The CENP-A NAC/CAD kinetochore complex controls chromosome congression and spindle bipolarity
2007; Springer Nature; Volume: 26; Issue: 24 Linguagem: Inglês
10.1038/sj.emboj.7601927
ISSN1460-2075
AutoresSarah E. McClelland, Satyarebala Borusu, Ana C. Amaro, Jennifer R. Winter, Mukta Belwal, Andrew D. McAinsh, Patrick Meraldi,
Tópico(s)RNA Research and Splicing
ResumoArticle15 November 2007free access The CENP-A NAC/CAD kinetochore complex controls chromosome congression and spindle bipolarity Sarah E McClelland Sarah E McClelland Chromosome Segregation Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK Search for more papers by this author Satyarebala Borusu Satyarebala Borusu Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Molecular Life Sciences PhD Program, Zurich, Switzerland Search for more papers by this author Ana C Amaro Ana C Amaro Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Molecular Life Sciences PhD Program, Zurich, Switzerland Search for more papers by this author Jennifer R Winter Jennifer R Winter Chromosome Segregation Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK Search for more papers by this author Mukta Belwal Mukta Belwal Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Molecular Life Sciences PhD Program, Zurich, Switzerland Search for more papers by this author Andrew D McAinsh Corresponding Author Andrew D McAinsh Chromosome Segregation Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK Search for more papers by this author Patrick Meraldi Corresponding Author Patrick Meraldi Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Search for more papers by this author Sarah E McClelland Sarah E McClelland Chromosome Segregation Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK Search for more papers by this author Satyarebala Borusu Satyarebala Borusu Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Molecular Life Sciences PhD Program, Zurich, Switzerland Search for more papers by this author Ana C Amaro Ana C Amaro Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Molecular Life Sciences PhD Program, Zurich, Switzerland Search for more papers by this author Jennifer R Winter Jennifer R Winter Chromosome Segregation Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK Search for more papers by this author Mukta Belwal Mukta Belwal Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Molecular Life Sciences PhD Program, Zurich, Switzerland Search for more papers by this author Andrew D McAinsh Corresponding Author Andrew D McAinsh Chromosome Segregation Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK Search for more papers by this author Patrick Meraldi Corresponding Author Patrick Meraldi Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Search for more papers by this author Author Information Sarah E McClelland1,‡, Satyarebala Borusu2,3,‡, Ana C Amaro2,3, Jennifer R Winter1, Mukta Belwal2,3, Andrew D McAinsh 1 and Patrick Meraldi 2 1Chromosome Segregation Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK 2Institute of Biochemistry, ETH Zurich, Zurich, Switzerland 3Molecular Life Sciences PhD Program, Zurich, Switzerland ‡These authors contributed equally to this work *Corresponding authors: Chromosome Segregation Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, UK. Tel.: +44 1883 722306; Fax: +44 1883 714375; E-mail: [email protected] Institute of Biochemistry, ETH Zurich, Zurich 8093, Switzerland. Tel.: +41 44 632 63 47; Fax: +41 44 632 12 69; E-mail: [email protected] The EMBO Journal (2007)26:5033-5047https://doi.org/10.1038/sj.emboj.7601927 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Kinetochores are complex protein machines that link chromosomes to spindle microtubules and contain a structural core composed of two conserved protein–protein interaction networks: the well-characterized KMN (KNL1/MIND/NDC80) and the recently identified CENP-A NAC/CAD. Here we show that the CENP-A NAC/CAD subunits can be assigned to one of two different functional classes; depletion of Class I proteins (Mcm21RCENP−O and Fta1RCENP−L) causes a failure in bipolar spindle assembly. In contrast, depletion of Class II proteins (CENP-H, Chl4RCENP−N, CENP-I and Sim4RCENP−K) prevents binding of Class I proteins and causes chromosome congression defects, but does not perturb spindle formation. Co-depletion of Class I and Class II proteins restores spindle bipolarity, suggesting that Class I proteins regulate or counteract the function of Class II proteins. We also demonstrate that CENP-A NAC/CAD and KMN regulate kinetochore–microtubule attachments independently, even though CENP-A NAC/CAD can modulate NDC80 levels at kinetochores. Based on our results, we propose that the cooperative action of CENP-A NAC/CAD subunits and the KMN network drives efficient chromosome segregation and bipolar spindle assembly during mitosis. Introduction Human kinetochores are complex molecular machines that assemble on a centromere that spans mega bases of DNA and ensure bi-orientation, congression and disjunction of sister chromatids during mitosis (Cleveland et al, 2003). Kinetochores execute three main functions: (1) they form a structure that is compatible with tight, but dynamic binding to the plus-end of spindle microtubules (MTs); (2) they modulate MT dynamics and kinesin-like proteins to generate the forces necessary to drive chromosome movement and (3) they generate spindle checkpoint signals that block cells before initiation of anaphase in the presence of unattached or tension-free kinetochores (Musacchio and Hardwick, 2002; Musacchio and Salmon, 2007). Bioinformatic and biochemical studies have demonstrated that kinetochores contain a structural core composed of two conserved protein–protein interaction networks called KMN and CENP-A NAC/CAD (De Wulf et al, 2003; Nekrasov et al, 2003; Cheeseman et al, 2004, 2006; Obuse et al, 2004; Liu et al, 2005; Foltz et al, 2006; Meraldi et al, 2006; Okada et al, 2006). The KMN network is composed of three complexes that are required for distinct functions: the NDC80 complex that is essential for MT–kinetochore attachment and spindle checkpoint signaling (Kline-Smith et al, 2005), the MIND/Mis12 complex that is essential for spindle checkpoint signaling and contributes to kinetochore-based force generation (Kline et al, 2006; McAinsh et al, 2006), and KNL-1/Spc105 that links MIND and NDC80 (Cheeseman et al, 2006). Importantly, the KMN network binds to MTs directly in vitro and is conserved in all eukaryotes (Cheeseman et al, 2006; Meraldi et al, 2006; Wei et al, 2007). The CENP-A NAC/CAD network is only partially conserved with corresponding protein-protein interaction networks identified in Saccharomyces cerevisiae (CTF19 complex) and Schizosaccharomyces pombe (Sim4 complex; Cheeseman et al, 2002; Liu et al, 2005). These three networks require the centromeric histone-H3 variant CENP-A for kinetochore binding. Moreover, bioinformatic analysis reveals that they are built up from a set of common orthologues (Figure 1A; Supplementary Figures S1 and S2; Meraldi et al, 2006). CENP-A NAC/CAD components have been subdivided into either NAC proteins (nucleosome-associated complex; CENP-C, CENP-H, CENP-50CENP−U, CENP-M, CENP-T and Chl4RCENP−N) or CAD proteins (CENP-A Distal; CENP-I, Mcm21RCENP−O, Fta1RCENP−L, Sim4RCENP−K, CENP-P, CENP-Q, CENP-R and CENP-S). This designation is based on their biochemical isolation: NAC proteins were purified in association with CENP-A nucleosomes, whereas CAD proteins could only be purified with NAC proteins (Foltz et al, 2006). Work with S. cerevisiae has demonstrated that the counterpart of CENP-A NAC/CAD, the CTF19 complex, consists of several stable biochemical subcomplexes that denote distinct functional units of the kinetochore (De Wulf et al, 2003; McAinsh et al, 2003). However, it is unknown whether designation of proteins as 'NAC' or 'CAD' reflects a similar functional organization, since the exact function(s) of CENP-A NAC/CAD proteins is uncertain; depletion of NAC and CAD proteins in human or chicken cells causes chromosome congression defects (Liu et al, 2003; Minoshima et al, 2005; Foltz et al, 2006; Okada et al, 2006). At the same time, depletion of the CAD protein Mcm21RCENP−O leads to kinetochore–MT attachment defects that cause monopolar spindles (McAinsh et al, 2006). The human CENP-A NAC/CAD network has also been proposed to play a role in loading CENP-A at centromeres; however, the depletion of CENP-A NAC/CAD subunits does not affect the recruitment of endogenous CENP-A (Fukagawa et al, 2001; Okada et al, 2006), in contrast to other bona fide CENP-A loading factors such as hMis18alpha, Mis18beta and M18BP1 (Fujita et al, 2007). Figure 1.Localization of CENP-H and Chl4RCENP−N. (A) Schematic diagram illustrating the conservation of KMN and CENP-A NAC/CAD protein interaction network subunits in S. cerevisiae, S. pombe and Homo sapiens. Proteins conserved in all organisms (black text), absent in S. cerevisiae (red text), absent in S. pombe (green text) or restricted to a single organism (blue text) are indicated. (B, C) Immunoblots of whole-cell lysates from cells transfected with control (siLaminA), siChl4R or siCENP-H RNA and probed with antibodies as indicated. Download figure Download PowerPoint It is also unclear to what degree the CENP-A NAC/CAD network interacts with the KMN network. Some studies reported that CENP-A NAC/CAD is required for loading of the MIND and NDC80 subunits onto kinetochores (Hori et al, 2003; Liu et al, 2006; Okada et al, 2006; Kwon et al, 2007), while other studies found no such effect (Foltz et al, 2006; McAinsh et al, 2006). Another group even observed the opposite effect in which CENP-A NAC/CAD components displayed reduced kinetochore binding in the absence of the MIND complex (Kline et al, 2006). Thus, an important challenge in the kinetochore field is to define the precise function of CENP-A NAC/CAD and to understand how it interacts with the core KMN network at the functional level. We have therefore addressed the following three key questions in this paper by combining functional cell biological assays, small interfering RNA (siRNA)-mediated protein depletion, high-resolution microscopy and live-cell imaging: (1) are components of the human CENP-A NAC/CAD network responsible for different kinetochore functions? (2) How would these different CENP-A NAC/CAD components cooperate with one another to modulate kinetochore function? (3) How does CENP-A NAC/CAD functionally interact with the KMN network? Results Loading of CENP-A NAC/CAD kinetochore components during the cell cycle To investigate the function and organization of CENP-A NAC/CAD, we first focused on two NAC proteins (CENP-H and Chl4RCENP−N) and one CAD protein (Mcm21RCENP−O). We chose these proteins because different phenotypes have been reported for the depletion of CENP-H and Chl4R (congression defects; Fukagawa et al, 2001; Foltz et al, 2006), or Mcm21R (monopolar spindles; McAinsh et al, 2006). We first raised polyclonal antibodies against human CENP-H and Chl4R (antibodies against Mcm21R were available; McAinsh et al, 2006). By immunoblotting, CENP-H antisera recognized a 34-kDa band in whole-cell extracts and Chl4R antisera recognized a 38-kDa band. Depletion of CENP-H or Chl4R with siRNA abolished the 34- or 38-kDa bands, confirming the specificity of our antibodies (Figure 1B and C). These antibodies were next tested by immunofluorescence on HeLa cells in conjunction with CENP-A antisera (kinetochore marker) and DAPI (DNA marker). Previous studies demonstrated that CENP-H localizes to kinetochores (Fukagawa et al, 2001), but Chl4R has been only examined as a GFP-tagged fusion protein (Foltz et al, 2006). Both CENP-H and Chl4R antisera (but not the corresponding preimmune sera) recognized kinetochores in interphase and mitotic cells (Figure 1D and E). This staining was specific, as in both cases depletion of CENP-H or Chl4R by siRNA reduced the kinetochore signal more than 20-fold in over 95% of the cells, whereas a control siRNA had no effect (Figure 1D and E, and data not shown). These results validated the specificity of our CENP-H and Chl4R antibodies and siRNAs. Figure 2.(D, E) Immunofluorescence images of mitotic or interphase HeLa cells transfected with control or siChl4R RNA and stained with DAPI (DNA), CENP-A antisera (kinetochores; red) and Chl4R preimmune or antisera (green) (D); or transfected with control or siCENP-H RNA and stained with DAPI (DNA), CENP-A antisera (kinetochores; red) CENP-H pre-immune or antisera (green) (E). Scale bar=10 μm. Download figure Download PowerPoint Previous studies have found that CENP-H levels at kinetochores are constant during the cell cycle (Fukagawa et al, 2001), whereas Mcm21R levels are highest during interphase and diminished by 50% during mitosis (McAinsh et al, 2006; Figure 2C). This suggested that different members of CENP-A NAC/CAD might accumulate with distinct dynamics on kinetochores during the cell cycle. We therefore quantified the Chl4R levels on kinetochores for each cell-cycle stage by 3D-deconvolution microscopy with respect to CREST levels, which are constant throughout the cell cycle and act as an internal control, and compared them to CENP-H levels (Hoffman et al, 2001; see Materials and methods for details). We found that the levels of kinetochore-bound Chl4R decreased markedly as cells entered mitosis (>80% decrease at metaphase), and increased again as cells exit mitosis, whereas CENP-H levels remained constant (Figures 1D, 2A–C). To exclude that this decrease was caused by epitope masking, we confirmed the results with a second polyclonal Chl4R rabbit antibody (Supplementary Figure S3A and B). Thus, the dynamic accumulation of Chl4R onto kinetochores during the cell cycle resembles Mcm21R but is distinct from CENP-H. Figure 3.Cell-cycle loading and recruitment dependencies of Chl4RCENP−N, CENP-H and Mcm21RCENP−O. Levels of (A) Chl4R or (B) CENP-H on kinetochores were determined from deconvolved 3D reconstructions of cells stained with DAPI (DNA), CREST antisera or CENP-A antibodies (kinetochores) and Chl4R or CENP-H antisera. The intensity of the kinetochore signal was determined in interphase (I), prophase (P), prometaphase (PM), metaphase (M), anaphase (A) or telophase (T) relative to CREST/CENP-A after background correction (see Materials and methods for details). (C) Comparison of cell-cycle loading profiles for CENP-H and Chl4R with Mcm21R, Nuf2R (NDC80 complex) and Nnf1R (MIND complex; McAinsh et al, 2006). (D–H) Levels of CENP-H, Chl4R, Mcm21R, Nnf1R, Nuf2R and Ndc80 on kinetochores were determined in control, siCENP-H-, siChl4R- or siMcm21R-transfected cells from deconvolved 3D reconstructions of cells stained with DAPI (DNA), CREST antisera or CENP-A antibodies (kinetochores) and the corresponding antisera. The intensity of kinetochore signal was determined relative to CREST/CENP-A after background correction. (I) Representative images of experiments described in (D–H) with CREST/CENP-A (green) and anti-CENP-H, Chl4R, Mcm21R, Nnf1R, Nuf2R or Ndc80 (red). Note that Ndc80 (mouse) and Nuf2R (rabbit) antibody staining was carried out on the same cells. Scale bar=10 μm, scale bar in zoom=1 μm. Download figure Download PowerPoint Binding dependencies between CENP-A NAC/CAD, MIND and NDC80 subunits We next tested by quantitative immunofluorescence to what extent Mcm21R, CENP-H or Chl4R are required for the kinetochore binding of one another and of MIND (Nnf1R) and NDC80 (Ndc80 and Nuf2R) components. Depletion of CENP-H or Chl4R had little effect on the levels of MIND and NDC80, but abolished the kinetochore association of both CENP-A NAC/CAD proteins (Figure 2D–I). Surprisingly, depletion of Mcm21R caused up to a two-fold increase in the levels of kinetochore-bound Chl4R, CENP-H, Ndc80 and Nuf2R while leaving Nnf1R levels unaffected (Figure 2D–H). These increases in kinetochore-bound levels are unlikely to be caused by epitope masking, as in each case we obtained similar results with several antibodies (antibodies against two proteins in the NDC80 complex, two polyclonal antibodies for CENP-H and two polyclonal antibodies for Chl4R; Figure 2; Supplementary Figure S3D). Based on these results we conclude that CENP-A NAC/CAD contains at least two classes of proteins: Mcm21R belongs to a first class of proteins (Class I), which appears to modulate the kinetochore-bound levels of the NDC80 complex, and a second class (Class II) of protein that includes CENP-H and Chl4R. Class II proteins are not required for binding of the MIND and NDC80 complexes, but are required for kinetochore binding of at least two other CENP-A NAC/CAD subunits. Functional independence of CENP-A NAC/CAD, MIND and NDC80 We next investigated how the dependency relationships between the CENP-A NAC/CAD, MIND and NDC80 complexes reflect their kinetochore functions. Our dependency experiments predict that MIND functions independently of Chl4R/CENP-H, while Chl4R and CENP-H should be interdependent at the functional level. Previous studies have reported that depletion of CENP-A NAC/CAD proteins causes chromosome congression defects (Fukagawa et al, 2001; Foltz et al, 2006; Kline et al, 2006; McAinsh et al, 2006). To test their functional relationship, we quantitatively compared the effect of Chl4R, CENP-H and Nnf1R depletion on chromosome congression, using single and double siRNA transfections. Immunofluorescence and immunoblotting demonstrated that Chl4R, CENP-H and Nnf1R were depleted to the same level in double transfections as in single siRNA transfections (Figure 3B–E; Supplementary Figure S4). As a first functional assay, we performed live-cell imaging of HeLa cells stably expressing Histone2B-GFP to monitor chromosome congression. Specifically, depleted cells were imaged every 3 min for 8 h (Figure 3A), with nuclear breakdown (NBD) set as T=0 min in each cell and the time of chromosome congression (defined as the time at which the last unaligned chromosome had congressed to the metaphase plate) recorded relative to NBD. When control cells (n=45) were monitored, 93% of cells completed chromosome congression by T=24 min (Figure 3A and D). In contrast, 50% of siCENP-H (n=95) and 70% of siChl4R (n=80)-transfected cells had not yet completed congression by T=24 min (Figure 3A and D). Importantly, when cells were transfected with both siChl4R and siCENP-H oligonucleotides (n=96), we observed no additive effect when compared to either single depletion (55% cells with uncongressed chromosomes at T=24 min; Figure 3A and D). Nnf1R depletion by itself caused congression errors in 46% of the cells at T=24 min (n=104; Figure 3A and D). Strikingly, co-depletion of CENP-H and Nnf1R produced an additive effect, as 80% of the cells had not yet completed chromosome congression by T=24 min (n=186; Figure 3A and D). Figure 4.CENP-H/Chl4RCENP−N and Nnf1R have separate roles in chromosome congression. (A) Successive frames every 3 min from live-cell movies of H2B-GFP-expressing HeLa cells transfected with control, siCENP-H, siChl4R or siChl4R+siCENP-H, siNnf1R-3 or siCENP-H+siNnf1R-3 RNA. NBD was set as T=0 min. Scale bar=10 μm. (B, D) Immunoblots showing protein levels of CENP-H, Chl4R or Nnf1R after treatment with siRNA as indicated. (C, E) Protein levels of CENP-H, Chl4R and Nnf1R as measured by quantitative immunofluorescence as in Figure 2D–H (see Supplementary Figure S4 for representative images). (F) Percentage of cells, treated as described in panel A, with uncongressed chromosomes at T=24 min. (G) Percentage of cells with one (dark blue), two (blue), three (red) or more than three (dark red) uncongressed chromosomes following 30-min treatment with MG132 as determined from images such as shown in (I) Error bars indicate the s.d. for the total number percentage of cells with uncongressed chromosomes. (H) Chromosomes in metaphase cells were counted as unaligned if they were located outside of the central 30% of the mitotic spindle, or if their kinetochores were aligned perpendicular to the spindle axis. (I) Representative images of cells treated for 30 min with MG132 and stained with DAPI (DNA; blue), anti-α-tubulin (green) and CENP-E antisera (red). Scale bar=10 μm, scale bar in zoom=1μm. Download figure Download PowerPoint To confirm these results and quantify the number of unaligned chromosomes at higher resolution, we used immunofluorescence on HeLa cells treated with MG132 for 30 min before fixation. The proteasome inhibitor MG132 blocks cells at multiple points in the cell cycle, including an arrest at the metaphase–anaphase transition independent of the spindle checkpoint (Rock et al, 1994). The fixed cells were stained with DAPI and sera against α-tubulin (MT marker) and CENP-E (kinetochore marker). In cells containing a metaphase plate indicating arrest at the metaphase–anaphase transition, we quantified the number of uncongressed chromosomes. Chromosomes were considered uncongressed if they were outside of a rectangular area encompassing the central 30% of the spindle (Figure 3H). We found that CENP-H or Chl4R depletion caused chromosome congression defects in 43 and 56% of metaphase cells, respectively (versus 18.5% following control depletion; Figure 3G and I). Simultaneous depletion of CENP-H and Chl4R did not result in any additive effect (53% of cells with unaligned chromosomes; Figure 3G and I). In contrast, co-depletion of CENP-H and Nnf1R resulted in an additive effect when compared with single depletions (80% of cells with unaligned chromosomes following siNnf1R+siCENP-H treatment versus 54% in Nnf1R-depleted cells; Figure 3G and I). We therefore conclude that CENP-H and Chl4R are required to efficiently align the chromosomes on a metaphase plate as part of a single process that is distinct from MIND function. Our dependency experiments also indicated that the NDC80 complex remains kinetochore bound following depletion of CENP-H. To confirm that NDC80 is still functional in the absence of CENP-A NAC/CAD, we tested whether kinetochores in control, siCENP-H, siNuf2R or siCENP-H+siNuf2R treated cells bind to MTs in a cold-stable assay. This assay is based on the observation that abrupt cooling of cells to 4°C causes the depolymerization of MTs that are not bound to kinetochores, while kinetochore-bound MTs (k-fibers) remain stable (Salmon and Begg, 1980). We first confirmed by quantitative immunofluorescence that Nuf2R and CENP-H were depleted to the same level in double transfections as in single siRNA transfections (Figure 4A). Cells were then treated with ice-cold medium and processed for immunofluorescence using α-tubulin and CREST antibodies. Mitotic control cells subjected to such a treatment maintained a high number of stable k-fibers, demonstrating a robust MT–kinetochore attachment (Figure 4B). In contrast, most MTs of Nuf2R-depleted mitotic cells were depolymerized, as previously found (Figure 4B; Kline-Smith et al, 2005). Importantly, the MTs were stable in CENP-H-depleted cells but depolymerized in cells co-depleted for CENP-H and Nuf2R (Figure 4B). The fact that CENP-H-depleted cells only lose stable MT–kinetochore attachment following Nuf2R depletion confirms that CENP-H depletion alone does not abrogate the MT-binding function of the NDC80 complex at kinetochores, and that active NDC80 is loaded on kinetochores independently of CENP-H. Figure 5.The Ndc80 complex is functional in the absence of CENP-H/Chl4RCENP−N. (A) Quantification of Nuf2R and CENP-H levels on kinetochores in cells treated with control, siCENP-H, siNuf2R or siCENP-H+siNuf2R RNAs (as described in Figure 2). (B) Cells treated with siRNAs as described in panel A were subjected to cold treatment for 10 min before fixation and staining with anti-α-tubulin (green) and CREST antisera (red). Scale bar=10 μm. Download figure Download PowerPoint CENP-H-Chl4R depletion suppresses the monopolar spindle phenotype caused by Mcm21R depletion How does the phenotype of CENP-H-Chl4R depletion (Class II) relate to that of Mcm21R (Class I)-depleted cells? The striking feature of Mcm21R-depleted cells is that their kinetochores inhibit bipolar spindle formation, causing an increase in monopolar spindles (McAinsh et al, 2006). Consistently, monitoring of spindle assembly in HeLa cells stably expressing H2B-GFP (chromosome marker) and α-tubulin-mRFP (spindle marker) revealed that 38% of Mcm21R-depleted cells fail to establish a bipolar spindle within the first 12 min after NBD (versus 5% in control depleted cells; Figure 5A and B). However, live-cell imaging also showed that CENP-H or Chl4R depletion does not perturb bipolar spindle assembly (Figure 5A and B), even though CENP-H-, Chl4R- or Mcm21R-depleted cells have similar low levels of Mcm21R at kinetochores (Figure 5C; Supplementary Figure S5). This discrepancy could be due to three possible causes: (1) a cytoplasmic pool of Mcm21R could rescue bipolar spindle assembly in CENP-H-Chl4R-depleted cells, (2) excessive levels of kinetochore-bound CENP-H-Chl4R in Mcm21R-depleted cells could interfere with bipolar spindle formation (Figures 2D and E, 5C) and (3) the mere presence of CENP-H/Chl4R on kinetochores lacking Mcm21R could be sufficient to disrupt bipolar spindle assembly. Figure 6.Chl4RCENP−N or CENP-H depletion rescues the bipolar spindle defect in Mcm21RCENP−O-depleted cells. (A) Successive frames every 3 min from live-cell movies of H2B-GFP/α-tubulin-mRFP-expressing HeLa cells transfected with control, siMcm21R, siCENP-H, siChl4R, siMcm21R+Chl4R, siMcm21R+siCENP-H or partial siCENP-H (50% siRNA)+siMcm21R RNA. The composite images for H2B-GFP (green) and α-tubulin-mRFP (red) are shown. Scale bar=10 μm. (B) Quantification of bipolar spindle formation errors at T=12 min after NBD in cells treated with siRNA as indicated. (C) Protein levels of Mcm21R, CENP-H and Chl4R in cells treated with siRNA as indicated as measured by quantitative immunofluorescence. (D) Protein levels of Mcm21R and CENP-H in cells treated with control or partial siCENP-H (50% siRNA)+siMcm21R RNAs. Download figure Download PowerPoint To distinguish between these possibilities, we first depleted both CENP-H or Chl4R and Mcm21R by siRNA, and followed spindle assembly by time-lapse microscopy. We found that the vast majority (91% compared with 95% in control cells) of double siChl4R+siMcm21R- or siCENP-H+siMcm21R-transfected cells assembled a bipolar spindle within the first 12 min after NBD (Figure 5A and B). Quantification of Mcm21R levels by immunofluorescence and immunoblotting in single and double depleted cells confirmed that this rescue was not due to incomplete depletion of Mcm21R (Figure 5C; Supplementary Figure S5). We therefore can exclude that cytoplasmic Mcm21R rescues bipolar spindle assembly, and conclude that the monopolar spindles observed in Mcm21R depleted cells require CENP-H/Chl4R at kinetochores. We next specifically removed the excessive amount of kinetochore-bound CENP-H in cells depleted of Mcm21R, using a partial CENP-H depletion combined with a full Mcm21R depletion (Figure 5D; for experimental details see Materials and methods). We found that 42% of the cells failed to form a bipolar spindle within 12 min after NBD (Figure 5A and B). We conclude that in the absence of Mcm21R even normal levels of CENP-H are sufficient to disrupt bipolar spindle formation. CENP-A NAC/CAD modulates NDC80 levels at kinetochores While the MT-binding NDC80 complex is loaded independently of CENP-A NAC/CAD, our dependency experiments also showed that the levels of the NDC80 were elevated in Mcm21R-depleted cells and normal in CENP-H-depleted cells (Figures 2H, I and 6A). Co-depletion of Mcm21R and CENP-H restored normal Ndc80 levels at kinetochores, indicating that kinetochore binding by Ndc80 can be modulated by CENP-A NAC/CAD (Figure 6A). This effect was specific for NDC80, as we found no evidence that the levels of another MT-binding kinetochore protein, CENP-E kinesin, were regulated by CENP-H or Mcm21R (Figure 6B). A key regulator of the NDC80 complex is the Aurora B kinase (DeLuca et al, 2006). It is therefore possible that an increase in the kinetochore binding of NDC80 reflects a misregulation of Aurora B, which could potentially cause monopolar spindles. However, we again found no evidence that CENP-A NAC/CAD regulates the levels of Aurora B at kinetochores (Figure 6C). In addition, treatment of Mcm21R-depleted cells with the Aurora B kinase inhibitor ZM1 at concentrations that inhibited cytokinesis (data not shown; see also Ditchfield et al, 2003) did not rescue the monopolar spindle phenotype (Figure 6D and E). Importantly ZM1 treatment by itself did not cause monopolar spindles (data not shown; Ditchfield et al, 2003). This indicates that failure in bipolar spindle formation in Mcm21R-depleted cells is not mediated by Aurora B activity. Overall, we conclude that CENP-A NAC/CAD can modulate NDC80 levels at kinetochores, even though at this point we have no evidence that these changes are linked to the monopolar spindles in Mcm21R-depleted cells. Figure 7.The CENP-A NAC/CAD controls the levels of kinetochore-bound NDC80 complex. (A–C) Protein levels of Ndc80, CENP-E and Aurora B in cells treated with siRNAs as indicated by quantitative immunofluorescence as described in Figure 2. (D) Quantification of bipolar spindle formation errors at T=12 min after NBD in cells treated with siMcm21R RNA and DMSO or 2 μM ZM1. (E) Successive frames every 3 min from live-cell movies of H2B-GFP/α-tubulin-mRFP-expressing HeLa cells transfected with siMcm21R RNA and 2 μM ZM1. The composite images for H2B-GFP (green) a
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