Internalization of anti-nucleolin antibody into viable HEp-2 cells

Anti-nucleolin antibodies have been detected in patients with systemic connective tissue diseases (SCTD) including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). In vivo bound autoantibodies to nucleoli of epidermal keratinocytes have been demonstrated in skin from patients with SCTD. In this study, monoclonal antibody to nucleolin (D-3) was used to determine the distribution of nucleolin in different culture cells including HEp-2, HepG2, HRCC, Molt-4 and Wil2 cells. Nucleolin was found to be present on the surface of HEp-2 and HepG2 cells, but not on the surface of HRCC and lymphoblastoid (Molt-4 and Wil2) cells; in contrast, nucleolin was detected in the nucleoli of all permeabilized cells examined. In immunoprecipitation, using extracts from ³²P-labeled HEp-2 cells as antigenic source, cell membrane as well as nuclear nucleolins were found to be phosphorylated with a molecular weight of 105 kDa. Viable HEp-2 and HepG2 cells were cocultured with IgG fraction of D-3 in a CO₂ incubator for 1 to 24 h, and then permeabilized with acetone followed by immunofluorescence staining with FITC-labeled goat anti-mouse IgG antibodies. Nucleolar staining was observed in cells after 10 h or longer of coculture. These data indicated that D-3 antibody reacted with cell membrane nucleolin and subsequently gain access into cells in a process related to pinocytosis.     

Identification of Novel Markers That Demarcate the Nucleolus during Severe Stress and Chemotherapeutic Treatment

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 high confidence under a variety of experimental settings.     

Intermediate filament proteins in tanycytes of the third cerebral ventricle in rats during postnatal ontogenesis

Cytoskeletal intermediate filaments (IF) are composed of proteins able to form homo- and heterodimers, while their repertoire can change during cell differentiation. Data on the IF protein composition in tanycytes lining the mammalian third cerebral ventricle are still discrepant. The aim of this study was to investigate age-related changes in the IF protein composition in tanycytes of the third cerebral ventricle in Wistar rats at different ages (7-, 14-, and 30-day-old pups and 4–5-month-old adults; n = 26), using immunocytochemistry and confocal laser microscopy. In adult animals, tanycytes were shown to express IF proteins vimentin, GFAP, and nestin. In different types of tanycytes GFAP and nestin begin to be synthesized at different postnatal ages. For example, in α1 tanycytes GFAP is already present in 7-day-old animals, while in β1 tanycytes it appears only by day 30 of postnatal development. Meanwhile, vimentin is an essential IF component at all ages studied. A comparison of our data with the results obtained on other animal models suggests the existence of species-specific differences in the IF protein repertoire in tanycytes.     

The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.

Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.     

Upon intracellular processing, the C-terminal death domain-containing fragment of the p53-inducible PIDD/LRDD protein translocates to the nucleoli and interacts with nucleolin

The p53-inducible and death domain-containing PIDD/LRDD protein has been described as an adaptor protein, which forms large protein complexes with RAIDD, another death domain-containing protein, leading to recruitment, and activation of the initiator caspase-2, and p53-mediated apoptosis. Here, we describe in further detail the proteolytic processing of PIDD/LRDD that occurs in healthy cells before induction of apoptosis. We could demonstrate that the C-terminal fragment containing the PIDD death domain shuttles into the nucleoli. This translocation is mediated by or leads to the interaction of the PIDD death domain with nucleolin, a protein important for rRNA processing within nucleoli and possibly involved in the DNA damage response. Ectopically expressed LRDD and endogenous nucleolin co-localized within the nucleoli, and overexpression of both full-length LRDD and the LRDD death domain sensitized cells for UV-induced apoptosis. When expressed alone, the PIDD/LRDD death domain tended to form large filamentous structures resembling so-called death filaments. The functional consequences of the identified PIDD/nucleolin interaction remain to be elucidated, but may be related to a recently discovered new role for PIDD in the activation of NF-kappaB upon genotoxic stress.     

Nucleolin, defective for MPF phosphorylation, localizes normally during mitosis and nucleologenesis

To determine what effect maturation promoting factor (MPF, p34 ^cdc2 kinase/cyclin B) phosphorylation has on nucleolin’s distribution during mitotic nucleolar disassembly and reassembly, we altered Chinese hamster ovary (CHO) nucleolin (the N protein) such that it cannot be phosphorylated by p34 ^cdc2 . As expected, the transiently expressed epitope-tagged N protein showed no apparent defect in nucleolar localization in interphase CHO cells, even after hypotonic shock and recovery to quickly disassemble and then reassemble interphase nucleoli. In mitotic CHO cells, the N protein localized to the perichromosomal sheath and the cytoplasm, as is typical for nucleolin. Similar to epitope-tagged wild-type nucleolin, the N protein also maintained its association with persistent nucleoli characteristic of mitotic Chinese hamster lung (Dede) cells. In synchronized HeLa cells, the N protein again localized to the perichromosomal sheath and the cytoplasm as nucleoli disassembled during prophase. In HeLa cell telophase, the N protein localized normally to nucleolus-derived foci within the cytoplasm and prenucleolar bodies within reforming nuclei. The observations indicate that MPF phosphorylation is not essential for nucleolin’s localizations to the perichromosomal sheath and the cytoplasm during prophase and metaphase, and that functional MPF phosphorylation sites are not essential for nucleolin’s localizations during nucleologenesis. Accepted: 15 April 1999     

The new face of nucleolin in human melanoma

Nucleolin is multifunctional protein mainly present in nucleoli but also detected in cytoplasm and plasma membranes. Extranuclear nucleolin differs from the nuclear form by its glycosylation. Studies on expression of nucleolin in breast cancer suggest a possible association to the metastatic cascade. In the present study, Vicia villosa lectin (VVL) precipitation followed by subsequent polyacrylamide gel electrophoresis and mass spectrometry analysis demonstrates nucleolin as a VVL-positive glycoprotein expressed in melanoma. The presence of VVL-positive nucleolin in the melanoma cell membrane and cytoplasm was confirmed by confocal microscopy. Using bioinformatic peptide prediction programs, nucleolin was shown to contain multiple possible MHC class-I binding peptides in its sequence which makes nucleolin an interesting melanoma marker and target for immunodiagnostic and possibly therapeutic purposes. This paper is an original contribution from the meeting which took place on 28–and 29 May, 2008, in Nottingham, UK, celebrating the contribution of Professor I.A. “Tony” Dodi (+29.1.2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.     

Pharmacological AMP Kinase Activators Target the Nucleolar Organization and Control Cell Proliferation

Aims

Phenformin, resveratrol and AICAR stimulate the energy sensor 5′-AMP activated kinase (AMPK) and inhibit the first step of ribosome biogenesis, de novo RNA synthesis in nucleoli. Nucleolar activities are relevant to human health, because ribosome production is crucial to the development of diabetic complications. Although the function of nucleoli relies on their organization, the impact of AMPK activators on nucleolar structures is not known. Here, we addressed this question by examining four nucleolar proteins that are essential for ribosome biogenesis.

Methods

Kidney cells were selected as model system, because diabetic nephropathy is one of the complications associated with diabetes mellitus. To determine the impact of pharmacological agents on nucleoli, we focused on the subcellular and subnuclear distribution of B23/nucleophosmin, fibrillarin, nucleolin and RPA194. This was achieved by quantitative confocal microscopy at the single-cell level in combination with cell fractionation and quantitative Western blotting.

Results

AMPK activators induced the re-organization of nucleoli, which was accompanied by changes in cell proliferation. Among the compounds tested, phenformin and resveratrol had the most pronounced impact on nucleolar organization. For B23, fibrillarin, nucleolin and RPA194, both agents (i) altered the nucleocytoplasmic distribution and nucleolar association and (ii) reduced significantly the retention in the nucleus. (iii) Phenformin and resveratrol also increased significantly the total concentration of B23 and nucleolin.

Conclusions

AMPK activators have unique effects on the subcellular localization, nuclear retention and abundance of nucleolar proteins. We propose that the combination of these events inhibits de novo ribosomal RNA synthesis and modulates cell proliferation. Our studies identified nucleolin as a target that is especially sensitive to pharmacological AMPK activators. Because of its response to pharmacological agents, nucleolin represents a potential biomarker for the development of drugs that diminish diabetic renal hypertrophy.     

Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression.     

Subcellular Distribution of Distinct Nucleolin Subfractions Recognized by Two Monoclonal Antibodies

Monoclonal antibodies binding to different domains of nucleolin have been used to localize nucleolin in tissue culture cells ofXenopus laevis.The monoclonal antibody b6-6E7 binds to an epitope in the N-terminal domain, which contains arrays of phosphorylation consensus sites. This monoclonal antibody binds to nucleolin of oocytes and of eggs with high affinity. In contrast, the monoclonal antibody Nu-1H6 binds poorly to the modified forms of nucleolin arising during meiosis and mitosis. In interphase cells, monoclonal antibody b6-6E7 preferentially stains the periphery of the nucleoli, where most of the rRNA accumulates. Staining by monoclonal antibody Nu-1H6 complements this pattern by staining mainly the center of the nucleoli. The epitope of monoclonal antibody Nu-1H6 is within the central domain of nucleolin, which contains the first two RNA binding domains. RNase treatment of cells results in loss of nucleolin from nucleoli. In mitotic cells, both monoclonal antibodies decorate the surface of condensing chromosomes in prophase. The periphery of the condensed chromosomes in metaphase and anaphase is preferentially stained by monoclonal antibody b6-6E7.     

Nucleolin in Neurons of the Human Substantia Nigra

Using immunocytochemistry and confocal laser microscopy, the distribution of nucleolin (protein C23) in neurons of the human substantia nigra was investigated. Fragments of the human midbrain (n = 8) with the verified lack of neurodegeneration were used in the study. The material was fixed in zinc ethanol formaldehyde, a special fixative that provides high preservation of antigenic determinants. New morphological data on the distribution of nucleolin in neurons of the human substantia nigra were obtained. Nucleolin was found in the nucleolus and nucleoplasm of dopaminergic neurons but not in the cytoplasm or cell surface. In the nucleolus and nucleoplasm, protein C23 is distributed irregularly in the form of some accumulations (clumps) with fuzzy borders. Moreover, nucleolin distribution varied in structurally similar neurons containing cytoplasmic neuromelanin. The Marinesco bodies typically not exhibiting nucleolin expression were identified in the nucleus, in addition to nucleoli, by confocal laser microscopy with simultaneous use of transmitted light detector. The findings can contribute to elucidation of the role of nucleolin in the specific functions of normal dopaminergic neurons in the human substantia nigra and assessment of probable involvement of C23 in the development of nucleolar stress and neurodegeneration.     

Morphometry of nucleoli and expression of nucleolin analyzed by laser scanning cytometry in mitogenically stimulated lymphocytes.

BACKGROUND: Various attributes of nucleoli, including abundance of the nucleolar product (rRNA), correlate with cell-proliferative status and are useful markers for tumor diagnosis and prognosis. However, there is a paucity of methods that can quantitatively probe nucleolus. The aim of the present study was to utilize the morphometric capacity of the laser scanning cytometer (LSC) to analyze nucleoli and measure expression of the nucleolar protein nucleolin (NCL) in individual cells and correlate it with their state of proliferation. MATERIALS AND METHODS: Human lymphocytes were mitogenically stimulated, and at different time points their nucleoli were detected immunocytochemically using NCL Ab. The frequency of nucleoli per nucleus, their area, and the level of expression of NCL, separately in the nuclear and nucleolar compartments, were estimated in relation to the G(0) to G(1) transition and the cell cycle progression. RESULTS: During the first 24 h of stimulation, when the cells underwent G(0) to G(1) transition, their RNA content was increased nearly 8-fold, the level of NCL per nucleus also increased 8-fold, the NCL per nucleolus increased 12-fold, nucleolear area increased 3-fold, and NCL/nucleolar area increased nearly 4-fold. During the subsequent 24-48 h of stimulation, when cells were progressing through S, G(2), and M and reentering the next cycle, the number of nucleoli per nucleus was increased and a massive translocation of NCL from nucleoli to nucleoplasm was observed; its overall level per nucleus, however, still remained high, at 6-fold above of that of G(0) cells. CONCLUSIONS: While high expression of NCL in the nucleolar compartment correlates with the rate of rRNA accumulation in the cell and is a sensitive marker of the G(0) to G(1) transition, the cells progressing through the remainder of the cycle are better distinguished from G(0) cells by high overall level of NCL within the nucleus. Such an analysis, when applied to tumors, may be helpful in obtaining the quantitative parameters related to the kinetic status of the tumor-cell population and tumor prognosis. The capability of LSC to measure the protein translocation between nucleolus and nucleoplasm can be used to study the function and regulatory mechanisms of other proteins that reside in these compartments.