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PubMed1998

Molecular segments of protein Tpr that confer nuclear targeting and association with the nuclear pore complex.

Tpr is a 267-kDa protein of unknown function recently identified as a constitutive component of the nuclear pore complex (NPC2)-attached intranuclear filaments. Secondary structure predictions suggest that the protein is divided into a large, coiled-coil forming aminoterminal domain and a shorter, highly acidic carboxyterminal domain. To identify which of Tpr's molecular segments determine its specific intranuclear localization, we have constructed expression vectors encoding various Tpr deletion mutants as well as chimeric combinations of Tpr sequences with the soluble cytoplasmic protein pyruvate kinase. Following transfection of cultured mammalian cells, we have identified a short region within Tpr's carboxyterminal domain that is essential and sufficient to mediate nuclear import of Tpr and which can also confer nuclear accumulation of pyruvate kinase. Tpr deletion mutants that contain this nuclear targeting segment, but lack the aminoterminal domain, appear evenly dispersed throughout the nucleus without any noticeable association to the NPC. In contrast, the aminoterminal domain lacking the carboxyterminal region remains located within the cytoplasm, forming aggregate-like structures not associated with the nuclear envelope. However, when tagged to Tpr's short nuclear targeting segment or to the nuclear localization signal of the SV40 large T protein, the aminoterminal domain is imported into the nucleus, where it then associates with the NPC. This association is mediated by shorter molecular segments within the aminoterminal domain which contain clusters of heptad repeats, whereas other regions are dispensable. This assignment of different topogenetic properties to distinct molecular segments of Tpr will now allow the design of future experiments to study the protein's structural properties further and determine its actual function.

Amino Acid Sequence↗
PubMed2007

Telomere attachment and clustering during meiosis.

Telomeres are important segments of chromosomes that protect chromosome ends from nucleolytic degradation and fusion. At meiosis telomeres display an unprecedented behavior which involves their attachment and motility along the nuclear envelope. The movements become restricted to a limited nuclear sector during the so-called bouquet stage, which is widely conserved among species. Recent observations suggest that telomere clustering involves actin and/or microtubules, and is altered in the presence of impaired recombinogenic and chromosome related functions. This review aims to provide an overview of what is currently known about meiotic telomere attachment, dynamics and regulation in synaptic meiosis.

Actins↗
PubMed2003

Virtual gating and nuclear transport: the hole picture.

The eukaryotic nucleus is surrounded by a protective nuclear envelope, which is perforated by trafficking machines termed nuclear pore complexes (NPCs). The NPCs are the sole mediators of exchange between the nucleus and the cytoplasm. Small molecules pass through the NPCs unchallenged; however, large macromolecules are excluded unless chaperoned across by transport factors. Here, we suggest a model, termed 'virtual gating', to explain the mechanism of this rapid and selective macromolecular trafficking.

Animals↗
PubMed2002

Slipping through the door: HIV entry into the nucleus.

HIV infection of non-dividing cellular targets like macrophages requires successful passage of the viral preintegration complex (PIC) across an intact nuclear envelope. Unique but redundant nuclear import signals reside within the HIV integrase, matrix, and Vpr proteins as well as the 'DNA flap'; these signals appear to facilitate PIC transport through the limiting nuclear pores. We discuss recent studies that have advanced our understanding of this key step in the HIV life cycle.

HIV Infections↗
PubMed1998

On the history of nuclear matrix manifestation.

The nonchromatin proteinous residue of the cell nucleus was revealed in our laboratory as early as in 1948 and then identified by light and electron microscopy as residual nucleoli, intranuclear network and nuclear envelope before 1960. This structure termed afterwards as "nuclear residue", "nuclear skeleton", "nuclear cage", "nuclear carcass" etc., was much later (in 1974) isolated, studied and entitled as "nuclear matrix" by Berezney and Coffey, to whom the discovery of this residual structure is often wrongly ascribed. The real history of nuclear matrix manifestation is reported in this paper.

Cell Fractionation↗
PubMed1998

Cytoplasmic control of nuclear assembly.

The reconstitution of a replication-competent, transcriptionally active nucleus following mitosis, fertilization or nuclear transplantation involves a stepwise series of reactions, most (if not all) of which are controlled by the cytoplasmic environment. This review discusses the nature of cytoplasmic contributions to the development of the male pronucleus at fertilization, and the effect of altering the cytoplasmic environment on nuclear assembly. The system used to investigate these regulations consists of permeabilized sea urchin sperm nuclei incubated under controlled conditions in a cell-free extract of fertilized sea urchin eggs. (1) In egg cytoplasmic extract, male pronuclear formation is initiated by the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation by a cytosolic protein kinase C. (2) Sperm histones are phosphorylated by an as yet unidentified soluble kinase. (3) The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-and cytosolic pH-dependent manner. (4) Chromatin decondensation is associated with the replacement of sperm histones by maternal histones. (5) Nuclear membranes form by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of these vesicles to one another. (6) Three cytoplasmic vesicle populations with distinct biochemical, chromatin-binding and fusion properties are required for nuclear envelope assembly. (7) Targeting of the bulk of nuclear membrane vesicles to chromatin is mediated by an integral membrane protein similar to human lamin B receptor. (8) The last step of male pronuclear formation, nuclear swelling, is promoted by the assembly of nuclear pores, nuclear import of soluble lamins and growth of the nuclear membranes. (9) Once inside the nucleus, lamin B associates with lamin B receptors, presumably to tether the inner nuclear membrane with the lamina. Overall, these processes are similar to those characterizing nuclear reconstitution after mitosis in somatic cells or nuclear remodeling following transplantation into oocytes or eggs. The influence of the egg cytoplasmic environment on some aspects of nuclear remodeling after nuclear transplantation is also discussed.

Animals↗
PubMed2004

Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LADelta50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LADelta50. Introduction of LADelta50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LADelta50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication.

Aged↗
PubMed1996

Dynamic localization of the nuclear import receptor and its interactions with transport factors.

Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rna1-1 strain, we have identified a genetic interaction between Rna1p, a GTPase activating protein required for nuclear transport, and yeast importin-beta, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rna1p physically interacts with importin-beta. Mutants in importin-beta exhibit in vivo nuclear protein import defects, and importin-beta localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-alpha, but not importin-beta, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-alpha and the nuclear localization signal-bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-alpha from the nucleus.

Amino Acid Sequence↗
PubMed1996

Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins.

Macromolecular trafficking across the nuclear envelope involves interactions between cytosolic transport factors and nuclear pore complex proteins. The p62 complex, an assembly of 62, 58, 54, and 45-kD O-linked glycoproteins-localized near the central gated channel of the nuclear pore complex, has been directly implicated in nuclear protein import. The cDNA cloning of rat p62 was reported previously. We have now carried out cDNA cloning of rat p58, p54, and p45. We found that p58 contains regions with FG (Phe, Gly) and PA (Pro, Ala) repeats at both its NH2 and COOH termini separated by a predicted alpha-helical coiled-coil region, while p54 has an NH2-terminal FG and PA repeat region and a COOH-terminal predicted coiled-coil region. p45 and p58 appear to be generated by alternative splicing, with p45 containing the NH2-terminal FG repeat region and the coiled-coil region of p58. Using immunogold electron microscopy, we found that p58/p45 and p54 are localized on both sides of the nuclear pore complex, like p62. Previous studies have shown that immobilized recombinant p62 can bind the cytosolic nuclear import factor NTF2 and thereby deplete transport activity from cytosol. We have now found that immobilized recombinant p58 and p54 also can deplete nuclear transport activity from cytosol, and that p62, p58, and p54 bind directly to the cytosolic nuclear import factors p97 and NTF2. At least in the case of p58, this involves FG repeat regions. Moreover, p58 can bind to a complex containing transport ligand, the nuclear localization sequence receptor (Srp1 alpha) and p97. These data support a model in which the p62 complex binds to a multicomponent particle consisting of transport ligand and cytosolic factors to achieve accumulation of ligand near the central gated channel of the nuclear pore complex.

Amino Acid Sequence↗
PubMed1963

STUDIES ON ISOLATED NUCLEI. I. ISOLATION AND CHEMICAL CHARACTERIZATION OF A NUCLEAR FRACTION FROM GUINEA PIG LIVER.

This article describes a method for the isolation of nuclei from guinea pig liver. It involves the homogenization of the tissue in 0.88 M sucrose-1.5 mM CaCl(2) followed by centrifugation in a discontinuous density gradient in which the upper phase is the homogenate and the lower phase is 2.2 M sucrose-0.5 mM CaCl(2). Based on DNA recovery, the isolated fraction contains 25 to 30 per cent of the nuclei of the original homogenate. Electron microscopical observations showed that approximately 88 per cent of the isolated nuclei come from liver cells (the rest from von Kupffer cells and leucocytes) and that approximately 90 per cent of the nuclei appear intact, with well preserved nucleoli, nucleoplasm, nuclear envelope, and pores. Cytoplasmic contamination is minimal and consists primarily of the nuclear envelope and its attached ribosomes. The nuclear fraction consists of approximately 22.3 per cent DNA, approximately 4.7 per cent RNA, and approximately 73 per cent protein, the DNA/RNA ratio being 4.7. Data on RNA extractibility by phosphate and salt and on the base composition of total nuclear RNA are included.

Animals↗
PubMed2006

Oogenesis in pig ovaries during the prenatal period: ultrastructure and morphometry.

Oogenesis in fetal pig ovaries comprises the successive changes from the primordial germ cells to the dictyotene oocytes in primordial ovarian follicles. In this study the observations were carried out with an electron microscope and stereological analysis was performed. At the ultrastructural level there are no differences between the primordial germ cells and oogonia, but oogonia are connected with the intercellular bridges. The onset of the dictyotene phase was accompanied by the changes in the cytoplasm of oocytes. Near the nucleus, the yolk nucleus is formed containing numerous Golgi bodies, endoplasmic reticulum (ER), mitochondria and granules. ER proliferates in contact with the external leaflet of the nuclear envelope forming the narrow ER cisterns. Between the nuclear envelope and ER cisterns, the vesicles with grey content are visible. The proliferating ER forms numerous concentric cisterns around the nucleus. Next, the most external cisterns fragment, detach, and then form the cup-like structures. These structures separate the distinct areas of cytoplasm-compartments, which contain mitochondria, ribosomes and lipid droplets. The cells of cortical sex cords of the ovary, which encloses the oocyte, form the follicles. The volume of oocytes in forming follicle increases due to the increase in the number of the cell inclusions: lipid droplets, vacuoles and yolk globules. In the oocytes of primordial ovarian follicles, the compartments are transformed into the yolk globules, which are encountered by a sheath of ER cisterns and the grey vesicles; they contain the mitochondria, lipid droplets and light vacuoles. The role of the compartments and yolk globules as metabolic units is discussed in comparison with similar structures of the mature eggs of pigs and other mammal species.

Animals↗
PubMed1997

The DNA replication licensing system.

The Xenopus cell free system has proved a good model system to study in vitro DNA replication and the mechanism preventing rereplication in a single cell cycle. Studies using this system resulted in the development of a model postulating the existence of a replication licensing factor (RLF), which binds to the chromatin before the G1-S transition of the cell cycle and is displaced during replication. The nuclear envelope prevents rebinding of RLF and hence relicensing. Nuclear envelope breakdown at mitosis is required to allow another round of replication. Protein kinase inhibitors block licensing factor activity and arrest Xenopus extracts in a G2 like state. These kinase inhibitors have allowed the development of an in vitro assay leading to the biochemical purification of RLF components. RLF can be separated into RLF-B and RLF-M, the latter consisting of several members of the MCM/P1 class of replication proteins. In Xenopus as well as in many other eukaryotes, the binding of MCM/P1 proteins to chromatin before S phase is essential for replication to occur. The proteins are then displaced as replication proceeds. These changes in subnuclear distribution are reflected by changes in the phosphorylation status. MCM/P1 proteins do not bind to the DNA on their own but need RLF-B to be loaded onto the chromatin. Their cycling behaviour is reminiscent of the existence of a prereplicative complex at the origins of replication in yeast, suggesting that the licensing mechanism is ubiquitous in eukaryotes.

Animals↗
PubMed2000

Fate of meiotic lamin C2 in rat spermatocytes cultured in the presence of okadaic acid.

The fate of nuclear envelope proteins during the pachytene/metaphase I transition was investigated in rat spermatocytes cultured in vitro in the presence of the phosphatase inhibitor okadaic acid (OA). Under these experimental conditions lamin B1 and the lamina-associated proteins 2 (LAPs2) behave as already described in other cell types. In contrast to these results, meiotic lamin C2 appears to be degraded after addition of OA to the spermatocyte culture medium as this lamin was no longer detectable by immunofluorescence microscopy or by immunoblotting. Taking into account the peculiarities of the lamin C2 primary structure, it is tempting to speculate that degradation of this protein represents a critical step in the process of disassembly of the spermatocyte nuclear envelope.

Animals↗
PubMed1997

The role of the nuclear pore complex in adenovirus DNA entry.

Adenovirus targets its genome to the cell nucleus by a multistep process involving endocytosis, membrane penetration and cytoplasmic transport, and finally imports its DNA into the nucleus. Using an immunochemical and biochemical approach combined with inhibitors of nuclear import, we demonstrate that incoming viral DNA and DNA-associated protein VII enter the nucleus via nuclear pore complexes (NPCs). Depletion of calcium from nuclear envelope and endoplasmic reticulum cisternae by ionophores or thapsigargin blocked DNA and protein VII import into the nucleus, but had no effect on virus targeting to NPCs. Calcium-depleted cells were capable of disassembling incoming virus. In contrast, inhibitors of cytosolic O-linked glycoproteins of the NPC blocked virus attachment to the nuclear envelope, capsid disassembly and also nuclear import of protein VII. The data indicate that NPCs have multiple roles in adenovirus entry into cells: they contain a virus-binding and/or dissociation activity and provide a gateway for the incoming DNA genome into the nucleus.

Adenoviridae↗
PubMed2000

P granules in the germ cells of Caenorhabditis elegans adults are associated with clusters of nuclear pores and contain RNA.

The germ cells, and germ cell precursors, in the nematode Caenorhabditis elegans contain distinctive granules called P granules. During early embryogenesis, P granules are segregated asymmetrically into those blastomeres that eventually produce the germ line. Because of the correlation between P granule distribution and the development of the germ line, P granules are widely thought to function in some aspect of germ line specification or differentiation. Most of the analysis of P granule structure and localization has focused on the early embryo, when P granules are located in the cytoplasm. However, during most of development P granules are associated with germ cell nuclei. We report here an ultrastructural analysis of the nuclear-associated P granules in the germ cells of the adult hermaphrodite gonad. We show that P granules are tightly associated with nuclear pores and that the positions of certain structures within the P granules correspond to the positions of pores on the nuclear envelope. We present immunocytochemical and ultrastructural data suggesting that P granules can associate, or remain associated, with pore-like structures even after they detach from the nuclear envelope during oogenesis. Finally, we show that nuclear-associated P granules in the gonad contain RNA, complementing previous studies showing that cytoplasmic P granules in embryos contain RNA.

Animals↗
PubMed1989

Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy.

Nuclear pore complexes (NPCs) play a central role in mediating nucleocytoplasmic transport and exchange processes in eukaryotic cells. The arrangement and interactions of NPCs within amphibian nuclear envelopes have been studied using cryo-electron microscopy of unfixed and frozen hydrated specimens. The nuclear lamina in Necturus forms an orthogonal network with crossover distances which vary between 1,600 and 4,000 A and which may be related to the basic filament repeat of lamins. Furthermore, the NPCs are attached randomly within the confines of the lamin network, presumably by their nucleoplasmic rings. Image analysis of edge-on and en face projections of detergent-extracted NPCs has been combined with data on the coaxial thin rings to provide a quantitative evaluation of the triple ring model of NPC architecture proposed previously (Unwin, P. N. T., and R. Milligan. 1982. J. Cell Biol. 93:63-75). Additional details of the complex have been visualized including an intimate association of the inner spoke domains as an inner spoke ring, extensive domains within the spokes and coaxial thin rings, and interestingly, a central channel-like feature. Membrane-associated NPCs and detergent-extracted NPCs both possess peripherally located radial arms resulting in an effective diameter of approximately 1,450-1,500 A. In projection, the radial arms possess approximate mirror symmetry suggesting that they originate from both sides of the assembly. Moreover, membrane-associated NPCs are asymmetric at most radii and right-handed as viewed from the cytoplasm; detergent-extracted NPCs appear to be symmetric and have approximately 822 symmetry. Taken together, the data suggests that the framework of membrane-associated NPCs is perturbed from a symmetrical configuration, either during isolation of nuclei or by interactions with the lamina and the nuclear envelope in vivo. However, detergent extraction of nuclei appears to result in a more symmetrical alignment of components in apposing halves of the assembly.

Animals↗
PubMed1991

Human autoantibodies to lamin B receptor are also anti-idiotypic to certain anti-lamin B antibodies.

Autoantibodies reactive with nuclear envelope proteins are mainly detected in human sera from patients with liver diseases. Some of these antibodies are directed to lamin B, lamins A and C, or to the lamin B receptor (LBR). We show here that the latter one are anti-idiotypic to certain anti-lamin B antibodies. Using an enzyme-linked immunosorbent assay specific for lamins we found that serum M containing anti-LBR antibodies inhibited the binding to lamins of anti-lamin B autoantibodies from three of five sera tested. Similar results were obtained using patient's M purified IgG. The binding of monoclonal IgM, lambda anti-lamin B antibodies produced by a lymphoblastoid cell line derived from the patient's blood lymphocytes was also inhibited. Absorption of serum M with nuclei abolished the inhibitory activity. No inhibition was recorded with normal sera or sera containing other antinuclear specificities. Anti-LBR antibodies did not alter the binding to lamins of sera containing anti-lamins A and C antibodies. Altogether these findings demonstrate that anti-LBR antibodies are also combining site related anti-idiotypic antibodies (Ab2) to certain anti-lamin B antibodies, provide further evidence for discrete specificities among anti-lamin B antibodies and suggest that the occurrence of autoantibodies to nuclear envelope antigens may be under idiotypic regulation.

Animals↗
PubMed1998

Regulated chromosomal DNA replication in the absence of a nucleus.

Using Xenopus egg extracts, we have developed a completely soluble system for eukaryotic chromosomal DNA replication. In the absence of a nuclear envelope, a single, complete round of ORC-dependent DNA replication is catalyzed by cytosolic and nuclear extracts added sequentially to demembranated sperm chromatin or prokaryotic plasmid DNA. The absence of rereplication is explained by an activity present in the nucleus that prevents the binding of MCM to chromatin. Our results indicate that the role of the nuclear envelope in DNA replication is to concentrate activators and inhibitors of replication inside the nucleus. In addition, they provide direct evidence that metazoans use the same strategy as yeast to activate DNA replication and to restrict it to a single round per cell cycle.

Animals↗