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Vol. 20, Issue 6, 1652-1660, March 15, 2009
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Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112
Submitted August 28, 2008;
Revised November 25, 2008;
Accepted January 9, 2009
Monitoring Editor: Sandra L. Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Approximately 30 proteins (termed nucleoporins), each present in multiple copies, comprise this macromolecular structure (Hetzer et al., 2005), providing a vital link between intracellular environments. In higher eukaryotes, once chromosomes undergo condensation, nuclear membranes and nuclear pore complexes are disassembled. A series of hallmark mitotic events ensue, including segregation of chromosomal content, rapid rebuilding of nuclei and, finally, abscission near the midbody, a structure that connects the daughter cells until cytokinesis is complete. Nucleoporins were originally thought to be stored inertly once released from the NPC at the entry into mitosis until once again incorporated into pores during late mitosis; however, recent evidence suggests that certain nucleoporins perform important mitotic functions independently of their roles during interphase. For example, the Nup107-160 complex as well as Nup358 (RanBP2) localize in part to kinetochores after their release from the NPC, and they function in chromosome congression (Zuccolo et al., 2007), maintenance of tension between sister kinetochores (Zuccolo et al., 2007), and sister chromatid segregation (Salina et al., 2003; Joseph et al., 2004; Dawlaty et al., 2008). Other mitotic functions of nucleoporins include roles in proper spindle assembly (Nup107-160 complex; Orjalo et al., 2006) and anaphase onset via regulation of APC/C (Nup98; Jeganathan et al., 2005).
Disassembly of the NPC occurs in coordination with nuclear membrane dispersal (Burke and Ellenberg, 2002; Hetzer et al., 2005; Prunuske and Ullman, 2006; Antonin et al., 2008); thus, the availability of nucleoporins for mitotic roles coincides with nuclear remodeling events that are important prerequisites for chromosome dynamics in prometaphase and metaphase. Before full dispersal, the NPC itself is remodeled (Lenart et al., 2003), suggesting that nucleoporins could also take on new roles during this brief window of transition. Indeed, distinct cell cycle-driven functions for nucleoporins can couple morphological remodeling of the nucleus and NPC with regulation of chromosomal inheritance and other mitotic processes. The precedents to date indicate that understanding the role of individual nucleoporins throughout the cell cycle is important to deciphering the network of interactions that orchestrate accurate cell division in mammalian cells.
Studies performed in Xenopus egg extracts, a system that recapitulates embryonic cell division, have implicated Nup153 in the process of nuclear envelope breakdown (Liu et al., 2003; Prunuske et al., 2006), indicating that this nucleoporin plays a specialized role at mitosis. Although mammalian Nup153 has been suggested to be essential for cell survival (Harborth et al., 2001), previous studies in which it was targeted for small interfering RNA (siRNA)-mediated depletion in cultured cells (Harborth et al., 2001; Hase and Cordes, 2003; Sabri et al., 2007) did not address a role for Nup153 in proper mitotic execution. More recently, Nup153 was identified in an unbiased genome-wide screen for proteins with roles in early mitosis (Rines et al., 2008). Defects in the absence of Nup153 were not characterized in detail, but the discovery of Nup153 in this context suggests a link to mitosis in mammalian cells. To gain insight into the mitotic role of Nup153, we have used graded levels of depletion to reveal functions of this nucleoporin that become impaired at different thresholds of expression. Additionally, using a rescue strategy, we have found that Nup153 plays distinct roles that impact both early and late stages of mitosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), 153-2 (GGACUUGUUAGAUCUAGUU), and a scrambled version of 153-1, Scr-1 (GCAAAUCUCCGAUCGUAGA), which was used as a control treatment.
Cell Culture, RNA Interference (RNAi) Depletion of Nup153, and Stable Cell Line Generation
HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). Transfection with siRNA was achieved using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, except that oligo 153-1 was used at either 0.17 nM (153-1lo) or 10 nM, whereas 153-2 and Scr-1 oligos were used at 10 nM. Cells were harvested 48 h after transfection for further analysis. Stable cell lines were generated by transfecting the Nup153 constructs into HeLa cells by using Lipofectamine LTX (Invitrogen). Twenty-four hours after transfection, cells were split 1:30 and incubated with 1 mg/ml Geneticin (Invitrogen) for 10 d, after which single colonies were purified and expanded. The histone H2B-CFP cell line was generated in a similar manner, except that individual clones were not isolated.
Immunoblotting and Immunofluorescence
Whole cell extracts were prepared by cell lysis in radioimmunoprecipitation assay buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and clarified by centrifugation. Samples were resolved by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, probed with the appropriate antibodies, and developed using the Western Lightning Chemiluminescence Reagent (PerkinElmer Life and Analytical Sciences, Boston, MA). For immunofluorescence analysis, cells were fixed either in methanol for 5 min at –20°C or in 4% paraformaldehyde for 10 min on ice. In the latter case, cells were then permeabilized by 5-min incubation in 0.5% Triton X-100 in phosphate-buffered saline (PBS) on ice. Samples were blocked by incubation in 3% bovine serum albumin, 0.02% Triton X-100 (in PBS), and then incubated with the appropriate antibodies. DNA was detected by incubation with 0.2 µg/ml Hoechst 33258 (Calbiochem, San Diego, CA) during the final wash. Coverslips were mounted using ProLong Gold anti-fade (Invitrogen).
Antibodies used in this study include anti-nucleoporins (mAb414; Covance Research Products, Princeton, NJ), anti-
-tubulin (YL1/2; Accurate Chemical & Scientific, Westbury, NY), anti-green fluorescent protein (GFP) (JL-8; Clontech, Mountain View, CA; and ab290, Abcam, Cambridge, MA), anti-Nup153-Z (Liu et al., 2003), anti-Nup153 (SA-1; a gift from Brian Burke, University of Florida, Gainesville, FL), anti-lamin B2 (LN43; Abcam), anti-CENP-E (1H12; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Nup133 (gift from D. Forbes, University of California, San Diego, La Jolla, CA), and anti-Nup62 (BD Biosciences, San Jose, CA). All secondary antibodies were obtained from Invitrogen.
Nuclear Transport and mRNA Export Assays
Twenty-four hours after siRNA treatment, HeLa cells grown in DMEM + 10% charcoal-stripped FBS were transfected (Lipofectamine LTX; Invitrogen) with a plasmid encoding a chimeric Rev-GFP-Glucocorticoid Receptor protein (RGG; Love et al., 1998; provided by M. Dasso), which can be induced to import into the nucleus upon addition of dexamethasone and to export to the cytoplasm upon removal of dexamethasone. An additional 24 h later, cells were treated with 250 nM dexamethasone and fixed at the indicated times. For protein export assays, cells treated for 120 min with 250 nM dexamethasone were washed and incubated in fresh culture medium for the indicated times. Export of poly(A)+ RNA was assessed by fluorescence in situ hybridization by using a biotinylated oligo(dT) 50mer probe as described previously (Bastos et al., 1996).
Live Cell Time-Lapse Imaging and Analysis
Forty-eight hours after siRNA transfection, cells stably expressing histone H2B-CFP were imaged in 24-well plastic dishes in a stage-top incubator (OKO Lab, Ottaviano, Italy), which maintained appropriate temperature, humidity, and CO2 levels. Images (CFP and phase contrast) were acquired every 6–10 min from five to 15 different stage positions for 15–45 h by using a 40x PLANAPO numerical aperture 0.9 objective (with correction collar) on an Olympus IX81 microscope equipped with a motorized XY stage and a Hamamatsu Orca ER camera, all controlled using the MetaMorph software (Molecular Devices, Sunnyvale, CA). Time-lapse image stacks were converted to QuickTime movies by using MatLab-based software developed at the University of Utah Fluorescence Microscopy Core Facility (Salt Lake City, UT). Statistical analysis was performed using the GraphPad Prism software (GraphPad Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30%) with severely multilobed nuclei (Figure 1G), whereas the number of midbodies was unaffected (Figure 1F). The distinctive appearance of the nuclei was readily observed by tracking either DNA (Figure 1D) or the nuclear lamina (Figure 1E). Although this nuclear morphology is somewhat reminiscent of apoptosis, Nup153-depleted cells showed no increase in activated caspase-3 (data not shown) and displayed viability over time in live imaging (Supplemental Video 4). Thus, these alterations in nuclear architecture likely reflect a role for Nup153 directly in forming nuclei or in influencing chromosomal dynamics during mitosis; deregulation of the latter process has previously been found to result in a very similar nuclear appearance (Draviam et al., 2007; Stegmeier et al., 2007; Ohsugi et al., 2008).
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). The results of Nup153 depletion in HeLaGFP cells mirrored those obtained with the parental HeLa cells: a three- to fourfold increase in the number of unresolved midbodies upon reduction of Nup153 and a dramatic increase in the number of multilobed nuclei upon further depletion (Figure 2, C and D). In contrast, when HeLa153GFP cells were transfected with siRNA oligonucleotides directed against endogenous Nup153, very little increase in either midbodies or multilobed nuclei was observed (Figure 2, C and D), indicating that these phenotypes result specifically from alterations in the level of Nup153.
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; Walther et al., 2001; Sabri et al., 2007), we next assessed nuclear import and export in Nup153-depleted cells. siRNA-treated cells were transfected with a plasmid encoding a nucleocytoplasmic transport reporter (RGG) (Love et al., 1998). Treatment with dexamethasone induces RGG to translocate to the nucleus and concentrate in nucleoli, and only a subtle delay in protein import was seen between control and Nup153-depleted cells (Figure 3A). After removal of dexamethasone, efficient protein export was observed in both treatments (Figure 3B).
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Live Imaging Reveals Distinct Effects on Cell Cycle Timing That Correspond to the Level of Nup153 Depletion
To gain additional insight into the mitotic roles of Nup153, we performed time-lapse imaging of cells expressing histone H2B-CFP. Lowering the levels of Nup153 prolonged the total duration of mitosis, from an average time of 86 min (Scr-1) to 106 or 112 min (153-2 and 153-1lo, respectively; Figure 4A, Supplemental Table 1, and Supplemental Videos 1–3). Not only was the time in mitosis extended upon Nup153 reduction, but there was notably higher variation in mitotic timing, consistent with miscoordination of mitotic progression (Meraldi et al., 2004). Live imaging also revealed an increase in cortical blebbing during interphase. However, this was much more pronounced with 153-1lo treatment compared with 153-2 (Supplemental Videos 2 and 3), indicating that blebbing activity at the cell periphery does not correlate with changes in mitotic timing. We further analyzed hallmark events during mitosis to determine whether the mitotic delay was due to an overall lengthening of mitosis or whether it was specific to certain mitotic stages. Very little difference was found between control and Nup153-depleted cells during prophase/prometaphase (Figure 4B) or metaphase (Figure 4C). Progression from anaphase through cytokinesis, however, was markedly prolonged in Nup153-depleted cells (153-1lo and 153-2; Figure 4D; Supplemental Table 1).
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; Salina et al., 2002; Loiodice et al., 2004). Furthermore, despite the delays observed in midbody resolution when Nup153 levels are reduced, this nucleoporin was not detected at midbodies (Figure 6). Although we did not find evidence for kinetochore or midbody targeting of Nup153, it is known to be very dynamically associated with the NPC (Daigle et al., 2001; Griffis et al., 2004) and could likewise dynamically associate with these mitotic structures, perhaps escaping detection.
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). At these expression levels, which did not exceed the endogenous amount of Nup153, the N+Z, N+C, and N constructs alone did not dominantly affect the number of midbodies or multilobed nuclei (Figure 7, C and D; data not shown). Upon reduction of endogenous Nup153 in HeLaN+Z and HeLaN cells, an increase in midbodies similar to that seen when Nup153 was reduced in HeLaGFP cells was observed (compare Figure 7C with Figure 2C). In contrast, an increase in midbodies was not observed after reducing endogenous Nup153 in HeLaN+C cells (Figure 7C), indicating that this late mitotic phenotype is complemented only if the rescue construct includes the Nup153 C-terminal region.
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; Prunuske et al., 2006). However, expression of the N-terminal domain of Nup153 was found to rescue the multilobed nuclei phenotype (Figure 7D). This pattern of complementation indicates that robust depletion of Nup153 results in mitotic defects that are more complex than can be resolved by our current strategy, either involving interplay between domains and/or a balance of more than one Nup153 activity. Yet, a clear point revealed by this rescue strategy is that the phenotypes resulting from graded Nup153 depletion are complemented by different regions of this protein and thus reflect at least two separable roles for Nup153 in mitosis.
Domain Analysis of Nup153 Function
A role for the Nup153 zinc-finger domain in mitotic events is underscored by the observation that ectopic expression of this domain in the HeLaZ cell line results in a significant presence (10%) of multinucleated cells (Figure 8), which likely indicates failure to initiate or complete cytokinesis. Interestingly, the zinc finger domain itself was also found to be sufficient to rescue the multilobed nuclei phenotype (Figure 9B). Further work will be needed to investigate how these activities are related.
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; Ben-Efraim and Gerace, 2001; Ullman and Higa, 2008), our results indicate that protein import and export are not significantly perturbed under these conditions of depletion (Figure 3). Because importin-β is also an important mitotic regulator (Harel and Forbes, 2004), we considered whether the Nup153 FG-domain exerts its effects during late mitosis by controlling the availability or activity of either importin-β or other potential mitotic partners. If so, one would predict that the presence of the FG-domain itself would supply the essential function of Nup153. Midbodies remained significantly increased, however, in HeLaC cells after endogenous Nup153 reduction (Figure 9A). Thus, an activity associated with the N-terminal domain works in conjunction with the C-terminal region of Nup153 to contribute to mitotic exit.
One major difference between the Nup153 C construct versus N+C is subcellular localization, because the NPC targeting region is located within the N-terminal domain (amino acids 39-339; Enarson et al., 1998). To determine whether delivery to the NPC is the critical function conferred by the Nup153 N-terminal domain in the context of this late mitotic role, we assessed whether the midbody phenotype could be rescued by expression of an N1-339+C construct. As expected, this construct localized to the nuclear rim in stably transfected cells and was expressed in similar amounts as the N+C construct (Figure 9, C and D). When endogenous Nup153 was depleted to levels that give rise to an increase in midbodies (153-1lo and 153-2), expression of the N1-339+C construct rescued this phenotype somewhat more robustly than the C-terminal domain alone (Figure 9D), consistent with a role for NPC targeting. However, the number of midbodies in Nup153 depleted HeLaN1-339+C remained higher than in the control (Scr-1) treatment of this same cell line (Figure 9D). The reproducible persistence of a late mitotic defect suggests that NPC targeting is not the sole function conferred by the N-terminal domain.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It is notable that even in the case where Nup153 depletion was found to result in deficient NLS-mediated import in Drosophila S2 cells, the reduction measured was significant, but not acute (25%) (Sabri et al., 2007). The exact effect on transport may depend on the extent of Nup153 reduction or the method by which its activity is impaired. Overall, transport is a very robust process that can proceed with relative efficiency even when changes in pore composition are introduced (Stavru et al., 2006; Dawlaty et al., 2008).
Although our results do not link the mitotic functions of Nup153 to a role in bulk transport of protein or mRNA, certain observations raise the question of whether a more specific alteration in nucleocytoplasmic trafficking underlies the accumulation of unresolved midbodies when Nup153 levels are reduced. The C-terminal region of Nup153 implicated in our rescue analysis of this phenotype houses a highly repetitive motif consisting of (FX)FG-repeats known to aide in mediating transport (Terry et al., 2007). Furthermore, the finding that the N-terminal region of Nup153 is needed in conjunction with C-terminal sequences indicates that this FG-rich domain does not carry out a mitotic role simply by sequestering and down-regulating a mitotic factor as cells progress to G1. This suggests that the N-terminal domain may be required to direct the C-terminal domain of Nup153 to the pore for this FG-rich region to participate in the transport of cargo particularly important to mitotic exit and/or cell cycle control. Such a role was recently identified for Nup96 (Chakraborty et al., 2008). To test this, the pore targeting region (amino acids 1-339) of Nup153 was linked directly to its C-terminal domain. In comparison to the C-terminal domain alone, however, this construct provided only a modest improvement in complementing the late mitotic phenotype. Although expression of N+C resulted in midbody numbers that were not significantly different in control versus Nup153 depletion, expression of N1-339+C did not fully attenuate the increase in midbodies that arise during Nup153 depletion. This indicates that pore targeting is not the sole function of the N-terminal domain and suggests that a transport role—at least one directed by the FG-rich domain—does not account for the activity of Nup153 in late mitosis. These results also implicate additional contributions made by the distal half of the N-terminal domain of Nup153.
While this manuscript was in preparation, a study was published in which Tpr, another component of the NPC, was found to bind directly to both Mad1 and Mad2 and to play a role in proper spindle checkpoint activation (Lee et al., 2008). The NPC localization of Tpr is known to be directed by its interaction with Nup153 (specifically amino acids 228-439 of the Nup153 N-terminal domain; Hase and Cordes, 2003). Tpr is an excellent candidate for collaborating with Nup153 in mitotic function, but it is important to note that loss of Tpr from the NPC, per se, would not account for the phenotypes observed upon Nup153 depletion. First, Tpr depletion was not found to have an effect on mitotic timing, whereas Nup153 levels were found critical for timely progression through both early and late mitosis. Second, although expression of a Nup153 construct lacking the C terminus has been shown previously to be sufficient to restore Tpr localization to the NPC (Sabri et al., 2007), here we found that expression of this construct does not rescue the increase in midbodies seen after Nup153 depletion. Although the phenotypes and rescue patterns observed in this study point toward contributions of Nup153 that are independent of Tpr, these two components of the nuclear pore basket are likely to participate in concert to provide a newly-emerging layer in mitotic regulation. The pore basket may act as a scaffold on which mitotic factors are sequestered and regulated. In addition to Mad1 and Mad2, the SUMO protease Ulp1p, required for late mitosis in yeast, is localized to the nuclear pore during interphase (Makhnevych et al., 2007). It is interesting to note that the mammalian homologue of Ulp1p, SENP2, also localizes to the nuclear pore and has been found to associate with the C-terminal domain of Nup153 (Hang and Dasso, 2002; Zhang et al., 2002).
Additional observations indicate that a subset of nucleoporins converge in a regulatory pathway during late mitosis and/or mitotic exit. Depletion of either Nup133, a member of the Nup107-160 complex, or ELYS, a protein associated with this complex, also results in accumulation of unresolved midbodies (Rasala et al., 2006). However, Nup153 is the only FG-containing nucleoporin so far identified to impinge on this stage of the cell cycle. Our finding that the FG-domain is required for rescue of midbody accumulation suggests that Nup153 contributes in a unique manner to this regulatory network.
When Nup153 levels are further depleted, many cells display abnormal, multilobed nuclei. This phenotype could reflect a direct role for Nup153 in forming nuclear structure. Alternatively, disruption of proper chromosome dynamics during mitosis is known to result in a similar nuclear morphology and indeed has been the basis of a screen for mitotic regulators (Draviam et al., 2007; Stegmeier et al., 2007; Ohsugi et al., 2008). We found in live imaging analysis that significant delays occur early in mitosis under these conditions of Nup153 depletion. This supports a model in which Nup153 function impacts progression out of prophase and/or chromosome segregation itself, and the miscoordination that follows results in aberrant nuclear morphology. More extensive imaging will be needed to distinguish where the defect in early mitosis lies more precisely. Further investigation into whether Nup153 has a separable role in assembly of proper nuclear architecture is also warranted.
In summary, we have found that two distinct phenotypes result from depleting Nup153 to different levels and, further, that rescue of these phenotypes requires distinct regions within Nup153. This graded response to disruption of Nup153 expression likely reflects different thresholds at which Nup153 becomes limiting for one or more processes. Taken together, our results indicate that Nup153 has at least two functions important for proper mammalian cell division and that the FG-rich region of Nup153 plays an important role at mitosis. Given the complicated multifunctional nature of nucleoporins, the strategy we have developed here, combining graded depletion and domain rescue, will aid in further elucidating how nucleoporins contribute to mitosis.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Katharine S. Ullman (katharine.ullman{at}hci.utah.edu)
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