Higher-order protein assembly controls kinetochore formation

doi:10.1038/s41556-023-01313-7

. 2024 Jan;26(1):45-56.

doi: 10.1038/s41556-023-01313-7. Epub 2024 Jan 2.

Higher-order protein assembly controls kinetochore formation

Gunter B Sissoko^#^{1

2}, Ekaterina V Tarasovetc^#³, Océane Marescal^{1

2}, Ekaterina L Grishchuk⁴, Iain M Cheeseman^{5

6}

Affiliations

¹ Whitehead Institute for Biomedical Research, Cambridge, MA, USA.
² Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
³ Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁴ Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. gekate@pennmedicine.upenn.edu.
⁵ Whitehead Institute for Biomedical Research, Cambridge, MA, USA. icheese@wi.mit.edu.
⁶ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. icheese@wi.mit.edu.

^# Contributed equally.

PMID: 38168769
PMCID: PMC10842828
DOI: 10.1038/s41556-023-01313-7

Higher-order protein assembly controls kinetochore formation

Gunter B Sissoko et al. Nat Cell Biol. 2024 Jan.

. 2024 Jan;26(1):45-56.

doi: 10.1038/s41556-023-01313-7. Epub 2024 Jan 2.

Authors

Gunter B Sissoko^#^{1

2}, Ekaterina V Tarasovetc^#³, Océane Marescal^{1

2}, Ekaterina L Grishchuk⁴, Iain M Cheeseman^{5

6}

Affiliations

¹ Whitehead Institute for Biomedical Research, Cambridge, MA, USA.
² Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
³ Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁴ Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. gekate@pennmedicine.upenn.edu.
⁵ Whitehead Institute for Biomedical Research, Cambridge, MA, USA. icheese@wi.mit.edu.
⁶ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. icheese@wi.mit.edu.

^# Contributed equally.

PMID: 38168769
PMCID: PMC10842828
DOI: 10.1038/s41556-023-01313-7

Abstract

To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent outer kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with outer kinetochore proteins, oligomers that mimic centromeric CENP-T density trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Our work suggests that the density dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of inner kinetochore proteins.

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Conflict of interest statement

Competing Interests Statement

The authors have no competing interests.

Figures

**Extended Data Fig. 1. CENP-T¹⁻²⁴² oligomers interact with spindles and recruit additional outer kinetochore proteins, but control oligomers do not.**
(A) Co-localization of outer kinetochore proteins with GFP-CENP-T¹⁻²⁴²-I3–01 oligomers by immunofluorescence. Identical linear brightness adjustments were used for GFP and kinetochore protein channels for each pair of experimental and control samples. Regions enlarged in insets are indicated by dashed boxes. Full-size image scale bars=5 μm. Inset scale bars=2 μm. SKA3 experiment was repeated 4 times with similar results. CENP-A and ZW10 experiments were repeated twice with similar results. (B) Pearson correlations between GFP and kinetochore protein signals for GFP-I3–01 and GFP-CENP-T¹⁻²⁴²-I3–01. Each point is a cell; n=number of cells measured in a single experiment. Bars represent mean ± SEM.; each experiment was performed 2 times with similar results. Statistical analysis of replicates and sample sizes can be found in Supplementary table 4. P-values were calculated with Welch’s two-tailed t-tests: ZW10: p<0.0001; SKA3: p<0.0001; CENPA: p=0.0809. Source numerical data are available in Source Data.

**Extended Data Fig. 2. CENP-T¹⁻²⁴² oligomers recruit additional outer kinetochore proteins, but control oligomers do not.**
(A) Outer kinetochore and kinetochore-associated proteins detected in immuno-precipitation mass spectrometry of GFP-I3–01 control oligomers. This experiment was performed twice with similar results. (B) Peptides counts for inner kinetochore proteins detected in immunoprecipitation mass spectrometry of GFP-I3–01 and GFP-CENP-T¹⁻²⁴²-I3–01 oligomers. This experiment was performed twice with similar results.

**Extended Data Fig. 3. Characterization of GFP CENP-T¹⁻²⁴² and control GFP oligomers isolated from HeLa cells.**
(A) Workflow to isolate GFP-CENP-T¹⁻²⁴²-I3–01 and GFP-I3–01 from mitotic cells. Left: representative images of HeLa cells expressing GFP-CENP-T¹⁻²⁴² or GFP oligomers. Cells were arrested in mitosis by expression of GFP-CENP-T¹⁻²⁴²-I3–01 or with the Eg5 inhibitor S-Trityl-L-Cysteine (see Methods for details). (B) Quantification of the number of GFP molecules. Left: representative image of a microscope field with single GFP molecules immobilized on plasma-cleaned coverslip. Repeated 3 times with similar results. Middle: Example photobleaching curve for a single molecule of GFP. Right: Histogram of integral intensities collected from 60 bleaching GFP dots from N=3 independent experiments. Each point represents the frequency in one independent repeat. Red line is fit to Gaussian function. Bars represent mean ± SEM. Peak value of 1.56 ± 0.04×10⁴ a.u. is the integral intensity of a single GFP fluorophore under our imaging conditions. This intensity was used to estimate number of GFP fluorophores in oligomers and complexes, see Methods for details. (C) Representative fluorescence microscopy images of the indicated GFP-labelled oligomers immobilized on coverslips; identical microscopy settings and brightness adjustments were used. Repeated 5 times with similar results. (D) Immunofluorescence measurements of NDC80 intensity associated with CENP-T¹⁻²⁴² oligomers and GFP oligomers that bound to taxol-stabilized microtubules or did not bind to microtubules. Each point represents the median value from an independent experiment. Bars represent mean ± SEM. For microtubule-bound GFP-CENP-T¹⁻²⁴²-I3–01, N=2; for other conditions N=5. Two-tailed Welch’s t-test: GFP-CENP-T¹⁻²⁴²-I3–01 unbound vs. GFP-I3–01 unbound: p=0.2129. (E) Kymographs illustrating complex motions of CENP-T¹⁻²⁴² oligomers on dynamic microtubules. Top left: an CENP-T¹⁻²⁴² oligomer diffuses on the microtubule wall and tracks the polymerizing plus-end. Bottom left: CENP-T¹⁻²⁴² oligomer tracks a depolymerize end, then tracks the end when it reverts to polymerization. Right: Processive plus-end-directed movement. Velocity on the GMPCPP-containing seed (red): 0.7 μm/min; on GDP-containing lattice (blue) 3 μm/min. Plus-end directed motion was observed in 8 out of 80 total observations. Observations were made over 8 independent experiments. Source numerical data are available in Source Data.

**Extended Data Fig. 4. CENP-T¹⁻²⁴² oligomers and monomers have distinct localization, are expressed at comparable levels, and do not reduce outer kinetochore protein expression.**
(A) Pearson correlation and Manders overlap coefficient for GFP and α-tubulin co-localization in cells expressing GFP-CENP-T¹⁻²⁴² or GFP-CENP-T¹⁻²⁴²-I3–01. Datapoints are cells from a single experiment. Bars represent mean ± SEM. Each experiment was performed 2 times with similar results. Statistical analysis of replicates can be found in Supplementary table 4. Two-tailed Welch’s t-tests: Pearson correlation: p=0.0793; Overlap: p<0.0001. (B) Normalized GFP signals from GFP-positive cells analyzed for DNA content in Fig. 4E as measured by flow cytometry. Each point is the mean GFP signal from 3 independent experiments. Bars represent mean ± SEM. The same cell lines were used for other assays with these three constructs. (C) Histograms showing the distribution of GFP expression levels in cells from cell line in (B) as measured by flow cytometry. Repeated 3 times with similar results. (D) Western Blot for expression levels of the NDC80 complex component NDC80 in cells expressing different constructs. NDC80 was detected using an antibody against the whole complex. Anti-GFP antibody was used to show expression of the expected construct in each cell line. Beta-Actin was used as a loading control. The NDC80 complex is an obligate complex, so depletion of one component, Spc24, with siRNA resulted in a reduction in NDC80 levels. This experiment was repeated 3 times with similar results. Source numerical data and unprocessed blots are available in Source Data.

**Extended Data Fig. 5. CENP-T¹⁻²⁴² oligomers recruit kinetochore-associated proteins and spindle assembly checkpoint proteins more robustly than monomeric CENP-T^1–242.**
(A) Comparison of protein co-immunoprecipitation by CENP-T¹⁻²⁴² oligomers and control GFP oligomers by quantitative mass spectrometry. Each point represents a biological replicate from a single multiplexed mass spectrometry run. Bars represent mean ± SEM. Analysis details can be found in materials and methods. (B) Comparison of protein co-immuno-precipitation by CENP-T¹⁻²⁴² oligomers and CENP-T¹⁻²⁴² monomers by TMT-based quantitative immune-precipitation mass spectrometry. Presented and analyzed as described in (A). Two-tailed Welch’s t-test: Astrin-SKAP: p=0.2383; Spindly: p=0.0094; Mad2L1: p=0.0002; Bub1: p=0.0506; Bub3: p=0.1151; chTOG: p=0.001. (C) Comparison of protein co-immuno-precipitation by control GFP oligomers and CENP-T¹⁻²⁴² monomers by TMT-based quantitative immuno-precipitation mass spectrometry. Presented and analyzed as described in (A). Source numerical data are available in Source Data.

**Extended Data Fig. 6. Additional SunTag mass spectrometry, centromere depletion, and controls.**
(A) Normalized GFP signals from Sun Tag cells in Fig. 5C DNA content analysis as measured by flow cytometry. Each point is the mean from N=3 independent experiments. Bars represent mean ± SEM. (B) Anti-GFP western blot of cell lines expressing scFv-sfGFP-CENP-T¹⁻²⁴² different tdTomato-tagged GCN4pep scaffolds. β-Actin was used as a loading control here and in all subsequent western blots. (C) Same analysis as in (A) for tdTomato. (D) Anti-T2A western blots of Sun Tag cell lines with different tdTomato-tagged GCN4pep scaffolds. Anti-T2A antibody binds to the C-terminus of the scaffolds. Experiments in panels (A-D) were performed on cells from Fig. 5C. (E) and (F) Anti-RFP and Anti-GFP westerns blots of Sun Tag cell lines expressing tdTomato-tagged GCN4pep scaffolds used in Fig. 5D, Extended Data Fig. 6H. (G) Validation western of anti-mCherry antibodies for immunoprecitation. IN=Input, IP=Immunoprecipitation, FT=Flow-through. (H) Comparison of MIS12 and KNL1 complex abundances in anti-mCherry quantitative immunoprecipitation mass spectrometry with different Sun Tag scaffolds. Each point represents a biological replicate from 2 multiplexed experiments. Each bar represents the mean ± SEM Two-tailed Welch’s t-test: MIS12: 1 vs. 6: p=0.1083; 6 vs. 10: p=0.7135; 10 vs. 18: p=0.0011; 1 vs. 18: p=0.0008. KNL1: 1 vs. 6: p=0.3592; 6 vs. 10: p=0.1559; 10 vs. 18: p=0.0605; 1 vs. 18: p=0.003. (I) Quantification of MIS12 levels at centromeres in cells expressing the scFv-sfGFP-CENP-T¹⁻²⁴² with different GCN4pep scaffolds. Each point is a cell. Each bar represents the mean ± SEM. Measurements were pooled from 3 independent experiments. 1: n=49; 2: n=45; 3: n=47; 4: n=25; 6: n=47; 8: n=47; 10: n=51; 12: n=47. Two-tailed Welch’s t test : 1 v. 12: p<0.0001. Welch’s ANOVA test: P<0.0001. (J) and (K) Anti-NDC80 Complex and anti-GFP western blots of Sun Tag cell lines expressing scFv-sfGFP-CENP-T¹⁻²⁴² alongside different GCN4pep scaffolds. (L) Same as in (D). Experiments in panels (J-L) were performed on cells lines used in Fig. 5E, 5F, Extended Data Fig. 5I. Scaffolds in these cell lines lack the tdTomato tag. Source numerical data and unprocessed blots are available in Source Data.

**Extended Data Fig. 7. Flowcytometry gating strategy for DNA content analysis of CENP-T¹⁻²⁴² SunTag oligomers.**
Gating strategy to select the population of cells to be analyzed for DNA content analysis in Fig. 5C. A similar gating strategy was used in Fig. 4E without the tdTomato-Area parameter.

**Extended Data Fig. 8. Known N-terminal NDC80 phosphorylation sites are required for NDC80 recruitment to oligomers.**
(A) Representative images of colocalization of GFP and the NDC80 complex in cells expressing scFv-sfGFP-CENP-T¹⁻²⁴² with either wild-type (WT) CENP-T¹⁻²⁴² or CENP-T¹⁻²⁴² with T11A and T85A mutations (2A). These constructs were expressed alongside 12xGCN4pep scaffolds. (B) Pearson correlations between GFP and NDC80 signal for experiment in (A). Datapoints are cells from a single experiment. Bars represent mean ± SEM. Repeated 2 times with similar results. Statistical analysis of replicates and sample sizes can be found in Source Data. Two-tailed Welch’s t-test: p<0.0001. (C) Quantification of NDC80 levels at centromeres in cells expressing scFv-sfGFP-CENP-T^1−242/2A with different GCN4pep scaffolds. Each bar represents the mean ± SEM of NDC80 signal from cells expressing the designated construct. Measurements were pooled from 2 different experiments. n=number of cells pooled from 2 independent experiments. 1: n=27; 2: n=33; 3: n=28; 4: n=27; 6: n=31; 8: n=30; 10: n=31; 12: n=33. Welch’s ANOVA: p<0.0001. Two-tailed Welch’s t-test: 1 v. 12: p=0.0237. (D) Anti-GFP and Anti-T2A western blots of cell lines expressing different GCN4pep scaffolds alongside scFv-sfGFP-CENP-T^1−242/2A. Anti-T2A antibody binds to the C-terminus of the scaffolds. β-Actin was used as a loading control. These cell lines were used in for all experiments in the figure. This was a cell line validation experiment that was only performed once. Source numerical data and unprocessed blots are available in Source Data.

**Extended Data Fig. 9. Additional fluorescence intensity quantifications for *in vitro* CENP-T-NDC80 binding assay using recombinant oligomers and NDC80 proteins.**
(A) Top: Representative images of purified recombinant GFP-CENP-T^1−242/3D-I3–01 oligomers attached to a coverslip. Bottom: histogram of the distribution of the number of GFP molecules per oligomer as a percentage of the total number of examined oligomers. Each point represents an independent measurement. Each bar represents the mean ± SEM from 3 independent experiments. Distribution mean ± SEM: 66 ± 10 GFP molecules. (B) Same as (A) for GFP-I3–01. 3 independent experiments. Distribution mean ± SEM: 44 ± 4 GFP molecules. (C) Same as (A) for GFP-CENP-T^1−242/3D. 3 independent experiments. Distribution mean ± SEM: 1.23 ± 0.05 molecules. (D) Single molecule binding experiment with NDC80^ΔSpc24/25. Top: Experimental workflow. Bottom: representative images of GFP-CENP-T^1−242/3TD-I3–01 oligomers at each experimental stage. (E) Efficiency of NDC80^Bonsai or NDC80^ΔSpc24/25 recruitment to GFP-CENP-T^1−242/3D-I3–01 oligomers. Bars represent mean ± SEM. Each point is the median result from 3 independent experiments with >12 oligomers. Data for GFP-CENP-T^1−242/3D-I3–01 oligomer is duplicated from Fig. 6D. Two-tailed Welch’s t-test: p=0.0054. (F) Graph of the stoichiometry of binding. Final GFP signal intensity as function of initial GFP signal intensity for individual oligomers. Each point represents the measurement for one oligomer pooled from N=3 independent experiments per data set. GFP-CENP-T^1−242/3D-I3–01+NDC80^Bonsai: n=85 Oligomers; GFP-CENP-T^1−242/3D-I3–01+NDC80 ^ΔSpc24/25: n=79 Oligomers; GFP-I3–01+NDC80^Bonsai: n=91 Oligomers. Data are fitted to linear functions. The slopes (± standard fitting error) correspond to the number of NDC80 molecules recruited per GFP-containing monomer for each combination of oligomer and NDC80 complex. (G) Photobleaching curve taken with identical microscope settings to those used for experiments with GFP-CENP-T^1−242/3D (Fig. 6B, E, F). The number of GFP puncta per imaging field at each time point was normalized to the number at t=0. Data were fitted to an exponential decay function to estimate the probability of bleaching during imaging time. Each point represents the mean ± SEM from N=3 independent measurements. Dashed line indicates experimental exposure time in Fig. 6E,F. Source numerical data are available in source data.

**Fig. 1:. I3–01 oligomerization strategy generates particles that interact with mitotic spindles.**
(A) Left top: diagram of endogenous CENP-T, its key phosphorylation sites, and the sites of established interactions. Left bottom: construct used to generate CENP-T¹⁻²⁴² oligomers in cells. Right: diagrams of the expected oligomers and their predicted interactions with the outer kinetochore. (B) Representative images of CENP-T¹⁻²⁴² oligomers and control GFP oligomers in interphase HeLa cells. Scalebars=10 μm. Image brightness is not scaled identically to make the appearance and localization of constructs visible despite large differences in brightness. This experiment was repeated 7 times with similar results. (C) Representative image of control GFP oligomers and examples of CENP-T¹⁻²⁴² oligomers in mitotic HeLa cells. Scalebars=5 μm. As in (B), image brightnesses are not scaled identically. This experiment was repeated 7 times with similar results. (D) Pearson correlation and Manders overlap coefficient for GFP and α-tubulin co-localization in cells expressing GFP-I3–01 or GFP-CENP-T¹⁻²⁴²-I3–01. Each point is a biologically independent cell; n=number of cell measure in a single experiment. Bars represent mean ± SEM. Each experiment was performed 2 times with similar results. Two-tailed Welch’s t-tests: Pearson Correlation: p<0.0001; Overlap: p<0.0001. Statistical analysis of replicates and source numerical data are available in the source data.

**Fig. 2:. CENP-T¹⁻²⁴² oligomers recruit almost the entire outer kinetochore.**
(A) Representative immunofluorescence images of co-localization of outer kinetochore proteins with GFP-CENP-T¹⁻²⁴²-I3–01 oligomers and GFP-I3–01 controls. Identical linear brightness adjustments were used for GFP and kinetochore protein channels for each pair of experimental and control samples. Regions enlarged in insets are indicated by dashed boxes. Scalebars=5 μm. These experiments were repeated 5 times with similar results. (B) Pearson correlations for the co-localization between GFP and outer kinetochore signals for GFP-I3–01 and GFP-CENP-T¹⁻²⁴²-I3–01. Each point is a cell; n=number of cells measured in a single experiment. Each experiment was performed 2 times with similar results. Bars represent mean ± SEM. P-values were calculated with two-tailed Welch’s t-tests: ****=p<0.0001. Statistical analysis of replicates is available in the source data. (C) Outer kinetochore and outer kinetochore-associated proteins detected in immunoprecipitation mass spectrometry of GFP-CENP-T¹⁻²⁴²-I3–01. This experiment was performed twice with similar results. Source numerical data is available in source data.

**Fig. 3:. Isolated CENP-T¹⁻²⁴² oligomers bind to microtubules and track dynamic microtubule ends.**
(A) Representative images of GFP-CENP-T¹⁻²⁴²-I3–01 and GFP-I3–01 isolated from mitotic cells. Scalebar=5 μm. Repeated 5 times with similar results. (B) Histogram showing the distribution of the number of molecules in each oligomer plotted as a percentage of the total number observed of oligomers. Each point represents mean ± SEM from 5 independent experiments, in which more than 180 oligomers were analyzed. Control oligomers contained 51 ± 8 GFP molecules. For more detailed statistics for this and other graphs, see Source data. (C) Representative images of fluorescent microtubules (red) incubated with GFP-tagged CENP-T¹⁻²⁴² oligomers and control GFP oligomers (green). Scalebar=5 μm. Repeated 3 times with similar results. (D) Average number of microtubule-bound oligomers in a 10 μm length of microtubule. Bars represent mean ± SEM from 3 independent experiments. Each point represents the mean of an independent experiment in which at least 10 microscopy fields were analyzed. Two-tailed Welch’s t-test: p=0.0418. (E) Schematic of the in vitro assay used to study interactions between CENP-T¹⁻²⁴² oligomers and dynamic microtubules. (F) Representative kymographs of dynamic microtubules (tubulin, blue in merge) grown from coverslip-bound microtubule seeds (red in merge) and CENP-T¹⁻²⁴² oligomers (GFP, green in merge). Top: CENP-T¹⁻²⁴² oligomer binds directly to polymerizing microtubule end, then tracks the end during polymerization. Bottom: CENP-T¹⁻²⁴² oligomer binds the wall of a microtubule, diffuses on the microtubule lattice, and then tracks the depolymerizing microtubule end. End tracking during polymerization and depolymerization were observed in 20 and 63 out of 80 total observations, respectively. Observations were made over 8 independent experiments. (G) Polymerization and depolymerization rates measured for free microtubule ends and microtubule ends coupled to CENP-T¹⁻²⁴² oligomers. Points represent individual microtubule ends pooled from 3 experiments without GFP-CENP-T¹⁻²⁴²-I3–01 and 8 experiments with GFP-CENP-T¹⁻²⁴²-I3–01. Polymerization rate: n=89 free ends, n=15 coupled ends; depolymerization rate: n=73 free ends, n=62 coupled ends. Bars show the mean ± SEM. Two-tailed Welch’s t-tests: Polymerization rate: p=0.9377; Depolymerization rate: p<0.0001. Source numerical data are available in source data.

**Fig. 4:. CENP-T¹⁻²⁴² oligomerization promotes outer kinetochore recruitment.**
(A) Representative images of CENP-T¹⁻²⁴² and GFP-CENP-T¹⁻²⁴²-I3–01 during mitosis. Image brightness is not scaled identically due to large differences in brightness. Repeated 4 times with similar results. (B) Representative immunofluorescence of NDC80 at centromeres in mitotic cells expressing each construct. Cells are arrested in mitosis with S-Trityl-L-Cysteine. GFP, centromere, and NDC80 channels were adjusted identically. Inset regions are indicated by dashed boxes. NDC80 insets are brighter than full-size images. Centromeres were stained with Anti-Centromere Antibodies. Scalebars=5 μm. Inset scalebars=2 μm. (C) Quantification of outer kinetochore complex signals from (B) and equivalent experiments. Bars represent mean ± SEM. NDC80: n=30 cells for each condition pooled from 2 experiments. Mis12: GFP-I3–01: n=42, CENP-T¹⁻²⁴²: n=57, GFP-CENP-T¹⁻²⁴²-I3–01: n=61 cells pooled from 3 experiments. KNL1: GFP-I3–01: n=34, CENP-T¹⁻²⁴²: n=27, GFP-CENP-T¹⁻²⁴²-I3–01: n=30 cells pooled from 2 experiments. Two-tailed Welch’s t-test: NDC80: GFP-I3–01 vs. GFP-CENP-T¹⁻²⁴²: p<0.0001; GFP-CENP-T¹⁻²⁴² vs. GFP-CENP-T¹⁻²⁴²-I3–01: p<0.0001. MIS12: GFP-I3–01 vs. GFP-CENP-T¹⁻²⁴²: p<0.0001; GFP-CENP-T¹⁻²⁴² vs. GFP-CENP-T¹⁻²⁴²-I3–01: p=0.0047. KNL1: GFP-I3–01 vs. GFP-CENP-T¹⁻²⁴²: p<0.0001; GFP-CENP-T¹⁻²⁴² vs. GFP-CENP-T¹⁻²⁴²-I3–01: p<0.0001. (D) Representative fields of cells expressing each construct. Scalebar=5 μm. Repeated 4 times with similar results. (E) Distribution of mitotic errors in metaphase cells upon expression of each construct. n=number of cells in a single experiment. Repeated twice with similar results. Chi-squared test: GFP-I3–01 vs. GFP-CENP-T¹⁻²⁴²: p=0.0348; GFP-CENP-T¹⁻²⁴² vs. GFP-CENP-T¹⁻²⁴²-I3–01: p<0.0001. (F) Percentage of cells in G2/M based on DNA content measurements by flow cytometry in cell lines expressing each construct. Expression of the constructs was induced with doxycycline. Bars represent mean ± SEM. Each point represents a measurement from 3 independent experiments. Statistical analysis was performed on the differences between induced and uninduced for each condition. Two-tailed Welch’s t-test: GFP-I3–01 vs. GFP-CENP-T¹⁻²⁴²-I3–01: p=0.0097; GFP-CENP-T¹⁻²⁴² vs. GFP-CENP-T¹⁻²⁴²-I3–01: p=0.009. (G) and (H) Comparison of outer kinetochore protein co-immunoprecipitation as measured by quantitative mass spectrometry. Each point represents a biological replicate from 1 multiplexed experiment. Bars represent mean ± SEM. (H) Two-tailed Welch’s t-test: NDC80: p=0.0257; MIS12: p=0.0025; KNL1: p=0.0037; SKA1: p=0.0021; RZZ: p=0.0004. Source numerical data are available in source data.

**Fig. 5:. Each additional CENP-T¹⁻²⁴² molecule incrementally increases outer kinetochore recruitment of neighboring molecules.**
(A) Diagram of Sun Tag oligomerization strategy. (B) Representative immunofluorescence images of SunTag oligomer localization with different numbers of GCN4pep on the scaffold. GFP signal in all images is scaled the same. Scalebar=5 μm. This experiment was repeated 3 times with similar results. (C) Percentage of cells in G2/M based on DNA content measurements by flow cytometry in cell lines expressing SunTag with scaffolds with different numbers of GCN4pep. scFv-sfGFP-CENP-T¹⁻²⁴² expression was induced with doxycycline. Bars represent mean percentage of cells in G2/M ± SEM from 3 repeats. Welch’s ANOVA test was performed on the differences between means of induced and uninduced to calculate a P-value: p<0.0001. (D) Comparison of NDC80 complex co-immunoprecipitation by scaffolds with 1, 6, 10, or 18 GCN4pep copies when they were expressed alongside.scFv-sfGFP-CENP-T¹⁻²⁴² NDC80 complex immunoprecipitation was measured by TMT-based quantitative mass spectrometry. TdTomato-tagged scaffolds were immunoprecipitated, then abundances were normalized and calculated as described in Methods. Each point represents a biological replicate from 2 multiplexed mass spectrometry runs. Each bar represents the mean ± SEM. Two-tailed Welch’s t-test: 1 vs. 6: p=0.0228; 6 vs. 10: p=0.0063; 10 vs. 18: p=0.0036. (E) Representative immunofluorescence images of NDC80 levels at centromeres in cell expressing the scFv-sfGFP-CENP-T¹⁻²⁴² with scaffolds with different numbers of GCN4pep. All cells are mitotically arrested with S-Trityl-L-Cysteine. All images use the same linear image adjustments. Scalebar=5 μm. (F) Quantification of NDC80 complex levels from (E). Each point is a cell. Each bar represents the mean ± SEM. 1: n=45; 2: n=45; 3: n=40; 4: n=40; 6: n=46; 8: n=45; 10: n=45; 12: n=40. n=cells measured over 3 independent experiments. Welch’s ANOVA test was performed to calculate P-value for the whole dataset (p<0.0001). Two-tailed Welch’s t test: 4 vs. 6 and 8 vs. 10: p<0.0001. Source numerical data are available in source data.

**Fig. 6:. Oligomerization of CENP-T is required to saturate NDC80 binding sites.**
(A) Diagram of recombinant constructs. Both constructs contain CENP-T¹⁻²⁴² region with activating phospho-mimetic substitutions at sites T11, T27, and T85. (B) Images of GFP-CENP-T^1−242/3D-I3–01 oligomers or GFP-CENP-T^1−242/3D monomers taken with identical microscope settings and brightness adjustments. This direct comparison was performed once. Scalebar=2 μm. (C) Top: Diagram of single molecule experimental approach. Experimental details are described in Methods. Bottom: representative images of GFP-CENP-T^1−242/3TD-I3–01 and GFP-I3–01 oligomers at each step. Scalebar=5 μm. (D) Efficiency of NDC80 recruitment to GFP-CENP-T^1−242/3D-I3–01 and control GFP-I3–01 from (C). The result is the number of NDC80 molecules bound per molecule in an oligomer. Each point is the median result from 3 independent trials with at least 12 oligomers. Bars are mean ± SEM. Two-tailed Welch’s t-test: p=0.0051. (E) Top: diagram of single molecule experimental approach with GFP-CENP-T^1−242/3D monomers. Experimental details are described in materials and methods. Bottom: representative examples of GFP-CENP-T^1−242/3D monomers before and after interaction with 100 nM GFP-tagged NDC80^Bonsai. Scalebar=5 μm. (F) Efficiency of NDC80 recruitment to GFP-CENP-T^1−242/3D-I3–01 oligomers and GFP-CENP-T^1−242/3TD monomers from (E). Each point is the median result from 3 independent trials with at least 12 oligomers or 33 monomers analyzed. Bars are mean ± SEM. Data for GFP-CENP-T^1−242/3D-I3–01 oligomer is duplicated from (D). Two-tailed Welch’s t-test: p=0. 0014. (G) Model of the role of higher order oligomerization in kinetochore assembly. In regions where CENP-A nucleosomes are at a high density they recruit inner kinetochore components that form higher-order assemblies. Those oligomers cluster multiple inner kinetochore modules, resulting in a high local concentration of CENP-T, which can robustly recruit the outer kinetochore during mitosis, generating complete kinetochores. When CENP-A is deposited at a low density (bottom), it may be able to recruit some inner kinetochore components, but it cannot generate a higher-order assembly. As a result, it is unable to generate the local concentration of CENP-T necessary to recruit the rest of the kinetochore. Source numerical data are available in source data.

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References

1. Cheeseman IM The kinetochore. Cold Spring Harb Perspect Biol (2014) doi:10.1101/cshperspect.a015826. - DOI - PMC - PubMed
1. Musacchio A & Desai A A Molecular View of Kinetochore Assembly and Function. Biology (Basel) 6, 5 (2017). - PMC - PubMed
1. Suzuki A, Badger BL & Salmon ED A quantitative description of Ndc80 complex linkage to human kinetochores. Nat Commun (2015) doi:10.1038/ncomms9161. - DOI - PMC - PubMed
1. Brinkley BR & Cart Wright J Ultrastructural analysis of mitotic spindle elongation in mammalian cells in vitro. Direct microtubule counts. J Cell Biol 50, 416–431 (1971). - PMC - PubMed
1. Zhou K et al. CENP-N promotes the compaction of centromeric chromatin. Nature Structural & Molecular Biology 2022 29:4 29, 403–413 (2022). - PMC - PubMed

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[1] Cheeseman IM The kinetochore. Cold Spring Harb Perspect Biol (2014) doi:10.1101/cshperspect.a015826. - DOI - PMC - PubMed

[2] Cheeseman IM The kinetochore. Cold Spring Harb Perspect Biol (2014) doi:10.1101/cshperspect.a015826. - DOI - PMC - PubMed

[3] Musacchio A & Desai A A Molecular View of Kinetochore Assembly and Function. Biology (Basel) 6, 5 (2017). - PMC - PubMed

[4] Musacchio A & Desai A A Molecular View of Kinetochore Assembly and Function. Biology (Basel) 6, 5 (2017). - PMC - PubMed

[5] Suzuki A, Badger BL & Salmon ED A quantitative description of Ndc80 complex linkage to human kinetochores. Nat Commun (2015) doi:10.1038/ncomms9161. - DOI - PMC - PubMed

[6] Suzuki A, Badger BL & Salmon ED A quantitative description of Ndc80 complex linkage to human kinetochores. Nat Commun (2015) doi:10.1038/ncomms9161. - DOI - PMC - PubMed

[7] Brinkley BR & Cart Wright J Ultrastructural analysis of mitotic spindle elongation in mammalian cells in vitro. Direct microtubule counts. J Cell Biol 50, 416–431 (1971). - PMC - PubMed

[8] Brinkley BR & Cart Wright J Ultrastructural analysis of mitotic spindle elongation in mammalian cells in vitro. Direct microtubule counts. J Cell Biol 50, 416–431 (1971). - PMC - PubMed

[9] Zhou K et al. CENP-N promotes the compaction of centromeric chromatin. Nature Structural & Molecular Biology 2022 29:4 29, 403–413 (2022). - PMC - PubMed

[10] Zhou K et al. CENP-N promotes the compaction of centromeric chromatin. Nature Structural & Molecular Biology 2022 29:4 29, 403–413 (2022). - PMC - PubMed

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Higher-order protein assembly controls kinetochore formation

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Higher-order protein assembly controls kinetochore formation

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