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PubMed2006

Nuclear pore complex proteins in Alzheimer disease.

Ultrastructural studies of neurofibrillary tangles in Alzheimer disease (AD) have demonstrated a close relationship between nuclear pores and the cytoplasmic paired helical filaments comprising the tangles, as well as nuclear irregularity in many tangle-bearing neurons; nuclear pore aggregation has been observed in nearby neurons. These observations prompted examination of the nuclear pore complex (NPC) and proteins critical to nucleocytoplasmic transport in neurons with and without tangles in AD and control cases. Light microscopic study of hippocampus and neocortex in AD and controls revealed that all nuclei were labeled by antibodies to NPC proteins, including the central transporter nucleoporin Nup62. Nucleoporin and tau label revealed significantly more nuclear irregularity in AD, often associated with neurofibrillary tangles. Double label of Nup62 with apoptotic markers (TUNEL and active caspase-3) and a cell-cycle protein (cyclin B1) revealed no clear relationship of nuclear irregularity to apoptosis or cell-cycle protein expression. However, cytoplasmic accumulation of nuclear transport factor 2 (NTF2), a protein that transports cargo from the cytoplasm into the nucleus, was observed in a subset of hippocampal neurons with and without tangles in AD but not control cases. Further investigation of the NPC and nucleocytoplasmic transport in AD is warranted.

Alzheimer Disease↗
PubMed1998

Autoantibodies against a 210 kDa glycoprotein of the nuclear pore complex as a prognostic marker in patients with primary biliary cirrhosis.

It has been reported that the presence of anti-nuclear antibody against a 210 kDa glycoprotein of nuclear pore complex (anti-gp210) is highly specific for primary biliary cirrhosis (PBC). The aim of the present study was to investigate the significance of anti-gp210, especially as a prognostic marker. The presence of anti-gp210 was ascertained in 113 patients with PBC and 162 controls by indirect immunofluorescence assay using HepG2 cells and immunoblotting analysis using nuclear extracts from HeLa cells. Anti-gp210 was detected in 25 of the 113 (22.1%) patients. None of the 162 controls was positive for anti-gp210. The appearance and titre of anti-gp210 in the patients with PBC did not vary from the time of diagnosis and through their clinical course. Anti-mitochondrial antibodies (AMA), including antibodies against pyruvate dehydrogenase complex, branched chain alpha-ketoacid dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex, were not detected by enzyme-linked immunosorbent assay in five of the 113 (4.4%) patients with PBC. However, anti-gp210 alone was positive in one of these five patients. The difference in prognosis was statistically significant; patients with PBC positive for anti-gp210 died from hepatic failure more frequently than those who were negative (P < 0.01), although there were no statistically significant differences in the frequency of jaundice and the histological stage at the time of diagnosis between the two groups. We suggest that the presence of anti-gp210 is one of the independent prognostic markers able to predict, at the time of diagnosis, a poor outcome in patients with PBC.

Adult↗
PubMed2002

Enzymes of the SUMO modification pathway localize to filaments of the nuclear pore complex.

SUMOs are small ubiquitin-related polypeptides that are reversibly conjugated to many nuclear proteins. Although the number of identified substrates has grown rapidly, relatively little is still understood about when, where, and why most proteins are modified by SUMO. Here, we demonstrate that enzymes involved in the SUMO modification and demodification of proteins are components of the nuclear pore complex (NPC). We show that SENP2, a SUMO protease that is able to demodify both SUMO-1 and SUMO-2 or SUMO-3 protein conjugates, localizes to the nucleoplasmic face of the NPC. The unique amino-terminal domain of SENP2 interacts with the FG repeat domain of Nup153, indicating that SENP2 associates with the nucleoplasmic basket of the NPC. We also investigated the localization of the SUMO conjugating enzyme, Ubc9. Using immunogold labeling of isolated nuclear envelopes, we found that Ubc9 localizes to both the cytoplasmic and the nucleoplasmic filaments of the NPC. In vitro binding studies revealed that Ubc9 and SUMO-1-modified RanGAP1 bind synergistically to form a trimeric complex with a component of the cytoplasmic filaments of the NPC, Nup358. Our results indicate that both SUMO modification and demodification of proteins may occur at the NPC and suggest a connection between the SUMO modification pathway and nucleocytoplasmic transport.

Active Transport, Cell Nucleus↗
PubMed1980

The polypeptides of rat liver nuclear envelope. I. Examination by nuclear pore complex polypeptides by solid-state lactoperoxidase labelling.

Purified nuclei retaining a high degree of ultrastructural integrity were isolated by conventional centrifugation techniques. The cytoplasmic surface of these nuclei was iodinated using lactoperoxidase immobilized onto giant Sepharose beads; thus the outer nuclear membrane and the cytoplasmic surface of nuclear pore complexes were selectively labelled. Pore complexes in association with a fibrous lamina were isolated from these nuclei by removal of the nucleoplasm and extraction with Triton X-100. The chemical composition of the pore-lamina fraction was 93.6% protein, 6% RNA, 0.4% phospholipid. The labelling suggests that major polypeptides N1 (70 000) and N2 (67 000) and more than 10 other more minor polypeptides, ranging from 33 000 to 200 000 mol. wt, as being components of the nuclear pore complex. Polypeptide N3 (58 000) is shown to be present only on the nucleoplasmic face of nuclear envelopes, probably in the fibrous lamina.

Animals↗
PubMed1995

Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine.

Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized. As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc). Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs. Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays. Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues. Most of these appear to be bound to proteins via a hydroxyl group. This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs.

Acetylglucosamine↗
PubMed1997

Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional reconstruction from electron micrographs of nuclear envelopes.

The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells. Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs. Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined. NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G1-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle. However, accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells. Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance. However, NPCs were found in large clusters over regions of the nuclear envelope. Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.

Cell Cycle↗
PubMed1995

The essential yeast nucleoporin NUP159 is located on the cytoplasmic side of the nuclear pore complex and serves in karyopherin-mediated binding of transport substrate.

We have identified a new yeast nucleoporin of 159 kDa that we term NUP159. Immunofluorescence microscopy with a monospecific monoclonal antibody against NUP159 gave the punctate nuclear rim staining characteristic of nucleoporins. Immunogold electron microscopy with isolated yeast NEs yielded decoration of only the cytoplasmic side of the nuclear pore complex. The gene encoding NUP159 is essential, and, like some other nucleoporins, NUP159 contains a coiled-coil domain as well as a domain of repeated motifs. Five segments of NUP159, covering its entire length, were expressed in Escherichia coli. The repeat motif-containing segment was found to bind a nuclear transport substrate in the presence of vertebrate cytosolic extract containing nuclear transport factors. This segment also bound 35S-labeled mammalian karyopherin beta, one such transport factor that mediates the docking of substrates to the nuclear pore complex. These data establish a direct biochemical link between the repeat motif domain of a yeast nucleoporin, transport factors, (specifically karyopherin beta), and nuclear transport substrates. Its cytoplasmic aspect implies a role for NUP159 in nuclear import.

Amino Acid Sequence↗
PubMed2004

Dynamics of nuclear pore complex organization through the cell cycle.

In eukaryotic cells, all macromolecules that traffic between the nucleus and the cytoplasm cross the double nuclear membrane through nuclear pore complexes (NPCs). NPCs are elaborate gateways that allow efficient, yet selective, translocation of many different macromolecules. Their protein composition has been elucidated, but how exactly these nucleoporins come together to form the pore is largely unknown. Recent data suggest that NPCs are composed of an extremely stable scaffold on which more dynamic, exchangeable parts are assembled. These could be targets for molecular rearrangements that change nuclear pore transport properties and, ultimately, the state of the cell.

Active Transport, Cell Nucleus↗
PubMed2006

Single hepatitis-B virus core capsid binding to individual nuclear pore complexes in Hela cells.

We investigate the interaction of hepatitis B virus capsids lacking a nuclear localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa cells. Confocal and wide-field optical images of the nuclear envelope show well-spaced individual NPCs. Specific interactions of capsids with single NPCs are characterized by extended residence times of capsids in the focal volume which are characterized by fluorescence correlation spectroscopy. In addition, single-capsid-tracking experiments using fast wide-field fluorescence microscopy at 50 frames/s allow us to directly observe specific binding via a dual-color colocalization of capsids and NPCs. We find that binding occurs with high probability on the nuclear-pore ring moiety, at 44 +/- 9 nm radial distance from the central axis.

Capsid↗
PubMed2000

The nuclear pore complex: structure, function, and dynamics.

A full understanding of nucleocytoplasmic transport depends on knowledge of nuclear pore complex (NPC) structure, the functional roles of NPC components, their interactions during transport and dynamics during the cell cycle. NPC structure is conserved, flexible, and is not simply a tunnel between the nucleus and cytoplasm but appears to be actively involved in the transport process by a series of structural modifications. Transport through the NPC begins in either of its asymmetrical peripheral compartments that are both structurally reorganized during transport in different ways. The central compartment is composed of two symmetrical halves, and functions as a system of transiently open, discrete gates that is not believed to play a role in determining direction. Each NPC subunit has a specific morphology that corresponds to the functional role it plays. A complicated system of vertical and horizontal connections may allow one part of the NPC to transmit a signal to other parts, leading to an ordered series of conformational changes that drive translocation. High-resolution scanning electron microscopy has identified sequential stages of NPC assembly in vitro and revealed how the individual NPC components are assembled into a mature NPC. This review focuses on structural events during transport and on possible mechanisms of NPC assembly.

Animals↗
PubMed2000

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates.

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.

Amino Acid Sequence↗
PubMed1998

Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications.

We have calculated a three-dimensional map of the yeast nuclear pore complex (yNPC) from frozen-hydrated specimens, thereby providing a direct comparison with the vertebrate NPC. Overall, the smaller yNPC is comprised of an octagonal inner spoke ring that is anchored within the nuclear envelope by a novel membrane-interacting ring. In addition, a cylindrical transporter is located centrally within the spokes and exhibits a variable radial expansion in projection that may reflect gating. The inner spoke ring, a transmembrane spoke domain, and the transporter are conserved between yeast and vertebrates; hence, they are required to form a functional NPC. However, significant alterations in NPC architecture have arisen during evolution that may be correlated with differences in nuclear transport regulation or mitotic behavior.

Animals↗
PubMed2001

Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase.

Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of Mad1 (HsMAD1), we have begun to characterize this protein in mammalian cells. HsMad1 is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find HsMad1 associated with HsMad2. Association with p55CDC, a protein previously shown to bind HsMad2, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, HsMad1 and HsMAD2 were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. HsMad1 was identified serendipitously by its binding to a viral protein, HTLV-1 Tax, which affects transcription of viral and human proteins. The localization of HsMad1 to nuclear pore complexes suggests an alternate, non-mitotic role for the Mad1/Tax interaction in the viral transformation of cells.

Amino Acid Sequence↗
PubMed2005

Ran GTPase regulates Mad2 localization to the nuclear pore complex.

In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the nuclear pore complex (NPC) during interphase. Deletion of MAD1 or MAD2 did not affect steady-state nucleocytoplasmic distribution of a classical nuclear localization signal-containing reporter, a nuclear export signal-containing reporter, or Ran localization. We utilized cells with conditional mutations in the yeast Ran GTPase pathway to examine the relationship between Ran and targeting of checkpoint regulators to the NPC. Mutations that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. Our observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.

Animals↗
PubMed2003

Preferentially localized dynein and perinuclear dynactin associate with nuclear pore complex proteins to mediate genomic union during mammalian fertilization.

Fertilization is complete once the parental genomes unite, and requires the migration of the egg nucleus to the sperm nucleus (female and male pronuclei, respectively) on microtubules within the inseminated egg. Neither the molecular mechanism of pronucleus binding to microtubules nor the role of motor proteins in regulating pronuclear motility has been fully characterized, and the failure of zygotic development in some patients suggests that they contribute to human infertility. Based on the minus-end direction of female pronuclear migration, we propose a role for cytoplasmic dynein and dynactin in associating with the pronuclear envelope and mediating genomic union. Our results show that dynein intermediate and heavy chains preferentially concentrate around the female pronucleus, whereas dynactin subunits p150Glued, p50 and p62 localize to the surfaces of both pronuclei. Transfection of antibodies against dynein and dynactin block female pronuclear migration in zygotes. Both parthenogenetic activation in oocytes and microtubule depolymerization in zygotes significantly reduce the localization of dynein to the female pronucleus but do not inhibit the pronuclear association of dynactin. When immunoprecipitated from zygotes, p150Glued associates with nuclear pore complex proteins, as well as the intermediate filament vimentin and dynein. Antibodies against nucleoporins and vimentin inhibit pronuclear apposition when transfected into zygotes. We conclude that preferentially localized dynein and perinuclear dynactin associate with the nuclear pore complex and vimentin and are required to mediate genomic union. These data suggest a model in which dynein accumulates and binds to the female pronucleus on sperm aster microtubules, where it interacts with dynactin, nucleoporins and vimentin.

Animals↗
PubMed2006

Nuclear pore complex structure and plasticity revealed by electron and atomic force microscopy.

To study the ultrastructure of nuclear pore complexes (NPCs), a wide spectrum of different electron microscopy (EM) or atomic force microscopy (AFM) techniques can be employed. The combination of these methods can reveal new insights into the structural and functional organization of this important supramolecular machine through which nucleocytoplasmic transport occurs. Negative staining, quick freezing/freeze-drying/rotary metal shadowing, embedding and thin sectioning, cryoelectron microscopy and tomography, scanning electron microscopy, or combination with immunolabeling techniques are tools for collecting data and information about the three-dimensional structure and architecture of the NPCs. AFM enables investigation of the functional dynamics of native NPCs under physiological conditions.

Animals↗
PubMed1993

Nup180, a novel nuclear pore complex protein localizing to the cytoplasmic ring and associated fibrils.

Using an autoimmune serum from a patient with overlap connective tissue disease we have identified by biochemical and immunocytochemical approaches an evolutionarily conserved nuclear pore complex (NPC) protein with an estimated molecular mass of 180 kD and an isoelectric point of approximately 6.2 which we have designated as nup180. Extraction of isolated nuclear envelopes with 2 M urea and chromatography of the solubilized proteins on WGA-Sepharose demonstrated that nup180 is a peripheral membrane protein and does not react with WGA. Affinity-purified antibodies yielded a punctate immunofluorescent pattern of the nuclear surface of mammalian cells and stained brightly the nuclear envelope of cryosectioned Xenopus oocytes. Nuclei reconstituted in vitro in Xenopus egg extract were also stained in the characteristic punctate fashion. Immunogold EM localized nup180 exclusively to the cytoplasmic ring of NPCs and short fibers emanating therefrom into the cytoplasm. Antibodies to nup180 did not inhibit nuclear protein transport in vivo nor in vitro. Despite the apparent lack of involvement in NPC assembly or nucleocytoplasmic transport processes, the conservation of nup180 across species and its exclusive association with the NPC cytoplasmic ring suggests an important, though currently undefined function for this novel NPC protein.

Animals↗
PubMed1999

The nuclear pore complex: from molecular architecture to functional dynamics.

Toward dissecting the molecular composition and architecture of the nuclear pore complex (NPC), over the past 18 months novel nucleoporins and NPC subcomplexes were identified and characterized. The three-dimensional structure of isolated yeast NPCs was determined by electron cryomicroscopy. New specimen preparation and labeling protocols localized a number of nucleoporins and NPC subcomplexes within the three-dimensional architecture of the yeast NPC. Structural changes of native NPCs mediated by physiological effectors such as calcium or ATP were monitored by time-lapse atomic force microscopy, thus revealing a first glimpse of the NPC's functional dynamics.

Animals↗