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PubMed1994

A conserved epitope on nuclear pore phosphoproteins reflects cell division status.

The nuclear pore complexes mediate the selective nuclear import of proteins in a signal- and energy-dependent process. We have earlier reported the characterization of a monoclonal antibody, Mab E2, that recognizes a novel class of nuclear pore phosphoproteins involved in signal-binding and protein transport. In the present study, we have analyzed the pattern of immunoreactivity of Mab E2 in cultured rat fibroblasts and have observed significant differences in the expression of epitopes in proliferating and quiescent cells. Furthermore, the common epitope recognized by Mab E2 is conserved across species, consistent with its essential role in nuclear protein import.

Amino Acid Sequence↗
PubMed1999

Pictures in cell biology. Structures of nuclear-transport components.

The past three years have seen the solution of several nuclear transport component structures and recently of the structure of a regulator bound to part of a nuclear pore complex (NPC) protein. These structures have provided a wealth of valuable information about the proteins involved and suggested strategies for further investigation of their properties. We do not have space here to go into detail about this information, so instead we are illustrating the structures and providing primary references enabling interested readers to find further information. On this page, we are concentrating on the GTPase Ran and proteins that modulate its activity, and on the facing page are the other transport factors, some of which also interact directly with Ran. Notably absent at the moment are the nuclear pore complex component s, apart from one domain of RanBP2. Only when theses are characterized fully will we really be able to understand how transport substrates move across the nuclear envelope.

Animals↗
PubMed1995

Analysis of nucleocytoplasmic transport and nuclear envelope structure in yeast disrupted for the gene encoding the nuclear pore protein Nup1p.

Nup1p and Nsp1p are structurally related nuclear pore complex (NPC) proteins which contain many degenerate repeat sequences of the FSFG type in their central domains. To find out whether this similarity also reflects a functional overlap at the NPC, we analyzed delta nup1 cells in comparison to ts nsp1 cells for defects in nucleocytoplasmic transport. When the NUP1 gene was disrupted in two different laboratory yeast strains, haploid delta nup1 progeny was viable, showing that NUP1 is not essential for vegetative cell growth; delta nup1 strains, however, exhibited a strongly reduced growth rate at 37 degrees C as compared to 23 degrees C. When analyzed by thin section electron microscopy, delta nup1 cells show a normal nuclear envelope morphology and the number and appearance of nuclear pore complexes apparently was not altered. Whereas delta nup1 cells grown at 37 degrees C could still accumulate a lacZ reporter protein carrying a nuclear localization sequence (NLS) inside the nucleus, poly(A)+ RNA export was significantly inhibited. Our data suggest that Nup1p which is not physically associated with Nsp1p, is required for efficient nucleocytoplasmic transport reactions.

Biological Transport↗
PubMed2003

Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes.

Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to approximately 40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.

Animals↗
PubMed1986

Nuclear actin and myosin as control elements in nucleocytoplasmic transport.

Fluorescence redistribution after photobleaching (FRAP) was used to examine the role of actin and myosin in the transport of dextrans through the nuclear pore complex. Anti-actin antibodies added to isolated rat liver nuclei significantly reduced the flux rate of fluorescently labeled 64-kD dextrans. The addition of 3 mM ATP to nuclei, which enhances the flux rate in control nuclei by approximately 250%, had no enhancement effect in the presence of either anti-actin or anti-myosin antibody. Phalloidin (10 microM) and cytochalasin D (1 micrograms/ml) individually inhibited the ATP stimulation of transport. Rabbit serum, anti-fibronectin, and anti-lamins A and C antibodies had no effect on transport. These results suggest a model for nuclear transport in which actin/myosin are involved in an ATP-dependent process that alters the effective transport rate across the nuclear pore complex.

Actins↗
PubMed2001

A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope.

The nuclear pore complex (NPC) is a multicomponent structure containing a subset of proteins that bind nuclear transport factors or karyopherins and mediate their movement across the nuclear envelope. By altering the expression of a single nucleoporin gene, NUP53, we showed that the overproduction of Nup53p altered nuclear transport and had a profound effect on the structure of the nuclear membrane. Strikingly, conventional and immunoelectron microscopy analysis revealed that excess Nup53p entered the nucleus and associated with the nuclear membrane. Here, Nup53p induced the formation of intranuclear, tubular membranes that later formed flattened, double membrane lamellae structurally similar to the nuclear envelope. Like the nuclear envelope, the intranuclear double membrane lamellae enclosed a defined cisterna that was interrupted by pores but, unlike the nuclear envelope pores, they lacked NPCs. Consistent with this observation, we detected only two NPC proteins, the pore membrane proteins Pom152p and Ndc1p, in association with these membrane structures. Thus, these pores likely represent an intermediate in NPC assembly. We also demonstrated that the targeting of excess Nup53p to the NPC and its specific association with intranuclear membranes were dependent on the karyopherin Kap121p and the nucleoporin Nup170p. At the nuclear envelope, the abilities of Nup53p to associate with the membrane and drive membrane proliferation were dependent on a COOH-terminal segment containing a potential amphipathic alpha-helix. The implications of these results with regards to the biogenesis of the nuclear envelope are discussed.

Active Transport, Cell Nucleus↗
PubMed1975

Synaptic fine structure and nuclear, cytoplasmic and extracellular networks: The stereoframework concept.

When certain intracellular and extracellular localities known to be rich in protein complexes are fixed and processed for electron microscopy, they show a reticulate precipitate which represents three-dimensional framework of material that forms the wall of polygonal lacunae. This is referred to as a stereoframework. Examples of a stereoframework described her include the presynaptic dense projections, cleft substance, postsynaptic density, the cytonet, coats of coated vesicles, reticulosomes, 'microfilamentous' network of growth cones, the glycocalyx of gut microvilli, blood plasma, precipitates of the Golgi apparatus, the chromatin of nuclei and the nuclear pore complex. The stereoframeworkappears most electron-dense when it has a very close mesh, e.g. as in the case of the dense projections. The stereoframework is assumed to have no direct relationship with themolecular architecture of the protein complexes in vivo and so can be regarded as a denaturization and precipitation artifact. This being so, attempts to elucidate the substructure of the above entities simply by inspection are fruitless. Furthermore, evidence is given that stereoframework precipitation can distort or completely obliterate organelles occupying the same locality, for example this could apply to structures such as actin filaments (perhaps running into the locality marked by a dense projection), microtubules(running into the presynaptic bag), smooth ER, tenuous connections between synaptic vesicles and the presynaptic membrane, structures within the nuclear pore complex and chromosome substructures in the nucleus. Finally it is suggested that the flat shape of synaptic vesicles (at inhibitory synapses) may be a distortion effect imposed upon the synaptic vesicles not as a result of osmotic effects, but as a conformation to the shape of a stereoframework which has been precipitated from protein complexes in the vicinity ofthe synaptic vesicles.

Animals↗
PubMed2025

Nuclear basket proteins Nup2 and Mlp1 drive heat shock-induced 3D genome restructuring downstream of transcriptional activation.

The nuclear pore complex (NPC), a multisubunit complex located within the nuclear envelope, regulates RNA export and the import and export of proteins. Here we address the role of the NPC in driving thermal stress-induced 3D genome repositioning of Heat Shock Responsive (HSR) genes in budding yeast. We found that two nuclear basket proteins, Nup2 and Mlp1, although dispensable for NPC integrity, are required for driving HSR genes into coalesced chromatin clusters, consistent with their strong, heat shock-dependent recruitment to HSR gene regulatory and coding regions. HSR gene clustering occurs predominantly within the nucleoplasm and is independent of the essential scaffold-associated proteins Nup1 and Nup145. Notably, acute double depletion of Nup2 and Mlp1 has little effect on the formation of Heat Shock Factor 1 (Hsf1)-containing transcriptional condensates, Hsf1 and Pol II recruitment to HSR genes, or HSR mRNA abundance. Our results define a 3D genome restructuring role for nuclear basket proteins extrinsic to the NPC and downstream of HSR gene activation.

3D genome architecture↗
PubMed2006

Inositol hexakisphosphate and Gle1 activate the DEAD-box protein Dbp5 for nuclear mRNA export.

Regulation of nuclear mRNA export is critical for proper eukaryotic gene expression. A key step in this process is the directional translocation of mRNA-ribonucleoprotein particles (mRNPs) through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope. Our previous studies in Saccharomyces cerevisiae defined an in vivo role for inositol hexakisphosphate (InsP6) and NPC-associated Gle1 in mRNA export. Here, we show that Gle1 and InsP6 act together to stimulate the RNA-dependent ATPase activity of the essential DEAD-box protein Dbp5. Overexpression of DBP5 specifically suppressed mRNA export and growth defects of an ipk1 nup42 mutant defective in InsP6 production and Gle1 localization. In vitro kinetic analysis showed that InsP6 significantly increased Dbp5 ATPase activity in a Gle1-dependent manner and lowered the effective RNA concentration for half-maximal ATPase activity. Gle1 alone had minimal effects. Maximal InsP6 binding required both Dbp5 and Gle1. It has been suggested that Dbp5 requires unidentified cofactors. We now propose that Dbp5 activation at NPCs requires Gle1 and InsP6. This would facilitate spatial control of the remodelling of mRNP protein composition during directional transport and provide energy to power transport cycles.

Active Transport, Cell Nucleus↗
PubMed2001

Nuclear envelope disorganization in fibroblasts from lipodystrophic patients with heterozygous R482Q/W mutations in the lamin A/C gene.

Dunnigan-type familial partial lipodystrophy (FPLD), characterized by an abnormal body fat redistribution with insulin resistance, is caused by missense heterozygous mutations in A-type lamins (lamins A and C). A- and B-type lamins are ubiquitous intermediate filament proteins that polymerize at the inner face of the nuclear envelope. We have analyzed primary cultures of skin fibroblasts from three patients harboring R482Q or R482W mutations. These cells were euploid and able to cycle and divide. A subpopulation of these cells had abnormal blebbing nuclei with A-type lamins forming a peripheral meshwork, which was frequently disorganized. Inner nuclear membrane protein emerin, an A-type lamin-binding protein, strictly colocalized with this abnormal meshwork. Cells from lipodystrophic patients often had other nuclear envelope defects, mainly consisting of nuclear envelope herniations that were deficient in B-type lamins, nuclear pore complexes, lamina-associated protein 2 beta, and chromatin. The mechanical properties of nuclear envelopes were altered, as judged from the extensive deformations observed in nuclei from heat-shocked cells, and from the low stringency of extraction of their components. These structural nuclear alterations were caused by the lamins A/C mutations, as the same changes were introduced in human control fibroblasts by ectopic expression of R482W mutated lamin A.

Adult↗
PubMed2005

Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex.

SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein. Here we describe the 3.0-A crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO-E2-thioester in an optimal orientation to enhance conjugation.

Amino Acid Sequence↗
PubMed2001

Caspases mediate nucleoporin cleavage, but not early redistribution of nuclear transport factors and modulation of nuclear permeability in apoptosis.

In eukaryotic cells, both soluble transport factors and components of the nuclear pore complex mediate protein and RNA trafficking between the nucleus and the cytoplasm. Here, we investigated whether caspases, the major execution system in apoptosis, target the nuclear pore or components of the nuclear transport machinery. Four nucleoporins, Nup153, RanBP2, Nup214 and Tpr are cleaved by caspases during apoptosis. In contrast, the nuclear transport factors, Ran, importin alpha and importin beta are not proteolytically processed, but redistribute across the nuclear envelope independently and prior to caspase activation. Also, mRNA accumulates into the nucleus before caspases become active. Microinjection experiments further revealed that early in apoptosis, the nucleus becomes permeable to dextran molecules of 70 kD molecular weight. Redistribution of import factors and mRNA, as well as nuclear permeabilisation, occur prior to caspase-mediated nucleoporin cleavage. Our findings suggest that the apoptotic programme includes modifications in the machinery responsible for nucleocytoplasmic transport, which are independent from caspase-mediated degradation of nuclear proteins.

Active Transport, Cell Nucleus↗
PubMed1996

Distinctive features of idiopathic inflammatory myopathies in French Canadians.

This is the first report on idiopathic inflammatory myopathies (IIM) in French Canadians. We reviewed retrospectively 30 French Canadian adults (20 women and 10 men) with IIM seen consecutively over 12 years. The median age at diagnosis was 45 years. The IIM were 8 (27%) primary polymyositis (PM), 9 (30%) primary dermatomyositis (DM), 5 (17%) IIM with neoplasia (lymphoma, breast, esophageal, colonic, and skin cancer) and 8 (27%) IIM with a connective tissue disease (4 with systemic sclerosis, 2 with mixed connective tissue disease, and 2 with rheumatoid arthritis). The most common presenting symptom was proximal muscle weakness (n = 10,33%). Of the remaining 20 patients, 6 (20%) had the onset of their weakness within 1 month of the presenting symptom. Only 3 (10%) patients did not have proximal muscle weakness. Twenty-six (87%) patients had weakness in the pelvic girdle, 25 (83%) in the shoulder girdle, and 7 (23%) in the neck muscles. Other common symptoms included dyspnea on exertion and dysphagia, each present in 13 (43%) patients. Gottron's papules and the heliotrope rash were the most common skin lesions documented in 11 (37%) and 10 (33%) patients, respectively. The serum creatine kinase (CK) level was between 171 and 1,000 U/L in 13 (43%) patients and between 1,001 and 6,000 U/L in 13 (43%) patients. Antinuclear antibodies (ANA) on HEp-2 cells were positive in 16 (53%) patients, of which 2 (13%) expressed autoantibodies to nuclear pore complexes. Autoantibody specificities were anti-La (n = 4, 13%), anti-U1RNP (n = 3, 10%), and anti-Ro (n = 2, 7%). None of the patients expressed anti-Jo-1, anti-topoisomerase I, or anticentromere antibodies. Twenty-eight (93%) patients received corticosteroid therapy, and 8 (27%) patients responded to prednisone alone. Thirteen (43%) patients were treated with methotrexate, and 9 (69%) responded. The mean follow-up was 62 months: 23 (77%) had their disease controlled, 3 (10%) patients were lost to follow-up, and 4 (13%) died (no death occurred because of IIM or its treatment). Therapy was discontinued because of remission in 5 (17%) patients. Cumulative survival rates at 2, 5, and 10 years were 89%, 89%, and 85%, respectively. The presence of autoantibodies to nuclear pore complexes and anti-La autoantibodies, the rare occurrence of anti-Jo-1 autoantibodies, the response to conventional therapies, and a high survival rate may distinguish IIM in French Canadians from that of other reported series.

Adult↗
PubMed1985

Correlation of surface topography of metal-shadowed specimens with their negatively stained reconstructions.

We present a comparison of surface reconstructions from three different freeze-dried and unidirectionally metal-shadowed specimens (i.e. bacteriophage T4 polyheads, crystalline actin sheets and nuclear pore complexes) with two- or three-dimensional reconstructions of the same specimens when prepared by negative staining. Based on these and many published results, the following conclusions have been reached: With a "cooperative" specimen (e.g. the polyheads), the surface reconstruction computed from a metal-shadowed replica compares favorably with two- or three-dimensional reconstructions obtained from the same specimen after negative staining at the 3-4 nm resolution level. This relatively "poor" level at which the surface topographies of the two preparations can be compared appears to be set by a "practical" resolution limit (i.e. of distinct and reproducible structural detail) of metal replicas of biological specimens, despite the appearance of weak higher-order diffraction spots (i.e. corresponding to 2-3 nm). While in some cases (e.g. the crystalline actin sheets) the surface reliefs of metal replicas may bear little resemblance to the actual structure under investigation, the replicas may still contain sufficient features to establish the polarity or handedness of the structure (i.e., the "top" and "bottom" surfaces of a crystalline sheet). Information from negatively stained specimens is usually complementary with information from freeze-dried and metal-shadowed specimens. However, there are artifacts in both techniques, and we present an example with the nuclear pore complex, where these techniques yield confusing results.

Actins↗
PubMed1995

The Ran/TC4 GTPase-binding domain: identification by expression cloning and characterization of a conserved sequence motif.

Ran/TC4 is an essential, nuclear GTPase implicated in the initiation of DNA replication, entry into and exit from mitosis, and in nuclear RNA and protein transport through the nuclear pore complex. This diversity of functions suggests that Ran interacts with a large number of down-stream targets. Using an overlay assay, we detected a family of putative target proteins that associate with GTP-bound Ran. The sequence of only one such protein, HTF9a/RanBP1, is known. We have now cloned two additional Ran-binding proteins, allowing identification of a distinctive, highly conserved sequence motif of approximately 150 residues. This motif represents a minimal Ran-binding domain that stabilizes the GTP-bound state of Ran. The isolated domain also functions as a coactivator of Ran-GTPase-activating protein. Mutation of a conserved residue within the Ran-binding domain of HTF9a protein drastically reduced Ran binding. Ran-binding proteins coimmunoprecipitated with epitope-tagged Ran from cell lysates, suggesting that these proteins may associate in vivo. A previously uncharacterized Caenorhabditis elegans gene could encode a protein (96 kDa) possessing two Ran-binding domains. This open reading frame also contains similarities to nucleoporins, suggesting a functional link between Ran and nuclear pore complexes.

Amino Acid Sequence↗
PubMed2001

An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells.

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.

Evolution, Molecular↗
PubMed2004

Annulate lamellae play only a minor role in the storage of excess nucleoporins in Drosophila embryos.

The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleo-cytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.

Amino Acid Sequence↗
PubMed1999

Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP.

Nuclear transport factor 2 (NTF2) is a small, homodimeric protein that binds to both RanGDP and xFxFG repeat-containing nucleoporins, such as yeast Nsp1p and vertebrate p62. NTF2 is required for efficient nuclear protein import and has been shown to mediate the nuclear import of RanGDP. We have used the crystal structures of rat NTF2 and its complex with RanGDP to design a mutant, W7A-NTF2, in which the affinity for xFxFG-repeat nucleoporins is reduced while wild-type binding to RanGDP is retained. The 2.5 A resolution crystal structure of W7A-NTF2 is virtually superimposable upon the wild-type protein structure, indicating that the mutation had not introduced a more general conformational change. Therefore, our data suggest that the exposed side-chain of residue 7 is crucial to the interaction between NTF2 and xFxFG repeat-containing nucleoporins. Consistent with its reduced affinity for xFxFG nucleoporins, fluorescently labelled W7A-NTF2 binds less strongly to the nuclear envelope of permeabilized cultured cells than wild-type NTF2 and, when microinjected into Xenopus oocytes, colloidal gold coated with W7A-NTF2 binds less strongly to the central channel of nuclear pore complexes than wild-type NTF2-coated gold. Significantly, W7A-NTF2 only weakly stimulated the nuclear import of fluorescein-labelled RanGDP, providing direct evidence that an interaction between NTF2 and xFxFG repeat-containing nucleoporins is required to mediate the nuclear import of RanGDP.

Animals↗