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PubMed2006

Nuclear exclusion of separase prevents cohesin cleavage in interphase cells.

During mitosis, equal transmission of the duplicated chromosomes demands a strict regulation of separase, which cleaves cohesin and triggers sister chromatid separation in anaphase. Vertebrate separase is inhibited by securin and the inhibitory phosphorylation of separase. However, knockout experiments indicate that securin is dispensable and the inhibitory phosphorylation was observed only in M phase cells. This begs the question how cohesin cleavage by separase is prevented in the absence these two mechanisms. Here we show that separase is excluded from cohesin by the nuclear envelope, which forms in telophase and disassembles in mitosis. The exclusion is achieved passively by its large physical mass and may be backed up by the CRM1-dependent nuclear export. A functional NES motif is identified in separase. We demonstrated that the nuclear envelope is sufficient to prevent active separase from cleaving nuclear cohesin. We propose that the nuclear exclusion is important to prevent cohesin cleavage during interphase in the absence of securin and the phosphorylation inhibition.

Active Transport, Cell Nucleus↗
PubMed1975

Amoeba proteus: the nuclear periphery.

This study extends previous work on the nuclear envelope and associated structures. It illustrates that the cylindrical structures of the honeycomb lattice are not attached to the nuclear envelope, although generally perpendicular and closely apposed to it, and that there is a complex arrangement of fibrillar material between the cylinders of the lattice. The relationship of nuclear helices to these structures is described and the possible mode of their transfer from nucleus to cytoplasm is discussed.

Amoeba↗
PubMed1975

A study on the different types of spermatogonia in buffalo (Bubalus bubalis).

As a first step to understanding spermatogenesis in the buffalo bull the cytological details of different types of spermatogonia were determined in adult buffalo bulls. Morphological changes in the nuclear details were used as a basis for classifying the different types of spermatogonia. The type A spermatogonia had a spherical to ovoid nucleus with finely granulated chromatin, homogeneously dispersed in the nucleoplasm and having one to two nucleoli adhering to the nuclear membrane. The type A0 spermatogonia were characterized by nuclei containing moderately stained, finely granulated chromatin and a nucleolus attached to the nuclear envelope. The A1 type spermatogonia, on the other hand, have pale stained, finely granulated chromatin with the nucleolus adhering to the nuclear membrane. The nuclei of A2 type spermatogonia resembled those of type A1, but contained coarse granular chromatin dispersed in the pale nucleoplasm. The intermediate type of spermatogonia acquired a central position of the nucleolus, but the chromatin remained coarsely granulated and non-clumped. Three classes of type B (B1-B3) spermatogonia were determined on the degree of clumping of the chromatin and the central position of the nucleolus. The type B1 cells were characterized by nuclei containing a few flakes of lightly stained chromatin and a centrally located nucleolus. The type B2 cells showed comparatively more clumping of chromatin than type B1 spermatogonia, which was dispersed at random in the pale nucleoplasm and along the nuclear envelope. The type B3 spermatogonia demonstrated chromophilic chromatin dispersed in the slightly grey nucleoplasm and adhering along the nuclear membrane. Since there seems to be a succession of events following differentiation of type A1 spermatogonia till the last type B cell differentiates into resting primary spermatocytes, may intermediate stages between the presently described classes of type A (A0-A2) and type B (B1-B3) could also be located in sections of the seminiferous tubules.

Animals↗
PubMed1986

Analysis of the internal nuclear matrix. Oligomers of a 38 kD nucleolar polypeptide stabilized by disulfide bonds.

When rat liver nuclei are treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to nuclease treatment and extraction with 1.6 M NaCl, residual nucleoli and an extensive non-chromatin intranuclear network remain associated with the nuclear envelope. Subsequent treatment of this structure with 1 M NaCl containing 20 mM dithiothreitol (DTT) solubilizes the intranuclear material, while the nuclear envelope remains structurally intact. We have isolated and partially characterized a major polypeptide of the disulfide-stabilized internal nuclear matrix. The polypeptide, which has an apparent molecular mass 38 kD and isoelectric point 5.3, has been localized to the nucleolus of rat liver nuclei by indirect immunofluorescence using a specific polyclonal chicken antiserum. Based on its molecular mass, isoelectric point, intracellular localization and amino acid composition, the 38 kD polypeptide appears to be analogous to the nucleolar phosphoprotein B23 described by Prestayko et al. (Biochemistry 13 (1974) 1945) [20]. Immunologically related polypeptides have likewise been localized to the nucleoli of both hamster and human tissue culture cell lines as well as the cellular slime mold Physarum polycephalum. By immunoblotting, a single 38 kD polypeptide is recognized by the antiserum in rat, mouse, hamster and human cell lines. The antiserum has been utilized to investigate the oligomeric structure of the 38 kD polypeptide and the nature of its association with the rat liver nuclear matrix. By introducing varying numbers of disulfide bonds, we have found that the 38 kD polypeptide becomes incorporated into the internal nuclear matrix in a two-step process. Soluble disulfide-bonded homodimers of the polypeptide are first formed and then are rendered salt-insoluble by more extensive disulfide cross-linking.

Animals↗
PubMed1999

Lamin A is part of the internal nucleoskeleton of human erythroleukemia cells.

Nuclear lamins are the most abundant components of the nuclear lamina, a 10-50-nm-thick fibrous layer underlying the inner nuclear envelope membrane. Nevertheless, a number of recent investigations performed on epithelial and fibroblast cells have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nucleoskeleton. We have studied the subnuclear distribution of lamins A and B1 in human erythroleukemia cells by using immunoblotting analysis and immunofluorescent staining of fractionated nuclei. In intact cells and isolated nuclei, antibodies to lamins A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. However, when chromatin was removed by nuclease digestion and extraction with nonionic detergent or solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix or scaffold. Preferential localization of lamin A to the inner part of the nucleus was also demonstrated by the presence of the majority of lamin A in the solubilized inner nuclear network subfraction. In contrast, lamin B1 was mainly recovered in the fraction corresponding to the nuclear periphery. Double labeling experiments showed that lamin A, but not lamin B1, colocalized with coiled and GATA-1 bodies. Thus, our results support the hypothesis that lamin A, but not lamin B1, may be a component of an internal nucleoskeleton in human erythroleukemia cells.

Cell Cycle↗
PubMed1987

Distribution of microtubules during the first meiotic cell division in the mouse oocyte: effect of taxol.

Mouse oocytes were stained by antitubulin antibody and by an anti-MAP1 antibody (JA2) during the first meiotic cell division. At germinal vesicle stage, JA2 antibody exclusively stains the nucleoplasm. When the nuclear envelope breaks down (GVBD) (2-3 h of culture), numerous foci of microtubules appear around the disrupting nuclear envelope; they are decorated by anti-MAP1 antibody. After 10-12 h of culture, the antigen component detected with anti-MAP1 antibody is present in the meiotic spindle. The antimitotic agent taxol (10 microns) induces microtubule formation, mainly at the periphery of the germinal vesicle. At GVBD, taxol provokes the formation of a large microtubular array stained with both antibodies, which is associated with the condensed chromosomes. Furthermore, numerous cytoplasmic asters become visible in the cytoplasm. At metaphases I and II, taxol induces an important enlargement of the metaphase spindles and increases the number of cytasters.

Alkaloids↗
PubMed1989

Endogenous opioids and neural cancer: an immunoelectron microscopic study.

[Met5]-enkephalin, an endogenous opioid peptide derived from proenkephalin A, participates in tumorigenic events by serving as a natural trophic factor that inhibits cell replication. In order to understand how endogenous opioids function in modulating neoplasia, the present study examined the fine structural association of enkephalin with the cellular components of a tumor cell. Immunoelectron microscopic studies were undertaken using antibodies recognizing [Met5]-enkephalin-like substances, and murine S20Y neuroblastoma cells that are known to be responsive to endogenous opioid modulation. Enkephalin was found throughout the cell body and process. Immunoreactivity was associated with the plasma membrane, outer nuclear envelope, and a variety of organelles. With the exception of aggregates of immunoreactivity subjacent to the inner nuclear envelope, the nucleus was not reactive. These results establish that growth-related enkephalins are localized discretely within neuroblastoma cells. Since neuroblastoma cells produce and secrete enkephalins, and enkephalins interact with receptors to mediate actions on cell replication, this study examined enkephalins involved in two different patterns of traffic; further work will be needed to examine each aspect.

Animals↗
PubMed2006

Ultrastructural nuclear import assay.

Electron microscopy (EM) has been used for several decades to study the mechanisms of nuclear transport. In early studies of nuclear import, gold-conjugated nuclear proteins were microinjected into cells and followed by EM. As the components of the nuclear pore complex (NPC) and soluble mediators of nuclear import were cloned and characterized, gold-conjugated antibodies were utilized to sublocalize the components of the nuclear transport machinery by immuno-EM. Further, gold-conjugated recombinant proteins were used to probe permeabilized cells or isolated nuclear envelopes and characterize binding sites for these proteins at the NPC. More recently, recombinant gold-conjugated nuclear proteins were used in in vitro nuclear import assays to help dissect the mechanisms of nuclear import. We have used this ultrastructural nuclear import assay to study the nuclear import of the transcription factor PU.1. The results showed that this import requires energy but is carrier-independent. In the presence of energy, gold-conjugated PU.1 shifted to the nuclear side of the NPC and the inside of the nucleus. In conjunction with biochemical assays, these results indicated that this shift involved Ran-dependent binding of PU.1 to NUP153, a nucleoporin situated at the nuclear side of the NPC. Here we describe in detail the methods used in the ultrastructural nuclear import assay including preparation of recombinant protein, gold conjugation, in vitro nuclear import assay, electron microscopy, and data analysis.

Active Transport, Cell Nucleus↗
PubMed1995

Distinct functions for the two importin subunits in nuclear protein import.

The import of nuclear proteins proceeds through the nuclear pore complex and requires nuclear localization signals (NLSs), energy and soluble factors, namely importin-alpha (M(r) 60K), importin-beta (90K) and Ran. Importin-alpha is primarily responsible for NLS recognition and is a member of a protein family that includes the essential yeast nuclear pore protein SRP1p (ref. 16). As the first event, the complex of importin-alpha and importin-beta binds the import substrate in the cytosol. Here we show that this nuclear pore targeting complex initially docks as a single entity to the nuclear pore via importin-beta. Then the energy-dependent, Ran-mediated translocation through the pore results in the accumulation of import substrate and importin-alpha in the nucleus. In contrast, importin-beta accumulates at the nuclear envelope, but not in the nucleoplasm. Immunoelectron microscopy detects importin-beta on both sides of the nuclear pore. This suggests that the nuclear pore targeting complex might move as a single entity from its initial docking site through the central part of the nuclear pore before it disassembles on the nucleoplasmic side.

Adenosine Triphosphate↗
PubMed2003

Partial iodide organification defect caused by a novel mutation of the thyroid peroxidase gene in three siblings.

BACKGROUND: Three siblings with goitre and latent to mild hypothyroidism were suspected of having thyroid peroxidase (TPO) abnormality. Direct sequencing of their genomic DNAs showed two novel mutations of the TPO gene, one of which was G1687T (Gly533Cys; exon 9) and the other 1808-13del (Asp574/Leu575del; exon 10). The two mutations were compound heterozygous, as the former was found in their father's DNA as heterozygous, and the latter was found in DNA from their mother, also as heterozygous. As Gly533 and Asp574/Leu575 were well-conserved amino acids in the peroxidase superfamily, Gly533Cys- and Asp574/Leu575del-TPOs were thought to be affected structurally or functionally. In expression studies using CHO-Kl cells and mRNAs introduced with individual mutations, both mutated TPO proteins were expressed at the same molecular size as wild-type TPO and had enzyme activity, although Gly533Cys-TPO was slightly lower in efficiency of expression and more degenerative than wild-type TPO. METHODS: We examined the localization of both mutated TPOs. Gly533Cys-TPO was located on the endoplasmic reticulum (ER) and nuclear envelope but not on the plasma membrane, whereas Asp574/Leu575del-TPO was located not only on the ER and nuclear envelope but also on the plasma membrane, as wild-type TPO. Nevertheless, only one point differed between Asp574/Leu575del- and wild-type TPOs: the mutated TPO was expressed on the plasma membrane surface at less than half the rate of wild-type TPO. RESULTS: Gly533Cys-TPO synthesized almost no thyroid hormone because of its defective localization on the apical membrane surface of thyrocytes, whereas Asp574/Leu575del-TPO performed thyroid hormone synthesis at a rate of less half that of wild-type TPO. In cotransfection experiments using three combinations of wild-type and G1687T-mRNAs, wild-type and 1808-13del-mRNAs, and G1687T-, 1808-13del-mRNAs, the three kinds of mRNAs were considered to have no influence on cell surface TPO expression of another mRNA when a 50%-maximal amount of each mRNA was transfected. When a larger amount of each mRNA was transfected, the former two combinations showed the level of cell surface TPO expression obtained from the saturating amount of wild-type mRNA, whereas the last combination of mutated mRNAs covered only about half of the expression level. CONCLUSION: Defective thyroid hormone production resulting from the abnormal TPOs was at a level that caused latent hypothyroidism when the patients were born. With their growth, thyroid hormone volume gradually became inadequate and their thyroid gland enlarged compensatorily.

Animals↗
PubMed1969

The mitotic apparatus in fungi, Ceratocystis fagacearum and Fusarium oxysporum.

Vegetative nuclei of fungi Ceratocystis fagacearum and Fusarium oxysporum were studied both in the living condition with phase-contrast microscopy and after fixation and staining by HCl-Giemsa, aceto-orcein, and acid fuchsin techniques. Nucleoli, chromosomes, centrioles, spindles, and nuclear envelopes were seen in living hyphae of both fungi. The entire division process occurred within an intact nuclear envelope. Spindles were produced between separating daughter centrioles. At metaphase the chromosomes became attached to the spindle at different points. In F. oxysporum the metaphase chromosomes were clear enough to allow counts to be made, and longitudinal splitting of the chromosomes into chromatids was observed. Anaphase was characterized in both fungi by separation of chromosomes to poles established by the centrioles, and in F. oxysporum anaphase separation of chromosomes was observed in vivo. Continued elongation of the spindles further separated the daughter nuclei. Maturing daughter nuclei of both fungi were quite motile; and in C. fagacearum the centriole preceded the bulk of the nucleus during migration. The above observations on living cells were corroborated by observations on fixed and stained material.

Ascomycota↗
PubMed2006

Identification of tyrosine-phosphorylation sites in the nuclear membrane protein emerin.

Although several proteins undergo tyrosine phosphorylation at the nuclear envelope, we achieved, for the first time, the identification of tyrosine-phosphorylation sites of a nuclear-membrane protein, emerin, by applying two mass spectrometry-based techniques. With a multiprotease approach combined with highly specific phosphopeptide enrichment and nano liquid chromatography tandem mass spectrometry analysis, we identified three tyrosine-phosphorylation sites, Y-75, Y-95, and Y-106, in mouse emerin. Stable isotope labeling with amino acids in cell culture revealed phosphotyrosines at Y-59, Y-74, Y-86, Y-161, and Y-167 of human emerin. The phosphorylation sites Y-74/Y-75 (human/mouse emerin), Y-85/Y-86, Y-94/Y-95, and Y-105/Y-106 are located in regions previously shown to be critical for interactions of emerin with lamin A, actin or the transcriptional regulators GCL and Btf, while the residues Y-161 and Y-167 are in a region linked to binding lamin-A or actin. Tyrosine Y-94/Y-95 is located adjacent to a five-residue motif in human emerin, whose deletion has been associated with X-linked Emery-Dreifuss muscle dystrophy.

Amino Acid Sequence↗
PubMed2004

Ultrastructural and morphometric studies of normal rat hepatocytes.

To investigate the arrangement of organelles in normal rat liver hepatocytes, we examined liver tissues by electron microscopy. We also analyzed hepatocyte organelles by morphometry using the NIH image software. The smooth ER area appears as a network of branching tubules and contains many glycogen rosettes. The rough ER area presents lamellar plates and contains free polyribosomes. The Golgi complex area comprises the parallel smooth surface of associated vesicles and contains secretory granules, lysosomes and a few glycogens. Ratio of the cross-sectional area of hepatocytes shows the smooth ER area (60 +/- SD 1.5%), the rough ER area (35 +/- SD 1.4%) and the Golgi complex area (4 +/- SD 1.7%) of the total cytoplasm. The rough ER shows a dendrite pattern in a hepatocyte. Many mitochondria are spread between the smooth ER and the rough ER area. The smooth ER directly communicates with the rough ER, the Golgi complex cisternae at a forming face and the nuclear envelope. Anastomosing tubules with polyribosomes between the smooth ER and rough ER, between smooth ER and the nuclear envelope frequently. These results provide a basics at the cellular level for better understanding the experimentally induced changes and for improving the pathological hepatic diagnosis.

Animals↗
PubMed1996

Characterization of a sperm nuclear protein.

PROBLEM: The molecular identity of sperm DNA-binding structural proteins contributing to the integrity of a sperm residual high salt/nuclease resistant nuclear structure is studied by cDNA cloning and monoclonal antibodies to the recombinant polypeptide. This structure, which is likely to be transferred unimpaired in the oocyte and is anticipated as a molecular correlate of the nuclear scaffold (nuclear matrix/envelope) in somatic cells, may be essential with respect to its DNA organization for the recovery and assembly of somatic-type chromatin in the zygote. Recently, a cDNA encoding one of these proteins has been cloned and the recombinant polypeptide expressed in E. coli as a beta-galactosidase fusion protein. The main objective of the present study is the identification of the native sperm antigen by monoclonal antibodies raised against the recombinant molecule. METHODS: We evaluated the possibility of immunizing by direct intrasplenic deposition in BALB/c mice of the recombinant fusion protein available as transblotted on nitrocellulose membrane carriers or as nitrocellulose protein-bearing particles. Isolated sperm DNA/tight binding protein complexes were used in ELISA and Western blotting for selection of monoclonal antibodies specific to self epitopes of the nuclear antigen, as well as immunofluorescence of swollen human spermatozoa subjected to in situ extraction with high salt/beta-mercaptoethanol/DNase I and proteolysis, and of a cultured fibroblast cell line L-929. RESULTS: A monoclonal antibody, Mab 2C4, was selected which recognized a 52 kDa protein in the fraction of sperm high salt/urea resistant proteins. The target polypeptide was detected on swollen spermatozoa primarily to the post-acrosomal and/or equatorial regions whereas in nonextracted sperm cells the epitope was exceedingly unavailable. The somatic cell location of the cognate epitope was confined to the nuclear envelope displaying a cap-like pattern of staining, and also in a juxtanuclear cytoplasmic randomly coiled filamentous network and in compact bodies. CONCLUSIONS: A nuclear protein salt-stably bound to the sperm residual structure has been identified. The antigen appears localized in sperm exclusively to perinuclear subacrosomal sites that may be anchored at the male nuclear envelope, given the occurrence of the target epitope in somatic cells as well in nuclear and cytoplasmic sites adjacent to the nuclear membrane.

Animals↗
PubMed1983

Evidence for mediated protein uptake by amphibian oocyte nuclei.

The objective of this investigation was to determine whether there is mediated transport of endogenous proteins across the nuclear envelope. For this purpose, we studied the nuclear uptake of a 148,000-dalton Rana oocyte polypeptide (RN1) and compared its actual uptake rate with the rate that would be expected if RN1 crossed the envelope by simple diffusion through the nuclear pores. Nuclear uptake was studied in two ways: first, oocytes were incubated in L-[3H]leucine for 1 h and, at various intervals after labeling, the amount of 3H-RN1 present in the nucleoplasm was determined. Second, L-[3H]leucine-labeled nuclear extracts, containing RN1, were microinjected into the cytoplasm of nonlabeled cells, and the proportion of 3H-RN1 that subsequently entered the nucleus was measured. It was found that RN1 can readily penetrate the nuclear envelope; for example, after 6 h, approximately 36% of the newly synthesized RN1 and 17% of the injected RN1 had entered the nucleus. The diffusion rate through pores having a radius of 45 A was calculated for several possible molecular configurations of RN1. Using axial ratios of 34, 7.5, 2, and 1, the estimated times required to reach 63% of diffusion equilibrium are 757, 468, 6,940 h, and infinity, respectively. Even assuming an axial ratio of 7.5 (the most diffusive configuration) and an equilibrium distribution of 45, simple diffusion through the pores could account for only approximately 1/20 the observed nuclear uptake of RN1. This and other comparisons indicate that some form of mediated transport is involved in the nucleocytoplasmic exchange of this polypeptide.

Animals↗
PubMed2002

Genome-wide nuclear morphology screen identifies novel genes involved in nuclear architecture and gene-silencing in Saccharomyces cerevisiae.

Organisation of the cell nucleus is crucial for the regulation of gene expression but little is known about how nuclei are structured. To address this issue, we designed a genomic screen to identify factors involved in nuclear architecture in Saccharomyces cerevisiae. This screen is based on microscopic monitoring of nuclear pore complexes and nucleolar proteins fused with the green fluorescent protein in a collection of approximately 400 individual deletion mutants. Among the 12 genes identified by this screen, most affect both the nuclear envelope and the nucleolar morphology. Corresponding gene products are localised preferentially to the nucleus or close to the nuclear periphery. Interestingly, these nuclear morphology alterations were associated with chromatin-silencing defects. These genes provide a molecular context to explore the functional link between nuclear architecture and gene silencing.

Cell Nucleolus↗
PubMed2005

Nucleocytoplasmic trafficking in plant cells.

In a eukaryotic cell, the nuclear envelope (NE) separates genetic information from the environment of biosynthesis and metabolism. Transfer of macromolecules across the NE involves the nuclear pores--large multisubunit protein complexes--and machinery that facilitates rapid, directional, and selective transport. While core elements of the transport process are conserved between kingdoms, different solutions to similar problems have also evolved. Although the structure and composition of the yeast and mammalian nuclear pore have been unraveled recently, the plant nuclear pore remains largely enigmatic. Like any other process, nucleocytoplasmic transport can be regulated. Several examples from plants are discussed that promise insights into the regulation of signaling pathways. While controlling the partitioning of cellular components, the nuclear envelope also presents an obstacle to viruses and transforming agents that need access to the genome, and different mechanisms have evolved to overcome this obstacle. Finally, the recent recognition of the importance of small RNAs for gene regulation emphasizes the need to understand small RNA nuclear export and the levels of its regulation. This review attempts to wed our molecular-mechanistic understanding of nucleocytoplasmic trafficking drawn from all model systems with the intriguing examples of regulated nucleocytoplasmic partitioning in plants.

Active Transport, Cell Nucleus↗