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A Study of Nuclear Assembly

 

I. Nuclear Pore Assembly

Removal of a Single Pore Subcomplex Results in Vertebrate Nuclei Devoid of Nuclear Pores

(Molecular Cell, 2003)

The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins. Using immunodepletion of   Xenopus nuclear reconstitution extracts, it has previously been possible to assemble   nuclei lacking pore subunits tied to protein import, export, or mRNA export. However,   these altered pores all still possessed the bulk of pore structure. Here, we   immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85   and Nup133, two of its components. The resulting reconstituted nuclei are severely   defective for NLS import and DNA replication. Strikingly, they show a profound defect   for  every tested nucleoporin. Even the integral membrane proteins POM121 and   gp210   are absent or unorganized. Scanning electron microscopy reveals pore-free   nuclei,  while adding back of the Nup107-160 complex restores functional pores. We   conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear   pore complex assembly.

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ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division

(Proceedings of the National Academy of Sciences, 2006)

At mitosis, the Nup107-160 complex, which consists of nine proteins, localizes to kinetochores, suggesting that it may also function in chromosome segregation. To investigate the dual roles of the Nup107-160 complex at the pore and during mitosis, we set out to identify binding partners by immunoprecipitation from both interphase and mitotic Xenopus egg extracts and mass spectrometry. ELYS, a putative transcription factor, was discovered to copurify with the Nup107-160 complex in Xenopus interphase extracts, Xenopus mitotic extracts, and human cell extracts. Indeed, a large fraction of ELYS localizes to the nuclear pore complexes of HeLa cells. Importantly, depletion of ELYS by RNAi leads to severe disruption of nuclear pores in the nuclear envelope, whereas lamin, Ran, and tubulin staining appear normal. At mitosis, ELYS targets to kinetochores, and RNAi depletion from HeLa cells leads to an increase in cytokinesis defects. Thus, we have identified an unexpected member of the nuclear pore and kinetochore that functions in both pore assembly at the nucleus and faithful cell division.

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Capture of AT-rich Chromatin by ELYS Recruits POM121 and NDC1 to Initiate Nuclear Pore Assembly

(Molecular Biology of the Cell, 2008)

Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A3 does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the
forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121- and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits.

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The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly

(Molecular Biology of the Cell, 2006)

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.

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II. Regulating the Assembly of a Nucleus

Importin Beta Negatively Regulates Nuclear Membrane Fusion and NPC Assembly

(Molecular Biology of the Cell, 2003)

Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. Here we report that importin b negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin b is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin b downregulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellaelike structures. We propose that a precise balance of importin b to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin b 45-462 allows membrane fusion, but produces nuclei lacking any NPCs. This reveals distinct importin b-regulation of NPC assembly. Excess full-length importin b and b 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin b NPC block, which maps downstream of GTPgS- and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 mM RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin b. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.

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The roles of importin b in regulating multiple key cellular processes are reviewed in Harel and Forbes, Mol. Cell, 2004.

 

Xenopus importin beta validates human importin beta as a cell cycle negative regulator

(BMC Cell Biology, 2008)

Human importin beta has been used in all Xenopus laevis in vitro nuclear assembly and spindle assembly studies. This disconnect between species raised the question for us as to whether importin beta was an authentic negative regulator of cell cycle events, or a dominant negative regulator due to a difference between the human and Xenopus importin beta sequences. No Xenopus importin beta gene was yet identified at the time of those studies. Thus, we first cloned, identified, and tested the Xenopus importin beta gene to address this important mechanistic difference. If human importin beta is an authentic negative regulator then we would expect human and Xenopus importin beta to have identical negative regulatory effects on nuclear membrane fusion and pore assembly. If human importin beta acts instead as a dominant negative mutant inhibitor, we should then see no inhibitory effect when we added the Xenopus homologue.

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Nucleoporin Levels Regulate Cell Cycle Progression and Phase-Specific Gene Expression

(Developmental Cell, 2008)

The Nup107-160 complex, the largest subunit of the nuclear pore, is multifunctional. It mediates mRNA export in interphase, and has roles in kinetochore function, spindle assembly, and postmitotic nuclear pore assembly. We report here that the levels of constituents of the Nup107-160 complex are coordinately cell cycle-regulated. At mitosis, however,
a member of the complex, Nup96, is preferentially downregulated. This occurs via the ubiquitin-proteasome pathway. When the levels of Nup96 are kept high, a significant delay in G1/S progression occurs. Conversely, in cells of Nup96+/ mice, which express low levels of Nup96, cell cycle progression is accelerated. These lowered levels of Nup96 yield specific defects in nuclear export of certain mRNAs and protein expression, among which are key cell cycle regulators. Thus, Nup96 levels regulate differential gene expression in a phase-specific manner, setting the stage for proper cell cycle progression
.

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III. Nuclear Transport

Centrin 2 Localizes to the Vertebrate Nuclear Pore and Plays a Role in mRNA and Protein Export

(Molecular and Cellular Biology, 2008)

Centrins in vertebrates have traditionally been associated with microtubule-nucleating centers such as the centrosome. Unexpectedly, we found centrin 2 to associate biochemically with nucleoporins, including the Xenopus laevis Nup107-160 complex, a critical subunit of the vertebrate nuclear pore in interphase and of the kinetochores and spindle poles in mitosis. Immunofluorescence of Xenopus cells and in vitro reconstituted nuclei indeed revealed centrin 2 localized at the nuclear pores. Use of the mild detergent digitonin in immunofluorescence also allowed centrin 2 to be clearly visualized at the nuclear pores of human cells. Disruption of nuclear pores using RNA interference of the pore assembly protein ELYS/MEL-28 resulted in a specific decrease of centrin 2 at the nuclear rim of HeLa cells. Functionally, excess expression of either the N or C-terminal calcium-binding domains of human centrin 2 caused a dominant-negative effect on both mRNA and protein export, leaving protein import intact. The mRNA effect mirrors that found for the Saccharomyes cerevisiae centrin Cdc31p at the yeast nuclear pore, a role until now thought to be unique to yeast. We conclude that in vertebrates, centrin 2 interacts with major subunits of the nuclear pore, exhibits nuclear pore
localization, and plays a functional role in multiple nuclear export pathways.

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Novel Vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export

(Journal of Cell Biology, 2001)

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.

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Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors

(Current Biology, 1998)

Proteins generally enter or exit the nucleus as cargo of one of a small family of import and export receptors. These receptors bear distant homology to importin beta, a subunit of the receptor for proteins with classical nuclear localisation sequences (NLSs). To understand the mechanism of nuclear transport, the next question involves identifying the nuclear pore proteins that interact with the different transport receptors as they dock at the pore and translocate through it. RESULTS: Two pathways of nuclear import were found to intersect at a single nucleoporin, Nup153, localized on the intranuclear side of the nuclear pore. Nup153 contains separate binding sites for importin alpha/beta, which mediates classical NLS import, and for transportin, which mediates import of different nuclear proteins. Strikingly, a Nup153 fragment containing the importin beta binding site acted as a dominant-negative inhibitor of NLS import, with no effect on transportin-mediated import. Conversely, a Nup153 fragment containing the transportin binding site acted as a strong dominant-negative inhibitor of transportin import, with no effect on classical NLS import. The interaction of transportin with Nup153 could be disrupted by a non-hydrolyzable form of GTP or by a GTPase-deficient mutant of Ran, and was not observed if transportin carried cargo. Neither Nup153 fragment affected binding of the export receptor Crm1 at the nuclear rim. CONCLUSIONS: Two nuclear import pathways, mediated by importin beta and transportin, converge on a single nucleoporin, Nup153. Dominant-negative fragments of Nup153 can now be used to distinguish different nuclear import pathways and, potentially, to dissect nuclear export.

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IV. New Roles for Nuclear Pores and Nucleoporins

Global histone acetylation induces functional genomic reorganization at mammalian nuclear pore complexes

(Genes and Development, 2008)

The nuclear localization of genes is intimately tied to their transcriptional status in Saccharomyces cerevisiae, with populations of both active and silent genes interacting with components of the nuclear envelope. We investigated the relationship between the mammalian nuclear pore and the human genome by generating high-resolution, chromosome-wide binding maps of human nucleoporin 93 (Nup93) in the presence and absence of a potent histone deacetylase inhibitor (HDACI). Here, we report extensive genomic reorganization with respect to the nuclear pore following HDACI treatment, including the recruitment of promoter regions, euchromatin-rich domains, and differentially expressed genes. In addition to biochemical mapping, we visually demonstrate the physical relocalization of several genomic loci with respect to the nuclear periphery. Our studies show that inhibiting HDACs leads to significant changes in genomic organization, recruiting regions of transcriptional regulation to mammalian nuclear pore complexes.

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V. Mapping The Vertebrate Nuclear Pore

Topology of yeast Ndc1p: predictions for the human NDC1/NET3 homologue

(The Anatomical Record Part A, 2006)

The nuclear pore complex is the predominant structure in the nuclear envelope that spans the double nuclear membranes of all eukaryotes. Yeasts have one additional organelle that is also embedded in the nuclear envelope: the spindle pole body, which functions as the microtubule organizing center. The only protein known to localize to and be important in the assembly of both of these yeast structures is the integral membrane protein, Ndc1p. However, no homologues of Ndc1p had been characterized in metazoa. Here, we identify and analyze NDC1 homologues that are conserved throughout evolution. We show that the overall topology of these homologues is conserved. Each contains six transmembrane segments in its N-terminal half and has a large soluble C-terminal half of approximately 300 amino acids. Charge distribution analysis infers that the N- and C-termini are exposed to the cytoplasm. Limited proteolysis of yeast Ndc1p in cellular membranes confirms the orientation of its C-terminus. Although it is not known whether vertebrate NDC1 protein localizes to nuclear pores like its yeast counterpart, the human homologue contains three FG repeats in the C-terminus, a feature of many nuclear pore proteins. Moreover, a small region containing mutations that affect assembly of the nuclear pore in yeast is highly conserved throughout evolution. Lastly, we bring together data from another study to demonstrate that the human homologue of NDC1 is the known inner nuclear membrane protein, NET3.

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Purification of the vertebrate nuclear pore complex by biochemical criteria

(Traffic, 2000)

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.

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Identification of a new vertebrate nucleoporin, Nup188, with the use of a novel organelle trap assay

(Molecular Biology of the Cell, 2000)

The study of the nuclear pore in vertebrates would benefit from a strategy to directly identify new nucleoporins and interactions between those nucleoporins. We have developed a novel two-step "organelle trap" assay involving affinity selection and in vitro pore assembly. In the first step, soluble proteins derived from Xenopus egg extracts are applied to a column containing a ligand of interest. The bound proteins are then tagged by biotinylation and eluted. In the second step, potential nucleoporins are selected for by virtue of their ability to assemble into annulate lamellae, a cytoplasmic mimic of nuclear pores. The incorporated proteins are then recognized by their biotin tag. Here we use the lectin wheat germ agglutinin (WGA) as ligand; WGA inhibits nuclear transport and has been shown to directly bind three known nucleoporins from Xenopus extract, Nup62, Nup98, and Nup214, all of which contain N-acetylglucosamine residues. Under reduced-stringency conditions, three additional proteins bind to WGA-Sepharose and are revealed by the organelle trap assay. We identified all three as partner nucleoporins. Two were discovered to be Xenopus Nup93 and Nup205. The third is a novel vertebrate nucleoporin, Nup188. This new vertebrate protein, Xenopus Nup188, exists in a complex with xNup93 and xNup205. The Nup93-Nup188-Nup205 complex does not bind directly to WGA but binds indirectly via the N-acetylglucosamine-modified nucleoporins. A gene encoding human Nup188 was also identified. The discovery of vertebrate Nup188, related to a yeast nucleoporin, and its novel protein-protein interactions illustrates the power of the two-step organelle trap assay and identifies new building blocks for constructing the nuclear pore.

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Preparation of Xenopus Extract

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