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Address correspondence to Brian Burke, Department of Anatomy and Cell Biology, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610-0235. Tel.: (352) 392-0040. Fax: (352) 392-3305. email: bburke{at}anatomy.med.ufl.edu
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Abstract |
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Key Words: nuclear pore complex; kinetochore; mitosis; Nup358; mitotic checkpoint
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Introduction |
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In all eukaryotes, the NE features a pair of inner and outer nuclear membranes that are joined in places where they are spanned by nuclear pore complexes (NPCs) (Gerace and Burke, 1988; Gant and Wilson, 1997). Metazoan NEs contain an additional structural element, the nuclear lamina, which is associated with the nuclear face of the inner nuclear membrane. The lamina consists of a thin (2050 nm) filamentous meshwork composed primarily of A- and B-type nuclear lamin proteins (Gerace and Burke, 1988; Gant and Wilson, 1997). During mitosis in mammalian somatic cells, the nuclear lamina and NPCs are disassembled. At the same time, nuclear membrane components disperse within the ER, which itself exhibits numerous connections with the outer nuclear membrane (Ellenberg et al., 1997; Yang et al., 1997; Ostlund et al., 1999). Disassembly of the lamina and NPCs occurs in response to phosphorylation of both lamina and NPC subunits (Gerace and Blobel, 1980; Heald and McKeon, 1990; Macaulay et al., 1995). Although the majority of these components become distributed throughout the mitotic cytoplasm, certain NPC proteins (nucleoporins or Nups) and associated molecules, including Rae1, Nup107, and Nup133, become preferentially associated with kinetochores (Belgareh et al., 2001; Wang et al., 2001; Babu et al., 2003). Another nucleoporin, Nup358, which is a component of the short (100 nm) filaments that extend from the cytoplasmic face of the NPC during interphase, relocates to both spindle microtubules and kinetochores (Joseph et al., 2002). This relocation occurs in association with Ran GTPase activating protein 1 (RanGAP1), a molecule with which Nup358 also interacts during interphase. Conversely, certain mitotic checkpoint proteins, such as Mad1 and Mad2, that are kinetochore associated during mitosis are found at the nuclear face of NPCs during interphase (Campbell et al., 2001). In yeast, this localization is mediated by Nup53p, part of a larger complex of NPC proteins that includes Nup157p and Nup170p (Iouk et al., 2002). Remarkably, yeast strains deficient in Mad1p exhibit a reduced rate of nuclear protein import as well as decreased stability of the Nup53p complex (Iouk et al., 2002). The implication is that there is a functional relationship between the mitotic apparatus and the NE. However, the significance of this has only recently become a focus of investigation.
The interplay between the NE and the mitotic spindle is further highlighted by findings that the spindle itself plays an active role in nuclear membrane dispersal during prometaphase (Beaudouin et al., 2002; Salina et al., 2002). The entire process is driven by cytoplasmic dynein, a microtubule minus enddirected motor protein, which concentrates on the NE during late G2/early prophase (Salina et al., 2002). By engaging with spindle microtubules, NE-linked dynein causes the deformation and rupture of the nuclear membranes, leading to release of the condensed chromosomes into the cytoplasm. This process, in effect, represents a mechanical checkpoint because it provides a means to delay NE breakdown (NEBD) until functional spindle microtubules have been assembled.
Although the identity of the dynein-binding partner on the NE remains unknown, cytoplasmically exposed NPC subunits have been suggested as possible candidates. One such protein, Nup358, is of particular interest because it is known to associate with both the mitotic spindle and kinetochores. To determine whether Nup358 does indeed play a role in early mitotic progression, we employed an siRNA approach to deplete cells of this particular nucleoporin. Although we were unable to find any evidence for an involvement in dynein binding and NEBD (unpublished data), we did observe a surprising effect on chromosome congression. Our data suggest that Nup358 plays an essential role in kinetochore function and chromatid segregation.
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Results |
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Until at least the fourth day of Nup358 siRNA treatment, some members of the unusual prometaphase population display the ability to escape mitotic arrest. In these cells, an NE reforms around individual chromosomes and groups of chromosomes, giving rise to the multiple micronuclei described above. These cells invariably show reduced labeling with anti-Nup358 antibodies (Fig. 2 C and Fig. 3 D). Surprisingly, many of these cells form an intracellular bridge and undergo cytokinesis (Fig. 5 C). Indeed at the 96-h time point, 34% of "telophase" or early G1 cells (defined by the presence of an intracellular bridge) were found to contain multiple micronuclei. Few such cells were observed in corresponding mock-treated populations. The ultimate fate of these unusual cells seems to be death, because, as pointed out above, the frequency of apoptosis increases steadily up to 5 d after siRNA treatment. After this time point, the occurrence of cells containing multiple micronuclei generally declines (Fig. 1 B). Remarkably, a virtually identical effect, including micronuclear formation and aberrant cytokinesis, has recently been reported in cells depleted of CENP-A and hMis12, a human kinetochore protein (Goshima et al., 2003).
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A final issue that arises is whether Nup358 itself might have a role as a spindle assembly checkpoint protein. To address this, we examined the effects of simultaneous depletion of Nup358 and the bona fide checkpoint protein Mad1 (Fig. 8). Depletion of Mad1 has previously been shown to result in premature anaphase and the appearance of lagging chromosomes (Luo et al., 2002; Martin-Lluesma et al., 2002). The prediction is that if the spindle assembly checkpoint remains functional in Nup358-depleted cells, then loss of Mad1 should result in a decline in the number of prometaphase/metaphase cells. At the same time, given the Nup358-associated congression defect, there should be an increase in the number of cells containing multiple micronuclei. As shown in Fig. 8, this is precisely what occurs. In cultures depleted only of Nup358, there is a 70% increase in the number metaphase and premetaphase cells over mock-treated cultures. It must be emphasized that this figure represents a minimum value, given the heterogeneity of the Nup358 siRNAtreated cells. If we had only counted cells overtly depleted of Nup358, this increase would be on the order of 150300%. Simultaneous depletion of both Nup358 and Mad1 yielded a sevenfold decline in the number of metaphase and premetaphase cells (compared with Nup358 depletion alone). Taken together, these results indicate that Nup358 is unlikely to possess a checkpoint function.
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Discussion |
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Although we observed little effect on nuclear protein import in Nup358 siRNAtreated HeLa cells, the effects on mitotic progression were quite dramatic. We noted the emergence of two unusual cell populations: prometaphase cells in which there was a failure in chromosome congression and interphase cells containing multiple micronuclei. The most reasonable explanation for the appearance of these cell populations is a failure of spindle microtubules to capture chromosomes followed eventually by mitotic exit and NE reformation around dispersed chromosomes or groups of chromosomes. In this way, the defective prometaphase cells would represent the precursors of the multinucleate cells. That this is indeed the case is suggested by the increasing numbers of multinucleate telophase and early G1 cells that can be seen in the siRNA-treated cultures. In many anaphase and telophase cells in 4-d siRNA-treated cultures, the presence of lagging chromosomes and chromatin strands spanning the intracellular bridge is yet further indication of congression failure and eventual mitotic exit.
The findings that Nup358 is associated with both spindle microtubules and kinetochores in mitotic cells are consistent with a number of recent studies that have revealed a direct role for components of the Ran system in mitotic spindle assembly. Members of the importin/karyopherin family are also implicated in these processes. A role for Nup358 in chromatid segregation has been highlighted in a recent study on Caenorhabditis elegans early embryos (Askjaer et al., 2002). An RNA interference approach has revealed that depletion of Nup358/RanBP2 leads to inhibition of chromosome congression associated with aberrant spindle morphology, very similar to the situation described here in HeLa cells. Although asters do form, chromosome capture does not occur and bipolar spindles are not seen. Identical effects have also been observed in C. elegans embryos that have been depleted of CENP-A, a protein required for normal kinetochore formation (Oegema et al., 2001). These observations confirm a role for kinetochores in spindle organization and indicate that Nup358 depletion could be interfering with either kinetochore or spindle microtubule function, or indeed both. Clearly, defects in either of these structures could in principle give rise to the types of aberrations in chromatid segregation that both we and Askjaer et al. (2002) have observed.
Taken together, our data suggest that the primary effects of Nup358 depletion on spindle assembly and function are operating at the level of kinetochore formation and maturation. EM studies of Nup358-depleted prometaphase cells reveal aberrant kinetochore morphology that features partial or complete loss of the trilaminar plate structure as well as incomplete condensation of subjacent centromeric heterochromatin. The C-shaped kinetochore morphology has also been reported after premature chromatin condensation in cell fusion experiments (Rattner and Wang, 1992), as well as after exposure to caffeine (Brinkley et al., 1988), whereas the expanded morphology is characteristic of prekinetochores (He and Brinkley, 1996). Of particular significance is our finding that certain kinetochore components, including the checkpoint proteins Mad1, Mad2, and Zw10, are mislocalized in prometaphase cells depleted of Nup358. Studies by Chan et al. (2000) using an antibody microinjection strategy have demonstrated quite convincingly that interference with one of these, Zw10, leads to bypass of the spindle assembly checkpoint, appearance of lagging chromatids, and aneuploidy (Chan et al., 2000). More recently, Yao et al. (2000) have shown that depletion of CENP-E in mammalian cells gives rise to a spectrum of anomalies that is virtually identical to what we have reported in this paper. This is consistent with our own finding that CENP-E is mislocalized in Nup358-depleted cells. Very similar defects have also been reported in studies involving depletion of several other kinetochore proteins, including Hec1, hMis12, and Drosophila Mast/Orbit (Maiato et al., 2002; Martin-Lluesma et al., 2002; Goshima et al., 2003).
The finding that depletion of Nup358 perturbs kinetochore structure and interferes with microtubule binding suggests that Nup358 has an important function in the assembly of the kinetochore. Such a role for Nup358 might be related to its ability to attract and bind other proteins of the Ran system. Indeed, RanGap1 and SUMO-I have both been found at the kinetochore (Joseph et al., 2002). Given that SUMO-modified RanGAP1 binds Nup358 at the NPC, it is tempting to imagine that Nup358 performs a similar function at the kinetochore during mitosis. In fact, a population of Ran is found at the kinetochore during mitosis while its nucleotide exchange factor, RCC1, remains chromatin associated (Moore et al., 2002). In C. elegans, depletion of RCC1 by RNA interference produces effects similar, although less severe, to those observed after Nup358 depletion (Askjaer et al., 2002). As Ran has been shown to be essential for kinetochoremicrotubule interaction, these various Ran system components could well function to modulate the cycling of proteins on and off the kinetochore. Interference with one branch of the Ran system might then result in the structural and compositional defects detected in our study. Such a view is lent considerable support by the recent findings of Arnaoutov and Dasso (2003) on the critical role of the Ran GTPase in kinetochore function. A second possibility is that since Nup358 is a SUMO ligase (Pichler et al., 2002), this activity might be required for proper kinetochore organization and function. In this regard, it is intriguing that SUMO-1 can act as a suppressor of certain CENP-C mutations in vertebrate cells (Fukagawa et al., 2001).
Our findings clearly indicate that Nup358 has an important role in the recruitment of kinetochore proteins, including those involved in the spindle assembly checkpoint. However, Nup358 itself appears unlikely to be a checkpoint protein per se. Rather, our data suggest that the spindle assembly checkpoint remains substantially intact in cells depleted of Nup358, and that such cells display only a relatively slow escape from the mitotic arrest. This escape could, however, be accelerated by codepletion of Mad1. Conversely, nocodazole treatment of both mock- and Nup358-depleted cells yielded little difference in the numbers of cells arrested in prometaphase (unpublished data). Given the substantial, albeit incomplete, mislocalization of kinetochore-associated proteins in cells depleted of Nup358, these results involving Mad1 codepletion are consistent with the notion that checkpoint complexes may remain functional at other cytoplasmic sites.
Could Nup358 depletion be having an indirect effect on kinetochore function? We know that Nup358 is part of the nucleocytoplasmic transport machinery. It is formally possible, therefore, that the mitotic defects we have observed could be a consequence of failure to import crucial kinetochore components into the nucleus in late G2. This scenario, however, seems very unlikely. All the evidence that we and others have available suggests that nuclear protein import is not seriously perturbed by Nup358 depletion (Walther et al., 2002). Indeed, if it were, we would expect cells to arrest in interphase and not to enter mitosis. Given its localization during mitosis, the most reasonable model remains that Nup358 is actually functional at the kinetochore.
Why should NPC or NE components play any role at all during mitosis? Joseph et al. (2002) have made the interesting proposal that the reciprocal relationship between the NPC and the mitotic spindle, represented by the cycling of proteins between these two structures, provides a fail-safe signal that defines the interphase versus mitotic status of the cell. Our data on Nup358 would suggest that this idea can also be extended to the kinetochore, which can only become functional once NEBD, disassembly of NPCs, and transfer of some NPC components to the kinetochores has commenced. In this way, an orderly and stepwise progression of mitotic events is ensured. This relationship between the NPC (or at least the NE) and the kinetochore may have its roots in the evolutionary history of these two structures. Many primitive cell types that undergo a closed mitosis, dinoflagellates for example, have well-differentiated kinetochores that remain closely associated with the nuclear surface of the NE (Kubai, 1975; Ris, 1975). In Trichonympha agilis, spindle microtubules, which are exclusively cytoplasmic, make contact not with the kinetochore itself, but with the patch of nuclear membrane that overlies the kinetochore (Kubai, 1975). In this way, chromosome segregation, although driven by the mitotic spindle, is actually mediated by NE components. It is possible that this mechanism has been conserved in organisms that have evolved an open mitosis such that disassembled NE components still maintain their ancient role.
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Materials and methods |
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Antibodies
The following antibodies were used in this study. Antibodies against LAP 2ß and Nup358 were obtained from L. Gerace (Scripps Research Institute, La Jolla, CA). An additional antibody against Nup358 was also obtained from T. Nishimoto (Kyushu University, Fukuoka-shi, Japan). AntiCENP-E, -Zw10, and -Mad1/2 antibodies were obtained from G. Chan (University of Alberta, Alberta, Canada). Antibodies against Mad1 were also provided by T. Yen (Fox Chase Cancer Center, Philadelphia, PA). The antibody against importin ß was obtained from D. Gorlich (ZMBH, Heidelberg, Germany). Antibodies against various nucleoporins (QE5), including Nup153 (SA1) and Nup214, have been described elsewhere (Panté et al., 1994; Bodoor et al., 1999). The antibody against CENP-F as well as the human autoimmune anticentromere antibody (ACA) have also been described previously (Kingwell and Rattner, 1987; Liao et al., 1995). The antiß-tubulin antibody was obtained from Sigma-Aldrich. The monoclonal antibody 74.1 against dynein intermediate chain was obtained from BabCo. Antiß-galactocidase antibody was obtained from Promega. Secondary antibodies were from Biosource International.
Immunofluorescence microscopy
HeLa cells were grown on glass coverslips and fixed in either 100% methanol at -20°C or 3% paraformaldehyde for 10 min followed by a 5-min permeabilization with 0.5% Triton X-100. The cells were then labeled with the appropriate antibodies plus the DNA-specific Hoechst dye 33258. For the experiment in Fig. 6, the cells were preextracted with 0.005% digitonin and later fixed in -20°C methanol exactly as previously described (Joseph et al., 2002). Specimens were observed using a Carl Zeiss MicroImaging, Inc. Axiophot microscope. Images were collected using a Photometrics CoolSnap HQ CCD camera linked to an Apple Macintosh G4 computer running IP Lab Spectrum software (Spectrum Analytics, Inc.). Fluorescence intensity measurements were performed using IP Lab (Salina et al., 2002).
EM
Cells grown and treated in 35-mm Petri dishes were fixed in 3% glutaraldehyde and 0.2% tannic acid in 200 mM sodium cacodylate buffer for 1 h at room temperature. Postfixation was in 2% OsO4 for 20 min. The cells were dehydrated in ethanol, lifted from the culture dish using propylene oxide, and then infiltrated with Polybed 812 resin. Polymerization was performed at 60°C for 24 h. Silver-gray sections were cut using a Leica ultramicrotome equipped with a diamond knife. The sections were stained with uranyl acetate and lead citrate and examined in a JEOL JEM-100CXII electron microscope.
In vivo nuclear import assay
To examine the effects of Nup358 depletion on nuclear protein import, HeLa cells, grown on glass coverslips, were exposed to Nup358 siRNA for 3 d. At this time, the cells were transfected with an expression plasmid encoding GRß (Picard and Yamamoto, 1987). Transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer's recommendations. 24 h after transfection, dexamethasone was added to the medium to a final concentration of 10 µg/ml. The cells were returned to the incubator for a period of up to 30 min and then fixed with 3% formaldehyde in PBS. Finally, The cells were processed for immunofluorescence microscopy using antibodies against both ß-galactosidase and Nup358.
siRNA methods
HeLa cells were depleted of Nup358 using siRNA corresponding to nucleotides 76327654 of human Nup358 (Dharmacon). The cells were exposed to the Nup358 siRNA in the presence of Oligofectamine (Invitrogen) precisely as described by Harborth et al. (2001). As a control, cells were exposed either to Oligofectamine alone, to Nup153 siRNA, or to an ineffective RNA duplex. For Mad1 depletion, the procedures and oligonucleotide sequences described by Martin-Lluesma et al. (2002) were followed precisely. In codepletion experiments, cells were first exposed to Nup358 siRNA. After 48 h, a combination of both Mad1 and Nup358 siRNAs was introduced to the cells. After an additional 48 h incubation, the cells were fixed and processed for immunofluorescence microscopy.
Immunoblotting and gel electrophoresis
Cells (siRNA or mock treated) grown in 35-mm tissue culture dishes were washed once in PBS and then lysed in a buffer containing 50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.5% Triton X-100, 1 mM DTT, 1 mM PMSF, and 1:1,000 CLAP (10 mg/ml in DMSO of each of the following: chymostatin, leupeptin, antipain, and pepstatin). The lysate was centrifuged for 5 min in an Eppendorf centrifuge at 4°C. Proteins in the supernatant were precipitated by the addition of TCA to a final concentration of 10%. The precipitate was washed with ethanol/ether and then solubilized in sodium dodecyl sulfatePAGE sample buffer. Protein samples were fractionated on 8% polyacrylamide gels and then transferred onto nitrocellulose filters, usually BA85 from Schleicher & Schuell (Burnette, 1981), using a semi-dry blotting apparatus manufactured by Hoeffer Scientific Instruments, Inc. Filters were blocked, labeled with primary antibodies, and then developed with peroxidase-conjugated secondary antibodies exactly as previously described (Burke et al., 1982).
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health to B. Burke. D. Salina was supported by studentships from the Alberta Heritage Foundation for Medical Research, University Technologies International, and the National Science and Engineering Research Council of Canada (NSERC). J.B. Rattner is supported by grants from the Canadian Institutes of Heath Research, NSERC, and the Arthritis Society.
Submitted: 15 April 2003
Accepted: 28 July 2003
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