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J. Biol. Chem., Vol. 279, Issue 13, 13076-13085, March 26, 2004
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From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
Received for publication, September 15, 2003 , and in revised form, December 22, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Kinetochores also play important roles in the spindle checkpoint (3). Premature separation of sister chromatids prior to their proper attachment to the mitotic spindle results in chromosome missegregation and aneuploidy, contributing to tumorigenesis or birth defects (4, 5). To avoid these dire consequences, cells employ a surveillance mechanism called the spindle checkpoint to monitor the status of microtubule attachment and tension at the kinetochores of sister chromatids (6-11). The spindle checkpoint ensures that chromosome segregation ensues only after all the chromosomes have congressed to the metaphase plate. Unattached kinetochores generate a diffusible signal that inhibits a ubiquitin-protein isopeptide ligase called the anaphase-promoting complex or cyclosome (APC/C)1 and in turn prevents sister chromatid separation (6-13). It has been postulated that the APC/C inhibitory signal generated by unattached kinetochores consists of a complex of MAD2, CDC20, BUBR1 and BUB3, known as the mitotic checkpoint complex (11). Many checkpoint proteins exhibit dynamic kinetochore localization in mitosis (14, 15). For example, MAD2 localizes only to unattached kinetochores, and the kinetochore-bound pool of MAD2 exchanges rapidly (t1/2 = 24-28 s) with the cytoplasmic pools (14). MAD2 recruitment to kinetochores depends upon another checkpoint protein, called MAD1, which is constitutively bound to MAD2 and shows the same kinetochore localization pattern as MAD2 (16-18). An attractive model for spindle checkpoint signaling is that MAD2 is recruited to kinetochores by MAD1 (11, 19). Through a poorly understood mechanism, possibly mediated by other checkpoint proteins, MAD2 dissociates from MAD1 and acquires a conformation that is compatible with its incorporation into the mitotic checkpoint complex (11, 19). Little is known about the mechanisms by which the spindle checkpoint proteins are recruited to kinetochores during mitosis. Detailed immunofluorescence analysis revealed that these proteins are recruited to the kinetochores with slightly different timing (20). Moreover, their kinetochore localization patterns vary in response to different spindle-damaging agents (21-23). This suggests that the spindle checkpoint proteins might be recruited to the kinetochores through different mechanisms and via interactions with specific kinetochore proteins. Identification of the kinetochore protein interface to which MAD1-MAD2 associates might provide insights into the mechanism of the generation of the mitotic checkpoint complex.
Genetic and biochemical studies in different organisms have led to the identification of a large number of kinetochore proteins, many of which are conserved during evolution (3). A physical association map for the kinetochore proteins is beginning to emerge through biochemical purification of kinetochore subcomplexes and through analysis of the interdependencies of various proteins for kinetochore localization (3, 24, 25). Despite the vast divergence of the centromeric sequences among organisms and the limited sequence similarities among kinetochore proteins of different species, the basic architecture of kinetochores appears to be conserved (3). Kinetochore proteins can be divided into three groups: the inner kinetochore proteins that associate with the centromeric DNA, the outer kinetochore proteins that interact with the microtubules, and the central kinetochore proteins that lie in between (3). The inner kinetochore is formed in part by a histone H3 variant, CENP-A/Cse4, and a coiled-coil protein, CENP-C/Mif2 (3). The central kinetochore consists of at least three subcomplexes in yeast: the Ndc80 complex, the Mtw1 complex, and the Ctf19 complex (3, 26). The central kinetochore proteins are responsible for the recruitment of the outer kinetochore proteins that, among other functions, regulate microtubule dynamics (3). These include microtubule motors, such as dynein and CENP-E, and non-motor proteins, such as CLIP-170 and the Dam1 complex (3, 27). Nearly all of the spindle checkpoint proteins localize to the outer kinetochore (3). Many central kinetochore proteins, including components of the Ndc80, Mis12/Mtw1, and Ctf19 complexes, remain to be identified in mammals. Identification of these components will shed light on the structure, assembly, and function of mammalian kinetochores.
The yeast Ndc80 complex consists of four coiled-coil proteins: Ndc80, Nuf2, Spc25, and Spc24 (28). Homologs of Ndc80 and Nuf2 have been identified in various organisms and shown to perform important functions in processes including kinetochore assembly, microtubule attachment, chromosome congression, and the spindle checkpoint (29-32). This suggests that the Ndc80 complex is an evolutionarily conserved kinetochore component. Several observations support a role of the Ndc80 complex in the spindle checkpoint. In budding yeast, temperature-sensitive SPC25 and SPC24 mutants are checkpoint-deficient (31). Simultaneous disruption of both NDC80 and NUF2 genes also abrogates the spindle checkpoint (31). Recently, human MAD1 has been shown to interact with HEC1 in a yeast two-hybrid screen (29). MAD1 and MAD2 fail to localize to kinetochores in mitotic HeLa cells depleted of HEC1 by RNA interference (RNAi), suggesting that HEC1 is required for the kinetochore recruitment of MAD1 and MAD2 (29). However, direct binding between HEC1 and MAD1 cannot be detected in vitro.
Given the importance of the NDC80 complex in both chromosome congression and the spindle checkpoint, we sought to characterize the composition and function of the human NDC80 complex. In particular, despite their likely existence, the mammalian functional homologs of yeast Spc25 and Spc24 have not been identified due to the lack of clear sequence homologs in the data bases. We have now purified the human NDC80 complex using an immunoaffinity approach and identified the SPC25 and SPC24 components of the human NDC80 complex. Human SPC25 (hSPC25) interacted with HEC1 in vitro and in vivo. It localized to the kinetochores during mitosis. RNAi-mediated depletion of hSPC25 in HeLa cells caused a plethora of mitotic defects and the loss of kinetochore localization of MAD1 and HEC1 in the absence of spindle-damaging agents. Therefore, despite the lack of significant sequence similarity, hSPC25 is a functional homolog of yeast Spc25 and a component of the conserved NDC80 complex. Interestingly, the NDC80 complex was not required for the kinetochore localization of MAD1 in nocodazole-arrested mitotic cells. This suggests that the NDC80 complex is not absolutely required for the recruitment of MAD1 to kinetochores upon checkpoint activation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-D-thiogalactopyranoside (250 µM final concentration) to the bacterial culture at A600 = 0.6. The cells were allowed to grow for another 3 h at room temperature. Soluble GST-HEC1 fusion proteins were isolated on glutathione-agarose beads (Amersham Biosciences), eluted with 10 mM glutathione (Sigma), and exchanged into phosphate-buffered saline (PBS) using PD-10 columns (Amersham Biosciences). The two GST-HEC1 fusion proteins were mixed and injected into rabbits for antibody production (Zymed Laboratories Inc.). Crude antibody sera were first precleared with GST-coupled Affi-Gel beads (Bio-Rad) to remove anti-GST antibody and subsequently affinity-purified using either GST-HEC1N or GST-HEC1C fusion protein-coupled Affi-Gel beads. For immunoblotting, affinity-purified anti-HEC1 antibodies were used at a final concentration of 1 µg/ml. For immunoprecipitation, affinity-purified anti-HEC1 antibodies were coupled to Affi-Prep-protein A beads (Bio-Rad) at a concentration of 1 mg of antibody/ml of protein A beads. Immunopurification of the Human NDC80 Complex—HeLa Tet-On cells (Clontech) were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 µg/ml each penicillin and streptomycin. At 80% confluency, the cells were treated with nocodazole at a final concentration of 100 ng/ml for 18 h. For each immunoprecipitation experiment, 20 plates (150 mm) of nocodazole-arrested HeLa cells were used. The cell pellet was resuspended in 12 ml of Nonidet P-40 lysis buffer (50 mM Tris, 150 mM KCl, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1 µM okadaic acid, 10 µg/ml cytochalasin B, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml chymostatin). The suspension was kept on ice for 20 min, followed by brief sonication. The lysate was cleared by centrifugation at top speed in a microcentrifuge for 1.5 h. The supernatant was filtered with 0.4-µm filters and precleared using 200 µl of anti-GST antibody-coupled protein A beads. The NDC80 complex was immunoprecipitated by incubating the lysate with 200 µl of either anti-HEC1N or anti-HEC1C antibody at 4 °C for 2 h. The antibody beads were washed four times with Nonidet P-40 lysis buffer plus 400 mM KCl and once with the lysis buffer. Immunoprecipitated proteins were then eluted with 1 ml of 100 mM glycine HCl (pH 2.5) and concentrated using Microcon centrifugation filter devices (Millipore Corp.). The concentrated proteins were resolved by SDS-PAGE and detected by silver staining. The protein bands were excised and subjected to trypsin digestion, followed by mass spectrometry analysis.
Cloning, Expression, and Purification of Recombinant hSPC25 and Antibody Production—The coding region of hSPC25 was amplified from a human fetal thymus cDNA library (Clontech) by PCR and cloned into the pCS2-Myc and pCS2-HA vectors. The clones were sequenced to verify the identity of hSPC25. A cDNA fragment of hSPC25 containing nucleotides 1-450 of the hSPC25 coding region (hSPC25N) was cloned into the pGEX4T-1 vector. The GST-hSPC25N protein was expressed and purified essentially as described above for the purification of GST-HEC1 fusion proteins, except that expression of GST-hSPC25N was induced by the addition of isopropyl--D-thiogalactopyranoside (250 µM final concentration) to the bacterial culture at A600 = 0.9. The purified GST-hSPC25N fusion protein was injected into rabbits for antibody production (Zymed Laboratories Inc.). Crude antibody sera were first precleared with GST-coupled Affi-Gel beads to remove anti-GST antibody and subsequently affinity-purified using GST-hSPC25N-coupled Affi-Gel beads.
Tissue Culture and Transfection—HeLa Tet-On cells were grown to 40-50% confluency and transfected with the pCS2-Myc or pCS2-HA plasmid containing the HEC1 or hSPC25 coding region using Effectene reagent (QIAGEN Inc.) according to the manufacturer's protocol. After 24 h, cells were treated with 100 ng/ml nocodazole for 16 h, harvested by trypsinization, and pelleted by centrifugation at 500 x g for 5 min. After washing with PBS, the cells were lysed and subjected to immunoprecipitation and immunoblotting experiments.
For RNAi experiments, the siRNA oligonucleotides specific for human HEC1, hSPC25, and human MAD2 were chemically synthesized. They contained sequences corresponding to nucleotides 344-366 and 1517-1539 of the coding region of human HEC1, nucleotides 161-183 of the coding region of hSPC25, and nucleotides 143-165 of the coding region of MAD2, respectively. The annealing of the siRNAs and subsequent transfection of the RNA duplexes into HeLa cells were performed exactly as described (33). In the case of hSPC25, at 48 h after the first round of RNAi transfection, cells were replated to 30% confluency. At 12 h after replating, the cells were transfected again with either hSPC25 siRNA oligonucleotides alone or with both hSPC25 and MAD2 siRNA duplexes. The time points mentioned in this study for these RNAi experiments refer to the time after the second round of RNAi transfection. Cells transfected with OligofectAMINE alone were used as controls. To determine the cell cycle status, the transfected cells were fixed with 70% ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry (FACS). The phenotypes of these cells were also analyzed by indirect immunofluorescence.
Immunoprecipitation, Immunoblotting, and Protein Binding Assays—Lysates of HeLa Tet-On cells transfected with HEC1 and hSPC25 plasmids were prepared and immunoprecipitated as described for the immunopurification of the NDC80 complex. Myc-HEC1 or Myc-hSPC25 was immunoprecipitated from lysates with mouse monoclonal anti-Myc antibody (Roche Applied Science) coupled to protein A beads. After washing, samples were resolved by SDS-PAGE and analyzed by immunoblotting with anti-hemagglutinin (HA) antibody at a final concentration of 1 µg/ml. For analysis of the interactions between endogenous HEC1 and hSPC25 proteins, the anti-HEC1 immunoprecipitates were blotted with anti-hSPC25 antibody at a 1 µg/ml final concentration. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (Amersham Biosciences) were used as a secondary antibody, and the immunoblots were developed using ECL reagent (Amersham Biosciences) following the manufacturer's protocols.
Plasmids encoding Myc-HEC1, human NUF2 (hNUF2), and hSPC25 were cotranslated in rabbit reticulocyte lysate in the presence of [35S]methionine. The lysate was immunoprecipitated using either anti-Myc or anti-GST (as a negative control) antibody. The beads were washed four times with Nonidet P-40 lysis buffer. The immunoprecipitated proteins were dissolved in SDS sample buffer, separated by SDS-PAGE, and analyzed by autoradiography.
Immunofluorescence—For the immunostaining of endogenous HEC1, hSPC25, MAD1, BUB1, and BUBR1, HeLa cells were grown to 40% confluency and transfected with different plasmids or siRNA duplexes on chambered cover slides. Cells were washed with PBS and extracted with 0.5% Triton X-100 in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 4 mM MgSO4) for 5 min at 37 °C. Cells were then fixed with 4% paraformaldehyde in PHEM buffer for 20 min at 37 °C, washed three times with 0.2%Triton X-100 in PBS, and incubated with blocking solution containing 3% bovine serum albumin in PHEM buffer for 1 h. Cells were incubated with primary antibodies in 3% bovine serum albumin in PHEM buffer for 1 h, washed three times with PBS plus 0.2% Triton X-100, and further incubated with fluorescent secondary antibodies (Molecular Probes, Inc.) at 1:500 dilution. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). Cells were washed three times with PBS, mounted, and viewed with a x63 objective on a Zeiss Axiovert 200M fluorescence microscope. Images were acquired with a CCD camera using the Intelligent Imaging software and further processed with Adobe Photoshop. The staining protocol for cyclin A1, cyclin B1, securin, and Myc-hSPC25 was essentially the same as described above, except that cells were not detergent-extracted prior to fixation. The immunostaining of tubulin and poly(ADP-ribose) polymerase-1 (PARP1) in the hSPC25/HEC1 RNAi cells was performed as described above, except that the cells were fixed with methanol at -20 °C. The antibodies used were as follows: mouse monoclonal anti-Myc antibody at 1 µg/ml, CREST serum at 1:1000 dilution (ImmunoVision), rabbit anti-cyclin A1 antibody at 1 µg/ml (Santa Cruz Biotechnology), rabbit anti-cyclin B1 antibody at 1 µg/ml (Santa Cruz Biotechnology), rabbit anti-securin antibody at 1 µg/ml (a gift from Hui Zou), mouse monoclonal anti-PARP1 antibody at 1:1000 dilution (Cell Signaling), rabbit anti-MAD1 antibody at 4 µg/ml, rabbit anti-HEC1 antibody at 2 µg/ml, rabbit anti-BUB1 antibody at 1 µg/ml, rabbit anti-BUBR1 antibody at 1 µg/ml, and mouse anti-tubulin antibody at 1:1000 dilution. Annexin V staining was performed on HEC1 RNAi cells that were released from thymidine arrest for 16 h using a fluorescein isothiocyanate-conjugated annexin V apoptosis detection kit (Pharmingen) according to the manufacturer's protocols. Briefly, cells were washed twice with annexin V binding buffer and incubated with fluorescein isothiocyanate-conjugated annexin V and PI for 15 min. Apoptotic cells were scored by positive annexin V staining and negative PI staining.
Cold- or Calcium-sensitive Microtubule Staining—For cold-sensitive microtubule staining, at 48 h after hSPC25 siRNA treatment, cells were incubated with ice-cold medium for 10 min, followed by extraction with 0.5% Triton X-100 in PHEM buffer for 5 min. For calcium treatment, samples were permeabilized with 100 mM PIPES, 1 mM MgCl2, 0.1 mM CaCl2, and 0.1% Triton X-100 (pH 6.9). Cells were subsequently fixed with methanol and immunostained with anti-tubulin antibody and CREST as described above.
Cell Synchronization Experiments—At 24 h after transfection of HeLa cells with HEC1 siRNA oligonucleotides, the mitotic cells were removed by mitotic shake off, and thymidine (2 mM final concentration) was added to the remaining cells. Another 18 h later, the mitotic and dead cells were again washed off, and the remaining cells were released into fresh medium. Samples were taken at 2-h intervals for 16 h and stained with annexin V or anti-PARP1 antibody to determine the percentage of apoptotic cells. To determine the timing of the appearance of spindle defects, samples were taken at 1-h intervals starting at 9 h after the release from thymidine arrest. These cells were fixed and stained with anti-tubulin antibody and DAPI. The effect of microtubule depolymerization on MAD1 staining and apoptosis in HEC1 RNAi cells was examined by adding nocodazole to these cells at 7 h after thymidine release. Immunostaining was performed at 7 h after nocodazole addition.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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hSPC25 Binds to HEC1 in Vitro and Interacts with HEC1 throughout the Cell Cycle in Vivo—To confirm the interaction between HEC1 and hSPC25, we transfected HeLa cells with plasmids encoding Myc-tagged HEC1 and HA-tagged hSPC25. The lysates of the transfected cells were then immunoprecipitated using anti-Myc antibody and blotted with anti-HA antibody (Fig. 2A). The HA-hSPC25 protein was present in the anti-Myc immunoprecipitates (Fig. 2A), indicating that HA-hSPC25 interacted with Myc-HEC1 in these cells. In a complementary experiment, plasmids encoding Myc-hSPC25 and HA-HEC1 were cotransfected into HeLa cells. The cell lysates were again immunoprecipitated with anti-Myc antibody and blotted with anti-HA antibody. HA-HEC1 was again found to interact with Myc-hSPC25 (Fig. 2B). These results demonstrated that, when overexpressed, HEC1 and hSPC25 formed a complex in living cells. In another experiment, we cotranslated plasmids encoding HA-HEC1 and Myc-hSPC25 in rabbit reticulocyte lysate in vitro in the presence of [35S]methionine. HA-HEC1 and Myc-hSPC25 were co-immunoprecipitated in these lysates, indicating that HEC1 and hSPC25 also interacted with each other in vitro (Fig. 2C).
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hSPC25 Localizes to Kinetochores during Mitosis—HEC1 and hNUF2, two known components of the human NDC80 complex, localize to kinetochores during mitosis (29, 30). We next examined whether hSPC25 exhibits a similar localization pattern. To determine the cellular localization of hSPC25, we transfected HeLa cells with plasmids encoding Myc-tagged hSPC25 and stained the transfected cells with anti-Myc antibody and a human autoimmune serum (CREST) that labeled the kinetochores (Fig. 3, A-C). Myc-hSPC25 exhibited a punctate staining pattern that matched that of CREST during all stages of mitosis, including prometaphase, metaphase, and anaphase (Fig. 3, A-C). This indicated that Myc-hSPC25 localized to kinetochores in mitosis. We next determined the cellular localization of the endogenous hSPC25 protein during mitosis. As shown in Fig. 3D, the endogenous hSPC25 protein also showed a punctate staining pattern that was similar to that of CREST, indicating that endogenous hSPC25 also localized to kinetochores during mitosis.
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30% of these cells possessed 4N DNA contents, whereas only 15% of the control cells had 4N DNA contents (Fig. 4B). About 30% of the hSPC25 RNAi cells possessed less than 2N DNA contents (Fig. 4B), suggesting that they had undergone cell death. Thus, depletion of hSPC25 from HeLa cells caused an accumulation of cells in G2/M and cell death. To determine whether the hSPC25 RNAi cells accumulated in G2 or mitosis, we examined the cellular and DNA morphology of these cells using a fluorescence microscope. Cells in mitosis were typically round with condensed DNA. At 24 h after RNAi treatment, we noticed an accumulation of hSPC25 RNAi cells in mitosis. The mitotic index of the hSPC25 RNAi cells was 32%, compared with a mitotic index of 10% in control cells (Fig. 4C). At 48 h after RNAi treatment, the mitotic index was 28% (Fig. 4C). This indicated that the hSPC25 RNAi cells accumulated in mitosis and that the majority of the 4N cells as determined by FACS represented cells in mitosis, rather than G2 (Fig. 4, B and C). Although the mitotic index of the hSPC25 RNAi cells did not increase at later time points, there was a dramatic increase in cell death (Fig. 4C). For example, at 72 h after transfection, 75% of the hSPC25 RNAi cells had undergone cell death, whereas <10% of the control cells had died (Fig. 4C). Therefore, our results are consistent with the notion that inactivation of hSPC25 causes a delay or arrest in mitosis, followed by cell death. Very similar phenotypes were observed with cells depleted of HEC1 by RNAi (Fig. 4C) (29). This indicated that inactivation of either hSPC25 or HEC1 had similar effects on the cell cycle, further supporting that hSPC25 is a component of the NDC80 complex. Mitotic Arrest in hSPC25-depleted Cells Depends upon a Functional Spindle Checkpoint—The mitotic arrest of hSPC25 cells can be explained by two equally plausible scenarios. First, inactivation of hSPC25 causes a defect in kinetochore function and arrests cells in prometaphase. Second, the hSPC25 RNAi cells undergo mitotic arrest mediated by an active spindle checkpoint. One way to distinguish between these two possibilities is to determine the protein level of cyclin A1 in these cells. As shown previously, the level of cyclin A1 is high in prometaphase cells, and it is already degraded by the metaphase-anaphase transition (34). Interestingly, although an active spindle checkpoint blocks the APC/C-mediated degradation of cyclin B1 and securin, it does not prevent cyclin A1 degradation (34). Therefore, mitotic cells arrested with an active spindle checkpoint are expected to contain low levels of cyclin A1 and high levels of cyclin B1 and securin. On the other hand, cells in prometaphase are expected to contain high levels of all three proteins. To determine the status of the spindle checkpoint and the stage of mitotic arrest of mitotic hSPC25 RNAi cells, we examined the protein levels of cyclin A1, cyclin B1, and securin in these cells by immunostaining with the corresponding antibodies. As shown in Fig. 5, the levels of cyclin B1 and securin were high in mitotic hSPC25 RNAi cells, whereas the levels of cyclin A were low. This suggests that these cells might be arrested in mitosis by an active spindle checkpoint, instead of a mechanical slowdown in prometaphase.
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30% of the hSPC25 RNAi cells were mitotic (Fig. 4C). At 72 h after RNAi treatment, the majority of the hSPC25 RNAi cells had undergone cell death, whereas >70% of the hSPC25/MAD2 double RNAi cells were viable (Fig. 4C). It is worth noting that many of the hSPC25/MAD2 double RNAi cells escaped from mitosis abnormally, as a vast majority of the hSPC25/MAD2-depleted interphase cells contained multiple or aberrant multilobed nuclei (data not shown). Therefore, similar to HEC1-depleted cells (29), the mitotic delay or arrest caused by the inactivation of hSPC25 also requires a functional spindle checkpoint.
Inactivation of the Human NDC80 Complex Causes Apoptosis after Transient Mitotic Arrest—To determine the timing of cell death in response to the loss of NDC80 function, we followed HEC1 siRNA-transfected cells after synchronization at the G1/S boundary by thymidine treatment. The percentage of cell death was significantly lower in thymidine-treated HEC1 RNAi cells (12%) compared with untreated RNAi samples (28%), suggesting that blocking cells at G1/S reduces cell death in HEC1-depleted cells. We next washed off the mitotic and dead cells that had accumulated during the 18-h thymidine treatment and examined HEC1-depleted cells and mock-transfected cells for 16 h after release from thymidine arrest (Fig. 6). As expected, a major increase in the mitotic index was observed at 9-10 h after thymidine release in both samples (Fig. 6A). Comparison of the mitotic index at later time points revealed that mitotic progression was much slower in the HEC1-depleted cells compared with the mock-transfected cells (Fig. 6A). After 14 h of thymidine release, only 10-15% of the control cells were in mitosis, whereas 30-35% of the HEC1-depleted cells were mitotic (Fig. 6A). There was no significant cell death in the HEC1 siRNA-treated cells until 14 h after thymidine release, at which time a sharp increase in cell death was observed (Fig. 6B). By 16 h, 50% of the HEC1-depleted cells were dead (Fig. 6B). This indicates that HEC1-depleted cells die following transient arrest in mitosis. During the same period, no significant cell death was observed for the control cells. These results are consistent with time lapse video microscopy by us (data not shown) and DeLuca et al. (30). A large percentage of the cells with a compromised NDC80 function died following a 5-8-h arrest in mitosis.
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Inactivation of hSPC25 Leads to Multiple Spindle Abnormalities—Several groups have observed various mitotic defects in vertebrate cells depleted of HEC1 or NUF2 (29-32). We also analyzed the DNA and spindle morphology of the hSPC25 RNAi cells. As shown in Fig. 7, the hSPC25 RNAi cells also exhibited defects in spindle morphology and chromosome congression. First, 18% of the mitotic hSPC25 RNAi cells contained multipolar spindles (Fig. 7A). In the mitotic cells with bipolar spindles, there were also clear defects: these spindles were more elongated and often distorted (Fig. 7A). Second, despite the presence of bipolar spindles, the chromosomes in most mitotic hSPC25-depleted cells were scattered throughout the cell, indicating failure of the chromosomes to align at the metaphase plate. In a few cells, although most of the chromosomes were aligned at the metaphase plate, some chromosomes were still lagging behind (Fig. 7A). These results indicate that hSPC25 is required for the proper execution of many mitotic processes, again consistent with its being a subunit of the NDC80 complex.
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25% of these mitotic cells contained multipolar spindles (see Supplemental Fig. S2). There was no significant increase in the number of cells with multipolar spindle at later time points (see Supplemental Fig. S2). This indicates that these spindle defects occur shortly after the entry into mitosis and are unlikely to be a nonspecific consequence of prolonged mitotic arrest. However, we did observe an increase in the number of cells with fractured spindles at later time points (see Supplemental Fig. S2), suggesting that this defect might be an indirect consequence of prolonged mitotic arrest. hSPC25-deficient Kinetochores Retain the Ability to Form Microtubule Attachments—The chromosome congression defect observed in hSPC25-depleted cells might be due to their inability to form functional kinetochore-microtubule connections. The presence of kinetochore-bound microtubules can be specifically detected by incubating cells for a short period with ice-cold medium or by permeabilizing them in the presence of high levels of calcium. Both of these treatments destabilize non-kinetochore microtubules, whereas kinetochore-bound microtubules are preserved. Microtubules appeared to be stable under both of these circumstances in hSPC25-deficient cells (Fig. 7B), suggesting that the NDC80 complex is not completely indispensable for the formation of kinetochore-microtubule attachments. However, we do not know whether these kinetochore-microtubule attachments formed in hSPC25 RNAi cells are functional.
Depletion of hSPC25 Leads to the Loss of Kinetochore Localization of MAD1 in a Microtubule-dependent Manner—Martin-Lluesma et al. (29) showed that HEC1 interacts with MAD1 in yeast two-hybrid assays and is essential for the localization of MAD1 to kinetochores during mitosis. We next tested whether hSPC25 is also required for the kinetochore recruitment of MAD1. We examined the kinetochore localization of three spindle checkpoint proteins (MAD1, BUB1, and BUBR1) in the hSPC25-depleted cells (Fig. 8). In control cells, MAD1 was clearly observed at the kinetochores during prometaphase (Fig. 8). However, the kinetochore staining of MAD1 was undetectable in prometaphase cells depleted of hSPC25 (Fig. 8). Depletion of hSPC25 did not adversely affect the integrity of the kinetochores in a general way, as CREST staining at the kinetochores was normal. The kinetochore staining of other checkpoint proteins, such as BUB1 and BUBR1, was also largely unaffected by the RNAi-mediated depletion of hSPC25 (Fig. 8). This indicates that inactivation of hSPC25 specifically abolishes the kinetochore localization of MAD1. Martin-Lluesma et al. (29) showed that the intensity of the kinetochore staining of BUB1 is reduced by 50% in HEC1 RNAi cells. In our hands, the BUB1 and BUBR1 kinetochore staining did not appear to be weaker in either HEC1 or hSPC25 RNAi cells. We do not know the underlying reason for this discrepancy, although it may result from the use of different antibodies and different fixation protocols.
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We next tested whether the NDC80 complex is also required for the kinetochore localization of MAD1 in nocodazole-arrested mitotic cells. As shown in Fig. 9, MAD1 localized to kinetochores in mock-transfected mitotic cells treated with nocodazole. As expected, the kinetochore localization of MAD1 was absent in mitotic HEC1 RNAi cells in the absence of nocodazole (Fig. 9). Surprisingly, the kinetochore localization of MAD1 was restored in HEC1 RNAi cells upon nocodazole treatment (Fig. 9). Although we could not determine whether a particular cell indeed received RNAi, we did analyze >100 mitotic cells, and the vast majority of them exhibited positive MAD1 staining. Therefore, it appears that the NDC80 complex is not absolutely required for MAD1 kinetochore localization in nocodazole-arrested mitotic cells that lack a functional spindle.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Roles of the NDC80 Complex in Chromosome Congression and Spindle Morphology—The Ndc80 complex has now been characterized in several organisms, including S. cerevisiae, Xenopus, chicken, and mammals (28-32). In all the systems studied so far, the NDC80 complex proteins localize to kinetochores in mitosis, and their inactivation leads to a defect in chromosome congression. We have observed a similar phenotype for HeLa cells transfected with either hSPC25 or HEC1 siRNA. The exact function of the NDC80 components in this process is not yet understood. The simplest explanation for the chromosome congression defect in these cells would be their inability to form functional kinetochore-microtubule attachments. But the resistance of microtubules to cold- or calcium-mediated destabilization in these cells indicates that these cells can achieve a certain degree of kinetochore-microtubule attachment. Moreover, some degree of chromosome alignment can be observed in many mitotic hSPC25/HEC1-depleted cells. The inability of these cells to align their chromosomes might then stem from their unstable or improper kinetochore-microtubule connections.
Apart from defects in chromosome alignment, cells with a compromised NDC80 function exhibit multiple spindle aberrations, including abnormally elongated spindles, multipolar spindles, and misaligned bipolar spindles. These abnormalities might be an indirect consequence of prolonged mitosis. Alternatively, certain kinetochore components, such as the NDC80 complex, are essential to maintain the integrity of the spindle. Our experiments with synchronized cells treated with HEC1 RNAi support the second possibility. Elongated and multipolar spindles could be observed at early stages of mitosis in HEC1-depleted cells, and no significant increase in the frequency of these abnormal spindles was observed at later time points. In addition, functional disruption of other kinetochore components, such as Mis12, Mis6, CENP-A, and CENP-C, also leads to abnormally elongated spindles (35). Multipolar and fractured spindles are also observed in response to inactivation of other kinetochore proteins. For example, disruption of the dynactin complex by overexpressing p50 dynamitin leads to fractured spindles (36). Similarly, depletion of human CENP-I/Mis6 results in multipolar spindles (35). The underlying mechanism for these spindle aberrations in cells with defective kinetochores is not known. However, it is conceivable that a functional kinetochore might regulate spindle length by counteracting the forces that tend to extend the spindle. It is also possible that kinetochores might have checkpoint functions that prevent precocious elongation of spindles and the formation of additional spindle poles.
Loss of NDC80 Function and Apoptosis—Another important phenotype of cells depleted of components of the NDC80 complex is that these cells died after a mitotic arrest of 5-8 h. The majority of these dead cells showed a cellular and nuclear morphology characteristic of apoptosis, including membrane blebbing and fragmented nuclei. Furthermore, these cells were positively stained with annexin V and an antibody against cleaved PARP1, indicating that the NDC80-deficient cells die by apoptosis. Thus, the mitotic arrest observed in these NDC80-depleted cells differs from that caused by CENP-E depletion or by microtubule poisons, such as nocodazole. In the latter cases, cells could stay arrested in mitosis for as long as 18 h without undergoing apoptosis. In addition, HEC1 RNAi cells died with the same kinetics in the presence of nocodazole. Therefore, it appears that apoptosis of HEC1 RNAi cells is not a secondary consequence of cells attempting to divide in the presence of a defective kinetochore but functional spindle. In the future, it will be interesting to determine whether and how the NDC80-deficient kinetochores actively trigger apoptosis.
Role of NDC80 in the Spindle Checkpoint—Martin-Lluesma et al. (29) have reported that HEC1 interacts with MAD1 in yeast two-hybrid assays and is required for the localization of MAD1 to kinetochores during mitosis. These observations have been confirmed by our findings in this study and by Hori et al. (32) using Nuf2-deleted chicken DT40 cell lines. There are two possible explanations for the lack of MAD1 kinetochore localization in NDC80-deficient cells. The NDC80 complex might form the docking surface through which MAD1 latches onto the kinetochores. Alternatively, it could be a secondary consequence of an aberrant mitosis with a defective kinetochore, but functional spindle. For example, HEC1-deficient kinetochores might be able to attach to microtubules to some extent, but cannot undergo chromosome congression due to the lack of tension.
The existence of kinetochore-microtubule attachment in these cells as detected by cold- or calcium-sensitive staining prompted us to examine MAD1 localization in hSPC25/HEC1-depleted cells after treatment with a microtubule-depolymerizing agent, such as nocodazole. Surprisingly, MAD1 localization to kinetochores remained unaffected in HEC1-depleted cells treated with nocodazole. Our findings are consistent with those of DeLuca et al. (30, 37). This suggests that HEC1-deficient kinetochores might still retain the ability to recruit MAD1. As mentioned above, HEC1-depleted cells can achieve a certain degree of kinetochore-microtubule attachment. This attachment might be sufficient for the dissociation of MAD1 from kinetochores. A similar situation has been observed in Ptk1 cells treated with hypothermia or in HeLa cells treated with microtubule-stabilizing agents, such as Taxol (38, 39). In all these situations, kinetochores are attached to microtubules, but lack tension. These kinetochores also lack MAD1 and MAD2 staining, suggesting that microtubule attachment to kinetochores is sufficient to trigger the dissociation of MAD1 from the kinetochores, even in the absence of tension. Despite the lack of MAD1 and MAD2 localization to kinetochores, these cells maintain mitotic arrest in a spindle checkpoint-dependent manner. Although the kinetochore localization of MAD1 and MAD2 appears to be dispensable for mitotic arrest in these cells, the cytoplasmic pool of MAD2 is absolutely necessary for the checkpoint activity. Microinjection of anti-MAD2 antibodies results in escape from mitotic arrest in all these circumstances. Similarly, the spindle checkpoint is active in NDC80-deficient cells, as indicated by low levels of cyclin A1 and high levels of cyclin B1 and securin; and this checkpoint activity is dependent upon proper MAD2 function. Simultaneous depletion of hSPC25/HEC1 and MAD2 from HeLa cells abolishes the mitotic arrest observed with the depletion of hSPC25/HEC1 alone.
Studies on the NDC80 complex and all the above-mentioned scenarios, where kinetochore attachment is established without tension, pose an important question: how do these cells maintain mitotic arrest despite a lack of MAD1 and MAD2 localization to the kinetochores? We suggest two possible explanations. First, it is possible that small amounts of MAD1, undetectable by our microscopy techniques, are still present on the kinetochores in the NDC80-deficient cells and are responsible for an active checkpoint. Alternatively, it is possible that MAD1 recruitment to kinetochores is required for the initial generation of the APC/C inhibitory checkpoint complexes and the establishment of the spindle checkpoint, but not for its maintenance. Other checkpoint proteins are then responsible for the maintenance of these APC/C inhibitory signals through mechanisms that do not require the kinetochore localization of MAD1 and MAD2. Clearly, more studies are needed to clarify these issues.
In conclusion, we have identified two novel components of the human NDC80 complex. Detailed characterization of hSPC25 revealed that it plays a pivotal role in chromosome congression and the maintenance of spindle integrity. Our studies also suggest that loss of NDC80 function in cells might serve as a direct signal to trigger apoptosis. Finally, we have also demonstrated that HEC1 depletion results in microtubule-dependent dissociation of MAD1 from kinetochores, reinforcing the notion that kinetochore localization of MAD1 and MAD2 is dependent primarily upon the microtubule occupancy status of kinetochores.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1 and S2.
Michael L. Rosenberg Scholar in biomedical research. To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390. Tel.: 214-648-9697; Fax: 214-648-2971; E-mail: hongtao.yu{at}utsouthwestern.edu.
1 The abbreviations used are: APC/C, anaphase-promoting complex or cyclosome; RNAi, RNA interference; hSPC25, human SPC25; CENP, centromere protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline; siRNA, small interfering RNA; PI, propidium iodide; FACS, fluorescence-activated cell sorting; HA, hemagglutinin; hNUF2, human NUF2; PIPES, 1,4-piperazinediethanesulfonic acid; DAPI, 4',6-diamidino-2-phenylindole; PARP1, poly(ADP-ribose) polymerase-1.
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