Cohesin Defects Lead to Premature Sister Chromatid Separation, Kinetochore Dysfunction, and Spindle-assembly Checkpoint Activation
2002; Elsevier BV; Volume: 277; Issue: 44 Linguagem: Inglês
10.1074/jbc.m206836200
ISSN1083-351X
AutoresMd. Tozammel Hoque, Fuyuki Ishikawa,
Tópico(s)Nuclear Structure and Function
ResumoScc1/Mcd1 is a component of the cohesin complex that plays an essential role in sister chromatid cohesion in eukaryote cells. Knockout experiments of this gene have been described in budding yeast, fission yeast, and chicken cells, but no study has been reported on human Scc1 thus far. In this study, we found that an N-terminally truncated human Scc1 shows a dominant-negative effect, and we examined the phenotypes of human cells defective in Scc1 function. Scc1 defects led to failure of sister chromatid cohesion in both interphase and mitotic cells. Interestingly, four chromatids derived from two homologues occupied four distinct territories in the nucleus in chromosome painting experiments. In mitotic Scc1-defective cells, chromatids were disjoined with normal condensation, and the spindle-assembly checkpoint was activated. We also found that, although the disjoined kinetochore (half-kinetochore) in Scc1-defective cells contains CENP-A, -B, -C, and -E normally, it apparently does not establish the kinetochore-microtubule association. These results indicate that Scc1 is essential for the association of kinetochores with microtubules. Scc1/Mcd1 is a component of the cohesin complex that plays an essential role in sister chromatid cohesion in eukaryote cells. Knockout experiments of this gene have been described in budding yeast, fission yeast, and chicken cells, but no study has been reported on human Scc1 thus far. In this study, we found that an N-terminally truncated human Scc1 shows a dominant-negative effect, and we examined the phenotypes of human cells defective in Scc1 function. Scc1 defects led to failure of sister chromatid cohesion in both interphase and mitotic cells. Interestingly, four chromatids derived from two homologues occupied four distinct territories in the nucleus in chromosome painting experiments. In mitotic Scc1-defective cells, chromatids were disjoined with normal condensation, and the spindle-assembly checkpoint was activated. We also found that, although the disjoined kinetochore (half-kinetochore) in Scc1-defective cells contains CENP-A, -B, -C, and -E normally, it apparently does not establish the kinetochore-microtubule association. These results indicate that Scc1 is essential for the association of kinetochores with microtubules. amino acid(s) fluorescence in situ hybridization immunofluorescence phosphate-buffered saline propidium iodide enhanced green fluorescent protein wild-type In eukaryotic cells, the replicated DNA (sister chromatid) remains connected to each other from the end of the S phase until the onset of anaphase. This sister chromatid cohesion ensures that the two daughter cells inherit an identical set of genetic information. It is well established that the sister chromatid cohesion is accomplished by a phylogenetically conserved protein complex called cohesin (reviewed in Ref. 1Nasmyth K. Annu. Rev. Genet. 2001; 35: 673-745Crossref PubMed Scopus (586) Google Scholar). Cohesin was identified genetically for the first time in budding yeast and contains at least four components: Scc1/Mcd1, Scc3, Smc1, and Smc3 (2Guacci V. Koshland D. Strunnikov A. Hogan E. Cell. 1997; 91: 47-57Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, 3Michaelis C. Ciosk R. Nasmyth K. Hogan E. Koshland D. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1165) Google Scholar, 4Toth A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (499) Google Scholar). Subsequent studies revealed that similar complexes are responsible for cohesion in diverse eukaryotic organisms including fission yeast, Xenopus, chicken, and human (5Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (516) Google Scholar, 6Tomonaga T. Nagao K. Kawasaki Y. Furuya K. Murakami A. Morishita J. Yuasa T. Sutani T. Kearsey S.E. Uhlmann F. Nasmyth K. Yanagida M. Genes Dev. 2000; 14: 2757-2770Crossref PubMed Scopus (240) Google Scholar, 7Sumara I. Vorlaufer E. Gieffers C. Peters B.H. Peters J.M. J. Cell Biol. 2000; 151: 749-762Crossref PubMed Scopus (336) Google Scholar, 8Sonoda E. Matsusaka T. Morrison C. Vagnarelli P. Hoshi O. Ushiki T. Nojima K. Fukagawa T. Waizenegger I.C. Peters J.M. Earnshaw W.C. Takeda S. Dev. Cell. 2001; 1: 759-770Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). However, the exact behaviors of cohesin differ among species. Specifically, Scc1 dissociates from chromosomes in a single step of the metaphase-to-anaphase transition in budding yeast, mediated by its proteolysis by a protease called Esp1 or separase (9Uhlmann F. Lottspeich F. Nasmyth K. Nature. 1999; 400: 37-42Crossref PubMed Scopus (750) Google Scholar). In contrast, the dissociation occurs in two steps in vertebrates, from the arm regions in prophase and from the centromeric region in the metaphase-to-anaphase transition (5Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (516) Google Scholar, 10Waizenegger I.C. Hauf S. Meinke A. Peters J.M. Cell. 2000; 103: 399-410Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 11Hoque M.T. Ishikawa F. J. Biol. Chem. 2001; 276: 5059-5067Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The bulk Scc1 dissociation in prophase occurs independently of separase, and is regulated by Polo-like kinase in Xenopus (12Sumara I. Vorlaufer E. Stukenberg P.T. Kelm O. Redemann N. Nigg E.A. Peters J.M. Mol. Cell. 2002; 9: 515-525Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), whereas the Scc1 dissociation from kinetochores at the metaphase-to-anaphase transition in human cells is achieved by the proteolysis of Scc1 by Esp1 (13Hauf S. Waizenegger I.C. Peters J.M. Science. 2001; 293: 1320-1323Crossref PubMed Scopus (376) Google Scholar). Scc1 also plays an important role in kinetochore function. In budding yeast, it was reported that kinetochore-microtubule association can be established, but sister kinetochores frequently associate with monopoles (14Tanaka T. Fuchs J. Loidl J. Nasmyth K. Nat. Cell Biol. 2000; 2: 492-499Crossref PubMed Scopus (263) Google Scholar). It was therefore concluded that Scc1 is important for the bipolar attachment of the sister kinetochores. A similar observation was reported in Scc1-depleted chicken DT40 cells, albeit the number of sister chromatids showing monopolar attachment was small (8Sonoda E. Matsusaka T. Morrison C. Vagnarelli P. Hoshi O. Ushiki T. Nojima K. Fukagawa T. Waizenegger I.C. Peters J.M. Earnshaw W.C. Takeda S. Dev. Cell. 2001; 1: 759-770Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Because the centromeres in higher eukaryotes have a much larger size and are much more complex than those in budding yeast, it is possible that Scc1 plays different roles in kinetochore function in higher eukaryotes. In this study, we found that the N-terminally truncated hScc1 (human Scc1) shows a dominant-negative effect in human cells. By exploiting this allele, we examined the phenotypes of Scc1-defective human cells. The full-length hScc1 cDNA and the deletion mutants, hScc1ΔN (287–631 aa)1 and hScc1ΔC (1–415 aa), were subcloned into pEGFP expression vectors (Clontech) and pMX-puro retroviral expression vector (15Kitamura T. Onishi M. Kinoshita S. Shibuya A. Miyajima A. Nolan G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9146-9150Crossref PubMed Scopus (223) Google Scholar). The retrovirus vectors were transfected into φNX amphotropic packaging cells (a gift from Dr. G. P. Nolan), and allowed to produce infectious retroviruses. HT1080 Tet-On cells, which constitutively express thetet-responsive trans-activator, were originally established and kindly provided by Dr. Y. Ishizaka (International Medical Center, Tokyo, Japan) (16Shimura M. Tanaka Y. Nakamura S. Minemoto Y. Yamashita K. Hatake K. Takaku F. Ishizaka Y. FASEB J. 1999; 13: 621-637Crossref PubMed Scopus (43) Google Scholar). An effector plasmid, pTO/hScc1ΔN, was constructed by inserting hScc1ΔN (287–631 aa) to pTO that possesses the tet-responsive DNA element and the hygromycin selection marker gene (16Shimura M. Tanaka Y. Nakamura S. Minemoto Y. Yamashita K. Hatake K. Takaku F. Ishizaka Y. FASEB J. 1999; 13: 621-637Crossref PubMed Scopus (43) Google Scholar). HT1080 Tet-On cells were transfected with pTO/hScc1ΔN and pTO-mock. Cells were cultured in the presence of G418 (400 μg/ml) and hygromycin (50 μg/ml). Twenty clones showing resistance to both drugs were selected and analyzed for inducible expression of hScc1ΔN after incubation with 1 μg/ml doxycycline for 48 h. One clone that showed the lowest background expression without induction and the highest expression with induction was used in this study. Western blotting was performed according to Ref. 11Hoque M.T. Ishikawa F. J. Biol. Chem. 2001; 276: 5059-5067Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar. In all IF analyses, cells were grown in chamber slide glasses (Falcon). Cells were fixed with 100% methanol for 20 min at −20 °C and permeabilized with 0.1% Triton X-100 for 10 min. Fixed cells were pre-incubated in blocking solution (0.1% bovine serum albumin and 0.1% skim milk in PBS), followed by incubation with primary antibodies for 1 h at 37 °C in the blocking solution. Cells were then washed three times with shaking in PBS, and incubated with secondary antibodies for 1 h at 37 °C. The cells that were washed with PBS three times were mounted in the mounting solution containing 0.25 μg/ml propidium iodide (PI) or TOTO3, and examined by laser confocal microscopy (Zeiss). IF analysis for 3F3/2 antigen was performed according to Ref. 17Gorbsky G.J. Chen R.H. Murray A.W. J. Cell Biol. 1998; 141: 1193-1205Crossref PubMed Scopus (199) Google Scholar. Two cosmid clones, cCI12–156 and pBR12, specific for chromosome 12 short arm telomere and the chromosome 12 centromere, respectively, were kindly provided by Dr. K. Okumura (Mie University, Mie, Japan) (18Nogami M. Nogami O. Kagotani K. Okumura M. Taguchi H. Ikemura T. Okumura K. Chromosoma. 2000; 108: 514-522Crossref PubMed Scopus (24) Google Scholar). FISH experiments were performed using samples fixed with methanol:acetic acid essentially as described in Ref. 19Lichter P. Cremer T. Tang C.J. Watkins P.C. Manuelidis L. Ward D.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9664-9668Crossref PubMed Scopus (245) Google Scholar with slight modifications. Slides were taken from −20 °C storage and dried at room temperature for 30 min. Nuclei were treated with 1 mg/ml pepsin (Sigma) in 10 mm HCl solution. Cells were washed with PBS and dehydrated gradually by rinsing them in succession with 70, 85, and 100% ethanol. Cellular DNA was denatured at 80 °C in 50% formamide in 2× SSC solution and dehydrated in succession with 70, 85, and 100% ice-cold ethanol. cCI12–156 and pBR12 probes were labeled with biotin-16-dUTP by the nick-translation method according to the supplier's protocol (BMY). Biotin-labeled probes were precipitated in presence of carrier salmon sperm DNA (Sigma) and competitor human Cot-1 DNA (BMY). Probes were then dissolved in hybridization solution (50% formamide (v/v), 10% dextran sulfate in 2× SSC), and denatured at 75 °C for 10 min. Denatured probes were pre-annealed at 37 °C for 30 min. Then, they were added to denatured nuclei and hybridized at 37 °C for 16 h in a humidified chamber. After hybridization, the slides were washed with 50% formamide in 2× SSC solution at 42 °C (three times, 5 min each) and with 2× SSC solution at 62 °C (three times, 5 min each). The biotinylated probe was detected using fluorescein-labeled avidin Fab fragment (Vector Laboratories), and finally nuclei were washed three times with 0.1% Tween 20 in 4× SSC solution at 42 °C. The nuclei were counterstained with PI and maintained in anti-fade medium (Vector Laboratories). The images were examined by laser confocal microscopy (Zeiss). Cells were fixed with methanol:acetic acid and denatured in 50% deionized formamide in 2× SSC solution for 2 min at 70 °C. Slides were transferred quickly to a coplin jar containing 70% ice-cold ethanol and incubated for 5 min. Then, the slides were incubated in succession with 85 and 100% ice-cold ethanol, each for 5 min, and finally air-dried. An aliquot of the digoxigenin-labeled whole chromosome 12 painting probe (BMY) was denatured for 10 min at 75 °C and pre-annealed at 37 °C for 30 min. The probe was added to the denatured nuclei, covered with a cover glass, and sealed with rubber cement. The slides were then transferred to a humidified chamber and incubated therein at 37 °C for 16 h. Then, the slides were washed twice with 50% formamide in 2× SSC solution for 15 min in a shaking water bath at 42 °C, followed by washing two times with 0.1% Tween 20 in 2× SSC solution at room temperature. Anti-digoxigenin-rhodamine Fab fragment (BMY) was diluted with PBS/bovine serum albumin solution and incubated with nuclei for 60 min in a humidified chamber at 37 °C. Slides were washed twice with 0.1% Tween 20 in 2× SSC solution, and DNA was counterstained with 0.1 μg/ml PI in PBS solution. The images were examined by laser confocal microscopy (Zeiss). Because Scc1 functions by forming a complex with Scc3, Smc1, and Smc3, it was expected that some deletion mutants of Scc1 might show a dominant-negative effect. Accordingly, we transfected the hScc1 cDNA encoding full-length, N-terminally or C-terminally truncated Scc1 (deleted for aa 1–286 or 416–631, respectively; Fig.1 A) fused with the N-terminal EGFP into 293T cells (EGFP-hScc1, EGFP-hScc1ΔN, or EGFP-hScc1ΔC, respectively). Scc1 contains a proline-rich region and a glutamate-rich region at the C-terminal half, which are absent in hScc1ΔC and retained in hScc1ΔN. The two truncated Scc1 proteins contain the putative nuclear localization sequence at their centers. Consistently, all EGFP-fused hScc1 proteins localized in the nuclei, where endogenous Scc1 is present (11Hoque M.T. Ishikawa F. J. Biol. Chem. 2001; 276: 5059-5067Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), whereas EGFP-mock protein found in the cytoplasm (Fig. 1 B). We next analyzed the cell cycle profiles of these transfectants. Cells at 48 h after the transfection were fixed, stained with PI, and analyzed by FACScan. To examine specifically the cells expressing the recombinant proteins, we first sorted the EGFP-positive cells and then analyzed the cell cycle profile of this population. Although overexpressing EGFP-hScc1 and EGFP-hScc1ΔC as well as EGFP-mock did not change the cell cycle profile, a significant decrease of the G1 fraction and an increase of the G2+M fraction were observed in EGFP-hScc1ΔN-overexpressing 293T cells (Fig. 1 C). The fraction of G2+M cells in EGFP-hScc1ΔN-overexpressing 293T cells was ∼40%, which is more than twice of those observed in the other transfectants. These results suggest that overexpressing hScc1ΔN interferes with the normal cell cycle progression, leading to cell cycle arrest at G2/M. The above results suggested that hScc1ΔN has a dominant effect on endogenous hScc1 protein. To investigate this possibility, we examined the sister chromatid cohesion in hScc1ΔN-expressing normal human fibroblast MRC-5 cells. Cells were infected with retroviral expression vector encoding FLAG-tagged wild-type hScc1 (wt-hScc1), hScc1ΔN, or hScc1ΔC mutant protein. The infected cells were analyzed at the indicated time after the completion of infection without drug selection (0 h). In a parallel control experiment, the transfection efficiency was estimated to be ∼40%. First, we monitored the proliferation rates. The wt-hScc1- and hScc1ΔC-expressing MRC-5 cells grew as well as the mock-infected cells (Fig. 2 A). In contrast, the hScc1ΔN-expressing MRC-5 cells grew less efficiently, which was evident at 72 h. We speculated that the impaired cell growth was a result of the cohesion defect. To test this possibility, asynchronously growing infected MRC-5 cells at 48 h were examined by FISH experiments using pBR12, a probe specific for the chromosome 12 centromere (referred to as 12CEN probe) (18Nogami M. Nogami O. Kagotani K. Okumura M. Taguchi H. Ikemura T. Okumura K. Chromosoma. 2000; 108: 514-522Crossref PubMed Scopus (24) Google Scholar). We found that the 12CEN signals in the wt-hScc1- and hScc1ΔC-expressing populations showed either one of the following three patterns: two separate spots, one spot and one set of twin spots, and two sets of twin spots. These three patterns were interpreted as two 12CENs unreplicated, one 12CEN replicated and the other unreplicated, and both 12CENs replicated, respectively. Fig. 2 B (a) shows one representative wt-hScc1-expressing cell that remained to replicate two homologous 12CENs. In contrast, we found that a significant fraction of the hScc1ΔN-expressing populations showed three or four separately located FISH spots, suggesting failure of cohesion at the chromosome 12 centromere most possibly in G2 cells (Fig. 2 B, b). We next examined the spread chromosomes in mitotic cells. In mitotic cells in wt-hScc1-expressing populations, two 12CEN-positive chromosomes were observed in all cases examined (Fig. 2 B,c), suggesting that the ectopic expression of wt-hScc1 does not interfere with the normal chromatid cohesion in mitosis. However, in hScc1ΔN-expressing populations, the number of condensed chromosomes was frequently larger than those found in the parental MRC-5 cells and wt-hScc1-expressing populations (Fig. 2 B,d–f). Most importantly, four 12CEN-positive chromosomes were frequently observed separately in hScc1ΔN-expressing populations. It was often observed that one of the 12CEN-positive chromosomes was completely separated from others (for example,d and f). However, it was unlikely that the four FISH signals represented four pairs of chromatids in tetraploid cells, because we did not find any significant increase in the number of hScc1ΔN-expressing cells with hyperploidy (data not shown). These observations indicate that two pairs of sister chromatids are disjoined in hScc1ΔN-expressing cells. We also noticed that the chromosomes in hScc1ΔN-expressing populations were condensed normally as in parent MRC-5 cells. To determine the fate of these cells, we monitored the later time points after retrovirus infection. When DNA and actin were stained with PI and anti-actin antibodies, respectively, we found that a significant number of cells in hScc1ΔN-expressing populations contained multiple nuclei in a single cell after 72 h of infection (Fig.2 C). In contrast, neither wt-hScc1- nor hScc1ΔC-expressing populations displayed such phenotypes (data not shown). The amounts of DNA in these nuclei varied. These results suggest that cohesin-defective cells show defects in chromosome segregation and form multiple nuclei in a single cell. To investigate the effect of hScc1ΔN in greater detail, we used an inducible expression system in human fibrosarcoma HT1080 cells that we confirmed to contain near-diploid chromosomes (data not shown). Using this approach, we were able to induce hScc1ΔN expression by the addition of doxycycline to the culture medium (hScc1ΔN-HT1080 cells; see "Materials and Methods"). hScc1ΔN was endowed with a C-terminal FLAG epitope that allowed its detection by FLAG-monoclonal antibodies. No detectable hScc1ΔN was expressed in the absence of doxycycline; in contrast, hScc1ΔN was induced upon the addition of doxycycline, which reached a plateau 2–3 days after the addition (Fig.3 A). By immunostaining with anti-FLAG antibody, we found that the hScc1ΔN protein was localized in the hScc1ΔN-HT1080 cell nuclei (data not shown). The uninduced hScc1ΔN-HT1080 cells, as well as mock-treated HT1080 cells with or without doxycycline addition, proliferated at a similar rate (data not shown). However, as expected, hScc1ΔN-HT1080 cells that had been induced to express hScc1ΔN protein showed a significantly reduced growth rate (data not shown). In the following experiments, hScc1ΔN-HT1080 cells grown in the presence or absence of doxycycline for 48 h were analyzed. Sister chromatid cohesion in these cells was examined by FISH experiment using cosmid clones cCI12–156 (Fig. 3 B) and pBR12 (Fig. 3 C), which recognize the telomere at the short arm of chromosome 12 and the centromere of the same chromosome, respectively. The cell cycle stage was determined by using a laser scanning cytometer (reviewed in Ref. 20Darzynkiewicz Z. Bedner E., Li, X. Gorczyca W. Melamed M.R. Exp. Cell Res. 1999; 249: 1-12Crossref PubMed Scopus (263) Google Scholar). This apparatus scans and records the two-dimensional position and the PI intensity, thereby determining the cell cycle stage for individual cells in a population on a slide glass. After the recording, cells of interest can be recalled and analyzed for a second chromo-probe. In both experiments using cCI12–156 or pBR12, two single spots and two sets of twin spots were observed in the uninduced hScc1ΔN-HT1080 cells at G1and G2 phases, respectively (Fig. 3 B,a and b; data not shown). In contrast, four completely separated FISH spots were frequently observed in induced G2 cells (Fig. 3 B, c). These four spots were not derived from four homologous chromosomes in tetraploid cells, because no significant fraction of tetraploid cells was detected. As for the pBR12 probe specific for the chromosome 12 centromere, four FISH spots were observed in 25% of the total population of the induced hScc1ΔN-HT1080 cells and in 3% of the uninduced cells (Fig. 3 C). We therefore concluded that the expression of hScc1ΔN also caused premature sister chromatid separation in this system. The four spots did not show any tendency of clustering into two groups; rather, they appeared to distribute randomly. The average distances between one FISH spot to the nearest one in the induced cells with four spots were 10.87 and 10.84 μm for cCI12–156 and pBR12 probes, respectively (data not shown). We noticed that the areas occupied by nuclei on the glass slide were larger in the induced G2 cells with four FISH spots (504.85 μm2) than in the uninduced G2 cells with two set of twin spots (346.14 μm2) (Fig. 3 D). No significant difference in nuclear area was observed between the induced and uninduced G1 cells, suggesting that the increase in nuclear area was related to the failure of sister chromatid cohesion. It was interesting to know if the entire chromatids of chromosome 12 were disjoined in the hScc1ΔN-expressing cells. To address this question, a similar analysis was conducted using a chromosome 12 painting probe. The uninduced G2 cells showed two separate chromosome 12 territories (Fig. 3 E, a). In contrast, four distinct territories were observed in a significant fraction of the induced G2 cells (Fig. 3 E,b). These results demonstrate that the four sister chromatids of two chromosomes 12 are separated in the G2phase. The four chromatids appeared to be randomly positioned in nuclei. There was no recognizable DNA connection between these remote chromatin masses, suggesting that the chromatids were completely separated without major DNA tangling. Next, we analyzed the M phase of the induced hScc1ΔN-HT1080 cells. Because it was not possible to synchronize HT1080 cells, we examined asynchronous cells by PI staining and immunostaining with anti-α-tubulin and anti-γ-tubulin antibodies (Fig.4 A). In uninduced cells, we observed normal metaphase and anaphase cells (Fig.4 A, a, d, and e). In contrast, we frequently observed abnormal anaphase-like configurations (hereafter referred to as pseudo-anaphase) in induced cells (Fig.4 A, b, c, and f). Despite the fact that the bipolar spindle was evident in IF experiments using anti-α- and anti-γ-tubulin antibodies, the chromosome DNA was excluded from the area occupied by the spindle. Indeed, it appeared that there was no kinetochore-microtubule association, and that the chromosome mass was mechanistically divided into two or three groups by the spindle. Moreover, the orientation of the polar spindle was perpendicular instead of parallel to the splitting orientation of chromosome masses. To interpret these results quantitatively, we measured the relative frequencies of cells showing the metaphase, anaphase, or pseudo-anaphase configurations in induced and uninduced populations. The pseudo-anaphase was defined by the appearance that the orientation of interpolar microtubules (when stained with anti-α-tubulin) or the interpolar axis (when stained with anti-γ-tubulin) was perpendicular to the splitting orientation of chromosomes. When more than 500 cells that appeared in metaphase, anaphase, or pseudo-anaphase were scored, 18% of induced cells, in contrast to 3∼4% of uninduced cells, showed the pseudo-anaphase configuration (Fig. 4 B). We therefore concluded that pseudo-anaphase cells were specifically observed in cells expressing hScc1ΔN. To examine kinetochore microtubule association in the induced hScc1ΔN-HT1080 cells in greater detail, we double-stained the cells with anti-CENP-C and anti-α-tubulin antibodies along with the DNA dye TOTO3 (Fig. 4 C). CENP-C is a protein that associates with functional kinetochores (21Pluta A.F. Mackay A.M. Ainsztein A.M. Goldberg I.G. Earnshaw W.C. Science. 1995; 270: 1591-1594Crossref PubMed Scopus (306) Google Scholar). In uninduced cells in metaphase and anaphase (Fig. 4 C, a and b, respectively), most kinetochores were found to associate with microtubules. In contrast, many kinetochores, if not all, appeared to associate with no microtubule in induced pseudo-anaphase cells (Fig.4 C, c). It was also noted that few kinetochores were positioned closely to monopoles, and that most kinetochores were excluded from the spindle periphery. These results strongly suggest that the kinetochores cannot establish microtubule association. We further examined the existence of other centromere-binding proteins CENP-A, -B, and -E at the kinetochores in the induced hScc1ΔN-HT1080 cells. CENP-E is a kinesin-like protein that functions as a kinetochore motor (22Wood K.W. Sakowicz R. Goldstein L.S. Cleveland D.W. Cell. 1997; 91: 357-366Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). All of these proteins were detected at the kinetochores in induced pseudo-anaphase cells. Therefore, we suggest that at least some aspects of the structural integrity of the kinetochores are maintained. Because the induced hScc1ΔN-HT1080 cells did not enter normal anaphase, and appeared defective in establishing microtubule-kinetochore connection, we were interested in whether the spindle-assembly checkpoint was activated in these cells or not. To address this question, we investigated the localization of the key spindle-assembly checkpoint proteins Mad2 and 3F3/2 in these cells. As shown previously (for review see Refs. 23Rieder C.L. Salmon E.D. Trends Cell Biol. 1998; 8: 310-318Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar and 24Maney T. Ginkel L.M. Hunter A.W. Wordeman L. Int. Rev. Cytol. 2000; 194: 67-131Crossref PubMed Google Scholar), both the mitotic checkpoint proteins Mad2 and 3F3/2 phosphoepitope are accumulated on kinetochores until they establish stable bipolar microtubule attachment. When the chromosomes are aligned on the metaphase plate by equal tensions applied by bipolar spindles, these proteins disappear from the kinetochores (25Gorbsky G.J. Ricketts W.A. J. Cell Biol. 1993; 122: 1311-1321Crossref PubMed Scopus (210) Google Scholar, 26Waters J.C. Chen R.H. Murray A.W. Salmon E.D. J. Cell Biol. 1998; 141: 1181-1191Crossref PubMed Scopus (395) Google Scholar). When the uninduced hScc1ΔN-HT1080 cells were examined for human Mad2 (hMad2) and 3F3/2 localizations by using specific antibodies, these proteins were found to localize at the kinetochores until metaphase and to disappear at anaphase, as expected (Fig. 5, A (a andb) and B (a–c)). In contrast, the kinetochores of the induced pseudo-anaphase cells were positive for these proteins (Fig. 5, A (c) and B(d)). If the spindle-assembly checkpoint was indeed activated in these induced cells in the M phase, it was expected that these cells would still maintain a high level of cyclin B1, because of the failure of anaphase-promoting complex activation (27Holloway S.L. Glotzer M. King R.W. Murray A.W. Waters J.C. Chen R.H. Salmon E.D. Cell. 1993; 73: 1393-1402Abstract Full Text PDF PubMed Scopus (489) Google Scholar, 28King R.W. Peters J.M. Tugendreich S. Rolfe M. Hieter P. Kirschner M.W. Cell. 1995; 81: 279-288Abstract Full Text PDF PubMed Scopus (825) Google Scholar). Indeed, we found a high level of cyclin B1 in the induced pseudo-anaphase cells (Fig. 5 C, c), but not in the uninduced anaphase cells (Fig. 5 C, a and b). To confirm these results, we measured the frequencies of hMad2-, 3F3/2-, and cyclin-B1-positive cells in different stages of cell cycle (Fig. 5 D). More than 250 cells each showing metaphase, anaphase, or pseudo-anaphase in the hScc1ΔN-expressing HT1080 population were examined by IF analyses for hMad2, 3F3/2, and cyclin B1, and the frequency of IF-positive cells was deduced. We found that 56, 4.5, and 92% of metaphase cells were positive for hMad2, 3F3/2, and cyclin B1, respectively. This result is consistent with previous studies showing that hMad2 remains to associate with kinetochores until later metaphase stages than the 3F3/2 epitope does (25Gorbsky G.J. Ricketts W.A. J. Cell Biol. 1993; 122: 1311-1321Crossref PubMed Scopus (210) Google Scholar, 26Waters J.C. Chen R.H. Murray A.W. Salmon E.D. J. Cell Biol. 1998; 141: 1181-1191Crossref PubMed Scopus (395) Google Scholar, 29Nicklas R.B. Ward S.C. Gorbsky G.J. J. Cell Biol. 1995; 130: 929-939Crossref PubMed Scopus (309) Google Scholar). As expected, these three marker proteins were not detected in anaphase cells. Significantly, we found that these proteins were positive in mo
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