Correspondence to Don W. Cleveland: dcleveland{at}ucsd.edu
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Abbreviations used in this paper: ACA, anti-centromere antiserum; CENP, centromere-associated protein; CSF, cytostatic factor; EYFP, enhanced YFP; KCR, kinetochore-containing region; LAP, localization and affinity purification; siRNA, small interfering RNA; Zwint-1, ZW10-interacting protein 1.
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Introduction |
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A role in generating this complex has been described for most proteins previously implicated in mitotic checkpoint control. Mad1 is required for the stable kinetochore association of a pool of Mad2 that in turn recruits and activates additional Mad2 molecules (Chen et al., 1998; Shah et al., 2004), and the kinesin-like microtubule motor centromere-associated protein (CENP)-E directly activates BubR1 kinase activity at the unattached kinetochore (Mao et al., 2003; Weaver et al., 2003). Two additional proteins, Zeste White 10 (ZW10) and Rough Deal (Rod), have been implicated in generating a sustained mitotic checkpoint; however, no molecular function in mitotic checkpoint signaling has thus far been suggested (Basto et al., 2000; Chan et al., 2000). It also remains unclear how any of these checkpoint components are connected to the underlying structural elements of the outer kinetochore.
ZW10 and Rod were originally identified in screens for mutations in Drosophila that interfere with the faithful transmission of chromosomes to daughter cells during mitosis (Smith et al., 1985; Karess and Glover, 1989), but orthologues have been recognized in C. elegans, A. thaliana, G. gallus, M. musculus, and humans (Starr et al., 1997; Chan et al., 2000; Okamura et al., 2001). The Drosophila larval neuroblasts of either mutant display identical phenotypes: anaphase figures with misaligned chromosomes and premature sister chromatid separation in cells treated with microtubule poisons (Williams et al., 1992; Scaerou et al., 1999). Several additional lines of evidence indicate that ZW10 and Rod function together. A Drosophila zw10;rod double mutant has a mitotic phenotype that is indistinguishable from the single mutants (Scaerou et al., 2001), and both proteins are found in a single complex in Drosophila embryos and human cells (Chan et al., 2000; Scaerou et al., 2001; Williams et al., 2003). In addition, ZW10 localization to kinetochores depends on Rod (Williams and Goldberg, 1994; Chan et al., 2000) and vice versa (Chan et al., 2000; Scaerou et al., 2001).
ZW10 has been implicated as a binding partner of several other proteins besides Rod. Immuno-purification of ZW10 from fly embryos isolated Zwilch, and a yeast-two-hybrid screen for human proteins that bind ZW10 identified human ZW10-interacting protein 1 (HZwint-1, hereafter referred to as Zwint-1; Starr et al., 2000; Williams et al., 2003). ZW10, Rod, and Zwilch are interdependent for kinetochore localization, can be found in a single complex in flies and human cells, and Drosophila mutants show identical mitotic phenotypes (Williams et al., 2003). No Drosophila Zwint-1 mutant has thus far been identified, but Zwint-1 is also localized to kinetochores, although it arrives there in prophase, before the arrival of ZW10 in early prometaphase (Starr et al., 2000). Another protein found to interact with ZW10 in the yeast-two-hybrid system is p50 dynamitin, a component of the cytoplasmic dynein activator dynactin (Starr et al., 1998). ZW10 can be found to interact with dynein weakly in Drosophila embryos (Williams et al., 2003), and ZW10 and Rod relocalization to Drosophila spindle microtubules during metaphase depends on functional dynein heavy chain (Wojcik et al., 2001).
ZW10 and Rod are essential for mitotic checkpoint signaling. Injection of antibodies to human ZW10 and Rod abrogates checkpoint function in human cells treated with nocodazole (Chan et al., 2000), and Drosophila mutant rod or zw10 neuroblasts are unable to elicit a checkpoint response induced by misattachment in an asp mutant strain or upon a colchicine-induced block in mitotic spindle assembly (Basto et al., 2000). Screening for mutations in alleles encoding putative chromosome instability genes in human colorectal cancers has recently identified mutations in ZW10, Rod, and Zwilch, hinting at the possibility that deregulation of ZW10 function may contribute to the chromosome instability phenotype of tumor cells (Wang et al., 2004b).
We now use immunodepletion of the ZW10Rod complex from Xenopus egg extracts and reduction of ZW10 accumulation in mammalian cells to identify ZW10 as a bridge between the structural outer kinetochorethrough its interaction with Zwint-1and the mitotic checkpoint, where it recruits a stably bound Mad1Mad2 complex to unattached kinetochores.
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Results |
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Zwint-1 was not found stably associated with our complexes containing ZW10 in the stringent LAPtag purifications. To test whether ZW10 and Zwint-1 interacted transiently or with a weak affinity, Zwint-1LAPtag was rapidly immunoprecipitated from mitotically arrested HeLa cells (clone LINT2.8; see Materials and methods) and tested for presence of endogenous ZW10. A small but significant fraction of ZW10 remained associated with Zwint-1 under these conditions (Fig. 2 a). Recombinant ZW10 also interacts with recombinant Zwint-1, and an association between the two can be found in human cultured cells that express high levels of epitope-tagged versions of both proteins (Wang et al., 2004a). That Zwint-1 is part of the structural kinetochore and binds ZW10 implies that Zwint-1 may recruit ZW10 to mitotic kinetochores. This was tested by reduction of Zwint-1 protein levels by plasmid-based small interfering RNA (siRNA) expression to 15% of Zwint-1 levels in cells transfected with mock siRNA (as indicated using an antibody to GFP for immunoblotting serial dilutions of LINT2.8 cell lysates expressing Zwint-1LAPtag; Fig. 2 b). Reduction of endogenous Zwint-1 yielded absence of endogenous ZW10 at kinetochores (Fig. 2 c). Because Zwint-1 apparently arrives at the kinetochore before ZW10 (Starr et al., 2000), and kinetochore localization of ZW10 depends on the presence of its Zwint-1 interaction domain (Wang et al., 2004a), these data reveal a crucial role for Zwint-1 in the recruitment of ZW10 to unattached kinetochores.
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ZW10Rod recruit the Mad1Mad2 complex and BubR1 to kinetochores in vitro
To determine why extracts depleted of the ZW10Rod complex were mitotic checkpoint-deficient, continued recruitment of various checkpoint proteins to the unattached kinetochore was compared in the presence or absence of X-ZW10X-Rod. X-BubR1 colocalized with X-Rod and X-ZW10 on kinetochores as expected in mock-depleted extracts, but did not bind to kinetochores depleted of the X-ZW10X-Rod complex (Fig. 5, ac). Similarly, X-Mad1 was absent from X-ZW10X-Roddepleted kinetochores (Fig. 5, ac), as was Mad2 (Fig. 5, ac), whose recruitment to unattached kinetochores depends on Mad1 (Chen et al., 1998). The absence of all three of these proteins at kinetochores was not due to co-depletion with X-ZW10X-Rod, as their protein levels in depleted versus control extracts were indistinguishable (Fig. 5 d). The absence at kinetochores was selective for components of the checkpoint signaling pathway: both the inner kinetochore histone H3 variant X-CENP-A (Maddox et al., 2003) and the kinetochore microtubule depolymerase X-KCM1 (also known as MCAK; Walczak et al., 1996) were present at undiminished levels at kinetochores after X-ZW10X-Rod depletion (Fig. 5, ac).
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ZW10 recruits Mad1Mad2 to unattached human kinetochores
How ZW10 contributes to mitotic checkpoint signaling in human cells was determined by depletion of it using expression of siRNA duplexes. Knockdown was partial and heterogeneous at 23 d after transfection (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200411118/DC1). Significant reduction of ZW10 levels was apparent 5 d after transfection of HeLa cells, with no detectable levels of ZW10 at unattached mitotic kinetochores in 80% of cells (Fig. 6, a and b). Although most cells looked relatively healthy at this time point, a fraction was dying, most likely because they had sufficient knockdown at an earlier time and had undergone multiple divisions in the absence of ZW10 (see below). Phospho-histone H3 levels measured by flow cytometry in cells transfected with control siRNA constructs and treated with nocodazole for 16 h showed a 10-fold elevation in mitotic cells when compared with asynchronously growing cells (Fig. 6, c and d). In contrast, mitotic checkpoint signaling was severely compromised by reduction in ZW10, as shown previously for cells injected with anti-ZW10 antibodies (Chan et al., 2000), with the ZW10 depleted cell population yielding only a twofold increase in mitotic index (Fig. 6, c and d). Similar results were found in cells depleted of Zwint-1 (Fig. 6, c and d). Moreover, because
20% of the cells transfected with ZW10 siRNA had residual ZW10 at kinetochores, even this modest increase in mitotic index almost certainly reflected continued mitotic checkpoint signaling in the ZW10-containing proportion of cells and a complete absence of sustained checkpoint signaling in ZW10-depleted cells. ZW10-deficient cells underwent aberrant mitoses, which resulted in cell death after several divisions, as indicated by markedly diminished colony formation in continued presence of ZW10 siRNA (Fig. 6 e) and aberrant chromosome distribution yielding chromatin bridges and micronuclei (Fig. 6 f). This is consistent with a mitotic checkpoint defect, as the same effect was seen in HeLa cells with an inactive mitotic checkpoint after reduction in levels of BubR1 or Mad2 (Kops et al., 2004).
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Discussion |
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Thus, the Zwint-1containing kinetochore subcomplex spans from the innermost components of the kinetochore across all layers of the centromere. Ultimately, mitosis-specific assembly of Zwint-1 recruits ZW10RodZwilch that serve to anchor attachment of Mad1 and Mad2 (Fig. 7 f). Although a previous study reported that ZW10Rod inhibition after injection of Rod antibody left Mad1 and Mad2 levels still detectable on mitotic kinetochores (Chan et al., 2000), it is now clear that removal of ZW10 or Rod inactivates the mitotic checkpoint with essentially no Mad1Mad2 recruited to unattached kinetochores in both Xenopus extracts and human cells. A requirement of ZW10Rod for Mad1Mad2 recruitment is further supported by genetics in Drosophila: GFP-Mad2 no longer attaches at kinetochores of colchicine-treated rod or zw10 mutant neuroblast cells (R. Karess, personal communication).
Although the ZW10 complex could bind Mad1 or a Mad1Mad2 heterodimer directly, this seems unlikely because Mad1 (as well as the Mad2 directly bound to it) is a stable component of the kinetochore before microtubule attachment (Howell et al., 2004; Shah et al., 2004), but was not found in the affinity purification of ZW10. Moreover, such a static, scaffold-like function for the ZW10 complex is hard to reconcile with the reported highly dynamic behavior of Rod in syncytial Drosophila embryos (Basto et al., 2004), albeit ZW10 and Rod dynamics have not been investigated in the context of mitotic human cells. More likely, ZW10 may affect Mad1 binding in a number of ways as only one component required for creating the Mad1-binding site(s) at the kinetochore. Ndc80HEC1 was found in a yeast-two-hybrid screen for interactors with human Mad1, and Mad1Mad2 kinetochore localization depends on a functional Ndc80HEC1 complex (Martin-Lluesma et al., 2002; DeLuca et al., 2003; McCleland et al., 2003, 2004; Bharadwaj et al., 2004). No binding between Ndc80HEC1 and Mad1 could be reconstituted in vitro with recombinant proteins (Martin-Lluesma et al., 2002). Perhaps the interaction found in the yeast-two-hybrid is too unstable to be identified by the assays we have used in vitro, which would support the idea that additional factors are required for establishing such an interaction. The ZW10 complex may fulfill that role. Tandem affinity purification of Mad1 and Ndc80HEC1-associating factors and reconstitution using recombinant proteins or purified subcellular complexes could now directly test this.
The dependency on ZW10 of BubR1 kinetochore localization differs in frog extracts and HeLa cells. Immunodepletion of the ZW10RodZwilch complex from frog extracts may affect an activity required for proper BubR1 kinetochore localization that is unaffected by RNAi of one component (ZW10) in HeLa cells. Alternatively, the frog kinetochore in extracts may behave like an all-or-none system more so than the human kinetochore. It is newly assembled upon addition of the sperm DNA to the extract and may therefore be less mature than that of human mitotic cells. Perhaps ZW10 depletion marginally affects BubR1 localization in human cells, an effect readily detected in the less rigidly structured kinetochores that must assemble and disassembly rapidly in the very short embryonic cell cycles. One way to distinguish between these possibilities is to examine checkpoint status and checkpoint protein localization in frog extracts to which the ZW10 complex, reconstituted from recombinant proteins, was restored, a daunting task that we have not been able to achieve since even though we identified a Xenopus Zwilch homologue (GenBank/EMBL/DDBL accession no. BC070818), we have been unsuccessful in identifying full-length XRod.
Despite linkage of dynein at kinetochores to the ZW10 complex through the dynactin subunit p50 dynamitin, it is unlikely that dynein plays any role in the establishment of mitotic checkpoint signaling by ZW10. Whereas depletion of ZW10 prevents the checkpoint from being activated, inhibition of dynein function chronically activates it: overexpression of p50 dynamitin to disrupt the dynein activator dynactin in vertebrate cells or hypomorphic dynein heavy chain mutants in flies both cause checkpoint-dependent mitotic arrest that is abrogated by mutation in Drosophila Rod (Wojcik et al., 2001) or injection of antibodies to Mad2 into PtK1 cells (Howell et al., 2001). Thus, ZW10 and Rod play a role in establishing checkpoint signaling and this is independent of functional dynein.
Although mitotic arrest after inhibition of dynein has been explained by the lack of tension between sister centromeres, perhaps due to improper spindle assembly (Echeverri et al., 1996; Gaglio et al., 1997; Merdes et al., 2000), it seems most likely to us that the probable role for dynein in checkpoint signaling is a role in directly silencing it, through dynein's microtubule-dependent removal of mitotic checkpoint proteins (including the ZW10Rod complex along with Mad1Mad2) from kinetochores after stable microtubule capture (Howell et al., 2001; Wojcik et al., 2001). This then leads to our proposal (modeled in Fig. 7 g) that establishment and silencing of mitotic checkpoint signaling at the kinetochore is achieved by linking an activator of the checkpoint (ZW10) with a silencer (dynein) whose inhibitor activity requires microtubule capture. At unattached kinetochores, the ZW10 complex recruits checkpoint components which dynein is unable to remove. Upon bi-oriented attachment, dynein powers their removal from both sister kinetochores, breaking the relatively weak binding between the tightly kinetochore-bound Zwint-1 and Zw10 and with dynein thus pulling the ZW10RodZwilch complex poleward with it. Loss of the ZW10 complex and its contribution to tethering the stably bound Mad1Mad2 complex serves to mediate rapid checkpoint signal silencing. Re-recruitment of the ZW10 complex during anaphase A may continue its role as a linker mediating dynein attachment at kinetochores, as poleward movement of chromosomes during anaphase is severely affected in Drosophila zw10 mutant cells (Savoian et al., 2000; Sharp et al., 2000).
Lastly, recognizing a bi-functional role for the ZW10 complex in recruitment of Mad1Mad2 heterodimers to unattached kinetochores and their removal after attachment, combined with a role as a dynein linker for anaphase chromosome movement, it is clear that compromised function of the complex would be predicted to accelerate chromosome missegregation. Indeed, mutations in all three components of the complex are found in highly aneuploid colorectal cancers (Wang et al., 2004b), implicating disruption of one (or both) roles of this bi-functional complex as likely contributors to generation of aneuploidy in human disease.
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Materials and methods |
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Identification of XZW10 and XRod cDNA
A radiolabeled 1.2-kb fragment of an EST clone containing 798 bp of 3' mouse ZW10 cDNA sequence was used to probe 106 plaques from a ZAP II cDNA library constructed from stage 2830 (tailbud tadpole) head. One clone containing a 1.2-kb fragment of X-ZW10 cDNA was used to rescreen the same library and identified a clone containing an additional 700 bp of X-ZW10 cDNA. This clone was then used to rescreen the library which resulted in the identification of a clone containing full-length X-ZW10 cDNA, which was subcloned into pGEX. Two Xenopus sequences for X-Rod were identified in the EST database (clones XL107l09 [GenBank/EMBL/DDBL accession no. BJ068773] and 4202179 [GenBank/EMBL/DDBL accession no. BG233529] and were found to contain 1,716 bp or 1,836 bp of the most 3' X-Rod coding sequence, respectively. The most 5' 400-bp fragment of clone 4202179 was used to screen the cDNA library described above and several other libraries, but repeatedly failed to identify additional clones. All clones were verified by automated sequencing.
Antibody production
X-ZW10 and XL107l09 were subcloned into pFastBac-HT (Invitrogen) to produce (His)6-X-ZW10 and (His)6-X-RodCOOH baculovirus, respectively, that were used to infect Hi5 insect cells. 48 h after infection, cells were lysed and X-ZW10 and X-RodCOOH were purified using nickel agarose and injected into New Zealand rabbits to produce pAb 1348 and 1351, respectively. Serum from these rabbit was affinity purified by binding to recombinant protein immobilized on CNBr-activated sepharose (Sigma-Aldrich). pAb to human Mad2 was produced by injecting rabbits with (His)6-tagged full-length human Mad2 purified from bacteria. The resulting serum was affinity purified on a HiTrap-NHS column (Amersham Biosciences).
siRNA-mediated depletion of ZW10 and Zwint-1
Double-stranded 19 nt RNA duplex to ZW10 (5'-UGAUCAAUGUGCUGUUCAA-3') and Control siRNA duplex IX were purchased from Dharmacon. Duplexes were transfected at 200 nM using Oligofectamine (Invitrogen) according to the manufacturer's protocol. pSUPERpuro-Zwint-1 was constructed as described previously (Brummelkamp et al., 2002) using the sequence: 5'-GCACGUAGAGGCCAUCAAA-3'. Plasmids were transfected using Effectene reagent (QIAGEN). Untransfected cells were removed from the cell population 24 h after transfection by treatment with puromycin (1 µg/ml) for 2 d.
Affinity purification of ZW10 and Zwint-1 complexes
HeLa cells stably expressing ZW10LAPtag or Zwint-1LAPtag were established as described previously (Shah et al., 2004). In short, ZW10LAPtag and Zwint-1LAPtag were subcloned to pBabe-blasticidin, and retrovirus was produced from 293-GP cells and used to infect HeLa cells. After 2 wk of selection with blasticidin (2 µg/ml), single cells clones were selected by flow cytometry and analyzed for EYFP localization. Clones LZ5, LZ7, LZ2.8 (ZW10LAPtag), LINT2.8, and LINT 2.14 (Zwint-1LAPtag) were grown in 20 15-cm dishes each and blocked in 200 ng/ml nocodazole for 20 h. Cells were collected and mixed for further purification of native complexes as described previously (Cheeseman et al., 2004). Mass spectrometry was conducted essentially as described previously (Cheeseman et al., 2002). The samples were analyzed using a ThermoFinnigan LTQ mass spectrometer using Mudpit with the following gradients of 500 mM ammonium acetate: 10, 25, 35, 50, 65, 80, and 100%. Results were searched by Sequest against an ncbi human-mouse-rat database and filtered with DTA Select.
Cell culture
HeLa cells were grown in DME supplemented with 5 µg/ml pen/strep (Invitrogen) and 10% FBS. Nocodazole was used at 200 ng/ml.
Xenopus extracts
CSF-arrested extracts were prepared from unfertilized Xenopus eggs as described previously (Murray, 1991). Checkpoint extracts were prepared from these by a 30-min incubation with 12,000 demembranated sperm nuclei/µl and 10 µg/ml nocodazole (Chen and Murray, 1997). Exit from CSF arrest was induced by addition of 0.5 mM CaCl2. For immunodepletion, 100 µg (antiX-ZW10) or 50 µg (antiX-RodCOOH) of affinity-purified antibody or nonimmune rabbit IgG were bound to 100 µl Dynabeads protein A (Dynal). 100 µl CSF egg extract was added for 1 h at 4°C. Immunodepletion of X-BubR1 was done as described previously (Mao et al., 2003). Checkpoint establishment experiments were done by depleting CSF extract for 1 h before adding sperm and nocodazole, whereas checkpoint maintenance experiments were done by depleting extracts for 1 h after sperm and nocodazole were added. Immunofluorescence with Xenopus egg extracts was performed as described previously (Abrieu et al., 2000), using absence of X-BubR1 signal at kinetochores as a marker for the successful depletion of X-ZW10 or X-Rod. Immunoprecipitation was performed as described previously (Mao et al., 2003). Antibodies to X-CENPE (1:200), X-BubR1 (1:1,000), X-Mps1 (1:200), and X-Mad2 (1:200) have been described previously (Abrieu et al., 2000, 2001; Mao et al., 2003). AntiX-CENPA (1:100) was a gift of A. Straight (Stanford University, Stanford, CA), antiX-KCM1 (1:100) was a gift of A. Desai (Ludwig Institute for Cancer Research, La Jolla, CA), and antiX-Mad1 (1:200) was a gift of R.-H. Chen (Cornell University, Ithaca, NY).
Immunolocalization
Images were acquired on a DeltaVision deconvolution microscope (Applied Precision) equipped with a CoolSnap CCD camera (Roper Scientific) at 20°C. z-sections were acquired at 2-µm steps using a 100x, 1.3 NA Olympus U-Planapo objective with 1 x 1 binning and 640 x 640 pixel area. All images are z-stack projections. HeLa cells, grown on poly-L-lysinecoated coverslips, were washed once in MTSB buffer (100 mM Pipes, 1 mM EGTA, 1 mM MgSO4, 30% glycerol) and preextracted in MTSB/0.5% Triton X-100 at 37°C for 5 min, unless stated otherwise. Cells were fixed with ice-cold methanol for 10 min and all coverslips were blocked in PBS/3% BSA for 1 h. Primary antibody incubations were in PBS/3% BSA for 1 h at RT, followed by four washes of PBS/0.1% Triton X-100. Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories and diluted 1:200 in PBS/3% BSA. Coverslips were washed, submerged in PBS containing DAPI, washed once with PBS, and mounted using ProLong antifade reagent (Molecular Probes). Anti-Bub1 (1:500) was purchased from Abgent. Anti-GFP was used at 1:1,000. Anti-centromere antiserum (ACA; 1:100) was purchased from Antibodies, Inc. Anti-hZw10 (1:50) was described previously (Hirose et al., 2004), anti-hZwint1 (1:100) was a gift of M. Goldberg (Cornell University), anti-BubR1 (5F9; 1:500) was a gift of S. Taylor (University of Manchester, Manchester, UK), and mouse anti-Mad1 (9B10; 1:100) was a gift of T. Yen (Fox Chase Cancer Center, Philadelphia, PA).
Quantitation of immunofluorescence
Images acquired on the DeltaVision microscope (see previous section) were quantitatively deconvolved, converted to maximum projections and saved as 16-bit TIFF files without scaling, after which ACA staining was used to draw regions containing all kinetochores using MetaMorph software. Regions were then transferred onto images of Mad1, Mad2, or Bub1 to be quantitated. Area, average pixel intensity, and integrated intensity of each kinetochore-containing region (KCR) was acquired and exported to Microsoft Excel. Values for a 400-pixel square background region from each cell were also obtained and several background values for each cell were averaged. Integrated intensity was calculated by multiplying the area of the KCR times the average intensity of the background square and subtracting this value from the total integrated intensity of the KCR. Resulting integrated intensities were normalized against the highest value obtained and then averaged and graphed.
Immunoprecipitation and immunoblotting
Immunoprecipitation of Zwint-1LAPtag was done as described for the affinity purification, using rabbit anti-GFP antibody coupled and cross-linked to Affi-Prep protein A support (Bio-Rad Laboratories). The matrix was washed and resuspended in 2x Laemmli sample buffer. SDS-PAGE and Western blotting were standard. Antibodies and dilutions used were anti-GFP (1:1,000), anti-tubulin (DM1a; 1:2,000), and anti-hZW10 (1:500). All antibodies to Xenopus proteins were at 1:1,000.
Flow cytometry
Cells were washed once with PBS and fixed in 70% ice-cold ethanol for 16 h. Cells were washed with PBS/0.1% Triton X-100 (PBST), incubated with antiphospho-Ser10-Histone H3 (Upstate Biotechnology) in PBST (final concentration of 3 µg/ml) for 1 h on ice and washed again with PBST. Incubation with FITC-conjugated donkey-antirabbit secondary antibody (Jackson ImmunoResearch Laboratories) was for one hour on ice and in the dark. After a final wash with PBST, DNA was stained with propidium iodide and measured on a FACS-Vantage flow cytometer (Becton Dickinson).
Colony outgrowth assay
To measure general toxicity of ZW10 and Zwint-1 depletion, cells were transfected with siRNA duplexes and 4 d after transfection cells were split to different confluencies. 16 h after that, cells were retransfected with the oligos. Surviving colonies were stained with crystal violet and counted 7 d after retransfection.
Online supplemental material
Fig. S1 shows the alignment of Xenopus ZW10 and Rod-COOH with human orthologues. Fig S2 shows the characterization of subcellular location of X-Rod in mitotic Xenopus XL177 cells. Fig. S3 shows the immunolabeling of the X-ZW10X-Rod complex on Xenopus sperm DNA in X-Roddepleted Xenopus egg extracts. Fig. S4 shows the time course of siRNA-mediated depletion of ZW10 in HeLa cells. Fig. S5 shows the CENP-E kinetochore localization in ZW10-depleted HeLa cells. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200411118/DC1.
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Acknowledgments |
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This work has been supported by grants from the National Institutes of Health to D.W. Cleveland (GM 29513) and to J.R. Yates III (RR11823-09). G.J.P.L. Kops is supported by a fellowship from the Dutch Cancer Society (KWF Kankerbestrijding). Salary support for D.W. Cleveland is provided by the Ludwig Institute for Cancer Research.
Submitted: 19 November 2004
Accepted: 4 March 2005
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