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PubMed2000

Purification of the vertebrate nuclear pore complex by biochemical criteria.

The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of approximately 120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin beta, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals approximately 40-50 distinct protein bands.

Animals↗
PubMed2006

Functional analysis of nuclear pore complex protein Nup62/p62 using monoclonal antibodies.

The nuclear pore complex (NPC) is an enormous structure embedded in the double membrane of the nuclear envelope that acts as a passageway for nucleocytoplasmic transport. The vertebrate NPC is comprised of about 30 unique proteins. Nup62/p62, a major component of the NPC, has been reported to interact directly with several nuclear transport factors, including importin-beta and NTF2. However, it has not been shown how the interaction of Nup62/p62 with transport factors is involved in nucleocytoplasmic transport. The present study reports on the preparation of monoclonal antibodies (MAbs) directed against human Nup62/p62 and a functional analysis of Nup62/p62 using antibodies in living cells. Hybridomas producing the antibodies were produced by the hybridization of mouse myeloma cells with medial iliac lymph node cells from an immunized rat. These MAbs specifically recognized Nup62/p62 as evidenced by immunoblotting analysis using a nuclear membrane fraction. In the immunostaining using MAbs, a punctuate nuclear rim staining pattern was observed. Moreover, cytoplasmic injected-anti-Nup62/p62 MAbs were rapidly targeted to the nuclear pore of cultured cells and some of them inhibited normal cell division, causing the formation of abnormal nuclei. The antibodies described in this study provide the means for immunochemical analyses of the NPC protein Nup62/p62 in mammalian cells, and represent useful molecular tools that should permit a better understanding of the biological roles and cellular dynamics of this protein in nucleocytoplasmic transport, cell division, and nuclear organization.

Active Transport, Cell Nucleus↗
PubMedDate not supplied

High resolution analysis of mammalian nuclear structure throughout the cell cycle: implications for nuclear pore complex assembly during interphase and mitosis.

Nuclear pore complexes (NPCs) are the gateways for both active and passive bidirectional molecular transport between the nucleoplasm and cytoplasm. These mega-dalton assemblies are composed of multiple copies of approximately 30 distinct proteins termed nucleoporins. Higher eukaryotes display an "open" mitosis in which the NPCs, nuclear envelope, and lamina disassemble. During mitosis several nucleoporins are redistributed to kinetochores until they are recruited back to the periphery of chromatin as the NPCs are reassembled. Within this study we have developed and optimized the visualization of mammalian cells and their chromosome profiles throughout the cell-cycle. Close attention has been paid to the preservation of chromatin, membranes, and NPC structure to investigate the ultrastructural locations of specific proteins in both interphase and mitosis.

Animals↗
PubMed1995

Identification of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes.

Targeting of import substrate to nuclear pore complexes of permeabilized vertebrate cells was previously shown to require a protein complex composed of two subunits, termed karyopherin. Yeast contain a homologue of karyopherin alpha named Srp1p, which was initially identified as a genetic suppressor of mutations in a subunit of RNA polymerase I. To determine whether yeast contain a karyopherin complex that includes Srp1p as the karyopherin alpha homologue, we genetically replaced Srp1p with a Srp1-Protein A chimera. Cytosol from this strain contained a complex, composed of the chimera and a protein of 95 kDa, that was purified using affinity chromatography on IgG Sepharose. Microsequence analysis showed that the 95-kDa protein was identical with a yeast protein encoded by gene L8300.15 on chromosome XII. Sequence comparison revealed that the L8300.15 gene product is the closest structural homologue of vertebrate karyopherin beta. The yeast alpha and beta karyopherin subunits were expressed in Escherichia coli and were purified. When combined, they formed a heterodimeric complex and were active in targeting import substrate to nuclear envelopes of mammalian cells. We propose that all karyopherins function as alpha/beta heterodimers.

Amino Acid Sequence↗
PubMed2003

RanGTP mediates nuclear pore complex assembly.

In metazoa, the nuclear envelope breaks down and reforms during each cell cycle. Nuclear pore complexes (NPCs), which serve as channels for transport between the nucleus and cytoplasm, assemble into the reforming nuclear envelope in a sequential process involving association of a subset of NPC proteins, nucleoporins, with chromatin followed by the formation of a closed nuclear envelope fenestrated by NPCs. How chromatin recruitment of nucleoporins and NPC assembly are regulated is unknown. Here we demonstrate that RanGTP production is required to dissociate nucleoporins Nup107, Nup153 and Nup358 from Importin beta, to target them to chromatin and to induce association between separate NPC subcomplexes. Additionally, either an excess of RanGTP or removal of Importin beta induces formation of NPC-containing membrane structures--annulate lamellae--both in vitro in the absence of chromatin and in vivo. Annulate lamellae formation is strongly and specifically inhibited by an excess of Importin beta. The data demonstrate that RanGTP triggers distinct steps of NPC assembly, and suggest a mechanism for the spatial restriction of NPC assembly to the surface of chromatin.

Amino Acid Substitution↗
PubMed1995

Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy.

The nuclear envelope functions as a selective barrier separating the nuclear from the cytosolic compartment. Nuclear pore complexes (NPCs) mediate nuclear import and export of macromolecules and, therefore, are potential regulators of gene expression. In this study we applied atomic force microscopy (AFM) to visualize the three dimensional (3D) structure of individual NPCs in the absence and presence of two different antibodies, one directed against a pore protein (gp62) and another directed against Xenopus lamin LIII, a component of the nuclear lamina, a filament meshwork localized on the nucleoplasmic side of the nuclear envelope (NE) adjacent to and interacting with NPCs. Using 12-nm gold-labelled secondary antibodies and transmission electron microscopy we could clearly localize the primary single anti-gp62 antibody on NPCs and the primary single anti-LIII antibody between NPCs. Using AFM, the secondary antibodies against anti-gp62 could be detected as particles 7 nm in height on the nucleoplasmic face of NPCs. The secondary antibodies against anti-LIII could be clearly identified between NPCs. The secondary antibodies, attached to a 12-nm colloidal gold particle and visualized on glass, revealed similar shapes and heights as found on NEs. According to the 3D images, the volume of a single gold particle conjugated with secondary antibodies was 10203 nm3. This volume is equivalent to the volume of 38 IgG molecules associated with one individual gold particle. A similar volume of 11987 nm3 was calculated from a model assuming that the 150-kDa IgG molecules perfectly cover the spherical gold particle. We conclude that AFM can be used for identifying antibodies or other macromolecules associated with biomembranes.

Animals↗
PubMedDate not supplied

Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex.

Nup153, one of the best characterized nuclear pore complex proteins (nucleoporins), plays a critical role in the import of proteins into the nucleus as well as in the export of RNAs and proteins from the nucleus. Initially an epitope of Nup153 was found to reside at the distal ring of the NPC, whereas more recently another epitope was localized to the nuclear ring moiety of the NPC. In an effort to more definitively determine the location of Nup153 within the 3-D architecture of the NPC we have generated domain-specific antibodies against distinct domains of Xenopus Nup153. With this approach we have found that the N-terminal domain is exposed at the nuclear ring of the NPC, whereas the zinc-finger domain of Nup153 is exposed at the distal ring of the NPC. In contrast, the C-terminal domain of Nup153 is not restricted to one particular subdomain of the NPC but rather appears to be highly flexible. Exogenous epitope-tagged hNup153 incorporated into Xenopus oocyte NPCs further underscored these findings. Our data illustrate that multiple domain-specific antibodies are essential to understanding the topology of a nucleoporin within the context of the NPC. Moreover, this approach has revealed new clues to the mechanisms by which Nup153 may contribute to nucleocytoplasmic transport.

Animals↗
PubMed1996

A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.

Ran is a nuclear Ras-like GTPase that is required for the bidirectional transport of proteins and ribnucleoproteins across the nuclear pore complex (NPC). A key regulator of the Ran GTP/GDP cycle is the 70-kD Ran-GTPase-activating protein RanGAP1. Here, we report the identification and localization of a novel form of RanGAP1. Using peptide sequence analysis and specific mAbs, RanGAP1 was found to be modified by conjugation to a ubiquitin-like protein. Immunoblot analysis and immunolocalization by light and EM demonstrated that the 70-kD unmodified from of RanGAP1 is exclusively cytoplasmic, whereas the 90-kD modified form of RanGAP1 is associated with the cytoplasmic fibers of the NPC. The modified form of RanGAP1 also appeared to associated with the mitotic spindle apparatus during mitosis. These findings have specific implications for Ran function and broad implications for protein regulation by ubiquitin-like modifications. Moreover, the variety and function of ubiquitin-like protein modifications in the cell may be more diverse than previously realized.

Amino Acid Sequence↗
PubMed1997

Nuclear calcium and the regulation of the nuclear pore complex.

In eukaryotic cells the nucleus and its contents are separated from the cytoplasm by the nuclear envelope. Macromolecules, as well as smaller molecules and ions, can cross the nuclear envelope through the nuclear pore complex. Molecules greater than approx. 60 kDa and containing a nuclear localization signal are actively transported across the nuclear membranes, but there has been little evidence for regulatory mechanisms for smaller molecules and ions. Recently, diffusion across the nuclear envelope has been observed to be regulated by nuclear cisternal Ca2+ concentrations. Following depletion of Ca2+ from the nuclear store by inositol 1,4,5-trisphosphate or Ca2+ chelators, a fluorescent 10 kDa marker molecule was no longer able to enter the nucleus. Distinct conformational states of the nuclear pore complexes depended on the Ca2+ filling state of the nuclear envelope, supporting the assumption that a switch in the conformation of the nuclear pore complex may control the transport of intermediate-sized molecules across the nuclear envelope. Thus nuclear Ca2+ stores may regulate the conformational state of the nuclear pore complex, and thereby passive diffusion of molecules between the cytosol and the nucleoplasm. The physiological significance of this finding is currently unknown.

Animals↗
PubMed2000

Ran alters nuclear pore complex conformation.

Transport across the nuclear membranes occurs through the nuclear pore complex (NPC), and is mediated by soluble transport factors including Ran, a small GTPase that is generally GDP-bound during import and GTP-bound for export. The dynamic nature of the NPC structure suggests a possible active role for it in driving translocation. Here we show that RanGTP but not RanGDP causes alterations of NPC structure when injected into the cytoplasm of Xenopus oocytes, including compaction of the NPC and extension of the cytoplasmic filaments. RanGTP caused accumulation of nucleoplasmin-gold along the length of extended cytoplasmic filaments, whereas RanGDP caused accumulation around the cytoplasmic rim of the NPC. This suggests a possible role for Ran in altering the conformation of the cytoplasmic filaments during transport.

Amino Acid Substitution↗
PubMed1995

Relationships between the nuclear membrane, nuclear pore complexes, and organelles in the type II pneumocyte.

Functional relationships among organelles of the type II cell are suggested based upon the proximity of organelles to specialized areas of the plasma- and nuclear membranes. In a two-dimensional morphometric analysis of the profiles of organelles in type II cells of the ferret and rat (and beagle dog), lamellar bodies were more likely to be located near the nuclear membrane than at the alveolar space (where exocytosis occurs). The size of lamellar body profiles was not correlated with distance from the nuclear membrane; however, large profiles were nearer the apical membrane, and smaller ones nearer the basement membrane. Profiles of highly branched mitochondria were 10 times more frequently associated with nuclear pore complexes than with the inter-pore nuclear membrane. Forty percent of all mitochondrial profiles were within 0.25 microns of the nucleus, 5% were within 0.02 microns and half of these appeared to touch the filaments of the nuclear pore complexes. The size of mitochondrial profiles was not correlated with distribution. In the ferret and rat, 8.6% and 2.5% respectively, of the nuclear pore complexes were associated with mitochondria. Sebaceous cells, from control mice, demonstrated a spatial distribution of granules which was size dependent but unrelated to the nuclear membrane.

Animals↗
PubMed2006

A point mutation in the Aspergillus nidulans sonBNup98 nuclear pore complex gene causes conditional DNA damage sensitivity.

The nuclear pore complex (NPC) is embedded in the nuclear envelope where it mediates transport between the cytoplasm and nucleus and helps to organize nuclear architecture. We previously isolated sonB1, a mutation encoding a single amino acid substitution within the Aspergillus nidulans SONBnNup98 NPC protein (nucleoporin). Here we demonstrate that this mutation causes marked DNA damage sensitivity at 42 degrees . Although SONBnNup98 has roles in the G2 transition, we demonstrate that the G2 DNA damage checkpoint is functional in the sonB1 mutant at 42 degrees . The MRN complex is composed of MRE11, RAD50, and NBS1 and functions in checkpoint signaling, DNA repair, and telomere maintenance. At 42 degrees we find that the DNA damage response defect of sonB1 mutants causes synthetic lethality when combined with mutations in scaANBS1, the A. nidulans homolog of NBS1. We provide evidence that this synthetic lethality is independent of MRN cell cycle checkpoint functions or MREAMRE11-mediated DNA repair functions. We also demonstrate that the single A. nidulans histone H2A gene contains the C-terminal SQE motif of histone H2AX isoforms and that this motif is required for the DNA damage response. We propose that the sonB1 nucleoporin mutation causes a defect in a novel part of the DNA damage response.

Arabidopsis Proteins↗
PubMed1977

Nuclear pore complexes. Elimination and reconstruction during mitosis.

Nuclear structures similar to those of the nuclear pore complex were found on chromosomes. This finding indicates that part of the pore complex is retained by the chromosomes through mitosis in the absence of the nuclear membrane. The formation of approximately the same number of pore complexes in the presence and absence of protein synthesis during the first 4 h after mitosis proves the reassembly rather than new synthesis of the pore complex. The structure of pore complexes reconstructed in the absence of protein synthesis cannot be distinguished from the structure of those of control cells.

Cell Nucleus↗
PubMed2005

Specific monoclonal antibody against the nuclear pore complex protein, nup98.

Nup98 is a component of nuclear pore complexes, which are large protein assemblies embedded in the nuclear envelope. Previous studies have shown that Nup98 interacts with several transport factors and plays a critical part in nuclear trafficking. However, the mechanism by which Nup98 contributes to nuclear trafficking is not clear. The present study reports on the preparation of a monoclonal antibody (MAb) directed against human Nup98. The antibody was produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. This antibody, MAb 2H10, specifically recognized Nup98, as evidenced by immunoblotting using a nuclear membrane fraction. In immunostaining using MAb 2H10, a punctuate nuclear rim staining pattern was observed. This MAb will be useful in immunoblotting and immunolocalization experiments in various cells and tissues, as well as further analyses of the biological function and cellular dynamics of this protein.

Animals↗
PubMed2005

Caspases target only two architectural components within the core structure of the nuclear pore complex.

Caspases were recently implicated in the functional impairment of the nuclear pore complex during apoptosis, affecting its dual activity as nucleocytoplasmic transport channel and permeability barrier. Concurrently, electron microscopic data indicated that nuclear pore morphology is not overtly altered in apoptotic cells, raising the question of how caspases may deactivate nuclear pore function while leaving its overall structure largely intact. To clarify this issue we have analyzed the fate of all known nuclear pore proteins during apoptotic cell death. Our results show that only two of more than 20 nuclear pore core structure components, namely Nup93 and Nup96, are caspase targets. Both proteins are cleaved near their N terminus, disrupting the domains required for interaction with other nucleoporins actively involved in transport and providing the permeability barrier but dispensable for maintaining the nuclear pore scaffold. Caspase-mediated proteolysis of only few nuclear pore complex components may exemplify a general strategy of apoptotic cells to efficiently disable huge macromolecular machines.

Apoptosis↗
PubMed2005

High-resolution near-field optical imaging of single nuclear pore complexes under physiological conditions.

Scanning near-field optical microscopy (SNOM) circumvents the diffraction limit of conventional light microscopy and is able to achieve optical resolutions substantially below 100 nm. However, in the field of cell biology SNOM has been rarely applied, probably because previous techniques for sample-distance control are less sensitive in liquid than in air. Recently we developed a distance control based on a tuning fork in tapping mode, which is also well-suited for imaging in solution. Here we show that this approach can be used to visualize single membrane protein complexes kept in physiological media throughout. Nuclear envelopes were isolated from Xenopus laevis oocytes at conditions shown recently to conserve the transport functions of the nuclear pore complex (NPC). Isolated nuclear envelopes were fluorescently labeled by antibodies against specific proteins of the NPC (NUP153 and p62) and imaged at a resolution of approximately 60 nm. The lateral distribution of epitopes within the supramolecular NPC could be inferred from an analysis of the intensity distribution of the fluorescence spots. The different number densities of p62- and NUP153-labeled NPCs are determined and discussed. Thus we show that SNOM opens up new possibilities for directly visualizing the transport of single particles through single NPCs and other transporters.

Animals↗
PubMed1988

Filamentous cross-bridges link intermediate filaments to the nuclear pore complexes.

Intermediate filament-nuclear matrix interactions were studied in cultured rat ventral prostate cells and isolated rat uterine epithelial cells. Cytokeratin filaments were identified by immunoelectron microscopy. In addition to conventional thin section of Triton X-100 treated cells, subcellular residues composed of intermediate filaments and nuclear matrix were critical-point dried and platinum-carbon replicated. The results demonstrate the presence of a previously unrecognized type of filamentous cross-bridges that link intermediate filaments to the nuclear pore complexes.

Animals↗
PubMed1981

Morphometry of nuclear pore complexes in thyroid cells during hyperplasia and involution.

Nuclear pore complexes were analyzed in freeze-fractured replicas of thyroid follicular cells of C3H mice in different physiological states. Thyroid stimulation induced a rapid and simultaneous increase of the nuclear surface and volume and of the total number of pore complexes. The numerical density (Na) of pore complexes increased at the 6th day of stimulation, but after that time the proportion of cells with an increased Na was always higher than the proportion of 3H-labelled nuclei. During thyroid involution, all the nuclear parameters, including the Na, returned to normal values. These results indicate that the total number of pore complexes and their Na are correlated with the cellular activity rather than with the cell cycle. They also suggest that 2 different mechanisms are involved in the generation of pore complexes: first, an addition of new membranes with a low density of pore complexes; later, a formation of new pore complexes in preexisting membranes. However, during involution, parts of the nuclear membranes and pore complexes in the remaining parts disappear synchronously. In freeze-fractured thyroid nuclear membranes, 2 neighbouring pore complexes were always separated by a distance of 105 nm. Clusters of pore complexes were not observed. A comparison of the distances between pore complexes and between randomly generated points never showed any significant differences indicating that pore complexes were randomly distributed.

Animals↗