The nucleolar structure and the activity of NopA100, a nucleolin-like protein, during the cell cycle in proliferating plant cells

For the purpose of gaining knowledge of the relationships between cell proliferation and ribosome biogenesis, as two fundamental mutually interconnected cellular processes, studies were performed on cell populations synchronized in their cell-cycle progression by treatment with hydroxyurea, followed by sampling at different times after its removal. A structural rearrangement of the nucleolus was observed throughout the interphase, along with changes in the relative amounts of different nucleolar subcomponents. A structural model of nucleolar organization was associated with each interphase period. Throughout interphase, the nucleolin-like protein, NopA100, was immunodetected in the dense fibrillar component of the nucleolus, preferentially near fibrillar centers and its levels were shown to increase from G1 to G2. A western blotting analysis of soluble nuclear protein extracts with anti-NopA100 antibody resulted in the intense labeling of a 100-kDa band, but also of a series of proteins related to it, suggesting that NopA100 undergoes a physiological process of proteolytic maturation, similar to that described for mammalian nucleolin, but not reported in other biological model systems. Physiological proteolysis of NopA100, related to cell-cycle progression, was confirmed after the nuclei extracted from synchronized cells were treated with the protease inhibitor, leupeptin, which resulted in an increase of the 100-kDa band at the expenses of the decrease of some other bands, according to the cell-cycle stages. We therefore conclude that there is a relationship between the increase in nucleolar activity, cell-cycle progression, nucleolar structure, the activity of NopA100, and the proteolysis of this nucleolin-like protein.     

NopA64, a novel nucleolar phosphoprotein from proliferating onion cells, sharing immunological determinants with mammalian nucleolin

Five major soluble nuclear proteins associated with cell proliferation were identified in Allium cepa L. root cells. One of them, of 64 kDa, was revealed by Western blotting with anti-mammalian nucleolin antibodies. A polyclonal antibody raised against this protein, which we have named NopA64, localised it in the nucleolus as well as in nuclear coiled bodies. Together with NopA64, the antibody also revealed a smaller form, called NopA61. Both proteins were present in the soluble ribonucleoprotein fraction and in the nuclear matrix of proliferating cells, but NopA61 was the only form revealed in differentiated cells. NopA64 contained epitopes also present in other plants, in mammalian nucleolin and in its yeast homologue, gar2. In mammals, the highest homology was with 50-kDa nucleolin fragments containing the RNA-binding motifs and the glycine-arginine-rich (GAR) domain. NopA64 was moderately phosphorylated in vitro by exogenous casein kinase II and cdc2 kinase, whereas NopA61 was highly phosphorylated by casein kinase II. Furthermore, NopA61 was the only band detected after dephosphorylation as well as after endoproteolysis of NopA64. This protein could be one of the various functional homologues of mammalian nucleolin in plant cells. Received: 2 July 1996 / Accepted: 15 October 1996     

Subnucleolar location of fibrillarin and NopA64 in Lepidium sativum root meristematic cells is changed in altered gravity

Summary. Fibrillarin and the plant nucleolin homolog NopA64 are two important nucleolar proteins involved in pre-rRNA processing. In order to determine the effects of the altered gravity environment on the nucleolus, we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under clinorotation, which reproduces an important feature of microgravity, namely, the absence of the orienting action of a gravity vector, and compared it to the location in control cells grown in normal 1 g conditions. Prior to these experiments, we report here the characterization of cress fibrillarin as a 41 kDa protein which can be isolated from meristematic cells in three nuclear fractions, namely, the soluble ribonucleoprotein fraction, the chromatin fraction, and the nuclear-matrix fraction. Furthermore, as reported for other species, the location of both fibrillarin and NopA64 in the cress cell nucleolus was in zones known to contain complex ribonucleoprotein particles involved in early pre-rRNA processing, i.e., processomes. Under altered gravity, a decrease in the quantity of both fibrillarin and NopA64 compared to controls was observed in the transition zone between fibrillar centers and the dense fibrillar component, as well as in the bulk of the dense fibrillar component. These data suggest that altered (reduced) gravity results in a lowered level of functional activity in the nucleolus. Correspondence and reprints: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu 9, 28040 Madrid, Spain.     

Proteomic analysis of root meristems and the effects of acetohydroxyacid synthase-inhibiting herbicides in the root of Medicago truncatula

Quantitative proteome analyses of meristematic and nonmeristematic tissues from Medicago truncatula primary and lateral roots and meristem tissues from plants treated with acetohydroxyacid synthase-inhibiting herbicides were made. The accumulation of 81 protein spots changed in meristematic and nonmeristematic tissues and 51 protein spots showed significant changes in accumulation in herbicide-treated meristems. Identified proteins indicate two trends, (i) increased accumulation of cell division and redox-mediating proteins in meristems compared to nonmeristematic tissues and (ii) increased accumulation of pathogenesis-related and decreased accumulation of metabolic proteins in herbicide-treated roots.     

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

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

Phosphorylation of nuclear and DNA-binding proteins in proliferating and quiescent mammalian cells.

The dependence of cell proliferation on nuclear protein phosphorylation was studied with exponential-phase and stationary-phase cultures of Chinese-hamster ovary cells. Nuclear proteins were fractionated, according to their DNA-binding affinities, by using sequential extractions of isolated nuclei with increasing concentrations of NaCl. When viable whole cells were labelled with H332PO4, phosphorylation of nuclear proteins was found to be lower in quiescent cells than in proliferating cells. Phosphorylation of nuclear proteins soluble in 0.30M-NaCl (less than 50% of these proteins bind to DNA) was greater than for those proteins soluble in higher salt concentrations (80-100% of these proteins bind to DNA). Cyclic AMP enhanced the phosphorylation of nuclear proteins soluble in 0.3 m-NaCl by 40-50%, and this stimulation was independent of cell growth. Cyclic AMP also increased the phosphorylation of nuclear proteins soluble in 0.6M-NaCl and 2.0M-NaCl by 40-50% in exponential-phase cultures, but not in stationary-phase cultures. Several examples of specific phosphorylation in response to cyclic AMP were observed, including a 35000-mol.wt. protein in the 0.30 M-NaCl-soluble fraction and several proteins larger than 100000 molecular weight within this fraction. A major peptide of molecular weight approx. 31000 extracted with 0.6M-NaCl was also phosphorylated. Its phosphorylation was independent of cyclic AMP in exponential-phase cultures, and it was not phosphorylated in plateau-phase cells. These changes in cell-growth-dependent phosphorylation occurred in the absence of any apparent qualitative changes in the nuclear protein molecular-weight distributions. These data demonstrate that (1) phosphorylation of nuclear proteins is dependent on the culture's proliferative status, (2) both cyclic AMP-dependent and cyclic AMP-independent specific phosphorylation occurs, and (3) the cyclic AMP-dependent growth-independent phosphorylation that occurs does not appear to be a modification of DNA-binding proteins, whereas the cyclic AMP-dependent growth-dependent phosphorylation does involve modification of DNA binding proteins.     

Coiled bodies in nuclei from plant cells evolving from dormancy to proliferation

To characterise the coiled bodies in meristematic nuclei of Saccharum officinarum, immunofluorescence labelling with antibodies against components of the splicing (U2B' and Sm core protein B) and pre-rRNA processing (fibrillarin) complexes was used in cells from the dormant root primordia and from roots at different times after activation to the steady state of proliferation. The number, size and distribution of coiled bodies varied in the meristematic tissue depending on cell activity. While G0 cells in the dry primordia and proliferating cells showed a similar number of coiled bodies attached to their nucleoli, the number of nucleoplasmic coiled bodies greatly increased after the primordia were stimulated to proliferate. Their number remained steady from the time the meristematic population reached the steady state of proliferation, as estimated by flow cytometry. Fractionation studies demonstrated that coiled bodies are a part of the underlying nuclear matrix. Comparison of immunocytochemical and cytochemical data from confocal and electron microscopical studies demonstrated that the nucleolar and nucleoplasmic coiled bodies detected by confocal microscopy shared many features, suggesting that they form a family of closely related structures.     

Altered gravity affects subnucleolus localization of fibrillarin and NopA64, the most important proteins of rRNA processing

Fibrillarin and plant nucleolin homologue NopA64 are two important nucleolar proteins involved in pre-rRNA processing. To understand better the effects of the altered gravity environment on the nucleolus functioning we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under simulated microgravity that was compared to the control cells grown in normal conditions at I g. Cress fibrillarin was first shown to have the molecular weight 41 kDa. Both fibrillarin and NopA64 in the cress cell nucleolus are located in the zones known to contain processing pre-rRNA molecules as it has been previously reported in other species. The data confirm participation of these proteins in processomes--RNP complex particles involved in pre-rRNA processing. Under altered gravity a decrease in the quantity of both fibrillarin and NopA64 in the transition zone between fibrillar centres and the dense fibrillar component was observed, compared to control, which could point out to a lowering of the level of early pre-rRNA processing in these experimental conditions. This decrease was also detected in the bulk of the dense fibrillar component. These data support the idea that altered (reduced) gravity results in lowering the level of functional activity of the nucleolus.     

Nuclear matrix proteins specific for subtypes of human hematopoietic cells

Nuclear matrices were prepared from isolated subtypes of human hematopoietic cells and from cultured leukemia cells. The nuclear matrix proteins were analyzed by high-resolution two-dimensional gel electrophoresis and computer-assisted image analysis. While more than 200 protein spots were shared among the cells, about 50 distinct spots were found characteristic for individual cells or groups of related cells. This allowed to differentiate between hematopoietic cells and nonhematopoietic cells, lymphocytes and myeloid cells, monocytes, neutrophils, and promyelocytic leukemia cells. B and T lymphocytes could not be differentiated. Myeloid cells with their polymorph nuclei were characterized by the presence of 13 and by the absence of seven distinct spots, as well as by low concentrations of nuclear lamins and of heterogeneous nuclear ribonucleoproteins. Neutrophils with multilobular nuclei displayed six additional spots, while lacking 18 nuclear matrix protein spots. The nuclear matrix of proliferating cells showed three distinct spots in addition to proliferating cell nuclear antigen, increased concentrations of numatrin (B23), and heterogeneous nuclear ribonucleoproteins. The described cell-specific nuclear matrix proteins may represent new markers for hematopoietic cells.     

NUCLEAR LOCALIZATION OF S-100 PROTEIN

Abstract— S-100 protein has been found in the nuclei isolated from the brain cortex of rabbit. The nuclear S-100 constitutes a small portion (0.55 per cent) of the S-100 present in the cytosol. Most of the large and pale nuclei appear to contain much more S-100 than the small and dark ones. The nuclear membrane is permeable in vitro to S-100 in presence of divalent cations. Three forms of S-100 occur in subnuclear fractions: free S-100, present in the soluble protein fraction; labile-bound S-100, present in the deoxyribonucleoprotein fraction and stable-bound S-100, present in the residual or‘nucleolar’fraction. The localization of the S-100 in those regions of the nucleus that are most active in RNA synthesis provides basic information for further studies on the possible role of this protein on genomic expression in nervous tissue.     

Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein

We have produced monoclonal antibodies against purified nuclei from the yeast Saccharomyces cerevisiae and have characterized three different antibodies that recognize a protein with an apparent molecular weight of 38,000, termed p38. Subcellular fractionation shows that virtually all of p38 occurs in the nuclear fraction. High concentrations of salt (1 M) or urea (6 M) effectively solubilize p38 from a nuclear envelope fraction prepared by digestion of nuclei with DNase. Indirect immunofluorescence demonstrates a crescent shaped distribution of p38 at the inner periphery of the nucleus, with p38 extending between dividing pairs of cells during (closed) mitosis. Postembedding immunogold electron microscopy shows decoration of the densely stained "crescent" region of the yeast nucleus, confirming the localization of p38 to the nucleolus. One of the monoclonals, D77, cross reacts on immunoblots with a single protein of molecular weight 37,000 from purified rat liver nuclei. Indirect immunofluorescence localizes this protein to the nucleolus, and shows that it is dispersed throughout the cell during mitosis. The yeast and rat liver nucleolar proteins behave similarly when electrophoresed in two dimensions, and appear to have basic pI values. Analysis of immunological cross-reactivity using D77, and antibodies specific for nucleolar proteins from other sources, suggests that the rat liver protein is fibrillarin, and demonstrates that p38 shares epitopes with fibrillarin, as well as with other vertebrate nucleolar proteins.     

Carbohydrate-binding protein 35. Isoelectric points of the polypeptide and a phosphorylated derivative

Purified carbohydrate-binding protein 35 (CBP35) and extracts of mouse cells containing CBP35 were analyzed by two-dimensional gel electrophoresis. Such an analysis on recombinant CBP35, obtained by expression of a cDNA clone in Escherichia coli, yielded a pI value of 8.7. When extracts of mouse 3T3 cells were subjected to two-dimensional gel electrophoresis and immunoblotting, two spots were observed, corresponding to pI values of 8.7 and 8.2. The pI 8.2 species represents post-translational modification of the CBP35 polypeptide (pI 8.7) by the addition of a single phosphate group. This conclusion is based on the finding that purified CBP35 contained a pI 8.2 species that was labeled with 32PO4 and could be converted to the unlabeled pI 8.7 species by alkaline phosphatase treatment. The phosphorylated (pI 8.2) form of CBP35 is found in both the cytosolic and nuclear fractions, whereas the unphosphorylated (pI 8.7) species is found exclusively in the nuclei. Quiescent populations of 3T3 fibroblasts (confluent monolayers or serum-starved sparse cultures) are characterized by the predominance of phosphorylated CBP35. Stimulation of the same cells into the proliferative state resulted in an increase in the amount of the phosphorylated species; more dramatic, however, is the elevation of the level of the unphosphorylated form, which is barely detectable in quiescent cells.     

Relations between nucleolar morphometric parameters and pre-rRNA synthesis in animal and plant cells

In order to study if there are differences between cells of the same tissue with one and two nucleoli, nuclear and nucleolar volume, density of tritiated uridine incorporation, amount of DNA per nucleus and intensity of cytoplasmic basophilia were measured in mononucleolated and binucleolated rat epithelial endometrial cells, in onion root meristematic cells and in chick embryo matrix cells of the central nervous system, neuroblasts and neurons. No significant differences in nuclear volume, density of tritiated uridine incorporation and amount of DNA per nucleus were found between cells of the same type with diverse numbers of nucleoli. Binucleolated endometrial cells, matrix cells, and root meristematic cells have biphasic distributions of nucleolar volumes. One peak of this distribution roughly coincides with the nucleolar volume of mononucleolated cells, the other peak corresponds almost to double the volume. As the density of uridine incorporation is the same irrespective of the nucleolar number and volume, the cells with larger nucleolar volumes have higher pre-rRNA synthesis. These cells also have higher amounts of ribosomes in the cytoplasm, as revealed by the photometric study of basophilia. It is concluded that in this population of cells the ribosomal production is regulated to a higher steady equilibrium than in the general population. This difference is not due to polyploidism or to the increased DNA content of G2 phase cells. Binucleolated neuroblasts and neurons have nucleolar volumes similar to those of mononucleolated ones.     

Monoclonal antibody against a nuclear matrix antigen in proliferating human cells

A monoclonal antibody of the IgG2a subclass was isolated from the supernate of a hybridoma line obtained with splenocytes from a mouse immunized with a crude nucleolar fraction of human Namalwa cells. This antibody identifies a single nuclear polypeptide antigen characterized by: (a) presence in proliferating human cell lines and phytohemagglutinin-stimulated lymphocytes, but absence in resting lymphocytes; (b) appearance in stimulated lymphocytes in parallel with the onset of DNA synthesis; (c) a speckled distribution in the nucleoplasm; (d) tight association with nuclear matrix structures identified by both biochemical and in situ extraction and enzyme treatment procedures; (e) mol wt of 125 kDa and pI 6.5 as determined by immunoprecipitation or immunoblotting of nuclear or nuclear matrix proteins fractionated by gel electrophoresis. The above characteristics identify the p125/6.5 nuclear matrix protein recognized by the isolated monoclonal antibody as belonging to the class of proliferating cell nuclear antigens.     

Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.     

An autoimmune antibody from scleroderma patients recognizes a component of the plant cell nucleolus

Summary An auto-antibody from human serum of patients with the autoimmune disease scleroderma was used to localize the nucleolus in meristematic cells of onion and soybean roots using indirect immunofluorescence microscopy. Similar lots of antiserum recognized a single 34 kD, nucleolar protein, fibrillarin, in a variety of animal cells (Ochs, et al. 1984, 1985). In both plants, antibody linked fluorescence is associated with the one to several nucleoli present in the interphase nucleus. The fluorescence becomes diffuse around condensing prophase chromosomes and becomes more diffused at metaphase with slightly more intense fluorescence surrounding the chromosomes. At anaphase-telophase the fluorescence is localized in dense areas within the chromosomes, presumably representing prenucleolar bodies which will form the interphase nucleoli of the daughter nuclei. This antiserum provides a new, valuable tool for the study of the nucleolus and the highly conversed nucleolar antigen(s) that it recognizes.     

Heat-induced morphological and biochemical changes in the nuclear lamina from Ehrlich ascites tumor cells in vivo

Membrane-depleted nuclei from Ehrlich ascites tumor (EAT) cells isolated at low ionic strength in the presence of EDTA exhibit highly decondensed chromatin fibers and a loss of morphologically identifiable nucleoli. Treatment of these nuclei with nucleases and 2 M NaCl followed by low-speed centrifugation permitted the facile isolation of the nuclear lamina layer. Under the same conditions, but after heat-shock treatment of the living cells, the chromatin appears in a more condensed state, the nucleoli are well-defined, and the nuclear lamina layer was destabilized in concert with the appearance of an internal nuclear matrix and nucleolar skeleton. Furthermore, we also found both an increase in the protein mass as well as the appearance of a relatively large number of new proteins in this fraction, which are phosphorylated. The major proteins of the nuclear lamina, the lamins, and the residual vimentin remained insoluble. These heat-shock-induced changes were also accompanied by a dephosphorylation of lamins A and C but not of lamin B.     

Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1-7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.     

Characterisation and high-resolution distribution of a matrix attachment region-binding protein (MFP1) in proliferating cells of onion

The first matrix attachment region (MAR)-binding protein sequenced in plants, MFP1, has been characterised in two dicot species. Based on their antigenic relationship, we report here the conservation of MFP1-like proteins in proliferating root cells of onion (Allium cepa L). Two MFP1-like proteins with different molecular masses and solubilities were detected. The most abundant was a 90-kDa basic protein, presenting several separate spots in two-dimensional blots. The MFP1 was partially soluble and, similar to the proliferating cell nuclear antigen (PCNA)-labelled replication factories in the nucleus and nuclear matrix, was localised at discrete foci as detected by confocal microscopy. High-resolution immunolocalisation of MFP1 by electron microscopy identified the foci as nuclear structures, some of them containing PCNA, which are ultrastructurally similar to the replication factories described in animal cells. Our data provide the first report on MFP1-like proteins in the Alliaceae. In addition, we present evidence of the presence of AcMFP1 in the putative replication factories. Received: 12 May 2000 / Accepted: 13 September 2000