Decision letter: Molecular basis of outer kinetochore assembly on CENP-T

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm. https://doi.org/10.7554/eLife.21007.001 Introduction Accurate chromosome segregation in eukaryotes requires the coordinated action of hundreds of proteins. Subsets of these assemble on centromeric chromatin that is epigenetically specified by the enrichment of centromeric protein A (CENP-A), a variant of Histone H3 (Guse et al., 2011). These assemblies, named kinetochores, form the major point of attachment between centromeres and the mitotic or meiotic spindle and couple the force of depolymerizing microtubules to chromosome movement (Cheeseman, 2014). Kinetochores also function as signaling platforms for the spindle assembly checkpoint and delay the onset of chromosome segregation in the presence of erroneous chromosome-spindle attachments (Musacchio, 2015). The structural core of the kinetochore assembles from two large multi-subunit complexes, the CCAN (constitutive centromere associated network), and the KMN (KNL1 complex, MIS12 complex, NDC80 complex, abbreviated as KNL1C, MIS12C, and NDC80C), which are respectively proximal and distal to the centromeric chromatin (Pesenti et al., 2016). The affinity of the kinetochore for microtubules in the KMN is predominantly mediated by the NDC80 complex, a heterotetramer with an approximately 50 nm long coiled coil region that separates the microtubule-binding calponin homology (CH) domains of the NDC80 and NUF2 subunits from the SPC24 and SPC25 subunits (Ciferri et al., 2005; Wei et al., 2005, 2007; Ciferri et al., 2008). NDC80C is essential to form stable regulated kinetochore-microtubule interactions and its localization at the outer kinetochore is thus a prerequisite for faithful chromosome segregation (DeLuca et al., 2005, 2006; Cheeseman et al., 2006). The recruitment of NDC80C requires the CCAN subunits CENP-C and CENP-T (Liu et al., 2006; Okada et al., 2006; Kwon et al., 2007; Carroll et al., 2010; Gascoigne et al., 2011; Hori et al., 2013) as well as a nuclear envelope breakdown and the activity of mitotic kinases (Gascoigne and Cheeseman, 2013). Whereas CENP-C directly binds to CENP-A containing nucleosomes (Carroll et al., 2010) and has been proposed to act as a blueprint for further kinetochore assembly (Klare et al., 2015), CENP-T is integrated in a CENP-TWSX complex that requires its DNA-binding activity (Hori et al., 2008; Nishino et al., 2012), as well as interactions with the CCAN (Carroll et al., 2010; Basilico et al., 2014; Logsdon et al., 2015; Samejima et al., 2015; Suzuki et al., 2015a; Pekgöz Altunkaya et al., 2016), to localize to the kinetochore. Depletions of CENP-C or CENP-T, or prevention of Cyclin-dependent kinase (CDK) phosphorylation of CENP-T all affect proper recruitment of MIS12C and NDC80C to the kinetochore (Gascoigne et al., 2011; Gascoigne and Cheeseman, 2013; Hori et al., 2013; Kim and Yu, 2015; Rago et al., 2015; Suzuki et al., 2015a). Studying the respective contribution of CENP-C and CENP-T to outer kinetochore assembly, however, has been complicated by their interdependent localization and their distinct regulation by post-translational modifications. Experiments that targeted either CENP-C or CENP-T to an ectopic chromatin array uncoupled these requirements and demonstrated the ability of both pathways to recruit outer kinetochore components (Gascoigne et al., 2011; Hori et al., 2013). An important conclusion from these previous studies is that CENP-C and CENP-T recruit NDC80C in distinct ways (Figure 1A). The CENP-C-dependent axis, which is now understood in detail, relies on a direct interaction of CENP-C with the MIS12 complex (MIS12C) (Screpanti et al., 2011; Przewloka et al., 2011; Dimitrova et al., 2016; Petrovic et al., 2016), which further recruits NDC80C via a direct interaction (Cheeseman et al., 2006; Petrovic et al., 2010). Figure 1 with 1 supplement see all Download asset Open asset Phosphorylation of CENP-T2-101 at T11 or T85 is sufficient for the binding of SPC24:SPC25. (A) Schematic representation of CENP-C and CENP-T recruiting MIS12C:NDC80C and NDC80C. (B) Besides the histone-fold domain (HFD) at the carboxy terminus, CENP-T mainly consists of regions of compositional bias, rich in polar residues, and likely to be intrinsically disordered. The boundaries of CENP-T constructs used in this study and domains involved in the binding of NDC80C and DNA/CCAN are indicated. In vitro phosphorylated residues in CENP-T1-101 are marked by a P (see also Table 1). (C) The binding between SPC24:SPC25 and CENP-T phosphopeptides was determined by isothermal titration calorimetry. The y-axis indicates kcal/mole of injectant. Dissociation constants (Kd) between SPC24:SPC25 and phosphopeptides containing T11 and T85 were determined to be 45 μM and 4 μM respectively. (D) SDS-PAGE analysis of various CENP-T2-101 mutants that were incubated with SPC24:SPC25 and separated by analytical size-exclusion chromatography. Red asterisks indicate mutated phosphorylation sites. See Figure 1—figure supplement 1 for the entire dataset. https://doi.org/10.7554/eLife.21007.002 The CENP-T-dependent pathway, on the other hand, remains less well characterized. It is firmly established that CENP-T interacts directly with NDC80C, with (humans and chicken) or without (budding yeast) prior phosphorylation by CDK1:Cyclin B of sequence motifs in the CENP-T N-terminal region (Gascoigne et al., 2011; Schleiffer et al., 2012; Malvezzi et al., 2013; Nishino et al., 2013; Pekgöz Altunkaya et al., 2016). Complicating the picture, however, CENP-T also contributes to kinetochore recruitment of MIS12C (Kim and Yu, 2015; Rago et al., 2015). This may appear surprising, because MIS12C and CENP-T bind to NDC80C in a competitive manner (Schleiffer et al., 2012; Nishino et al., 2013), suggesting that they can only recruit NDC80C independently from each other. Whether the interaction of CENP-T with MIS12C is direct, and compatible with CENP-T binding to NDC80C, is therefore currently unclear. The ability of kinetochores to form multiple low-affinity linkages with microtubules is at the basis of popular models of kinetochore-microtubule attachment such as the Hill’s sleeve (Hill, 1985). It is also plausible that crucial regulatory aspects of kinetochore-microtubule attachment, such as the tension-dependent regulation required for correcting erroneous attachments or for stabilization of correct ones, depend on the effective number and distribution of linkages, as recently proposed in elegant modeling studies (Zaytsev et al., 2014). A detailed understanding of the stoichiometry of outer kinetochore composition and of its regulation is therefore crucial. To address this question, we have embarked on an effort of biochemical reconstitution of kinetochores. This recently allowed us to report that the CENP-A nucleosome recruits two copies of the CCAN complex (Weir et al., 2016). In this study, we address instead the composition and regulation of the interactions between CCAN and the outer kinetochore, focusing on the interactions of CENP-T, MIS12C, and NDC80C. CDK1:Cyclin B phosphorylation reactions with systematically mutated CENP-T substrates revealed that the phosphorylation of CENP-T at Thr11 and Thr85 results in the recruitment of two NDC80Cs. We also report that CENP-T interacts directly with MIS12C. This requires phosphorylation of CENP-T at Ser201, which is encompassed within a non-canonical CDK1 consensus sequence. This CENP-T-bound MIS12C recruits additional NDC80C and KNL1C complexes. Importantly, CENP-C and CENP-T bind MIS12C in a competitive manner and form separate outer kinetochore recruitment modules. Thus, we have reconstituted and visualized the molecular basis of CENP-T mediated assembly of the outer kinetochore and its phospho-regulation. Results Phosphorylation of CENP-T at Thr11 or Thr85 is sufficient to recruit SPC24:SPC25 To obtain insight into the molecular mechanism of CENP-T mediated recruitment of the human outer kinetochore, we set out to reconstitute this process using purified components. Previous studies identified multiple phosphorylation sites in CENP-T and showed that its phosphorylation contributes to the recruitment of NDC80C (Gascoigne et al., 2011; Kettenbach et al., 2011; Gascoigne and Cheeseman, 2013; Nishino et al., 2013; Rago et al., 2015). Confirming this, we identified by mass spectrometry numerous residues in CENP-T that were in vitro phosphorylated by CDK1:Cyclin B (Table 1). These included the CDK target sites Thr11, Thr27, Ser47, and Thr85 in the N-terminal region of CENP-T, a region known to interact with the SPC24:SPC25 subunits of NDC80C (Malvezzi et al., 2013; Nishino et al., 2013) (Figure 1B). We used isothermal titration calorimetry to test the ability of four short synthetic phosphopeptides encompassing these residues to bind SPC24:SPC25. Peptides containing phosphorylated Thr11 or Thr85 showed a binding affinity of 45 μM and 4 μM, respectively, whereas peptides containing phosphorylated Thr27 or Ser47 did not show an interaction with SPC24:SPC25 (Figure 1C). Although the determined affinities are modest and unlikely to fully represent the binding affinities between full-length CENP-T and SPC24:SPC25, they distinguish bona fide binding sites from sites that do not interact. Table 1 Phospho-sites on CENP-T2-373 in vitro phosphorylated by CDK1:Cyclin B. Identified phospho-peptides with a localization probability for the phospho-site >80% and an Andromeda search engine score >100 are shown. All peptides have a posterior error probability (PEP) below 1 × 10−25. The last column indicates if sites were identified as single (S) and/or double (D) phospohorylated peptides. https://doi.org/10.7554/eLife.21007.004 PositionPeptide with pS or pTScoreP-sites8MADHNPDpSDSpTPRTLLR144S and D10MADHNPDSDpSTPRTLLR209S11MADHNPDpSDpSpTPRTLLRR209S and D27VLDTADPRpTPR135S45RALLEpTApSPRK112D47RALLEpTApSPRK112S and D78SAHIQApSGHLEEQpTPR101S and D85SAHIQApSGHLEEQpTPR167S and D184LSVFQQGVDQGLSLSQEPQGNADASpSLTR129S195SLNLTFApTPLQPQpSVQRPGLAR152S and D201SLNLTFApTPLQPQpSVQRPGLAR186S and D Confirming these equilibrium binding experiments, the interaction between an N-terminal fragment of purified CENP-T and SPC24:SPC25 depended on the phosphorylation of CENP-T by CDK1:Cyclin B (Figure 1D and Figure 1—figure supplement 1). We next generated a set of CENP-T2-101 constructs in which three of the four CDK phosphorylation sites were mutated to Alanine. Constructs retaining either Thr11 or Thr85 bound to SPC24:SPC25 in a phosphorylation dependent manner, whereas constructs retaining either Thr27 or Ser47, or having all four sites mutated to Alanine, did not bind SPC24:SPC25 (Figure 1D and Figure 1—figure supplement 1). Phosphorylation of CENP-T at Thr11 or Thr85 is thus sufficient to bind SPC24:SPC25. These results are consistent with in vivo experiments showing that ectopic chromosome anchoring of a CENP-T1-250 fragment results in the recruitment of NDC80C when either Thr11 or Thr85 were mutated to Alanine, but not when both were mutated (Rago et al., 2015). CENP-T coordinates two NDC80 complexes in a single complex The finding that phosphorylation of CENP-T at either Thr11 or Thr85 is sufficient to recruit SPC24:SPC25, both in vivo (Rago et al., 2015) and in vitro (Figure 1), raises the question whether phosphorylated CENP-T can bind two SPC24:SPC25 units simultaneously. This possibility is supported by the change in retention volume and the apparent stoichiometry of the CENP-T2-101:SPC24:SPC25 complex (Figure 1D). We set out to test the stoichiometry of CENP-T:SPC24:SPC25 complexes further using a CENP-T2-373 fragment that we C-terminally labeled with a fluorescent dye to specifically follow CENP-T during chromatography and SDS-PAGE (Figure 2). This is helpful because CENP-T2-373, which lacks Tryptophan and has only one Tyrosine, absorbs poorly at 280 nm. Fluorescently labeled CENP-T2-373 (hereafter CENP-T) was in vitro phosphorylated by CDK1:Cyclin B and subsequently incubated with a threefold molar excess of SPC24:SPC25. CENP-T was part of a broad peak with two SPC24:SPC25-containing species with approximate retention volumes of 1.2 and 1.35 ml (Figure 2, green traces). Consistent with the idea that the complex eluting at 1.2 mL contained two SPC24:SPC25 molecules per CENP-T, this species was not observed for CENP-T containing the T11A or the T85A mutation (Figure 2, orange traces). CENP-T that was mutated at both Thr11 and Thr85 or that was not phosphorylated, did not form a complex with SPC24:SPC25 (Figure 2, red and black traces). Figure 2 Download asset Open asset CENP-T phosphorylated at T11 and T85 binds two copies of SPC24:SPC25. Analytical size-exclusion chromatography (Superdex 200 5/150 increase) and SDS-PAGE show that CENP-T2-373 phosphorylated by CDK1:Cyclin B at positions T11 and T85 can bind two copies of SPC24:SPC25. CENP-T was fluorescently labeled with FAM (wt) or TMR (mutants) and monitored specifically during chromatography and after SDS-PAGE. Red asterisks indicate mutated phosphorylation sites. https://doi.org/10.7554/eLife.21007.005 Using a similar experimental setup, we determined that CENP-T also binds full-length NDC80Cs in a manner that depends on the phosphorylation of Thr11 and Thr85 (Figure 3A and Figure 3—figure supplement 1). We next set out to visualize these assemblies directly by electron microscopy (EM) after low-angle metal shadowing. This technique enhances the contrast of the coiled-coil regions of NDC80C, enabling a characterization of the overall dimensions of complexes containing NDC80C. The coiled-coil of NDC80C spans 51 nm and the NDC80:NUF2 - SPC24:SPC25 end-to-end length is 62 nm (corrected for an estimated 3 nm shadow contribution at both ends) (Figure 3B and Figure 3—figure supplement 2). This is slightly longer than the 57 nm of yeast NDC80C previously determined using the same technique (Wei et al., 2005). The difference might be explained by the use of full-length human NDC80C whereas the used yeast version lacked the N-terminal 100 residues of the Ndc80p subunit, although interspecies or technical differences cannot be excluded. The low-resolution and the flexible appearance of the NDC80C prevent unambiguous assignment of the SPC24:SPC25 and the NDC80:NUF2 modules of the complexes, although a clear kink in the coiled coil, previously attributed to a sequence insertion in NDC80 (Ciferri et al., 2008) marks NDC80:NUF2 in a number of cases (Figure 3B and Figure 3—figure supplement 2). Figure 3 with 4 supplements see all Download asset Open asset Phosphorylated CENP-T recruits two full-length NDC80 complexes. (A) Analytical size-exclusion chromatography (Superose 6 5/150) and SDS-PAGE show that CENP-T2-373 phosphorylated by CDK1:Cyclin B at positions T11 and T85 can bind two full-length NDC80 complexes. The mixture of CENP-T and NDC80C contained NDC80C, CENP-T:NDC80C, as well as CENP-T:NDC80C2 species (green trace). The specific monitoring of fluorescently labeled CENP-T was used to distinguish these species. Red asterisks indicate mutated phosphorylation sites. Analysis of the single T11A and T85A CENP-T mutants and additional controls are included in Figure 3—figure supplement 1. (B) NDC80C (top row) and CENP-T:NDC80C2. (middle and bottom rows) were visualized by electron microscopy after glycerol spraying and low-angle platinum shadowing. Asterisks mark the kink in the NDC80:NUF2 coiled coil region. The first micrographs show a representative field of view at a lower magnification. All scale bars represent 50 nm. More micrographs of NDC80C and CENP-T:NDC80C2 as well as sample preparation information are included in Figure 3—figure supplements 2–4. https://doi.org/10.7554/eLife.21007.006 Metal-shadowed particles from an early eluting size-exclusion chromatography (SEC) fraction of the CENP-T:NDC80C mixture had a more heterogeneous appearance than NDC80C in isolation (Figure 3B and Figure 3—figure supplements 3 and 4). We were able to distinguish at least two different kinds of complexes, containing either one or two NDC80Cs. Though it is not possible to ascertain if complexes with one discernible NDC80 contain CENP-T, those containing two NDC80Cs are presumably assembled on phosphorylated CENP-T. This interpretation is further supported by the distance between the base of the complexes and the characteristic kinks in the coiled coil of NDC80:NUF2 (Figure 3B and Figure 3—figure supplement 4). The apparent orientation of the two CENP-T-bound NDC80Cs widely varies, presumably because the CENP-T11-85 stretch spanning the two NDC80C binding sites is flexible. In brief, we visualized how CENP-T recruits two NDC80Cs. The SPC24:SPC25 modules of these complexes are coordinated by the relative proximity of the phosphorylated Thr11 and Thr85 of CENP-T, but the microtubule-binding CH domains can be well over 100 nm apart. CENP-T phosphorylated at Ser201 stoichiometrically binds the MIS12 complex It had previously been shown that MIS12C and the N-terminal region of CENP-T bind SPC24:SPC25 in a competitive manner (Schleiffer et al., 2012; Nishino et al., 2013). Consistent with these previous reports, MIS12C bound SPC24:SPC25, displacing CENP-T2-101, already at equimolar concentrations, indicative of higher binding affinity (unpublished data). In apparent contradiction with these observations, however, it has also been reported that the phosphorylation of human CENP-T on Thr195, within a weakly conserved CDK1 consensus motif (Figure 4A), contributes to the recruitment of MIS12C to CENP-T in vivo (Rago et al., 2015). Furthermore, the CENP-T200-230 region has been identified as a crucial determinant of the CENP-T:MIS12C interaction in vivo (Rago et al., 2015), indicating that residues downstream of Thr195 are also important for MIS12C binding. Figure 4 with 4 supplements see all Download asset Open asset CENP-T phosphorylated by CDK1 at position S201 binds the MIS12 complex. (A) Multiple sequence alignment of the CENP-T184-215 region. The CENP-T length in the various species, as well as the T195 and S201 phosphosites are indicated. The indicated non-canonical CDK1 recognition site, to which S201 and neighboring residues conform perfectly, and the shown CDK1 minimal canonical T/SP site were found to be equally good CDK1 substrates (Suzuki et al., 2015b). (B) Analytical size-exclusion chromatography (Superose 6 5/150 increase) shows that CENP-T184-215 binds to MIS12C when phosphorylated at position S201. Dashed traces indicate absorption at 495 nm and represent peptide that co-elutes with MIS12C. Free peptides have a retention volume of 2.6 ml. Peak fractions were analyzed by Coomassie staining and in-gel fluorescence after SDS-PAGE. (C) Analytical size-exclusion chromatography (Superdex 200 5/150 increase) and SDS-PAGE show that CENP-T2-373 phosphorylated by CDK1:Cyclin B at S201 binds to MIS12C. Red asterisks indicate mutated phosphorylation sites. Additional controls are included in Figure 4—figure supplement 2. (D) Sedimentation velocity analytical ultracentrifugation of MIS12C, CENP-T, and MIS12C:CENP-T show that MIS12C and CENP-T bind in a 1:1 complex. MIS12C was monitored by its absorbance at 280 nm whereas CENP-T and the CENP-T:MIS12C mixture were followed using the absorbance of the fluorescently labeled CENP-TTMR at 555 nm. https://doi.org/10.7554/eLife.21007.011 Thr195 is flanked by a well-conserved downstream motif responding to the consensus PXSVXR, where X are positions where significant sequence variability is observed (Figure 4A). This motif conforms to a recently identified non-consensus CDK1 substrate signature (Suzuki et al., 2015b). Furthermore, the presence of Pro at the −2 position relative to the phospho-acceptor site has been previously recognized as a favorable signature for CDK1 phosphorylation, while Val at the +1 position is among the least unfavorable residues (Alexander et al., 2011). Both Thr195 and Ser201 have been previously identified as mitotic CENP-T phosphorylation sites (Kettenbach et al., 2011), and we confirmed that these residues are also found phosphorylated in CENP-T pulled down with a specific antibody from mitotic HeLa cells (Figure 4—figure supplement 1). Both sites were effectively phosphorylated by CDK1 in vitro (Table 1 and Figure 4—figure supplement 1). To assess if CENP-T binds directly to MIS12C, and if phosphorylation of Thr195 or Ser201 is important for this interaction, we generated synthetic fluorescent peptides encompassing residues CENP-T184-215 and containing phosphorylated Thr195 or Ser201, incubated them with MIS12C, and analyzed the mixture by SEC. The peptide carrying phosphorylated Ser201 bound directly to MIS12C in vitro, whereas the equivalent peptide containing phosphorylated Thr195 did not (Figure 4B). The importance of CENP-T phosphorylation at position Ser201 was recapitulated for the longer CENP-T2-373 construct, where mutation of this residue was shown to abrogate binding to MIS12C (Figure 4C and Figure 4—figure supplement 2). Alanine mutation of the phospho-targets Thr11 and Thr85 (Figure 4C) or Thr195 (Figure 4—figure supplement 3) did not affect the binding between CENP-T and MIS12C. Phosphorylation of CENP-T at position Ser201 is thus both required and sufficient to mediate the MIS12C:CENP-T interaction in vitro. Although results the possibility that Ser201 is phosphorylated in vitro by a than by CDK1:Cyclin to this the CDK1:Cyclin B sample we used is (Figure 4—figure supplement and of other as by mass spectrometry (unpublished data). phosphorylation of CENP-T Ser201 was also observed using a CDK1:Cyclin B preparation (Figure 4—figure supplement the of the CDK1 specific the overall phosphorylation of CENP-T by CDK1:Cyclin B by the of CENP-T on Figure 4—figure supplement 4B). the specific phosphorylation of CENP-T at Ser201 and the CENP-T:MIS12C interaction, Figure 4—figure supplement We further the interaction between CENP-T and MIS12C using velocity analytical ultracentrifugation Both MIS12C and CENP-T with indicative of an or of of a respectively (Figure To the of the CENP-T:MIS12C we specifically monitored absorbance of the fluorescently labeled CENP-T and thus analyzed of CENP-T:MIS12C without MIS12C. The CENP-T:MIS12C complex with a of and a determined mass in with the mass of a 1:1 complex (Figure Phosphorylation of CENP-T Ser201 is important for MIS12C recruitment in vivo To how CENP-T phosphorylation at various positions kinetochore recruitment of NDC80C and MIS12C in we a previously established (Gascoigne et al., and proteins to an integrated array in previously reported (Gascoigne et al., 2011; Rago et al., 2015), NDC80C and MIS12C were to CENP-T in and not in (Figure in with the observed recruitment of these complexes to CENP-T in vitro 3 and 4). The NDC80C binding binds MIS12C in vitro (Figure but in the of a it to recruit not only NDC80C, but also MIS12C, to the as previously reported (Rago et al., 2015) (Figure on the other hand, NDC80C to the but longer with MIS12C (Figure This is consistent with the for phosphorylation of Ser201 for MIS12C recruitment in vitro (Figure and with the previous of the CENP-T200-230 region as a crucial determinant of the CENP-T:MIS12C interaction in vivo (Rago et al., 2015). an of Thr195 of CENP-T recruitment of both the MIS12C and the NDC80C to the ectopic site, if this residue is for MIS12C or NDC80C binding in vitro (Figure Figure 4—figure supplement and Figure We therefore that Thr195 contributes to the of phosphorylation or to the of CENP-T in an that is not recapitulated in vitro, where this residue to be for MIS12C binding. data the importance of Ser201 of CENP-T as a MIS12C binding site both in vitro and in Figure Download asset Open asset MIS12C and NDC80C are to CENP-T in (A) cells that contain a array recruit constructs the cell NDC80C and MIS12C are to ectopic CENP-T in but not in kinetochores are marked by was used to the and the indicates or constructs and marks the position of the bars represent for the and 2 for the (B) cells or or (C) The and were determined from three experiments and to the for the A of cells were analyzed per and error bars indicate error of CENP-T recruits one MIS12 complex and three NDC80 complexes To test directly if MIS12C that is bound to phosphorylated CENP-T its ability to bind NDC80C, we to reconstituted and incubated different constructs of CENP-T with MIS12C and a molar excess of NDC80C. Phosphorylated CENP-T integrated in large complexes volume from the column was than that of MIS12:NDC80 complexes. Our set of CENP-T mutants a analysis of these In to observations for CENP-T:NDC80C (Figure 3) and CENP-T:MIS12C (Figure these experiments showed that CENP-T with mutated motifs and associated with MIS12C:NDC80C (Figure red trace). This depended on the phosphorylation of CENP-T at Ser201 because the CENP-T longer a complex with MIS12C or NDC80C (Figure trace). results that with MIS12C and NDC80C in or with MIS12C and NDC80C in and 3) with MIS12C and NDC80C in The of the set of CENP-T mutants upon with MIS12C and/or NDC80C is in Figure supplement 1). Thus, MIS12C that interacts with CENP-T phosphorylated at Ser201 can also interact with NDC80C. This complex was also able to KNL1C, indicating that it can bind an entire KMN assembly, as is the for MIS12C bound to CENP-C et al., (Figure supplement 2). KNL1C is to the outer kinetochore in a and manner in and if this contributes to spindle assembly checkpoint remains to be Figure 6 with 4 supplements see all Download asset Open asset and of CENP-T:MIS12C bound to three full-length NDC80 complexes. (A) Analytical size-exclusion chromatography (Superose 6 5/150 increase) and SDS-PAGE show that CENP-T2-373 phosphorylated by CDK1:Cyclin B can bind MIS12C and three full-length NDC80 complexes. The mutation of and S201 is sufficient to prevent CENP-T from interact