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Higher-order protein assembly controls kinetochore formation

Nature Cell Biology volume 26, pages 45–56 (2024)Cite this article

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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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Fig. 1: I3-01 oligomerization strategy generates particles that interact with mitotic spindles.
Fig. 2: CENP-T1–242 oligomers recruit almost the entire outer kinetochore.
Fig. 3: Isolated CENP-T1–242 oligomers bind to microtubules and track dynamic microtubule ends.
Fig. 4: CENP-T1–242 oligomerization promotes outer kinetochore recruitment.
Fig. 5: Each additional CENP-T1–242 molecule incrementally increases outer kinetochore recruitment of neighbouring molecules.
Fig. 6: Oligomerization of CENP-T is required to saturate NDC80 binding sites.

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Data availability

The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE90 partner repository with the dataset identifiers PXD042174 and https://doi.org/10.6019/PXD042174. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank J. Ly and S. Bell for feedback on the paper, and members of the Cheeseman and Grishchuk labs for feedback throughout the process. This work was supported by grants to I.M.C. from the NIH/NIGMS (R35GM126930), including diversity supplement funding for G.B.S., and the Gordon and Betty Moore Foundation, a grant to E.L.G. from NIH/NIGMS (R35-GM141747), and grants to both I.M.C. and E.L.G. from the American Cancer Society Theory Lab Collaborative Grant (TLC-20-117-01-TLC) and NSF (2029868).

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Author notes

  1. These authors contributed equally: Gunter B. Sissoko, Ekaterina V. Tarasovetc.

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Gunter B. Sissoko, Océane Marescal & Iain M. Cheeseman

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Gunter B. Sissoko, Océane Marescal & Iain M. Cheeseman

  3. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ekaterina V. Tarasovetc & Ekaterina L. Grishchuk

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  1. Gunter B. Sissoko
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Contributions

Conceptualization—G.B.S., I.M.C. and E.L.G.; methodology, validation and investigation—G.B.S. for reagent generation, experimental design and all in vivo experiments, E.V.T. for all in vitro experiments, and O.M. for western blots and additional support; writing, original draft preparation—G.B.S. and I.M.C.; writing, review and editing—E.L.G., E.V.T. and O.M.; visualization—G.B.S., E.V.T. and O.M.; supervision—I.M.C. and E.L.G.; funding acquisition—I.M.C. and E.L.G.

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Correspondence to Ekaterina L. Grishchuk or Iain M. Cheeseman.

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Extended data

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.

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.

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.

Source data

Extended Data Fig. 5 CENP-T1–242 oligomers recruit kinetochore-associated proteins and spindle assembly checkpoint proteins more robustly than monomeric CENP-T1–242.

(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 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.

Source data

Extended Data Fig. 6 Additional SunTag mass spectrometry, centromere depletion, and controls.

(a) Normalized GFP signals from SunTag cells in Fig. 5c 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 with 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 SunTag 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 SunTag 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 SunTag 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 SunTag 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, f, Extended Data Fig. 6i. Scaffolds in these cell lines lack the tdTomato tag. Source numerical data and unprocessed blots are available in Source Data.

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 (2 A). 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-T1-242/2A 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-T1-242/2A. 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.

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-T1-242/3D-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-T1-242/3D. 3 independent experiments. Distribution mean ± SEM: 1.23 ± 0.05 molecules. (d) Single molecule binding experiment with NDC80ΔSpc24/25. Top: Experimental workflow. Bottom: representative images of GFP-CENP-T1-242/3TD-I3-01 oligomers at each experimental stage. (e) Efficiency of NDC80Bonsai or NDC80ΔSpc24/25 recruitment to GFP-CENP-T1-242/3D-I3-01 oligomers. Bars represent mean ± SEM. Each point is the median result from 3 independent experiments with >12 oligomers. Data for GFP-CENP-T1-242/3D-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-T1-242/3D-I3-01+NDC80Bonsai: n = 85 Oligomers; GFP-CENP-T1-242/3D-I3-01 + NDC80ΔSpc24/25: n = 79 Oligomers; GFP-I3-01+NDC80Bonsai: 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-T1-242/3D (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.

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Supplementary information

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Supplementary Tables

Supplementary Table 1. Cell lines. Supplementary Table 2. Primary antibodies. Supplementary Table 3. Immunofluorescence fixation conditions. Supplementary Table 4. Primers and small interfering RNAs.

Supplementary Video 1

Kinetochore-like particle tracking a polymerizing microtubule end. This video shows a dynamic microtubule (blue) growing from the coverslip-bound GMPCPP microtubule seed (red) in the presence of soluble tubulin and GFP–CENP-T1–242–I3-01 oligomers (green) isolated from mitotic HeLa cells. The isolated oligomer with associated proteins (that is, kinetochore-like particle) binds to the end of a polymerizing microtubule and moves processively with the end as it polymerizes. This video plays at 7 fps, and events are shown at 3.5 times actual speed.

Supplementary Video 2

A kinetochore-like particle exhibits plus-end-directed processive movement. A larger green particle remains stationary, while a smaller kinetochore-like particle moves processively and unidirectionally towards the growing microtubule plus-end. Initial motion on the GMPCPP-containing microtubule seed is slow, but the particle speeds up on the GDP-containing microtubule wall (for more details, see Extended Data Fig. 3d). These plus-end-directed motions were rarer than the other types of motion. This video plays at 7 fps, and events are shown at 3.5 times actual speed.

Supplementary Video 3

Kinetochore-like particle diffusing along a microtubule wall and tracking a depolymerizing microtubule end. Video shows a dynamic microtubule interacting with two kinetochore-like particles. A larger particle associates with the microtubule wall and remains stationary for the duration of the video. A smaller particle lands on the microtubule wall at the site indicated with an arrow. The particle diffuses along the microtubule wall, then tracks the depolymerizing end of the microtubule. This video plays at 7 fps, and events are shown at 3.5 times actual speed.

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Sissoko, G.B., Tarasovetc, E.V., Marescal, O. et al. Higher-order protein assembly controls kinetochore formation. Nat Cell Biol 26, 45–56 (2024). https://doi.org/10.1038/s41556-023-01313-7

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