Article |
Address correspondence to Valerie Doye, UMR 144 CNRS-Institut Curie, Section Recherche, 26 rue d'Ulm, 75248 Paris cedex 05, France. Tel.: 33-1-42-34-64-10. Fax: 33-1-42-34-64-21. E-mail: vdoye{at}curie.fr
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: nucleoporin; nuclear pore; mitosis; kinetochores; GFP
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An extensively characterized building block of the S. cerevisiae NPC is the scNup84 subcomplex, which consists of scNup84, scNup85, scNup120, scNup145-C (the in vivocleaved COOH-terminal half of scNup145), scSeh1 (Sec13 homologue 1), and a fraction of scSec13 (Siniossoglou et al., 1996; Teixeira et al., 1997). This complex has been shown recently to exhibit a Y-shaped structure with an average diameter of 25 nm (Siniossoglou et al., 2000). Mutations of nucleoporins that belong to the scNup84 complex lead to specific defects in the export of mRNA and to a constitutive clustering of the NPCs (for review see Doye and Hurt, 1997). Recently, the vertebrate homologue of scNup145-C, Nup96, was shown to comigrate on a sucrose gradient with Nup107 (the vertebrate homologue of scNup84) (Radu et al., 1994; Siniossoglou et al., 1996), mammalian sec13, and p37 (a sec13-related protein), suggesting that the scNup84 complex might have been conserved partly during evolution (Fontoura et al., 1999).
Although scNup133 shares very similar phenotypes and is genetically linked to the members of the scNup84 complex (Doye et al., 1994; Siniossoglou et al., 1996), biochemical approaches did not previously allow the characterization of any interaction between scNup133 and constituents of the scNup84 complex. To address this question in another organism and get access to the dynamics of these nucleoporins at the various stages of the cell cycle, we have characterized the human homologue of scNup133. Two hybrid screens and immunoprecipitation experiments indicated that hNup133 is part of a vertebrate NPC subcomplex that also contains hNup107, hNup96, and a novel mammalian nucleoporin homologous to scNup120. Immunoelectron microscopy revealed that like their S. cerevisiae counterparts hNup107 and hNup133 are localized on both sides of the NPC. Using immunofluorescence and in vivo analysis of GFP-tagged hNup133 and hNup107, we demonstrate that hNup133 and hNup107 are stable components of the NPC in interphase, remain associated with each other during mitosis, and are targeted at early stages to the reforming nuclear envelope. Unexpectedly, this study further revealed that a fraction of hNup133 and hNup107 associates with the kinetochores from prophase to late anaphase and that the interaction of hNup133 with kinetochores is dynamic.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
hNup133 and hNup107 belong to an NPC subcomplex that contains hNup96 and a novel protein homologous to scNup120
Although our data indicate that the two-hybrid interaction between Nup133 and Nup84/107 is highly specific and may also take place in wild-type yeast and human cells, scNup133 has never been reported to be present in the scNup84 complex (Siniossoglou et al., 1996, 2000; see Discussion). Therefore, we decided to characterize the interaction between hNup133 and hNup107 in human cells. Immunoprecipitation experiments performed on HeLa cell extracts using anti-hNup107 or anti-hNup133 antibodies revealed that these two nucleoporins coprecipitate efficiently with each other, whereas neither p62 nor any other nucleoporin recognized by the mAb414 antibody could be detected in the immune pellets (Fig. 2 A). Silver-staining analysis of the immunoprecipitates obtained with anti-hNup107 (Fig. 2 B) and anti-hNup133 (Fig. 2 E) reproducibly revealed four specific bands of similar intensity that migrated in the 100150-kD range. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and quadrupole time-of-flight (Q-TOF) spectrometry analysis confirmed the identity of bands 2 and 4 as hNup133 and hNup107 and further identified band 3 as hNup96 (the human homologue of scNup145-C) and band 1 as an uncharacterized 149-kD protein referred to in the database as KIAA0197 (sequence data available from GenBank/EMBL/DDBJ under accession no. G02870). Blast searches revealed that the KIAA0197 protein showed significant homology to an S. pombe ORF coding for a 130-kD protein designated as SPBC3B9.16C (sequence data available from GenBank/EMBL/DDBJ under accession no. T40355) and to scNup120 (Fig. S2 available at http://www.jcb.org/content/vol154/issue6). Accordingly and irrespective of their apparent molecular weights, we have designated the human and the S. pombe proteins as hNup120 and spNup120, respectively.
|
During mitosis, the soluble components of mammalian NPCs are partially disassembled into subcomplexes and dispersed throughout the mitotic cytosol. To analyze the biological relevance of this biochemically identified NPC subcomplex in mitotic cells, we performed immunoprecipitation experiments from interphase and mitotic populations of HeLa cells. The four major constituents of the hNup133-hNup107containing complex were found to coprecipitate in mitotic cell extracts with a stoichiometry similar in both interphasic and mitotic precipitates (Fig. 2, CE), demonstrating that these nucleoporins remain as a stable subcomplex during mitosis.
hNup133 and hNup107 are localized on both sides of the nuclear pore complex
We determined the localization of these human nucleoporins within the NPC using at first a differential permeabilization protocol based on the fact that low concentrations of digitonin permeabilize the plasma membrane but leave the nuclear membrane intact (Adam et al., 1990). Although lamin B or Nup153, used as controls, were detected only in Triton X-100permeabilized cells, anti-hNup133 and anti-hNup107 antibodies labeled the nuclear envelope in both digitonin- and Triton X-100permeabilized cells, indicating that these nucleoporins are at least located at the cytoplasmic face of the NPC (unpublished data).
To determine whether these two nucleoporins are also present on the nuclear side of the NPC, ultrathin sections of HeLa cells fixed and processed for ultracryomicrotomy were immunogold-labeled using as primary antibodies an affinity purified anti-hNup133 antibody (Fig. 3 A) or two distinct anti-hNup107 antibodies (Fig. 3, B and C). Analysis of the distribution of the gold particles revealed that like their S. cerevisiae homologues hNup133 and hNup107 are localized to both the cytoplasmic and nucleoplasmic faces of the nuclear envelope (Fig. 3, right).
|
|
|
|
|
|
Dynamics of hNup133 at the kinetochore
The low turnover of hNup107/hNup133 in the nuclear envelope prompted us to investigate the dynamics of their interaction with kinetochores. To this end, NRK cells stably expressing GFP3-hNup133 that gave a slightly higher kinetochore signal than GFP3-hNup107 cells were used. From prometaphase to early anaphase, these cells contained only two pools of hNup133: diffuse cytoplasmic and kinetochore localized. Four-dimensional FRAP experiments (see Materials and methods) in which the entire kinetochore fraction of GFP3-hNup133 was photobleached were carried out to see if this pool could recover from the cytoplasmic reservoir. FRAPs were started in prometaphase and completed before anaphase onset when the imminent nuclear assembly would have complicated the measurement. All cells used for photobleaching initiated anaphase normally, serving as a control for photo damage (Fig. 9 A, fifth panel). Recovery of kinetochore hNup133 was observed after several minutes (Fig. 9 A) with a halftime of 132 ± 64 s (Fig. 9 C), meaning that it took over 2 min to exchange half of the GFP3-hNup133 molecules bound to kinetochores with freely diffusing molecules in the cytoplasm. An immobile fraction of 34 ± 16% that did not exchange at all over the course of 10 min was also characterized in these experiments (Fig. 9 C). Using a simple kinetic model for the exchange of the mobile fraction of GFP3-hNup133 with a single binding site on kinetochores (Fig. S3 and supplemental methods available at http://www.jcb.org/content/vol154/issue6), we could estimate its dissociation constant as koff = 0.0058-1 ± 0.0026-1 s. Thus 1/koff, the mean residence time, was 172 ± 77 s, meaning on average each GFP3-hNup133 molecule spent
3 min bound to kinetochores before it exited to the cytoplasm.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although hNup133 thus appears as a constitutive subunit of the hNup107 complex, scNup133 had not been detected previously in the scNup84 complex (Siniossoglou et al., 1996, 2000). This could result from the high sensitivity of scNup133 to proteolysis. Alternatively, considering that scNup133 interacts directly with scNup84 (as demonstrated by the species-specific two-hybrid interaction) and that scNup84 resides peripherally within the scNup84 complex (Siniossoglou et al., 2000), scNup133 might be associated loosely to the periphery of the scNup84 complex. Conversely, although scNup85 is an essential constituent of the scNup84 complex (Siniossoglou et al., 2000), no major band could be detected in the 75-kD range (the theoretical molecular weight of the putative human homologue of scNup85; unpublished data) on silver-stained gels from hNup133 or hNup107 immune pellets. In addition, no specific protein migrating at 75 kD was recorded to cosediment specifically on the sucrose gradient with Nup96 and Nup107 (Fontoura et al., 1999). If present in the hNup107 complex, the putative mammalian homologue of scNup85 may, as hypothesized for scNup133, be either loosely associated with this complex or more sensitive to proteolysis. Alternatively, hNup85 may not migrate at its expected molecular weight. Finally, although mSec13 and a novel sec13-related protein were found also to cosediment with rat Nup107 and Nup96 (Fontoura et al., 1999), their presence in our immune pellets could not be assessed so far, since their putative signals could not be resolved from the immunoglobulin light chains.
Immunofluorescence experiments on semipermeabilized cells and immunoelectron microscopy revealed that hNup133 and hNup107 are localized on both sides of the NPC. However, previous studies performed with an antibody directed against a 200amino acid domain of hNup96 revealed its specific localization on the nucleoplasmic side of the NPC (Fontoura et al., 1999). This could result from the specific enrichment on the nucleoplasmic side of the NPC of a fraction of Nup96, which would not associate with the hNup107 complex. Conversely, Nup96 may be present only in a subfraction of hNup107 complexes containing the other components and localized on the nucleoplasmic side of the NPC. However, the fact that Nup96 sediments as a unique peak on the sucrose gradient (Fontoura et al., 1999) together with the previously described localization of scNup133 and the various members of the scNup84 complex (Rout et al., 2000) rather suggests that the entire hNup107 NPC subcomplex displays a symmetric localization on both sides of the NPC (Fontoura et al., 1999). Thus, the observed discrepancy could be due to a biased accessibility of the epitope recognized by this anti-Nup96 antibody. Together, these data indicate that the scNup84- and hNup107-containing complexes are structurally and possibly functionally related.
The hNup107 complex behaves as a structural building block of the mammalian NPCs
The characterization in mammalian cells of this evolutionarily conserved NPC complex enabled us to address the dynamics of its subunits in both interphase and mitosis thereby providing a set of functional data that could not be obtained in yeast. Immunofluorescence studies and four-dimensional in vivo imaging during nuclear envelope reassembly revealed an early recruitment of both nucleoporins from the soluble cytoplasmic pool to the chromosome periphery, producing a well-defined rim in late anaphase. In vivo imaging also indicated that a fraction of GFP3-hNup107 was recruited diffusely to the chromosome surface 23 min earlier than hNup133, a rather unexpected difference, since our biochemical data indicate that hNup133 and hNup107 remain in a stable complex in metaphase-arrested cells. Because the in vivo approach relies on the use of GFP-tagged nucleoporins, this discrepancy could be due to the mild overexpression inherent to the use of stably transfected tissue culture cells. Alternatively, there might also be a pool of uncomplexed subunits for both proteins. The chromosome-recruited fraction of hNup107 might then provide a template for the formation of additional hNup107/hNup133 complexes during anaphase.
In either case, the early chromosome labeling, which precedes by several minutes the formation of the nuclear membrane (unpublished data) and the recruitment of many other nucleoporin characterized to date (Bodoor et al., 1999), makes the hNup107 complex a likely key player in an early step of NPC reassembly after mitosis. In addition, FRAP experiments revealed an extremely low turnover of both nucleoporins in single NPCs during interphase. The identical behavior of these two soluble nucleoporins strongly suggests that the entire hNup107 complex is bound tightly to the NPC and is exchanged only once per cell cycle, consistent with a structural function of this complex within the NPC. In comparison, Nup153 has a high turnover rate in the order of seconds, whereas such a stable association with the NPC was so far observed only for the membrane-anchored POM121 nucleoporin (Daigle et al., 2001).
Thus, in the future it will be of interest to correlate these data with the proposed structural intermediates in NPC assembly that have been imaged in Xenopus egg extracts using high resolution scanning EM (Goldberg et al., 1997). In particular, because of the Y shape of the scNup84 complex (Siniossoglou et al., 2000) it is tempting to speculate that this conserved NPC subcomplex might build up the star-ring region of the NPC, a structure consisting of eight triangular subunits that appears as an early intermediate in NPC assembly (Goldberg et al., 1997).
A novel link between NPC constituents and kinetochores
The most unexpected result arising from this study was the localization of a pool of both hNup133 and hNup107 to the mitotic kinetochores. This dual localization does not reflect the existence of specific splice variants recognized by the antibodies, since a fraction of transfected GFP-tagged hNup133 or hNup107 is recruited similarly to the kinetochores during mitosis. Our immunofluorescence data and in vivo labeling studies indicate that only a minor fraction of hNup133 and hNup107 is localized at the kinetochores, the remaining pool giving rise to a diffuse staining throughout the mitotic cytoplasm. The overall stability of the hNup107 complex in metaphase-arrested cells suggests that all of the constituents of the hNup107 complex may associate with kinetochores. However, since this kinetochore-associated fraction would not affect the overall yield of our immunoprecipitation experiments we cannot rule out that only hNup133 and hNup107 (which directly interact with each other) associate with the kinetochores.
Although we demonstrated that hNup133 is a stable constituent of NPC during interphase, both FRAP and FLIP experiments showed that the kinetochore fraction of GFP3-hNup133 exchanges with the diffuse cytoplasmic pool. 30% of the kinetochore-bound pool exchanged with the cytoplasm every minute, and we detected no change in the rate of this exchange from prometaphase to anaphase. The recovery during FRAP experiments shows clearly that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that interaction of this nucleoporin with kinetochores is dynamic. In addition, these photobleaching experiments also pointed to a significant immobile fraction of 34% of hNup133 on kinetochores, suggesting the presence of a second higher affinity binding site for this nucleoporin on kinetochores.
Interestingly, while this work was in progress Mad1 and Mad2, two mitotic checkpoint proteins that transiently associate with the kinetochores during mitosis (for review see Shah et al., 2000), were described as being localized to the nucleocytoplasmic face of the NPC throughout interphase (Campbell et al., 2001). Conversely, the mRNA export factor hRae1 was shown to interact with the mitotic checkpoint protein mBUB1 and to be localized at kinetochores of prometaphase chromosome (Wang et al., 2001). In addition, a few other proteins including a fraction of the SUMO-1modified form of RanGAP1 (Matunis et al., 1996), a fraction of tankyrase (Smith and de Lange, 1999), and the PCB68 antigen (Theodoropoulos et al., 1999), have been reported previously to relocalize from the NPCs to microtubule-associated structures during mitosis. Finally, it was demonstrated recently that a mutation in the S. cerevisiae nucleoporin gene NUP170 leads to defects in chromosome transmission fidelity and kinetochore integrity in this organism (Kerscher et al., 2001). Together with the involvement of the nuclear transport factor Ran in mitotic spindle formation (for review see Azuma and Dasso, 2000), these data support the notion that the nucleocytoplasmic transport and mitotic machineries contain multiple shared components. They further suggest that the shared localization of a subset of NPC/kinetochore constituents may be an important and so far neglected aspect of cell division or nuclear transport.
What could be the function(s) of these structural nuclear pore constituents at the kinetochores? One hypothesis is that this fraction of hNup133 and hNup107 may act as "kinetochore-associated passenger proteins" that use kinetochores as a means of conveyance to be positioned correctly during the subsequent mitotic events (for review see Adams et al., 2001). Unlike the currently described bona fide "passenger proteins," this minor pool of nucleoporins does not redistribute to the spindle midzone in anaphase but persists as a distinctly localized pool during nuclear envelope assembly in late anaphase and would possibly be positioned properly to allow the assembly of a specific subset of NPCs in late anaphase or early telophase. Alternatively, hNup133 and hNup107 may play a specific role on kinetochores in mitotic cells. Although the dynamics of kinetochores constituents had been addressed so far only for Mad2 (Howell et al., 2000), our photobleaching experiments, demonstrating that the interaction of hNup133 with kinetochores is dynamic, argues against hNup133 as a structural constituent of kinetochores. Conversely, the dissociation rate of hNup133 from kinetochores is about fivefold slower than the one measured previously for GFP-Mad2 (Howell et al., 2000). This and the fact that hNup133 kinetochore localization is maintained even through the early stages of nuclear assembly in late anaphase actually argues against a direct involvement of hNup133 in the "spindle assembly" checkpoint. Although the function of the kinetochore fraction of hNup107/hNup133 still remains to be determined (a study that will probably be complicated by their dual NPC/kinetochore localization), it is intriguing to speculate that these nucleoporins could provide a link between cell cycle control and NPC disassembly/assembly, a question currently under investigation in our laboratories.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A human Nup107 cDNA was obtained from total human RNA by reverse transcriptase-PCR amplification with PCR primers designed by using human EST sequences homologous to the rat NUP107 cDNA sequence (Radu et al., 1994). The primary sequence of hNup107 (which we found identical to NP_065134) revealed 91.6% identity to the published rat Nup107 peptide sequence (Radu et al., 1994). For in vivo studies, the hNup107 cDNA was subcloned into pEGFP3-C1.
Yeast two-hybrid screening
Two-hybrid screens using as bait either full-length scNup133 or scN-Nup133 (Doye et al., 1994) cloned into pAS
or hNup133 cloned into pLex12 were performed by a mating strategy as described (Fromont-Racine et al., 1997, 2000) with the FRYL S. cerevisiae genomic library (Fromont-Racine et al., 1997) or a human Jurkat cell line cDNA library (Jullien-Flores et al., 1995). To investigate pairwise interactions between Nup133 and Nup84/Nup107 from various organisms, the spNup133a, spNup133b, and hNup133 cDNAs were also cloned into the pAS
vector. Two-hybrid preys either recovered from the previous screens or constructed using pACTII were transformed into the Y187 strain (Fromont-Racine et al., 1997). Bait and prey strains were mated in rich medium, and diploids were tested on histidine-free medium containing 5 mM 3-aminotriazole.
Antibodies, immunofluorescence, and immunoelectron microscopy
Polyclonal antibodies against hNup133 and hNup107 were obtained by injecting recombinant GST-hNup133, 6His-hNup107-N, or 6His-hNup107-C into rabbits (Agrobio). The anti-hNup133 serum was depleted of GST antibodies and affinity purified using GST-hNup133 immobilized on an NHS-activated column. The antihNup107-N and antihNup107-C antibodies were affinity purified against recombinant GST-hNup107-N and GST-hNup107-C, respectively. Antilamin B (Guilly et al., 1987) and autoimmune CREST serum were obtained from J.C. Courvalin (Institut J. Monad, Paris, France), guinea pig anti-p62 (Cordes et al., 1991) was a gift from G. Krohne (Biocenter of the University, Würzburg, Germany); SA1 monoclonal antibody against Nup153 (Bodoor et al., 1999) was provided by Ricardo Bastos (University of Barcelona, Barcelona, Spain); monoclonal anti-p150Glued was from Transduction Laboratories. Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc.
For immunofluorescence, cells were either fixed for 20 min in 3% fresh paraformaldehyde and permeabilized with 0.5% Triton X-100 or fixed for 5 min in methanol at -20°C. Ultracryomicrotomy and immunogold labeling of HeLa cells fixed with 4% paraformaldehyde was performed as described (Raposo et al., 1997).
Immunoprecipitation, MALDI-TOF, and Q-TOF spectrometry identification
Immunoprecipitation experiments from HeLa whole cell extracts (2 x 106 cells) were performed essentially as described (Grandi et al., 1997) using 25 µl of crude sera or 6 µg of affinity purified anti-hNup133 antibody bound to 25 µl Affi-prep protein A matrix (Bio-Rad Laboratories). For some experiments, HeLa cells synchronized using a double thymidine block were accumulated in prometaphase using 1µM nocodazole and collected by shake-off 4 h after nocodazole addition.
For MALDI-TOF or Q-TOF spectrometry analysis, protein spots were cut off from silver-stained polyacrylamide gels and digested in gel slices with trypsin as described (Shevchenko et al., 1996). Digests were resuspended in 20 µl formic acid 1%, desalted using Zip Tips C18 from Millipore, dried, and dissolved in 3 µl of formic acid 1%. The sample and the matrix (a saturated solution of 2,5-dihydroxybenzoic acid in TFA 0.1%) were loaded on the target using the dried droplet method. MALDI-TOF spectra of the peptides were obtained with a Voyager-DE STR Biospectrometry Workstation mass spectrometer (PE Corp.) and were calibrated externally using Des-Arg bradykin and ACTH peptides and internally using the trypsin autoproteolysis products. Data mining was performed using the ProFound software. A mass deviation of 0.1 D was allowed in the database searches. For Q-TOF spectrometry identification, the dried trypsin digests were dissolved in 3 µl of a mixture of water, formic acid, and methanol (49:1:50) and were analyzed by MS/MS on a nanoESI Q-TOF mass spectrometer (Micromass).
Photobleaching experiments and four-dimensional confocal microscopy
Photobleaching experiments and four-dimensional confocal microscopy were performed on a custom made ZEISS LSM510 system essentially as described (Zaal et al., 1999; Daigle et al., 2001). For three-dimensional reconstruction of confocal z-stacks, individual images were processed with an anisotropic diffusion filter to remove background noise while preserving edge information (Tvarusko et al., 1999). Then, chromosomes and kinetochores were segmented by thresholding and surface rendered using Amira (Template Graphics Software, Inc.).
FRAP experiments on kinetochores were started in prometaphase to allow maximum recovery time before anaphase onset. Bleaching was performed on the outlined regions in three optical sections (field width half maximum = 1.6 µm) distributed over the metaphase plate and completely bleached all kinetochores (unpublished data). Recovery was monitored every minute on three different focal planes spaced 1.5 µm apart in the center of the metaphase plate. In FLIP experiments, bleaching was repeated on the outlined cytoplasmic regions every 10 s from early metaphase to the onset of anaphase. One image was acquired after each bleach to monitor depletion of fluorescence from cytoplasm and kinetochores. Mean fluorescence intensity of the cytoplasm and the kinetochores was quantitated using the public domain software ImageJ (http://rsb.info.nih.gov/ij). Kinetochores were measured with constant circular regions centered on their peak intensity using an ImageJ macro. The values measured for all kinetochores at the same time point were averaged. Since optical slice thickness of 1.6 µm far exceeds the diameter of a kinetochore, we assumed that cytoplasmic GFP3-hNup133 contributes equally to kinetochore regions. Thus, to obtain GFP3-hNup133 bound to kinetochores mean cytoplasmic intensity was subtracted from mean kinetochore intensity. Bleaching due to the acquisition was corrected and was <10% in all experiments. For comparison, prebleach intensities were normalized to 1.
Online supplemental material
ClustalW alignment of scNup133 and scNup120 homologues (Figs. S1 and S2), Quicktime videos of the sequences shown in Fig. 4 A (videos 1 and 2) and Fig. 6 (videos 3 and 4), representative data set with theoretical fit from FRAP and FLIP experiment shown in Fig. 9 (Fig. S3), and the corresponding kinetic analysis that allowed us to determine the koff from FRAP and FLIP experiments are provided as supplemental material. Supplemental material is available at http://www.jcb.org/content/vol154/issue6.
![]() |
Footnotes |
---|
O.V. Zatsepina's present address is A.N. Belozersky Institute of Physical and Chemical Biology, Moscow State University, Moscow 119899, Russia.
* Abbreviations used in this paper: FLIP, fluorescence loss in photobleaching; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; NPC, nuclear pore complex; Q-TOF, quadrupole time-of-flight.
![]() |
Acknowledgments |
---|
This work was supported by Centre National de la Recherche Scientifique, the Institut Curie, and the Association pour la Recherche contre le Cancer (grants to V. Doye and fellowship to N. Belgareh). J. Beaudouin was supported by a fellowship through the European Molecular Biology Laboratory International Ph.D. Program.
Submitted: 24 January 2001
Accepted: 2 August 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adam, S.A., R.S. Marr, and L. Gerace. 1990. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111:807816.[Abstract]
Adams, R.R., M. Carmena, and W.C. Earnshaw. 2001. Chromosomal passengers and the (aurora) ABCs of mitosis. Trends Cell Biol. 11:4954.[Medline]
Azuma, Y., and M. Dasso. 2000. The role of Ran in nuclear function. Curr. Opin. Cell Biol. 12:302330.[Medline]
Belgareh, N., and V. Doye. 1999. Yeast and vertebrate nuclear-pore complexes: evolutionary conserved, yet divergent macromolecular assemblies. Protoplasma. 209:133143.
Bodoor, K., S. Shaikh, D. Salina, W.H. Raharjo, R. Bastos, M. Lohka, and B. Burke. 1999. Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis. J. Cell Sci. 112:22532264.
Busson, S., D. Dujardin, A. Moreau, J. Dompierre, and J.R. De Mey. 1998. Dynein and dynactin are localized to astral microtubules and at cortical sites in mitotic epithelial cells. Curr. Biol. 8:541544.[Medline]
Campbell, M., G. Chan, and T. Yen. 2001. Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase. J. Cell Sci. 114:953963.
Chan, G.K., B.T. Schaar, and T.J. Yen. 1998. Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1. J. Cell Biol. 143:4963.
Cordes, V., I. Waizenegger, and G. Krohne. 1991. Nuclear pore complex glycoprotein p62 of Xenopus laevis and mouse: cDNA cloning and identification of its glycosylated region. Eur. J. Cell Biol. 55:3147.[Medline]
Daigle, N., J. Beaudouin, L. Hartnell, G. Imreh, E. Hallberg, J. Lippincott-Schwartz, and J. Ellenberg. 2001. Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. J. Cell Biol. 154:763774.
Doye, V., and E. Hurt. 1997. From nucleoporins to nuclear pore complexes. Curr. Opin. Cell Biol. 9:401411.[Medline]
Doye, V., R. Wepf, and E.C. Hurt. 1994. A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution. EMBO J. 13:60626075.[Abstract]
Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J. Cell Biol. 132:617633.[Abstract]
Fontoura, B.M., G. Blobel, and M.J. Matunis. 1999. A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186-kilodalton precursor generates Nup98 and the novel nucleoporin, Nup96. J. Cell Biol. 144:10971112.
Fromont-Racine, M., J.C. Rain, and P. Legrain. 1997. Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat. Genet. 16:277282.[Medline]
Fromont-Racine, M., A.E. Mayes, A. Brunet-Simon, J.C. Rain, A. Colley, I. Dix, L. Decourty, N. Joly, F. Ricard, J.D. Beggs, et al. 2000. Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins. Yeast. 17:95110.[Medline]
Goldberg, M.W., C. Wiese, T.D. Allen, and K.L. Wilson. 1997. Dimples, pores, star-rings, and thin rings on growing nuclear envelopes: evidence for structural intermediates in nuclear pore complex assembly. J. Cell Sci. 110:409420.
Grandi, P., T. Dang, N. Pane, A. Shevchenko, M. Mann, D. Forbes, and E. Hurt. 1997. Nup93, a vertebrate homologue of yeast Nic96p, forms a complex with a novel 205-kDa protein and is required for correct nuclear pore assembly. Mol. Biol. Cell. 8:20172038.
Guilly, M.N., F. Danon, J.C. Brouet, M. Bornens, and J.C. Courvalin. 1987. Autoantibodies to nuclear lamin B in a patient with thrombopenia. Eur. J. Cell Biol. 43:266272.[Medline]
Howell, B.J., D.B. Hoffman, G. Fang, A.W. Murray, and E.D. Salmon. 2000. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells. J. Cell Biol. 150:12331250.
Jullien-Flores, V., O. Dorseuil, F. Romero, F. Letourneur, S. Saragosti, R. Berger, A. Tavitian, G. Gacon, and J.H. Camonis. 1995. Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity. J. Biol. Chem. 270:2247322477.
Kerscher, O., P. Hieter, M. Winey, and M.A. Basrai. 2001. Novel role for a Saccharomyces cerevisiae nucleoporin, Nup170p, in chromosome segregation. Genetics. 157:15431553.
Lennon, G.G., C. Auffray, M. Polymeropoulos, and M.B. Soares. 1996. The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics. 33:151152.[Medline]
Matunis, M.J., E. Coutavas, and G. Blobel. 1996. A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPaseactivating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135:14571470.[Abstract]
Radu, A., G. Blobel, and R.W. Wozniak. 1994. Nup107 is a novel nuclear pore complex protein that contains a leucine zipper. J. Biol. Chem. 269:1760017605.
Raposo, G., M.J. Kleijmeer, G. Posthuma, J.W. Slot, and H.J. Geuze. 1997. Immunoglod labeling of ultrathin cryosections: application in immunology. Handbook of Exp. Immunol. Vol. 4. L.A. Herzenberg, D. Weir, and C. Blackwell, editors. Blackwell Science, Inc., Cambridge, MA. 111.
Rout, M.P., J.D. Aitchison, A. Suprapto, K. Hjertaas, Y. Zhao, and B.T. Chait. 2000. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148:635651.
Shah, J.V., D.W. Cleveland, G.K. Chan, S.A. Jablonski, D.A. Starr, M.L. Goldberg, and T.J. Yen. 2000. Waiting for anaphase: Mad2 and the spindle assembly checkpoint. Cell. 103:9971000.[Medline]
Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68:850858.[Medline]
Siniossoglou, S., C. Wimmer, M. Rieger, V. Doye, H. Tekotte, C. Weise, S. Emig, A. Segref, and E.C. Hurt. 1996. A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores. Cell. 84:265275.[Medline]
Siniossoglou, S., M. Lutzmann, H. Santos-Rosa, K. Leonard, S. Mueller, U. Aebi, and E. Hurt. 2000. Structure and assembly of the Nup84p complex. J. Cell Biol. 149:4154.
Smith, S., and T. de Lange. 1999. Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes. J. Cell Sci. 112:36493656.
Stoffler, D., B. Fahrenkrog, and U. Aebi. 1999. The nuclear pore complex: from molecular architecture to functional dynamics. Curr. Opin. Cell Biol. 11:391401.[Medline]
Teixeira, M.T., S. Siniossoglou, S. Podtelejnikov, J.C. Benichou, M. Mann, B. Dujon, E. Hurt, and E. Fabre. 1997. Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins. EMBO J. 16:50865097.
Theodoropoulos, P.A., H. Polioudaki, M. Koulentaki, E. Kouroumalis, and S.D. Georgatos. 1999. PBC68: a nuclear pore complex protein that associates reversibly with the mitotic spindle. J. Cell Sci. 112:30493059.
Tran, P.T., L. Marsh, V. Doye, S. Inoué, and F. Chang. 2001. Mechanism of nuclear positioning in fission yeast based upon microtubule pushing. J. Cell Biol. 153:397411.
Tvarusko, W., M. Bentele, T. Misteli, R. Rudolf, C. Kaether, D.L. Spector, H.H. Gerdes, and R. Eils. 1999. Time-resolved analysis and visualization of dynamic processes in living cells. Proc. Natl. Acad. Sci. USA. 96:79507955.
Vasu, S.K., and D.J. Forbes. 2001. Nuclear pores and nuclear assembly. Curr. Opin. Cell Biol. 13:363375.[Medline]
Wang, X., J.R. Babu, J.M. Harden, S.A. Jablonski, M.H. Gazi, W.L. Lingle, P.C. de Groen, T.J. Yen, and J.M. van Deursen. 2001. The mitotic checkpoint protein hBUB3 and the mRNA export factor hRAE1 interact with GLEBS-containing proteins. J. Biol. Chem. 276:2655926567.
Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Golgi membranes are absorbed into and reemerge from the ER during mitosis. Cell. 99:589601.[Medline]
Related Article