Nucleolar Morphology Research Articles

Nuclear control of early embryonic development in domestic pigs

In mammals, growing oocytes have characteristically high levels of RNA synthesis. After the initiation of meiosis, that is germinal vesicle breakdown, this RNA synthesis ceases. Although there is limited evidence for RNA synthesis by the zygote, significant amounts of RNA synthesis do not occur until a species-specific cell stage. In pigs, significant amounts of mRNA synthesis cannot be detected before the four-cell stage. There appear to be three qualitatively different periods of transcription during the four-cell stage. The first occurs during a short (< 2 h) G1 phase. The second, occurs after completion of DNA synthesis (S phase) about 16 h after cleavage to the four-cell stage, and the third occurs about 24 h after cleavage to the four-cell stage. Correlated with these changes in RNA synthesis are changes in nucleolar morphology, amino acid transport characteristics, protein production, mitochondrial morphology, and metabolism of the embryo. The mechanisms that regulate initiation of RNA synthesis in early mammalian embryos appear to repress transcription. A state of transcription permissiveness then follows that sets into motion the differentiation programme.

Clinical Significance of Nucleoli Cytomorphology Assessment in Patients With Uveal Melanoma.

To assess the prognostic significance of nucleolar morphological parameters in a large cohort of patients with uveal melanoma. The presence, size and number of nucleoli of cancer cells were assessed in haematoxylin and eosin (HE)-stained slides of 164 formalin-fixed paraffin-embedded primary uveal melanoma tissue specimens. The results were correlated with clinicopathological features and patient survival. The presence of macronucleoli and multiple nucleoli significantly correlated with the epithelioid type of uveal melanoma, high mitotic rate, and marked pleomorphism. There was a positive correlation between the presence of macronucleoli as well as the number of nucleoli and the largest tumour basal diameter. The increased nucleolus count in tumour cells positively correlated with primary tumour (pT) staging. The presence of both prominent and multiple nucleoli was associated with significantly reduced overall and disease-free survival. Histological assessment of nucleolar morphology in routine HE staining would be a helpful low-cost method to obtain reliable prognostic information.

Comparative analysis of MACROD1, MACROD2 and TARG1 expression, localisation and interactome

The posttranslational modification ADP-ribosylation is involved in many cellular processes, with distinct roles for poly- and mono(ADP-ribosyl)ation (PAR- and MARylation, respectively). Reversibility of intracellular MARylation was demonstrated with the discovery of MACROD1, MACROD2 and TARG1, three macrodomain-containing enzymes capable of reversing MARylation of proteins and RNA. While the three enzymes have identical activities in vitro, their roles in cells are unclear and published data are partially contradictory, possibly due to a lack of validated reagents. We developed monoclonal antibodies to study these proteins and analysed their tissue distribution and intracellular localisation. MACROD1 is most prevalent in mitochondria of skeletal muscle, MACROD2 localises to nucleo- and cytoplasm and is found so far only in neuroblastoma cells, whereas the more ubiquitously expressed TARG1 is present in nucleoplasm, nucleolus and stress granules. Loss of MACROD1 or loss of TARG1 leads to disruption of mitochondrial or nucleolar morphology, respectively, hinting at their importance for these organelles. To start elucidating the underlying mechanisms, we have mapped their interactomes using BioID. The cellular localisation of interactors supports the mitochondrial, nucleolar and stress granule localisation of MACROD1 and TARG1, respectively. Gene ontology analysis suggests an involvement of MACROD1 and TARG1 in RNA metabolism in their respective compartments. The detailed description of the hydrolases’ expression, localisation and interactome presented here provides a solid basis for future work addressing their physiological function in more detail.

Che-1/AATF binds to RNA polymerase I machinery and sustains ribosomal RNA gene transcription.

Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.

Moringa oleifera leaf flavonoids protect bovine mammary epithelial cells from hydrogen peroxide-induced oxidative stress in vitro.

Bovine mammary epithelial cells (BMECs) of high-producing dairy cows are subject to constant oxidative stress as a result of high metabolic rate and physiological adaptation to intensive farming. Moringa (Moringa oleifera) leaf has been proposed to have the antioxidant potential in scavenging free radicals due to the presence of flavonoids. In this study, we investigated the cytoprotective effects of moringa leaf flavonoids in alleviating oxidative stress in BMECs in vitro. Oxidative stress was established by exposing isolated BMECs to H2 O2 for 2hr. Doses of moringa leaf flavonoids were evaluated by treating BMECs for 12hr. The optimal concentrations of H2 O2 and moringa leaf flavonoids were 500μmol/L and 1.0mg/ml, respectively. The results showed that moringa leaf flavonoids increased the activities of superoxide dismutase, catalase, and glutathione peroxidase; and reduced malondialdehyde activity and intracellular accumulation of reactive oxygen species (ROS) in the H2 O2 -induced oxidative stress system. A Hoechst33258 staining assay revealed that moringa leaf flavonoids decreased the apoptosis rate in BMECs, while leaving membrane integrity and nucleolar morphology unchanged. We concluded that moringa leaf flavonoids have the antioxidant capacity to effectively reduce oxidative stress in BMECs.

Generation and characterization of a mitotane-resistant adrenocortical cell line.

Mitotane is the only drug approved for the therapy of adrenocortical carcinoma (ACC). Its clinical use is limited by the occurrence of relapse during therapy. To investigate the underlying mechanisms in vitro, we here generated mitotane-resistant cell lines. After long-term pulsed treatment of HAC-15 human adrenocortical carcinoma cells with 70 µM mitotane, we isolated monoclonal cell populations of treated cells and controls and assessed their respective mitotane sensitivities by MTT assay. We performed exome sequencing and electron microscopy, conducted gene expression microarray analysis and determined intracellular lipid concentrations in the presence and absence of mitotane. Clonal cell lines established after pulsed treatment were resistant to mitotane (IC50 of 102.2 ± 7.3 µM (n = 12) vs 39.4 ± 6.2 µM (n = 6) in controls (biological replicates, mean ± s.d., P = 0.0001)). Unlike nonresistant clones, resistant clones maintained normal mitochondrial and nucleolar morphology during mitotane treatment. Resistant clones largely shared structural and single nucleotide variants, suggesting a common cell of origin. Resistance depended, in part, on extracellular lipoproteins and was associated with alterations in intracellular lipid homeostasis, including levels of free cholesterol, as well as decreased steroid production. By gene expression analysis, resistant cells showed profound alterations in pathways including steroid metabolism and transport, apoptosis, cell growth and Wnt signaling. These studies establish an in vitro model of mitotane resistance in ACC and point to underlying molecular mechanisms. They may enable future studies to overcome resistance in vitro and improve ACC treatment in vivo.

Abstract LB-A12: A synthetic lethal drug screen identifies exploitable vulnerabilities in cohesin-deficient cells

Abstract The cohesin complex comprises four core subunits: RAD21, SMC3, SMC1A, and STAG2. Cohesin is required for many biological processes, including sister chromatid cohesion, DNA damage response, regulation of gene expression, and ribosomal biogenesis. Germline mutations of cohesin subunits or their regulators give rise to human developmental disorders, while somatic mutations in cohesin subunits are found in a wide range of human cancers, including acute myeloid leukemia (AML) (12%), Ewing sarcoma (15%) and glioblastoma (19%). In particular, cohesin subunit STAG2, has been identified as one of only 12 genes that are significantly mutated in more than 4 human cancers. Currently, there is no targeted therapeutic strategy for cancer patients with cohesin mutations. Synthetic lethality has emerged as a promising approach for targeted therapy. While synthetic lethal interactions of STAG2 have been demonstrated with its paralog, STAG1, and with the DNA damage repair pathway, synthetic lethal interactions with other pathways have not been explored. To identify synthetic lethal drug activity with cohesin mutations, we generated three isogenic cohesin-deficient MCF10A cell lines (RAD21+/-, SMC3+/-, and STAG2-/-) using CRISPR/Cas9. Cohesin-deficient MCF10A cell lines are sufficiently similar to their parental line concerning growth rate and chromosome stability. However, these cell lines exhibit alterations in nucleolar morphology and increased nuclear location of γH2AX and p53 (except in STAG2-/-). RNA-sequencing analysis of the cell lines showed that cohesin subunit mutation led to downregulation of genes involved in regulation of ribosome biogenesis and RNA processing. Furthermore, ribosomal stress mediated by Actinomycin D led to severe nucleolar fragmentation in cohesin-deficient MCF10A, but not MCF10A parental, suggesting that cohesin mutation might confer vulnerability to perturbation of ribosome biogenesis. Using MCF10A parental and isogenic cohesin-deficient MCF10A cell lines, we performed a quantitative high-throughput drug screen against 3,009 compounds of FDA-approved drugs, kinase, and epigenetic inhibitors. Several classes of inhibitors targeting mTOR signaling, Wnt signaling and chromatin modification were found to selectively inhibit the growth of cohesin-deficient MCF10A cells by 30% or more. WAY-600 (mTORC1/mTORC2 inhibitor) and I-BET-762 (Bromodomain inhibitor) were among the top hits. Mechanistic studies on how these drugs preferentially kill cohesin-deficient cells are under way. In summary, we have identified potential synthetic lethal compounds that may provide the basis for development of targeted therapies of cancers with cohesin deficiency or mutation. Citation Format: Chue Vin Chin, Jisha Antony, Amee J George, JinShu He, Kate Parson, Ross D Hannan, Julia A Horsfield. A synthetic lethal drug screen identifies exploitable vulnerabilities in cohesin-deficient cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A12. doi:10.1158/1535-7163.TARG-19-LB-A12

Novel anti-flavivirus drugs targeting the nucleolar distribution of core protein

The risk of infectious diseases caused by Flavivirus is increasing globally. Here, we developed a novel high-throughput screening (HTS) system to evaluate the inhibitory effects of compounds targeting the nuclear localization of the flavivirus core protein. We screened 4000 compounds based on their ability to inhibit the nuclear localization of the core protein, and identified over 20 compounds including inhibitors for cyclin dependent kinase and glycogen synthase kinase. The efficacy of the identified compounds to suppress viral growth was validated in a cell-based infection system. Remarkably, the nucleolus morphology was affected by the treatment with the compounds, suggesting that the nucleolus function is critical for viral propagation. The present HTS system provides a useful strategy for the identification of antivirals against flavivirus by targeting the nucleolar localization of the core protein.

The long-term monitoring of sympathetic ophthalmia in the diagnostic and terapeutic view. Review Department of Ophthalmology.

To evaluate options of diagnostic and therapeutic procedures of sympathetic ophthalmia (SO) compared with literature data. Backgroud: SO is an ocular autoimmune disease. It is characterized by disbalance in the imunoregulatory T-subsets within cell mediated immune response. File examination of SO by evaluation humoral and cellular immunity was ranked nukleolar test (NT). It evaulates the current status of lymphocyte activation based on the nucleolar morphology and RNA transcription aktivity. The classical histological examination was supported by immunohistochemical analysis of lymphocytic subpopulations in the eyeball enucleated for. SO in one case. Five boys and men overall were monitored and treated in two studies from 1979 to 1994 and from 1999 to 2017 with SO In four cases it was subsequent after penetrating injuries and once after lensectomy with vitrectomy. The age of patients in the time of onset of SO was between 4 and 24 years (average 12 years). The time interval between insult and onset of SO varied between 10 days and 3 months (average 1.7 months). The relaps of disease appeared in the all cases in the time interval from 3 months to 38 years (average 15 years). Another two patients with SO were examined only in consultation: 16 years old boy with relaps of SO after cataract extraction and 71 years old women with SO subsequent after lensectomy and vitrectomy. There was examined and compared group of 19 patients with other types of uveitis in the same time. The lens-associated uveitis were caused after ocular contusion and penetrating eye injury in 16 patients (13 male patients). In another three cases (young women) with uveitid underlined by II. or III. type of hypersenzitivity the enucleation of dolorous eyeball calmed down the secondary uveitis on their second eye without any change of immunosupressive treatment and without change in NT. Therewere changes in the complex immunological laboratory tests results in the SO cases in the counts of activated lymphocytes in the peripheral blood. The count of activated lymphocytes was increased in SO cases unlike in lens-associated uveitis. There was detected in NT statistically significant difference (p = 0,0134) between the two groups of uveitis. The histological examination (5 eyes with SO and 7 eyes without sympathetic uveitis) confirmed the diagnoses, supporting basically the clinical diagnosis. The immunohistochemical examination corfirmed the presence of populations of T-lymphocytes, macrophages and also B-lymphocytes. Abasis of immunosuppressive therapy was the combination of prednisone and azathioprin at the first time. Effective therapy featured cyklosporine later. The nucleolar test of lymphocytes draws attention of their up-to-day increased activity without the diferentiation of subpopulations and their absolute number increase related to the current activation of type IV. hypersensitivity (cell-mediated) in uveitis mechanism. The immunosupressive therapy calmes down this activation predominantly in SO, but also in other case sof uveitis with different types of hypersensivity. The immunohistochemical examination illustrates different presence of lymphocytic types according to the stage of SO.

P53 deficiency augments nucleolar instability after ionizing irradiation.

Ribosomes are important cellular components that maintain cellular homeostasis through overall protein synthesis. The nucleolus is a prominent subnuclear structure that contains ribosomal DNA (rDNA) encoding ribosomal RNA (rRNA), an essential component of ribosomes. Despite the significant role of the rDNA-rRNA-ribosome axis in cellular homeostasis, the stability of rDNA in the context of the DNA damage response has not been fully investigated. In the present study, the number and morphological changes of nucleolin, a marker of the nucleolus, were examined following ionizing radiation (IR) in order to investigate the impact of DNA damage on nucleolar stability. An increase in the number of nucleoli per cell was found in HCT116 and U2OS cells following IR. Interestingly, the IR-dependent increase in nucleolar fragmentation was enhanced by p53 deficiency. In addition, the morphological analysis revealed several distinct types of nucleolar fragmentation following IR. The pattern of nucleolar morphology differed between HCT116 and U2OS cells, and the p53 deficiency altered the pattern of nucleolar morphology. Finally, a significant decrease in rRNA synthesis was observed in HCT116 p53−/− cells following IR, suggesting that severe nucleolar fragmentation downregulates rRNA transcription. The findings of the present study suggest that p53 plays a key role in protecting the transcriptional activity of rDNA in response to DNA damage.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

NOL12 Repression Induces Nucleolar Stress-Driven Cellular Senescence and Is Associated with Normative Aging.

The nucleolus is a subnuclear compartment with key roles in rRNA synthesis and ribosome biogenesis, complex processes that require hundreds of proteins and factors. Alterations in nucleolar morphology and protein content have been linked to the control of cell proliferation and stress responses and, recently, further implicated in cell senescence and ageing. In this study, we report the functional role of NOL12 in the nucleolar homeostasis of human primary fibroblasts. NOL12 repression induces specific changes in nucleolar morphology, with increased nucleolar area but reduced nucleolar number, along with nucleolar accumulation and increased levels of fibrillarin and nucleolin. Moreover, NOL12 repression leads to stabilization and activation of p53 in an RPL11-dependent manner, which arrests cells at G2 phase and ultimately leads to senescence. Importantly, we found NOL12 repression in association with nucleolar stress-like responses in human fibroblasts from elderly donors, disclosing it as a biomarker in human chronological aging.

Impact of intron removal from tRNA genes on Saccharomyces cerevisiae.

In eukaryotes and archaea, tRNA genes frequently contain introns, which are removed during maturation. However, biological roles of tRNA introns remain elusive. Here, we constructed a complete set of Saccharomyces cerevisiae strains in which the introns were removed from all the synonymous genes encoding 10 different tRNA species. All the intronless strains were viable, but the tRNAPheGAA and tRNATyrGUA intronless strains displayed slow growth, cold sensitivity and defective growth under respiratory conditions, indicating physiological importance of certain tRNA introns. Northern analyses revealed that removal of the introns from genes encoding three tRNAs reduced the amounts of the corresponding mature tRNAs, while it did not affect aminoacylation. Unexpectedly, the tRNALeuCAA intronless strain showed reduced 5.8S rRNA levels and abnormal nucleolar morphology. Because pseudouridine (Ψ) occurs at position 34 of the tRNAIleUAU anticodon in an intron-dependent manner, tRNAIleUAU in the intronless strain lost Ψ34. However, in a portion of tRNAIleUAU population, position 34 was converted into 5-carbamoylmethyluridine (ncm5U), which could reduce decoding fidelity. In summary, our results demonstrate that, while introns are dispensable for cell viability, some introns have diverse roles, such as ensuring proper growth under various conditions and controlling the appropriate anticodon modifications for accurate pairing with the codon.

Heat and chilling stress induce nucleolus morphological changes

The nucleolus, where components of the ribosome are constructed, is known to play an important role in various stress responses in animals. However, little is known about the role of the plant nucleolus under environmental stresses such as heat and chilling stress. In this study, we analyzed nucleolus morphology by determining the distribution of newly synthesized rRNAs with an analog of uridine, 5-ethynyl uridine (EU). When EU was incorporated into the root of the Arabidopsis thaliana, EU signals were strongly localized in the nucleolus. The results of the short-term incorporation of EU implied that there is no compartmentation among the processes of transcription, processing, and construction of rRNAs. Nevertheless, under heat and chilling stress, EU was not incorporated into the center of the nucleolus. Morphological analyses using whole rRNA staining and differential interference contrast observations revealed speckled and round structures in the center of the nucleolus under heat and chilling stress, respectively.

The intracellular domain of \u03b2-dystroglycan mediates the nucleolar stress response by suppressing UBF transcriptional activity

β-dystroglycan (β-DG) is a key component of multiprotein complexes in the plasma membrane and nuclear envelope. In addition, β-DG undergoes two successive proteolytic cleavages that result in the liberation of its intracellular domain (ICD) into the cytosol and nucleus. However, stimuli-inducing ICD cleavage and the physiological relevance of this proteolytic fragment are largely unknown. In this study we show for the first time that β-DG ICD is targeted to the nucleolus where it interacts with the nuclear proteins B23 and UBF (central factor of Pol I-mediated rRNA gene transcription) and binds to rDNA promoter regions. Interestingly DG silencing results in reduced B23 and UBF levels and aberrant nucleolar morphology. Furthermore, β-DG ICD cleavage is induced by different nucleolar stressors, including oxidative stress, acidosis, and UV irradiation, which implies its participation in the response to nucleolar stress. Consistent with this idea, overexpression of β-DG elicited mislocalization and decreased levels of UBF and suppression of rRNA expression, which in turn provoked altered ribosome profiling and decreased cell growth. Collectively our data reveal that β-DG ICD acts as negative regulator of rDNA transcription by impeding the transcriptional activity of UBF, as a part of the protective mechanism activated in response to nucleolar stress.

Influence of seed priming with iron and/or zinc in the nucleolar activity and protein content of bread wheat.

Seed priming with iron (Fe) and/or zinc (Zn) can overcome the reduced availability of these micronutrients in soils and crops, but suitable dosages should be predetermined. Nucleolus responds to stress, such as cytotoxicity, with alterations perceivable by cytogenetic analyses. This work intends to study how seed priming with Fe and/or Zn affects the nucleolar activity in roots and the total soluble protein content in the flour of bread wheat cv. 'Jordão'. Seven priming treatments with 0mgL-1 to 8mgL-1 of Fe and/or Zn were performed. In all treatments, each metaphase cell presented a maximum of six nucleolar organizer regions positively stained with silver nitrate (Ag-NORs). Also, a maximum number of six nucleoli per nucleus were observed in all treatments, except in the hydroprimed seeds (used as control) that showed a maximum of five nucleoli, probably due to nucleolar fusion. Irregular interphases were frequent in treatments with the highest dosage of micronutrients (8mgL-1 Fe and/or 8mgL-1 Zn). The nucleolar area reduced (p < 0.001) as the number of nucleoli increased, and it was lower in treatments with a combination of Fe and Zn. However, the combinations of Fe and Zn showed the highest concentrations of total soluble protein (p ≤ 0.001). Although a reduced nucleolar area represents low ribosomal RNA gene transcription and ribosomal production, the significant increase of the number of nucleoli in the seeds primed with Fe and Zn enhanced the total soluble protein content as compared to the hydroprimed seeds (control) probably due to an increase of nucleolar surface-to-volume ratio that improved the protein synthesis. Overall, this work revealed that priming of bread wheat seeds with suited dosages of Fe and Zn can improve the nutritional value of flour, and the nucleolar number, morphology, and area can be useful biomarkers in cytotoxicity studies.

Use of the iNo score to discriminate normal from altered nucleolar morphology, with applications in basic cell biology and potential in human disease diagnostics.

Ribosome biogenesis is initiated in the nucleolus, a cell condensate essential to gene expression, whose morphology informs cancer pathologists on the health status of a cell. Here, we describe a protocol for assessing, both qualitatively and quantitatively, the involvement of trans-acting factors in the nucleolar structure. The protocol involves use of siRNAs to deplete cells of factors of interest, fluorescence imaging of nucleoli in an automated high-throughput platform, and use of dedicated software to determine an index of nucleolar disruption, the iNo score. This scoring system is unique in that it integrates the five most discriminant shape and textural features of the nucleolus into a parametric equation. Determining the iNo score enables both qualitative and quantitative factor classification with prediction of function (functional clustering), which to our knowledge is not achieved by competing approaches, as well as stratification of their effect (severity of defects) on nucleolar structure. The iNo score has the potential to be useful in basic cell biology (nucleolar structure-function relationships, mitosis, and senescence), developmental and/or organismal biology (aging), and clinical practice (cancer, viral infection, and reproduction). The entire protocol can be completed within 1 week.

High-Content Imaging Approaches to Quantitate Stress-Induced Changes in Nucleolar Morphology.

The nucleolus is a dynamic subnuclear compartment that has a number of different functions, but its primary role is to coordinate the production and assembly of ribosomes. For well over 100 years, pathologists have used changes in nucleolar number and size to stage diseases such as cancer. New information about the nucleolus' broader role within the cell is leading to the development of drugs which directly target its structure as therapies for disease. Traditionally, it has been difficult to develop high-throughput image analysis pipelines to measure nucleolar changes due to the broad range of morphologies observed. In this study, we describe a simple high-content image analysis algorithm using Harmony software (PerkinElmer), with a PhenoLOGIC™ machine-learning component, that can measure and classify three different nucleolar morphologies based on nucleolin and fibrillarin staining ("normal," "peri-nucleolar rings" and "dispersed"). We have utilized this algorithm to determine the changes in these classes of nucleolar morphologies over time with drugs known to alter nucleolar structure. This approach could be further adapted to include other parameters required for the identification of new therapies that directly target the nucleolus.

Prohibitin 2 localizes in nucleolus to regulate ribosomal RNA transcription and facilitate cell proliferation in RD cells

Prohibitin 2 (PHB2), as a conserved multifunctional protein, is traditionally localized in the mitochondrial inner membrane and essential for maintenance of mitochondrial function. Here, we investigated the role of PHB2 in human rhabdomyosarcoma (RMS) RD cells and found substantial localization of PHB2 in the nucleolus. We demonstrated that PHB2 knockdown inhibited RD cell proliferation through inducing cell cycle arrest and suppressing DNA synthesis. Meanwhile, down-regulation of PHB2 also induced apoptosis and promoted differentiation in fractions of RD cells. In addition, PHB2 silencing led to altered nucleolar morphology, as observed by transmission electron microscopy, and impaired nucleolar function, as evidenced by down-regulation of 45S and 18S ribosomal RNA synthesis. Consistently, upon PHB2 knockdown, occupancy of c-Myc at the ribosomal DNA (rDNA) promoter was attenuated, while more myoblast determination protein 1 (MyoD) molecules bound to the rDNA promoter. In conclusion, our findings suggest that nucleolar PHB2 is involved in maintaining nucleolar morphology and function in RD cells by regulating a variety of transcription factors, which is likely to be one of the underlying mechanisms by which PHB2 promotes tumor proliferation and represses differentiation. Our study provides new insight into the pathogenesis of RMS and novel characterizations of the highly conserved PHB2 protein.

Lamin B2 Modulates Nucleolar Morphology, Dynamics, and Function.

ABSTRACTThe nucleolus is required for ribosome biogenesis. Human cells have 2 or 3 nucleoli associated with nucleolar organizer region (NOR)-bearing chromosomes. An increase in number and altered nucleolar morphology define cancer cells. However, the mechanisms that modulate nucleolar morphology and function are unclear. Here we show that in addition to localizing at the nuclear envelope, lamin B2 localizes proximal to nucleolin at the granular component (GC) of the nucleolus and associates with the nucleolar proteins nucleolin and nucleophosmin. Lamin B2 knockdown severely disrupted the nucleolar morphology, which was rescued to intact and discrete nucleoli upon lamin B2 overexpression. Furthermore, two mutually exclusive lamin B2 deletion mutants, ΔHead and ΔSLS, rescued nuclear and nucleolar morphology defects, respectively, induced upon lamin B2 depletion, suggesting independent roles for lamin B2 at the nucleolus and nuclear envelope. Lamin B2 depletion increased nucleolin aggregation in the nucleoplasm, implicating lamin B2 in stabilizing nucleolin within the nucleolus. Lamin B2 knockdown upregulated nucleolus-specific 45S rRNA and upstream intergenic sequence (IGS) transcripts. The IGS transcripts colocalized with aggregates of nucleolin speckles, which were sustained in the nucleoplasm upon lamin B2 depletion. Taken together, these studies uncover a novel role for lamin B2 in modulating the morphology, dynamics, and function of the nucleolus.