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Vol. 19, Issue 2, 587-594, February 2008
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*Ludwig Institute for Cancer Research, and Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093; and Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan
Submitted October 19, 2007;
Revised November 8, 2007;
Accepted November 20, 2007
Monitoring Editor: Kerry Bloom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Okada et al., 2006). The CCAN includes a subgroup of three proteins (CENP-H, -I, and -K) that are interdependent for localization and play essential roles in kinetochore structure and function (Okada et al., 2006). Additional proteins begin to associate with the CENP-A/CCAN platform during mitotic entry to generate the mature mitotic kinetochore structure that forms a dynamic interface with spindle microtubules and serves as a platform for signaling pathways controlling the fidelity of chromosome segregation.
Previous work in the nematode Caenorhabditis elegans has examined pairwise localization dependency relationships for the vast majority of kinetochore proteins (Oegema et al., 2001; Desai et al., 2003; Cheeseman et al., 2004). These studies have revealed a largely linear hierarchy of kinetochore assembly in this organism. A key player in kinetochore assembly in C. elegans is KNL-1, which functions downstream of the inner kinetochore components such as CENP-A, but upstream of all known outer kinetochore proteins (Desai et al., 2003; Cheeseman et al., 2004). KNL-1 belongs to a conserved protein family that includes budding yeast Spc105 (Nekrasov et al., 2003), fission yeast Spc7 (Kerres et al., 2004), Drosophila Spc105R (Przewloka et al., 2007), and a novel protein previously identified in human cells known as AF15q14, D40, CASC5, or Blinkin (Cheeseman et al., 2004; Obuse et al., 2004; Kiyomitsu et al., 2007). AF15q14 is present in all vertebrates including chicken (Gallus gallus). Here we will refer to the human and chicken proteins as hKNL1 and ggKNL1, respectively. The C. elegans, human, Drosophila, and fission yeast KNL-1 proteins directly interact with the Ndc80 and Mis12 complexes (Cheeseman et al., 2004; Obuse et al., 2004; Liu et al., 2005; Przewloka et al., 2007). We have termed this interacting protein set that is conserved throughout eukaryotes the KNL-1/Mis12 complex/Ndc80 complex (KMN) network (Cheeseman et al., 2006). The KMN network is a key structural component of the kinetochore, but also directly binds to microtubules via the Ndc80 subunit of the Ndc80 complex and KNL-1 (Cheeseman et al., 2006).
Here, we focus on the contribution of KNL1 to kinetochore assembly in vertebrates. We demonstrate that KNL1 localizes to kinetochores throughout mitosis and is required for the localization of a subset of outer kinetochore proteins. However, in contrast to C. elegans KNL-1, depletion of hKNL1 or ggKNL1 does not abolish Ndc80 complex localization. This apparent difference was explained by analysis of the CCAN subunit CENP-K. KNL1 and CENP-K appear to work coordinately to target the Ndc80 complex to kinetochores. Simultaneously reducing the levels of both hKNL1 and the CENP-K results in a failure of kinetochore assembly downstream of CENP-A and -C and severe defects in chromosome segregation. These results reveal that the KMN network and the CENP-K subgroup of the CCAN coordinately direct kinetochore assembly in vertebrates. The system is evolutionarily plastic, because the CCAN pathway appears to have been lost in C. elegans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), YFP-CENP-A (Kops et al., 2005), and GFP-CENP-H, and GFP-CENP-Q (Okada et al., 2006) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, and L-glutamine (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere with 5% CO2. RNA interference (RNAi) experiments were conducted as described previously (Kline et al., 2006). CENP-K (Okada et al., 2006), hDsn1 (Kline et al., 2006), and hNuf2 (DeLuca et al., 2002) small interfering RNAs (siRNAs) have been previously described. For hKNL1, a combination of four pooled sequences were predesigned by Ambion (Austin, Tx). These include GGAAUCCAAUGCUUUGAGAtt, GAAAUAAGAAAAACUCUCGtt, GAUACUAUAAAGGUAUUCCtt, and GGAAGCUUCAGAUAAUUGUtt.
The chicken KNL1 target disruption construct was generated such that exons 8 and 9 were replaced with a histidinol- (hisD) or puromycin- (puro) resistance cassette under control of the β-actin promoter resulting in a severely truncated ggKNL1. Target constructs were transfected with a Gene Pulser II electroporator (Bio-Rad, Richmond, CA). Chicken DT40 cells were cultured and transfected as described previously (Fukagawa et al., 2001, 2004). All DT40 cells were cultured at 38°C in Dulbecco's modified medium supplemented with 10% fetal calf serum, 1% chicken serum, and penicillin/streptomycin. To repress the expression of the tetracycline-responsive transgenes, tetracycline (tet; Sigma, St. Louis, MO) was added to the culture medium to a final concentration of 2 µg/ml.
Immunofluorescence
Immunofluorescence in human cells was conducted as described previously (Kline et al., 2006). For fluorescence of microtubules, DM1
(Sigma) was used at 1:500. For visualization of kinetochore proteins, mouse anti-HEC1 (9G3; Abcam, Cambridge, MA) was used at 1:1000, mouse anti-CENP-C (a generous gift of Lars Jensen and Don Cleveland, Ludwig Institute for Cancer Research, La Jolla, CA) was used at 1:200, and human anti-centromere antibodies (ACA; Antibodies, Davis, CA) were used at 1:100. YFP-CENP-A–, GFP-CENP-H–, or GFP-CENP-Q–expressing HeLas were counterstained with goat anti-green fluorescent protein (GFP; a generous gift of Andres Ladurner, European Molecular Biology Laboratory, Heidelberg, Germany) at 1:1000. Affinity-purified rabbit polyclonal antibodies were generated against hKNL1 (residues 1413-1624) as described previously (Desai et al., 2003). Polyclonal antibodies against hDsn1 and hKNL1 were used at 1 µg/ml. Cy2-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used at 1/100, although in some cases directly labeled antibodies against hDsn1 and hKNL1 were used without secondaries. DNA was visualized using 10 µg/ml Hoechst in Tris-buffered saline-TX.
Immunofluorescent staining of DT40 cells was performed as described previously (Okada et al., 2006). DT40 cells were collected onto slides with a cyto-centrifuge and fixed in 3% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature or 100% methanol for 15 min at –20°C, permeabilized in 0.5% NP-40 in PBS for 15 min at room temperature, rinsed three times in 0.5% bovine serum albumin (BSA) in PBS, and incubated for 1 h at 37°C with primary antibody. Binding of primary antibody was then detected with fluorescein isothiocyanate–conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) diluted to an appropriate concentration in 0.5% BSA in PBS. Affinity-purified rabbit polyclonal antibodies were generated against recombinant chicken CENP-C (Fukagawa et al., 1999), chicken CENP-I (Nishihashi et al., 2002), chicken CENP-O (Okada et al., 2006), chicken Mis12 (Kline et al., 2006), and chicken Ndc80 (Hori et al., 2003). To examine localization of ggKNL1-FLAG, anti-FLAG antibody (Sigma) was used. Chromosomes and nuclei were counterstained with DAPI at 0.2 µg/ml in Vectashield Antifade (Vector Labs, Burlingame, CA).
Microscopy and Image Acquisition
Images of fixed cells were acquired on a DeltaVision deconvolution microscope (Applied Precision Instruments, Issaquah, WA) equipped with a CoolSnap CCD camera (Roper Scientific, Tucson, AZ). Z-sections (n = 30–40) were acquired at 0.2-µm steps using a 100x, 1.3 NA Olympus U-PlanApo objective with 1 x 1 binning (Melville, NY). For analysis of microtubules and spatial localization within the kinetochore, images were deconvolved using the DeltaVision software. Measurements of the intensity of kinetochore localization were conducted on nondeconvolved images. All images for a specific experiment used identical exposure settings and scaling. Quantitative analysis of kinetochore localization intensities were performed using MetaMorph software as described previously (Universal Imaging, West Chester, PA; Kline et al., 2006). Images of DT40 cells were collected with a cooled CCD camera (Cool Snap HQ, Photometrics Image Point; Roper Scientific, Woburn, MA) mounted on an Olympus IX71 inverted microscope with a 60x objective lens (PlanApo 60x, NA 1.40) together with a filter wheel. Images were analyzed with an IPLab software (Signal Analytics, Vienna, VA) or DeltaVision deconvolution system (Applied Precision).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To monitor localization of hKNL1 and ggKNL1 throughout the cell cycle, we generated affinity-purified antibodies against amino acids 1413-1624 of hKNL1 and a DT40 chicken cell line stably expressing FLAG-ggKNL1. The FLAG-ggKNL1 fusion is fully functional based on its ability to rescue the depletion of untagged ggKNL1 (not shown). Immunofluorescence analysis indicated that hKNL1 and ggKNL1 localize to kinetochores throughout mitosis, but begin to disappear from kinetochores during telophase (Figure 1, A and B). Localization was also observed in a subset of interphase cells, which likely correspond to cells in G2. KNL1 exhibited similar temporal localization to subunits of the human Mis12 complex (not shown; (Goshima et al., 2003; Kline et al., 2006); both KNL1 and the Mis12 complex localize to kinetochores before the Ndc80 complex subunit Ndc80HEC1 (Figure 1, A and B; top row).
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Analysis of hKNL1-depleted cells indicated that many chromosomes were able to align in the middle of the cell. However, misaligned chromosomes were always detected at spindle poles or in an inappropriate orientation relative to the spindle axis (Figure 1C). This phenotype is reminiscent of that previously described after depletion of components of the hMis12 complex (Obuse et al., 2004; Kline et al., 2006), or human CENP-H/I/K (Okada et al., 2006), but is much less severe than depletion of the hNdc80 complex (DeLuca et al., 2002). Depletion of ggKNL1 also resulted in severe consequences in chromosome alignment and penetrant cell lethality (Figure 1D; Supplementary Figure 1). These results indicate that KNL1 is essential for cell viability and proper chromosome segregation in vertebrates.
KNL1 Does not Contribute to Localization of Constitutive Centromere Proteins But Is Required to Localize CENP-F and Zwint
We next examined the localization of a diverse collection of kinetochore proteins after hKNL1 RNAi. Although the CCAN has not been identified in C. elegans, previous work has demonstrated an important role for its subunits, especially of the CENP-H/I/K subgroup, in kinetochore assembly in budding yeast, fission yeast, and vertebrates (De Wulf et al., 2003; Kerres et al., 2006; Liu et al., 2006; Okada et al., 2006). Thus, we examined the same range of kinetochore proteins in cells where CENP-K levels were reduced using previously established RNAi conditions (Okada et al., 2006). We also analyzed conditional DT40 knockout cell lines of ggKNL1, ggCENP-K, and ggCENP-H. To detect the localization of different kinetochore proteins, we used a combination of affinity-purified antibodies and clonal cell lines stably expressing GFP fusions. The monitored fusion proteins are at least partially functional based on their similar localization to the endogenous protein and their copurification in multisubunit kinetochore subcomplexes (Cheeseman et al., 2004; Kops et al., 2005; Kline et al., 2006; Okada et al., 2006).
Depletion of hKNL1 abolished the localization of the outer kinetochore proteins Zwint and CENP-F and reduced the localization of hDsn1, a component of the Mis12 complex (Figure 2A). However, there was no significant effect observed on the localization of CENP-A, -C, -H, or -Q. CENP-Q is in a distinct subgroup of the CCAN from CENP-H/I/K (Okada et al., 2006). Similarly, ggKNL1 depletion reduced the localization of ggMis12, but did not strongly affect ggCENP-C, -I, or -O (Figure 2C). CENP-I and -O are present in the same subgroups of the CCAN as CENP-H and -Q, respectively. Finally, both hKNL1 and ggKNL1 require subunits of the Mis12 complex (hDsn1 or ggMis12, respectively) for their localization to kinetochores (Figure 2, B and D), similar to what is observed in C. elegans (Cheeseman et al., 2004).
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; Kwon et al., 2007) and with the close association of CENP-H/I/K with CENP-C (Foltz et al., 2006; Izuta et al., 2006; Figure 2A). Because CENP-H and CENP-Q localization is strongly affected after CENP-K RNAi (Figure 2A; also see quantitative analysis in Okada et al., 2006), this suggests that the RNAi conditions used here do significantly reduce CENP-K levels. However, we do not currently have the means to directly examine the depletion of CENP-K in human cells. In contrast to what we observed here, a previous study found that RNAi-mediated depletion of CENP-I did affect CENP-F localization (Liu et al., 2003). In addition, in chicken cells CENP-K or -H depletion reduced the localization of a GFP-ggKNL1 fusion protein (Figure 2E). Preliminary results in chicken cells also indicate that the localization of ggMis12 to mitotic kinetochores is reduced after CENP-H or -K depletion (Hori and Fukagawa., unpublished observations), in contrast to the normal hDsn1 localization observed after CENP-K RNAi in human cells (Figure 2A). In light of these observations, we acknowledge the possibility that the CENP-K knockdown in HeLa cells in this study may be partial. All the experiments including CENP-K RNAi are interpreted taking this point into consideration. In the human cell RNAi experiments, hKNL1 appears to be significantly depleted because both endogenous hKNL1 and endogenous CENP-F signals were eliminated. CENP-K is at least partially depleted with clear consequences on localization of its closely associated CCAN subunits, but no significant effect on hKNL1, CENP-F, or Zwint localization. Under these conditions, largely nonoverlapping protein groups downstream of CENP-A and -C depend on hKNL1 or CENP-K for their kinetochore localization. This finding predicts that codepletion of hKNL1 and CENP-K should prevent localization of all of the components tested above except for CENP-A and -C. This was indeed the case: no localization of hDsn1, CENP-H, CENP-Q, Zwint, or CENP-F was observed after the double RNAi against hKNL1 and CENP-K (Figure 2A). The dependency relationships for kinetochore localization observed in this work, together with the relationships defined by other recent studies, are summarized in Figure 5.
hKNL1 and CENP-K Act Coordinately to Recruit the Ndc80 Complex to the Outer Kinetochore
The Ndc80 complex plays a critical role in interactions with spindle microtubules at kinetochores (Cheeseman et al., 2006; DeLuca et al., 2006; Wei et al., 2007). Thus, it is of fundamental importance to properly recruit the Ndc80 complex during kinetochore assembly. The Ndc80 complex localizes external to KNL1 and CENP-K within the kinetochore for a reduced window of the cell cycle (Figure 1, A and B; Okada et al., 2006). In addition, the Ndc80 complex physically associates with hKNL1 and the Mis12 complex within the KMN network (Cheeseman et al., 2004; Obuse et al., 2004) and has been reported to associate with the CENP-H subunit of the CCAN (Mikami et al., 2005; Okada et al., 2006). In C. elegans, where the equivalent of the CENP-H/I/K protein group is likely missing, depletion of KNL-1 abolishes the localization of the Ndc80 complex. These prior results left open the question as to how KNL1 influences kinetochore localization of the Ndc80 complex in vertebrates. We found that hNdc80 localized at normal levels to kinetochores in human cells throughout mitosis after hKNL1 depletion (Figure 3A; data not shown). CENP-K RNAi reduced, but did not abolish, hNdc80 localization (Figure 3A; Okada et al., 2006). A reduction in ggNdc80 localization was observed in both ggKNL1 and ggCENP-K depleted chicken cells (Figure 3, B and C; Okada et al., 2006), although a more severe reduction was observed after ggCENP-K depletion, consistent with prior work (Liu et al., 2006). Thus, these results demonstrate that, unlike in C. elegans, depletion of KNL1 in vertebrates does not abolish kinetochore localization of the Ndc80 complex.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Our results reveal that KNL1 and the CENP-H/I/K complex coordinately direct kinetochore assembly in vertebrate cells. Combined with other recent work (Kline et al., 2006; Liu et al., 2006; Okada et al., 2006), these results lead to a model for assembly of the vertebrate kinetochore depicted in Figure 5A. For comparison, the kinetochore assembly hierarchy established in C. elegans is also depicted.
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; Cheeseman et al., 2004) and the close biochemical association between KNL-1 and the Ndc80 complex that is observed in C. elegans, fission yeast, Drosophila, and humans, we expected vertebrate KNL1 to function upstream of the Ndc80 complex. In agreement with this expectation, in the DT40 chicken cell conditional knockout analysis, we observed a twofold reduction in Ndc80 levels at the kinetochore after KNL1 depletion. However, in both the chicken cell analysis and in the human RNAi experiments significant amounts of Ndc80 were still able to localize to kinetochores after KNL1 depletion. Our work suggests that at least one explanation for this is a contribution from the CENP-H/I/K complex to the targeting of the Ndc80 complex. The CENP-H/I/K complex is likely to be upstream of KNL1 in vertebrate kinetochore assembly and the analysis in chicken cells with genetic CENP-H or -K deletions is consistent with this notion. However, Ndc80 complex targeting downstream of the CENP-H/I/K complex does not appear to work exclusively via KNL1 in vertebrates. This finding is supported not only by the localization analysis in human and chicken cells but also by the dramatic synergy in phenotypic defects in the KNL1/CENP-K double RNAi experiments. Work in fission yeast is also consistent with this conclusion as discussed further below.
Thus far, despite genome-wide RNAi screening and extensive biochemical analysis, we have been unable to identify C. elegans counterparts to any of the subunits of the CCAN, with the exception of CENP-C. In light of the results presented here, the apparent absence of the CENP-H/I/K group proteins is consistent with KNL-1 being absolutely required for Ndc80 complex localization in C. elegans. Because C. elegans chromosomes are holocentric, with diffuse kinetochores assembling along the length of each sister chromatid, one possibility is that loss of the CCAN is a necessary prerequisite for holocentricity. However, with the exception of CENP-C, no counterparts to the CCAN have been found to date in Drosophila, which has monocentric chromosomes. The reported dependency of Ndc80 complex localization on the KNL1 ortholog in Drosophila (Przewloka et al., 2007) suggests that, as in C. elegans, the CCAN protein group may have been lost in this lineage, further work is needed to test if this indeed the case.
The CENP-H/I/K class of proteins and other components of the CCAN are present in fungi, albeit weakly conserved, suggesting that they arose early in eukaryotic evolution and have been lost in specific lineages. Recent work in fission yeast demonstrated that the KNL-1 homologue Spc7 is not required for Ndc80 localization, but also reported synthetic lethal genetic interactions between spc7 and mis6 (CENP-I) or sim4 (CENP-K) mutants (Kerres et al., 2004), consistent with the synergistic defects we describe here for hKNL1/CENP-K double inhibitions. Overall, these results suggest evolutionary plasticity in kinetochore assembly, specifically in the recruitment of the critical Ndc80 complex. Because orthologues of KNL1, the Ndc80 complex and the Mis12 complex—the three parts of the KMN network—are found throughout eukaryotes and are essential in all systems where they have been tested, whereas the CCAN appears to have been lost in specific lineages, the future challenge will be to elucidate the mechanism of CCAN action and the reason for its dispensability in specific organisms.
There are several possibilities that could explain the synergistic defect in Ndc80 complex localization after hKNL1 and CENP-K inhibitions in human cells. KNL1 and CENP-H/I/K proteins may act in parallel pathways to direct Ndc80 localization or may function coordinately in intersecting pathways. The reduction in KNL1 and Mis12 complex localization after CENP-K depletion in chicken cells as well as the reduction in CENP-H/I/K and KNL1 localization after Mis12 complex inhibition is more consistent with the latter. In addition to providing a structural scaffold for the association of the Ndc80 complex, these proteins sets could also function via a regulatory mechanism directing Ndc80 complex localization to kinetochores. On the basis of the physical associations that have been previously reported for these proteins, we speculate that one possible explanation for our results is that hKNL1 and the CENP-H/I/K subgroup of the CCAN directly associate with distinct regions of the four-subunit Ndc80 complex (Figure 5B). KNL1 is likely to associate with the Spc24/Spc25 dimer that is oriented toward the inner kinetochore, as suggested by reconstitution experiments with C. elegans proteins (Cheeseman et al., 2006), whereas CENP-H/I/K may associate with the Ndc80/Nuf2 dimer that is projecting out toward the spindle microtubules, as suggested by two-hybrid analysis in chickens (Mikami et al., 2005). In this scenario, loss of either hKNL1 or CENP-K would partially compromise Ndc80 complex localization, whereas loss of both would dramatically inhibit localization. Such a "two-hand" mechanism is evolutionarily plastic, in the sense that one interaction may be compensated for by a change in the affinity of the other. We note that interactions with KNL-1 modulate the microtubule-binding activity of the C. elegans Ndc80 complex (Cheeseman et al., 2006). Thus, although a substantial amount of Ndc80 complex persists at kinetochores after depletion of KNL1, its function in generating interactions with microtubules may be compromised. Such a "two-hand" mechanism should be testable in future work using reconstituted complexes in vitro.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Whitehead Institute for Biomedical Research, and Department of Biology, Massachusetts Institute of Technology, Suite 401, Nine Cambridge Center, Cambridge, MA 02142. 
Address correspondence to: Arshad Desai (abdesai{at}ucsd.edu) or Iain Cheeseman (icheese{at}wi.mit.edu)
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