-sensing machinery for nutrient deficiency. Thus, GCN2 may play pivotal roles in multiple protein translation checkpoints in both the nucleolus and cytosol. Copyright © 2017 Elsevier Inc. All rights reserved.

Nucleation by rRNA Dictates the Precision of Nucleolus Assembly.

Science.gov (United States)

Falahati, Hanieh; Pelham-Webb, Bobbie; Blythe, Shelby; Wieschaus, Eric

2016-02-08

Membrane-less organelles are intracellular compartments specialized to carry out specific cellular functions. There is growing evidence supporting the possibility that such organelles form as a new phase, separating from cytoplasm or nucleoplasm. However, a main challenge to such phase separation models is that the initial assembly, or nucleation, of the new phase is typically a highly stochastic process and does not allow for the spatiotemporal precision observed in biological systems. Here, we investigate the initial assembly of the nucleolus, a membrane-less organelle involved in different cellular functions including ribosomal biogenesis. We demonstrate that the nucleolus formation is precisely timed in D. melanogaster embryos and follows the transcription of rRNA. We provide evidence that transcription of rRNA is necessary for overcoming the highly stochastic nucleation step in the formation of the nucleolus, through a seeding mechanism. In the absence of rDNA, the nucleolar proteins studied are able to form high-concentration assemblies. However, unlike the nucleolus, these assemblies are highly variable in number, location, and time at which they form. In addition, quantitative study of the changes in the nucleoplasmic concentration and distribution of these nucleolar proteins in the wild-type embryos is consistent with the role of rRNA in seeding the nucleolus formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Composition and Dynamics of the Nucleolinus, a Link between the Nucleolus and Cell Division Apparatus in Surf Clam (Spisula) Oocytes*

Science.gov (United States)

Alliegro, Mark C.; Hartson, Steven; Alliegro, Mary Anne

2012-01-01

The nucleolinus is a little-known cellular structure, discovered over 150 years ago (Agassiz, L. (1857) Contributions to the Natural History of the United States of America, First Monograph, Part IIL, Little, Brown and Co., Boston) and thought by some investigators in the late 19th to mid-20th century to function in the formation of the centrosomes or spindle. A role for the nucleolinus in formation of the cell division apparatus has recently been confirmed in oocytes of the surf clam, Spisula solidissima (Alliegro, M. A., Henry, J. J., and Alliegro, M. C. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 13718–13723). However, we know so little about the composition and dynamics of this compartment, it is difficult to construct mechanistic hypotheses or even to be sure that prior reports were describing analogous structures in the cells of mammals, amphibians, plants, and other organisms where it was observed. Surf clam oocytes are an attractive model to approach this problem because the nucleolinus is easily visible by light microscopy, making it accessible by laser microsurgery as well as isolation by common cell fractionation techniques. In this report, we analyze the macromolecular composition of isolated Spisula nucleolini and examine the relationship of this structure to the nucleolus and cell division apparatus. Analysis of nucleolinar RNA and protein revealed a set of molecules that overlaps with but is nevertheless distinct from the nucleolus. The proteins identified were primarily ones involved in nucleic acid metabolism and cell cycle regulation. Monoclonal antibodies generated against isolated nucleolini revealed centrosomal forerunners in the oocyte cytoplasm. Finally, induction of damage to the nucleolinus by laser microsurgery altered the trafficking of α- and γ-tubulin after fertilization. These observations strongly support a role for the nucleolinus in cell division and represent our first clues regarding mechanism. PMID:22219192

Composition and dynamics of the nucleolinus, a link between the nucleolus and cell division apparatus in surf clam (Spisula) oocytes.

Science.gov (United States)

Alliegro, Mark C; Hartson, Steven; Alliegro, Mary Anne

2012-02-24

The nucleolinus is a little-known cellular structure, discovered over 150 years ago (Agassiz, L. (1857) Contributions to the Natural History of the United States of America, First Monograph, Part IIL, Little, Brown and Co., Boston) and thought by some investigators in the late 19th to mid-20th century to function in the formation of the centrosomes or spindle. A role for the nucleolinus in formation of the cell division apparatus has recently been confirmed in oocytes of the surf clam, Spisula solidissima (Alliegro, M. A., Henry, J. J., and Alliegro, M. C. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 13718-13723). However, we know so little about the composition and dynamics of this compartment, it is difficult to construct mechanistic hypotheses or even to be sure that prior reports were describing analogous structures in the cells of mammals, amphibians, plants, and other organisms where it was observed. Surf clam oocytes are an attractive model to approach this problem because the nucleolinus is easily visible by light microscopy, making it accessible by laser microsurgery as well as isolation by common cell fractionation techniques. In this report, we analyze the macromolecular composition of isolated Spisula nucleolini and examine the relationship of this structure to the nucleolus and cell division apparatus. Analysis of nucleolinar RNA and protein revealed a set of molecules that overlaps with but is nevertheless distinct from the nucleolus. The proteins identified were primarily ones involved in nucleic acid metabolism and cell cycle regulation. Monoclonal antibodies generated against isolated nucleolini revealed centrosomal forerunners in the oocyte cytoplasm. Finally, induction of damage to the nucleolinus by laser microsurgery altered the trafficking of α- and γ-tubulin after fertilization. These observations strongly support a role for the nucleolinus in cell division and represent our first clues regarding mechanism.

Bioinformatic analysis of the nucleolus

DEFF Research Database (Denmark)

Leung, Anthony K L; Andersen, Jens S; Mann, Matthias

2003-01-01

The nucleolus is a plurifunctional, nuclear organelle, which is responsible for ribosome biogenesis and many other functions in eukaryotes, including RNA processing, viral replication and tumour suppression. Our knowledge of the human nucleolar proteome has been expanded dramatically by the two r...

ARF tumor suppression in the nucleolus.

Science.gov (United States)

Maggi, Leonard B; Winkeler, Crystal L; Miceli, Alexander P; Apicelli, Anthony J; Brady, Suzanne N; Kuchenreuther, Michael J; Weber, Jason D

2014-06-01

Since its discovery close to twenty years ago, the ARF tumor suppressor has played a pivotal role in the field of cancer biology. Elucidating ARF's basal physiological function in the cell has been the focal interest of numerous laboratories throughout the world for many years. Our current understanding of ARF is constantly evolving to include novel frameworks for conceptualizing the regulation of this critical tumor suppressor. As a result of this complexity, there is great need to broaden our understanding of the intricacies governing the biology of the ARF tumor suppressor. The ARF tumor suppressor is a key sensor of signals that instruct a cell to grow and proliferate and is appropriately localized in nucleoli to limit these processes. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease. Copyright © 2014 Elsevier B.V. All rights reserved.

NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus.

Science.gov (United States)

Ando, Kiyohiro; Parsons, Melissa J; Shah, Richa B; Charendoff, Chloé I; Paris, Sheré L; Liu, Peter H; Fassio, Sara R; Rohrman, Brittany A; Thompson, Ruth; Oberst, Andrew; Sidi, Samuel; Bouchier-Hayes, Lisa

2017-06-05

The PIDDosome (PIDD-RAIDD-caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2-dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function. © 2017 Ando et al.

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage.

Science.gov (United States)

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein-protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage.

The actin family protein ARP6 contributes to the structure and the function of the nucleolus.

Science.gov (United States)

Kitamura, Hiroshi; Matsumori, Haruka; Kalendova, Alzbeta; Hozak, Pavel; Goldberg, Ilya G; Nakao, Mitsuyoshi; Saitoh, Noriko; Harata, Masahiko

2015-08-21

The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. Copyright © 2015 Elsevier Inc. All rights reserved.

Bankruptcy and the Per Capita Nucleolus

NARCIS (Netherlands)

Huijink, S.; Borm, P.E.M.; Reijnierse, J.H.; Kleppe, J.

2013-01-01

Abstract: This article characterizes the per capita nucleolus for bankruptcy games as a bankruptcy rule. This rule, called the cligths rule, is based on the wellknown constrained equal awards principle. The essential feature of the rule however is that, for each bankruptcy problem, it takes into

Localization in the Nucleolus and Coiled Bodies of Protein Subunits of the Ribonucleoprotein Ribonuclease P

Science.gov (United States)

Jarrous, Nayef; Wolenski, Joseph S.; Wesolowski, Donna; Lee, Christopher; Altman, Sidney

1999-01-01

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors. PMID:10444065

Nucleolus-like morphology produced during the in vitro reassociation of nucleolar components

OpenAIRE

1993-01-01

Nucleoli, the sites of rRNA synthesis, rRNA processing, and the assembly of ribosomes, are dynamic organelles that, in most cells, disperse and reform during mitosis. The mechanisms that regulate nucleolar formation are unknown as is the relationship between nucleolar morphology and the pathway of ribosome biogenesis. In this report we describe the in vitro formation of nucleolus-like particles (NLPs) from soluble extracts of nucleoli. NLPs, which reached sizes comparable to nucleoli (1-3 mic...

Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization.

Science.gov (United States)

Gouzil, Julie; Fablet, Aurore; Lara, Estelle; Caignard, Grégory; Cochet, Marielle; Kundlacz, Cindy; Palmarini, Massimo; Varela, Mariana; Breard, Emmanuel; Sailleau, Corinne; Viarouge, Cyril; Coulpier, Muriel; Zientara, Stéphan; Vitour, Damien

2017-01-01

Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this

Analysis of the C. elegans Nucleolus by Immuno-DNA FISH.

Science.gov (United States)

Lanctôt, Christian

2016-01-01

Caenorhabditis elegans is a well-established model organism which allows, among others, to investigate the link between nucleolar structure/function on the one hand and cell fate choices and cellular differentiation on the other. In addition, C. elegans can be used to study the role of the nucleolus in processes that can be difficult to faithfully reproduce in vitro, such as gametogenesis, disease development, and aging. Here I present two complementary techniques, immunofluorescent staining and DNA fluorescence in situ hybridization, that have been adapted to label nucleolar components at various stages of the life cycle of the worm.

El nucléolo como un regulador del envejecimiento celular The nucleolus as a regulator of cellular senescence

Directory of Open Access Journals (Sweden)

María Rosete

2007-04-01

codifying for damaged rRNA, and the mutations in DNA helicases, which minimizes the formation of DNA extra-chromosomal circles codifying for rRNA, modify the nucleolar structure and induce premature senescence in yeast. Similarly, in humans, the reduction of these DNA helicases levels, which are localized in the nucleoli and participate in maintenance of genomic integrity, helps to the development of those diseases associated with premature senescence. Furthermore, the presence in the nucleolus of some telomerase components, indicates that part of the biosynthesis of this enzyme occurred in this nuclear structure; suggesting a communication between the nucleolus and the synthesis of the telomeres in the regulation of cell senescence. On the other hand, the nucleolus sequesters proteins to regulate its own biological activity, from the start to the end of cellular replication. In addition this nuclear structure is involved in the biosynthesis of most cellular ribonucleoprotein particles, as well as in cell cycle regulation, making it central to gene expression. In conclusion, the nucleolus became a multifunctional subnuclear structure involved from cell proliferation to cell senescence.

Role of the nucleolus in neurodegenerative diseases with particular reference to the retina: a review.

Science.gov (United States)

Sia, Paul I; Wood, John Pm; Chidlow, Glyn; Sharma, Shiwani; Craig, Jamie; Casson, Robert J

2016-04-01

The nucleolus has emerged as a key regulator of cellular growth and the response to stress, in addition to its traditionally understood function in ribosome biogenesis. The association between nucleolar function and neurodegenerative disease is increasingly being explored. There is also recent evidence indicating that the nucleolus may well be crucial in the development of the eye. In this present review, the role of the nucleolus in retinal development as well as in neurodegeneration with an emphasis on the retina is discussed. © 2015 Royal Australian and New Zealand College of Ophthalmologists.

Effects of Lead on the Morphology and Structure of the Nucleolus in the Root Tip Meristematic Cells of Allium cepa L.

Directory of Open Access Journals (Sweden)

Ze Jiang

2014-07-01

Full Text Available To study the toxic mechanisms of lead (Pb in plants, the effects of Pb on the morphology and structure of the nucleolus in root tip meristematic cells of Allium cepa var. agrogarum L. were investigated. Fluorescence labeling, silver-stained indirect immunofluorescent microscopy and western blotting were used. Fluorescence labeling showed that Pb ions were localized in the meristematic cells and the uptake and accumulation of Pb increased with treatment time. At low concentrations of Pb (1–10 μM there were persistent nucleoli in some cells during mitosis, and at high concentration (100 μM many of the nucleolar organizing regions were localized on sticky chromosomes in metaphase and anaphase cells. Pb induced the release of particles containing argyrophilic proteins to be released from the nucleus into the cytoplasm. These proteins contained nucleophosmin and nucleolin. Pb also caused the extrusion of fibrillarin from the nucleus into the cytoplasm. Western blotting demonstrated the increased expression of these three major nucleolar proteins under Pb stress.

The nucleolus is well-posed

Science.gov (United States)

Fragnelli, Vito; Patrone, Fioravante; Torre, Anna

2006-02-01

The lexicographic order is not representable by a real-valued function, contrary to many other orders or preorders. So, standard tools and results for well-posed minimum problems cannot be used. We prove that under suitable hypotheses it is however possible to guarantee the well-posedness of a lexicographic minimum over a compact or convex set. This result allows us to prove that some game theoretical solution concepts, based on lexicographic order are well-posed: in particular, this is true for the nucleolus.

Insulin/IGF1-PI3K-dependent nucleolar localization of a glycolytic enzyme--phosphoglycerate mutase 2, is necessary for proper structure of nucleolus and RNA synthesis.

Science.gov (United States)

Gizak, Agnieszka; Grenda, Marcin; Mamczur, Piotr; Wisniewski, Janusz; Sucharski, Filip; Silberring, Jerzy; McCubrey, James A; Wisniewski, Jacek R; Rakus, Dariusz

2015-07-10

Phosphoglycerate mutase (PGAM), a conserved, glycolytic enzyme has been found in nucleoli of cancer cells. Here, we present evidence that accumulation of PGAM in the nucleolus is a universal phenomenon concerning not only neoplastically transformed but also non-malignant cells. Nucleolar localization of the enzyme is dependent on the presence of the PGAM2 (muscle) subunit and is regulated by insulin/IGF-1-PI3K signaling pathway as well as drugs influencing ribosomal biogenesis. We document that PGAM interacts with several 40S and 60S ribosomal proteins and that silencing of PGAM2 expression results in disturbance of nucleolar structure, inhibition of RNA synthesis and decrease of the mitotic index of squamous cell carcinoma cells. We conclude that presence of PGAM in the nucleolus is a prerequisite for synthesis and initial assembly of new pre-ribosome subunits.

Identification of novel markers that demarcate the nucleolus during severe stress and chemotherapeutic treatment.

Science.gov (United States)

Su, Haitong; Kodiha, Mohamed; Lee, Sunghoon; Stochaj, Ursula

2013-01-01

The nucleolus, the ribosomal factory of the cell, has emerged as a key player that regulates many aspects of cell biology. Several thousand proteins associate at least transiently with nucleoli, thereby generating a highly dynamic compartment with a protein profile which is sensitive to changes in cell physiology and pharmacological agents. Powerful tools that reliably demarcate the nucleoli are a prerequisite to measure their composition and activities. Previously, we developed quantitative methods to measure fluorescently labeled molecules in nucleoli. While these tools identify nucleoli under control and mild stress conditions, the accurate detection of nucleolar boundaries under harsh experimental conditions is complicated by the lack of appropriate markers for the nucleolar compartment. Using fluorescence microscopy we have now identified new marker proteins to detect nucleoli upon (a) severe stress and (b) drug treatments that trigger a pronounced reorganization of nucleoli. Our results demonstrate that nucleolin is an ideal marker to delimit nucleoli when cells are exposed to heat or oxidative stress. Furthermore, we show for the first time that cellular apoptosis susceptibility protein (CAS) and human antigen R protein (HuR) are excluded from nucleoli and can be employed to delimit these compartments under severe conditions that redistribute major nucleolar proteins. As proof-of-principle, we used these markers to demarcate nucleoli in cells treated with pharmacological compounds that disrupt the nucleolar organization. Furthermore, to gain new insights into the biology of the nucleolus, we applied our protocols and quantified stress- and drug-induced changes in nucleolar organization and function. Finally, we show that CAS, HuR and nucleolin not only identify nucleoli in optical sections, but are also suitable to demarcate the nucleolar border following 3D reconstruction. Taken together, our studies present novel marker proteins that delimit nucleoli with

Identification of novel markers that demarcate the nucleolus during severe stress and chemotherapeutic treatment.

Directory of Open Access Journals (Sweden)

Haitong Su

Full Text Available The nucleolus, the ribosomal factory of the cell, has emerged as a key player that regulates many aspects of cell biology. Several thousand proteins associate at least transiently with nucleoli, thereby generating a highly dynamic compartment with a protein profile which is sensitive to changes in cell physiology and pharmacological agents. Powerful tools that reliably demarcate the nucleoli are a prerequisite to measure their composition and activities. Previously, we developed quantitative methods to measure fluorescently labeled molecules in nucleoli. While these tools identify nucleoli under control and mild stress conditions, the accurate detection of nucleolar boundaries under harsh experimental conditions is complicated by the lack of appropriate markers for the nucleolar compartment. Using fluorescence microscopy we have now identified new marker proteins to detect nucleoli upon (a severe stress and (b drug treatments that trigger a pronounced reorganization of nucleoli. Our results demonstrate that nucleolin is an ideal marker to delimit nucleoli when cells are exposed to heat or oxidative stress. Furthermore, we show for the first time that cellular apoptosis susceptibility protein (CAS and human antigen R protein (HuR are excluded from nucleoli and can be employed to delimit these compartments under severe conditions that redistribute major nucleolar proteins. As proof-of-principle, we used these markers to demarcate nucleoli in cells treated with pharmacological compounds that disrupt the nucleolar organization. Furthermore, to gain new insights into the biology of the nucleolus, we applied our protocols and quantified stress- and drug-induced changes in nucleolar organization and function. Finally, we show that CAS, HuR and nucleolin not only identify nucleoli in optical sections, but are also suitable to demarcate the nucleolar border following 3D reconstruction. Taken together, our studies present novel marker proteins that

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus

International Nuclear Information System (INIS)

Reimer, G.; Raska, I.; Scheer, U.; Tan, E.M.

1988-01-01

Certain autoimmune sera contain antibodies against a nucleolar ribonucleotprotein particle associated with 7-2-RNA. In this study, the authors showed by immunofluorescence microscopy that antibodies reactive with 7-2-ribonucleoprotein immunolocalized in the granular regions of actinomycin D and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB)--segregated nucleoli from Vero cells. By electron microscopic immunocytochemistry, antigen-antibody complexes were located in the granular component of transcriptionally active nucleoli from rat liver hepatocytes and HeLa cells. Anti-7-2-RNP antibodies from two autoimmune sera immunoprecipitated a major protein of M r 40,000 from [ 35 S] methionine-labeled HeLa cell extract. The immunolocalization data suggest that 7-2-ribonucleoprotein may be involved in stages of ribosome biogenesis which take place in the granular component of the nucleolus, i.e., assembly, maturation, and/or transport of preribosomes

SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage.

Science.gov (United States)

Brun, Sonia; Abella, Neus; Berciano, Maria T; Tapia, Olga; Jaumot, Montserrat; Freire, Raimundo; Lafarga, Miguel; Agell, Neus

2017-01-01

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.

Tissue-specific features of the X chromosome and nucleolus spatial dynamics in a malaria mosquito, Anopheles atroparvus.

Science.gov (United States)

Bondarenko, Semen M; Artemov, Gleb N; Sharakhov, Igor V; Stegniy, Vladimir N

2017-01-01

Spatial organization of chromosome territories is important for maintenance of genomic stability and regulation of gene expression. Recent studies have shown tissue-specific features of chromosome attachments to the nuclear envelope in various organisms including malaria mosquitoes. However, other spatial characteristics of nucleus organization, like volume and shape of chromosome territories, have not been studied in Anopheles. We conducted a thorough analysis of tissue-specific features of the X chromosome and nucleolus volume and shape in follicular epithelium and nurse cells of the Anopheles atroparvus ovaries using a modern open-source software. DNA of the polytene X chromosome from ovarian nurse cells was obtained by microdissection and was used as a template for amplification with degenerate oligo primers. A fluorescently labeled X chromosome painting probe was hybridized with formaldehyde-fixed ovaries of mosquitoes using a 3D-FISH method. The nucleolus was stained by immunostaining with an anti-fibrillarin antibody. The analysis was conducted with TANGO-a software for a chromosome spatial organization analysis. We show that the volume and position of the X chromosome have tissue-specific characteristics. Unlike nurse cell nuclei, the growth of follicular epithelium nuclei is not accompanied with the proportional growth of the X chromosome. However, the shape of the X chromosome does not differ between the tissues. The dynamics of the X chromosome attachment regions location is tissue-specific and it is correlated with the process of nucleus growth in follicular epithelium and nurse cells.

The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway.

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Bailly, A; Perrin, A; Bou Malhab, L J; Pion, E; Larance, M; Nagala, M; Smith, P; O'Donohue, M-F; Gleizes, P-E; Zomerdijk, J; Lamond, A I; Xirodimas, D P

2016-01-28

The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.

Matching games: the least core and the nucleolus

NARCIS (Netherlands)

Kern, Walter; Paulusma, Daniël

2003-01-01

A matching game is a cooperative game defined by a graph G = (N, E). The player set is N and the value of a coalition S ⫅ N is defined as the size of a maximum matching in the subgraph induced by S. We show that the nucleolus of such games can be computed efficiently. The result is based on an

An Algorithm for the Nucleolus of Airport Profit Problems

NARCIS (Netherlands)

Brânzei, R.; Inarra, E.; Tijs, S.H.; Zarzuelo, J.

2003-01-01

Airport profit games are a generalization of airport cost games as well as of bankruptcy games.In this paper we present a simple algorithm to compute the nucleolus of airport profit games.In addition we prove that there exists an unique consistent allocation rule in airport profit problems, and it

Crosstalk between the nucleolus and the DNA damage response.

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Ogawa, L M; Baserga, S J

2017-02-28

Nucleolar function and the cellular response to DNA damage have long been studied as distinct disciplines. New research and a new appreciation for proteins holding multiple functional roles, however, is beginning to change the way we think about the crosstalk among distinct cellular processes. Here, we focus on the crosstalk between the DNA damage response and the nucleolus, including a comprehensive review of the literature that reveals a role for conventional DNA repair proteins in ribosome biogenesis, and conversely, ribosome biogenesis proteins in DNA repair. Furthermore, with recent advances in nucleolar proteomics and a growing list of proteins that localize to the nucleolus, it is likely that we will continue to identify new DNA repair proteins with a nucleolar-specific role. Given the importance of ribosome biogenesis and DNA repair in essential cellular processes and the role that they play in diverse pathologies, continued elucidation of the overlap between these two disciplines will be essential to the advancement of both fields and to the development of novel therapeutics.

The nucleolus directly regulates p53 export and degradation.

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Boyd, Mark T; Vlatkovic, Nikolina; Rubbi, Carlos P

2011-09-05

The correlation between stress-induced nucleolar disruption and abrogation of p53 degradation is evident after a wide variety of cellular stresses. This link may be caused by steps in p53 regulation occurring in nucleoli, as suggested by some biochemical evidence. Alternatively, nucleolar disruption also causes redistribution of nucleolar proteins, potentially altering their interactions with p53 and/or MDM2. This raises the fundamental question of whether the nucleolus controls p53 directly, i.e., as a site where p53 regulatory processes occur, or indirectly, i.e., by determining the cellular localization of p53/MDM2-interacting factors. In this work, transport experiments based on heterokaryons, photobleaching, and micronucleation demonstrate that p53 regulatory events are directly regulated by nucleoli and are dependent on intact nucleolar structure and function. Subcellular fractionation and nucleolar isolation revealed a distribution of ubiquitylated p53 that supports these findings. In addition, our results indicate that p53 is exported by two pathways: one stress sensitive and one stress insensitive, the latter being regulated by activities present in the nucleolus.

Nucleolus Precursor Bodies and Ribosome Biogenesis in Early Mammalian Embryos: Old Theories and New Discoveries.

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Fulka, Helena; Aoki, Fugaku

2016-06-01

In mammals, mature oocytes and early preimplantation embryos contain transcriptionally inactive structures termed nucleolus precursor bodies instead of the typical fibrillo-granular nucleoli. These nuclear organelles are essential and strictly of maternal origin. If they are removed from oocytes, the resulting embryos are unable to replace them and consequently fail to develop. Historically, nucleolus precursor bodies have been perceived as a passive repository site of nucleolar proteins that are required for embryos to form fully functional nucleoli. Recent results, however, contradict this long-standing dogma and show that these organelles are dispensable for nucleologenesis and ribosome biogenesis. In this article, we discuss the possible roles of nucleolus precursor bodies and propose how they might be involved in embryogenesis. Furthermore, we argue that these organelles are essential only shortly after fertilization and suggest that they might actively participate in centromeric chromatin establishment. © 2016 by the Society for the Study of Reproduction, Inc.

Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA.

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Mitrea, Diana M; Cika, Jaclyn A; Guy, Clifford S; Ban, David; Banerjee, Priya R; Stanley, Christopher B; Nourse, Amanda; Deniz, Ashok A; Kriwacki, Richard W

2016-02-02

The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus.

A note on the nucleolus for 2-convex TU games

NARCIS (Netherlands)

Driessen, Theo; Hou, D.

For 2-convex n-person cooperative TU games, the nucleolus is determined as some type of constrained equal award rule. Its proof is based on Maschler, Peleg, and Shapley’s geometrical characterization for the intersection of the prekernel with the core. Pairwise bargaining ranges within the core are

Intertissue Control of the Nucleolus via a Myokine-Dependent Longevity Pathway

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Fabio Demontis

2014-06-01

Full Text Available Recent evidence indicates that skeletal muscle influences systemic aging, but little is known about the signaling pathways and muscle-released cytokines (myokines responsible for this intertissue communication. Here, we show that muscle-specific overexpression of the transcription factor Mnt decreases age-related climbing defects and extends lifespan in Drosophila. Mnt overexpression in muscle autonomously decreases the expression of nucleolar components and systemically decreases rRNA levels and the size of the nucleolus in adipocytes. This nonautonomous control of the nucleolus, a regulator of ribosome biogenesis and lifespan, relies on Myoglianin, a myokine induced by Mnt and orthologous to human GDF11 and Myostatin. Myoglianin overexpression in muscle extends lifespan and decreases nucleolar size in adipocytes by activating p38 mitogen-activated protein kinase (MAPK, whereas Myoglianin RNAi in muscle has converse effects. Altogether, these findings highlight a key role for myokine signaling in the integration of signaling events in muscle and distant tissues during aging.

Tobacco etch virus protein P1 traffics to the nucleolus and associates with the host 60S ribosomal subunits during infection.

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Martínez, Fernando; Daròs, José-Antonio

2014-09-01

The genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells. In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly

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

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Hernández-Ibarra, Jose Anselmo; Laredo-Cisneros, Marco Samuel; Mondragón-González, Ricardo; Santamaría-Guayasamín, Natalie; Cisneros, Bulmaro

2015-12-01

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. © 2015 Wiley Periodicals, Inc.

A new level set model for cell image segmentation

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Ma, Jing-Feng; Hou, Kai; Bao, Shang-Lian; Chen, Chun

2011-02-01

In this paper we first determine three phases of cell images: background, cytoplasm and nucleolus according to the general physical characteristics of cell images, and then develop a variational model, based on these characteristics, to segment nucleolus and cytoplasm from their relatively complicated backgrounds. In the meantime, the preprocessing obtained information of cell images using the OTSU algorithm is used to initialize the level set function in the model, which can speed up the segmentation and present satisfactory results in cell image processing.

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells

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N.P. Silva

2004-07-01

Full Text Available Cajal bodies (CB are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

Label-free imaging of mammalian cell nucleoli by Raman microspectroscopy.

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Schulze, H Georg; Konorov, Stanislav O; Piret, James M; Blades, Michael W; Turner, Robin F B

2013-06-21

The nucleolus is a prominent subnuclear structure whose major function is the transcription and assembly of ribosome subunits. The size of the nucleolus varies with the cell cycle, proliferation rate and stress. Changes in nucleolar size, number, chemical composition, and shape can be used to characterize malignant cells. We used spontaneous Raman microscopy as a label-free technique to examine nucleolar spatial and chemical features. Raman images of the 1003 cm(-1) phenylalanine band revealed large, well-defined subnuclear protein structures in MFC-7 breast cancer cells. The 783 cm(-1) images showed that nucleic acids were similarly distributed, but varied more in intensity, forming observable high-intensity regions. High subnuclear RNA concentrations were observed within some of these regions as shown by 809 cm(-1) Raman band images. Principal component analyses of sub-images and library spectra validated the subnuclear presence of RNA. They also revealed that an actin-like protein covaried with DNA within the nucleolus, a combination that accounted for 64% or more of the spectral variance. Embryonic stem cells are another rapidly proliferating cell type, but their nucleoli were not as large or well defined. Estimating the size of the larger MCF-7 nucleolus was used to show the utility of Raman microscopy for morphometric analyses. It was concluded that imaging based on Raman microscopy provides a promising new method for the study of nucleolar function and organization, in the evaluation of drug and experimental effects on the nucleolus, and in clinical diagnostics and prognostics.

Colouring of PK cells nucleoli with silver in case of hyperactivation and activation of RNA nucleoli synthesis under ultraviolet radiation

International Nuclear Information System (INIS)

Sacharov, V.N.; Voronkova, L.N.; Valova, T.M.

1988-01-01

Localization and redistribution of silver-coloured nucleolus proteins in pig embryo kidney cell nuclei under postmitotic formation and evolution of nucleoli in order and under UV-microirradiation stimulated degradation and neature nucleolus compensator hyperactivation in the interphase are studied. In the anaphase chromosome set only 4 silver granules colouring the nucleolus organizer proteins were usually detected. At the initial stages of nucleolus postmitotic formation the number of silver granules in nucleolus organizer increased rapidly up to 25-30 per a nucleus, and at subsequent cellular cycle stages it doubled gradually. Under the natural growth and stimulated modifications of nucleoli the change of silver granule number in nucleoli correlated with the changes of nucleolus sizes and with the change of nucleolus RNA synthesis level. A possible connection between the nucleolus functional activity and the presence of proteins coloured with silver is discussed

A new level set model for cell image segmentation

International Nuclear Information System (INIS)

Ma Jing-Feng; Chen Chun; Hou Kai; Bao Shang-Lian

2011-01-01

In this paper we first determine three phases of cell images: background, cytoplasm and nucleolus according to the general physical characteristics of cell images, and then develop a variational model, based on these characteristics, to segment nucleolus and cytoplasm from their relatively complicated backgrounds. In the meantime, the preprocessing obtained information of cell images using the OTSU algorithm is used to initialize the level set function in the model, which can speed up the segmentation and present satisfactory results in cell image processing. (cross-disciplinary physics and related areas of science and technology)

C. elegans nucleostemin is required for larval growth and germline stem cell division.

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Michelle M Kudron

2008-08-01

Full Text Available The nucleolus has shown to be integral for many processes related to cell growth and proliferation. Stem cells in particular are likely to depend upon nucleolus-based processes to remain in a proliferative state. A highly conserved nucleolar factor named nucleostemin is proposed to be a critical link between nucleolar function and stem-cell-specific processes. Currently, it is unclear whether nucleostemin modulates proliferation by affecting ribosome biogenesis or by another nucleolus-based activity that is specific to stem cells and/or highly proliferating cells. Here, we investigate nucleostemin (nst-1 in the nematode C. elegans, which enables us to examine nst-1 function during both proliferation and differentiation in vivo. Like mammalian nucleostemin, the NST-1 protein is localized to the nucleolus and the nucleoplasm; however, its expression is found in both differentiated and proliferating cells. Global loss of C. elegans nucleostemin (nst-1 leads to a larval arrest phenotype due to a growth defect in the soma, while loss of nst-1 specifically in the germ line causes germline stem cells to undergo a cell cycle arrest. nst-1 mutants exhibit reduced levels of rRNAs, suggesting defects in ribosome biogenesis. However, NST-1 is generally not present in regions of the nucleolus where rRNA transcription and processing occurs, so this reduction is likely secondary to a different defect in ribosome biogenesis. Transgenic studies indicate that NST-1 requires its N-terminal domain for stable expression and both its G1 GTPase and intermediate domains for proper germ line function. Our data support a role for C. elegans nucleostemin in cell growth and proliferation by promoting ribosome biogenesis.

Localization of rDNA in small, nucleolus-like structures in human diplotene oocyte nuclei

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Wolgemuth-Jarashow, D.J.; Jagiello, G.M.; Henderson, A.S.

1977-01-01

Small, nucleolus-like structures were demonstrated in the nuclei of human diplotene oocytes. At least some of these bodies were shown to be true micronucleoli by virtue of their ability to bind rRNA during RNA-DNA hybridization in situ.

Homologous subfamilies of human alphoid repetitive DNA on different nucleolus organizing chromosomes

International Nuclear Information System (INIS)

Joergensen, A.L.; Bostock, C.J.; Bak, A.L.

1987-01-01

The organization of alphoid repeated sequences on human nucleolus-organizing (NOR) chromosomes 13, 21, and 22 has been investigated. Analysis of hybridization of alphoid DNA probes to Southern transfers of restriction enzyme-digested DNA fragments from hybrid cells containing single human chromosomes shows that chromosomes 13 and 21 share one subfamily of alphoid repeats, whereas a different subfamily may be held in common by chromosomes 13 and 22. The sequences of cloned 680-base-pair EcoRI fragments of the alphoid DNA from chromosomes 13 and 21 show that the basic unit of this subfamily is indistinguishable on each chromosome. The sequence of cloned 1020-base-pair Xba I fragments from chromosome 22 is related to, but distinguishable from, that of the 680-base-pair EcoRI alphoid subfamily of chromosomes 13 and 21. These results suggest that, at some point after they originated and were homogenized, different subfamilies of alphoid sequences must have exchanged between chromosomes 13 and 21 and separately between chromosomes 13 and 22

Proteome distribution between nucleoplasm and nucleolus and its relation to ribosome biogenesis in Arabidopsis thaliana.

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Palm, Denise; Simm, Stefan; Darm, Katrin; Weis, Benjamin L; Ruprecht, Maike; Schleiff, Enrico; Scharf, Christian

2016-01-01

Ribosome biogenesis is an essential process initiated in the nucleolus. In eukaryotes, multiple ribosome biogenesis factors (RBFs) can be found in the nucleolus, the nucleus and in the cytoplasm. They act in processing, folding and modification of the pre-ribosomal (r)RNAs, incorporation of ribosomal proteins (RPs), export of pre-ribosomal particles to the cytoplasm, and quality control mechanisms. Ribosome biogenesis is best established for Saccharomyces cerevisiae. Plant ortholog assignment to yeast RBFs revealed the absence of about 30% of the yeast RBFs in plants. In turn, few plant specific proteins have been identified by biochemical experiments to act in plant ribosome biogenesis. Nevertheless, a complete inventory of plant RBFs has not been established yet. We analyzed the proteome of the nucleus and nucleolus of Arabidopsis thaliana and the post-translational modifications of these proteins. We identified 1602 proteins in the nucleolar and 2544 proteins in the nuclear fraction with an overlap of 1429 proteins. For a randomly selected set of proteins identified by the proteomic approach we confirmed the localization inferred from the proteomics data by the localization of GFP fusion proteins. We assigned the identified proteins to various complexes and functions and found about 519 plant proteins that have a potential to act as a RBFs, but which have not been experimentally characterized yet. Last, we compared the distribution of RBFs and RPs in the various fractions with the distribution established for yeast.

The Trypanosoma cruzi nucleolus: a morphometrical analysis of cultured epimastigotes in the exponential and stationary phases.

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Nepomuceno-Mejía, Tomás; Lara-Martínez, Reyna; Cevallos, Ana María; López-Villaseñor, Imelda; Jiménez-García, Luis Felipe; Hernández, Roberto

2010-12-01

Our group is interested in rRNA and ribosome biogenesis in the parasitic protozoan Trypanosoma cruzi. Epimastigotes represent an extracellular replicative stage of T. cruzi and can be cultured in axenic media. The growth curve of epimastigotes allows assessment of potential differences in the nucleoli of cells undergoing growth-rate transitions. To establish cellular parameters for studying ribosome biogenesis in T. cruzi, a morphometric analysis of the nucleoli of cultured cells in the exponential and stationary phases was conducted. Electron micrograph-based measurements of nuclear sections from independent cells demonstrated that the nucleolar area is over twofold higher in exponentially growing cells, as compared with epimastigotes in the stationary phase. The granular component of the nucleoli of actively growing cells was the main structural element. Cycloheximide moderately reduced the apparent size of the nucleoli without an apparent disruption of their architecture. Our results provide a firm basis for the establishment of an experimental model to study the organization of the nucleolus during the growth and development of T. cruzi. © 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved.

De-repression of RaRF-mediated RAR repression by adenovirus E1A in the nucleolus.

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Um, Soo-Jong; Youn, Hye Sook; Kim, Eun-Joo

2014-02-21

Transcriptional activity of the retinoic acid receptor (RAR) is regulated by diverse binding partners, including classical corepressors and coactivators, in response to its ligand retinoic acid (RA). Recently, we identified a novel corepressor of RAR called the retinoic acid resistance factor (RaRF) (manuscript submitted). Here, we report how adenovirus E1A stimulates RAR activity by associating with RaRF. Based on immunoprecipitation (IP) assays, E1A interacts with RaRF through the conserved region 2 (CR2), which is also responsible for pRb binding. The first coiled-coil domain of RaRF was sufficient for this interaction. An in vitro glutathione-S-transferase (GST) pull-down assay was used to confirm the direct interaction between E1A and RaRF. Further fluorescence microscopy indicated that E1A and RaRF were located in the nucleoplasm and nucleolus, respectively. However, RaRF overexpression promoted nucleolar translocation of E1A from the nucleoplasm. Both the RA-dependent interaction of RAR with RaRF and RAR translocation to the nucleolus were disrupted by E1A. RaRF-mediated RAR repression was impaired by wild-type E1A, but not by the RaRF binding-defective E1A mutant. Taken together, our data suggest that E1A is sequestered to the nucleolus by RaRF through a specific interaction, thereby leaving RAR in the nucleoplasm for transcriptional activation. Copyright © 2014 Elsevier Inc. All rights reserved.

Nucleolus-derived mediators in oncogenic stress response and activation of p53-dependent pathways.

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Stępiński, Dariusz

2016-08-01

Rapid growth and division of cells, including tumor ones, is correlated with intensive protein biosynthesis. The output of nucleoli, organelles where translational machineries are formed, depends on a rate of particular stages of ribosome production and on accessibility of elements crucial for their effective functioning, including substrates, enzymes as well as energy resources. Different factors that induce cellular stress also often lead to nucleolar dysfunction which results in ribosome biogenesis impairment. Such nucleolar disorders, called nucleolar or ribosomal stress, usually affect cellular functioning which in fact is a result of p53-dependent pathway activation, elicited as a response to stress. These pathways direct cells to new destinations such as cell cycle arrest, damage repair, differentiation, autophagy, programmed cell death or aging. In the case of impaired nucleolar functioning, nucleolar and ribosomal proteins mediate activation of the p53 pathways. They are also triggered as a response to oncogenic factor overexpression to protect tissues and organs against extensive proliferation of abnormal cells. Intentional impairment of any step of ribosome biosynthesis which would direct the cells to these destinations could be a strategy used in anticancer therapy. This review presents current knowledge on a nucleolus, mainly in relation to cancer biology, which is an important and extremely sensitive element of the mechanism participating in cellular stress reaction mediating activation of the p53 pathways in order to counteract stress effects, especially cancer development.

RNA content in the nucleolus alters p53 acetylation via MYBBP1A

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Kuroda, Takao; Murayama, Akiko; Katagiri, Naohiro; Ohta, Yu-mi; Fujita, Etsuko; Masumoto, Hiroshi; Ema, Masatsugu; Takahashi, Satoru; Kimura, Keiji; Yanagisawa, Junn

2011-01-01

A number of external and internal insults disrupt nucleolar structure, and the resulting nucleolar stress stabilizes and activates p53. We show here that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. We identified three nucleolar proteins, MYBBP1A, RPL5, and RPL11, involved in p53 acetylation and accumulation. MYBBP1A was tethered to the nucleolus through nucleolar RNA. When rRNA transcription was suppressed by nucleolar stress, MYBBP1A translocated to the nucleoplasm and facilitated p53–p300 interaction to enhance p53 acetylation. We also found that RPL5 and RPL11 were required for rRNA export from the nucleolus. Depletion of RPL5 or RPL11 blocked rRNA export and counteracted reduction of nucleolar RNA levels caused by inhibition of rRNA transcription. As a result, RPL5 or RPL11 depletion inhibited MYBBP1A translocation and p53 activation. Our observations indicated that a dynamic equilibrium between RNA generation and export regulated nucleolar RNA content. Perturbation of this balance by nucleolar stress altered the nucleolar RNA content and modulated p53 activity. PMID:21297583

The carboxyl-terminus directs TAF(I)48 to the nucleus and nucleolus and associates with multiple nuclear import receptors.

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Dynes, Joseph L; Xu, Shuping; Bothner, Sarah; Lahti, Jill M; Hori, Roderick T

2004-03-01

The protein complex Selectivity Factor 1, composed of TBP, TAF(I)48, TAF(I)63 and TAF(I)110, is required for rRNA transcription by RNA polymerase I in the nucleolus. The steps involved in targeting Selectivity Factor 1 will be dependent on the transport pathways that are used and the localization signals that direct this trafficking. In order to investigate these issues, we characterized human TAF(I)48, a subunit of Selectivity Factor 1. By domain analysis of TAF(I)48, the carboxyl-terminal 51 residues were found to be required for the localization of TAF(I)48, as well as sufficient to direct Green Fluorescent Protein to the nucleus and nucleolus. The carboxyl-terminus of TAF(I)48 also has the ability to associate with multiple members of the beta-karyopherin family of nuclear import receptors, including importin beta (karyopherin beta1), transportin (karyopherin beta2) and RanBP5 (karyopherin beta3), in a Ran-dependent manner. This property of interacting with multiple beta-karyopherins has been previously reported for the nuclear localization signals of some ribosomal proteins that are likewise directed to the nucleolus. This study identifies the first nuclear import sequence identified within the TBP-Associated Factor subunits of Selectivity Factor 1.

Emerging roles of the nucleolus in regulating the DNA damage response: the noncanonical DNA repair enzyme APE1/Ref-1 as a paradigmatical example.

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Antoniali, Giulia; Lirussi, Lisa; Poletto, Mattia; Tell, Gianluca

2014-02-01

An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world.

Quantitative analysis of thymus lymphoid cells during murine radioleukemogenesis

International Nuclear Information System (INIS)

Boniver, J.; Simar, L.J.; Courtoy, R.; Betz, E.H.

1978-01-01

Fractionated irradiation by four doses of 150 R leads to the development of lymphoma in the thymus of C57BL mice, after a long latent period (4 to 12 months) during which thymic subcapsular blast cells undergo neoplastic transformation. Electron microscope studies on this blast cell population have revealed several types that are distinguishable on the basis of nuclear ultrastructure and activity in the cell cycle. These blast cells are lymphoblasts, ring-shaped nucleolus cells, and x-cells. A stereological analysis demonstrates that the result of the irradiations is not an increase of the thymic blast cell number during the preleukemic period, as generally thought before, but important modifications in the relative proportion of the three cell types forming the blastic population. X-cells, which essentially appear in S phase, are more numerous after the third and fourth doses of radiation. They might be more ''sensitive'' to the action of the leukemogenic virus and facilitate the leukemogenic process. In contrast, in the preleukemic atrophic thymus just before the appearance of the lymphoma, x-cells are scarce, and ring-shaped nucleolus cells are more frequent than in the controls, suggesting important changes in thymus cell kinetics. The only modification detected in the blast cells at any time after irradiation concerns the surface of Golgi membranes, which decreases in x-cells and ring-shaped nucleolus cells of the atrophic thymus. Because the same change is observed later on in the lymphomatous cells, it is suggested that the change might be related to the transformation of the thymic blast cells into cancerous cells

A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

International Nuclear Information System (INIS)

You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

2008-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus

Interaction of Protein Phosphatase 1δ with Nucleophosmin in Human Osteoblastic Cells

International Nuclear Information System (INIS)

Haneji, Tatsuji; Teramachi, Jumpei; Hirashima, Kanji; Kimura, Koji; Morimoto, Hiroyuki

2012-01-01

Protein phosphorylation and dephosphorylation has been recognized as an essential mechanism in the regulation of cellular metabolism and function in various tissues. Serine and threonine protein phosphatases (PP) are divided into four categories: PP1, PP2A, PP2B, and PP2C. At least four isoforms of PP1 catalytic subunit in rat, PP1α, PP1γ1, PP1γ2, and PP1δ, were isolated. In the present study, we examined the localization and expression of PP1δ in human osteoblastic Saos-2 cells. Anti-PP1δ antibody recognized a protein present in the nucleolar regions in Saos-2 cells. Cellular fractionation revealed that PP1δ is a 37 kDa protein localized in the nucleolus. Nucleophosmin is a nucleolar phosphoprotein and located mainly in the nucleolus. Staining pattern of nucleophosmin in Saos-2 cells was similar to that of PP1δ. PP1δ and nucleophosmin were specifically stained as dots in the nucleus. Dual fluorescence images revealed that PP1δ and nucleophosmin were localized in the same regions in the nucleolus. Similar distribution patterns of PP1δ and nucleophosmin were observed in osteoblastic MG63 cells. The interaction of PP1δ and nucleophosmin was also shown by immunoprecipitation and Western analysis. These results indicated that PP1δ associate with nucleophosmin directly in the nucleolus and suggested that nucleophosmin is one of the candidate substrate for PP1δ

Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells

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Mazel-Sanchez Beryl

2010-03-01

Full Text Available Abstract In mammalian cells, nucleolar localization of influenza A NS1 requires the presence of a C-terminal nucleolar localization signal. This nucleolar localization signal is present only in certain strains of influenza A viruses. Therefore, only certain NS1 accumulate in the nucleolus of mammalian cells. In contrast, we show that all NS1 tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described C-terminal nucleolar localization signal. Thus, nucleolar localization of NS1 in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains.

C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

Science.gov (United States)

Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

2015-09-11

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Cajal body and the nucleolus: "In a relationship" or "It's complicated"?

Science.gov (United States)

Trinkle-Mulcahy, Laura; Sleeman, Judith E

2017-06-03

From their initial identification as 'nucleolar accessory bodies' more than a century ago, the relationship between Cajal bodies and nucleoli has been a subject of interest and controversy. In this review, we seek to place recent developments in the understanding of the physical and functional relationships between the 2 structures in the context of historical observations. Biophysical models of nuclear body formation, the molecular nature of CB/nucleolus interactions and the increasing list of joint roles for CBs and nucleoli, predominantly in assembling ribonucleoprotein (RNP) complexes, are discussed.

Transcriptome profiling confirmed correlations between symptoms and transcriptional changes in RDV infected rice and revealed nucleolus as a possible target of RDV manipulation.

Science.gov (United States)

Yang, Liang; Du, Zhenguo; Gao, Feng; Wu, Kangcheng; Xie, Lianhui; Li, Yi; Wu, Zujian; Wu, Jianguo

2014-05-06

Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which limits rice production in many areas of south East Asia. Transcriptional changes of rice in response to RDV infection have been characterized by Shimizu et al. and Satoh et al.. Both studies found induction of defense related genes and correlations between transcriptional changes and symptom development in RDV-infected rice. However, the same rice cultivar, namely Nipponbare belonging to the Japonic subspecies of rice was used in both studies. Gene expression changes of the indica subspecies of rice, namely Oryza sativa L. ssp. indica cv Yixiang2292 that show moderate resistance to RDV, in response to RDV infection were characterized using an Affymetrix Rice Genome Array. Differentially expressed genes (DEGs) were classified according to their Gene Ontology (GO) annotation. The effects of transient expression of Pns11 in Nicotiana benthaminana on the expression of nucleolar genes were studied using real-time PCR (RT-PCR). 856 genes involved in defense or other physiological processes were identified to be DEGs, most of which showed up-regulation. Ribosome- and nucleolus related genes were significantly enriched in the DEGs. Representative genes related to nucleolar function exhibited altered expression in N. benthaminana plants transiently expressing Pns11 of RDV. Induction of defense related genes is common for rice infected with RDV. There is a co-relation between symptom severity and transcriptional alteration in RDV infected rice. Besides ribosome, RDV may also target nucleolus to manipulate the translation machinery of rice. Given the tight links between nucleolus and ribosome, it is intriguing to speculate that RDV may enhance expression of ribosomal genes by targeting nucleolus through Pns11.

Nanoscopical dissection of ancestral nucleoli in Archaea: a case of study in Evolutionary Cell Biology

KAUST Repository

Islas Morales, Parsifal

2018-04-01

Is the nucleolus a sine qua non condition of eukaryotes? The present project starts from this central question to contribute to our knowledge about the origin and the evolution of the cells. The nucleolus is a cryptic organelle that plays a central role in cell function. It is responsible for the orchestration of ribosomal RNA expression, maturation and modification in the regulatory context of cellular homeostasis. Ribosomal expression is undoubtedly the greatest transcriptional and regulatory activity of any cell. The nucleolus is not just a conventional organelle –membrane-limited-, but a magnificent transcriptional puff: a dichotomy between structure and process, form and function. What is the minimum nucleolus? Evolution should bring some light into these questions. Evolutionary cell biology (ECB) has raised increasing attention in the last decades. Is this a new discipline and an historical opportunity to combine functional and evolutionary biology towards the insight that cell evolution underlies organismic complexity? In the post-genomic era, we have developed the potential of combining high throughput acquisition of data with functional in situ and in sillico approaches: integration understood as omics approaches. Can this provide a real consilience between evolutionary and functional approaches? The reduced number of model organisms and cultivation techniques still excludes the majority of the extant diversity of cells from the scope of experimental inquiry. Furthermore, at the conceptual level, the simplification of evolutionary processes in biosciences still limits the conformation of a successful disciplinary link between functional and evolutionary biology. This limits the formulation of questions and experiments that properly address the mechanistic nature of cellular events that underlie microbial and organismic diversity and evolution. Here we provide a critical and comparative review to the historical background of ECB. This project takes the

CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis.

Science.gov (United States)

Bai, Baoyan; Moore, Henna M; Laiho, Marikki

2013-01-01

CRM1 is an export factor that together with its adaptor NMD3 transports numerous cargo molecules from the nucleus to cytoplasm through the nuclear pore. Previous studies have suggested that CRM1 and NMD3 are detected in the nucleolus. However, their localization with subnucleolar domains or participation in the activities of the nucleolus are unclear. We demonstrate here biochemically and using imaging analyses that CRM1 and NMD3 co-localize with nucleolar marker proteins in the nucleolus. In particular, their nucleolar localization is markedly increased by inhibition of RNA polymerase I (Pol I) transcription by actinomycin D or by silencing Pol I catalytic subunit, RPA194. We show that CRM1 nucleolar localization is dependent on its activity and the expression of NMD3, whereas NMD3 nucleolar localization is independent of CRM1. This suggests that NMD3 provides nucleolar tethering of CRM1. While inhibition of CRM1 by leptomycin B inhibited processing of 28S ribosomal (r) RNA, depletion of NMD3 did not, suggesting that their effects on 28S rRNA processing are distinct. Markedly, depletion of NMD3 and inhibition of CRM1 reduced the rate of pre-47S rRNA synthesis. However, their inactivation did not lead to nucleolar disintegration, a hallmark of Pol I transcription stress, suggesting that they do not directly regulate transcription. These results indicate that CRM1 and NMD3 have complex functions in pathways that couple rRNA synthetic and processing engines and that the rRNA synthesis rate may be adjusted according to proficiency in rRNA processing and export.

Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus

International Nuclear Information System (INIS)

Rowland, Raymond R.R.; Schneider, Paula; Fang Ying; Wootton, Sarah; Yoo, Dongwan; Benfield, David A.

2003-01-01

The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the principal component of the viral nucleocapsid and localizes to the nucleolus. Peptide sequence analysis of the N protein of several North American isolates identified two potential nuclear localization signal (NLS) sequences located at amino acids 10-13 and 41-42, which were labeled NLS-1 and NLS-2, respectively. Peptides containing NLS-1 or NLS-2 were sufficient to accumulate enhanced green fluorescent protein (EGFP) in the nucleus. The inactivation of NLS-1 by site-directed mutagenesis or the deletion of the first 14 amino acids did not affect N protein localization to the nucleolus. The substitution of key lysine residues with uncharged amino acids in NLS-2 blocked nuclear/nucleolar localization. Site-directed mutagenesis within NLS-2 identified the sequence, KKNKK, as forming the core localization domain within NLS-2. Using an in vitro pull-down assay, the N protein was able to bind importin-α, importin-β nuclear transport proteins. The localization pattern of N-EGFP fusion peptides represented by a series of deletions from the C- and N-terminal ends of the N protein identified a region covering amino acids 41-72, which contained a nucleolar localization signal (NoLS) sequence. The 41-72 N peptide when fused to EGFP mimicked the nucleolar-cytoplasmic distribution of native N. These results identify a single NLS involved in the transport of N from the cytoplasm and into nucleus. An additional peptide sequence, overlapping NLS-2, is involved in the further targeting of N to the nucleolus

Raptor, a positive regulatory subunit of mTOR complex 1, is a novel phosphoprotein of the rDNA transcription machinery in nucleoli and chromosomal nucleolus organizer regions (NORs).

Science.gov (United States)

Vazquez-Martin, Alejandro; Cufí, Sílvia; Oliveras-Ferraros, Cristina; Menendez, Javier A

2011-09-15

Raptor is the key scaffolding protein that recruits mTOR substrates to rapamycin-sensitive mTOR complex 1 (mTORC1), a molecular integrator of mitogenic and nutrient/energy environmental inputs into protein translation and cell growth. Although Raptor phosphorylation on various sites is pivotal in the regulation of mTORC1 activity, it remains to be elucidated whether site-specific phosphorylation differentially distributes Raptor to unique subcellular compartments. When exploring the spatiotemporal cell cycle dynamics of six different phospho (P)-Raptor isoforms (Thr ( 706) , Ser ( 722) , Ser ( 863) , Ser ( 792) and Ser ( 877) ), a number of remarkable events differentially defined a topological resetting of P-RaptorThr706 on interphasic and mitotic chromosomes. In interphase nuclei, P-Raptor (Thr706) co-localized with fibrillarin, a component of the nucleolar small nuclear ribonucleoprotein particle, as well as with RNA polymerase I, the enzyme that transcribes nucleolar rRNA. Upon Actinomycin D-induced nucleolar segregation and disaggregation, P-RaptorThr706 was excluded from the nucleolus to accumulate at discrete nucleoplasmic bodies. During mitosis, CDK1 inhibition-induced premature assembly of nucleoli relocated fibrillarin to the surrounding regions of chromosomal-associated P-Raptor (Thr706) , suggesting that a subpopulation of mitotic P-Raptor (Thr706) remained targeted at chromosomal loops of rDNA or nuclear organizer regions (NORs). At the end of mitosis and cytokinesis, when reassembly of incipient nucleoli begins upon NORs activation of rDNA transcription, fibrillarin spatially reorganized with P-Raptor (Thr706) to give rise to daughter nucleoli. Treatment with IGF1 exclusively hyperactivated nuclear P-Raptor (Ser706) and concomitantly promoted Ser ( 2481) autophosphorylation of mTOR, which monitors mTORC1-associated catalytic activity. Nucleolar- and NOR-associated P-Raptor (Ser706) may physically link mTORC1 signaling to ever-growing nucleolus

Research on radiation-biology effects of 3H: Pt. 3

International Nuclear Information System (INIS)

Tang Danling; Qi Yuzao; Li Jingyan

1993-01-01

By using staining and electron microscopic examination, Spirogyra sp. in terphase cell nucleolus appeared as a globe in the nucleus, and was composed of nucleolar fibrous core and nucleolar granular cortex. After Cultured in 3H -thymidine (10 μCi/mL) for 1400 hours, Spirogyra sp. cells were radiated by 3 H in and out of cell in and out of nucleus, the nucleolus volume enlarged and a little of nucleolar fibrous core or nucleolar granular cortex left the nucleolus, forming 'micro-nucleolus' in the nucleus beside the nucleolus. this kind of micro-nucleolus induced by 3 H-thymidine was different from micro-nucleus

Electron microscope studies of methotrexate and radiation effects in human squamous cell carcinoma of the mouth

International Nuclear Information System (INIS)

De Martino, C.

1974-01-01

Serial biopsy specimens from two squamous cell carcinomas of the mouth were studied by electron microscopy. This report described the ultrastructural changes in the cells produced by treatment with methotrexate followed by irradiation. The main ultrastructural findings after treatment are: numerous autophagic lysosomes and residual bodies are visible in the cytoplasm of the tumor cells; mitochondria are swollen. The mitochondrial cristae are distorted and disrupted, and mitochondrial matrix disappears; the nucleolus shows a series of morphological changes such as development of a compact nucleolus, aggregation of granular elements, atrophy, dissolution and fragmentation of the nucleolar mass; infiltration of lymphocytes, granulocytes and macrophages in the tumor. The significance of these ultrastructural findings is discussed. (U.S.)

The Cajal body and the nucleolus: “In a relationship” or “It's complicated”?

Science.gov (United States)

2017-01-01

ABSTRACT From their initial identification as ‘nucleolar accessory bodies’ more than a century ago, the relationship between Cajal bodies and nucleoli has been a subject of interest and controversy. In this review, we seek to place recent developments in the understanding of the physical and functional relationships between the 2 structures in the context of historical observations. Biophysical models of nuclear body formation, the molecular nature of CB/nucleolus interactions and the increasing list of joint roles for CBs and nucleoli, predominantly in assembling ribonucleoprotein (RNP) complexes, are discussed. PMID:27661468

The actin family protein ARP6 contributes to the structure and the function of the nucleolus

Czech Academy of Sciences Publication Activity Database

Kitamura, H.; Matsumori, H.; Kalendová, Alžběta; Hozák, Pavel; Goldberg, I.G.; Nakao, M.; Saitoh, N.; Harata, M.

2015-01-01

Roč. 464, č. 2 (2015), s. 554-560 ISSN 0006-291X R&D Projects: GA MŠk EE2.3.30.0050; GA MŠk(CZ) ED1.1.00/02.0109 Grant - others:Human Frontier in Science programe(FR) RGP0017/2013 Institutional support: RVO:68378050 Keywords : Actin-related protein * ARP6 * Histone H2A.Z * Nucleolus * Wndchrm Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.371, year: 2015

The ultrastructure of interphase nucleoli of PK-cells during their compensatory hypertrophy and degradation as a result of local UV microirradiation

International Nuclear Information System (INIS)

Zatsepina, O.V.; Voronkova, L.N.; Sakharov, V.N.; Chentsov, Yu.S.; AN SSSR, Moscow

1988-01-01

The ultrastructure of degraded and hypertrophic nucleoli in pig embryo kidney cells was studied using serial superfine sections. It is shown that nucleolus compensator hypertrophy is accompanied by a sufficient increase of the number of fibrillary centers (FC) and by a reduction of their linear dimensions as compared to the control; in degraded nucleoli the FC number is reduced and their dimensions are increased. On the whole the degraded nucleolus structural changes after UV-micro-irradiation correspond to the changes caused by an action of other rRNA synthesis inhibitors known. The nucleolus ability to compensator hypertropy shows that apart from operating ribosome genes, latent p-gens, which cn be activated under extreme conditions to keep the necessary rRNA synthesis level, exist in a cell. An assumption is made that such an activation is accompanied by ''fragmentation'' of pre-existing FC into smaller and more multiple ones

Incorporation of uridine-H3 into healthy and tobacco necrosis virus-infected mesophyll cells of Chenopodium amaranticolor

International Nuclear Information System (INIS)

Faccioli, G.; Rubies-Autonel, C.

1975-01-01

Tritiated uridine was selectively incorporated into the nucleus, nucleolus and cytoplasm of actinomycin D-treated Chenopodium amaranticolor cells locally infected with a strain of tobacco necrosis virus (TNV), 3 days after inoculation. Healthy cells did not show such an incorporation. Chloroplasts, in both types of cells, were free of label. Treatment with pancreatic ribonuclease removed the label completely in the majority of nuclei and nucleoli of infected cells. Since infectivity tests showed that AMD treatment increased virus multiplication by 10-12%, it is conceivable to think that the incorporation observed was due to virus synthesis. Preliminary infectivity experiments also showed that treatment of the cells with cycloheximide inhibited virus multiplication up to 80%, while chloramphenicol increased such multiplication. Our results lead to the conclusion that nucleus, nucleolus and cytoplasm but not chloroplasts are the sites involved in the synthesis of TNV. (orig.) [de

Aspirin Prevention of Colorectal Cancer: Focus on NF-κB Signalling and the Nucleolus

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Jingyu Chen

2017-07-01

Full Text Available Overwhelming evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs have anti-tumour activity and the potential to prevent cancer, particularly colorectal cancer. However, the mechanisms underlying this effect remain hypothetical. Dysregulation of the nuclear factor-kappaB (NF-κB transcription factor is a common event in many cancer types which contributes to tumour initiation and progression by driving expression of pro-proliferative/anti-apoptotic genes. In this review, we will focus on the current knowledge regarding NSAID effects on the NF-κB signalling pathway in pre-cancerous and cancerous lesions, and the evidence that these effects contribute to the anti-tumour activity of the agents. The nuclear organelle, the nucleolus, is emerging as a central regulator of transcription factor activity and cell growth and death. Nucleolar function is dysregulated in the majority of cancers which promotes cancer growth through direct and indirect mechanisms. Hence, this organelle is emerging as a promising target for novel therapeutic agents. Here, we will also discuss evidence for crosstalk between the NF-κB pathway and nucleoli, the role that this cross-talk has in the anti-tumour effects of NSAIDs and ways forward to exploit this crosstalk for therapeutic purpose.

Cytogenetic and identification of the nucleolus organizer region in Heliconia bihai (L. L.

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Heloisa Rocha do Nascimento

2014-08-01

Full Text Available The genus Heliconia is not much studied and the number of existing species in this genus is still uncertain. It is known that this number relies between 150 to 250 species. In Brazil, about 40 species are native and known by many different names. The objective of this paper was to characterize morphometrically and to identify the NOR (active nucleolus organizer regions by Ag-NOR banding of chromosomes of Heliconia bihai (L L. Root meristems were submitted to blocking treatment in an amiprofos-methyl (APM solution, fixed in methanol-acetic acid solution for 24 hours, at least. The meristems were washed in distilled water and submitted to enzymatic digestion with pectinase enzyme. The slides were prepared by dissociation of the root meristem, dried in the air and also on hot plate at 50°C. Subsequently, some slides were submitted to 5% Giemsa stain for karyotype construction and to a solution of silver nitrate (AgNO3 50% for Ag-NOR banding. The species H. bihai has 2n = 22 chromosomes, 4 pairs of submetacentric chromosomes and 7 pairs of metacentric chromosomes, and graded medium to short (3.96 to 0.67 μM, with the presence of active NOR in pairs 1 and 2 and interphase cells with 2 nucleoli. These are the features of a diploid species.

The nucleolus as a fundamental regulator of the p53 response and a new target for cancer therapy.

Science.gov (United States)

Woods, Simone J; Hannan, Katherine M; Pearson, Richard B; Hannan, Ross D

2015-07-01

Recent studies have highlighted the fundamental role that key oncogenes such as MYC, RAS and PI3K occupy in driving RNA Polymerase I transcription in the nucleolus. In addition to maintaining essential levels of protein synthesis, hyperactivated ribosome biogenesis and nucleolar function plays a central role in suppressing p53 activation in response to oncogenic stress. Consequently, disruption of ribosome biogenesis by agents such as the small molecule inhibitor of RNA Polymerase I transcription, CX-5461, has shown unexpected, potent, and selective effects in killing tumour cells via disruption of nucleolar function leading to activation of p53, independent of DNA damage. This review will explore the mechanism of DNA damage-independent activation of p53 via the nucleolar surveillance pathway and how this can be utilised to design novel cancer therapies. Non-genotoxic targeting of nucleolar function may provide a new paradigm for treatment of a broad range of oncogene-driven malignancies with improved therapeutic windows. This article is part of a Special Issue entitled: Translation and Cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of loop structure of bovine lactoferricin on apoptosis in Jurkat cells.

Science.gov (United States)

Zhang, Tie-nan; Yang, Wei; Liu, Ning

2010-06-01

Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces apoptosis in Jurkat cells. However less is known about the influence of this kind of apoptosis on the intra-cellular ceramide metabolism and the structure-function relationship between the loop structure of LfcinB and its action of inducing apoptosis in Jurkat cells. In the present study, the artificially synthesized LfcinB and LfcinB-derived peptide (Cys 19 residue in LfcinB was replaced by Ala) was added in Jurkat cells, the nucleolus shape was observed by fluorescent microscopy, the ceramide concentration in Jurkat cells was determined by reversed phase high performance liquid chromatography (RP-HPLC). The results of MTT assay showed that LfcinB inhibited proliferation of Jurkat cells, and the inhibition rate was approximately 18.90%. Moreover, the inhibition rate of LfcinB together with MAPP was upto approximately 59.89%. The RP-HPLC result showed that LfcinB improved the ceramide level in Jurkat cells. By using the DNA fragmentation assay and observing the nucleolus shape, the result displayed deficiency of the loop structure could cause LfcinB losing the biological activity of inducing apoptosis in Jurkat cells.

The reaction of Lupinus angustifolius L. root meristematic cell nucleoli to lead.

Science.gov (United States)

Balcerzak, Lucja; Glińska, Sława; Godlewski, Mirosław

2011-04-01

The effect of 2-48 h treatment of Lupinus angustifolius L. roots with lead nitrate at the concentration of 10(-4) M on the nucleoli in meristematic cells was investigated. In the lead presence the number of ring-shaped as well as segregated nucleoli increased especially after 12-48 h of treatment, while spindle-shaped nucleoli appeared after 24 h and 48 h. Lead presence also increased the frequency of cells with silver-stained particles in the nucleus and the number of these particles especially from the 12th hour of treatment. It was accompanied by significant decline of nucleolar area. Analysis of these cells in transmission electron microscope confirmed the presence of ring-shaped and segregated nucleoli. Moreover, electron microscopy revealed compact structure nucleoli without granular component. Additionally, one to three oval-shaped fibrillar structures attached to nucleolus or lying free in the nucleoplasm were visible. The possible mechanism of lead toxicity to the nucleolus is briefly discussed.

HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus

International Nuclear Information System (INIS)

Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang

2016-01-01

The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. - Highlights: • HIV-1 NC possess a NLS and leads to nuclear and nucleolus localization. • Mutations in basic residues between two ZFs in NC decrease the nucleus localization. • ZFs of NC affect cytoplasmic organelles localization rather than nucleus localization.

HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus

Energy Technology Data Exchange (ETDEWEB)

Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang, E-mail: jiyou@catholic.ac.kr

2016-05-15

The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. - Highlights: • HIV-1 NC possess a NLS and leads to nuclear and nucleolus localization. • Mutations in basic residues between two ZFs in NC decrease the nucleus localization. • ZFs of NC affect cytoplasmic organelles localization rather than nucleus localization.

Genetics Home Reference: Bowen-Conradi syndrome

Science.gov (United States)

... are assembled in a cell compartment called the nucleolus . The particular EMG1 gene mutation known to cause ... of EMG1 protein that is available in the nucleolus. A shortage of this protein in the nucleolus ...

Detection of intranuclear forces by the use of laser optics during the recovery process of elongated interphase nuclei in centrifuged protonemal cells of Adiantum capillus-veneris

International Nuclear Information System (INIS)

Wunsch, C.; Kurachi, M.; Kikumoto, M.; Tashiro, H.; Wada, M.

1998-01-01

For the direct investigation of intranuclear dynamics in living cells, extremely deformed nuclei of basipetally centrifuged protonemal cells of the fern Adiantum capillusveneris were manipulated by the laser rap and the laser scalpel. Whereas the nucleolus was tightly fixed at the central position inside the non-centrifuged nucleus and proved to be immovable by the optical trap, it could easily be trapped and moved towards three directions inside the bubble-like terminal widening of the basal thread-like extension of centrifuged nuclei. Due to the connection of the nucleolus to the chromatin inside the nuclear thread (NT), moving was not possible against the direction of the nuclear apical main body. Nucleoli in recovered nuclei were again immovable, thus indicating the presence of a dynamic nucleolar anchoring system inside the nucleus. When the nucleolus in the bubble was arrested during the thread shortening process by the optical trap, the acropetal movement of the bubble continued. Probably dye to dragging forces, some nucleoli became stretched, and a thick strand of a still unknown composition stretched between the nucleolus and the insertion site of the shortening NT. To assess whether the shrinking of the nuclear envelop (NE) and the shortening of the chromatin inside the NT were independent processes, the chromatin above the bubble was cut inside the Nt by the laser scalpel. After severance, a gap between the nucleolus and the end of the chromatin strand in the NT indicated the shortening of the chromatin inside the Nt. From these findings it was concluded that a shortening force was existing in the chromatin of the NT and that probably no physical link existed between the chromatin and the NE

Nop2 is expressed during proliferation of neural stem cells and in adult mouse and human brain

Czech Academy of Sciences Publication Activity Database

Kosi, N.; Alic, I.; Kolacevic, M.; Vrsaljko, N.; Milosevic, N.J.; Sobol, Margaryta; Philimonenko, Anatoly; Hozák, Pavel; Gajovic, S.; Pochet, R.; Mitrecic, D.

2015-01-01

Roč. 1597, FEB 9 (2015), s. 65-76 ISSN 1872-6240 R&D Projects: GA TA ČR(CZ) TE01020118; GA MPO FR-TI3/588 Institutional support: RVO:68378050 Keywords : Nop2 * Brain * Stem cells * Stroke * Nucleolus * Cell cycle Subject RIV: EB - Genetics ; Molecular Biology

Structural features of nucleoli in blood, leukemic, lymphoma and myeloma cells

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K Smetana

2010-01-01

Full Text Available At present, it seems clear that the nucleolus is multifunctional and represents one of the key cell organelles that participate directly or indirectly in cell resting, proliferation, differentiation and maturation states, and possibly also in programmed cell death. Thus, the morphology and cytochemistry of nucleoli may represent a very useful tool not only for the evaluation of nucleolar biosynthetic activities but also for the evaluation of various cell states under physiological, experimental and pathological conditions.

RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs.

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Baoyan Bai

Full Text Available Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA. Sequences from 47 sdRNAs were identified, which mapped to both 5' and 3' ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha-mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.

Symmetric dimeric bisbenzimidazoles DBP(n reduce methylation of RARB and PTEN while significantly increase methylation of rRNA genes in MCF-7 cancer cells.

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Svetlana V Kostyuk

Full Text Available Hypermethylation is observed in the promoter regions of suppressor genes in the tumor cancer cells. Reactivation of these genes by demethylation of their promoters is a prospective strategy of the anticancer therapy. Previous experiments have shown that symmetric dimeric bisbenzimidazoles DBP(n are able to block DNA methyltransferase activities. It was also found that DBP(n produces a moderate effect on the activation of total gene expression in HeLa-TI population containing epigenetically repressed avian sarcoma genome.It is shown that DBP(n are able to penetrate the cellular membranes and accumulate in breast carcinoma cell MCF-7, mainly in the mitochondria and in the nucleus, excluding the nucleolus. The DBP(n are non-toxic to the cells and have a weak overall demethylation effect on genomic DNA. DBP(n demethylate the promoter regions of the tumor suppressor genes PTEN and RARB. DBP(n promotes expression of the genes RARB, PTEN, CDKN2A, RUNX3, Apaf-1 and APC "silent" in the MCF-7 because of the hypermethylation of their promoter regions. Simultaneously with the demethylation of the DNA in the nucleus a significant increase in the methylation level of rRNA genes in the nucleolus was detected. Increased rDNA methylation correlated with a reduction of the rRNA amount in the cells by 20-30%. It is assumed that during DNA methyltransferase activity inhibition by the DBP(n in the nucleus, the enzyme is sequestered in the nucleolus and provides additional methylation of the rDNA that are not shielded by DBP(n.It is concluded that DBP (n are able to accumulate in the nucleus (excluding the nucleolus area and in the mitochondria of cancer cells, reducing mitochondrial potential. The DBP (n induce the demethylation of a cancer cell's genome, including the demethylation of the promoters of tumor suppressor genes. DBP (n significantly increase the methylation of ribosomal RNA genes in the nucleoli. Therefore the further study of these compounds is needed

Rrp12 and the Exportin Crm1 participate in late assembly events in the nucleolus during 40S ribosomal subunit biogenesis.

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Moriggi, Giulia; Nieto, Blanca; Dosil, Mercedes

2014-12-01

During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes.

[Activation of nucleolar organizers during in vitro cultivation of mouse R1 embryonic stem cells].

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Kunafina, E R; Chaplina, M V; Filiasova, E I; Gibanova, N V; Khodarovich, Iu M; Larionov, O A; Zatsepina, O V

2005-01-01

We studies the activities of ribosomal genes (nucleolus forming regions of chromosomes) at successive stages of cultivation of the mouse R1 embryonic stem cells. The total number and number of active nucleolar organizers were estimated by means of in situ hybridization with mouse rDNA probes and argentophilic staining of nucleolus forming chromosomes regions from the 16th until the 32nd passages. The data we obtained suggest that the total number of nucleolar organizers per metaphase plate was constant (as a rule, eight), while the mean number of active nucleolar organizers progressively increased from the early (16th) to the late (32nd) passages: 5.2 +/- 0.4 versus 7.4 +/- 0.9 argentophilic organizers per cell. Cell heterogeneity by the number of active nucleolar organizers also increased during the late passages. Taken together, these data suggest activation of DNA transcription and synthesis of ribosomes during cultivation of mouse R1 embryonic stem cells. Based on the experimental and published data, it has been proposed that activation of ribosomal genes correlates in time with a decreased capacity of embryonic stem cells for pluripotent differentiation.

Reference: 481 [Arabidopsis Phenome Database[Archive

Lifescience Database Archive (English)

Full Text Available Frederic et al. 2007 Feb. Mol. Biol. Cell 18(2):369-79. Nucleolin is one of the most abundant protein in the nucleolus...t defects. AtNUC-L1 is localized in the nucleolus, mainly in the dense fibrillar ...component. Absence of this protein in Atnuc-L1 plants induces nucleolar disorganization, nucleolus organizer...th a disrupted nucleolin-like gene and defines a new role for nucleolin in nucleolus structure and rDNA chro...matin organization. Characterization of AtNUC-L1 reveals a central role of nucleolin in nucleolus organizati

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

Energy Technology Data Exchange (ETDEWEB)

Peng, Jamy C. [Univ. of California, Berkeley, CA (United States)

2007-01-01

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in

Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

Science.gov (United States)

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2012-11-01

Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

Nucleolar ultrastructure in bovine nuclear transfer embryos

DEFF Research Database (Denmark)

Kanka, J; Smith, S D; Soloy, E

1999-01-01

in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase...... at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary...... time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear...

Nucleolar remodeling in nuclear transfer embryos

DEFF Research Database (Denmark)

Laurincik, Jozef; Maddox-Hyttel, Poul

2007-01-01

Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate the devel......Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate...... nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail...

Nucleolar chromatin organization at different activities of soybean root meristematic cell nucleoli.

Science.gov (United States)

Stępiński, Dariusz

2013-06-01

Nucleolar chromatin, including nucleolus-associated chromatin as well as active and inactive condensed ribosomal DNA (rDNA) chromatin, derives mostly from secondary constrictions known as nucleolus organizer regions containing rDNA genes on nucleolus-forming chromosomes. This chromatin may occupy different nucleolar positions being in various condensation states which may imply different rDNA transcriptional competence. Sections of nucleoli originating from root meristematic cells of soybean seedlings grown at 25 °C (the control), then subjected to chilling stress (10 °C), and next transferred again to 25 °C (the recovery) were used to measure profile areas occupied by nucleolar condensed chromatin disclosed with sodium hydroxide methylation-acetylation plus uranyl acetate technique. The biggest total area of condensed chromatin was found in the nucleoli of chilled plants, while the smallest was found in those of recovered plants in relation to the amounts of chromatin in the control nucleoli. The condensed nucleolar chromatin, in the form of different-sized and different-shaped clumps, was mainly located in fibrillar centers. One can suppose that changes of condensed rDNA chromatin amounts might be a mechanism controlling the number of transcriptionally active rDNA genes as the nucleoli of plants grown under these experimental conditions show different transcriptional activity and morphology.

Cell cycle stage-specific differential expression of topoisomerase I in tobacco BY-2 cells and its ectopic overexpression and knockdown unravels its crucial role in plant morphogenesis and development.

Science.gov (United States)

Singh, Badri Nath; Mudgil, Yashwanti; John, Riffat; Achary, V Mohan Murali; Tripathy, Manas Kumar; Sopory, Sudhir K; Reddy, Malireddy K; Kaul, Tanushri

2015-11-01

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. Cell cycle coupled differential accumulation of topoisomerase I (Topo I) revealed biphasic expression maximum at S-phase and M/G1-phase of cultured synchronized tobacco BY-2 cells. This suggested its active role in resolving topological constrains during DNA replication (S-phase) and chromosome decondensation (M/G1 phase). Immuno-localization revealed high concentrations of Topo I in nucleolus. Propidium iodide staining and Br-UTP incorporation patterns revealed direct correlation between immunofluorescence intensity and rRNA transcription activity within nucleolus. Immuno-stained chromosomes during metaphase and anaphase suggested possible role of Topo I in resolving topological constrains during mitotic chromosome condensation. Inhibitor studies showed that in comparison to Topo I, Topo II was essential in resolving topological constrains during chromosome condensation. Probably, Topo II substituted Topo I functioning to certain extent during chromosome condensation, but not vice-versa. Transgenic Topo I tobacco lines revealed morphological abnormalities and highlighted its crucial role in plant morphogenesis and development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A ribosomal protein L23-nucleophosmin circuit coordinates Miz1 function with cell growth

DEFF Research Database (Denmark)

Wanzel, Michael; Russ, Annika C; Kleine-Kohlbrecher, Daniela

2008-01-01

by retaining nucleophosmin, an essential co-activator of Miz1 required for Miz1-induced cell-cycle arrest, in the nucleolus. Mutant forms of nucleophosmin found in acute myeloid leukaemia fail to co-activate Miz1 and re-localize it to the cytosol. As L23 is encoded by a direct target gene of Myc...

Activation of the ribosomal RNA genes late in the third cell cycle of porcine embryos

DEFF Research Database (Denmark)

Viuff, Dorthe; Greve, Torben; Holm, Peter

2002-01-01

; there was no silver staining at the sites of the rRNA genes and nucleolus precursor bodies. From 30 hpc onwards, most 4-cell embryos had medium size to large clusters of FITC-labeled areas colocalized with silver staining of rRNA gene clusters and fibrillogranular nucleoli. These observations indicate that r...

The development of fluorescence turn-on probe for Al(III) sensing and live cell nucleus-nucleoli staining

Science.gov (United States)

Saini, Anoop Kumar; Sharma, Vinay; Mathur, Pradeep; Shaikh, Mobin M.

2016-10-01

The morphology of nucleus and nucleolus is powerful indicator of physiological and pathological conditions. The specific staining of nucleolus recently gained much attention due to the limited and expensive availability of the only existing stain “SYTO RNA-Select”. Here, a new multifunctional salen type ligand (L1) and its Al3+ complex (1) are designed and synthesized. L1 acts as a chemosensor for Al3+ whereas 1 demonstrates specific staining of nucleus as well as nucleoli. The binding of 1 with nucleic acid is probed by DNase and RNase digestion in stained cells. 1 shows an excellent photostability, which is a limitation for existing nucleus stains during long term observations. 1 is assumed to be a potential candidate as an alternative to expensive commercial dyes for nucleus and nucleoli staining.

Nucleolus disassembly in mitosis and apoptosis: dynamic redistribution of phosphorylated-c-Myc, fibrillarin and Ki-67

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C Soldani

2009-06-01

Full Text Available The nucleolus may undergo disassembly either reversibly during mitosis, or irreversibly in apoptosis, thus allowing the redistribution of the nucleolar proteins.We investigated here by immunocytochemistry the fate of three representative proteins, namely phosphorylated c-Myc, fibrillarin and Ki-67, and found that they behave independently in both processes: they relocate in distinct compartments during mitosis, whereas during apoptosis they may either be cleaved (Ki-67 or be extruded into the cytoplasm with a different kinetics and following an ordered, non chaotic program. The separation of these nucleolar proteins which occurs in early apoptotic nuclei continues also in the cytoplasm, and culminates in the final formation of apoptotic blebs containing different nucleolar proteins: this evidence confirms that the apoptotic bodies may be variable in size, content and surface reactivity, and include heterogeneous aggregates of nuclear proteins and/or nucleic acids.

Application and potentiality of laser micro-irradiation of chromosomes in animal and plant cells

Energy Technology Data Exchange (ETDEWEB)

Lu, H Liang; Wang, L L [Laboratory of Laser and Genetics, Institute of Genetics, Academia Sinica, Beijing (China)

1990-01-01

Full text: The classical methods of inducing genetic variation use chemical or physical mutagens applied to populations of cells. Since the development of laser techniques allows focussing a beam to a submicron spot, laser microsurgery of chromosomes was attempted. In mammalian cells, the nucleolus formation could be prevented. Also several chromosome damages were produced by focussing on specific chromosomes in prophase, metaphase and anaphase. Chromosomes of broad bean, maize, wheat, barley were dissected into small fragments. (author)

Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

International Nuclear Information System (INIS)

Chandra, Subhash

2004-01-01

Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13 C and 15 N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13 C 15 N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39 K, 23 Na and 40 Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors

Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

Energy Technology Data Exchange (ETDEWEB)

Chandra, Subhash

2004-06-15

Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes {sup 13}C and {sup 15}N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK{sub 1} kidney cells at mass 28 ({sup 13}C{sup 15}N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of {sup 39}K, {sup 23}Na and {sup 40}Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

Ultrastructural Observations of the Vitelline Cells of Metamicrocotyla macracantha (Monogenea, Microcotylidae

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Maria de Fatima D Baptista-Farias

1998-07-01

Full Text Available An electron microscopic study of the vitelline follicles of Metamicrocotyla macracantha (Alexander, 1954 Koratha,1955 showed that they are composed of cells in different stages of development. The immature cells have a large nucleus, nucleolus, cytoplasm with free ribosomes and few mitochondria. The developing vitelline cells present granules which are small in the early stages, increasing with maturity. The mature cells have an extensive granular endoplasmic reticulum and droplets of shell-protein; with maturation, clusters of shell protein and yolk bodies are formed and released in the ciliated vitelline ducts. Vitelline development is continuous and all of the cellular stages involved can be found in each follicle.

Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

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C. Soldani

2010-05-01

Full Text Available Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP-containing components (PANA, hnRNP-core proteins, fibrillarin or RNP-associated nuclear proteins (SC-35 splicing factor. Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.

Prestress mediates force propagation into the nucleus

International Nuclear Information System (INIS)

Hu Shaohua; Chen Jianxin; Butler, James P.; Wang Ning

2005-01-01

Several reports show that the nucleus is 10 times stiffer than the cytoplasm. Hence, it is not clear if intra-nuclear structures can be directly deformed by a load of physiologic magnitudes. If a physiologic load could not directly deform intra-nuclear structures, then signaling inside the nucleus would occur only via the mechanisms of diffusion or translocation. Using a synchronous detection approach, we quantified displacements of nucleolar structures in cultured airway smooth muscle cells in response to a localized physiologic load (∼0.4 μm surface deformation) via integrin receptors. The nucleolus exhibited significant displacements. Nucleolar structures also exhibited significant deformation, with the dominant strain being the bulk strain. Increasing the pre-existing tensile stress (prestress) in the cytoskeleton significantly increased the stress propagation efficiency to the nucleolus (defined as nucleolus displacement per surface deformation) whereas decreasing the prestress significantly lowered the stress propagation efficiency to the nucleolus. Abolishing the stress fibers/actin bundles by plating the cells on poly-L-lysine-coated dishes dramatically inhibited stress propagation to the nucleolus. These results demonstrate that the prestress in the cytoskeleton is crucial in mediating stress propagation to the nucleolus, with implications for direct mechanical regulation of nuclear activities and functions

Assessment of Geographical Variation in the Respiratory Toxicity of Desert Dust Particles

Science.gov (United States)

2013-01-01

dense nucleolus (n). There is close contact between the cells (arrowhead). The cytoplasm has many lamellar bodies – small, spherical, membrane-bound...osmiophilic, lamellar structures that contain phospholipid. Note the intact nuclear membrane (white arrow) and nucleolus (black arrow) and several

Changes in biomolecular profile in a single nucleolus during cell fixation.

Science.gov (United States)

Kuzmin, Andrey N; Pliss, Artem; Prasad, Paras N

2014-11-04

Fixation of biological sample is an essential technique applied in order to "freeze" in time the intracellular molecular content. However, fixation induces changes of the cellular molecular structure, which mask physiological distribution of biomolecules and bias interpretation of results. Accurate, sensitive, and comprehensive characterization of changes in biomolecular composition, occurring during fixation, is crucial for proper analysis of experimental data. Here we apply biomolecular component analysis for Raman spectra measured in the same nucleoli of HeLa cells before and after fixation by either formaldehyde solution or by chilled ethanol. It is found that fixation in formaldehyde does not strongly affect the Raman spectra of nucleolar biomolecular components, but may significantly decrease the nucleolar RNA concentration. At the same time, ethanol fixation leads to a proportional increase (up to 40%) in concentrations of nucleolar proteins and RNA, most likely due to cell shrinkage occurring in the presence of coagulant fixative. Ethanol fixation also triggers changes in composition of nucleolar proteome, as indicated by an overall reduction of the α-helical structure of proteins and increase in the concentration of proteins containing the β-sheet conformation. We conclude that cross-linking fixation is a more appropriate protocol for mapping of proteins in situ. At the same time, ethanol fixation is preferential for studies of RNA-containing macromolecules. We supplemented our quantitative Raman spectroscopic measurements with mapping of the protein and lipid macromolecular groups in live and fixed cells using coherent anti-Stokes Raman scattering nonlinear optical imaging.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

Science.gov (United States)

Herce, Henry D; Schumacher, Dominik; Schneider, Anselm F L; Ludwig, Anne K; Mann, Florian A; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M Cristina; Hackenberger, Christian P R

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

Science.gov (United States)

Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Fbw7α and Fbw7γ Collaborate To Shuttle Cyclin E1 into the Nucleolus for Multiubiquitylation

Science.gov (United States)

Bhaskaran, Nimesh; van Drogen, Frank; Ng, Hwee-Fang; Kumar, Raman; Ekholm-Reed, Susanna; Peter, Matthias

2013-01-01

Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G1-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G1 and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCFFbw7 (also known as SCFCdc4), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCFFbw7α in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCFFbw7γ. Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCFFbw7γ prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation. PMID:23109421

Acrolein preferentially damages nucleolus eliciting ribosomal stress and apoptosis in human cancer cells.

Science.gov (United States)

Wang, Hsiang-Tsui; Chen, Tzu-Ying; Weng, Ching-Wen; Yang, Chun-Hsiang; Tang, Moon-Shong

2016-12-06

Acrolein (Acr) is a potent cytotoxic and DNA damaging agent which is ubiquitous in the environment and abundant in tobacco smoke. Acr is also an active cytotoxic metabolite of the anti-cancer drugs cyclophosphamide and ifosfamide. The mechanisms via which Acr exerts its anti-cancer activity and cytotoxicity are not clear. In this study, we found that Acr induces cytotoxicity and cell death in human cancer cells with different activities of p53. Acr preferentially binds nucleolar ribosomal DNA (rDNA) to form Acr-deoxyguanosine adducts, and induces oxidative damage to both rDNA and ribosomal RNA (rRNA). Acr triggers ribosomal stress responses, inhibits rRNA synthesis, reduces RNA polymerase I binding to the promoter of rRNA gene, disrupts nucleolar integrity, and impairs ribosome biogenesis and polysome formation. Acr causes an increase in MDM2 levels and phosphorylation of MDM2 in A549 and HeLa cells which are p53 active and p53 inactive, respectively. It enhances the binding of ribosomal protein RPL11 to MDM2 and reduces the binding of p53 and E2F-1 to MDM2 resulting in stabilization/activation of p53 in A549 cells and degradation of E2F-1 in A549 and HeLa cells. We propose that Acr induces ribosomal stress which leads to activation of MDM2 and RPL11-MDM2 binding, consequently, activates p53 and enhances E2F-1 degradation, and that taken together these two processes induce apoptosis and cell death.

Probing the stiffness of isolated nucleoli by atomic force microscopy.

Science.gov (United States)

Louvet, Emilie; Yoshida, Aiko; Kumeta, Masahiro; Takeyasu, Kunio

2014-04-01

In eukaryotic cells, ribosome biogenesis occurs in the nucleolus, a membraneless nuclear compartment. Noticeably, the nucleolus is also involved in several nuclear functions, such as cell cycle regulation, non-ribosomal ribonucleoprotein complex assembly, aggresome formation and some virus assembly. The most intriguing question about the nucleolus is how such dynamics processes can occur in such a compact compartment. We hypothesized that its structure may be rather flexible. To investigate this, we used atomic force microscopy (AFM) on isolated nucleoli. Surface topography imaging revealed the beaded structure of the nucleolar surface. With the AFM's ability to measure forces, we were able to determine the stiffness of isolated nucleoli. We could establish that the nucleolar stiffness varies upon drastic morphological changes induced by transcription and proteasome inhibition. Furthermore, upon ribosomal proteins and LaminB1 knockdowns, the nucleolar stiffness was increased. This led us to propose a model where the nucleolus has steady-state stiffness dependent on ribosome biogenesis activity and requires LaminB1 for its flexibility.

Ultrastructural Localization and Molecular Associations of HCV Capsid Protein in Jurkat T Cells

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Cecilia Fernández-Ponce

2018-01-01

Full Text Available Hepatitis C virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. When expressed in CD4+ T lymphocytes, it can induce modifications in several essential cellular and biological networks. To shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed HCV-core subcellular localization and its associations with host proteins in Jurkat T cells. In order to investigate the intracellular localization of Hepatitis C virus core protein, we have used a lentiviral system to transduce Jurkat T cells and subsequently localize the protein using immunoelectron microscopy techniques. We found that in Jurkat T cells, Hepatitis C virus core protein mostly localizes in the nucleus and specifically in the nucleolus. In addition, we performed pull-down assays combined with Mass Spectrometry Analysis, to identify proteins that associate with Hepatitis C virus core in Jurkat T cells. We found proteins such as NOLC1, PP1γ, ILF3, and C1QBP implicated in localization and/or traffic to the nucleolus. HCV-core associated proteins are implicated in RNA processing and RNA virus infection as well as in functions previously shown to be altered in Hepatitis C virus core expressing CD4+ T cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. Thus, in the current work, we show the ultrastructural localization of Hepatitis C virus core and the first profile of HCV core associated proteins in T cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of Hepatitis C virus core protein. Thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying HCV core mediated alterations that had been described in relevant biological processes in CD4+ T cells.

Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses

OpenAIRE

Iwai Ohbayashi; Munetaka Sugiyama

2018-01-01

The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized p...

Investigation of Energy Transfer Dynamics Between Target Contrast Agents and Prostate Cancer Cells Using Ultrafast Spectroscopy

Science.gov (United States)

2012-05-01

morphology, including changes in the shape and size of the nucleus, the nuclear envelope, the nucleolus , and the chromatin.21 Richards-Kortum et al. found...that light scattering is strongly influenced by the difference in morphology of nucleus and nucleolus , DNA content, and clumping and coarse chromatins

Cytoplasmic influence of nucleolar development

International Nuclear Information System (INIS)

Ghosh, Sibdas

1974-01-01

The role of cytoplasmic factors on the development of nucleolus in nucleus has been investigated in Ehrlich mouse ascites tumour cells using tritiated thymidine/uridine for autoradiography. It is inferred from the observations that the cytoplasmic factors has some but not absolute control over the development of nucleolus. (M.G.B.)

Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Medicago truncatula Cells Overexpressing the MtTdp2α Gene

Science.gov (United States)

Macovei, Anca; Faè, Matteo; Biggiogera, Marco; de Sousa Araújo, Susana; Carbonera, Daniela; Balestrazzi, Alma

2018-01-01

The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2) involved in the repair of 5′-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively) and a control (CTRL) line carrying the empty vector were investigated. Transmission electron microscopy (TEM) revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL), the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL) and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells. PMID:29868059

Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Medicago truncatula Cells Overexpressing the MtTdp2α Gene

Directory of Open Access Journals (Sweden)

Anca Macovei

2018-05-01

Full Text Available The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2 involved in the repair of 5′-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively and a control (CTRL line carrying the empty vector were investigated. Transmission electron microscopy (TEM revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL, the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells.

The mathematical cell model reconstructed from interference microscopy data

Science.gov (United States)

Rogotnev, A. A.; Nikitiuk, A. S.; Naimark, O. B.; Nebogatikov, V. O.; Grishko, V. V.

2017-09-01

The mathematical model of cell dynamics is developed to link the dynamics of the phase cell thickness with the signs of the oncological pathology. The measurements of irregular oscillations of cancer cells phase thickness were made with laser interference microscope MIM-340 in order to substantiate this model. These data related to the dynamics of phase thickness for different cross-sections of cells (nuclei, nucleolus, and cytoplasm) allow the reconstruction of the attractor of dynamic system. The attractor can be associated with specific types of collective modes of phase thickness responsible for the normal and cancerous cell dynamics. Specific type of evolution operator was determined using an algorithm of designing of the mathematical cell model and temporal phase thickness data for cancerous and normal cells. Qualitative correspondence of attractor types to the cell states was analyzed in terms of morphological signs associated with maximum value of mean square irregular oscillations of phase thickness dynamics.

Changes in the number and size of nucleoli of Chara vulgaris L. antheridial filament cells during the period preceding light-induced re-initiation of cell divisions following a mitodepressive effect of darkness

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Maria Kwiatkowska

2014-01-01

Full Text Available The changes in number and size of nucleoli of Chara vulgaris antheridial filament cells were monitored with the use of Howell and Black's silver staining method. After a 3-day mitodepressive treatment with darkness the cells were exposed to light which reactivated mitotic activity after 18-20 hours. Eight-celled antheridial filaments were observed. In the period preceding light-induced re-initiation of mitoses a gradual reconstruction of the number and size of nucleoli characteristic of control, as well as their total area per nucleus appeared. The obtained results indicate that one of the important conditions for a cell to be able to divide is accumulation of nucleolus components characteristic of a given developmental stage and this controls nucleologenesis of the subsequent cell cycle.

[Participation of the piRNA pathway in recruiting a component of RNA polymerase I transcription initiation complex to germline cell nucleoli].

Science.gov (United States)

Fefelova, E A; Stolyarenko, A D; Yakushev, E Y; Gvozdev, V A; Klenov, M S

2017-01-01

Proteins of the Piwi family and short Piwi-interacting RNAs (piRNAs) ensure the protection of the genome from transposable elements. We have previously shown that nuclear Piwi protein tends to concentrate in the nucleoli of the cells of Drosophila melanogaster ovaries. It could be hypothesized that the function of Piwi in the nucleolus is associated with the repression of R1 and R2 retrotransposons inserted into the rDNA cluster. Here, we show that Piwi participates in recruiting Udd protein to nucleoli. Udd is a component of the conserved Selectivity Factor I-like (SL1-like) complex, which is required for transcription initiation by RNA polymerase I. We found that Udd localization depends on Piwi in germline cells, but not in somatic cells of the ovaries. In contrast, knockdowns of the SL1-like components (Udd or TAF1b) do not disrupt Piwi localization. We also observed that the absence of Udd or TAF1b in germline cells, as well as the impairment of Piwi nuclear localization lead to the accumulation of late stage egg chambers in the ovaries, which could be explained by reduced rRNA transcription. These results allow us to propose for the first time a role for Piwi in the nucleolus that is not directly associated with transposable element repression.

Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

DEFF Research Database (Denmark)

Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

2009-01-01

Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

Analysis of nucleolar morphology and protein localization as an indicator of nuclear reprogramming

DEFF Research Database (Denmark)

Østrup, Olga; Pedersen, Hanne Skovsgaard; Holm, Hanne M.

2015-01-01

When a cell is reprogrammed to a new phenotype, the nucleolus undergoes more or less dramatic modulations, which can be used as a marker for the occurrence of the reprogramming. This phenomenon is most pronounced when differentiated cells are reprogrammed to totipotency when they are submitted...... of the nucleolus are summarized in this developmental context, but also as they occur in assisted reproductive technologies such as in vitro fertilization and somatic cell nuclear transfer. Moreover, detailed protocols for monitoring the nucleolar changes by transmission electron microscopy and immunocytochemistry...

Ultrastructural response of cabbage outer leaf mesophyll cells (Brassica oleracea L. to excess of nickel

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Jolanta Molas

2014-01-01

Full Text Available Changes in the structure and in the ultrastructure of cabbage outer leaf mesophyll cells [Brassica oleracea L.] cv. Sława from Enkhouizen were examined by means of light and electron microscopy. The examined plants were grown on the basic Murashige and Skoog medium with addition of excesive concentrations of nickel (added as NiSO4 x 7H2O,i.e. Ni 5, Ni 10 and Ni 20 mg/dm3. In Ni 5 mg samples mainly adaptation changes to the conditions of stress were observed. These changes were manifested by the increase of cytoplasm content and by cytoplasm vacuolization, by the increase of nucleus and nucleous volume, nucleolus vacuolization, the increase of plasmalemma invaginations and of the amount of rough ER, by the central arrangement of smooth ER and of the thylakoids of chloroplasts; it was also shown by the growth of the number of mitochondria and of peroxisomes in the cell. In Ni 10 mg samples, apart from adaptation changes, such as the increase of the nucleus volume, increase of plasmalemma invaginations, cytoplasm and nucleolus vacuolization, degeneration changes were also observed. They concerned mainly the nucleus (the increasing amount of condensed chromatin, ER (swelling and fragmentation of rER and sER, mitochondrium (swelling and reduction of cristae, Golgi apparatus (disintegration and decay and chloroplasts (changes of shape, swelling and reduction of thylakoids, disappearance of starch and presence of big plastoglobuli. In Ni 20 mg samples cell protoplasts were in different stages of degeneration and the cell organelles that were identifiable, were usually damaged.

Role of Nuclear Matrix in Estrogen Regulated Gene Expression in Human Breast Cancer Cells

Science.gov (United States)

1998-08-01

reticular pattern evenly distributed throughout the nucleus, excluding the nucleolus (Figure 4A). This is not so for T47D cells where a composite pattern...acetylation is required to maintain the unfolded nucleosome structure associated with transcribing DNA. Journal of Biological Chemistry 273:14516...nuclear matrix include ER, YY1, AML-1, Spl, Oct1, mutant p53, and Rb [25,28,31,34-40]. Appendix A, part 4 reviews alterations in nuclear matrix composition

Dynamic Nucleolar Targeting of Dengue Virus Polymerase NS5 in Response to Extracellular pH

Science.gov (United States)

Fraser, Johanna E.; Rawlinson, Stephen M.; Heaton, Steven M.

2016-01-01

ABSTRACT The nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment. IMPORTANCE Infections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond

Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine

International Nuclear Information System (INIS)

Uddin, M.; Altmann, G.G.; Leblond, C.P.

1984-01-01

The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [ 3 H]uridine injection or by the de novo pathway as shown after [ 3 H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium - that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5- 3 H]uridine or [5- 3 H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloroacetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [ 3 H]uridine and 72% after [ 3 H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A + RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [ 3 H]uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant

Structure of nucleoli in first-order spermatocytes of selected free-living animal species.

Science.gov (United States)

Andraszek, Katarzyna; Gryzińska, Magdalena; Ceranka, Mariola; Larisch, Agnieszka

2015-10-01

Nucleoli are the product of the activity of nucleolar organizer regions (NOR) in certain chromosomes. Their main functions are the formation of ribosomal subunits from ribosomal protein molecules and the transcription of genes encoding rRNA. Nucleoli are present in the nuclei of nearly all eukaryotic cells because they contain housekeeping genes. The size and number of nucleoli gradually decrease during spermatogenesis. Some of the material originating in the nucleolus probably migrates to the cytoplasm and takes part in the formation of chromatoid bodies (CB). Nucleolus fragmentation and CB assembly take place at the same stage of spermatogenesis. CB are involved in the formation of the acrosome, the migration of mitochondria to the midpiece, and the formation of the sperm tail fibrous sheath. The aim of the study was to characterize the nucleoli in the early prophase of spermatogenesis in the wild boar and the roe deer. The roe deer cells have larger nucleoli and a larger cell nucleus than the wild boar cells. The area of the nucleolus as a percentage of the total area of the nucleus was larger as well. The coefficients of variation for all parameters were higher in the roe deer. In the wild boar cells the nucleoli were mainly regularly shaped. The size of the nucleolus and the nucleus of the spermatocyte is a species-specific trait associated with karyotype and the number of nucleolar organizer regions in a given species. Copyright © 2015 Elsevier B.V. All rights reserved.

Electron microscopic in situ hybridization and autoradiography: Localization and transcription of rDNA in human lymphocyte nucleoli

International Nuclear Information System (INIS)

Wachtler, F.; Mosgoeller, W.S.; Schwarzacher, H.G.

1990-01-01

The distribution of ribosomal DNA (rDNA) in the nucleoli of human lymphocytes was revealed by in situ hybridization with a nonautoradiographic procedure at the electron microscopic level. rDNA is located in the dense fibrillar component of the nucleolus but not in the fibrillar centers. In the same cells the incorporation of tritiated uridine takes place in the dense fibrillar component of the nucleolus as seen by autoradiography followed by gold latensification. From these findings it can be concluded that the transcription of ribosomal DNA takes place in the dense fibrillar component of the nucleolus

Use of transmission electron microscope to assess the damage to Sarcoma 180 ascites tumour cells following in vivo treatment of mitomycin-C and gamma radiation

International Nuclear Information System (INIS)

Majumdar, S.K.; Bhattacharya, S.; Mitra, A.; Mukherji, S.

1991-01-01

Five day old Sarcoma 180 tumour bearing mice were exposed to different doses of mitomycin-C (4 mg or 7 mg per kg body weight of mouse) and gamma radiation (400 R or 800 R) applied singly or in combination. Surviving populations were collected after 5 days of treatment and processed for transmission electron microscopy. The control Sarcoma 180 tumour cells has the following characteristics: profused microvilli, different sized mitochondria with poorly developed internal structure, distinct endoplasmic reticulum studded with ribosomes, the large nucleus rich in chromatin materials and distinct nucleolus containing closely interwined granular and fibrillar components with associated chromatins. Damage to treated cells were ascertained by the reduction in microvilli, swelling of mitochondria with cloudy appearance, dilation and fragmentation of endoplasmic reticulum, blebbing of nuclear membrane, condensation of heterochromatin, appearance of perichromatin granules, segregation and fragmentation of nucleolus and invagination of plasma memebrane with increased intracellular spaces. With the help of transmission electron microscope it is thus possible to assess the nature of damage to organelles effected by mitomycin C and radiation both singly and in combination. Growth inhibition and damage in the cellular ultrastructure were maximum among tumour cells which survived with concomitant treatment with 7 mg MMC and 800 R. (author). 7 refs., 4 figs., 1 tab

Localized movement and morphology of UBF1-positive nucleolar regions are changed by -irradiation in G2 phase of the cell cycle

Czech Academy of Sciences Publication Activity Database

Sorokin, D.V.; Stixová, Lenka; Sehnalová, Petra; Legartová, Soňa; Suchánková, Jana; Šimara, P.; Kozubek, Stanislav; Matula, Pavel; Skalníková, M.; Raška, I.; Bártová, Eva

2015-01-01

Roč. 6, č. 4 (2015), s. 301-313 ISSN 1949-1034 R&D Projects: GA ČR GBP302/12/G157; GA ČR GA13-07822S; GA MŠk 7F14369; GA MŠk(CZ) EE2.3.30.0030 Institutional support: RVO:68081707 Keywords : DNA damage * live cells * nucleolus Subject RIV: BO - Biophysics Impact factor: 2.446, year: 2015

Altered Machinery of Protein Synthesis in Alzheimer's: From the Nucleolus to the Ribosome.

Science.gov (United States)

Hernández-Ortega, Karina; Garcia-Esparcia, Paula; Gil, Laura; Lucas, José J; Ferrer, Isidre

2016-09-01

Ribosomes and protein synthesis have been reported to be altered in the cerebral cortex at advanced stages of Alzheimer's disease (AD). Modifications in the hippocampus with disease progression have not been assessed. Sixty-seven cases including middle-aged (MA) and AD stages I-VI were analyzed. Nucleolar chaperones nucleolin, nucleophosmin and nucleoplasmin 3, and upstream binding transcription factor RNA polymerase I gene (UBTF) mRNAs are abnormally regulated and their protein levels reduced in AD. Histone modifications dimethylated histone H3K9 (H3K9me2) and acetylated histone H3K12 (H3K12ac) are decreased in CA1. Nuclear tau declines in CA1 and dentate gyrus (DG), and practically disappears in neurons with neurofibrillary tangles. Subunit 28 ribosomal RNA (28S rRNA) expression is altered in CA1 and DG in AD. Several genes encoding ribosomal proteins are abnormally regulated and protein levels of translation initiation factors eIF2α, eIF3η and eIF5, and elongation factor eEF2, are altered in the CA1 region in AD. These findings show alterations in the protein synthesis machinery in AD involving the nucleolus, nucleus and ribosomes in the hippocampus in AD some of them starting at first stages (I-II) preceding neuron loss. These changes may lie behind reduced numbers of dendritic branches and reduced synapses of CA1 and DG neurons which cause hippocampal atrophy. © 2015 International Society of Neuropathology.

Ultrastructure of central cell in female gametophyte of Castilleja wightii Elmer (Scrophulariaceae).

Science.gov (United States)

Ekici, Nuran; Dane, Feruzan; Olgun, Göksel

2013-09-01

Embryo sac cells are highly differentiated in plants. The central cell is one of the most important cells of the embryo sac. It forms endosperm by fusion with a sperm cell. Ultrastructure of the central cell in the mature embryo sac of Castilleja wightii was investigated in this study. Nucleolus which had a lot of vacuole in a large secondary nucleus and numerous dictyosomes, vesicles, mitochondria, amyloplasts in cytoplasm were seen in this cell. Also free ribosomes in the form of polysomes and large lipid bodies were detected in the cytoplasm. Numerous vacuoles of different size were observed and some of them had autophagic function. Both smooth and rough endoplasmic reticulums were seen. Although invaginations were seen in the plasmalemma of the central cell to the inside of the embryo sac, a thick cuticular layer was observed outer side on the cell wall. The aim of this study was to contribute studies about the ultrastructure of embryo sacs.

Nucleolar exit of RNF8 and BRCA1 in response to DNA damage

International Nuclear Information System (INIS)

Guerra-Rebollo, Marta; Mateo, Francesca; Franke, Kristin; Huen, Michael S.Y.; Lopitz-Otsoa, Fernando; Rodríguez, Manuel S.; Plans, Vanessa; Thomson, Timothy M.

2012-01-01

The induction of DNA double-strand breaks (DSBs) elicits a plethora of responses that redirect many cellular functions to the vital task of repairing the injury, collectively known as the DNA damage response (DDR). We have found that, in the absence of DNA damage, the DSB repair factors RNF8 and BRCA1 are associated with the nucleolus. Shortly after exposure of cells to γ-radiation, RNF8 and BRCA1 translocated from the nucleolus to damage foci, a traffic that was reverted several hours after the damage. RNF8 interacted through its FHA domain with the ribosomal protein RPSA, and knockdown of RPSA caused a depletion of nucleolar RNF8 and BRCA1, suggesting that the interaction of RNF8 with RPSA is critical for the nucleolar localization of these DDR factors. Knockdown of RPSA or RNF8 impaired bulk protein translation, as did γ-irradiation, the latter being partially countered by overexpression of exogenous RNF8. Our results suggest that RNF8 and BRCA1 are anchored to the nucleolus through reversible interactions with RPSA and that, in addition to its known functions in DDR, RNF8 may play a role in protein synthesis, possibly linking the nucleolar exit of this factor to the attenuation of protein synthesis in response to DNA damage. -- Highlights: ► RNF8 and BRCA1 are associated with the nucleolus of undamaged cells. ► Upon γ-radiation, RNF8 and BRCA1 are translocated from the nucleolus to damage foci. ► The ribosomal protein RPSA anchors RNF8 to the nucleolus. ► RNF8 may play previously unsuspected roles in protein synthesis.

Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

DEFF Research Database (Denmark)

Svarcova, Olga; Dinnyes, A.; Polgar, Z.

2009-01-01

displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

Ultrastructural changes in goat interspecies and intraspecies reconstructed early embryos

DEFF Research Database (Denmark)

Tao, Yong; Gheng, Lizi; Zhang, Meiling

2008-01-01

and dispered gradually from the 4-cell period. The nucleolus of GC and GG embryos changed from electron dense to a fibrillo-granular meshwork at the 16-cell stage, showing that nucleus function in the reconstructed embryos was activated. The broken nuclear envelope and multiple nucleoli in one blastomere......- and intraspecies reconstructed embryos have a similar pattern of developmental change to that of in vivo-produced embryos for ZP, rough ER, Gi and nucleolus, but differ for mitochondria, LD, vesicles, nucleus and gap junction development. In particular, the interspecies cloned embryos showed more severe...

Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25

International Nuclear Information System (INIS)

Fujiwara, Takashi; Suzuki, Shunji; Kanno, Motoko; Sugiyama, Hironobu; Takahashi, Hisaaki; Tanaka, Junya

2006-01-01

Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the mechanism of Nop25 localization in the nucleolus. Deletion experiments of Nop25 amino acid sequence showed Nop25 to contain a nuclear targeting sequence in the N-terminal and a nucleolar targeting sequence in the C-terminal. By expressing derivative peptides from the C-terminal as GFP-fusion proteins in the cells, a lysine and arginine residue-enriched peptide (KRKHPRRAQDSTKKPPSATRTSKTQRRRR) allowed a GFP-fusion protein to be transported and fully retained in the nucleolus. When the peptide was fused with cMyc epitope and expressed in the cells, a cMyc epitope was then detected in the nucleolus. Nop25 did not localize in the nucleolus by deletion of the peptide from Nop25. Furthermore, deletion of a subdomain (KRKHPRRAQ) in the peptide or amino acid substitution of lysine and arginine residues in the subdomain resulted in the loss of Nop25 nucleolar localization. These results suggest that the lysine and arginine residue-enriched peptide is the most prominent nucleolar targeting sequence of Nop25 and that the long stretch of basic residues might play an important role in the nucleolar localization of Nop25. Although Nop25 contained putative SUMOylation, phosphorylation and glycosylation sites, the amino acid substitution in these sites had no effect on the nucleolar localization, thus suggesting that these post-translational modifications did not contribute to the localization of Nop25 in the nucleolus. The treatment of the cells, which expressed a GFP-fusion protein with a nucleolar targeting sequence of Nop25, with RNase A resulted in a complete dislocation of the protein from the nucleolus. These data suggested that the nucleolar targeting sequence might therefore play an important role in the binding of Nop25 to RNA molecules and that the RNA binding of Nop25 might be essential for the nucleolar localization of Nop25

Cell survival following alpha particle irradiation: critical sites and implications for carcinogenesis

International Nuclear Information System (INIS)

Lloyd, E.L.; Gemmell, M.A.; Henning, C.B.; Gemmell, D.S.; Zabransky, B.J.

1976-01-01

In experiments in which mammalian cells were irradiated with 5.6 MeV alpha particles from a Tandem Van de Graaff machine we have confirmed the finding of others that the mean lethal dose (D 0 ) is about 100 rad, but by measurements of the area of the cell nuclei as irradiated we found that this mean lethal dose corresponds not to 1, as expected, but to about 27 alpha particles per cell nucleus. (The exact number appears to change slightly with cell passage number.) This allows for the possibility that the direct action of alpha particles on the nucleus may be the important event in carcinogenesis, a theory which was previously difficult to accept if a single particle hitting the nucleus anywhere was considered to be lethal. Evidence is presented to implicate the nucleolus as a possible critical site for the inhibition of reproductive integrity of the cell

Production of giant mouse oocyte nucleoli and assessment of their protein content.

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Fulka, Helena; Martinkova, Stanislava; Kyogoku, Hirohisa; Langerova, Alena; Fulka, Josef

2012-01-01

Compared with advanced developmental stage embryos and somatic cells, fully grown mammalian oocytes contain specific nucleolus-like structures (NPB - nucleolus precursor bodies). It is commonly accepted that they serve as a store of material(s) from which typical nucleoli are gradually formed. Whilst nucleoli from somatic cells can be collected relatively easily for further biochemical analyses, a sufficient number of oocyte nucleoli is very difficult to obtain. We have found that isolated oocytes nucleoli fuse very efficiently when contact is established between them. Thus, well visible giant nucleoli can be obtained, relatively easily handled and then used for further biochemical analyses. With the use of colloidal gold staining, we estimated that a single fully grown mouse oocyte nucleolus contains approximately 1.6 ng of protein. We do believe that this approach will accelerate further research aiming at analyzing the composition of oocyte nucleoli in more detail.

Werner complex deficiency in cells disrupts the Nuclear Pore Complex and the distribution of lamin B1.

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Li, Zhi; Zhu, Yizhou; Zhai, Yujia; R Castroagudin, Michelle; Bao, Yifei; White, Tommy E; Glavy, Joseph S

2013-12-01

From the surrounding shell to the inner machinery, nuclear proteins provide the functional plasticity of the nucleus. This study highlights the nuclear association of Pore membrane (POM) protein NDC1 and Werner protein (WRN), a RecQ helicase responsible for the DNA instability progeria disorder, Werner Syndrome. In our previous publication, we connected the DNA damage sensor Werner's Helicase Interacting Protein (WHIP), a binding partner of WRN, to the NPC. Here, we confirm the association of the WRN/WHIP complex and NDC1. In established WRN/WHIP knockout cell lines, we further demonstrate the interdependence of WRN/WHIP and Nucleoporins (Nups). These changes do not completely abrogate the barrier of the Nuclear Envelope (NE) but do affect the distribution of FG Nups and the RAN gradient, which are necessary for nuclear transport. Evidence from WRN/WHIP knockout cell lines demonstrates changes in the processing and nucleolar localization of lamin B1. The appearance of "RAN holes" void of RAN corresponds to regions within the nucleolus filled with condensed pools of lamin B1. From WRN/WHIP knockout cell line extracts, we found three forms of lamin B1 that correspond to mature holoprotein and two potential post-translationally modified forms of the protein. Upon treatment with topoisomerase inhibitors lamin B1 cleavage occurs only in WRN/WHIP knockout cells. Our data suggest the link of the NDC1 and WRN as one facet of the network between the nuclear periphery and genome stability. Loss of WRN complex leads to multiple alterations at the NPC and the nucleolus. © 2013. Published by Elsevier B.V. All rights reserved.

Ellagic Acid-Changed Epigenome of Ribosomal Genes and Condensed RPA194-Positive Regions of Nucleoli in Tumour Cells.

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Legartová, S; Sbardella, G; Kozubek, S; Bártová, E

2015-01-01

We studied the effect of ellagic acid (EA) on the morphology of nucleoli and on the pattern of major proteins of the nucleolus. After EA treatment of HeLa cells, we observed condensation of nucleoli as documented by the pattern of argyrophilic nucleolar organizer regions (AgNORs). EA also induced condensation of RPA194-positive nucleolar regions, but no morphological changes were observed in nucleolar compartments positive for UBF1/2 proteins or fibrillarin. Studied morphological changes induced by EA were compared with the morphology of control, non-treated cells and with pronounced condensation of all nucleolar domains caused by actinomycin D (ACT-D) treatment. Similarly as ACT-D, but in a lesser extent, EA induced an increased number of 53BP1-positive DNA lesions. However, the main marker of DNA lesions, γH2AX, was not accumulated in body-like nuclear structures. An increased level of γH2AX was found by immunofluorescence and Western blots only after EA treatment. Intriguingly, the levels of fibrillarin, UBF1/2 and γH2AX were increased at the promoters of ribosomal genes, while 53BP1 and CARM1 levels were decreased by EA treatment at these genomic regions. In the entire genome, EA reduced H3R17 dimethylation. Taken together, ellagic acid is capable of significantly changing the nucleolar morphology and protein levels inside the nucleolus.

Nucleolar exit of RNF8 and BRCA1 in response to DNA damage

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Guerra-Rebollo, Marta; Mateo, Francesca; Franke, Kristin [Department of Cell Biology, Molecular Biology Institute of Barcelona (IBMB), CSIC, Barcelona Science Park, Helix Building, Baldiri Reixac 15-21, 08028 Barcelona (Spain); Huen, Michael S.Y. [Department of Anatomy, Centre for Cancer Research, The University of Hong Kong, L1, Laboratory Block, 21 Sassoon Road, Hong Kong Special Administrative Region (Hong Kong); Lopitz-Otsoa, Fernando; Rodriguez, Manuel S. [Proteomics Unit, CIC bioGUNE CIBERehd, ProteoRed, Technology Park of Bizkaia, Building 801A, 48160 Derio (Spain); Plans, Vanessa [Department of Cell Biology, Molecular Biology Institute of Barcelona (IBMB), CSIC, Barcelona Science Park, Helix Building, Baldiri Reixac 15-21, 08028 Barcelona (Spain); Thomson, Timothy M., E-mail: titbmc@ibmb.csic.es [Department of Cell Biology, Molecular Biology Institute of Barcelona (IBMB), CSIC, Barcelona Science Park, Helix Building, Baldiri Reixac 15-21, 08028 Barcelona (Spain)

2012-11-01

The induction of DNA double-strand breaks (DSBs) elicits a plethora of responses that redirect many cellular functions to the vital task of repairing the injury, collectively known as the DNA damage response (DDR). We have found that, in the absence of DNA damage, the DSB repair factors RNF8 and BRCA1 are associated with the nucleolus. Shortly after exposure of cells to {gamma}-radiation, RNF8 and BRCA1 translocated from the nucleolus to damage foci, a traffic that was reverted several hours after the damage. RNF8 interacted through its FHA domain with the ribosomal protein RPSA, and knockdown of RPSA caused a depletion of nucleolar RNF8 and BRCA1, suggesting that the interaction of RNF8 with RPSA is critical for the nucleolar localization of these DDR factors. Knockdown of RPSA or RNF8 impaired bulk protein translation, as did {gamma}-irradiation, the latter being partially countered by overexpression of exogenous RNF8. Our results suggest that RNF8 and BRCA1 are anchored to the nucleolus through reversible interactions with RPSA and that, in addition to its known functions in DDR, RNF8 may play a role in protein synthesis, possibly linking the nucleolar exit of this factor to the attenuation of protein synthesis in response to DNA damage. -- Highlights: Black-Right-Pointing-Pointer RNF8 and BRCA1 are associated with the nucleolus of undamaged cells. Black-Right-Pointing-Pointer Upon {gamma}-radiation, RNF8 and BRCA1 are translocated from the nucleolus to damage foci. Black-Right-Pointing-Pointer The ribosomal protein RPSA anchors RNF8 to the nucleolus. Black-Right-Pointing-Pointer RNF8 may play previously unsuspected roles in protein synthesis.

Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation

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Duangmano Suwit

2012-10-01

Full Text Available Abstract Background Cucurbitacin B, an oxygenated tetracyclic triterpenoid compound extracted from the Thai medicinal plant Trichosanthes cucumerina L., has been reported to have several biological activities including anti-inflammatory, antimicrobial and anticancer. Cucurbitacin B is great of interest because of its biological activity. This agent inhibits growth of various types of human cancer cells lines. Methods In this study, we explored the novel molecular response of cucurbitacin B in human breast cancer cells, MCF-7 and MDA-MB-231. The growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay. The effects of cucurbitacin B on microtubules morphological structure and tubulin polymerization were analyzed using immunofluorescence technique and tubulin polymerization assay kit, respectively. Proteomic analysis was used to identify the target-specific proteins that involved in cucurbitacin B treatment. Some of the differentially expressed genes and protein products were validated by real-time RT-PCR and western blot analysis. Cell cycle distributions and apoptosis were investigated using flow cytometry. Results Cucurbitacin B exhibited strong antiproliferative effects against breast cancer cells in a dose-dependent manner. We show that cucurbitacin B prominently alters the cytoskeletal network of breast cancer cells, inducing rapid morphologic changes and improper polymerization of the microtubule network. Moreover, the results of 2D-PAGE, real-time RT-PCR, and western blot analysis revealed that the expression of nucleophosmin/B23 and c-Myc decreased markedly after cucurbitacin B treatment. Immunofluorescence microscopy showed that cucurbitacin B induced translocation of nucleophosmin/B23 from the nucleolus to nucleoplasm. Treatment with cucurbitacin B resulted in cell cycle arrest at G2/M phase and the enhancement of apoptosis. Conclusions Our findings suggest that cucurbitacin B may inhibit the

Subcellular localization of estradiol receptor in MCF7 cells studied with nanogold-labelled antibody fragments.

Science.gov (United States)

Kessels, M M; Qualmann, B; Thole, H H; Sierralta, W D

1998-01-01

Ultrastructural localization studies of estradiol receptor in hormone-deprived and hormone-stimulated MCF7 cells were done using F(ab') fragments of three different antibodies (#402, 13H2, HT277) covalently linked to nanogold. These ultra-small, non-charged immunoreagents, combined with a size-enlargement by silver enhancement, localized estradiol receptor in both nuclear and cytoplasmic areas of non-stimulated target cells; stimulation with the steroid induced a predominantly nuclear labelling. In the cytoplasm of resting cells, tagging was often observed at or in the proximity of stress fibers. In the nucleus a large proportion of receptor was found inside the nucleolus, specially with the reagent derived from antibody 13H2. We postulate that different accessibilities of receptor epitopes account for the different labelling densities observed at cytoskeletal elements and the nucleoli.

Ultrastructural study of the chromatoid body in planarian regenerative cells

Energy Technology Data Exchange (ETDEWEB)

Hori, I. (Kanazawa Medical Univ., Ishikawa (Japan))

1982-04-01

The present paper deals with the ultrastructural changes of chromatoid bodies in planarian regenerative cells under normal and experimental conditions. A close relationship was usually observed between chromatoid bodies and pore regions of the nuclear envelope in these cells. The chromatoid bodies continued to decrease in size during cytodifferentiation of regenerative cells, though they did not disappear entirely throughout the regeneration processes. Cytochemistry and (/sup 3/H)uridine autoradiography have shown that the chromatoid body contains RNA. The typical morphological effect of actinomycin D became apparent in three organelles, i.e., nucleolus, polysome and chromatoid body. Ultrastructural changes in nucleoli were observed to occur after actinomycin treatment (20 ..mu..g/ml). The exposure to a higher dose of actinomycin (50 ..mu..g/ml) caused a decay of chromatoid bodies while nuclear envelopes retained numerous pores. Both the nucleoli and the chromatoid bodies disappeared in the sequential stages. Within the cytoplasm of such cells disintegration of a polysomal pattern was correlated with the disappearance of chromatoid bodies. The significance of the planarian chromatoid body is discussed in relation to differentiation of the regenerative cells.

Nucleolus degradation and growth induced by uv-microbeam irradiation of interphase cells grown in culture

International Nuclear Information System (INIS)

Sakharov, V.N.; Voronkova, N.

1976-01-01

In contrast to total cell irradiation, local UV-microbeam irradiation can stimulate a significant diminution in the irradiated mature nucleoli in interphase mammalian cells in culture. This diminution is accompanied by the concomitant expansion of the unirradiated nucleoli within the same nucleus, and the total nucleolar volume per nucleus does not change appreciably. It is suggested that these nucleolar volume changes are the result of the dispersion, migration, and redistribution of the nucleolar material between competitive nucleolar organizer regions of the interphase nucleus

Characterization of the Variability of Nucleoli in the Cells of Panax ginseng Meyer In Vivo and In Vitro.

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Khrolenko, Yuliya A; Burundukova, Olga L; Lauve, Lyudmila S; Muzarok, Tamara I; Makhan'kov, Vyacheslav V; Zhuravlev, Yuri N

2012-07-01

Results of karyological study of intact plants and some callus lines of Panax ginseng are presented. In the native plants of P. ginseng the nucleus with 1 nucleolus (90%) dominate, and nucleus with 2 nucleoli is rare. One nucleolar nucleus also dominate in interphase nuclei of cells of cultivated P. ginseng (from 2006), but we also found nucleus with 2 to 3 nucleoli in the same cell lines. Interphase nuclei of P. ginseng in long cultivated lines (from 1988) contain 1 to 9 nucleoli, with a predominance of nuclei containing from 3 to 4 nucleoli. It was shown that long-time cells (cultivated since 1988) had cytogenetic changes such as increase level of polyploid and aneuploid cells, increase of nucleoli number into interphase nucleus and decrease of nuclei/nucleoli ratio. These long-time cultivated cells had very low ginsenoside content.

Number and size of nucleoli in the spermatocytes of chicken and Japanese quail.

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Andraszek, Katarzyna; Gryzińska, Magdalena; Knaga, Sebastian; Wójcik, Ewa; Smalec, Elzbieta

2012-01-01

Nucleoli are the product of nucleolus organizing region activity (NOR) of specific chromosomes. Their basic function is to synthetise ribosomal RNA precursors and promote the maturation and assemblage of preribosomal RNP molecules. Information on rRNA-coding gene activity can be provided by the analysis of the number and size of nucleoli in the prophase of the first meiotic division. The morphology and ultrastructure of a nucleolus depends, among others, on the species and cell growth cycle as well as the physiological and pathological state of an organism. The purpose of this research was to determine the number and size of nucleoli in the spermatocytes of the domestic chicken and the Japanese quail. Diverse numbers and sizes of nucleoli in the cells of the analysed birds were observed. 1-4 nucleoli were identified in chicken cells (1.91 +/- 0.63 on average) and 1-2 in quail cells (1.13 +/- 0.33 on average). For the total of 957 nucleoli observed in Gallus cells, 329 were classified as large and 628 as small. In Coturnix cells, 563 nucleoli were identified (66 large and 497 small ones). An analysis of the numbers and sizes of nucleoli can be performed at the cytogenetic level and serve as an alternative source of information on rRNA encoding gene and nucleolus organising region (NOR) activities.

Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro.

Science.gov (United States)

Gallenberger, Martin; Meinel, Dominik M; Kroeber, Markus; Wegner, Michael; Milkereit, Philipp; Bösl, Michael R; Tamm, Ernst R

2011-02-01

Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

International Nuclear Information System (INIS)

Vázquez-Iglesias, Lorena; Lostalé-Seijo, Irene; Martínez-Costas, José; Benavente, Javier

2012-01-01

A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

Energy Technology Data Exchange (ETDEWEB)

Vazquez-Iglesias, Lorena; Lostale-Seijo, Irene; Martinez-Costas, Jose [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain); Benavente, Javier, E-mail: franciscojavier.benavente@usc.es [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain)

2012-10-25

A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

Science.gov (United States)

2012-01-01

Background Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. Results Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. Conclusion NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin. PMID:22909121

Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

International Nuclear Information System (INIS)

Kim, Hye-Mi; Kang, Dong-Ku; Kim, Hak Yong; Kang, Sang Sun; Chang, Soo-Ik

2007-01-01

Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells

Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.

Science.gov (United States)

Li, Zhou Fang; Liang, Yi Min; Lau, Pui Ngan; Shen, Wei; Wang, Dai Kui; Cheung, Wing Tai; Xue, Chun Jason; Poon, Lit Man; Lam, Yun Wah

2013-01-01

Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

An ultrastructural study of the chromatoid body in planarian regenerative cells

International Nuclear Information System (INIS)

Hori, Isao

1982-01-01

The present paper deals with the ultrastructural changes of chromatoid bodies in planarian regenerative cells under normal and experimental conditions. A close relationship was usually observed between chromatoid bodies and pore regions of the nuclear envelope in these cells. The chromatoid bodies continued to decrease in size during cytodifferentiation of regenerative cells, though they did not disappear entirely throughout the regeneration processes. Cytochemistry and [ 3 H]uridine autoradiography have shown that the chromatoid body contains RNA. The typical morphological effect of actinomycin D became apparent in three organelles, i.e., nucleolus, polysome and chromatoid body. Ultrastructural changes in nucleoli were observed to occur after actinomycin treatment (20 μg/ml). The exposure to a higher dose of actinomycin (50 μg/ml) caused a decay of chromatoid bodies while nuclear envelopes retained numerous pores. Both the nucleoli and the chromatoid bodies disappeared in the sequential stages. Within the cytoplasm of such cells disintegration of a polysomal pattern was correlated with the disappearance of chromatoid bodies. The significance of the planarian chromatoid body is discussed in relation to differentiation of the regenerative cells. (author)

Characterization of Rat Hair Follicle Stem Cells Selected by Vario Magnetic Activated Cell Sorting System

International Nuclear Information System (INIS)

Huang, Enyi; Lian, Xiaohua; Chen, Wei; Yang, Tian; Yang, Li

2009-01-01

Hair follicle stem cells (HfSCs) play crucial roles in hair follicle morphogenesis and hair cycling. These stem cells are self-renewable and have the multi-lineage potential to generate epidermis, sebaceous glands, and hair follicle. The separation and identification of hair follicle stem cells are important for further research in stem cell biology. In this study, we report on the successful enrichment of rat hair follicle stem cells through vario magnetic activated cell sorting (Vario MACS) and the biological characteristics of the stem cells. We chose the HfSCs positive surface markers CD34, α6-integrin and the negative marker CD71 to design four isolation strategies: positive selection with single marker of CD34, positive selection with single marker of α6-integrin, CD71 depletion followed by CD34 positive selection, and CD71 depletion followed by α6-integrin positive selection. The results of flow cytometry analysis showed that all four strategies had ideal effects. Specifically, we conducted a series of researches on HfSCs characterized by their high level of CD34, termed CD34 bri cells, and low to undetectable expression of CD34, termed CD34 dim cells. CD34 bri cells had greater proliferative potential and higher colony-forming ability than CD34 dim cells. Furthermore, CD34 bri cells had some typical characteristics as progenitor cells, such as large nucleus, obvious nucleolus, large nuclear:cytoplasmic ratio and few cytoplasmic organelles. Our findings clearly demonstrated that HfSCs with high purity and viability could be successfully enriched with Vario MACS

Radiation-induced mutation frequency in marked chromosome of Macaca mulatta

International Nuclear Information System (INIS)

Dzhemilev, Z.A.; Machavariani, M.G.

1976-01-01

The symmetric and asymmetric exchange frequencies of marked (nucleolus forming) chromosomes were studied in the lymphocytes and epithelial kidney cells irradiated by X-rays at G 0 , both in vivo and in vitro. Symmetric and asymmetric exchange frequencies were found to be equal. In both the types of Macaca mulatta cells, the exchange frequency in the long arm appeared to be higher than theoretically expected. The increased exchange in the long arm is thought to be due to a greater quantity of late replicating heterochromatin in it. The short arm of marked chromosome of epithelial kidney cells enters the exchange in accordance to its length in mitosis, but exchange number in the short arm chromosome in lymphocytes is lower than in epithelial cells. This difference is caused likely by different functioning of the nucleolus forming heterochromatin. (author)

Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.

Directory of Open Access Journals (Sweden)

Zhou Fang Li

Full Text Available Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

Microscopic Aspects of Autoschizic Cell Death in Human Ovarian Carcinoma (2774) Cells Following Vitamin C, Vitamin K3 or Vitamin C:K3 Treatment

Science.gov (United States)

Gilloteaux, Jacques; Jamison, James M.; Arnold, David; Taper, Henryk S.; von Gruenigen, Vivian E.; Summers, Jack L.

2003-08-01

Human ovarian carcinoma cells (MDAH 2774) were treated with sodium ascorbate (VC), menadione (VK3), or with a VC:VK3 combination for 1 h and then studied using light microscopy (LM) and scanning (SEM) and transmission electron (TEM) microscopy. Plasma membrane damage (blisters and blebs, hairy aspect) results from vitamin C (VC) treatment, while cytoskeletal damage and self-morsellation are caused by vitamin K3 (VK3) treatment. VC:VK3-treated cells exhibit exacerbated injuries characteristic of both VC and VK3 treatment as well as a significant decrease in cell diameters from 20 35 [mu]m for control cells to 7 12 [mu]m for VC:VK3 treatment. Moreover, after a 1-h exposure to the vitamin combination, autoschizis (43%), apoptosis (3%), and oncosis (1.9%) are observed at the percentages indicated. All cellular changes associated with autoschizis observed with SEM were confirmed by LM and TEM observations and are consistent with cell death by autoschizis: decrease in cell size, cytoplasmic self-excisions, degradation of the nucleus and nucleolus without formation of apoptotic bodies and, ultimately, karyorrhexis and karyolysis. These results also suggest that the vitamin combination may find clinical use in the treatment of ovarian cancer.

X-ray microimaging of cisplatin distribution in ovarian cancer cells

International Nuclear Information System (INIS)

Kiyozuka, Yasuhiko; Tsubura, Airo; Takemoto, Kuniko; Kihara, Hiroshi; Yamamoto, Akitsugu; Guttmann, Peter

2000-01-01

X-ray microscopy has the possibility to be in use for elemental analysis of tissue and cells especially under physiological conditions with high lateral resolution. In X-ray microimaging cisdiamminedichloroplatinum II (cisplatin: CDDP), an anticancer agent, which has a platinum atom at its functional center gives sufficient contrast against organic material at sub-cellular level. We analyzed the enhance effect and intracellular distribution of CDDP in human ovarian cancer cells with the transmission X-ray microscope at BESSY, Berlin. Two human ovarian cancer cell lines (MN-1 and EC) were treated with 1 and 10 μg/ml of CDDP for 4 hours and compared with untreated cells X-ray images of CDDP-treated samples show clearly labeled nucleoli, periphery of the nucleus and mitochondria, in a concentration-dependent manner. CDDP binds to DNA molecules via the formation of intra- or-inter-strand cross-links. Higher contrasts at the periphery of nucleus and nucleoli suggest the distribution of tightly packed heterochromatin. In addition, results show the possibility that CDDP binds to mitochondrial DNA. Biological function of cisplatin is not only the inhibition of DNA replication but is suggested to disturb mitochondrial function and RNA synthesis in the nucleolus

Composition of ribonucleic acid from various parts of spider oocytes.

Science.gov (United States)

EDSTROM, J E

1960-09-01

Microphoretic purine-pyrimidine analyses of the ribonucleic acid (RNA) in nucleoli, nucleoplasm, cytoplasm, and yolk nuclei of spider oocytes have been carried out. The material necessary for the analyses was isolated by micromanipulation. Determinations of the amounts of RNA in the different parts of the cell were also performed. No differences between the composition of RNA in the nucleolus and the cytoplasm could be disclosed. Nucleoplasmic RNA was, on the other hand, distinctly different from that in the nucleolus and in the cytoplasm. The difference lies in the content of adenine, which is highest in nucleoplasmic RNA. The few analyses carried out on yolk nuclei showed their RNA to be variable in composition with a tendency to high purine values. The cytoplasm contains about 99 per cent of the total RNA in these cells, the nucleoplasm about 1 per cent, and the nucleolus not more than 0.3 per cent, although the highest concentrations are found in these latter structures. When considered in the light of other recent findings the results are compatible with the view that nucleolar RNA is the precursor of cytoplasmic RNA.

Phase Transitions in the Nucleus: the functional implications of concentration-dependent assembly of a Liquid-like RNA/Protein Body

Science.gov (United States)

Zhu, Lian; Weber, Stephanie; Berry, Joel; Vaidya, Nilesh; Haataja, Mikko; Brangwynne, Clifford

2015-03-01

The nucleolus is a liquid-like membrane-less nuclear body which plays an important role in cell growth and size control. By modulating nucleolar component concentration through RNAi conditions that change C. elegans cell size, we find that nucleoli only assemble above a threshold concentration; moreover, the ripening dynamics of nucleated droplets are consistent with the hypothesis that the assembly of the nucleolus represents an intracellular liquid-liquid phase transition. A key question is how this phase-transition is linked to the primary function of the nucleolus, in transcribing and processing ribosomal RNA. To address this, we characterize the localization of RNA Polymerase I, a key transcriptional enzyme, into nucleolar foci as a function of nucleolar component concentration. Our results suggest that there are a small number of key disordered phosphoproteins that may serve as a link between transcription and assembly. Finally, we present preliminary results using a reduced model system consisting of purified nucleolar proteins to assess the ability of nucleolar proteins to drive liquid-liquid phase separation in vitro. These results lay the foundation for a quantitative understanding of intracellular phase transitions and their impact on biomedically-critical RNA-processing steps.

Origin of Microcells in the Human Sarcoma Cell Line HT-1080

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Indulis Buiķis

1999-01-01

Full Text Available The aim of this study was to investigate the development of microcells in the human sarcoma cell line HT‐1080 after interference with thiophosphamidum. We found that damaged interphase macrocells located at the projection of the nucleolus may form one or several microcells. The micronuclei of the microcells intensively incorporate the thymidine analogue 5‐bromo‐2'‐deoxyuridine and strongly express argyrophilic nucleolar organiser region proteins. At an early phase of the development, the micronuclei contain fragmented DNA, but in subsequent phases, the micronuclei accumulate polymeric DNA, simultaneously with an increase in their size. After desintegration of the damaged macrocell, the microcells appear in the intercellular space. The microcells can enter mitosis and they strongly express the lung resistance protein. Electron microscopic observations suggest that coiled bodies are involved in the development of the microcells. Since the observed path of microcell formation differs from apoptotic cell fragmentation into apoptotic bodies, we propose a new term for this microcell development: sporosis. We suggest that self‐renewal of the tumour stem cells is likely based on sporosis.

Positioning of the NOR-bearing chromosomes in relation to nucleoli in daughter cells after mitosis.

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Kalmárová, M; Smirnov, E; Kovácik, L; Popov, A; Raska, I

2008-01-01

It is known that chromosomes occupy non-random positions in the cell nucleus. However, it is not clear to what extent their nuclear positions, together with their neighborhood, are conserved in daughter cells. To address specific aspects of this problem, we used the model of the chromosomes carrying ribosomal genes that are organized in clusters termed Nucleolus Organizer Regions (NORs). We compared the association of chosen NOR-bearing chromosomes (NOR-chromosomes) with nucleoli, as well as the numbers of nucleoli, in the pairs of daughter cells, and established how frequently the daughter cells had equal numbers of the homologs of certain NOR-chromosomes associated with individual nucleoli. The daughter cells typically had different numbers of nucleoli. At the same time, using immuno-FISH with probes for chromosomes 14 and 15 in HeLa cells, we found that the cell pairs with identical combinations appeared significantly more frequently than predicted by the random model. Thus, although the total number of chromosomes associated with nucleoli is variable, our data indicate that the position of the NOR-bearing chromosomes in relation to nucleoli is partly conserved through mitosis.

NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

International Nuclear Information System (INIS)

Shen, Qi; Zheng, Xingzheng; McNutt, Michael A.; Guang, Lizhao; Sun, Ying; Wang, Jiaochen; Gong, Yilei; Hou, Lin; Zhang, Bo

2009-01-01

The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated α-tubulin, suggesting that it plays a role in maintaining or enhancing stability of α-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated α-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.

NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

Energy Technology Data Exchange (ETDEWEB)

Shen, Qi; Zheng, Xingzheng; McNutt, Michael A.; Guang, Lizhao; Sun, Ying; Wang, Jiaochen; Gong, Yilei; Hou, Lin [Department of Pathology, Health Science Center of Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191 (China); Zhang, Bo, E-mail: zhangbo@bjmu.edu.cn [Department of Pathology, Health Science Center of Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191 (China)

2009-06-10

The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated {alpha}-tubulin, suggesting that it plays a role in maintaining or enhancing stability of {alpha}-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated {alpha}-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.

Ultrastructure of Endogenous Stages of Eimeria ninakohlyakimovae Yakimoff & Rastegaieff, 1930 Emend. Levine, 1961 in Experimentally Infected Goat

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Vieira Luiz S

1997-01-01

Full Text Available The ultrastructure of endogenous stages of Eimeria ninakohlyakimovae was observed in epithelial cells of cecum and colon crypts from a goat experimentally infected with 2.0 x 105 oocysts/kg. The secondary meronts developed above the nucleus of the host cell. The nucleus first divides and merozoites then form on the surface of multinucleated meronts. Free merozoites in the parasitophorous vacuole present a conoid, double membrane, one pair of rhoptries, micronemes, micropore, anterior and posterior polar ring, a nucleus with a nucleolus and peripheral chromatin. The microgamonts are located below the nucleus of the host cell and contain several nuclei at the periphery of the parasite. The microgametes consist of a body, a nucleus, three flagella and mitochondria. The macrogamonts develop below the nucleus of the host cell and have a large nucleus with a prominent nucleolus. The macrogametes contain a nucleus, wall-forming bodies of type I and type II. The young oocysts present a wall containing two layers and a sporont

Synergistic antitumor cytotoxic actions of ascorbate and menadione on human prostate (DU145) cancer cells in vitro: nucleus and other injuries preceding cell death by autoschizis.

Science.gov (United States)

Gilloteaux, Jacques; Jamison, James M; Neal, Deborah; Summers, Jack L

2014-04-01

Scanning (SEM) and transmission electron microscopy (TEM) were used to characterize the cytotoxic effects of ascorbate (VC), menadione (VK3), or a VC:VK3 combination on a human prostate carcinoma cell line (DU145) following a 1-h vitamin treatment and a subsequent 24-h incubation in culture medium. Cell alterations examined by light and electron microscopy were treatment-dependent with VC + VK3 >VK3 > VC > Sham. Oxidative stress-induced damage was found in most organelles. This report describes injuries in the tumor cell nucleus (chromatin and nucleolus), mitochondria, endomembranes, lysosomal bodies (autophagocytoses) and inclusions. Morphologic alterations suggest that cytoskeleton damage is likely responsible for the superficial cytoplasmic changes, including major changes in cell shape and size and the self-excising phenomena. Unlike apoptotic bodies, the excised pieces contain ribonucleoproteins, but not organelles. These deleterious events cause a progressive, significant reduction in the tumor cell size. During nuclear alterations, the nuclei maintain their envelope during chromatolysis and karyolysis until cell death, while nucleoli undergo a characteristic segregation of their components. In addition, changes in fat and glycogen storage are consistent the cytotoxic and metabolic alterations caused by the respective treatments. All cellular ultrastructural changes are consistent with cell death by autoschizis and not apoptosis or other kinds of cell death.

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora).

Science.gov (United States)

Leonova, Olga G; Karajan, Bella P; Ivlev, Yuri F; Ivanova, Julia L; Skarlato, Sergei O; Popenko, Vladimir I

2013-01-01

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

Molecular determinants of nucleolar translocation of RNA helicase A

International Nuclear Information System (INIS)

Liu Zhe; Kenworthy, Rachael; Green, Christopher; Tang, Hengli

2007-01-01

RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role

Identification of nuclear τ isoforms in human neuroblastoma cells

International Nuclear Information System (INIS)

Loomis, P.A.; Howard, T.H.; Castleberry, R.P.; Binder, L.I.

1990-01-01

The τ proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, τ has been shown to be an integral component of paired helical filaments, the principal constituent of the neurofibrillary tangles found in brains of patients with Alzheimer's disease and of most aged individuals with Down syndrome (trisomy 21). The authors report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, they further demonstrated the existence of the entire τ molecule in the isolated nuclei of neuroblastoma cells. Nuclear τ proteins, like the τ proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that τ may function in processes not directly associated with microtubules and that highly insoluble complexes of τ may also play a role in normal cellular physiology

Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos

DEFF Research Database (Denmark)

Svarcova, Olga; Laurincik, Jozef; Avery, Birthe

2007-01-01

and the early 8-cell stage, and the entire nucleoplasm as well as nucleolus precursor bodies (NBBs) were prominently labelled in all late 8-cell stages. The NPBs displayed initial transformation into fibrillo-granular nucleoli. In the a-amanitin group, lack of autoradiographic labelling was seen at all...

Chromosome characterization of two varieties of Mangifera indica L.¹

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Neiva Izabel Pierozzi

2011-10-01

Full Text Available Chromosome studies were performed in two varieties of Mangifera indica L. (mango, 'IAC-140 Espadona' and in its progenitor 'Espada Stahl'. Both varieties showed 2n=40 chromosomes though the karyotype formulae were 8m + 10sm + 2sm s for 'Stahl' and 7m + 11sm + 2sm s for 'IAC-140'. The varieties showed moderate karyotype asymmetry which was estimated according to four different indices. Both varieties exhibited three chromosome pairs with silver impregnation after NOR-banding. The number of nucleoli within interphase cells varied from one, the commonest, to eight. The nucleolus persistent phenomenon was observed in more than 22% of metaphase cells of both varieties, seeing that in 'Stahl', up to two nucleoli were evidenced. This variety also showed one nucleolus in several anaphase cells. The studies were suitable for evidencing diversity at chromosomal level between these two varieties.

Multiphoton microscopic imaging of histological sections without hematoxylin and eosin staining differentiates carcinoma in situ lesion from normal oesophagus

Science.gov (United States)

Chen, Jianxin; Xu, Jian; Kang, Deyong; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Jiang, Xingshan

2013-10-01

Multiphoton microscopy (MPM) has become a powerful, important tool for tissues imaging at the molecular level. In this paper, this technique was extended to histological investigations, differentiating carcinoma in situ (CIS) lesion from normal oesophagus by imaging histological sections without hematoxylin and eosin (H&E) staining. The results show that the histology procedures of dehydration, paraffin embedding, and de-paraffinizing highlighted two photon excited fluorescence of cytoplasm and nucleolus of epithelial cell and collagen in stroma. MPM has the ability to identify the characteristics of CIS lesion including changes of squamous cells and full epithelium, identification of basement membrane, especially prominent nucleolus. The studies described here show that MPM has the potential for future retrospective studies of tumor staging by employing on histological section specimens without H&E staining.

A cell cycle role for the epigenetic factor CTCF-L/BORIS.

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Manuel Rosa-Garrido

Full Text Available CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis.

Nucleoli in human early erythroblasts (K2, K1, K1/2 cells).

Science.gov (United States)

Smetana, K; Jirásková, I; Klamová, H

2005-01-01

Human early erythroid precursors classified according to the nuclear size were studied to provide information on nucleoli in these cells using simple cytochemical procedures for demonstration of RNA and proteins of silver-stained nucleolar organizers. K2 cells with nuclear diameter larger than 13 microm and K1 cells with nuclear diameter larger than 9 microm corresponding to proerythroblasts and macroblasts (large basophilic erythroblasts) mostly possessed large irregularly shaped nucleoli with multiple fibrillar centres representing "active nucleoli". K1/2 cells with nuclear diameter smaller than 9 microm corresponding to small basophilic erythroblasts were usually characterized by the presence of micronucleoli representing "inactive nucleolar types". On the other hand, a few K1/2 cells contained large nucleoli with multiple fibrillar centres similar to those present in K2 cells and thus appeared as "microproerythroblasts". The nucleolar asynchrony expressed by the presence of large irregularly shaped nucleoli with multiple nucleoli (active nucleoli) and ring-shaped nucleoli (resting nucleoli) in one and the same nucleus of K2 or K1 cells was not exceptional and might reflect a larger resistance of these cells to negative factors influencing the erythropoiesis. The intranucleolar translocation of silver-stained nucleolus organized regions was noted in K2 cells and might indicate the premature aging of these cells without further differentiation. More studies, however, are required in this direction.

Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities

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Uozumi, Naoki; Matsumoto, Hotaru [Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto (Japan); Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp [Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto (Japan); Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan)

2016-05-27

The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycin-conjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis. -- Highlights: •Click chemistry detects O-propargyl-puromycin (OP-Puro) signals in the nucleus. •OP-Puro accumulates at PML-NBs during abortive proteasome activities. •SUMO and ubiquitin are promiscuously associated with OP-Puro at PML-NBs. •The nucleus may function in immature protein homeostasis.

Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities

International Nuclear Information System (INIS)

Uozumi, Naoki; Matsumoto, Hotaru; Saitoh, Hisato

2016-01-01

The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycin-conjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis. -- Highlights: •Click chemistry detects O-propargyl-puromycin (OP-Puro) signals in the nucleus. •OP-Puro accumulates at PML-NBs during abortive proteasome activities. •SUMO and ubiquitin are promiscuously associated with OP-Puro at PML-NBs. •The nucleus may function in immature protein homeostasis.

Quantitative autoradiography at electronic microscopy level of tobacco cells (Nicotiana tabacum L.) infected by pepper ringspot virus

International Nuclear Information System (INIS)

Lage, G.

1980-06-01

RNA replication of the pepper ringspot virus, its translocation and its association with mitochondria are studied. Some basic aspects of the research are first examined: actinomycin D (AMD) effects on parts of the nucleolus, nucleus and cytoplasm of healthy - and infected cells; comparative study between the circle method and the planimetry method to determine the cell areas; determination of the proportion between the silver grain densities of nucleulus, nucleus and cytoplasm of the cells treated with AMD; determination of the HD (Half-Distance) for the working conditions. Use of the mathemathical model proposed by NADLER gives basic information with respect to the translocation and association of the virus with the mitochondria in the host cells: in the mitochondria associated system the silver grains covering the two components are predominantly constituted by the RNA of the radioactive virus (78%); the time necessary for the RNA synthesis, the virus maturity and its translocation to the mitochondria, (checked by U-5- 3 H treatment) can be shorter than 5 hours. (M.A.) [pt

A new rapid method for isolating nucleoli.

Science.gov (United States)

Li, Zhou Fang; Lam, Yun Wah

2015-01-01

The nucleolus was one of the first subcellular organelles to be isolated from the cell. The advent of modern proteomic techniques has resulted in the identification of thousands of proteins in this organelle, and live cell imaging technology has allowed the study of the dynamics of these proteins. However, the limitations of current nucleolar isolation methods hinder the further exploration of this structure. In particular, these methods require the use of a large number of cells and tedious procedures. In this chapter we describe a new and improved nucleolar isolation method for cultured adherent cells. In this method cells are snap-frozen before direct sonication and centrifugation onto a sucrose cushion. The nucleoli can be obtained within a time as short as 20 min, and the high yield allows the use of less starting material. As a result, this method can capture rapid biochemical changes in nucleoli by freezing the cells at a precise time, hence faithfully reflecting the protein composition of nucleoli at the specified time point. This protocol will be useful for proteomic studies of dynamic events in the nucleolus and for better understanding of the biology of mammalian cells.

Histological, Topographical and Ultrastructural Organization of Different Cells Lining the Olfactory Epithelium of Red Piranha, Pygocentrus nattereri (Characiformes, Serrasalmidae

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Ghosh S. K.

2016-10-01

Full Text Available The structural characterization of the olfactory epithelium in Pygocentrus nattereri Kner, 1858 was studied with the help of light as well as scanning and transmission electron microscope. The oval shaped olfactory rosette consisted of 26–28 primary lamellae radiated from midline raphe. The olfactory epithelium of each lamella was well distributed by sensory and non-sensory epithelium. The sensory epithelium contained morphologically distinct ciliated and microvillous receptor cells, supporting cells and basal cells. The non-sensory epithelium was made up of labyrinth cells, mucous cells and stratified epithelial cells. According to TEM investigation elongated rod emerging out from dendrite end of the receptor cells in the free space. The dendrite process of microvillous receptor cells contained microvilli. The supporting cells had lobular nucleus with clearly seen electron dense nucleolus. The apex of the ciliated non-sensory cells was broad and provided with plenty of kinocilia. Basal cells provided with oval nucleus and contained small number of secretory granules. The mucous cells were restricted to the non-sensory areas and the nuclei situated basally and filled with about two-third of the vesicles. The functional significance of various cells lining the olfactory epithelium was discussed with mode of life and living of fish concerned.

Cytomorphological and autoradiografical studies on the receptacle of the strawberry (Fragaria grandiflora Eherh. in the initial stages of development

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Jan Szymański

2013-12-01

Full Text Available The volume of the cortex and pith cells of strawberries from the cultivar Talizman gradually increased until ripening, especially in the cortex. The volume of the nucleus also increased. The nuclei in the pith were smaller then in the cortex. The volume of the nucleolus increased significantly between days 1 and 5 after pollination, after which it remained unchanged. At the end of the growth period, the volume of the nucleolus in cortical and pith cells in the lower and central part of the fruit increased. Endomitotic polyploidization which was responsible for the growth of the fruit, occurred during the development of the receptacle. The intensity of 3H actinomycin binding reached a maximum in the oldest stages. This is proof for the accumulation of active DNA during mitosis. Saturation with this antibiotic occurred in the early stages of fruit development. No dependence between the volume of cells and the number of nuclei binding 3H actinomycin was observed.

The nucleolus—guardian of cellular homeostasis and genome integrity.

Science.gov (United States)

Grummt, Ingrid

2013-12-01

All organisms sense and respond to conditions that stress their homeostasis by downregulating the synthesis of rRNA and ribosome biogenesis, thus designating the nucleolus as the central hub in coordinating the cellular stress response. One of the most intriguing roles of the nucleolus, long regarded as a mere ribosome-producing factory, is its participation in monitoring cellular stress signals and transmitting them to the RNA polymerase I (Pol I) transcription machinery. As rRNA synthesis is a most energy-consuming process, switching off transcription of rRNA genes is an effective way of saving the energy required to maintain cellular homeostasis during acute stress. The Pol I transcription machinery is the key convergence point that collects and integrates a vast array of information from cellular signaling cascades to regulate ribosome production which, in turn, guides cell growth and proliferation. This review focuses on the mechanisms that link cell physiology to rDNA silencing, a prerequisite for nucleolar integrity and cell survival.

Effect of expressing an anti-HIV lectin, Griffithsin, in different plant cellular compartments

CSIR Research Space (South Africa)

Stark, T

2010-08-01

Full Text Available of subcellular targeting of GRFT in tobacco on expression levels and plant cell viability • Integration vector • Deconstructed viral vector © CSIR 2010 Slide 8 Subcellular location Nucleus Nucleolus Endoplasmic reticulum Vacuole Mitochondria Golgi...

Preparation of nano-hydroxyapatite particles with different morphology and their response to highly malignant melanoma cells in vitro

Energy Technology Data Exchange (ETDEWEB)

Li Bo [National Engineering Research Center for Biomaterial, Sichuan University, Chengdu 610064 (China); Guo Bo [National Engineering Research Center for Biomaterial, Sichuan University, Chengdu 610064 (China); West China Eye Center of Huaxi Hospital, Sichuan University, Chengdu 610064 (China); Fan Hongsong [National Engineering Research Center for Biomaterial, Sichuan University, Chengdu 610064 (China)], E-mail: leewave@126.com; Zhang Xingdong [National Engineering Research Center for Biomaterial, Sichuan University, Chengdu 610064 (China)

2008-11-15

To investigate the effects of nano-hydroxyapatite (HA) particles with different morphology on highly malignant melanoma cells, three kinds of HA particles with different morphology were synthesized and co-cultured with highly malignant melanoma cells using phosphate-buffered saline (PBS) as control. A precipitation method with or without citric acid addition as surfactant was used to produce rod-like hydroxyapatite (HA) particles with nano- and micron size, respectively, and a novel oil-in-water emulsion method was employed to prepare ellipse-like nano-HA particles. Particle morphology and size distribution of the as prepared HA powders were characterized by transmission electron microscope (TEM) and dynamic light scattering technique. The nano- and micron HA particles with different morphology were co-cultured with highly malignant melanoma cells. Immunofluorescence analysis and MTT assay were employed to evaluate morphological change of nucleolus and proliferation of tumour cells, respectively. To compare the effects of HA particles on cell response, the PBS without HA particles was used as control. The experiment results indicated that particle nanoscale effect rather than particle morphology of HA was more effective for the inhibition on highly malignant melanoma cells proliferation.

The FEAR protein Slk19 restricts Cdc14 phosphatase to the nucleus until the end of anaphase, regulating its participation in mitotic exit in Saccharomyces cerevisiae.

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Ann Marie E Faust

Full Text Available In Saccharomyces cerevisiae mitosis, the protein Slk19 plays an important role in the initial release of Cdc14 phosphatase from the nucleolus to the nucleus in early anaphase, an event that is critical for proper anaphase progression. A role for Slk19 in later mitotic stages of Cdc14 regulation, however, has not been demonstrated. While investigating the role of Slk19 post-translational modification on Cdc14 regulation, we found that a triple point mutant of SLK19, slk19(3R (three lysine-to-arginine mutations, strongly affects Cdc14 localization during late anaphase and mitotic exit. Using fluorescence live-cell microscopy, we found that, similar to slk19Δ cells, slk19(3R cells exhibit no defect in spindle stability and only a mild defect in spindle elongation dynamics. Unlike slk19Δcells, however, slk19(3R cells exhibit no defect in Cdc14 release from the nucleolus to the nucleus. Instead, slk19(3R cells are defective in the timing of Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase. This mutant has a novel phenotype: slk19(3R causes premature Cdc14 movement to the cytoplasm prior to, rather than concomitant with, spindle disassembly. One consequence of this premature Cdc14 movement is the inappropriate activation of the mitotic exit network, made evident by the fact that slk19(3R partially rescues a mutant of the mitotic exit network kinase Cdc15. In conclusion, in addition to its role in regulating Cdc14 release from the nucleolus to the nucleus, we found that Slk19 is also important for regulating Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase.

Alanine Zipper-Like Coiled-Coil Domains Are Necessary for Homotypic Dimerization of Plant GAGA-Factors in the Nucleus and Nucleolus

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Bloss, Ulrich; Hecker, Andreas; Elgass, Kirstin; Hummel, Sabine; Hahn, Achim; Caesar, Katharina; Schleifenbaum, Frank; Harter, Klaus; Berendzen, Kenneth W.

2011-01-01

GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms. PMID:21347358

Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

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Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka, E-mail: kinjo@sci.hokudai.ac.jp

2015-07-31

Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS{sup SV40}) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.

Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

International Nuclear Information System (INIS)

Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka

2015-01-01

Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS SV40 ) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS SV40 in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS SV40 formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS SV40 likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS SV40 can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus

Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

International Nuclear Information System (INIS)

Kakuk, Annamaria; Friedlaender, Elza; Vereb, Gyoergy; Lisboa, Duarte; Bagossi, Peter; Toth, Gabor; Gergely, Pal; Vereb, Gyoergy

2008-01-01

PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence 916 NFNHIHKRIRRVADKYLSG 934 comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin α/β mechanism employing importins α1 and α3 but not α5. This transport was inhibited by wheat germ agglutinin and GTPγS. The sequence 1414 SKKTNRGSQLHKYYMKRRTL 1433 , a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin α1/β or α3/β complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS

Reprogramming of round spermatids by the germinal vesicle cytoplasm in mice.

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Peng-Cheng Kong

Full Text Available The birthrate following round spermatid injection (ROSI remains low in current and evidence suggests that factors in the germinal vesicle (GV cytoplasm and certain substances in the GV such as the nucleolus might be responsible for genomic reprogramming and embryonic development. However, little is known whether the reprogramming factors in GV oocyte cytoplasm and/or nucleolus in GV are beneficial to the reprogramming of round spermatids and development of ROSI embryos. Here, round spermatids were treated with GV cytolysates and injected this round spermatid alone or co-injected with GV oocyte nucleolus into mature metaphase II oocytes. Subsequent embryonic development was assessed morphologically and by Oct4 expression in blastocysts. There was no significant difference between experimental groups at the zygote to four-cell development stages. Blastocysts derived from oocytes which were injected with cytolysate treated-round spermatid alone or co-injected with nucleoli injection yielded 63.6% and 70.3% high quality embryos, respectively; comparable to blastocysts derived by intracytoplasmic sperm injection (ICSI, but higher than these oocytes which were co-injected with lysis buffer-treated round spermatids and nucleoli or injected with the lysis buffer-treated round spermatids alone. Furthermore, the proportion of live offspring resulting from oocytes which were co-injected with cytolysate treated-round spermatids and nucleoli or injected with cytolysate treated-round spermatids alone was higher than those were injected with lysis buffer treated-round spermaids, but comparable with the ICSI group. Our results demonstrate that factors from the GV cytoplasm improve round spermatid reprogramming, and while injection of the extra nucleolus does not obviously improve reprogramming its potential contribution, although which cannot be definitively excluded. Thus, some reprogramming factors are evidently present in GV oocyte cytoplasm and could

B23/nucleophosmin interacts with bovine immunodeficiency virus Rev protein and facilitates viral replication.

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Passos-Castilho, Ana Maria; Marchand, Claude; Archambault, Denis

2018-02-01

The bovine immunodeficiency virus (BIV) Rev shuttling protein contains nuclear/nucleolar localization signals and nuclear import/export mechanisms that are novel among lentivirus Rev proteins. Several viral proteins localize to the nucleolus, which may play a role in processes that are essential to the outcome of viral replication. Although BIV Rev localizes to the nucleoli of transfected/infected cells and colocalizes with one of its major proteins, nucleophosmin (NPM1, also known as B23), the role of the nucleolus and B23 in BIV replication remains to be determined. Here, we demonstrate for the first time that BIV Rev interacts with nucleolar phosphoprotein B23 in cells. Using small interfering RNA (siRNA) technology, we show that depletion of B23 expression inhibits virus production by BIV-infected cells, indicating that B23 plays an important role in BIV replication. The interaction between Rev and B23 may represent a potential new target for the development of antiviral drugs against lentiviruses. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of Lead on Ultrastructure of Isoetes sinensis Palmer (Isoetaceae, a Critically Endangered Species in China.

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Guohua Ding

Full Text Available Isoetes sinensis Palmer (Isoetaceae is a critically endangered fern that is a marsh plant (that is an aquatic or amphibious plant in China. To evaluate damage or influence of lead (Pb on cell ultrastructure in I. sinensis, we used 2000mg·L-1 Pb(NO32 solution to treat I. sinensis for 35d, and used transmission electron microscope (TEM to observe the cell ultrastructure of leaf blades and roots of the plant. Our results indicated that Pb induced distinct changes of the organelles including chloroplast, mitochondria, nucleolus and vacuole. The level of damage organ was lower leaf > upper leaf > root The typical performance of the damages caused by lead shown that part of the nucleolus cracked; the cristae dilated, matrix vacuolized and membrane structure blurred in mitochondria; the vacuole cracked; grana lamella decreased, stroma lamella loosed, starch grains decreased, and membrane structure was disrupted in chloroplasts; Pb deposits were present on cell wall. The damages to chloroplasts and mitochondria were relatively severe, while damage to the nucleus was relatively lighter. The damage to the cell ultrastructure of leaf blades with direct contact with Pb was more severe than that without direct contact with Pb.

Dynamics and cytochemistry of oogenesis in Astyanax fasciatus (Cuvier (Teleostei, Characiformes, Characidae from Rio Sapucaí, Minas Gerais State, Brazil

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José Antônio Dias Garcia

2001-12-01

Full Text Available Oogenesis involves a set of transformations which are undergone by female germ cells These cells change into oogonias and then into mature oocytes. Sexually mature female fish were collected monthly, during one year, from the Sapucaí River, a tributary of the Rio Grande, which is part of the Furnas Reservoir in the state of Minas Gerais. During the several stages of maturation, we observed small round oogonias with a large nucleus, a single nucleolus, and weakly stained cytoplasm with eosinophilic granules. The primary oocytes showed a large basophilic nucleus, with a developed peripheral nucleolus and a reduced cytoplasm. The previtellogenic oocytes presented voluminous cytoplasm and nucleus with several small peripheral nucleoli. The oocytes underwent vitellogenesis with the development of the zona radiata and the follicle cells. Their cytochemical reactions indicated that the two layers of the zona radiata of A. fasciatus contained proteins and polysaccharides. The initially squamous follicle cells, became cuboidal. In mature oocytes, the nucleus moved toward the periphery, next to the micropyle, and the yolk granules formed by proteins, fulfilled the cytoplasm. The clear unstained vesicles are likely to be the cortical alveoli in the perivitelline region.

Nucleolar targeting of proteins by the tandem array of basic amino acid stretches identified in the RNA polymerase I-associated factor PAF49

International Nuclear Information System (INIS)

Ushijima, Ryujiro; Matsuyama, Toshifumi; Nagata, Izumi; Yamamoto, Kazuo

2008-01-01

There is accumulating evidence to indicate that the regulation of subnuclear compartmentalization plays important roles in cellular processes. The RNA polymerase I-associated factor PAF49 has been shown to accumulate in the nucleolus in growing cells, but disperse into the nucleoplasm in growth-arrested cells. Serial deletion analysis revealed that amino acids 199-338 were necessary for the nucleolar localization of PAF49. Combinatorial point mutation analysis indicated that the individual basic amino acid stretches (BS) within the central (BS1-4) and the C-terminal (BS5 and 6) regions may cooperatively confer the nucleolar localization of PAF49. Addition of the basic stretches in tandem to a heterologous protein, such as the interferon regulatory factor-3, translocated the tagged protein into the nucleolus, even in the presence of an intrinsic nuclear export sequence. Thus, tandem array of the basic amino acid stretches identified here functions as a dominant nucleolar targeting sequence

Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III–transcribed genes in budding yeast

Science.gov (United States)

Belagal, Praveen; Normand, Christophe; Shukla, Ashutosh; Wang, Renjie; Léger-Silvestre, Isabelle; Dez, Christophe; Bhargava, Purnima; Gadal, Olivier

2016-01-01

The association of RNA polymerase III (Pol III)–transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III–transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements—centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III–transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III–transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III–dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III–transcribed genes required active transcription. We conclude that the association of Pol III–transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization. PMID:27559135

Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III-transcribed genes in budding yeast.

Science.gov (United States)

Belagal, Praveen; Normand, Christophe; Shukla, Ashutosh; Wang, Renjie; Léger-Silvestre, Isabelle; Dez, Christophe; Bhargava, Purnima; Gadal, Olivier

2016-10-15

The association of RNA polymerase III (Pol III)-transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III-transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements-centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III-transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III-transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III-dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III-transcribed genes required active transcription. We conclude that the association of Pol III-transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization. © 2016 Belagal et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

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Noonan James P

2001-07-01

Full Text Available Abstract Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10 or PML (promyelocytic leukemia nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.

Development of a randomized 3D cell model for Monte Carlo microdosimetry simulations

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Douglass, Michael; Bezak, Eva; Penfold, Scott [School of Chemistry and Physics, University of Adelaide, North Terrace, Adelaide 5005, South Australia (Australia) and Department of Medical Physics, Royal Adelaide Hospital, North Terrace, Adelaide 5000, South Australia (Australia)

2012-06-15

Purpose: The objective of the current work was to develop an algorithm for growing a macroscopic tumor volume from individual randomized quasi-realistic cells. The major physical and chemical components of the cell need to be modeled. It is intended to import the tumor volume into GEANT4 (and potentially other Monte Carlo packages) to simulate ionization events within the cell regions. Methods: A MATLAB Copyright-Sign code was developed to produce a tumor coordinate system consisting of individual ellipsoidal cells randomized in their spatial coordinates, sizes, and rotations. An eigenvalue method using a mathematical equation to represent individual cells was used to detect overlapping cells. GEANT4 code was then developed to import the coordinate system into GEANT4 and populate it with individual cells of varying sizes and composed of the membrane, cytoplasm, reticulum, nucleus, and nucleolus. Each region is composed of chemically realistic materials. Results: The in-house developed MATLAB Copyright-Sign code was able to grow semi-realistic cell distributions ({approx}2 Multiplication-Sign 10{sup 8} cells in 1 cm{sup 3}) in under 36 h. The cell distribution can be used in any number of Monte Carlo particle tracking toolkits including GEANT4, which has been demonstrated in this work. Conclusions: Using the cell distribution and GEANT4, the authors were able to simulate ionization events in the individual cell components resulting from 80 keV gamma radiation (the code is applicable to other particles and a wide range of energies). This virtual microdosimetry tool will allow for a more complete picture of cell damage to be developed.

Gonadal Morphogenesis and Sex Differentiation in Intraovarian Embryos of the Viviparous Fish Zoarces viviparus (Teleostei, Perciformes, Zoarcidae): A Histological and Ultrastructural Study

DEFF Research Database (Denmark)

Rasmussen, Tina H.; Jespersen, Åse; Korsgaard, Bodil

2006-01-01

undifferentiated paired bilobed gonad contains primordial germ cells. In the female embryos, ovarian differentiation, initiated 14 days posthatch (dph), is characterized by the initial formation of the endoovarian cavity of the single ovary as well as by the presence of some early meiotic oocytes in a chromatin-nucleolus...

17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha.

Science.gov (United States)

Batnasan, Enkhzaya; Wang, Ruoxi; Wen, Jitao; Ke, Yueshuang; Li, Xiaoxue; Bohio, Ameer Ali; Zeng, Xianlu; Huo, Hongliang; Han, Liping; Boldogh, Istvan; Ba, Xueqing

2015-01-05

Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H2O2). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H2O2-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Subcellular localization of casein kinase I

DEFF Research Database (Denmark)

Grankowski, N; Issinger, O G

1990-01-01

An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus....

STK35L1 associates with nuclear actin and regulates cell cycle and migration of endothelial cells.

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Pankaj Goyal

Full Text Available BACKGROUND: Migration and proliferation of vascular endothelial cells are essential for repair of injured endothelium and angiogenesis. Cyclins, cyclin-dependent kinases (CDKs, and cyclin-dependent kinase inhibitors play an important role in vascular tissue injury and wound healing. Previous studies suggest a link between the cell cycle and cell migration: cells present in the G(1 phase have the highest potential to migrate. The molecular mechanism linking these two processes is not understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we explored the function of STK35L1, a novel Ser/Thr kinase, localized in the nucleus and nucleolus of endothelial cells. Molecular biological analysis identified a bipartite nuclear localization signal, and nucleolar localization sequences in the N-terminal part of STK35L1. Nuclear actin was identified as a novel binding partner of STK35L1. A class III PDZ binding domains motif was identified in STK35L1 that mediated its interaction with actin. Depletion of STK35L1 by siRNA lead to an accelerated G(1 to S phase transition after serum-stimulation of endothelial cells indicating an inhibitory role of the kinase in G(1 to S phase progression. Cell cycle specific genes array analysis revealed that one gene was prominently downregulated (8.8 fold in STK35L1 silenced cells: CDKN2A alpha transcript, which codes for p16(INK4a leading to G(1 arrest by inhibition of CDK4/6. Moreover in endothelial cells seeded on Matrigel, STK35L1 expression was rapidly upregulated, and silencing of STK35L1 drastically inhibited endothelial sprouting that is required for angiogenesis. Furthermore, STK35L1 depletion profoundly impaired endothelial cell migration in two wound healing assays. CONCLUSION/SIGNIFICANCE: The results indicate that by regulating CDKN2A and inhibiting G1- to S-phase transition STK35L1 may act as a central kinase linking the cell cycle and migration of endothelial cells. The interaction of STK35L1 with nuclear

Phospho-eNOS Ser-1176 is associated with the nucleoli and the Golgi complex in C6 rat glioma cells.

Science.gov (United States)

Klinz, Franz-Josef; Herberg, Natalie; Arnhold, Stefan; Addicks, Klaus; Bloch, Wilhelm

2007-06-29

Enzymatic activity of endothelial nitric oxide synthase (eNOS) is controlled by posttranslational modifications, protein-protein interactions, and subcellular localization. For example, N-terminal fatty acid modifications target eNOS to the Golgi complex where it becomes phosphorylated. We show here by immunofluorescence analysis that phospho-eNOS Ser-1176 is enriched in the perinuclear region of interphase C6 rat glioma cells. Confocal double immunofluorescence microscopy with the Golgi marker protein 58K revealed that phospho-eNOS Ser-1176 is associated with the Golgi complex. Surprisingly, we observed several spots in the nucleus of C6 cells that were positive for phospho-eNOS Ser-1176. Confocal double immunofluorescence analysis with the nucleolus marker protein fibrillarin revealed that within the nucleus phospho-eNOS Ser-1176 is exclusively associated with the nucleoli. It is known that in mitotic cells nucleoli are lost during prophase and rebuild during telophase. In agreement with this, we find no nucleoli-like distribution of phospho-eNOS Ser-1176 in metaphase and anaphase C6 glioma cells. Our finding that phospho-eNOS Ser-1176 is selectively associated with the nucleoli points to a so far unknown role for eNOS in interphase glioma cells.

The Role of a Novel Myosin Isoform in Prostate Cancer Metastasis

Science.gov (United States)

2013-10-01

2013 Accepted 14 February 2013 Available online 21 February 2013 Keywords: Myosin IC Isoforms Nucleolar localization signal Nucleolus Nucleus RNA...polymerase I Fibrillarinnt matter & 2013 Elsevier 1016/j.yexcr.2013.02.008 S, nucleolar localization ; No, nucleolus ; N, nucle bovine serum albumin; S...the nucleus, and the nucleolus . In the cytoplasm, myosin IC associ- ates with membranes and is involved in vesicle transport of membrane proteins [2

Suppression of tumor growth and angiogenesis by a specific antagonist of the cell-surface expressed nucleolin.

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Damien Destouches

Full Text Available BACKGROUND: Emerging evidences suggest that nucleolin expressed on the cell surface is implicated in growth of tumor cells and angiogenesis. Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, here we show that the growth of tumor cells and angiogenesis are suppressed in various in vitro and in vivo experimental models. HB-19 inhibited colony formation in soft agar of tumor cell lines, impaired migration of endothelial cells and formation of capillary-like structures in collagen gel, and reduced blood vessel branching in the chick embryo chorioallantoic membrane. In athymic nude mice, HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in nude mice, and in some cases eliminated measurable tumors while displaying no toxicity to normal tissue. This potent antitumoral effect is attributed to the direct inhibitory action of HB-19 on both tumor and endothelial cells by blocking and down regulating surface nucleolin, but without any apparent effect on nucleolar nucleolin. CONCLUSION/SIGNIFICANCE: Our results illustrate the dual inhibitory action of HB-19 on the tumor development and the neovascularization process, thus validating the cell-surface expressed nucleolin as a strategic target for an effective cancer drug. Consequently, the HB-19 pseudopeptide provides a unique candidate to consider for innovative cancer therapy.

[The morphofunctional state of the bone marrow in lead and zinc intoxication].

Science.gov (United States)

Vladimtseva, T M; Pashkevich, I A; Salmina, A B

2006-01-01

The nucleolus is a compulsory nuclear structure of all cells of eukaryotes. The quantitative and qualitative characteristics of nuclei show the functional activity of a cell, the rate of its synthesis of RNA and portents, and its metabolic state. Heavy metals (zinc chloride and lead acetate) were comparatively investigated for their effects on the nucleolar apparatus of bone marrow cells in in vivo experiments. Zinc chloride and lead acetate were ascertained to damage the nucleolar apparatus of cells, thus decreasing their transcriptional activity or irreversibly damaging them.

Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

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Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C

2014-01-01

as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA...

Identification of an evolutionary conserved SURF-6 domain in a family of nucleolar proteins extending from human to yeast

International Nuclear Information System (INIS)

Polzikov, Mikhail; Zatsepina, Olga; Magoulas, Charalambos

2005-01-01

The mammalian SURF-6 protein is localized in the nucleolus, yet its function remains elusive in the recently characterized nucleolar proteome. We discovered by searching the Protein families database that a unique evolutionary conserved SURF-6 domain is present in the carboxy-terminal of a novel family of eukaryotic proteins extending from human to yeast. By using the enhanced green fluorescent protein as a fusion protein marker in mammalian cells, we show that proteins from distantly related taxonomic groups containing the SURF-6 domain are localized in the nucleolus. Deletion sequence analysis shows that multiple regions of the SURF-6 protein are capable of nucleolar targeting independently of the evolutionary conserved domain. We identified that the Saccharomyces cerevisiae member of the SURF-6 family, named rrp14 or ykl082c, has been categorized in yeast databases to interact with proteins involved in ribosomal biogenesis and cell polarity. These results classify SURF-6 as a new family of nucleolar proteins in the eukaryotic kingdom and point out that SURF-6 has a distinct domain within the known nucleolar proteome that may mediate complex protein-protein interactions for analogous processes between yeast and mammalian cells

Distribution of elements in rat peripheral axons and nerve cell bodies determined by x-ray microprobe analysis

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LoPachin, R.M. Jr.; Lowery, J.; Eichberg, J.; Kirkpatrick, J.B.; Cartwright, J. Jr.; Saubermann, A.J.

1988-09-01

X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.

Quantitative Immunofluorescence Analysis of Nucleolus-Associated Chromatin.

Science.gov (United States)

Dillinger, Stefan; Németh, Attila

2016-01-01

The nuclear distribution of eu- and heterochromatin is nonrandom, heterogeneous, and dynamic, which is mirrored by specific spatiotemporal arrangements of histone posttranslational modifications (PTMs). Here we describe a semiautomated method for the analysis of histone PTM localization patterns within the mammalian nucleus using confocal laser scanning microscope images of fixed, immunofluorescence stained cells as data source. The ImageJ-based process includes the segmentation of the nucleus, furthermore measurements of total fluorescence intensities, the heterogeneity of the staining, and the frequency of the brightest pixels in the region of interest (ROI). In the presented image analysis pipeline, the perinucleolar chromatin is selected as primary ROI, and the nuclear periphery as secondary ROI.

Comparison of the karyotypes ofPsathyrostachys juncea andP. huashanica (Poaceae) studied by banding techniques

DEFF Research Database (Denmark)

Linde-Laursen, Ib; Bothmer, R. von

1986-01-01

. The patterns of both taxa are polymorphic, supporting that both taxa are outbreeders. The karyotypic characters suggest that P. juncea is more closely related to P. fragilis than either is to P. huashanica. N-banding stains weakly. Silver nitrate staining demonstrates that nucleolus organizers of both species...... have different nucleolus forming capacities. The presence of micronucleoli suggests that both species have an extra unidentified chromosome with nucleolus forming capacity....

Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.

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Fred E Indig

Full Text Available The Werner protein (WRNp, a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL, an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA.These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes.

Ultrastructural changes in nucleoli and fibrillar centers under the effect of local ultraviolet microbeam irradiation of interphase culture cells

International Nuclear Information System (INIS)

Zatsepina, O.V.; Voronkova, L.N.; Sakharov, V.N.; Chentsov, Y.S.

1989-01-01

As shown previously, ultraviolet (uv) microbeam irradiation of one of the two mature nucleoli within an interphase cell nucleus causes significant diminution and inactivation of the irradiated nucleolus and compensatory growth and activation of the nonirradiated one. In the present work we describe the results of an ultrastructural study of this phenomenon. The changes in the nucleoli were examined by means of complete series of ultrathin sections obtained from seven irradiated pig kidney cells. The compensatory hypertrophy of the nonirradiated nucleoli is shown to be accompanied by a nearly twofold increase in the number of fibrillar centers (FCs) and by a decrease in their linear dimensions compared with the control cells of the same ploidy. In the degraded nucleoli the number of FCs decreases, but their dimensions increase. Ultraviolet microbeam irradiation causes dramatic diminution of the dense fibrillar component within the irradiated nucleoli as well. The nucleolar capacity for compensatory hypertrophy indicates that in addition to active ribosomal genes, mature nucleoli also contain silent genes capable of being activated under extreme conditions to sustain the required level of rRNA synthesis. It is assumed that activation of latent ribosomal genes is accompanied by FC fragmentation without a considerable increase in their total volume per cell

The Potential Mechanism of ZFX Involvement in the Cell Growth

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Mahboube Ganji arjenaki

2016-04-01

Full Text Available Background:The zinc-finger X linked (ZFX gene encodes a transcription factor that acts as a regulator of self-renewal of stem cells. Due to the role of ZFX in cell growth, understanding ZFX protein-protein interactions helps to clarify its proper biological functions in signaling pathways. The aim of this study is to define ZFX protein-protein interactions and the role of ZFX in cell growth. Materials and Methods: The PIPs output includes three interacting proteins with ZFX: eukaryotic translation initiation factor 3 subunit I(EIF3I, eukaryotic translation initiation factor 3 subunit G(EIF3G and protein nuclear pore and COPII coat complex component homolog isoform 3 (SEC13L1. Results: As a cargo and transmembrane protein interacting with Sec13,eIF3I and eIF3G, ZFX mediates cargo sorting in COPII vesicles at ER exit sites. While traveling to cis-Golgi, eIF3I is phosphorylated by the mechanistic target of rapamycin (mTOR. Proteins transport by COPI vesicles to the nucleusouter site layer containing SEC13 via the contribution of microtubules. EIF3G and eIF3I interact with coatomer protein complex subunit beta 2 (COPB2 that helps to enclose ZFX in COPI vesicle. ZFX and eIF3G enter nucleolus where activation of transcription from pre rDNA genes occurs. Conclusion:We proposed a model in which ZFX is involved in cell growth by promoting the transcription of rDNA genes.

Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses

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Wahyu eWulan

2015-06-01

Full Text Available Most viruses with non-segmented single stranded RNA genomes complete their life cycle in the cytoplasm of infected cells. However, despite undergoing replication in the cytoplasm, the structural proteins of some of these RNA viruses localize to the nucleus at specific times in the virus life cycle, primarily early in infection. Limited evidence suggests that this enhances successful viral replication by interfering with or inhibiting the host antiviral response. Nucleocapsid proteins of RNA viruses have a well-established, essential cytoplasmic role in virus replication and assembly. Intriguingly, nucleocapsid proteins of some RNA viruses also localize to the nucleus/nucleolus of infected cells. Their nuclear function is less well understood although significant advances have been made in recent years. This review will focus on the nucleocapsid protein of cytoplasmic enveloped RNA viruses, including their localization to the nucleus/nucleolus and function therein. A greater understanding of the nuclear localization of nucleocapsid proteins has the potential to enhance therapeutic strategies as it can be a target for the development of live-attenuated vaccines or antiviral drugs.

An extracellular DNA mediated bystander effect produced from low dose irradiated endothelial cells

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Ermakov, Aleksei V., E-mail: avePlato@mail.ru [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Konkova, Marina S.; Kostyuk, Svetlana V.; Smirnova, Tatiana D.; Malinovskaya, Elena M.; Efremova, Liudmila V.; Veiko, Natalya N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

2011-07-01

The human umbilical vein endothelial cells culture was exposed to X-ray radiation in a low dose of 10 cGy. The fragments of extracellular genomic DNA (ecDNA{sup R}) were isolated from the culture medium after the short-term incubation. A culture medium of unirradiated endothelial cells was then supplemented with ecDNA{sup R}, followed by analysing the cells along the series of parameters (bystander effect). The exposed cells and bystander endotheliocytes showed similar response to low doses: approximation of the 1q12 loci of chromosome 1 and their transposition into the cellular nucleus, change in shape of the endotheliocytic nucleus, activation of the nucleolus organizing regions (NORs), actin polymerization, and an elevated level of DNA double-stranded breaks. Following blockade of TLR9 receptors with oligonucleotide-inhibitor or chloroquine in the bystander cells these effects - except of activation of NORs - on exposure to ecDNA{sup R} disappeared, with no bystander response thus observed. The presence of the radiation-induced apoptosis in the bystander effect being studied suggests a possibility for radiation-modified ecDNA fragments (i.e., stress signaling factors) to be released into the culture medium, whereas inhibition of TLR9 suggests the binding these ligands to the recipient cells. A similar DNA-signaling pathway in the bystander effect we previously described for human lymphocytes. Integrity of data makes it possible to suppose that a similar signaling mechanism which we demonstrated for lymphocytes (humoral system) might also be mediated in a monolayer culture of cells (cellular tissue) after the development of the bystander effect in them and transfer of stress signaling factors (ecDNA{sup R}) through the culture medium.

An extracellular DNA mediated bystander effect produced from low dose irradiated endothelial cells

International Nuclear Information System (INIS)

Ermakov, Aleksei V.; Konkova, Marina S.; Kostyuk, Svetlana V.; Smirnova, Tatiana D.; Malinovskaya, Elena M.; Efremova, Liudmila V.; Veiko, Natalya N.

2011-01-01

The human umbilical vein endothelial cells culture was exposed to X-ray radiation in a low dose of 10 cGy. The fragments of extracellular genomic DNA (ecDNA R ) were isolated from the culture medium after the short-term incubation. A culture medium of unirradiated endothelial cells was then supplemented with ecDNA R , followed by analysing the cells along the series of parameters (bystander effect). The exposed cells and bystander endotheliocytes showed similar response to low doses: approximation of the 1q12 loci of chromosome 1 and their transposition into the cellular nucleus, change in shape of the endotheliocytic nucleus, activation of the nucleolus organizing regions (NORs), actin polymerization, and an elevated level of DNA double-stranded breaks. Following blockade of TLR9 receptors with oligonucleotide-inhibitor or chloroquine in the bystander cells these effects - except of activation of NORs - on exposure to ecDNA R disappeared, with no bystander response thus observed. The presence of the radiation-induced apoptosis in the bystander effect being studied suggests a possibility for radiation-modified ecDNA fragments (i.e., stress signaling factors) to be released into the culture medium, whereas inhibition of TLR9 suggests the binding these ligands to the recipient cells. A similar DNA-signaling pathway in the bystander effect we previously described for human lymphocytes. Integrity of data makes it possible to suppose that a similar signaling mechanism which we demonstrated for lymphocytes (humoral system) might also be mediated in a monolayer culture of cells (cellular tissue) after the development of the bystander effect in them and transfer of stress signaling factors (ecDNA R ) through the culture medium.

Regulation of c-Myc mRNA by L11 in Response to UV and Gamma Irradiation

Science.gov (United States)

2014-12-01

the nucleolus fol- lowed by their transport into the cytoplasm (50). This process requires coordinated transcription catalyzed by all three RNA...these RPs, including L11, are released from the nucleolus or from intact ribosomes to suppress MDM2 (68). However, whether L11 suppresses c-Myc in...centrifugation. For isolation of the nucleolus fraction, the nuclear pellets were resuspended in buffer S1 containing 0.25 M sucrose and 10 mM MgCl2, layered over

ORF Alignment: NC_003074 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available in CNBN0180 [Cryptococcus neoformans var. ... neoformans B-3501A] gb|AAW47011.1| nucleolus protein, ... ... ... putative [Cryptococcus neoformans var. neoformans JEC21] ... ref|XP_568528.1| nucleolus pro

Documentation of normal and leukemic myelopoietic progenitor cells with high-resolution phase-contrast time-lapse cinematography.

Science.gov (United States)

Boll, I T

2001-08-01

The high-resolution phase-contrast, time-lapse cinematography using oil immersion lenses and 16-mm film demonstrates the kinetic cell events as maturation, locomotion, mitosis, and apoptosis of cells cultivated at 37 degrees C for up to 10 days. 0.5 v/v frozen-thawed sera with presumably high cytokine concentrations were added to the plasma or agar clot. Vital progenitor cells from human bone marrow and blood have a large, bright, unstructured nucleus with a large nucleolus and a narrow rim of cytoplasm (nuclear/cytoplasmic volume ratio = 0.7). Their nuclei are 6-14 micrometer in diameter and double their volume within 8 h. Many (70%) move at a mean speed of 2 micrometer/min, and many (30%) multiply with alpha-2alpha mitoses, generating progenitor cell families. Various disturbances during the course of mitosis lead to the formation of polyploid cells, thereby yielding the megakaryocytic cell line. Some of the progenitor cells undergo asymmetric alpha-alphan mitoses: One of the two initially identical daughter cells remains a progenitor cell in the morphological sense, whereas the other daughter cell - depending on the size of its mother cell - matures in the same culture medium to form a granulocytopoietic, monocytopoietic or erythrocytopoietic cell line. - In acute myeloid leukemias (AML), the blasts and their nuclei are slightly larger than the corresponding progenitor cells and move faster (5 micrometer/min). Symmetric alpha-2alpha mitoses permit unlimited multiplication of the leukemic blasts if contact with cytotoxic lymphocytes does not render them apoptotic. This results in more stromal cells than normal. Granulocytopenia, monocytopenia, and anemia occur due to the genetic impairment of signaling control for asymmetric alpha-alphan mitoses, and thrombocytopenia occurs due to the reduction in polyploidization. Copyright 2001 S. Karger GmbH, Freiburg

Yeast Interacting Proteins Database: YOL006C, YMR233W [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available fusion protein localizes to the cytoplasm, nucleus and nucleolus Rows with this prey as prey (1) Rows with t...on protein localizes to the cytoplasm, nucleus and nucleolus Rows with this prey

Function of oval cells in hepatocellular carcinoma in rats.

Science.gov (United States)

Fang, Chi-Hua; Gong, Jia-Qing; Zhang, Wei

2004-09-01

To study oval cells' pathological characteristics and relationship with the occurrence of hepatocellular carcinoma (HCC); to observe the form and structural characteristics of oval cells; to explore the expression characteristics of C-kit, PCNA mRNA and c-myc gene during the occurrence and development of HCC and the effect of ulinastatin (UTI) on C-kit and PCNA expression. One hundred and twenty-five SD rats fed on 3,3'-diaminobenzidine (DAB) to construct HCC models were divided into control group, cancer-inducing group and UTI intervention group. In each group, rat liver samples were collected at weeks 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 respectively to study pathological distribution characteristics of oval cells in the process of carcinogenesis under optical microscope. Oval cells were separated by the methods of improved density gradient centrifugation and their structural characteristics were observed under optical microscope and electronic microscope respectively; the oval cells expressing C-kit and PCNA in the collected samples were observed by the methods of immunohistochemistry and image analysis and the expression of c-myc mRNA was also detected by reverse transcription polymerase chain reaction (RT-PCR). Oval cells proliferated firstly in the portal area then gradually migrated into hepatic parenchyma in the inducing group and intervention group. The oval cells distributed inside and outside the carcinoma nodes. The oval cells presented the characteristics of undifferentiated cells: a high ratio of nucleolus and cellular plasm and obvious nucleoli, rare organelle in plasm. Only a few mitochondria and endoplasmic reticulum and some villus-like apophysis on surface of cells could be seen. Cells stained with C-kit and PCNA antibody were mainly oval cells distributed in the portal area. The expression of c-myc mRNA increased with the progression of HCC. However, in the intervention group, UTI could retard its increase. Oval cells work throughout

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

Science.gov (United States)

Morovic, Martin; Strejcek, Frantisek; Nakagawa, Shoma; Deshmukh, Rahul S; Murin, Matej; Benc, Michal; Fulka, Helena; Kyogoku, Hirohisa; Pendovski, Lazo; Fulka, Josef; Laurincik, Jozef

2017-12-01

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

Interdependence of Pes1, Bop1, and WDR12 controls nucleolar localization and assembly of the PeBoW complex required for maturation of the 60S ribosomal subunit.

Science.gov (United States)

Rohrmoser, Michaela; Hölzel, Michael; Grimm, Thomas; Malamoussi, Anastassia; Harasim, Thomas; Orban, Mathias; Pfisterer, Iris; Gruber-Eber, Anita; Kremmer, Elisabeth; Eick, Dirk

2007-05-01

The PeBoW complex is essential for cell proliferation and maturation of the large ribosomal subunit in mammalian cells. Here we examined the role of PeBoW-specific proteins Pes1, Bop1, and WDR12 in complex assembly and stability, nucleolar transport, and pre-ribosome association. Recombinant expression of the three subunits is sufficient for complex formation. The stability of all three subunits strongly increases upon incorporation into the complex. Only overexpression of Bop1 inhibits cell proliferation and rRNA processing, and its negative effects could be rescued by coexpression of WDR12, but not Pes1. Elevated levels of Bop1 induce Bop1/WDR12 and Bop1/Pes1 subcomplexes. Knockdown of Bop1 abolishes the copurification of Pes1 with WDR12, demonstrating Bop1 as the integral component of the complex. Overexpressed Bop1 substitutes for endogenous Bop1 in PeBoW complex assembly, leading to the instability of endogenous Bop1. Finally, indirect immunofluorescence, cell fractionation, and sucrose gradient centrifugation experiments indicate that transport of Bop1 from the cytoplasm to the nucleolus is Pes1 dependent, while Pes1 can migrate to the nucleolus and bind to preribosomal particles independently of Bop1. We conclude that the assembly and integrity of the PeBoW complex are highly sensitive to changes in Bop1 protein levels.

Nucleolar proteins change in altered gravity

Science.gov (United States)

Sobol, M. A.; Kordyum, E. L.; Gonzalez-Camacho, F.; Medina, F. J.

Discovery of gravisensitivity of cells no specified to gravity perception focused continuous attention on an elucidation of mechanisms involved in altered gravity effects at the different levels of cellular organization A nucleolus is the nuclear domain in which the major portion of ribosome biogenesis takes place This is a basic process for cell vitality beginning with the transcription of rDNA followed by processing newly synthesized pre-rRNA molecules A wide range of nucleolar proteins plays a highly significant role in all stages of biosynthesis of ribosomes Different steps of ribosome biogenesis should respond to various external factors affecting generally the cell metabolism Nevertheless a nucleolus remains not enough studied under the influence of altered environmental conditions For this reason we studied root apices from 2-day old Lepidium sativum seedlings germinated and grown under slow horizontal clinorotation and stationary conditions in darkness The extraction of cell nuclei followed by sequential fractionation of nuclear proteins according to their solubility in buffers of increasing ionic strength was carried out This procedure gave rise to 5 distinct fractions We analyzed nuclear subproteomes of the most soluble fraction called S2 It is actually a functionally significant fraction consisting of ribonucleoproteins actively engaged in pre-rRNA synthesis and processing 2D-electrophoresis of S2 fraction proteins was carried out The gels were silver stained and stained gels were scanned and analyzed

Direct action of the X-ray on the submandibular and sublingual glands. Histologic and histochemical study in rats

International Nuclear Information System (INIS)

Santos-Pinto, M.C. dos; Martinelli, C.; Santos-Pinto, R. dos

1991-01-01

The submandibular and sublingual glands of rats are surgically exposed and irradiated by X-ray. A simulated group with surgical exposition of the glands is performed. In the irradiated submandibular glands occurred atrophy, architectural disorder and degenerative processes. RNA reduction in the nucleus, nucleolus and cytoplasm of the acini cells and a decrease of protein synthesis are described. (M.A.C.)

Yeast Interacting Proteins Database: YER127W, YLR423C [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available to inhibited pre-rRNA processing and reduced polysome levels; localizes primarily to the nucleolus Rows with...etion leads to inhibited pre-rRNA processing and reduced polysome levels; localizes primarily to the nucleolus

Nucleoli from growing oocytes inhibit the maturation of enucleolated, full-grown oocytes in the pig.

Science.gov (United States)

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi; Fulka, Josef

2011-06-01

In mammals, the nucleolus of full-grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full-grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non-treated or actinomycin D-treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re-injection of nucleoli from growing oocytes (23%), but not when nucleoli from full-grown oocytes were re-injected into enucleolated, growing oocytes (49%). When enucleolated, full-grown oocytes were injected with nucleoli from growing or full-grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full-grown oocytes injected with nucleoli from full-grown oocytes matured to metaphase II (56%), whereas injection with growing-oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing-oocyte nucleolus is involved in the oocyte's meiotic arrest, and that the full-grown oocyte nucleolus has lost the ability. Copyright © 2011 Wiley-Liss, Inc.

Reference: 607 [Arabidopsis Phenome Database[Archive

Lifescience Database Archive (English)

Full Text Available and function in mitosis. Our analysis of AtSYN3 shows that it localizes primarily in the nucleolus of both m...dopsis cohesin protein SYN3 localizes to the nucleolus and is essential for gamet

RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study

Energy Technology Data Exchange (ETDEWEB)

Motlik, J. (Institute of Animal Physiology and Genetics, Czechoslovak Academy of Sciences, CS, Libechov); Kopecny, V.; Travnik, P. (Faculty of Medecine, J.E. Purkyne University, Brno (Czechoslovakia)); Pivko, J. (Animal Production Research Institute, Nitra (Czechoslovakia))

1984-01-01

RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5-0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6-2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5-6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver-positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5-6 mm.

Ribosomal RNA and nucleolar proteins from the oocyte are to some degree used for embryonic nucleolar formation in cattle and pig

DEFF Research Database (Denmark)

Maddox-Hyttel, Poul; Svarcova, Olga; Laurincik, Josef

2007-01-01

The nucleolus is the site of ribosomal RNA (rRNA) and ribosome production. In the bovine primordial follicle oocyte, this organelle is inactive, but in the secondary follicle an active fibrillo-granular nucleolus develops and proteins involved in rDNA transcription (topoisomerase I, RNA polymerase...... I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) localize to it. At the end of the oocyte growth phase, the nucleolus is inactivated again and transforms into a solid remnant. The nucleolar remnant is dissolved when meiosis is resumed. Upon...... fertilization, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities are engaged in the re-establishment of fibrilo-granular nucleoli at the major activation of the embryonic genome. This nucleolar formation can...

RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study

International Nuclear Information System (INIS)

Motlik, J.; Pivko, J.

1984-01-01

RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5-0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6-2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5-6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver-positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5-6 mm

Early Contact Stage of Apoptosis: Its Morphological Features and Function

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Etheri Mikadze

2006-01-01

Full Text Available Apoptosis has been a biological phenomenon of intense interest for 20 years, but the earlier morphological features of apoptosis have not been determined hitherto. Using the methods of semi- and ultrathin sections, the livers of intact embryos and young rats have been studied under the effect of cycloheximide to determine morphological features of an early stage of apoptosis. It is discovered that both in hepatoblasts and hepatocytes, apoptosis, besides the well-known stages, also includes an early contact stage, distinguishing features of which are agglutination of bound ribosomes (breaking of translation, elimination of the nucleolus, reduction of free polysomes (and in hepatocytes, reduction of cisterns of rough endoplasmic reticulum, formation of cytoplasmic excrescences, and cell shape changes. The early stage of apoptosis is characterized by close contact with neighboring cells. At a certain phase of the contact stage of apoptosis, the nucleolus reappears in the nucleus and the number of free polysomes in the cytoplasm increases, which suggests the renewal of synthesis of new RNA and proteins. Close contact of differentiating and mitotic hepatoblasts with apoptotic cells indicates a certain functional relationship between these cells that is realized not only by micropinocytosis, but through gap junctions as well. We assume that the apoptotic cell, besides proteolytic products, can contain newly synthesized, low-molecular substances, the relocation of which from apoptotic to neighboring cells may contribute to both functional activity and proliferation of adjacent hepatoblasts and, therefore, the function of apoptosis may not be limited only to the elimination of harmful, damaged, and unwanted cells.

Yeast Interacting Proteins Database: YER127W, YDR299W [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available to inhibited pre-rRNA processing and reduced polysome levels; localizes primarily to the nucleolus Rows with...of 18S rRNA; depletion leads to inhibited pre-rRNA processing and reduced polysome levels; localizes primarily to the nucleolus

Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

Energy Technology Data Exchange (ETDEWEB)

Ostrup, Olga, E-mail: osvarcova@gmail.com [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway); Hyttel, Poul; Klaerke, Dan A. [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Collas, Philippe, E-mail: philc@medisin.uio.no [Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway)

2011-09-02

Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

Regulation of TRAIL-Medicated Apoptosis in Prostate Cancer by Overexpression of XIAP

Science.gov (United States)

2005-01-01

to a composite element, and YY1 and E1A cooperate to amplify p53 promoter activity. Mol Cell Biol 1996, 16:5933-5945 41. Guo J, Casolaro V, Seto E...Lian JB, Bushmeyer S, Seto E, Atchison ML, Penman S, van Wijnen AJ, Stein GS: Targeting of the YY1 transcription factor to the nucleolus and the...Multiple Cell Types and Tissues. The Journal of Biological Chemistry . 277: 9637-9640. 12. Lorch AS, Foust III R, Gow A, Arkoviz A, Salzman AL, Szabo

SmgGDS is a transient nucleolar protein that protects cells from nucleolar stress and promotes the cell cycle by regulating DREAM complex gene expression.

Science.gov (United States)

Gonyo, P; Bergom, C; Brandt, A C; Tsaih, S-W; Sun, Y; Bigley, T M; Lorimer, E L; Terhune, S S; Rui, H; Flister, M J; Long, R M; Williams, C L

2017-12-14

The chaperone protein and guanine nucleotide exchange factor SmgGDS (RAP1GDS1) is a key promoter of cancer cell proliferation and tumorigenesis. SmgGDS undergoes nucleocytoplasmic shuttling, suggesting that it has both cytoplasmic and nuclear functions that promote cancer. Previous studies indicate that SmgGDS binds cytoplasmic small GTPases and promotes their trafficking to the plasma membrane. In contrast, little is known about the functions of SmgGDS in the nucleus, or how these nuclear functions might benefit cancer cells. Here we show unique nuclear localization and regulation of gene transcription pathways by SmgGDS. Strikingly, SmgGDS depletion significantly reduces expression of over 600 gene products that are targets of the DREAM complex, which is a transcription factor complex that regulates expression of proteins controlling the cell cycle. The cell cycle regulators E2F1, MYC, MYBL2 (B-Myb) and FOXM1 are among the DREAM targets that are diminished by SmgGDS depletion. E2F1 is well known to promote G1 cell cycle progression, and the loss of E2F1 in SmgGDS-depleted cells provides an explanation for previous reports that SmgGDS depletion characteristically causes a G1 cell cycle arrest. We show that SmgGDS localizes in nucleoli, and that RNAi-mediated depletion of SmgGDS in cancer cells disrupts nucleolar morphology, signifying nucleolar stress. We show that nucleolar SmgGDS interacts with the RNA polymerase I transcription factor upstream binding factor (UBF). The RNAi-mediated depletion of UBF diminishes nucleolar localization of SmgGDS and promotes proteasome-mediated degradation of SmgGDS, indicating that nucleolar sequestration of SmgGDS by UBF stabilizes SmgGDS protein. The ability of SmgGDS to interact with UBF and localize in the nucleolus is diminished by expressing DiRas1 or DiRas2, which are small GTPases that bind SmgGDS and act as tumor suppressors. Taken together, our results support a novel nuclear role for SmgGDS in protecting malignant

Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

International Nuclear Information System (INIS)

Siyam, Arwa; Wang, Suzhen; Qin, Chunlin; Mues, Gabriele; Stevens, Roy; D’Souza, Rena N.; Lu, Yongbo

2012-01-01

Highlights: ► Nuclear localization of DMP1 in various cell lines. ► Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. ► Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

NCBI nr-aa BLAST: CBRC-DDIS-03-0047 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DDIS-03-0047 ref|XP_001383670.1| nonribosomal protein of the nucleolus and coi...led bodies [Pichia stipitis CBS 6054] gb|ABN65641.1| nonribosomal protein of the nucleolus and coiled bodies [Pichia stipitis CBS 6054] XP_001383670.1 3e-04 38% ...

Fragmentary model of exchange interactions

CERN Document Server

Kotov, V M

2000-01-01

This article makes attempt to refusal from using neutrino for explanation continuous distribution of beta particle energy by conversion to characteristic exchange interaction particles in nucleolus. It is taking formulation for nuclear position with many different fragments. It is computing half-value period of spontaneous fission of heavy nucleolus. (author)

The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells

Science.gov (United States)

Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan

2011-01-01

The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their ‘fresh’ host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its ‘fresh’ host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA. PMID:21729006

G2E3 is a nucleo-cytoplasmic shuttling protein with DNA damage responsive localization

International Nuclear Information System (INIS)

Brooks, William S.; Banerjee, Sami; Crawford, David F.

2007-01-01

G2E3 was originally described as a G2/M-specific gene with DNA damage responsive expression. The presence of a conserved HECT domain within the carboxy-terminus of the protein indicated that it likely functions as a ubiquitin ligase or E3. Although HECT domains are known to function in this capacity for many proteins, we demonstrate that a portion of the HECT domain from G2E3 plays an important role in the dynamic subcellular localization of the protein. We have shown that G2E3 is a nucleo-cytoplasmic shuttling protein with nuclear export mediated by a novel nuclear export domain that functions independently of CRM1. In full-length G2E3, a separate region of the HECT domain suppresses the function of the NES. Additionally, G2E3 contains a nucleolar localization signal (NoLS) in its amino terminus. Localization of G2E3 to the nucleolus is a dynamic process, and the protein delocalizes from the nucleolus rapidly after DNA damage. Cell cycle phase-specific expression and highly regulated subcellular localization of G2E3 suggest a possible role in cell cycle regulation and the cellular response to DNA damage

Follicle and oocyte growth in early postnatal calves: cytochemical, autoradiographical and electron microscopical studies

International Nuclear Information System (INIS)

Mhawi, A.J.; Kaňka, J.; Motlík, J.

1991-01-01

The initiation of oocyte and follicle growth was studied in 1- and 3-d-old calf ovaries using cytochemical, autoradiographical and electron microscopical approaches. Attention was only paid to unilaminar ovarian follicles that were classified into 3 categories: unilaminar flattened (UF), unilaminar flatto-cuboidal (UFC) and unilaminar cuboidal (UC) ovarian follicles when the oocyte was surrounded by 1 layer of flattened, a mixture of flattened and cuboidal and entirely cuboidal follicle cells, respectively. Our findings suggested that oocytes within each of these follicle categories were in different developmental stages. Furthermore, electron microscopic observations revealed that early after birth, oocyte nuclei characteristic of diplotene configuration (aggregation of the nuclear chromatin into moderately electron-dense small patches and fibrillo-granular texture of the nucleolus) were encountered in 41% of the UF follicles. The rest of the UF as well as all of the UFC and UC follicles were found to contain dictyate oocytes in which the chromatin was highly decondensed and the nucleolus differentiated into fibrillar, fibrillo-granular and granular components. The present results also indicated that the complete transition of the surrounding follicle cells from flattened to cuboidal shape and the morphological changes of the oocyte endoplasmic reticulum and mitochondria were 2 complementary events essential for initiation of oocyte growth

Dynamics and cytochemistry of oogenesis in Leporinus striatus Kner (Teleostei, Characiformes, Anostomidae from the Rio Sapucaí, Minas Gerais State, Brazil

Directory of Open Access Journals (Sweden)

Helena A.S. Chini

2001-12-01

Full Text Available Oogenesis involves a sequense of transformations which are undergone by female germ cells. These cells change into oogonias and then into mature oocytes. Sexually mature females were collected monthly, during one year, from the Rio Sapucaí, tributary of the Rio Grande, which is part of the Furnas Reservoir system in the state of Minas Gerais. The observed material showed that oogonias were small spherical cells, had a big spherical nucleus, with a single nucleolus, and weakly stained cytoplasm with eosinophilic granules (FG stained, which indicate their protein content. The primary oocytes showed a big basophilic nucleus, with a large peripheral nucleolus, and several smaller nucleoli. They show a reduced cytoplasmic content. The previtellogenic oocytes presented voluminous cytoplasm and nucleus with several small peripheral nucleoli. The oocytes underwent vitellogenesis with the development of the zona radiata and the follicle cells. The zona radiata had two layers, the outer and the inner, which showed its protein content when stained with CM and FG techniques. TB pH 2.5 and pH 4.0 staining showed that oocytes undergoing vitellogenesis presented weakly stained cytoplasm and peripheral cytoplasmic vesicles. The follicle cells that were squamous became cuboidal. In mature oocytes, the yolk granules that filled the cytoplasm became green and blue when stained with FG and CM techniques, indicating their protein content. The perivitclline region showed rosy stained vesicles (TB pH 2.5 and pH 4.0 spread among the weakly stained peripheral vesicles, which seemed to be the cortical alveoli. The zona radiata cells, CM and FG stained, still showed two layers like the oocytes from the previous stage, but thicker.

Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

Energy Technology Data Exchange (ETDEWEB)

Siyam, Arwa [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); Wang, Suzhen; Qin, Chunlin; Mues, Gabriele [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Stevens, Roy [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); D' Souza, Rena N. [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Lu, Yongbo, E-mail: ylu@bcd.tamhsc.edu [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)

2012-08-03

Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

AcEST: BP913209 [AcEST

Lifescience Database Archive (English)

Full Text Available terized protein OS=Magna... 40 0.10 tr|A3LQY9|A3LQY9_PICST Nonribosomal protein of the nucleolus and... 39 0...AESGKRSA 694 Query: 478 SK 483 SK Sbjct: 695 SK 696 >tr|A3LQY9|A3LQY9_PICST Nonribosomal protein of the nucleolus

Decreased Expression of the Early Mitotic Gene, CHFR, Contributes to the Acquisition of Breast Cancer Phenotypes

Science.gov (United States)

2008-03-01

A composite image of two separate Western blots. Whole-cell lysate from the MCF10A IHMEC line was used as a control on both blots in the composite ...which the nucleolus is depicted as a dark spot within the nucleus. Parental (far left ) and scramble shRNA controls (middle ) normally contain one or... Chemistry of Life Processes” Cumulative GPA: 3.97/4.0 Honors and Awards: University of Michigan Department of Defense Breast Cancer

Coilin, the signature protein of Cajal bodies, differentially modulates the interactions of plants with viruses in widely different taxa

OpenAIRE

Shaw, Jane; Love, Andrew J; Makarova, Svetlana S; Kalinina, Natalia O; Harrison, Bryan D; Taliansky, Michael E

2014-01-01

Cajal bodies (CBs) are distinct nuclear bodies physically and functionally associated with the nucleolus. In addition to their traditional function in coordinating maturation of certain nuclear RNAs, CBs participate in cell cycle regulation, development, and regulation of stress responses. A key “signature” component of CBs is coilin, the scaffolding protein essential for CB formation and function. Using an RNA silencing (loss-of-function) approach, we describe here new phenomena whereby coil...

Altered gravity influences rDNA and NopA100 localization in nucleoli

Science.gov (United States)

Sobol, M. A.; Kordyum, E. L.

Fundamental discovery of gravisensitivity of cells no specified to gravity perception focused increasing attention on an elucidation of the mechanisms involved in altered gravity effects at the cellular and subcellular levels. The nucleolus is the transcription site of rRNA genes as well as the site of processing and initial packaging of their transcripts with ribosomal and nonribosomal proteins. The mechanisms inducing the changes in the subcomponents of the nucleolus that is morphologically defined yet highly dynamic structure are still unknown in detail. To understand the functional organization of the nucleolus as in the control as under altered gravity conditions it is essential to determine both the precise location of rDNA and the proteins playing the key role in rRNA processing. Lepidium sativum seeds were germinated in 1% agar medium on the slow horizontal clinostat (2 rpm) and in the stationary conditions. We investigated the root meristematic cells dissected from the seedlings grown in darkness for two days. The investigations were carried out with anti-DNA and anti-NopA100 antibodies labeling as well as with TdT procedure, and immunogold electron microscopy. In the stationary growth conditions, the anti-DNA antibody as well TdT procedure were capable of detecting fibrillar centers (FCs) and the dense fibrillar component (DFC) in the nucleolus. In FCs, gold particles were revealed on the condensed chromatin inclusions, internal fibrils of decondensed rDNA and the transition zone FC-DFC. Quantitatively, FCs appeared 1,5 times more densely labeled than DFC. NopA100 was localized in FCs and in DFC. In FCs, the most of protein was revealed in the transition zone FC-DFC. After a quantitative study, FCs and the transition zone FC-DFC appeared to contain NopA100 1,7 times more than DFC. Under the conditions of altered gravity, quantitative data clearly showed a redistribution of nucleolar DNA and NopA100 between FCs and DFC in comparison with the control. In

Cell-cycle-dependent three-dimensional redistribution of nuclear proteins, P 120, pKi-67, and SC 35 splicing factor, in the presence of the topoisomerase I inhibitor camptothecin.

Science.gov (United States)

Elias, Emmanuel; Lalun, Nathalie; Lorenzato, Marianne; Blache, Laurent; Chelidze, Pavel; O'Donohue, Marie-Françoise; Ploton, Dominique; Bobichon, Hélène

2003-11-15

Topoisomerase I (Topo I) is mostly known for its role in DNA relaxation, which is required for duplication and transcription. Topo I acts as a protein kinase mainly directed to the mRNA splicing factor SC35. Camptothecin is one of the specific Topo I inhibitors and is effective on the two functions of the enzyme. In this study we demonstrated that treatment of KB cells with camptothecin for only 30 min induced the 3D reorganization and redistribution of three proteins involved in the nucleus machinery, P 120, pKi-67, and SC 35, and this occurred in a cell cycle-dependent manner. Our data were obtained from confocal microscopic studies after immunolabeling, 3D reconstruction, and measurement of the nuclear components volumes. In the presence of camptothecin, P 120, which occupied the nucleolar volume, lost its reticulation and pKi-67 was redistributed within the nucleoplasm and even into the cytoplasm. Finally, for SC 35 the fusion of its dots into bigger volumes was observed specifically during the G1 phase. Variations of volumes were also observed for the nucleolus and for the nucleus. These results pointed out that, depending on the cell cycle phase, Topo I functions were selective toward the three different proteins.

ClC-3 Promotes Osteogenic Differentiation in MC3T3-E1 Cell After Dynamic Compression.

Science.gov (United States)

Wang, Dawei; Wang, Hao; Gao, Feng; Wang, Kun; Dong, Fusheng

2017-06-01

ClC-3 chloride channel has been proved to have a relationship with the expression of osteogenic markers during osteogenesis, persistent static compression can upregulate the expression of ClC-3 and regulate osteodifferentiation in osteoblasts. However, there was no study about the relationship between the expression of ClC-3 and osteodifferentiation after dynamic compression. In this study, we applied dynamic compression on MC3T3-E1 cells to detect the expression of ClC-3, runt-related transcription factor 2 (Runx2), bone morphogenic protein-2 (BMP-2), osteopontin (OPN), nuclear-associated antigen Ki67 (Ki67), and proliferating cell nuclear antigen (PCNA) in biopress system, then we investigated the expression of these genes after dynamic compression with Chlorotoxin (specific ClC-3 chloride channel inhibitor) added. Under transmission electron microscopy, there were more cell surface protrusions, rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, abundant glycogen, and lysosomes scattered in the cytoplasm in MC3T3-E1 cells after dynamic compression. The nucleolus was more obvious. We found that ClC-3 was significantly up-regulated after dynamic compression. The compressive force also up-regulated Runx2, BMP-2, and OPN after dynamic compression for 2, 4 and 8 h. The proliferation gene Ki67 and PCNA did not show significantly change after dynamic compression for 8 h. Chlorotoxin did not change the expression of ClC-3 but reduced the expression of Runx2, BMP-2, and OPN after dynamic compression compared with the group without Cltx added. The data from the current study suggested that ClC-3 may promotes osteogenic differentiation in MC3T3-E1 cell after dynamic compression. J. Cell. Biochem. 118: 1606-1613, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Dynamics of the association of heat shock protein HSPA6 (Hsp70B') and HSPA1A (Hsp70-1) with stress-sensitive cytoplasmic and nuclear structures in differentiated human neuronal cells.

Science.gov (United States)

Shorbagi, Sadek; Brown, Ian R

2016-11-01

Heat shock proteins (Hsps) are cellular repair agents that counter the effects of protein misfolding that is a characteristic feature of neurodegenerative diseases. HSPA1A (Hsp70-1) is a widely studied member of the HSPA (Hsp70) family. The little-studied HSPA6 (Hsp70B') is present in the human genome and absent in mouse and rat; hence, it is missing in current animal models of neurodegenerative diseases. Differentiated human neuronal SH-SY5Y cells were employed to compare the dynamics of the association of YFP-tagged HSPA6 and HSPA1A with stress-sensitive cytoplasmic and nuclear structures. Following thermal stress, live-imaging confocal microscopy and Fluorescence Recovery After Photobleaching (FRAP) demonstrated that HSPA6 displayed a prolonged and more dynamic association, compared to HSPA1A, with centrioles that play critical roles in neuronal polarity and migration. HSPA6 and HSPA1A also targeted nuclear speckles, rich in RNA splicing factors, and the granular component of the nucleolus that is involved in rRNA processing and ribosomal subunit assembly. HSPA6 and HSPA1A displayed similar FRAP kinetics in their interaction with nuclear speckles and the nucleolus. Subsequently, during the recovery from neuronal stress, HSPA6, but not HSPA1A, localized with the periphery of nuclear speckles (perispeckles) that have been characterized as transcription sites. The stress-induced association of HSPA6 with perispeckles displayed the greatest dynamism compared to the interaction of HSPA6 or HSPA1A with other stress-sensitive cytoplasmic and nuclear structures. This suggests involvement of HSPA6 in transcriptional recovery of human neurons from cellular stress that is not apparent for HSPA1A.

CARM1 modulators affect epigenome of stem cells and change morphology of nucleoli.

Science.gov (United States)

Franek, M; Legartová, S; Suchánková, J; Milite, C; Castellano, S; Sbardella, G; Kozubek, S; Bártová, E

2015-01-01

CARM1 interacts with numerous transcription factors to mediate cellular processes, especially gene expression. This is important for the maintenance of ESC pluripotency or intervention to tumorigenesis. Here, we studied epigenomic effects of two potential CARM1 modulators: an activator (EML159) and an inhibitor (ellagic acid dihydrate, EA). We examined nuclear morphology in human and mouse embryonic stem cells (hESCs, mESCs), as well as in iPS cells. The CARM1 modulators did not function similarly in all cell types. EA decreased the levels of the pluripotency markers, OCT4 and NANOG, particularly in iPSCs, whereas the levels of these proteins increased after EML159 treatment. EML159 treatment of mouse ESCs led to decreased levels of OCT4 and NANOG, which was accompanied by an increased level of Endo-A. The same trend was observed for NANOG and Endo-A in hESCs affected by EML159. Interestingly, EA mainly changed epigenetic features of nucleoli because a high level of arginine asymmetric di-methylation in the nucleoli of hESCs was reduced after EA treatment. ChIP-PCR of ribosomal genes confirmed significantly reduced levels of H3R17me2a, in both the promoter region of ribosomal genes and rDNA encoding 28S rRNA, after EA addition. Moreover, EA treatment changed the nuclear pattern of AgNORs (silver-stained nucleolus organizer regions) in all cell types studied. In EA-treated ESCs, AgNOR pattern was similar to the pattern of AgNORs after inhibition of RNA pol I by actinomycin D. Together, inhibitory effect of EA on arginine methylation and effect on related morphological parameters was especially observed in compartment of nucleoli.

High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes.

Science.gov (United States)

Shishova, Kseniya V; Khodarovich, Yuriy M; Lavrentyeva, Elena A; Zatsepina, Olga V

2015-10-01

Nucleolus-like bodies (NLBs) of fully-grown (germinal vesicle, GV) mammalian oocytes are traditionally considered as morphologically distinct entities, which, unlike normal nucleoli, contain transcribed ribosomal genes (rDNA) solely at their surface. In the current study, we for the first time showed that active ribosomal genes are present not only on the surface but also inside NLBs of the NSN-type oocytes. The "internal" rRNA synthesis was evidenced by cytoplasmic microinjections of BrUTP as precursor and by fluorescence in situ hybridization with a probe to the short-lived 5'ETS segment of the 47S pre-rRNA. We further showed that in the NLB mass of NSN-oocytes, distribution of active rDNA, RNA polymerase I (UBF) and rRNA processing (fibrillarin) protein factors, U3 snoRNA, pre-rRNAs and 18S/28S rRNAs is remarkably similar to that in somatic nucleoli capable to make pre-ribosomes. Overall, these observations support the occurrence of rDNA transcription, rRNA processing and pre-ribosome assembly in the NSN-type NLBs and so that their functional similarity to normal nucleoli. Unlike the NSN-type NLBs, the NLBs of more mature SN-oocytes do not contain transcribed rRNA genes, U3 snoRNA, pre-rRNAs, 18S and 28S rRNAs. These results favor the idea that in a process of transformation of NSN-oocytes to SN-oocytes, NLBs cease to produce pre-ribosomes and, moreover, lose their rRNAs. We also concluded that a denaturing fixative 70% ethanol used in the study to fix oocytes could be more appropriate for light microscopy analysis of nucleolar RNAs and proteins in mammalian fully-grown oocytes than a commonly used cross-linking aldehyde fixative, formalin. Copyright © 2015 Elsevier Inc. All rights reserved.

Ovarian structure and oogenesis of the extremophile viviparous teleost Poecilia mexicana (Poeciliidae) from an active sulfur spring cave in Southern Mexico.

Science.gov (United States)

Torres-Martínez, Aarón; Hernández-Franyutti, Arlette; Uribe, Mari Carmen; Contreras-Sánchez, Wilfrido Miguel

2017-12-01

The structure of the ovary and oogenesis of Poecilia mexicana from an active sulfur spring cave is documented. Poecilia mexicana is the only poeciliid adapted to a subterranean environment with high hydrogen sulfide levels and extreme hypoxic conditions. Twenty females were captured throughout one year at Cueva del Azufre, located in the State of Tabasco in Southern Mexico. Ovaries were processed with histological techniques. P. mexicana has a single, ovoid ovary with ovigerous lamella that project to the ovarian lumen. The ovarian wall presents abundant loose connective tissue, numerous melanomacrophage centers and large blood vessels, possibly associated with hypoxic conditions. The germinal epithelium bordering the ovarian lumen contains somatic and germ cells forming cell nests projecting into the stroma. P. mexicana stores sperm in ovarian folds associated with follicles at different developmental phases. Oogenesis in P. mexicana consisted of the following stages: (i) oogonial proliferation, (ii) chromatin nucleolus, (iii) primary growth, subdivided into: (a) one nucleolus, (b) multiple nucleoli, (c) droplet oils-cortical alveoli steps; (iv) secondary growth, subdivided in: (a) early secondary growth, (b) late secondary growth, and (c) full grown. Follicular atresia was present in all stages of follicular development; it was characterized by oocyte degeneration, where follicle cells hypertrophy and differentiate in phagocytes. The ovary and oogenesis are similar to these seen in other poeciliids, but we found frequent atretic follicles, melanomacrophage centers, reduced fecundity and increased of offspring size. © 2017 Wiley Periodicals, Inc.

Silencing of ribosomal protein S9 elicits a multitude of cellular responses inhibiting the growth of cancer cells subsequent to p53 activation.

Directory of Open Access Journals (Sweden)

Mikael S Lindström

Full Text Available BACKGROUND: Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. CONCLUSIONS: p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

Conformational determinants of the intracellular localization of midkine

International Nuclear Information System (INIS)

Dai Lichen; Xu, Diyong; Yao Xing; Lu Yongliang; Xu Zhengping

2005-01-01

Midkine (MK) is a multifunctional growth factor and has been discovered to play important roles in carcinogenesis. MK has been reported to localize to the nucleus and nucleolus, however, the data are not consistent and the signals responsible for the localization are unknown. Here we reported that human MK exclusively localized to the nucleus and nucleolus in HepG2 cells by using GFP as a tracking molecule. In order to identify the motifs required for the nuclear localization and nucleolar accumulation, point- and deletion-mutations were introduced and the corresponding subcellular localizations were analyzed. Data revealed that (i) K79R81, K86K87, and the C-terminal tail of MK constitute the nuclear localization determinant of MK, and (ii) the C-terminal tail is the key element controlling MK nucleolar accumulation though the N-terminal tail, K79R81, and K86K87 also contribute to this process. Taken together, our results provide the first documentation about the determinants required for MK nuclear and nucleolar localization

Autoradiographic study of the nucleolar RNA metabolism during the oogenesis of Lineus ruber Mueller (Heteronemertes)

International Nuclear Information System (INIS)

Rue, Gerard; Gontcharoff, Marie

1975-01-01

During Lineus ruber oogenesis, there is a very high uridine uptake by the oocyte before the yolk formation. At the end of this stage, the nucleolus shows a special structure that seems to be related to a decrease of the ribosomal RNA synthesis. During the yolk formation, the nucleolus scatters in the nucleus, allowing ribonucleoprotein granules to go towards the cytoplasm [fr

Clinical analysis of the therapy for recurrent and intractable non-small cell lung cancer with combination of cryoablation and intervention

International Nuclear Information System (INIS)

Zhang Futong; Li Xiuli; Li Huijie; An Yonghui

2007-01-01

Objective: To explore the re-treatment approach for nonsmall-cell lung cancer (NSCLC )in clinic with therapeutic alliance of cryotherapy and interventional chemotherapy. Methods: Sixty two patients with uncontrolled NSCLC after common radiotherapy and (or)chemotherapy were re-treated with therapeutic alliance of cryotherapy and interventional chemotherapy, and their serum tumor markers of NSE, CEA, CY21- 1 and the value (I.S%,I.O.D%)of correlative protein in nucleolus region-Ag-NORs were tested before and after the re-treatment. Meanwhile, patients one year survival and therapeutic effect to NSCLC were recorded during the follow up. Results: After the re-treatment, distinguished decline of the serum tumor markers was observed (P<0.01)and values of Ag-NORs(I.S% and I.O.D%)were obviously lowered down. The therapeutic effect and one year survival were 79.03%(49/62)and 80.63%(50/62)respectively. Conclusion: Therapeutic alliance of cryotherapy and interventional chemotherapy is a kind of effective re-treatment for NSCLC and also as a new approach for the refractory NSCLC. (authors)

Nucleoli cytomorphology in cutaneous melanoma cells - a new prognostic approach to an old concept.

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Donizy, Piotr; Biecek, Przemyslaw; Halon, Agnieszka; Maciejczyk, Adam; Matkowski, Rafal

2017-12-29

The nucleolus is an organelle that is an ultrastructural element of the cell nucleus observed in H&E staining as a roundish body stained with eosin due to its high protein content. Changes in the nucleoli cytomorphology were one of the first histopathological characteristics of malignant tumors. The aim of this study was to assess the relationship between the cytomorphological characteristics of nucleoli and detailed clinicopathological parameters of melanoma patients. Moreover, we analyzed the correlation between cytomorphological parameters of nucleoli and immunoreactivity of selected proteins responsible for, among others, regulation of epithelial-mesenchymal transition (SPARC, N-cadherin), cell adhesion and motility (ALCAM, ADAM-10), mitotic divisions (PLK1), cellular survival (FOXP1) and the functioning of Golgi apparatus (GOLPH3, GP73). Three characteristics of nucleoli - presence, size and number - of cancer cells were assessed in H&E-stained slides of 96 formalin-fixed paraffin-embedded primary cutaneous melanoma tissue specimens. The results were correlated with classical clinicopathological features and patient survival. Immunohistochemical analysis of the above mentioned proteins was described in details in previous studies. Higher prevalence and size of nucleoli were associated with thicker and mitogenic tumors. All three nucleolar characteristics were related to the presence of ulceration. Moreover, microsatellitosis was strongly correlated with the presence of macronucleoli and polynucleolization (presence of two or more nucleoli). Lack of immunologic response manifested as no TILs in primary tumor was associated with high prevalence of melanoma cells with distinct nucleoli. Interestingly, in nodular melanoma a higher percentage of melanoma cells with prominent nucleoli was observed. In Kaplan-Meier analysis, increased prevalence and amount, but not size of nucleoli, were connected with shorter cancer-specific and disease-free survival. (1) High

Immunodetection of nucleolar proteins and ultrastructure of nucleoli of soybean root meristematic cells treated with chilling stress and after recovery.

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Stepiński, Dariusz

2009-03-01

The nucleolar proteins, fibrillarin and nucleophosmin, have been identified immunofluorescently in the root meristematic cells of soybean seedlings under varying experimental conditions: at 25 degrees C (control), chilling at 10 degrees C for 3 h and 4 days and recovery from the chilling stress at 25 degrees C. In each experimental variant, the immunofluorescence signals were present solely at the nucleolar territories. Fluorescent staining for both proteins was mainly in the shape of circular domains that are assumed to correspond to the dense fibrillar component of the nucleoli. The fewest fluorescent domains were observed in the nucleoli of chilled plants, and the highest number was observed in the plants recovered after chilling. This difference in the number of circular domains in the nucleoli of each variant may indicate various levels of these proteins in each variant. Both the number of circular domains and the level of these nucleolar proteins changed with changes in the transcriptional activity of the nucleoli, with the more metabolically active cell having higher numbers of active areas in the nucleolus and higher levels of nucleolar proteins, and conversely. Electron microscopic studies revealed differences in the ultrastructure of the nucleoli in all experimental variants and confirmed that the number of fibrillar centres surrounded by dense fibrillar component was the lowest in the nucleoli of chilled plants, and the highest in the nucleoli of recovered seedlings.

Nucleolin (C23), a physiological substrate for casein kinase II

DEFF Research Database (Denmark)

Schneider, H R; Issinger, O G

1988-01-01

Nucleolin (C23), a 110 kDa phosphoprotein, which is mainly found in the nucleolus has been shown to be a physiological substrate for casein kinase II (CKII). Nucleolin was identified and characterized by immunodetection using an anti-nucleolin antibody. Phosphopeptide patterns from nucleolin...... phosphorylated by purified casein kinase II and of phosphorylated nucleolin which had been isolated from tumor cells grown in the presence of [32P]-o-phosphate, were identical. The partial tryptic digest revealed nine phosphopeptides. Nucleolin isolated from Krebs II mouse ascites cells was phosphorylated...... by purified casein kinase II with about two moles phosphate per one mole of nucleolin....

Hypoxia and Prx1 in Malignant Progression of Prostate Cancer

Science.gov (United States)

2007-09-01

promoter composition of human prx1 gene and identified EpRE elements and Nrf2 as critical regulatory component of its up- regulation in prostate cancer...nucleus as well as in the cytoplasm in the rat kidney (42). The presence of Prx1 in the nucleolus of hepatic parenchymal cells has also been shown in the...Gpx; KO, knock-out; JNK, c-Jun N-terminal kinase. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 30, pp. 22011–22022, July 27, 2007 © 2007 by The

Effect of X- and γ-irradiation on the ultrastructure of Haematococcus pluvialis

International Nuclear Information System (INIS)

Jiang Xiamin; Zhai Xingwen; Dong Jiaodi; Wang Xun; Wang Jianhua

2003-01-01

Effects of X- and γ-irradiation on the ultrastructure change of Hematococcus pluvialis has been studied. The results show that X or γ irradiation causes different damages on different cell organelles such as the cytolemma be lacerated, the bioplasm excursed, the area of thin electron occurred in nucleus, the chloroplast lacerated, the thylakoids ruptured, the ribosome increased, the plastosome inflated and its membrane lacerated. In addition, X-irradiation causes perforation of starch sheath, decrease of SER and inducing of lycotropal pyrenoid. γ-irradiation causes dissolving of starch sheath, increase of SER and inflation of golgi body. The mutation effects of X- and γ-irradiation on the cell are probably caused by the laceration of some cell organelles like nucleolus, chloroplast and plastosome

Organization of the nucleoli of soybean root meristematic cells at different states of their activity.

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Stepiński, Dariusz

2010-06-01

Internal organization of a nucleolus changes along with rRNA transcriptional activity. These changes mainly concern qualitative and quantitative alternations of three main nucleolar components: fibrillar centres (FC), dense fibrillar component (DFC) and granular component (GC). In the present work quantitative measurements of the number and sizes of FCs and DFCs in nucleoli of root meristematic cells of soybean seedlings grown at (1) chilling conditions that reduce transcriptional activity of soybean nucleoli (temp. of 10 degrees C) and at (2) conditions that increase this activity (recovery at optimal temp. of 25 degrees C after previous chilling), even more than (3) the control, have been carried out. Morphometric measurements showed that the highest number of FCs and DFCs was in the most active nucleoli, while the smallest number - in those with the lowest activity. The average size of an individual FC was similar in all nucleoli regardless of their transcriptional activity, that of the individual DFC varied, being bigger in the nucleoli of the chilled plants and smallest in those of the recovered plants. The numbers of FCs and DFCs seem to be indicators of transcriptional activity of plant nucleoli - the higher number of FCs and DFCs the more active nucleoli. Copyright 2009 Elsevier Ltd. All rights reserved.

Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders.

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Calo, Eliezer; Gu, Bo; Bowen, Margot E; Aryan, Fardin; Zalc, Antoine; Liang, Jialiang; Flynn, Ryan A; Swigut, Tomek; Chang, Howard Y; Attardi, Laura D; Wysocka, Joanna

2018-02-01

Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and

Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses

Directory of Open Access Journals (Sweden)

Iwai Ohbayashi

2018-01-01

Full Text Available The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized pathway of this stress response involves p53 and MDM2 as key players. p53 is a crucial transcription factor that functions in response to not only nucleolar stress but also other cellular stresses such as DNA damage stress. These cellular stresses release p53 from the inhibition by MDM2, an E3 ubiquitin ligase targeting p53, in various ways, which leads to p53-dependent activation of a set of genes. In plants, genetic impairments of ribosome biogenesis factors or ribosome components have been shown to cause characteristic phenotypes, including a narrow and pointed leaf shape, implying a common signaling pathway connecting ribosomal perturbations and certain aspects of growth and development. Unlike animals, however, plants have neither p53 nor MDM2 family proteins. Then the question arises whether plant cells have a nucleolar stress response pathway. In recent years, it has been reported that several members of the plant-specific transcription factor family NAC play critical roles in the pathways responsive to various cellular stresses. In this mini review, we outline the plant cellular stress response pathways involving NAC transcription factors with reference to the p53-MDM2-dependent pathways of animal cells, and discuss the possible involvement of a plant-unique, NAC-mediated pathway in the nucleolar stress response in plants.

Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses.

Science.gov (United States)

Ohbayashi, Iwai; Sugiyama, Munetaka

2017-01-01

The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized pathway of this stress response involves p53 and MDM2 as key players. p53 is a crucial transcription factor that functions in response to not only nucleolar stress but also other cellular stresses such as DNA damage stress. These cellular stresses release p53 from the inhibition by MDM2, an E3 ubiquitin ligase targeting p53, in various ways, which leads to p53-dependent activation of a set of genes. In plants, genetic impairments of ribosome biogenesis factors or ribosome components have been shown to cause characteristic phenotypes, including a narrow and pointed leaf shape, implying a common signaling pathway connecting ribosomal perturbations and certain aspects of growth and development. Unlike animals, however, plants have neither p53 nor MDM2 family proteins. Then the question arises whether plant cells have a nucleolar stress response pathway. In recent years, it has been reported that several members of the plant-specific transcription factor family NAC play critical roles in the pathways responsive to various cellular stresses. In this mini review, we outline the plant cellular stress response pathways involving NAC transcription factors with reference to the p53-MDM2-dependent pathways of animal cells, and discuss the possible involvement of a plant-unique, NAC-mediated pathway in the nucleolar stress response in plants.

A new class of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in plant parasitic cyst nematodes.

Science.gov (United States)

Tytgat, Tom; Vanholme, Bartel; De Meutter, Jan; Claeys, Myriam; Couvreur, Marjolein; Vanhoutte, Isabelle; Gheysen, Greetje; Van Criekinge, Wim; Borgonie, Gaetan; Coomans, August; Gheysen, Godelieve

2004-08-01

By performing cDNA AFLP on pre- and early parasitic juveniles, we identified genes encoding a novel type of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in the cyst nematode Heterodera schachtii. The proteins consist of three domains, a signal peptide for secretion, a mono-ubiquitin domain, and a short C-terminal positively charged domain. A gfp-fusion of this protein is targeted to the nucleolus in tobacco BY-2 cells. We hypothesize that the C-terminal peptide might have a regulatory function during syncytium formation in plant roots.

A novel, mouse mammary tumor virus encoded protein with Rev-like properties

International Nuclear Information System (INIS)

Indik, Stanislav; Guenzburg, Walter H.; Salmons, Brian; Rouault, Francoise

2005-01-01

We have identified a novel, multiple spliced, subgenomic mRNA species in MMTV producing cells of different origin containing an open reading frame encoding a 39-kDa Rev-like protein, Rem (regulator of expression of MMTV). An EGFP-Rem fusion protein is shown to be predominantly in the nucleolus. Further leptomycin B inhibits the nuclear export of nonspliced MMTV transcripts, implicating Rem in nuclear export by the Crm1 pathway in MMTV. Rem is thus reminiscent of the Rec protein from the related endogenous human retrovirus, HERV-K

Functional genomics by mass spectrometry

DEFF Research Database (Denmark)

Andersen, Jens S.; Mann, M

2000-01-01

Systematic analysis of the function of genes can take place at the oligonucleotide or protein level. The latter has the advantage of being closest to function, since it is proteins that perform most of the reactions necessary for the cell. For most protein based ('proteomic') approaches to gene f...... numbers of intact proteins by mass spectrometry directly. Examples from this laboratory illustrate biological problem solving by modern mass spectrometric techniques. These include the analysis of the structure and function of the nucleolus and the analysis of signaling complexes....

RINT-1 interacts with MSP58 within nucleoli and plays a role in ribosomal gene transcription.

Science.gov (United States)

Yang, Chuan-Pin; Kuo, Yu-Liang; Lee, Yi-Chao; Lee, Kuen-Haur; Chiang, Chi-Wu; Wang, Ju-Ming; Hsu, Che-Chia; Chang, Wen-Chang; Lin, Ding-Yen

2016-09-16

The nucleolus is the cellular site of ribosomal (r)DNA transcription and ribosome biogenesis. The 58-kDa microspherule protein (MSP58) is a nucleolar protein involved in rDNA transcription and cell proliferation. However, regulation of MSP58-mediated rDNA transcription remains unknown. Using a yeast two-hybrid system with MSP58 as bait, we isolated complementary (c)DNA encoding Rad50-interacting protein 1 (RINT-1), as a MSP58-binding protein. RINT-1 was implicated in the cell cycle checkpoint, membrane trafficking, Golgi apparatus and centrosome dynamic integrity, and telomere length control. Both in vitro and in vivo interaction assays showed that MSP58 directly interacts with RINT-1. Interestingly, microscopic studies revealed the co-localization of MSP58, RINT-1, and the upstream binding factor (UBF), a rRNA transcription factor, in the nucleolus. We showed that ectopic expression of MSP58 or RINT-1 resulted in decreased rRNA expression and rDNA promoter activity, whereas knockdown of MSP58 or RINT-1 by siRNA exerted the opposite effect. Coexpression of MSP58 and RINT-1 robustly decreased rRNA synthesis compared to overexpression of either protein alone, whereas depletion of RINT-1 from MSP58-transfected cells enhanced rRNA synthesis. We also found that MSP58, RINT-1, and the UBF were associated with the rDNA promoter using a chromatin immunoprecipitation assay. Because aberrant ribosome biogenesis contributes to neoplastic transformation, our results revealed a novel protein complex involved in the regulation of rRNA gene expression, suggesting a role for MSP58 and RINT-1 in cancer development. Copyright © 2016 Elsevier Inc. All rights reserved.

The sub-nucleolar localization of PHF6 defines its role in rDNA transcription and early processing events

Science.gov (United States)

Todd, Matthew A M; Huh, Michael S; Picketts, David J

2016-01-01

Ribosomal RNA synthesis occurs in the nucleolus and is a tightly regulated process that is targeted in some developmental diseases and hyperactivated in multiple cancers. Subcellular localization and immunoprecipitation coupled mass spectrometry demonstrated that a proportion of plant homeodomain (PHD) finger protein 6 (PHF6) protein is localized within the nucleolus and interacts with proteins involved in ribosomal processing. PHF6 sequence variants cause Börjeson–Forssman–Lehmann syndrome (BFLS, MIM#301900) and are also associated with a female-specific phenotype overlapping with Coffin–Siris syndrome (MIM#135900), T-cell acute lymphoblastic leukemia (MIM#613065), and acute myeloid leukemia (MIM#601626); however, very little is known about its cellular function, including its nucleolar role. HEK 293T cells were treated with RNase A, DNase I, actinomycin D, or 5,6-dichloro-β-D-ribofuranosylbenzimadole, followed by immunocytochemistry to determine PHF6 sub-nucleolar localization. We observed RNA-dependent localization of PHF6 to the sub-nucleolar fibrillar center (FC) and dense fibrillar component (DFC), at whose interface rRNA transcription occurs. Subsequent ChIP-qPCR analysis revealed strong enrichment of PHF6 across the entire rDNA-coding sequence but not along the intergenic spacer (IGS) region. When rRNA levels were quantified in a PHF6 gain-of-function model, we observed an overall decrease in rRNA transcription, accompanied by a modest increase in repressive promoter-associated RNA (pRNA) and a significant increase in the expression levels of the non-coding IGS36RNA and IGS39RNA transcripts. Collectively, our results demonstrate a role for PHF6 in carefully mediating the overall levels of ribosome biogenesis within a cell. PMID:27165002

Endocytic mechanisms and osteoinductive profile of hydroxyapatite nanoparticles in human umbilical cord Wharton’s jelly-derived mesenchymal stem cells

Science.gov (United States)

Zhang, Juan; Wang, Chen

2018-01-01

Background As a potentially bioactive material, the widespread application of nanosized hydroxyapatite (nano-HAP) in the field of bone regeneration has increased the risk of human exposure. However, our understanding of the interaction between nano-HAP and stem cells implicated in bone repair remains incomplete. Methods Here, we characterized the adhesion and cellular internalization of HAP nanoparticles (HANPs) with different sizes (20 nm np20 and 80 nm np80) and highlighted the involved pathway in their uptake using human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs). In addition, the effects of HANPs on cell viability, apoptosis response, osteogenic differentiation, and underlying related mechanisms were explored. Results It was shown that both types of HANPs readily adhered to the cellular membrane and were transported into the cells compared to micro-sized HAP particles (m-HAP; 12 μm). Interestingly, the endocytic routes of np20 and np80 differed, although they exhibited similar kinetics of adhesion and uptake. Our study revealed involvement of clathrin- and caveolin-mediated endocytosis as well as macropinocytosis in the np20 uptake. However, for np80, clathrin-mediated endocytosis and some as-yet-unidentified important uptake routes play central roles in their internalization. HANPs displayed a higher preference to accumulate in the cytoplasm compared to m-HAP, and HANPs were not detected in the nucleolus. Exposure to np20 for 24 h caused a decrease in cell viability, while cells completely recovered with an exposure time of 72 h. Furthermore, HANPs did not influence apoptosis and necrosis of hWJ-MSCs. Strikingly, HANPs enhanced mRNA levels of osteoblast-related genes and stimulated calcium mineral deposition, and this directly correlated with the activation in c-Jun N-terminal kinases and p38 pathways. Conclusion Our data provide additional insight about the interactions of HANPs with MSCs and suggest their application

Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

International Nuclear Information System (INIS)

Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki

2006-01-01

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus

Ahp2 (Hop2) function in Arabidopsis thaliana (Ler) is required for stabilization of close alignment and synaptonemal complex formation except for the two short arms that contain nucleolus organizer regions.

Science.gov (United States)

Stronghill, P; Pathan, N; Ha, H; Supijono, E; Hasenkampf, C

2010-08-01

A cytological comparative analysis of male meiocytes was performed for Arabidopsis wild type and the ahp2 (hop2) mutant with emphasis on ahp2's largely uncharacterized prophase I. Leptotene progression appeared normal in ahp2 meiocytes; chromosomes exhibited regular axis formation and assumed a typical polarized nuclear organization. In contrast, 4',6'-diamidino-2-phenylindole-stained ahp2 pachytene chromosome spreads demonstrated a severe reduction in stabilized pairing. However, transmission electron microscopy (TEM) analysis of sections from meiocytes revealed that ahp2 chromosome axes underwent significant amounts of close alignment (44% of total axis). This apparent paradox strongly suggests that the Ahp2 protein is involved in the stabilization of homologous chromosome close alignment. Fluorescent in situ hybridization in combination with Zyp1 immunostaining revealed that ahp2 mutants undergo homologous synapsis of the nucleolus-organizer-region-bearing short arms of chromosomes 2 and 4, despite the otherwise "nucleus-wide" lack of stabilized pairing. The duration of ahp2 zygotene was significantly prolonged and is most likely due to difficulties in chromosome alignment stabilization and subsequent synaptonemal complex formation. Ahp2 and Mnd1 proteins have previously been shown, "in vitro," to form a heterodimer. Here we show, "in situ," that the Ahp2 and Mnd1 proteins are synchronous in their appearance and disappearance from meiotic chromosomes. Both the Ahp2 and Mnd1 proteins localize along the chromosomal axis. However, localization of the Ahp2 protein was entirely foci-based whereas Mnd1 protein exhibited an immunostaining pattern with some foci along the axis and a diffuse staining for the rest of the chromosome.

In vivo labelling of functional ribosomes reveals spatial regulation during starvation in Podospora anserina

Directory of Open Access Journals (Sweden)

Silar Philippe

2000-11-01

Full Text Available Abstract Background To date, in eukaryotes, ribosomal protein expression is known to be regulated at the transcriptional and/or translational levels. But other forms of regulation may be possible. Results Here, we report the successful tagging of functional ribosomal particles with a S7-GFP chimaeric protein, making it possible to observe in vivo ribosome dynamics in the filamentous fungus Podospora anserina. Microscopic observations revealed a novel kind of ribosomal protein regulation during the passage between cell growth and stationary phases, with a transient accumulation of ribosomal proteins and/or ribosome subunits in the nucleus, possibly the nucleolus, being observed at the beginning of stationary phase. Conclusion Nuclear sequestration can be another level of ribosomal protein regulation in eukaryotic cells.This may contribute to the regulation of cell growth and division.

In vivo labelling of functional ribosomes reveals spatial regulation during starvation in Podospora anserina

Science.gov (United States)

Lalucque, Hervé; Silar, Philippe

2000-01-01

Background To date, in eukaryotes, ribosomal protein expression is known to be regulated at the transcriptional and/or translational levels. But other forms of regulation may be possible. Results Here, we report the successful tagging of functional ribosomal particles with a S7-GFP chimaeric protein, making it possible to observe in vivo ribosome dynamics in the filamentous fungus Podospora anserina. Microscopic observations revealed a novel kind of ribosomal protein regulation during the passage between cell growth and stationary phases, with a transient accumulation of ribosomal proteins and/or ribosome subunits in the nucleus, possibly the nucleolus, being observed at the beginning of stationary phase. Conclusion Nuclear sequestration can be another level of ribosomal protein regulation in eukaryotic cells.This may contribute to the regulation of cell growth and division. PMID:11112985

Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat

DEFF Research Database (Denmark)

Jakociunas, Tadas; Domange Jordö, Marie Elise; Mebarek, Mazhoura Aït

2013-01-01

Our research addresses the relationship between subnuclear localization and gene expression in fission yeast. We observed the relocalization of a heterochromatic region, the mating-type region, from its natural location at the spindle-pole body to the immediate vicinity of the nucleolus. Relocali......Our research addresses the relationship between subnuclear localization and gene expression in fission yeast. We observed the relocalization of a heterochromatic region, the mating-type region, from its natural location at the spindle-pole body to the immediate vicinity of the nucleolus...

Mass spectrometry based proteomics in cell biology and signaling research

International Nuclear Information System (INIS)

Mann, M.; Andersen, J.; Ishihama, Y.; Rappsilber, J.; Ong, S.; Foster, L.; Blagoev, B.; Kratchmarova, I.; Lasonder, E.

2002-01-01

Full text: Proteomics is one of the most powerful post-genomics technologies. Recently accomplishments include large scale protein-protein interaction mapping, large scale mapping of phosphorylation sites and the cloning of key signaling molecules. In this talk, current state of the art of the technology will be reviewed. Applications of proteomics to the mapping of multiprotein complexes will be illustrated with recent work on the spliceosome and the nucleolus. More than 300 proteins have been mapped to each of these complexes. Quantitative techniques are becoming more and more essential in proteomics. They are usually performed by the incorporation of stable isotopes - a light form in cell state 'A' and a heavy form in cell state 'E' - and subsequent comparison of mass spectrometric peak heights. A new technique called, SILAC for Stable isotope Incorporation by Amino acids in Cell culture, has been applied to studying cell differentiation and mapping secreted proteins from adipocytes. A number of known and novel proteins important in adipocyte differentiation have been identified by this technique. Some of these proved to be upregulated at the 1 mRNA level, too, whereas others appear to be regulated post-translationally. We have also applied the SILAC method to protein-protein interaction mapping. For example, we compared immunoprecipitates from stimulated and non-stimulated cells to find binding partners recruited to the bait due to the stimulus. Several novel substrates in the EGF pathway were found in this way. An important application of proteomics in the signaling field is the mapping of post-translational modifications. In particular, there are a number of techniques for phosphotyrosine phosphorylation mapping which have proven very useful. Making use of the mass deficiency of the phosphogroup, 'parent ion scans' con be performed, which selectively reveal phosphotyrosine peptides from complex peptides mixtures. This technique has been used to clone several

Adaptive response to chronic mild ethanol stress involves ROS, sirtuins and changes in chromosome dosage in wine yeasts.

Science.gov (United States)

Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Kwiatkowska, Aleksandra; Potocki, Leszek; Rawska, Ewa; Pabian, Sylwia; Kaplan, Jakub; Lewinska, Anna; Wnuk, Maciej

2016-05-24

Industrial yeast strains of economic importance used in winemaking and beer production are genomically diverse and subjected to harsh environmental conditions during fermentation. In the present study, we investigated wine yeast adaptation to chronic mild alcohol stress when cells were cultured for 100 generations in the presence of non-cytotoxic ethanol concentration. Ethanol-induced reactive oxygen species (ROS) and superoxide signals promoted growth rate during passages that was accompanied by increased expression of sirtuin proteins, Sir1, Sir2 and Sir3, and DNA-binding transcription regulator Rap1. Genome-wide array-CGH analysis revealed that yeast genome was shaped during passages. The gains of chromosomes I, III and VI and significant changes in the gene copy number in nine functional gene categories involved in metabolic processes and stress responses were observed. Ethanol-mediated gains of YRF1 and CUP1 genes were the most accented. Ethanol also induced nucleolus fragmentation that confirms that nucleolus is a stress sensor in yeasts. Taken together, we postulate that wine yeasts of different origin may adapt to mild alcohol stress by shifts in intracellular redox state promoting growth capacity, upregulation of key regulators of longevity, namely sirtuins and changes in the dosage of genes involved in the telomere maintenance and ion detoxification.

Investigating the spectral characteristics of backscattering from heterogeneous spheroidal nuclei using broadband finite-difference time-domain simulations

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Chao, Guo-Shan; Sung, Kung-Bin

2010-02-01

Backscattered light spectra have been used to extract size distribution of cell nuclei in epithelial tissues for noninvasive detection of precancerous lesions. In existing experimental studies, size estimation is achieved by assuming nuclei as homogeneous spheres or spheroids and fitting the measured data with models based on Mie theory. However, the validity of simplifying nuclei as homogeneous spheres has not been thoroughly examined. In this study, we investigate the spectral characteristics of backscattering from models of spheroidal nuclei under plane wave illumination using three-dimensional finite-difference time-domain (FDTD) simulation. A modulated Gaussian pulse is used to obtain wavelength dependent scattering intensity with a single FDTD run. The simulated model of nuclei consists of a nucleolus and randomly distributed chromatin condensation in homogeneous cytoplasm and nucleoplasm. The results show that backscattering spectra from spheroidal nuclei have similar oscillating patterns to those from homogeneous spheres with the diameter equal to the projective length of the spheroidal nucleus along the propagation direction. The strength of backscattering is enhanced in heterogeneous spheroids as compared to homogeneous spheroids. The degree of which backscattering spectra of heterogeneous nuclei deviate from Mie theory is highly dependent on the distribution of chromatin/nucleolus but not sensitive to nucleolar size, refractive index fluctuation or chromatin density.

Comparison of the Giemsa C-banded karyotypes of the three subspecies of Psathyrostachys fragilis, subspp. villosus (2x), secaliformis (2x, 4x), and fragilis (2x) (Poaceae), with notes on chromosome pairing

DEFF Research Database (Denmark)

Linde-Laursen, I.; Baden, C.

1994-01-01

. Only the presence of 12 nucleoli in interphases identified 6 SAT-chromosome pairs. In subsp. villosus one or two extra micronucleoli indicated a chromosome pair with very low nucleolus-forming activity, bringing the number of SAT-chromosome pairs to 4. This number may be a characteristic...... of a common genome, N. NORs had different nucleolus-forming activities. Meiotic analysis demonstrated a high level of bivalent pairing in the three taxa. A chromosomal rearrangement was suggested in subsp. villosus. The low multivalent frequency in subsp. secaliformis indicates the presence of a pairing...

Evidence for nucleolar subcompartments in Dictyostelium

International Nuclear Information System (INIS)

Catalano, Andrew; O’Day, Danton H.

2015-01-01

Highlights: • Two nucleolar subcompartments (NoSC1, NoSC2) were found in Dictyostelium. • Specific nucleolar proteins localize to different nucleolar subcompartments. • Specific proteins exit NoSC1 and NoSC2 differently upon Actinomycin D treatment. • KRKR appears to function as an NoSC2 nucleolar subcompartment localization signal. - Abstract: The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during

Evidence for nucleolar subcompartments in Dictyostelium

Energy Technology Data Exchange (ETDEWEB)

Catalano, Andrew, E-mail: acatalano@ccny.cuny.edu [Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario L5L 1C6 (Canada); O’Day, Danton H., E-mail: danton.oday@utoronto.ca [Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario L5L 1C6 (Canada); Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, Ontario M5S 3G5 (Canada)

2015-01-24

Highlights: • Two nucleolar subcompartments (NoSC1, NoSC2) were found in Dictyostelium. • Specific nucleolar proteins localize to different nucleolar subcompartments. • Specific proteins exit NoSC1 and NoSC2 differently upon Actinomycin D treatment. • KRKR appears to function as an NoSC2 nucleolar subcompartment localization signal. - Abstract: The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during

Probing Intranuclear Environments at the Single-Molecule Level

Science.gov (United States)

Grünwald, David; Martin, Robert M.; Buschmann, Volker; Bazett-Jones, David P.; Leonhardt, Heinrich; Kubitscheck, Ulrich; Cardoso, M. Cristina

2008-01-01

Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins, when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentration of reactants and availability of binding sites. PMID:18065482

Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

DEFF Research Database (Denmark)

Østrup, Olga; Hyttel, Poul; Klærke, Dan Arne

2011-01-01

Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression....... This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling...... and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation...

Localization switching of a large object in a crowded cavity: A rigid/soft object prefers surface/inner positioning

Science.gov (United States)

Shew, Chwen-Yang; Oda, Soutaro; Yoshikawa, Kenichi

2017-11-01

For living cells in the real world, a large organelle is commonly positioned in the inner region away from membranes, such as the nucleus of eukaryotic cells, the nucleolus of nuclei, mitochondria, chloroplast, Golgi body, etc. It contradicts the expectation by the current depletion-force theory in that the larger particle should be excluded from the inner cell space onto cell boundaries in a crowding media. Here we simply model a sizable organelle as a soft-boundary large particle allowing crowders, which are smaller hard spheres in the model, to intrude across its boundary. The results of Monte Carlo simulation indicate that the preferential location of the larger particle switches from the periphery into the inner region of the cavity by increasing its softness. An integral equation theory is further developed to account for the structural features of the model, and the theoretical predictions are found consistent with our simulation results.

Differential Targeting of Hsp70 Heat Shock Proteins HSPA6 and HSPA1A with Components of a Protein Disaggregation/Refolding Machine in Differentiated Human Neuronal Cells following Thermal Stress

Directory of Open Access Journals (Sweden)

Ian R. Brown

2017-04-01

Full Text Available Heat shock proteins (Hsps co-operate in multi-protein machines that counter protein misfolding and aggregation and involve DNAJ (Hsp40, HSPA (Hsp70, and HSPH (Hsp105α. The HSPA family is a multigene family composed of inducible and constitutively expressed members. Inducible HSPA6 (Hsp70B' is found in the human genome but not in the genomes of mouse and rat. To advance knowledge of this little studied HSPA member, the targeting of HSPA6 to stress-sensitive neuronal sites with components of a disaggregation/refolding machine was investigated following thermal stress. HSPA6 targeted the periphery of nuclear speckles (perispeckles that have been characterized as sites of transcription. However, HSPA6 did not co-localize at perispeckles with DNAJB1 (Hsp40-1 or HSPH1 (Hsp105α. At 3 h after heat shock, HSPA6 co-localized with these members of the disaggregation/refolding machine at the granular component (GC of the nucleolus. Inducible HSPA1A (Hsp70-1 and constitutively expressed HSPA8 (Hsc70 co-localized at nuclear speckles with components of the machine immediately after heat shock, and at the GC layer of the nucleolus at 1 h with DNAJA1 and BAG-1. These results suggest that HSPA6 exhibits targeting features that are not apparent for HSPA1A and HSPA8.

Nucleoli from growing oocytes support the development of enucleolated full-grown oocytes in the pig.

Science.gov (United States)

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2010-02-01

Recent research has shown that the maternal nucleolus is essential for embryonic development. The morphology of the nucleolus in growing oocytes differs from that in full-grown oocytes. We determined the ability of nucleoli from growing oocytes to substitute for nucleoli of full-grown oocytes in terms of supporting embryonic development in this study. Growing (around 100 microm in diameter) and full-grown porcine oocytes (120 microm) were collected from small (0.6-1.0 mm) and large antral follicles (4-5 mm), respectively. The nucleolus was aspirated from full-grown oocytes by micromanipulation, and the resulting enucleolated oocytes were matured to metaphase II; the nucleoli originating from full-grown and growing oocytes were then injected into the oocytes. The Chromatin of growing oocytes was aspirated with the nucleolus during the enucleolation process. Growing oocytes were thus treated with actinomycin D to release the chromatin from their nucleoli, and the nucleoli were collected and transferred to the enucleolated and matured full-grown oocytes. After activation by electro-stimulation, nucleoli were formed in pronuclei of sham-operated oocytes. Enucleolated oocytes that had been injected with nucleoli from either full-grown or growing, however, did not form any nucleoli in the pronuclei. No enucleolated oocytes developed to blastocysts, whereas enucleolated oocytes injected with nucleoli from full-grown oocytes (15%) or growing oocytes (18%) developed to blastocysts. These results indicate that the nucleoli from growing oocytes can substitute for nucleoli from full-grown oocytes during early embryonic development. (c) 2009 Wiley-Liss, Inc.

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive.

Science.gov (United States)

Muro, Eleonora; Hoang, Thang Q; Jobart-Malfait, Aude; Hernandez-Verdun, Danièle

2008-05-01

The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.

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

Energy Technology Data Exchange (ETDEWEB)

Magalhaes, M.C.; Vitor, A.B.; Magalhaes, M.M.

1986-01-01

The effect of ACTH on the RNA synthesis in adrenal zona fasciculata cells of the young rat were studied by light and electron microscope radioautography. Two units of ACTH were administered sc to animals and immediately followed by an iv injection of (/sup 3/)uridine. ACTH-injected and control rats, which received the isotope alone, were sacrificed at various time intervals. Labelling over extranucleolar areas was higher in the ACTH-treated animals at 20 min, then becoming lower than in the controls at 60 min and 24 h. Nucleolar radioactivity, however, was consistently decreased by ACTH at all experimental times. Apart from these changes in the rate of synthesis, the over-all curves of labelling were similar to those in the control animals with a striking peak at 1 h. The short-term increase in extranucleolar RNA synthesis observed after ACTH injection was considered to be consistent with the hypothesis that an enhanced extranucleolar synthesis of mRNA takes place early in stimulated animals and is associated with the synthesis of steroidogenic proteins. On the other hand, the relatively decreased uridine uptake of the label by the nucleolus in ACTH-treated animals, suggests an inhibition of nucleolar transcription with diminished pre-rRNA formation in treated animals.

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

International Nuclear Information System (INIS)

Magalhaes, M.C.; Vitor, A.B.; Magalhaes, M.M.

1986-01-01

The effect of ACTH on the RNA synthesis in adrenal zona fasciculata cells of the young rat were studied by light and electron microscope radioautography. Two units of ACTH were administered sc to animals and immediately followed by an iv injection of [ 3 ]uridine. ACTH-injected and control rats, which received the isotope alone, were sacrificed at various time intervals. Labelling over extranucleolar areas was higher in the ACTH-treated animals at 20 min, then becoming lower than in the controls at 60 min and 24 h. Nucleolar radioactivity, however, was consistently decreased by ACTH at all experimental times. Apart from these changes in the rate of synthesis, the over-all curves of labelling were similar to those in the control animals with a striking peak at 1 h. The short-term increase in extranucleolar RNA synthesis observed after ACTH injection was considered to be consistent with the hypothesis that an enhanced extranucleolar synthesis of mRNA takes place early in stimulated animals and is associated with the synthesis of steroidogenic proteins. On the other hand, the relatively decreased uridine uptake of the label by the nucleolus in ACTH-treated animals, suggests an inhibition of nucleolar transcription with diminished pre-rRNA formation in treated animals. (author)

Cytochemical study of the nucleolus of the slime mold Dictyostelium discoideum

International Nuclear Information System (INIS)

Benichou, J.C.; Quiviger, B.; Ryter, A.

1983-01-01

The nucleus of the slime mold Dictyostelium discoideum is characterized by the presence of several large dense masses which are all in tight contact with the nuclear membrane. These dense masses, considered as nucleoli, present a rather homogeneous texture, in which dense chromatin, fibrillar, and granular material are not easily detected. The autoradiographic study of [ 3 H]uridine pulse-labeled cells showed that the majority of the silver grains were located inside these masses. The use of EDTA regressive-staining, acetylation and enzymatic digestion indicated that they are mostly composed of RNP and are totally devoid of dense chromatin as the rest of the nucleus is. After treatment with actinomycin D, fibrillar and granular material segregated but no chromatin could be found. All these observations confirmed that the dense masses correspond to nucleoli despite their peculiar ultrastructure. It can also be concluded that this type of nucleoli cannot be considered as a taxonomic character of the slime molds because it does not exist in all slime molds and was observed in some dinoflagellates, and ascomycetes

In situ hybridization of iodinated 5S and 18/25S RNA to Vicia faba metaphase chromosomes

International Nuclear Information System (INIS)

Schubert, I.; Baeumlein, H.; Wobus, U.

1978-01-01

In vitro labelled 125 I ribosomal RNA fractions (18/25S and 5S) were in situ hybridized to metaphase chromosomes of a reconstructed karyotype of Vicia faba (characterized by two translocations and one pericentric inversion, each being present homozygously). The sites of 18S and 25S RNA were found to be confined to the nucleolus organizing secondary constriction. Two loci of 5S RNA were recognized on the satellite of nucleolus bearing chromosome. Possible correlations between the location of ribosomal genes, heterochromatic G-bands and clusters of mutagen induced chromatid aberrations are discussed. (author)

Effects of aluminum on nucleoli in root tip cells and selected physiological and biochemical characters in Allium cepa var. agrogarum L.

Science.gov (United States)

Qin, Rong; Jiao, Yunqiu; Zhang, Shanshan; Jiang, Wusheng; Liu, Donghua

2010-10-21

Increased Al concentration causes reduction of mitotic activity, induction of nucleolar alteration, increase of the production of ROS and alteration of several antioxidant enzyme activities in plant cells. Allium cepa is an excellent plant and a useful biomarker for environmental monitoring. Limited information is available about the effects of Al on nucleoli, antioxidant enzyme system, contents of MDA and soluble protein in A. cepa. Therefore, we carried out the investigation in order to better understand the effects of Al on the growth, nucleoli in root tip cells and selected physiological and biochemical characters. The results showed that the root growth exposed to 50 μM Al was inhibited significantly. 50 μM Al could induce some particles of argyrophilic proteins scattered in the nuclei and extruded from the nucleoli into the cytoplasm. The nucleolus did not disaggregate normally and still remained its characteristic structure during metaphase. Nucleolar reconstruction was inhibited. 50 μM Al induced high activities of SOD and POD in leaves and roots significantly (P nucleoli and the alterations of antioxidant enzyme activities, MDA and soluble protein contents in Allium cepa can serve as useful biomarkers, which can provide valuable information for monitoring and forecasting effects of exposure to Al in real scenarios conditions. Among the antioxidant enzymes SOD and POD appear to play a key role in the antioxidant defense mechanism under Al toxicity condition. Data from MDA concentration show that Al indirectly produces superoxide radicals, resulting in increased lipid peroxidative products and oxidative stress.

Nucleolin is regulated both at the level of transcription and translation

International Nuclear Information System (INIS)

Bicknell, Katrina; Brooks, Gavin; Kaiser, Pete; Chen Hongying; Dove, Brian K.; Hiscox, Julian A.

2005-01-01

Nucleolin is a multi-functional protein that is located to the nucleolus. In tissue culture cells, the stability of nucleolin is related to the proliferation status of the cell. During development, rat cardiomyocytes proliferate actively with increases in the mass of the heart being due to both hyperplasia and hypertrophy. The timing of this shift in the phenotype of the myocyte from one capable of undergoing hyperplasia to one that can grow only by hypertrophy occurs within 4 days of post-natal development. Thus, cardiomyocytes are an ideal model system in which to study the regulation of nucleolin during growth in vivo. Using Western blot and quantitative RT-PCR (TaqMan) we found that the amount of nucleolin is regulated both at the level of transcription and translation during the development of the cardiomyocyte. However, in cells which had exited the cell cycle and were subsequently given a hypertrophic stimulus, nucleolin was regulated post-transcriptionally

Effects of growth hormone on morphology of cardiac muscle and skeletal muscle and hormone levels in rats

International Nuclear Information System (INIS)

Yang Ping; Liu Cong; Meng Fanbo; Zhu Jinming; Ni Jinsong; Zhou Hong; Tang Yubo

2005-01-01

Objective: To study the effects of growth hormone (GH) on morphology of cardiac muscle and skeletal muscle and hormone levels in Wistar rats. Methods: The GH was given with subcutaneous injection for 15 days, the level of serum GH was determined by radiation-immune method; the body weight and the ratio of organ weight to body weight were determined; the cell appearances of cardiac muscle and skeletal muscle were observed under microscope. the control group was set up. Results; The level of serum GH and rat body weight in experimental group were obviously higher than that in the control group, but the ratio of organ weight to body weight was not obviously different in two groups; musculature hypertrophy and cell nucleolus increasing were observed under microscopy, there were no capillary vessel hyperplasia and inflammatory soakage. Conclusion: GH can induce hypertrophy of cardiac muscle cells and skeletal muscle cells but not interstitial proliferation. (authors)

The influence of ionizing irradiation on functional state of chromatin neurons in mice living at the territory of the Chernobylsk NPP

International Nuclear Information System (INIS)

Mustafin, A.G.; Yarygin, V.N.; Vakhtel', N.M.; Bibaeva, L.V.

1996-01-01

Daily variations in activity of endo-RNA-polymerases were studied in neurocyte nuclei of the L v -cerebrospinal node, α-motoneurons of spinal cord, neurons of sensomotor and visual areas of the mice crust, subjected to radiation impact in the Chernobyl NPP zone. Changes in circadian time organization of nerve cells genetic apparatus functioning in various nerve system sections were observed in the irradiated mice. Shift of acrophases of nucleoplasmatic and nucleolus tracing was observed during daily cycle after 40-day exposure under conditions of increased radioactive background. Degree of synchronization of ribosome and structural genes transcription in the studied cell populations decreased. Mesors and amplitudes of circadian rhythms of chromatin matrix activity was reduced. The chromatin histon state in irradiated animals also changed. 8 refs.; 2 tabs

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods.

Science.gov (United States)

Goto, N

1987-09-01

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.

Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA of Haliotis discus hannai from Fujian, China.

Directory of Open Access Journals (Sweden)

Haishan Wang

Full Text Available We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82% and two rare types 2n = 36 = 11M + 7SM (14% and 2n = 36 = 10M + 7SM + 1ST (4%. The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA3 displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.

Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

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Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

2013-03-15

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

International Nuclear Information System (INIS)

Grierson, Patrick M.; Acharya, Samir; Groden, Joanna

2013-01-01

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription

Morphological and physiological aspects of the salivary gland chromosomes of Rhynchosciara in vitro

Energy Technology Data Exchange (ETDEWEB)

Simoes, L.C.G.; Cestari, A.N.; Uemura, G. (Sao Paulo Univ. (Brazil). Inst. de Biociencias)

1981-09-01

The behavior of the salivary gland chromosomes of Rhynchosciara americana was investigated under in vitro conditions for different periods of time. The salivary glands were incubated in a chemically defined medium supplemented with calf serum. The incubation of the salivary glands lasted from a few minutes to 30 days. RNA synthesis was studied by means of autoradiography and the DNA synthesis was studied both by autoradiography and liquid scintillation spectrometry. Puffing activity under in vitro conditions was studied as well as the activity of the nucleolus. The results suggest that the activity of the nucleolus and that of some of the RNA puffs studied is cyclical under in vitro conditions.

[Morpho-functional parameters of nucleoli in polyploid mucous and albumen cells of salivary gland in the snail Succinea lauta].

Science.gov (United States)

Anisimova, A A; Anisimov, A P

2005-01-01

Variation of some characteristics of nucleoli of polyploid mucous and albumen cells was examined in salivary glands of the snail Succinea lauta. The number, total area and Ag-protein content of nucleoli, and DNA content in each nucleus were estimated on squashed preparations incubated with AgNO3, decolorized and then Feulgen stained. The ultrastructure of nucleoli was studied by electron microscopy. Differentiated mucous cells had 4c-8c-16c-32c nuclei; albumen cells had 8c-16c-32c-64c-128c nuclei. The ultrastructure of nucleoli of the two cell types was essentially the same. Normally, a large fibrous to granular zone was observed in the nucleoli, without a clear distinction between fibrous and granular components. At the same time, aggregations of granular matter could be discerned at the periphery of nucleoli. No fibrous centers were observed. Occassionally, nucleolonema-like structures occurred. Normally each nucleolus contacted several chromosomes. On squashed preparations, the least size of nucleoli was 2-3 microm, and the largest size amounted to 14 microm in mucous cells, and to 50-80 microm in albumen cells. The number of nucleoli rose from 1-2 in tetraploid nuclei to 2-3 in 32c-nuclei, and to 5-7 in 128c-nuclei. The disparity between the ploidy levels of nuclei and the numbers of nucleoli may be due, presumably, to aggregation of chromosome NORs. The Ag-protein content in the nucleoli, and the total nucleolar area displayed a strong mutual correlation. Both parameters differed significantly by 1.5-2.2 times in mucous and albumen cells of the same ploidy level. Thus, in albumen and mucous cells the total Ag-protein content in octaploid nuclei was 3.3 and 2.2 relative units (r. u.), respectively. In 16c- and 32c-nuclei of albumen cells, it was 7.6 and 15.1 r. u.; and in the same nuclei of mucous cells--3.8 and 6.8 r. u., respectively. On the whole, in albumen cells, in the course of 4 endocycles (4c-128c), the total Ag-protein content increased by 17 times

The effects of acute morphine treatment on the incorporation of [3H]L-lysine by normal and regenerating facial nucleus neurons

International Nuclear Information System (INIS)

Sinatra, R.S.; Ford, D.H.; Rhines, R.K.

1979-01-01

The effect of morphine on the incorporation of [ 3 H]L-lysine into proteins of facial nucleus neurons was examined by light microscopic radioautography. Silver grains present within various compartments of normal and regenerating (3-, 7-. 14- and 21 days post-axotomy) neurons from saline-treated Wistar rats were compared with the amount present in similar cells from animals receiving 40 mg/kg morphine sulfate i.v. At 14- and 21-days post-axotomy, regenerating neurons were larger and the grain count in the emulsion over these cells was greater than that observed in normal (unoperated) neurons. In normal facial neurons, the accumulation of lysine into the nucleus and nucleolus was significantly lower 60 min after morphine adminstration. However, morphine's inhibition of lysine incorporation was even more pronounced in regenerating neurons. In these cells, nuclear lysine uptake was depressed at 3 and 7 days, while maximum inhibition of cytoplasmic incorporation occurred at 14-days post-axotomy. Morphine adminstration decreased nucleolar lysine incorporation at all survival intervals. (Auth.)

Microprobe PIXE analysis and EDX analysis on the brain of patients with Alzheimer`s disease

Energy Technology Data Exchange (ETDEWEB)

Yumoto, S. [Tokyo Univ. (Japan). Faculty of Medicine; Horino, Y.; Mokuno, Y.; Fujii, K.; Kakimi, S.; Mizutani, T.; Matsushima, H.; Ishikawa, A.

1996-12-31

To investigate the cause of Alzheimer`s disease (senile dementia of Alzheimer`s disease type), we examined aluminium (Al) in the brain (hippocampus) of patients with Alzheimer`s disease using heavy ion (5 MeV Si{sup 3+}) microprobe particle-induced X-ray emission (PIXE) analysis. Heavy ion microprobes (3 MeV Si{sup 2+}) have several times higher sensitivity for Al detection than 2 MeV proton microprobes. We also examined Al in the brain of these patients by energy dispersive X-ray spectroscopy (EDX). (1) Al was detected in the cell nuclei isolated from the brain of patients with Alzheimer`s disease using 5 MeV Si{sup 3+} microprobe PIXE analysis, and EDX analysis. (2) EDX analysis demonstrated high levels of Al in the nucleolus of nerve cells in frozen sections prepared from the brain of these patients. Our results support the theory that Alzheimer`s disease is caused by accumulation of Al in the nuclei of brain cells. (author)

Gemistocytic astrocytoma in the spinal cord in a dog: a case report

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R.O. Chaves

2016-08-01

Full Text Available ABSTRACT This paper reports a case of a rare variant of the cervical spinal cord astrocytoma diagnosed in a dog with progressive neurological signs, initially asymmetrical, not ambulatory tetraparesis, segmental reflexes and normal muscle tone in all four limbs and absence of pain upon palpation of the cervical spine. Myelography revealed attenuation of the ventral and dorsal contrast line in the third region of the fifth cervical vertebra. At necropsy intramedullary cylindrical mass that stretched from the third to the sixth cervical vertebra, which replaced all the gray matter of the spinal cord was observed. In the histological study, there was the replacement of the substance by neoplastic cells mantle arranged loosely. The cells were large and slightly rounded. The eosinophilic cytoplasm was well defined, sometimes forming processes interconnecting cells. The nucleus was eccentric, round, oval or kidney-shaped, and the nucleolus was evident. Thus, the microscopic changes observed in the cervical spinal cord were consistent with gemistocytic astrocytoma.

Microprobe PIXE analysis and EDX analysis on the brain of patients with Alzheimer's disease

International Nuclear Information System (INIS)

Yumoto, S.; Horino, Y.; Mokuno, Y.; Fujii, K.; Kakimi, S.; Mizutani, T.; Matsushima, H.; Ishikawa, A.

1996-01-01

To investigate the cause of Alzheimer's disease (senile dementia of Alzheimer's disease type), we examined aluminium (Al) in the brain (hippocampus) of patients with Alzheimer's disease using heavy ion (5 MeV Si 3+ ) microprobe particle-induced X-ray emission (PIXE) analysis. Heavy ion microprobes (3 MeV Si 2+ ) have several times higher sensitivity for Al detection than 2 MeV proton microprobes. We also examined Al in the brain of these patients by energy dispersive X-ray spectroscopy (EDX). (1) Al was detected in the cell nuclei isolated from the brain of patients with Alzheimer's disease using 5 MeV Si 3+ microprobe PIXE analysis, and EDX analysis. (2) EDX analysis demonstrated high levels of Al in the nucleolus of nerve cells in frozen sections prepared from the brain of these patients. Our results support the theory that Alzheimer's disease is caused by accumulation of Al in the nuclei of brain cells. (author)

Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing.

Science.gov (United States)

Liang, Xue-Hai; Crooke, Stanley T

2011-06-01

Little is known about whether components of the RNA-induced silencing complex (RISC) mediate the biogenesis of RNAs other than miRNA. Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells. In cells depleted of Drosha or Dicer, different precursors to 5.8S rRNA strongly accumulated, without affecting normal endonucleolytic cleavages. Moderate yet distinct processing defects were also observed in Ago2-depleted cells. Physical links between pre-rRNA and these proteins were identified by co-immunoprecipitation analyses. Interestingly, simultaneous depletion of Dicer and Drosha led to a different processing defect, causing slower production of 28S rRNA and its precursor. Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs. Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.

Morphological and physiological aspects of the salivary gland chromosomes of Rhynchosciara in vitro

International Nuclear Information System (INIS)

Simoes, L.C.G.; Cestari, A.N.; Uemura, G.

1981-01-01

The behavior of the salivary gland chromosomes of Rhynchosciara americana was investigated under in vitro conditions for different periods of time. The salivary glands were incubated in a chemically defined medium supplemented with calf serum. The incubation of the salivary glands lasted from a few minutes to 30 days. RNA synthesis was studied by means of autoradiography and the DNA synthesis was studied both by autoradiography and liquid scintillation spectrometry. Puffing activity under in vitro conditions was studied as well as the activity of the nucleolus. The results suggest that the activity of the nucleolus and that of some of the RNA puffs studied is cyclical under in vitro conditions. (Author) [pt

Internal Associations of the Acidic Region of Upstream Binding Factor Control Its Nucleolar Localization.

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Ueshima, Shuhei; Nagata, Kyosuke; Okuwaki, Mitsuru

2017-11-15

Upstream binding factor (UBF) is a member of the high-mobility group (HMG) box protein family, characterized by multiple HMG boxes and a C-terminal acidic region (AR). UBF is an essential transcription factor for rRNA genes and mediates the formation of transcriptionally active chromatin in the nucleolus. However, it remains unknown how UBF is specifically localized to the nucleolus. Here, we examined the molecular mechanisms that localize UBF to the nucleolus. We found that the first HMG box (HMG box 1), the linker region (LR), and the AR cooperatively regulate the nucleolar localization of UBF1. We demonstrated that the AR intramolecularly associates with and attenuates the DNA binding activity of HMG boxes and confers the structured DNA preference to HMG box 1. In contrast, the LR was found to serve as a nuclear localization signal and compete with HMG boxes to bind the AR, permitting nucleolar localization of UBF1. The LR sequence binds DNA and assists the stable chromatin binding of UBF. We also showed that the phosphorylation status of the AR does not clearly affect the localization of UBF1. Our results strongly suggest that associations of the AR with HMG boxes and the LR regulate UBF nucleolar localization. Copyright © 2017 American Society for Microbiology.

High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

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van Koningsbruggen, Silvana; Gierlinski, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J; Ariyurek, Yavuz; den Dunnen, Johan T; Lamond, Angus I

2010-11-01

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

Localization of SERBP1 in stress granules and nucleoli.

Science.gov (United States)

Lee, Yu-Jen; Wei, Hung-Ming; Chen, Ling-Yun; Li, Chuan

2014-01-01

SERPINE1 mRNA-binding protein 1 (SERBP1) is an arginine-methylated RNA-binding protein whose modification affects protein interaction and intracellular localization. In the present study, we show that, under normal growth conditions without stress, SERBP1 interacts with arginine-methylated and stress granule-associated proteins such as heterogeneous nuclear ribonucleoprotein A1, fragile X mental retardation protein and fragile X mental retardation syndrome-related protein 1 in an RNA-dependent manner. We also show that, after arsenite treatment, a proportion of full-length SERBP1 protein co-localizes with the typical stress granule marker T-cell intracellular antigen-1 in the cytoplasmic stress granules. Truncated SERBP1 with an N-terminal, central RG or C-terminal deletion, or single-domain segments comprising the N-terminal, central or C-terminal region, were recruited to stress granules upon arsenite treatment but with reduced efficiency. In addition, upon arsenite treatment, the localization of SERBP1 changed from a diffuse cytoplasmic localization to nuclear-dominant (concentrated in the nucleolus) A similar distribution was observed when cells were treated with the methylation inhibitor adenosine periodate, and was also detected for N- or C-terminal domain deletions and all three single-domain fragments even without stress induction. We further demonstrate that adenosine periodate treatment delays the association/dissociation of SERBP1 with stress granules. Hypomethylation retains SERBP1 in the nucleus/nucleolus regardless of arsenite treatment. Our study indicates that arginine methylation is correlated with recruitment of SERBP to stress granules and nucleoli and its retention therein. To our knowledge, this is the first report of an RNA-binding protein that is shifted simultaneously to cytoplasmic stress granules and nucleoli, two ribonucleoprotein-enriched subcellular compartments, upon stress. © 2013 FEBS.

RINT-1 interacts with MSP58 within nucleoli and plays a role in ribosomal gene transcription

International Nuclear Information System (INIS)

Yang, Chuan-Pin; Kuo, Yu-Liang; Lee, Yi-Chao; Lee, Kuen-Haur; Chiang, Chi-Wu; Wang, Ju-Ming; Hsu, Che-Chia

2016-01-01

The nucleolus is the cellular site of ribosomal (r)DNA transcription and ribosome biogenesis. The 58-kDa microspherule protein (MSP58) is a nucleolar protein involved in rDNA transcription and cell proliferation. However, regulation of MSP58-mediated rDNA transcription remains unknown. Using a yeast two-hybrid system with MSP58 as bait, we isolated complementary (c)DNA encoding Rad50-interacting protein 1 (RINT-1), as a MSP58-binding protein. RINT-1 was implicated in the cell cycle checkpoint, membrane trafficking, Golgi apparatus and centrosome dynamic integrity, and telomere length control. Both in vitro and in vivo interaction assays showed that MSP58 directly interacts with RINT-1. Interestingly, microscopic studies revealed the co-localization of MSP58, RINT-1, and the upstream binding factor (UBF), a rRNA transcription factor, in the nucleolus. We showed that ectopic expression of MSP58 or RINT-1 resulted in decreased rRNA expression and rDNA promoter activity, whereas knockdown of MSP58 or RINT-1 by siRNA exerted the opposite effect. Coexpression of MSP58 and RINT-1 robustly decreased rRNA synthesis compared to overexpression of either protein alone, whereas depletion of RINT-1 from MSP58-transfected cells enhanced rRNA synthesis. We also found that MSP58, RINT-1, and the UBF were associated with the rDNA promoter using a chromatin immunoprecipitation assay. Because aberrant ribosome biogenesis contributes to neoplastic transformation, our results revealed a novel protein complex involved in the regulation of rRNA gene expression, suggesting a role for MSP58 and RINT-1 in cancer development. - Highlights: • RINT-1 is a novel MSP58-interacting protein. • RINT-1 is a nucleolar protein that suppresses ribosomal RNA gene transcription. • RINT-1 and MSP58 cooperate to suppress ribosomal RNA gene transcription. • RINT-1, MSP58, and UBF form a complex on the rDNA promoter.

Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

International Nuclear Information System (INIS)

Kumari, Gita; Mahalingam, S.

2009-01-01

Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

Energy Technology Data Exchange (ETDEWEB)

Kumari, Gita [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Mahalingam, S., E-mail: mahalingam@iitm.ac.in [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Department of Biotechnology, Laboratory of Molecular Virology and Cell Biology, Indian Institute of Technology-Madras, Chennai 600 036 (India)

2009-10-01

Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

The ultrastructure of the mature embryo sac in the natural tetraploid of red clover (Trifolium pratense L.: that has a very low rate of seed formation

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Gönül Algan

2014-01-01

Full Text Available In this study, ultrastructural organization of cells in the mature embryo sac of natural tetraploid Trifolium pratense L. was investigated. The mature embryo sac of this plant contains an egg cell with two synergids at the micropylar end, and a central cell with two polar nuclei. The ultrastructure of these cells agrees with what is known for most angiosperms studied with the electron microscope. The egg cell is a large and highly vacuolate cell, partially surrounded by a wall. Much of the cytoplasm is located around the nucleus at the chalazal end and there are few numbers of channel-shaped endoplasmic reticulum, mitochondria, plastids and numerous ribosomes distributed throughout the cytoplasm. Unlike the egg cell, much of the cytoplasm in synergid cells is located at micropylar part of the cell and the synergid cytoplasm contains especially, large numbers of rough endoplasmic reticulum, free ribosomes, mitochondria and plastids. The central cell of T. pratense L. contains two large polar nuclei which lie close to the egg apparatus. Each polar nucleus has a single, large, dense nucleolus that contains several nucleolar vacuoles. Much of the central cell cytoplasm consisting of granular and agranular endoplasmic reticulum, mitochondria, plastids, ribosomes, dictyosomes and lipid bodies are placed around polar nuclei.

Progress in studies on nucleolus functions

International Nuclear Information System (INIS)

Chang Lei; Zhou Guangming

2011-01-01

Nucleoli is the sites for ribosome synthesis and processing, however, recent approaches have revealed that it is also involved in variety of cellular processes, especially the cellular stress response. As sensors, nucleoli regulate the localization of nucleolar proteins, such as (Alternate Reading Frame, ARF), and the activation of key factors, such as P53, and consequently mediate the cellular stress response.In this paper, recent progress in the studies on nucleolar functions in cellular stress response to radiation is reviewed. (authors)

On the Nucleolus of Neighbour Games

NARCIS (Netherlands)

Hamers, H.J.M.; Klijn, F.; Solymosi, T.; Tijs, S.H.; Vermeulen, D.

1999-01-01

The class of neighbour games is the intersection of the class of assignment games (cf. Shapley and Shubik (1972)) and the class of component additive games (cf. Curiel et al. (1994)). For assignment games and component additive games there exist polynomially bounded algorithms of order p4 for

Cytochemical and autoradiographic studies of meiosis and microsporanogenesis in tradescantia paludosa

International Nuclear Information System (INIS)

Dryanovska, O.

1981-01-01

Labelling experiments with H 3 -thymidine, H 3 -uridine and H 3 -leucine have been carried out with Tradescantia paludosa. The results of this study showed that from pre-meiosis to the pollen grain the chromatin, the nucleoli and the cytoplasm undergo alternative structural, cytochemical and functional changes connected with the differential functioning of the genes and the differentiation of the cells. Chromatin condensation is connected with DNA synthesis only in the pre-meiosis and in the microspore. Decondensation is connected with despiralization and with slight heterochromatization of the chromatin, with development and functioning of the nucleoli, slight DNA synthesis and intense synthesis of RNAs and proteins. Probably the nucleolus in meiosis is playing a certain role. (author)

Hard X-ray submicrometer tomography of human brain tissue at Diamond Light Source

Science.gov (United States)

Khimchenko, A.; Bikis, C.; Schulz, G.; Zdora, M.-C.; Zanette, I.; Vila-Comamala, J.; Schweighauser, G.; Hench, J.; Hieber, S. E.; Deyhle, H.; Thalmann, P.; Müller, B.

2017-06-01

There is a lack of the necessary methodology for three-dimensional (3D) investigation of soft tissues with cellular resolution without staining or tissue transformation. Synchrotron radiation based hard X-ray in-line phase contrast tomography using single-distance phase reconstruction (SDPR) provides high spatial resolution and density contrast for the visualization of individual cells using a standard specimen preparation and data reconstruction. In this study, we demonstrate the 3D characterization of a formalin-fixed paraffin-embedded (FFPE) human cerebellum specimen by SDPR at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, UK) at pixel sizes down to 0.45 μm. The approach enables visualization of cerebellar layers (Stratum moleculare and Stratum granulosum), the 3D characterization of individual cells (Purkinje, stellate and granule cells) and can even resolve some subcellular structures (nucleus and nucleolus of Purkinje cells). The tomographic results are qualitatively compared to hematoxylin and eosin (H&E) stained histological sections. We demonstrate the potential benefits of hard X-ray microtomography for the investigations of biological tissues in comparison to conventional histology.

Hard X-ray submicrometer tomography of human brain tissue at Diamond Light Source

International Nuclear Information System (INIS)

Khimchenko, A; Bikis, C; Schulz, G; Hieber, S E; Deyhle, H; Thalmann, P; Müller, B; Zdora, M-C; Zanette, I; Vila-Comamala, J; Schweighauser, G; Hench, J

2017-01-01

There is a lack of the necessary methodology for three-dimensional (3D) investigation of soft tissues with cellular resolution without staining or tissue transformation. Synchrotron radiation based hard X-ray in-line phase contrast tomography using single-distance phase reconstruction (SDPR) provides high spatial resolution and density contrast for the visualization of individual cells using a standard specimen preparation and data reconstruction. In this study, we demonstrate the 3D characterization of a formalin-fixed paraffin-embedded (FFPE) human cerebellum specimen by SDPR at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, UK) at pixel sizes down to 0.45 μm. The approach enables visualization of cerebellar layers ( Stratum moleculare and Stratum granulosum ), the 3D characterization of individual cells (Purkinje, stellate and granule cells) and can even resolve some subcellular structures (nucleus and nucleolus of Purkinje cells). The tomographic results are qualitatively compared to hematoxylin and eosin (H and E) stained histological sections. We demonstrate the potential benefits of hard X-ray microtomography for the investigations of biological tissues in comparison to conventional histology. (paper)

Critical pathogenic steps to high risk Helicobacter pylori gastritis and gastric carcinogenesis.

Science.gov (United States)

Lee, Inchul

2014-06-07

Helicobacter pylori (H. pylori) gastritis may progress to high risk gastropathy and cancer. However, the pathological progression has not been characterized in detail. H. pylori induce persistent inflammatory infiltration. Neutrophils are unique in that they directly infiltrate into foveolar epithelium aiming the proliferative zone specifically. Neutrophilic proliferative zone foveolitis is a critical pathogenic step in H. pylori gastritis inducing intensive epithelial damage. Epithelial cells carrying accumulated genomic damage and mutations show the Malgun (clear) cell change, characterized by large clear nucleus and prominent nucleolus. Malgun cells further undergo atypical changes, showing nuclear folding, coarse chromatin, and multiple nucleoli. The atypical Malgun cell (AMC) change is a novel premalignant condition in high risk gastropathy, which may progress and undergo malignant transformation directly. The pathobiological significance of AMC in gastric carcinogenesis is reviewed. A new diagnosis system of gastritis is proposed based on the critical pathologic steps classifying low and high risk gastritis for separate treatment modality. It is suggested that the regulation of H. pylori-induced neutrophilic foveolitis might be a future therapeutic goal replacing bactericidal antibiotics approach.

Karyopherin alpha2 is essential for rRNA transcription and protein synthesis in proliferative keratinocytes.

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Noriko Umegaki-Arao

Full Text Available Karyopherin proteins mediate nucleocytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest KPNA2 expression is induced in tumor cells and is strongly associated with prognosis, although the precise roles and mechanisms of KPNA2 overexpression in proliferative disorders have not been defined. We found that KPNA2 expression is induced in various proliferative disorders of the skin such as psoriasis, Bowen's disease, actinic keratosis, squamous cell carcinoma, Paget's disease, Merkel cell carcinoma, and mycosis fungoides. siRNA-mediated KPNA suppression revealed that KPNA2 is essential for significant suppression of HaCaT proliferation under starvation conditions. Ribosomal RNA transcription and protein synthesis were suppressed by starvation combined with knockdown of KPNA (including KPNA2 expression. KPNA2 localized to the nucleolus and interacted with proteins associated with mRNA processing, ribonucleoprotein complex biogenesis, chromatin modification, and transcription, as demonstrated by tandem affinity purification and mass spectrometry. KPNA2 may be an important promoter of ribosomal RNA and protein synthesis in tumor cells.

Mitotic effects of monochromatic ultraviolet radiation at 225, 265, and 280 nm on eleven stages of the cell cycle of the grasshopper neuroblast in culture. I. Overall retardation from the stage irradiated to nuclear membrane breakdown

International Nuclear Information System (INIS)

Carlson, J.G.

1976-01-01

Neuroblasts of Chortophaga viridifasciata (DeGeer) in culture were exposed to different doses of 225, 265, or 280 nm ultraviolet radiations at 11 different stages and substages of the mitotic cycle and individually selected cells were timed to breakdown of the nuclear membrane. Comparisons of the effectiveness of different wavelengths on the different stages were based on the dose that reduced the cell progression rate to 67 percent of normal (D 67 ) and the slope of the regression line, i.e., the control to treated time (C/T) ratio change/erg/mm 2 at the D 67 level. Cells of the prereplication period (metaphase + anaphase + early telophase) and the S phase (middle and late telophase + interphase + very early prophase) are equally sensitive to uv and contrast sharply with the much lower sensitivity of those in the postreplication period (early and middle prophase). This can best be interpreted if chromosomal DNA is the chromophore for uv-induced mitotic retardation. Cells in the prereplication period at exposure show no wavelength effect. In the S phase all stages except middle telophase and all stages combined are significantly more sensitive to 265 and 280 nm than to 225 nm. Of the postreplication stages, early prophase is retarded significantly more by 280 than by 225 or 265 nm. The C/T ratio/erg/mm 2 is greater after exposure to 265 nm at all prereplication and replication stages, but exhibits no consistent wavelength pattern during the postreplication period. Evidence based on the orientation of the neuroblast with respect to the uv-source suggests that the chromophore for mitotic retardation does not reside within the centrosome and related structures, but may be present, at least partly, in the nucleolus

Plasma cell morphology in multiple myeloma and related disorders.

Science.gov (United States)

Ribourtout, B; Zandecki, M

2015-06-01

Normal and reactive plasma cells (PC) are easy to ascertain on human bone marrow films, due to their small mature-appearing nucleus and large cytoplasm, the latter usually deep blue after Giemsa staining. Cytoplasm is filled with long strands of rough endoplasmic reticulum and one large Golgi apparatus (paranuclear hof), demonstrating that PC are dedicated mainly to protein synthesis and excretion (immunoglobulin). Deregulation of the genome may induce clonal expansion of one PC that will lead to immunoglobulin overproduction and eventually to one among the so-called PC neoplasms. In multiple myeloma (MM), the number of PC is over 10% in most patients studied. Changes in the morphology of myeloma PC may be inconspicuous as compared to normal PC (30-50% patients). In other instances PC show one or several morphological changes. One is related to low amount of cytoplasm, defining lymphoplasmacytoid myeloma (10-15% patients). In other cases (40-50% patients), named immature myeloma cases, nuclear-cytoplasmic asynchrony is observed: presence of one nucleolus, finely dispersed chromatin and/or irregular nuclear contour contrast with a still large and blue (mature) cytoplasm. A peculiar morphological change, corresponding to the presence of very immature PC named plasmablasts, is observed in 10-15% cases. Several prognostic morphological classifications have been published, as mature myeloma is related to favorable outcome and immature myeloma, peculiarly plasmablastic myeloma, is related to dismal prognosis. However, such classifications are no longer included in current prognostic schemes. Changes related to the nucleus are very rare in monoclonal gammopathy of unknown significance (MGUS). In contrast, anomalies related to the cytoplasm of PC, including color (flaming cells), round inclusions (Mott cells, Russell bodies), Auer rod-like or crystalline inclusions, are reported in myeloma cases as well as in MGUS and at times in reactive disorders. They do not correspond

Nullbasic, a potent anti-HIV tat mutant, induces CRM1-dependent disruption of HIV rev trafficking.

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Min-Hsuan Lin

Full Text Available Nullbasic, a mutant of the HIV-1 Tat protein, has anti-HIV-1 activity through mechanisms that include inhibition of Rev function and redistribution of the HIV-1 Rev protein from the nucleolus to the nucleoplasm and cytoplasm. Here we investigate the mechanism of this effect for the first time, establishing that redistribution of Rev by Nullbasic is not due to direct interaction between the two proteins. Rather, Nullbasic affects subcellular localization of cellular proteins that regulate Rev trafficking. In particular, Nullbasic induced redistribution of exportin 1 (CRM1, nucleophosmin (B23 and nucleolin (C23 from the nucleolus to the nucleus when Rev was coexpressed, but never in its absence. Inhibition of the Rev:CRM1 interaction by leptomycin B or a non-interacting RevM10 mutant completely blocked redistribution of Rev by Nullbasic. Finally, Nullbasic did not inhibit importin β- or transportin 1-mediated nuclear import, suggesting that cytoplasmic accumulation of Rev was due to increased export by CRM1. Overall, our data support the conclusion that CRM1-dependent subcellular redistribution of Rev from the nucleolus by Nullbasic is not through general perturbation of either nuclear import or export. Rather, Nullbasic appears to interact with and disrupt specific components of a Rev trafficking complex required for its nucleocytoplasmic shuttling and, in particular, its nucleolar accumulation.

ORF Sequence: NC_001137 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available turation of 18S rRNA; depletion leads to inhibited pre-rRNA processing and reduced polysome levels; localizes primarily to the nucleo...lus; Lcp5p [Saccharomyces cerevisiae] MSELNALLKDINGSLTATSESLERLSGIYSNSATDEIPESNQLHE

Sequence Classification: 893067 [

Lifescience Database Archive (English)

Full Text Available tuent of 66S pre-ribosomal particles, involved in 60S ribosomal subunit biogenesis; localizes to both nucleolus and cytoplasm; Nop15p || http://www.ncbi.nlm.nih.gov/protein/6324219 ...

Sequence Classification: 890578 [

Lifescience Database Archive (English)

Full Text Available ction, localized in the nucleoplasm and the nucleolus, genetically interacts with MTR2 in 60S ribosomal protein subunit export; Ecm1p || http://www.ncbi.nlm.nih.gov/protein/6319259 ...

ORF Sequence: NC_001134 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available nd concentrated in the nucleolus; Enp1p [Saccharomyces cerevisiae] MARASSTKARKQRHDPLLKDLDAAQGTLKKINKKKLAQNDA...14 snoRNAs, required for pre-rRNA processing and 40S ribosomal subunit synthesis; localized in the nucleus a

The vehicle routing game: An application of cost allocation in a gas and oil company

Energy Technology Data Exchange (ETDEWEB)

Engevall, Stefan; Goethe-Lundgren, Maud; Vaerbrand, Peter

2000-12-01

In this article we study a cost allocation problem that arises in a distribution planning situation at the Logistics department at Norsk Hydro Olje AB. The routes from one depot during one day are considered, for which the total distribution cost is to be divided among the customers that are visited. This cost allocation problem is formulated as a vehicle routing game, allowing the use of vehicles with different capacities. Cost allocation methods based on different concepts from cooperative game theory, such as the core and the nucleolus, are discussed. A procedure that can be used to investigate whether the core is empty or not is presented, as well as a procedure to compute the nucleolus. Computational results for the Norsk Hydro case are also presented and discussed.

Cytochemical and autoradiographic studies of meiosis and microsporanogenesis in tradescantia paludosa

Energy Technology Data Exchange (ETDEWEB)

Dryanovska, O. (Bylgarska Akademiya na Naukite, Sofia. Inst. po Genetika)

1981-01-01

Labelling experiments with H/sup 3/-thymidine, H/sup 3/-uridine and H/sup 3/-leucine have been carried out with Tradescantia paludosa. The results of this study showed that from pre-meiosis to the pollen grain the chromatin, the nucleoli and the cytoplasm undergo alternative structural, cytochemical and functional changes connected with the differential functioning of the genes and the differentiation of the cells. Chromatin condensation is connected with DNA synthesis only in the pre-meiosis and in the microspore. Decondensation is connected with despiralization and with slight heterochromatization of the chromatin, with development and functioning of the nucleoli, slight DNA synthesis and intense synthesis of RNAs and proteins. Probably the nucleolus in meiosis is playing a certain role.

Age-dependent change in the morphology of nucleoli and methylation of genes of the Nucleolar Organizer Region in Japanese quail Coturnix japonica) model (Temminck and Schlegel, 1849) (Galliformes: Aves).

Science.gov (United States)

Andraszek, Katarzyna; Gryzińska, Magdalena; Wójcik, Ewa; Knaga, Sebastian; Smalec, Elżbieta

2014-01-01

Nucleoli are the product of the activity of nucleolar organizer regions (NOR) in certain chromosomes. Their main functions are the formation of ribosomal subunits from ribosomal protein molecules and the transcription of genes encoding rRNA. The aim of the study was to determine the shape of nucleoli and analyse methylation in the gene RN28S in the spermatocytes of male Japanese quail (Coturnixjaponica) in two age groups. Nucleoli were analysed in cells of the first meiotic prophase. Their number and shape were determined and they were classified as regular, irregular or defragmented. In the cells of the young birds no defragmented nucleoli were observed, with regular and irregular nucleoli accounting for 97% and 3%, respectively. In the cells of older birds no regular nucleoli were observed, while irregular and defragmented nucleoli accounted for 37% and 67%, respectively. MSP (methylation-specific PCR) showed that the gene RN28S is methylated in both 15-week-old and 52-week-old quails. In recent years an association has been established between nucleolus morphology and cellular ageing processes.

Nuclear labelling after prolonged 3H-uridine incorporation as visualized by high resolution autoradiography

International Nuclear Information System (INIS)

Fakan, S.; Bernhard, W.

1973-01-01

Localization of radioactive labeling over the nuclei of BSC 1 cells is visualized after long periods of 3 H-5-uridine incubation followed or not followed by periods of postincubation in nonradioactive medium for up to several days, using high resolution autoradiography combined with a preferential staining method for ribonucleoproteins. It is shown that when cells are labeled for 1 or 6 h with 3 H-uridine and postincubated with a nonradioactive medium up to several days, there is always some radioactivity present in the nucleolus and nucleoplasm. When sections of cells fixed after 1 h of labeling followed by 24 h of postincubation are treated with RNase, part of the radioactivity found in the nucleus disappears almost completely only after a succeeding DNase digestion. The majority of interchromatin granules are weakly labeled after most incubation times, with the label localized rather at the periphery of clusters of granules, or are unlabeled. The results are discussed in the context of recent biochemical findings. It is proposed that interchromatin granules might represent a structure containing a limited quantity of slowly labeled nuclear RNA

Crystal Structure and Regulation of the Citrus Pol III Repressor MAF1 by Auxin and Phosphorylation.

Science.gov (United States)

Soprano, Adriana Santos; Giuseppe, Priscila Oliveira de; Shimo, Hugo Massayoshi; Lima, Tatiani Brenelli; Batista, Fernanda Aparecida Heleno; Righetto, Germanna Lima; Pereira, José Geraldo de Carvalho; Granato, Daniela Campos; Nascimento, Andrey Fabricio Ziem; Gozzo, Fabio Cesar; de Oliveira, Paulo Sérgio Lopes; Figueira, Ana Carolina Migliorini; Smetana, Juliana Helena Costa; Paes Leme, Adriana Franco; Murakami, Mario Tyago; Benedetti, Celso Eduardo

2017-09-05

MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrus. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress

International Nuclear Information System (INIS)

Woo, Leslie L.; Futami, Kazunobu; Shimamoto, Akira; Furuichi, Yasuhiro; Frank, Karen M.

2006-01-01

Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear localization regions in the N-terminal region of RECQL4 and a nucleolar localization signal at amino acids 376-386. RECQL4 localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and γ irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and signaling pathways. The RECQL4 nucleolar localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1

Design and Testing of Bi-Functional, P-Loop-Targeted MDM2 Inhibitors

National Research Council Canada - National Science Library

Prives, Carol L; Stockwell, Brent R

2007-01-01

Our proposal is to design and evaluate a novel class of bifunctional MDM2 inhibitors, based on the discovery that nucleotides can bind to the P-loop of MDM2 and cause its relocalization to the nucleolus...

Design and Testing of Bi-Functional, P-Loop-Targeted MDM2 Inhibitors

National Research Council Canada - National Science Library

Prives, Carol L

2006-01-01

This proposal is to design and evaluate a novel class of bifunctional MDM2 inhibitors, based on the discovery that nucleotides can bind to the P-loop of MDM2 and cause its relocalization to the nucleolus...

Scanning transmission ion microscopy mass measurements for quantitative trace element analysis within biological samples and validation using atomic force microscopy thickness measurements

Energy Technology Data Exchange (ETDEWEB)

Deves, Guillaume [Laboratoire de chimie nucleaire analytique et bioenvironnementale, UMR 5084, CNRS-Universite de Bordeaux 1, BP 120 Chemin du solarium, F33175 Gradignan cedex (France)]. E-mail: deves@cenbg.in2p3.fr; Cohen-Bouhacina, Touria [Centre de Physique Moleculaire Optique et Hertzienne, Universite de Bordeaux 1, 351, cours de la Liberation, F33405 Talence cedex (France); Ortega, Richard [Laboratoire de chimie nucleaire analytique et bioenvironnementale, UMR 5084, CNRS-Universite de Bordeaux 1, BP 120 Chemin du solarium, F33175 Gradignan cedex (France)

2004-10-08

We used the nuclear microprobe techniques, micro-PIXE (particle-induced X-ray emission), micro-RBS (Rutherford backscattering spectrometry) and scanning transmission ion microscopy (STIM) in order to perform the characterization of trace element content and spatial distribution within biological samples (dehydrated cultured cells, tissues). The normalization of PIXE results was usually expressed in terms of sample dry mass as determined by micro-RBS recorded simultaneously to micro-PIXE. However, the main limit of RBS mass measurement is the sample mass loss occurring during irradiation and which could be up to 30% of the initial sample mass. We present here a new methodology for PIXE normalization and quantitative analysis of trace element within biological samples based on dry mass measurement performed by mean of STIM. The validation of STIM cell mass measurements was obtained in comparison with AFM sample thickness measurements. Results indicated the reliability of STIM mass measurement performed on biological samples and suggested that STIM should be performed for PIXE normalization. Further information deriving from direct confrontation of AFM and STIM analysis could as well be obtained, like in situ measurements of cell specific gravity within cells compartment (nucleolus and cytoplasm)

Novel compound heterozygous frameshift mutations of C2orf37 in a ...

Indian Academy of Sciences (India)

uterus with two thin-walled cysts in the right half of the pelvis with no ... including intellectual disability, deafness, hypogonadism, alo- pecia and diabetes ... for more study and research on the key roles played by the nucleolus in humans.

The karyotype of Festucopsis serpentini (Poaceae Triticeae) from Albania studied by banding techniques and in situ hybridization

DEFF Research Database (Denmark)

Linde-Laursen, I.; Seberg, O.; Frederiksen, S.

1996-01-01

-chromosomes, a metacentric and a submetacentric pair. SAT-chromosomes from one population carried exclusively minute satellites, whereas SAT-chromosomes from another population also carried larger polymorphic satellites, suggesting a geographical differentiation. The existence of four chromosomes with nucleolus forming...

Visualization of the Nucleolus Using Ethynyl Uridine

Czech Academy of Sciences Publication Activity Database

Dvořáčková, M.; Fajkus, Jiří

2018-01-01

Roč. 9, FEB2018 (2018) ISSN 1664-462X Institutional support: RVO:68081707 Keywords : dense fibrillar components * ribosomal-rna * actinomycin-d * transcription sites Subject RIV: EF - Botanics OBOR OECD: Plant science s, botany Impact factor: 4.298, year: 2016

Perturbation of Ribosome Biogenesis Drives Cells into Senescence through 5S RNP-Mediated p53 Activation

Directory of Open Access Journals (Sweden)

Kazuho Nishimura

2015-03-01

Full Text Available The 5S ribonucleoprotein particle (RNP complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses.

Ribosomal genes as early targets of cadmium-induced toxicity in Chironomus riparius larvae

International Nuclear Information System (INIS)

Planello, R.; Martinez-Guitarte, J.L.; Morcillo, G.

2007-01-01

Cadmium is a widespread environmental pollutant that causes severe impacts in organisms. Although the effects of cadmium on aquatic insects have been studied in terms of their toxicity and changes in developmental parameters, little is known about its molecular and genetic effects. We have investigated the alterations in the pattern of gene expression provoked by acute exposure to cadmium in Chironomus riparius Mg. (Diptera, Chironomidae), a sentinel organism widely used in aquatic toxicity testing. The early cytotoxic effects were evaluated using immunocytochemistry and specific fluorescent probes in fourth instar larvae after 12 h of 10 mM cadmium treatments; under these conditions no significant effect on larvae mortality was detected until after 36 h of exposure. The changes in the pattern of gene expression were analysed by means of DNA/RNA hybrid antibodies in the polytene chromosomes from salivary gland cells. A decrease in the activity of the nucleolus is especially remarkable, accompanied by a significant reduction in size and the modification in nucleolar architecture, as shown by FISH. The inhibition of rDNA transcription was further evaluated by Northern blot analysis, which showed a marked decrease in the level of preribosomal rRNA (54% 45S 12 h). However, the BR genes, whose products are the giant polypeptides that constitute the silk-like secretion for constructing housing tubes, remain active. Simultaneously, decondensation and activation take place at some chromosomal regions, especially at the centromeres. The changes observed in the pattern of gene expression do not resemble those found after heat shock or other cell stressors. These data provide the first evidence that cadmium interacts with ribosomal genes and results in a drastic impairment of the functional activity of the nucleolus, an essential organelle for cellular survival. Therefore, the depletion of ribosomes would be a long-term effect of Cd-induced cellular damage. These findings may

Ribosomal genes as early targets of cadmium-induced toxicity in Chironomus riparius larvae

Energy Technology Data Exchange (ETDEWEB)

Planello, R. [Biologia Ambiental, Facultad de Ciencias, Universidad Nacional de Educacion a Distancia, Senda del Rey 9, 28040, Madrid (Spain); Martinez-Guitarte, J.L. [Biologia Ambiental, Facultad de Ciencias, Universidad Nacional de Educacion a Distancia, Senda del Rey 9, 28040, Madrid (Spain); Morcillo, G. [Biologia Ambiental, Facultad de Ciencias, Universidad Nacional de Educacion a Distancia, Senda del Rey 9, 28040, Madrid (Spain)]. E-mail: gmorcillo@ccia.uned.es

2007-02-01

Cadmium is a widespread environmental pollutant that causes severe impacts in organisms. Although the effects of cadmium on aquatic insects have been studied in terms of their toxicity and changes in developmental parameters, little is known about its molecular and genetic effects. We have investigated the alterations in the pattern of gene expression provoked by acute exposure to cadmium in Chironomus riparius Mg. (Diptera, Chironomidae), a sentinel organism widely used in aquatic toxicity testing. The early cytotoxic effects were evaluated using immunocytochemistry and specific fluorescent probes in fourth instar larvae after 12 h of 10 mM cadmium treatments; under these conditions no significant effect on larvae mortality was detected until after 36 h of exposure. The changes in the pattern of gene expression were analysed by means of DNA/RNA hybrid antibodies in the polytene chromosomes from salivary gland cells. A decrease in the activity of the nucleolus is especially remarkable, accompanied by a significant reduction in size and the modification in nucleolar architecture, as shown by FISH. The inhibition of rDNA transcription was further evaluated by Northern blot analysis, which showed a marked decrease in the level of preribosomal rRNA (54% 45S 12 h). However, the BR genes, whose products are the giant polypeptides that constitute the silk-like secretion for constructing housing tubes, remain active. Simultaneously, decondensation and activation take place at some chromosomal regions, especially at the centromeres. The changes observed in the pattern of gene expression do not resemble those found after heat shock or other cell stressors. These data provide the first evidence that cadmium interacts with ribosomal genes and results in a drastic impairment of the functional activity of the nucleolus, an essential organelle for cellular survival. Therefore, the depletion of ribosomes would be a long-term effect of Cd-induced cellular damage. These findings may

Interactive Purchasing Situations

NARCIS (Netherlands)

Groote Schaarsberg, M.; Borm, P.E.M.; Hamers, H.J.M.; Reijnierse, J.H.

2012-01-01

Abstract: This paper introduces a new class of interactive cooperative purchasing situations and provides an explicit alternative characterization of the nucleolus of cooperative games, which offers an alternative to Kohlberg (1971). In our cooperative purchasing situation, the unit price of a

Giemsa C-banded karyotypes of Hordeum taxa from North America

DEFF Research Database (Denmark)

Linde-Laursen, Ib; Bothmer, R. von; Jacobsen, N.

1986-01-01

submetacentrics than previously reported in Hordeum distinguished the genomes of H. arizonicum and H. brachyantherum (4x) from Newfoundland. A partial inactivation of the nucleolus organizers of one parental genome in interspecific hybrids is considered more common than generally appreciated....

Characterisation of transcriptionally active and inactive chromatin domains in neurons

NARCIS (Netherlands)

A.S. Akhmanova (Anna); T. Verkerk (Ton); A. Langeveld (An); N.J. Galjart (Niels); F.G. Grosveld (Frank)

2000-01-01

textabstractThe tandemly organised ribosomal DNA (rDNA) repeats are transcribed by a dedicated RNA polymerase in a specialised nuclear compartment, the nucleolus. There appears to be an intimate link between the maintenance of nucleolar structure and the presence of

To the nucleolar density and size in apoptotic human leukemic myeloblasts produced in vitro by Trichostatin A

Directory of Open Access Journals (Sweden)

K Smetana

2009-08-01

Full Text Available The present study was designed to provide more information on nucleoli in apoptotic cells, which were represented in the present study by cultured leukemic myeloblasts (Kasumi-1 cells. The apoptotic process in these cells was produced by trichostatin A (TSA that is a histone deacetylase inhibitor with strong cytostatic effects. The selected TSA concentration added to cultures facilitated to study apoptotic and notapoptotic cells in one and the same specimen. The nucleolar diameter and density were determined using computer assisted measurement and densitometry in specimens stained for RNA. In comparison with not-apoptotic cells, in apoptotic cells, nucleolar mean diameter did not change significantly and nucleolar RNA density was also not apparently different. On the other hand, the cytoplasmic RNA density in apoptotic cells was markedly reduced. Thus it seemed to be possible that the transcribed RNA remained “frozen” within the nucleolus but its transport to the cytoplasm decreased or stopped. However, the possibility of the RNA degradation in the cytoplasm of apoptotic cells based on the present study cannot be eliminated. At this occasion it should be added that AgNORs reflecting nucleolar biosynthetic and cell proliferation activity in apoptotic cells decreased in number or disappeared. The presented results also indicated that large nucleoli intensely stained for RNA need not be necessarily related to the high nucleolar biosynthetic or cell proliferation activity and may be also present in apoptotic cells responding to the cytostatic treatment.

Colleters in Caryocar brasiliense (Caryocaraceae ontogenesis, ultrastructure and secretion

Directory of Open Access Journals (Sweden)

E. A. S. Paiva

Full Text Available Colleters are widely distributed and have been reported in several taxa. However, the only report of colleters in the Caryocaraceae family is in Caryocar brasiliense. This study purports to describe the distribution, structure and ultrastructure of colleters in C. brasiliense. Vegetative shoot apices with stipules in different stages of development were collected, fixed, and processed by light, transmission and scanning electron microscopy. Colleters occur on the stipule adaxial face and are differentiated on younger stipules, remaining functional until senescence. Stipules are deciduous, falling before the beginning of leaf expansion. Colleter secretion is abundant, filling all the space delimited by the stipule. Colleter origin is mixed, involving the protoderm and ground meristem. In this species, colleters are finger-shaped, consisting of a column of non-secretory central cells and a secretory epithelium whose cells are arranged in palisades. Secretory epithelium cells show dense cytoplasm, rough endoplasmic reticulum (RER segments, free ribosomes, mitochondria, and abundant well developed dictyosomes. The colleters central cell shows a flocculated cytoplasm and developed vacuole. These cells have a small lobated nucleus with an inconspicuous nucleolus. Mitochondria and RER stand out in the organelles. There is evidence of granulocrine secretion and colleter involvement with protection of the apical meristem and leaves in the initial stages of development.

Influence of heart failure on nucleolar organization and protein expression in human hearts

International Nuclear Information System (INIS)

Roselló-Lletí, Esther; Rivera, Miguel; Cortés, Raquel; Azorín, Inmaculada; Sirera, Rafael; Martínez-Dolz, Luis; Hove, Leif; Cinca, Juan; Lago, Francisca; González-Juanatey, José R.; Salvador, Antonio; Portolés, Manuel

2012-01-01

Highlights: ► Heart failure alters nucleolar morphology and organization. ► Nucleolin expression is significant increased in ischemic and dilated cardiomyopathy. ► Ventricular function of heart failure patients was related with nucleolin levels. -- Abstract: We investigate for the first time the influence of heart failure (HF) on nucleolar organization and proteins in patients with ischemic (ICM) or dilated cardiomyopathy (DCM). A total of 71 human hearts from ICM (n = 38) and DCM (n = 27) patients, undergoing heart transplantation and control donors (n = 6), were analysed by western-blotting, RT-PCR and cell biology methods. When we compared protein levels according to HF etiology, nucleolin was increased in both ICM (117%, p < 0.05) and DCM (141%, p < 0.01). Moreover, mRNA expression were also upregulated in ICM (1.46-fold, p < 0.05) and DCM (1.70-fold, p < 0.05. Immunofluorescence studies showed that the highest intensity of nucleolin was into nucleolus (p < 0.0001), and it was increased in pathological hearts (p < 0.0001). Ultrastructure analysis by electron microscopy showed an increase in the nucleus and nucleolus size in ICM (17%, p < 0.05 and 131%, p < 0.001) and DCM (56%, p < 0.01 and 69%, p < 0.01). Nucleolar organization was influenced by HF irrespective of etiology, increasing fibrillar centers (p < 0.001), perinucleolar chromatin (p < 0.01) and dense fibrillar components (p < 0.01). Finally, left ventricular function parameters were related with nucleolin levels in ischemic hearts (p < 0.0001). The present study demonstrates that HF influences on morphology and organization of nucleolar components, revealing changes in the expression and in the levels of nucleolin protein.

Cost-allocation principles for pipeline capacity and usage

International Nuclear Information System (INIS)

Salant, D.J.; Watkins, G.C.

1996-01-01

The issue of cost sharing among multiple users of transmission facilities, such as pipelines, was discussed. The various ways in which a fair and reasonable pipeline cost-allocation scheme can be implemented were examined. It was suggested that no method exists for allocating costs that will achieve all major policy goals. The advantages and disadvantages of a system of uniform rates, such as postage stamp tolls, was discussed in the context of a natural gas pipeline system. A postage stamp system is one in which all users pay the same amount per unit of capacity, regardless of transport distances. This rate structure, while sometimes appropriate, is inefficient if total costs are distance-sensitive or if there is a significant variation in the sources of demand. Two commonly accepted minimal principles that a cost allocation should satisfy include: (1) the stand-alone cost test, and (2) the incremental cost test. This means that no one should pay costs in excess of their stand-alone costs and no one should pay less than their incremental costs. Postage stamp tolls were found to fail the minimal set of commonly applied principles. More optimal ways in which to allocate pipeline network costs among users were presented. The nucleolus is a unique cost allocation that is consistent, symmetric and homogeneous. The nucleolus is calculated by splitting the costs equally among users of a common facility. Another effective cost allocation system is the Shapley value which can be derived from a set of axioms which differ slightly from those that identify the nucleolus. 2 refs., 1 tab

Chromosomal characteristics and distribution of rDNA sequences in the brook trout Salvelinus fontinalis (Mitchill, 1814).

Science.gov (United States)

Śliwińska-Jewsiewicka, A; Kuciński, M; Kirtiklis, L; Dobosz, S; Ocalewicz, K; Jankun, Malgorzata

2015-08-01

Brook trout Salvelinus fontinalis (Mitchill, 1814) chromosomes have been analyzed using conventional and molecular cytogenetic techniques enabling characteristics and chromosomal location of heterochromatin, nucleolus organizer regions (NORs), ribosomal RNA-encoding genes and telomeric DNA sequences. The C-banding and chromosome digestion with the restriction endonucleases demonstrated distribution and heterogeneity of the heterochromatin in the brook trout genome. DNA sequences of the ribosomal RNA genes, namely the nucleolus-forming 28S (major) and non-nucleolus-forming 5S (minor) rDNAs, were physically mapped using fluorescence in situ hybridization (FISH) and primed in situ labelling. The minor rDNA locus was located on the subtelo-acrocentric chromosome pair No. 9, whereas the major rDNA loci were dispersed on 14 chromosome pairs, showing a considerable inter-individual variation in the number and location. The major and minor rDNA loci were located at different chromosomes. Multichromosomal location (3-6 sites) of the NORs was demonstrated by silver nitrate (AgNO3) impregnation. All Ag-positive i.e. active NORs corresponded to the GC-rich blocks of heterochromatin. FISH with telomeric probe showed the presence of the interstitial telomeric site (ITS) adjacent to the NOR/28S rDNA site on the chromosome 11. This ITS was presumably remnant of the chromosome rearrangement(s) leading to the genomic redistribution of the rDNA sequences. Comparative analysis of the cytogenetic data among several related salmonid species confirmed huge variation in the number and the chromosomal location of rRNA gene clusters in the Salvelinus genome.

Cost-allocation principles for pipeline capacity and usage

Energy Technology Data Exchange (ETDEWEB)

Salant, D.J.; Watkins, G.C. [Law and Economics Consulting Group, Emeryville, CA (United States)

1996-12-31

The issue of cost sharing among multiple users of transmission facilities, such as pipelines, was discussed. The various ways in which a fair and reasonable pipeline cost-allocation scheme can be implemented were examined. It was suggested that no method exists for allocating costs that will achieve all major policy goals. The advantages and disadvantages of a system of uniform rates, such as postage stamp tolls, was discussed in the context of a natural gas pipeline system. A postage stamp system is one in which all users pay the same amount per unit of capacity, regardless of transport distances. This rate structure, while sometimes appropriate, is inefficient if total costs are distance-sensitive or if there is a significant variation in the sources of demand. Two commonly accepted minimal principles that a cost allocation should satisfy include: (1) the stand-alone cost test, and (2) the incremental cost test. This means that no one should pay costs in excess of their stand-alone costs and no one should pay less than their incremental costs. Postage stamp tolls were found to fail the minimal set of commonly applied principles. More optimal ways in which to allocate pipeline network costs among users were presented. The nucleolus is a unique cost allocation that is consistent, symmetric and homogeneous. The nucleolus is calculated by splitting the costs equally among users of a common facility. Another effective cost allocation system is the Shapley value which can be derived from a set of axioms which differ slightly from those that identify the nucleolus. 2 refs., 1 tab.

The Integrative Studies of Genetic and Environmental Factors in Systemic Sclerosis

Science.gov (United States)

2011-05-01

in electronics , computer, aerospace and biomedical science. CNTs are commonly formed in ordinary flames (1), or by burning methane (2), ethylene (3...nucleolus to the nucleo- plasm due to mechanisms that are not clearly under- stood. [17-19]. Interestingly, SSc fibroblasts examined herein showed enhanced

ORF Sequence: NC_001135 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available h Lrs4p, located in the nucleolus; Lrs4p-Csm1p heterodimer binds to Mam1p at kinetochores during meiosis I t... NC_001135 gi|6319928 >gi|6319928|ref|NP_010009.1| Protein that forms a complex wit

Nucleologenesis and Nucleolotransfer in Mammalian Oocytes: A Review

Czech Academy of Sciences Publication Activity Database

Benc, M.; Strejček, F.; Murín, M.; Morovic, M.; Martínková, S.; Jettmarová, D.; Pendovski, L.; Fulka Jr, J.; Laurinčík, Jozef

2017-01-01

Roč. 40, č. 2 (2017), s. 117-124 E-ISSN 1857-7415 R&D Projects: GA MŠk EF15_003/0000460 Institutional support: RVO:67985904 Keywords : nucleous precursor body * nucleolus-like bodies * enucleolation Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Developmental biology

Extramedullary plasmacytoma. Fine needle aspiration findings.

Science.gov (United States)

Kumar, P V; Owji, S M; Talei, A R; Malekhusseini, S A

1997-01-01

To determine the role of fine needle aspiration cytology in the diagnosis of extramedullary plasmacytoma. The study group consisted of 13 patients with palpable masses at various sites. The tumors were aspirated for cytologic study. The smears revealed groups of mature and immature plasma cells at various stages of maturation. Mature plasma cells showed an eccentric nucleus and abundant, deep, basophilic cytoplasm with a paranuclear halo. Plasmablasts (immature plasma cells) showed a prominent, eccentric nucleus with single, large nucleolus and abundant, deep, basophilic cytoplasm with no paranuclear halo. Binucleate and multinucleate forms were also seen quite often. The tumors were excised, and the histologic sections confirmed the cytologic diagnosis. All the patients received radiotherapy. One patient (18 years old) developed recurrence and died due to extensive infiltration into the maxilla and mandible. Two patients (57 and 62 years) developed multiple myeloma one to two years after the excision of tumors, and both died two to three months later. The remaining 10 patients were alive and well at this writing. The smears from all 13 patients were diagnosed as extramedullary plasmacytomas by fine needle aspiration cytology.

Sequence Classification: 893448 [

Lifescience Database Archive (English)

Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|6322843|ref|NP_012916.1| Protein involved in mRNA turnov...er and ribosome assembly, localizes to the nucleolus; Mrt4p || http://www.ncbi.nlm.nih.gov/protein/6322843 ...

Computing an element in the lexicographic kernel of a game

NARCIS (Netherlands)

Faigle, U.; Kern, Walter; Kuipers, Jeroen

The lexicographic kernel of a game lexicographically maximizes the surplusses $s_{ij}$ (rather than the excesses as would the nucleolus). We show that an element in the lexicographic kernel can be computed efficiently, provided we can efficiently compute the surplusses $s_{ij}(x)$ corresponding to a

Computing an element in the lexicographic kernel of a game

NARCIS (Netherlands)

Faigle, U.; Kern, Walter; Kuipers, J.

2002-01-01

The lexicographic kernel of a game lexicographically maximizes the surplusses $s_{ij}$ (rather than the excesses as would the nucleolus). We show that an element in the lexicographic kernel can be computed efficiently, provided we can efficiently compute the surplusses $s_{ij}(x)$ corresponding to a

The complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection

International Nuclear Information System (INIS)

Qi Dong; Omarov, Rustem T.; Scholthof, Karen-Beth G.

2008-01-01

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus for replication and movement in host plants. The positive-sense single-stranded genomic RNA of SPMV encodes a 17-kDa capsid protein (CP) to form 16-nm virions. We determined that SPMV CP accumulates in both cytosolic and non-cytosolic fractions, but cytosolic accumulation of SPMV CP is exclusively associated with virions. An N-terminal arginine-rich motif (N-ARM) on SPMV CP is used to bind its cognate RNA and to form virus particles. Intriguingly, virion formation is dispensable for successful systemic SPMV RNA accumulation, yet this process still depends on an intact N-ARM. In addition, a C-terminal domain on the SPMV CP is necessary for self-interaction. Biochemical fractionation and fluorescent microscopy of green fluorescent protein-tagged SPMV CP demonstrated that the non-cytosolic SPMV CP is associated with the cell wall, the nucleus and other membranous organelles. To our knowledge, this is the first report that a satellite virus CP not only accumulates exclusively as virions in the cytosol but also is directed to the nucleolus and membranes. That SPMV CP is found both in the nucleus and the cell wall suggests its involvement in viral nuclear import and cell-to-cell transport

Site-specific accumulation and dynamic change of flavonoids in Apocyni Veneti Folium.

Science.gov (United States)

Chen, Cui-Hua; Xu, Hu; Liu, Xun-Hong; Zou, Li-Si; Wang, Mei; Liu, Zi-Xiu; Fu, Xing-Sheng; Zhao, Hui; Yan, Ying

2017-12-01

Site-specific accumulation of flavonoids in Apocyni Veneti Folium was determined by laser scanning confocal microscope (LSCM) and the localization of catechins also was observed via vanillin-HCl staining under the conventional optical microscope. The contents of five flavonoids in Apocyni Veneti Folium from different harvest times and growth parts were measured using HPLC method. LSCM observation showed that flavonoids are accumulated in cuticle of epidermal cells and vessel walls, especially in protoplasts and nucleolus of the collenchyma cells and the epidermal cells. Catechins are localized in the palisade parenchyma cells and vessel walls, particularly in the laticifers found in the phloem. On the basis of the difference of the maximal emission wavelength between quercetin and kaempferol derivatives which have fluorescence behavior by appropriate treatment, kaempferol and its derivatives are localized exclusively in the cuticle. Results showed that the content of astragalin in Apocyni Veneti Folium from different parts revealed the decreasing trend, while hyperin and isoquercitrin were higher in June and July analyzed by HPLC. In summary, the site-specific accumulation of flavonoids in Apocyni Veneti Folium can be determined by LSCM and vanillin-HCl staining. The contents of flavonoids in Apocyni Veneti Folium are correlated with harvest times and growth parts. © 2017 Wiley Periodicals, Inc.

Fibrillarin methylates H2A in RNA polymerase I trans-active promoters in Brassica oleracea

Directory of Open Access Journals (Sweden)

lloyd eLoza-Muller

2015-11-01

Full Text Available Fibrillarin is a well conserved methyltransferase involved in several if not all of the more than 100 methylations sites in rRNA which are essential for proper ribosome function. It is mainly localized in the nucleoli and Cajal bodies inside the cell nucleus where it exerts most of its functions. In plants, fibrillarin binds directly the guide RNA together with Nop56, Nop58 and 15.5ka proteins to form a snoRNP complex that selects the sites to be methylated in pre-processing of ribosomal RNA. Recently, the yeast counterpart NOP1 was found to methylate histone H2A in the nucleolar regions. Here we show that plant fibrillarin can also methylate histone H2A. In Brassica floral meristem cells the methylated histone H2A is mainly localized in the nucleolus but unlike yeast or human cells it also localize in the periphery of the nucleus. In specialized transport cells the pattern is altered and it exhibits a more diffuse staining in the nucleus for methylated histone H2A as well as for fibrillarin. Here we also show that plant fibrillarin is capable of interacting with H2A and carry out its methylation in the rDNA promoter.

Hepatocellular differentiation status is characterized by distinct subnuclear localization and form of the chanzyme TRPM7.

Science.gov (United States)

Ogunrinde, Adenike; Pereira, Robyn D; Beaton, Natalie; Lam, D Hung; Whetstone, Christiane; Hill, Ceredwyn E

The channel-kinase TRPM7 is important for the survival, proliferation, and differentiation, of many cell types. Both plasma membrane channel activity and kinase function are implicated in these roles. Channel activity is greater in less differentiated hepatoma cells compared with non-dividing, terminally differentiated adult hepatocytes, suggesting differences in protein expression and/or localization. We used electrophysiological and immunofluorescence approaches to establish whether hepatocellular differentiation is associated with altered TRPM7 expression. Mean outward current decreased by 44% in WIF-B hepatoma cells incubated with the established hepatic differentiating factors oncostatin M/dexamethasone for 1-8 days. Pre-incubation with pyridone 6, a pan-JAK inhibitor, blocked the current reduction. An antibody targeted to the C-terminus of TRPM7 labelled the cytoplasm in WIF-B cells and intact rat liver. Significant label also localized to the nuclear envelope (NE), with relatively more detected in adult hepatocytes compared with WIF-B cells. Hepatoma cells also exhibited nucleoplasmic labelling with intense signal in the nucleolus. The endogenous labelling pattern closely resembles that of HEK293T cells heterologously expressing a TRPM7 kinase construct containing a putative nucleolar localization sequence. These results suggest that TRPM7 form and distribution between the plasma membrane and nucleus, rather than expression, is altered in parallel with differentiation status in rat hepatic cells. Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Conserved regulators of nucleolar size revealed by global phenotypic analyses.

Science.gov (United States)

Neumüller, Ralph A; Gross, Thomas; Samsonova, Anastasia A; Vinayagam, Arunachalam; Buckner, Michael; Founk, Karen; Hu, Yanhui; Sharifpoor, Sara; Rosebrock, Adam P; Andrews, Brenda; Winston, Fred; Perrimon, Norbert

2013-08-20

Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I-mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry-based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules.

Conserved Regulators of Nucleolar Size Revealed by Global Phenotypic Analyses

Science.gov (United States)

Neumüller, Ralph A.; Gross, Thomas; Samsonova, Anastasia A.; Vinayagam, Arunachalam; Buckner, Michael; Founk, Karen; Hu, Yanhui; Sharifpoor, Sara; Rosebrock, Adam P.; Andrews, Brenda; Winston, Fred; Perrimon, Norbert

2014-01-01

Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I–mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I–mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry–based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules. PMID:23962978

Structure of the nucleoli in domestic cattle spermatocytes.

Science.gov (United States)

Andraszek, Katarzyna; Smalec, Elżbieta

2012-10-08

The work was aimed at determining the number and morphology of nucleoli in the prophase of the first meiotic division in domestic cattle males. The use of AgNO₃ staining, commonly applied in cytogenetics for the identification of nucleolar organiser regions, made it possible to identify nucleoli in first-order spermatocytes. One nucleolus was identified in each analysed cell. Considerable morphological differentiation of the nucleoli during the prophase of the first meiotic division, particularly in leptotene, unobserved in other farm animal species, was noticed. Dark-hued grain-like structures were found within the disintegrating nucleoli, corresponding approximately or exactly to the number of the nucleolar organiser regions in the domestic cattle karyotype. Dark areas were identified in the selected prometaphase chromosomes. Their number corresponded with the number of active NORs defined in the domestic cattle karyotype.

The dose effect of irradiated rice pollen on double fertilization

International Nuclear Information System (INIS)

Wang Houcong; Chen Zhengming; Chen Ruming; Qiu Simi; Yang Juemin; Yang Huijie

1995-01-01

The mature panicles of rice were treated with 60 Co γ-rays in the range of 0∼0.372 kGy. The male sterile line used as the female plants were fertilized with γ-irradiated pollen manually. The dose effect of the irradiated pollen on double fertilization was investigated. It was found that double fertilization of the irradiated pollen was suppressed to different degrees as compared with the control. The effect was noticeable as that the fusion time of the male nucleolus with the female one was delayed with the increasing of γ-radiation dose. The delayed time was less than 13 hours when the dose was below 0.186 kGy and it was more than 15 hours when the dose was above 0.279 kGy. Furthermore, several types of deformed embryonic cells and endosperm nuclei were observed

The incorporation of 14C-adenine into the oocytes of Asellus aquaticus as studied by autoradiography

NARCIS (Netherlands)

Broek, C.J.H. van den; Tates, A.D.

Asellus aquaticus females were injected with 8-14C-adenine, fixed after 3 hours and sectioned. In coated autoradiographs, the number of β-tracks from 14C were counted over nucleolus, nucleus and cytoplasm of the oocytes at various stages of their development. Incorporation into nucleolar RNA, being

Sequence Classification: 890890 [

Lifescience Database Archive (English)

Full Text Available lar protein of unknown function, positive regulator of exit from mitosis; involved in regulating the release of Cdc14p from the nucle...olus in early anaphase; proposed to play similar role in meiosis; Spo12p || http://www.ncbi.nlm.nih.gov/protein/6321946 ...

Cytological and cytochemical characterization of the polytene chromosomes of Chironomus sancticaroli (Diptera: chironomidae)

Energy Technology Data Exchange (ETDEWEB)

Freitas, F. de; Leoncini, O.; Floeter-Winter, L.M.

1985-03-01

The chromosome complement of a Brazilian Chironomus Species, C. Sancticaroli, was analyzed cytochemically. The polytene Chromosomes were identified and characterized and the nucleolus organizer regions (NORs) were located by a technique of in situ hybridization and immunofluorescence. Constitutive heterochromatin and its distribution in relation to the NORs were studied.

Expression of protein-coding genes embedded in ribosomal DNA

DEFF Research Database (Denmark)

Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik

2007-01-01

Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns that e...... in the nucleolus....

Perturbation of ribosome biogenesis drives cells into senescence through 5S RNP-mediated p53 activation.

Science.gov (United States)

Nishimura, Kazuho; Kumazawa, Takuya; Kuroda, Takao; Katagiri, Naohiro; Tsuchiya, Mai; Goto, Natsuka; Furumai, Ryohei; Murayama, Akiko; Yanagisawa, Junn; Kimura, Keiji

2015-03-03

The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Influence of heart failure on nucleolar organization and protein expression in human hearts

Energy Technology Data Exchange (ETDEWEB)

Rosello-Lleti, Esther; Rivera, Miguel; Cortes, Raquel [Cardiocirculatory Unit, Research Center, Hospital Universitario La Fe, Valencia (Spain); Azorin, Inmaculada [Experimental Neurology, Research Center, Hospital Universitario La Fe, Valencia (Spain); Sirera, Rafael [Biotechnology Department, Universidad Politecnica, Valencia (Spain); Martinez-Dolz, Luis [Cardiology Unit, Hospital Universitario La Fe, Valencia (Spain); Hove, Leif; Cinca, Juan [Cardiology Unit, Hospital San Pau, Barcelona (Spain); Lago, Francisca; Gonzalez-Juanatey, Jose R. [Cardiology Unit, Institute of Biomedical Research, Hospital Clinicode Santiagode Compostela (Spain); Salvador, Antonio [Experimental Neurology, Research Center, Hospital Universitario La Fe, Valencia (Spain); Portoles, Manuel, E-mail: portoles_man@gva.es [Cell Biology and Pathology Unit, Research Center, Hospital Universitario La Fe, Valencia (Spain)

2012-02-10

Highlights: Black-Right-Pointing-Pointer Heart failure alters nucleolar morphology and organization. Black-Right-Pointing-Pointer Nucleolin expression is significant increased in ischemic and dilated cardiomyopathy. Black-Right-Pointing-Pointer Ventricular function of heart failure patients was related with nucleolin levels. -- Abstract: We investigate for the first time the influence of heart failure (HF) on nucleolar organization and proteins in patients with ischemic (ICM) or dilated cardiomyopathy (DCM). A total of 71 human hearts from ICM (n = 38) and DCM (n = 27) patients, undergoing heart transplantation and control donors (n = 6), were analysed by western-blotting, RT-PCR and cell biology methods. When we compared protein levels according to HF etiology, nucleolin was increased in both ICM (117%, p < 0.05) and DCM (141%, p < 0.01). Moreover, mRNA expression were also upregulated in ICM (1.46-fold, p < 0.05) and DCM (1.70-fold, p < 0.05. Immunofluorescence studies showed that the highest intensity of nucleolin was into nucleolus (p < 0.0001), and it was increased in pathological hearts (p < 0.0001). Ultrastructure analysis by electron microscopy showed an increase in the nucleus and nucleolus size in ICM (17%, p < 0.05 and 131%, p < 0.001) and DCM (56%, p < 0.01 and 69%, p < 0.01). Nucleolar organization was influenced by HF irrespective of etiology, increasing fibrillar centers (p < 0.001), perinucleolar chromatin (p < 0.01) and dense fibrillar components (p < 0.01). Finally, left ventricular function parameters were related with nucleolin levels in ischemic hearts (p < 0.0001). The present study demonstrates that HF influences on morphology and organization of nucleolar components, revealing changes in the expression and in the levels of nucleolin protein.

Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

Science.gov (United States)

Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

2009-12-15

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

The dynamic nature and territory of transcriptional machinery in the bacterial chromosome

Directory of Open Access Journals (Sweden)

Ding Jun Jin

2015-05-01

Full Text Available Our knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

Cytological and cytochemical characterization of the polytene chromosomes of Chironomus sancticaroli (Diptera: chironomidae)

International Nuclear Information System (INIS)

Freitas, F. de; Leoncini, O.

1985-01-01

The chromosome complement of a Brazilian Chironomus Species, C. Sancticaroli, was analyzed cytochemically. The polytene Chromosomes were identified and characterized and the nucleolus organizer regions (NORs) were located by a technique of in situ hybridization and immunofluorescence. Constitutive heterochromatin and its distribution in relation to the NORs were studied. (Author) [pt

Effect of the number of portal area on modified histological activity ...

African Journals Online (AJOL)

2013-03-23

Mar 23, 2013 ... PA 11 and over group as compared to the PA 2‑4 group. PA. N. Mean. P. 2-4 group ... chronic at a rate of 60‑80% in adults.[9] .... [3] Nucleus and nucleolus sizes of ... A systematic review of the quality of liver biopsy specimens.

U3 snoRNP associates with fibrillarin a component of the scleroderma clumpy nucleolar domain

DEFF Research Database (Denmark)

Herrera-Esparza, Rafael; Kruse, Lars; von Essen, Marina

2002-01-01

by ELISA was recognized by the clumpy scleroderma serum from the majority of patients. In situ hybridization assays showed that the fibrillarin tagged by the elicited antibodies was colocalized with U3 snoRNP in the nucleolus in a clumpy manner and coprecipitated the U3 snoRNP. In conclusion...

1-concave basis for TU games and the library game

NARCIS (Netherlands)

Driessen, Theo; Khmelnitskaya, Anna Borisovna; Sales, J.

2012-01-01

The study of 1-convex/1-concave TU games possessing a nonempty core and for which the nucleolus is linear was initiated by Driessen and Tijs (Methods Oper. Res. 46:395–406, 1983) and Driessen (OR Spectrum 7:19–26, 1985). However, until recently appealing abstract and practical examples of these

The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus

DEFF Research Database (Denmark)

Torres-Rosell, Jordi; Sunjevaric, Ivana; De Piccoli, Giacomo

2007-01-01

at an extranucleolar site. The nucleolar exclusion of Rad52 recombination foci entails Mre11 and Smc5-Smc6 complexes and depends on Rad52 SUMO (small ubiquitin-related modifier) modification. Remarkably, mutations that abrogate these activities result in the formation of Rad52 foci within the nucleolus and cause r...

Two Nucleolar Proteins, GDP1 and OLI2, Function As Ribosome Biogenesis Factors and Are Preferentially Involved in Promotion of Leaf Cell Proliferation without Strongly Affecting Leaf Adaxial–Abaxial Patterning in Arabidopsis thaliana

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Koji Kojima

2018-01-01

Full Text Available Leaf abaxial–adaxial patterning is dependent on the mutual repression of leaf polarity genes expressed either adaxially or abaxially. In Arabidopsis thaliana, this process is strongly affected by mutations in ribosomal protein genes and in ribosome biogenesis genes in a sensitized genetic background, such as asymmetric leaves2 (as2. Most ribosome-related mutants by themselves do not show leaf abaxialization, and one of their typical phenotypes is the formation of pointed rather than rounded leaves. In this study, we characterized two ribosome-related mutants to understand how ribosome biogenesis is linked to several aspects of leaf development. Previously, we isolated oligocellula2 (oli2 which exhibits the pointed-leaf phenotype and has a cell proliferation defect. OLI2 encodes a homolog of Nop2 in Saccharomyces cerevisiae, a ribosome biogenesis factor involved in pre-60S subunit maturation. In this study, we found another pointed-leaf mutant that carries a mutation in a gene encoding an uncharacterized protein with a G-patch domain. Similar to oli2, this mutant, named g-patch domain protein1 (gdp1, has a reduced number of leaf cells. In addition, gdp1 oli2 double mutants showed a strong genetic interaction such that they synergistically impaired cell proliferation in leaves and produced markedly larger cells. On the other hand, they showed additive phenotypes when combined with several known ribosomal protein mutants. Furthermore, these mutants have a defect in pre-rRNA processing. GDP1 and OLI2 are strongly expressed in tissues with high cell proliferation activity, and GDP1-GFP and GFP-OLI2 are localized in the nucleolus. These results suggest that OLI2 and GDP1 are involved in ribosome biogenesis. We then examined the effects of gdp1 and oli2 on adaxial–abaxial patterning by crossing them with as2. Interestingly, neither gdp1 nor oli2 strongly enhanced the leaf polarity defect of as2. Similar results were obtained with as2 gdp1 oli2

Multifunctional Pt(II) Reagents: Covalent Modifications of Pt Complexes Enable Diverse Structural Variation and In-Cell Detection.

Science.gov (United States)

White, Jonathan D; Haley, Michael M; DeRose, Victoria J

2016-01-19

Pt(II)-neutral complex analogous in structure to the aforementioned difunctional azide-Pt(II) reagent. In all cases, significant accumulation of Pt in the nucleolus of cells was observed, in addition to broader localization in the nucleus and cytoplasm of the cell. Using the same strategy of postbinding click modification with fluorescent probes, Pt adducts were detected and roughly quantified on rRNA and tRNA from Pt-treated Saccharomyces cerevisiae; rRNA adducts were found to be relatively long-lived and not targeted for immediate degradation. Finally, the utility and feasibility of the alkyne-appended Pt(II) compound has been further demonstrated with a turn-on fluorophore, dansyl azide, in fluorescent detection of DNA in vitro. In all, these modifications utilizing reactive handles have allowed for the diversification of new Pt reagents, as well as providing cellular localization information on the modified Pt compounds.

Determining the sub-cellular localization of proteins within Caenorhabditis elegans body wall muscle.

Science.gov (United States)

Meissner, Barbara; Rogalski, Teresa; Viveiros, Ryan; Warner, Adam; Plastino, Lorena; Lorch, Adam; Granger, Laure; Segalat, Laurent; Moerman, Donald G

2011-01-01

Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive "localizome" for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function.

Structure of the nucleoli in domestic cattle spermatocytes