Tarasovetc EV et al. (2021), Permitted and restricted steps of human kinetoc...

XB-ART-58078

Mol Biol Cell 2021 Jun 15;3213:1241-1255. doi: 10.1091/mbc.E20-07-0461.

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Permitted and restricted steps of human kinetochore assembly in mitotic cell extracts.

Tarasovetc EV , Allu PK , Wimbish RT , DeLuca JG , Cheeseman IM , Black BE , Grishchuk EL .

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Mitotic kinetochores assemble via the hierarchical recruitment of numerous cytosolic components to the centromere region of each chromosome. However, how these orderly and localized interactions are achieved without spurious macromolecular assemblies forming from soluble kinetochore components in the cell cytosol remains poorly understood. We developed assembly assays to monitor the recruitment of green fluorescent protein-tagged recombinant proteins and native proteins from human cell extracts to inner kinetochore components immobilized on microbeads. In contrast to prior work in yeast and Xenopus egg extracts, we find that human mitotic cell extracts fail to support de novo assembly of microtubule-binding subcomplexes. A subset of interactions, such as those between CENP-A-containing nucleosomes and CENP-C, are permissive under these conditions. However, the subsequent phospho-dependent binding of the Mis12 complex is less efficient, whereas recruitment of the Ndc80 complex is blocked, leading to weak microtubule-binding activity of assembled particles. Using molecular variants of the Ndc80 complex, we show that auto-inhibition of native Ndc80 complex restricts its ability to bind to the CENP-T/W complex, whereas inhibition of the Ndc80 microtubule binding is driven by a different mechanism. Together, our work reveals regulatory mechanisms that guard against the spurious formation of cytosolic microtubule-binding kinetochore particles.

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Species referenced: Xenopus laevis
Genes referenced: cdk1 cenpc dnai1 dsn1 h2ac21 mis12 ndc80 nuf2 spc24 spc25
GO keywords: kinetochore [+]

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	FIGURE 1:. Strategies for reconstructing kinetochores and our experimental approach. (A) Principal architecture of mitotic kinetochore and its binding to microtubules; see the text for details. CCAN, constitutive centromere-associated network. Letters “P” on CENP-T indicate phosphorylation-dependent activation of its binding to Ndc80 and Mis12 complexes; letters “P” on Mis12 indicate phosphorylation-dependent activation of its binding to CENP-C. (B) Previous experimental approaches to reconstitute kinetochore assembly and function in vitro; see the text for references. (C) Schematic of GFP-fused proteins stably expressed in HeLa cells. Experiments with Ndc80 complexes used three different cell lines: Nuf2-GFP (shown), GFP-Spc24, and GFP-Spc25. (D) Key steps of our experimental approach. Left image shows representative HeLa cell that was fixed and stained with propidium iodide to reveal DNA; green signal is from Mis12-GFP. Graph shows concentration of GFP-fused kinetochore proteins in cell extracts. Each colored point represents average bead brightness from independent experiments, during which 50–100 beads were analyzed. For more detailed statistics, see the Supplemental Source data. Black lines show mean with SEM. The column for native Ndc80-GFP combines data from cell lines expressing GFP-fused Nuf2, Spc24, and Spc25.
	FIGURE 2:. Microtubule binding by native kinetochore complexes in mitotic cell extracts. (A) Scheme of TIRF-based assay to visualize binding of soluble GFP-fused kinetochore components (2.5–3 nM) to GMPCPP-stabilized and fluorescently labeled microtubules, which were immobilized on the coverslip via anti-tubulin antibodies. (B) Colored images show representative microtubules (red) with bound GFP-fused kinetochore complexes (green); the corresponding grayscale kymographs below reveal mobility of these complexes over 30-s observation time; “rec” correspondents to purified recombinant proteins. (C) Average GFP brightness of microtubule decoration normalized against the concentration of GFP-labeled kinetochore protein (means with SD); note semi-log scale. Each point represents an independent experiment in which brightness was collected from >18 microtubules. p values were calculated by unpaired t test: , p < 0.05; *, p <.0.01. For more detailed statistics, see the Supplemental Source data.
	FIGURE 3:. Microtubule binding by native kinetochore complexes clustered on microbeads. (A) Scheme of bead-based assay to test microtubule binding of kinetochore components. (B) Representative examples of microscopy fields showing microtubules (red) and GFP-fused kinetochore components conjugated to coverslip-immobilized beads, shown in the DIC (top) and GFP (bottom) channels. (C) Average GFP bead brightness corresponding to the coating density of GFP-fused kinetochore components on bead surfaces. Identical quantification procedures were used throughout this study, so GFP-bead brightness can be compared directly between different panels and graphs. (D) Average number of bead-bound microtubules normalized against the number of beads per imaging field. In panels C and D, means are shown with SD, and each point represents an independent experiment. p values were calculated by unpaired t test: n.s., p > 0.05; *, p < 0.05. For more detailed statistics, see the Supplemental Source data. Results for native Ndc80-GFP combine data from cell lines expressing GFP-fused Nuf2, Spc24, and Spc25.
	FIGURE 4:. Interactions between various Ndc80 complexes and recombinant CENP-T/W complex. (A) Schematic representation of recombinant CENP-T/W protein, which serves as inner kinetochore scaffold. The phosphomimetic substitutions in CENP-T/W are indicated by red dots (left). Schematic representation of recombinant Ndc80 constructs (right). (B) Representative images of coverslip-immobilized beads in bright-field and GFP channels, showing recruitment of GFP-fused Ndc80 complexes to recombinant wild-type (top) or phosphomimetic (bottom) CENP-T/W, which has no fluorescent tag. (C) Average GFP bead brightness (mean with SD) of wild-type (left) or phosphomimetic (right) CENP-T/W–coated beads incubated with various Ndc80 (orange) or Mis12 (blue) proteins, as in panel B. Each point is derived from an independent experiment and represents the average brightness of >30 beads. p values were calculated by unpaired t test: , p < 0.05; , p < 0.01; , p < 0.001; **, p < 0.0001. For more detailed statistics, see the Supplemental Source data. Concentrations of GFP-labeled soluble proteins, applied as minimally diluted mitotic cell extracts, were as follows: 50–190 nM Ndc80 and 10–80 nM Mis12 complexes. Recombinant proteins were used at 100–130 nM.
	FIGURE 5:. Reconstructions based on the CENP-A nucleosomes and mitotic cell extracts. (A) The DNA (147 base pairs) used in nucleosome reconstitution is a CENP-A positioning fragment from the human X-chromosome centromeric α-satellite repeat; the 5′ end is labeled with Cy-5. (B) Schematic and constituents required to reconstitute nucleosomes with labeled DNA and His-H2A protein. (C) Nucleosomes prepared by gradient salt dialysis were analyzed by separation on native PAGE followed by ethidium bromide and Coomassie staining. (D) Schematic of nucleosome immobilization on beads and an example of a mitotic HeLa cell stably expressing GFP-CENP-C, fixed and stained with anti-H3S28phos antibodies to visualize chromosomes. (E) Representative images of beads in DIC and fluorescent channels, showing DNA-Cy5 and GFP-CENP-C. (F) Ratio of brightness of CENP-A– vs. H3-coated beads in the Cy5 and GFP channels. The conjugation levels of different nucleosomes are similar, but only nucleosomes containing CENP-A recruit native GFP-CENP-C. Each point in panels F and G represents the average bead brightness obtained in one independent experiment; errors are SEM. p values were calculated by unpaired t test: , p < 0.01; *, p < 0.001, n.s., not significant. For more detailed statistics, see the Supplemental Source data. (G) Average GFP brightness of beads reflects the level of recruitment of the indicated kinetochore components to different nucleosomes. Means are shown with SD.
	FIGURE 6:. Reconstructions using autoinhibition-deficient Mis12 complex and different modes of CENP-C recruitment to microbeads. (A) Cartoon illustrating Mis12 binding to CENP-C and Mis12 activation by a truncation within the Dsn1 N-tail. (B) Representative images of beads in the DIC and GFP channels, showing recruitment of native mutant Mis12 complexes to different nucleosomes, or to beads coated with anti–CENP-C antibodies. (C) GFP bead brightness showing recruitment of different native Mis12 complexes to beads with indicated coatings. In panels C–F, horizontal lines and column bars show means with SD, each dot represents the average result from one independent experiment. p values were calculated by unpaired t test: , p < 0.05; , p < 0.01; p < 0.001; **, p < 0.0001. For more detailed statistics, see the Supplemental Source data. (D) GFP-brightness of beads showing recruitment of native CENP-C and Mis12 complexes to beads with indicated coatings; data for Dsn1-Δ91-113 is the same as on panel (C), plotted here to provide side-by-side comparison with the recruitment of native CENP-C. (E) GFP-bead brightness (left axis and green columns) and the number of bead-bound microtubules (right axis and gray columns) plotted for beads with indicated compositions. Data for GFP brightness are the same as in panel C. (F) Results of experiments similar to those in panel (E) but using recombinant Ndc80 (orange dots) in the presence of unlabeled cell extract. These data are the same as in Figure 3, C and D, and are shown here for side-by-side comparison with the recruitment activity of native kinetochore complexes.
	FIGURE 7:. Summary of the permitted and restricted assembly steps in human cell extracts. (A) Simplified scheme indicating results from our reconstitutions: permitted assembly steps (green check mark), moderately/weakly efficient reactions (blue check mark), and restricted interactions (red crosses). Yellow circles indicate phosphomimetic substitutions in CENP-T (T11D, T27D, and T85D); dashed line in Mis12 complex indicates truncation within the Dsn1 subunit (Δ91-113). (B) Scheme illustrating binding between human CENP-T and Ndc80 complexes found for different mutants in reconstructions in vitro and in mitotic cells. Blue arrows and red cross illustrate effect of CDK1-dependent phosphorylation on this binding. Summary scheme for native full-length Ndc80 complex in cells is based on Gascoigne et al. (2011), Nishino et al. (2013), Rago et al. (2015), and Huis In ‘t Veld et al. (2016).

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	FIGURE 1:. Strategies for reconstructing kinetochores and our experimental approach. (A) Principal architecture of mitotic kinetochore and its binding to microtubules; see the text for details. CCAN, constitutive centromere-associated network. Letters “P” on CENP-T indicate phosphorylation-dependent activation of its binding to Ndc80 and Mis12 complexes; letters “P” on Mis12 indicate phosphorylation-dependent activation of its binding to CENP-C. (B) Previous experimental approaches to reconstitute kinetochore assembly and function in vitro; see the text for references. (C) Schematic of GFP-fused proteins stably expressed in HeLa cells. Experiments with Ndc80 complexes used three different cell lines: Nuf2-GFP (shown), GFP-Spc24, and GFP-Spc25. (D) Key steps of our experimental approach. Left image shows representative HeLa cell that was fixed and stained with propidium iodide to reveal DNA; green signal is from Mis12-GFP. Graph shows concentration of GFP-fused kinetochore proteins in cell extracts. Each colored point represents average bead brightness from independent experiments, during which 50–100 beads were analyzed. For more detailed statistics, see the Supplemental Source data. Black lines show mean with SEM. The column for native Ndc80-GFP combines data from cell lines expressing GFP-fused Nuf2, Spc24, and Spc25.
	FIGURE 2:. Microtubule binding by native kinetochore complexes in mitotic cell extracts. (A) Scheme of TIRF-based assay to visualize binding of soluble GFP-fused kinetochore components (2.5–3 nM) to GMPCPP-stabilized and fluorescently labeled microtubules, which were immobilized on the coverslip via anti-tubulin antibodies. (B) Colored images show representative microtubules (red) with bound GFP-fused kinetochore complexes (green); the corresponding grayscale kymographs below reveal mobility of these complexes over 30-s observation time; “rec” correspondents to purified recombinant proteins. (C) Average GFP brightness of microtubule decoration normalized against the concentration of GFP-labeled kinetochore protein (means with SD); note semi-log scale. Each point represents an independent experiment in which brightness was collected from >18 microtubules. p values were calculated by unpaired t test: , p < 0.05; *, p <.0.01. For more detailed statistics, see the Supplemental Source data.
	FIGURE 3:. Microtubule binding by native kinetochore complexes clustered on microbeads. (A) Scheme of bead-based assay to test microtubule binding of kinetochore components. (B) Representative examples of microscopy fields showing microtubules (red) and GFP-fused kinetochore components conjugated to coverslip-immobilized beads, shown in the DIC (top) and GFP (bottom) channels. (C) Average GFP bead brightness corresponding to the coating density of GFP-fused kinetochore components on bead surfaces. Identical quantification procedures were used throughout this study, so GFP-bead brightness can be compared directly between different panels and graphs. (D) Average number of bead-bound microtubules normalized against the number of beads per imaging field. In panels C and D, means are shown with SD, and each point represents an independent experiment. p values were calculated by unpaired t test: n.s., p > 0.05; *, p < 0.05. For more detailed statistics, see the Supplemental Source data. Results for native Ndc80-GFP combine data from cell lines expressing GFP-fused Nuf2, Spc24, and Spc25.
	FIGURE 4:. Interactions between various Ndc80 complexes and recombinant CENP-T/W complex. (A) Schematic representation of recombinant CENP-T/W protein, which serves as inner kinetochore scaffold. The phosphomimetic substitutions in CENP-T/W are indicated by red dots (left). Schematic representation of recombinant Ndc80 constructs (right). (B) Representative images of coverslip-immobilized beads in bright-field and GFP channels, showing recruitment of GFP-fused Ndc80 complexes to recombinant wild-type (top) or phosphomimetic (bottom) CENP-T/W, which has no fluorescent tag. (C) Average GFP bead brightness (mean with SD) of wild-type (left) or phosphomimetic (right) CENP-T/W–coated beads incubated with various Ndc80 (orange) or Mis12 (blue) proteins, as in panel B. Each point is derived from an independent experiment and represents the average brightness of >30 beads. p values were calculated by unpaired t test: , p < 0.05; , p < 0.01; , p < 0.001; **, p < 0.0001. For more detailed statistics, see the Supplemental Source data. Concentrations of GFP-labeled soluble proteins, applied as minimally diluted mitotic cell extracts, were as follows: 50–190 nM Ndc80 and 10–80 nM Mis12 complexes. Recombinant proteins were used at 100–130 nM.
	FIGURE 5:. Reconstructions based on the CENP-A nucleosomes and mitotic cell extracts. (A) The DNA (147 base pairs) used in nucleosome reconstitution is a CENP-A positioning fragment from the human X-chromosome centromeric α-satellite repeat; the 5′ end is labeled with Cy-5. (B) Schematic and constituents required to reconstitute nucleosomes with labeled DNA and His-H2A protein. (C) Nucleosomes prepared by gradient salt dialysis were analyzed by separation on native PAGE followed by ethidium bromide and Coomassie staining. (D) Schematic of nucleosome immobilization on beads and an example of a mitotic HeLa cell stably expressing GFP-CENP-C, fixed and stained with anti-H3S28phos antibodies to visualize chromosomes. (E) Representative images of beads in DIC and fluorescent channels, showing DNA-Cy5 and GFP-CENP-C. (F) Ratio of brightness of CENP-A– vs. H3-coated beads in the Cy5 and GFP channels. The conjugation levels of different nucleosomes are similar, but only nucleosomes containing CENP-A recruit native GFP-CENP-C. Each point in panels F and G represents the average bead brightness obtained in one independent experiment; errors are SEM. p values were calculated by unpaired t test: , p < 0.01; *, p < 0.001, n.s., not significant. For more detailed statistics, see the Supplemental Source data. (G) Average GFP brightness of beads reflects the level of recruitment of the indicated kinetochore components to different nucleosomes. Means are shown with SD.
	FIGURE 6:. Reconstructions using autoinhibition-deficient Mis12 complex and different modes of CENP-C recruitment to microbeads. (A) Cartoon illustrating Mis12 binding to CENP-C and Mis12 activation by a truncation within the Dsn1 N-tail. (B) Representative images of beads in the DIC and GFP channels, showing recruitment of native mutant Mis12 complexes to different nucleosomes, or to beads coated with anti–CENP-C antibodies. (C) GFP bead brightness showing recruitment of different native Mis12 complexes to beads with indicated coatings. In panels C–F, horizontal lines and column bars show means with SD, each dot represents the average result from one independent experiment. p values were calculated by unpaired t test: , p < 0.05; , p < 0.01; p < 0.001; **, p < 0.0001. For more detailed statistics, see the Supplemental Source data. (D) GFP-brightness of beads showing recruitment of native CENP-C and Mis12 complexes to beads with indicated coatings; data for Dsn1-Δ91-113 is the same as on panel (C), plotted here to provide side-by-side comparison with the recruitment of native CENP-C. (E) GFP-bead brightness (left axis and green columns) and the number of bead-bound microtubules (right axis and gray columns) plotted for beads with indicated compositions. Data for GFP brightness are the same as in panel C. (F) Results of experiments similar to those in panel (E) but using recombinant Ndc80 (orange dots) in the presence of unlabeled cell extract. These data are the same as in Figure 3, C and D, and are shown here for side-by-side comparison with the recruitment activity of native kinetochore complexes.
	FIGURE 7:. Summary of the permitted and restricted assembly steps in human cell extracts. (A) Simplified scheme indicating results from our reconstitutions: permitted assembly steps (green check mark), moderately/weakly efficient reactions (blue check mark), and restricted interactions (red crosses). Yellow circles indicate phosphomimetic substitutions in CENP-T (T11D, T27D, and T85D); dashed line in Mis12 complex indicates truncation within the Dsn1 subunit (Δ91-113). (B) Scheme illustrating binding between human CENP-T and Ndc80 complexes found for different mutants in reconstructions in vitro and in mitotic cells. Blue arrows and red cross illustrate effect of CDK1-dependent phosphorylation on this binding. Summary scheme for native full-length Ndc80 complex in cells is based on Gascoigne et al. (2011), Nishino et al. (2013), Rago et al. (2015), and Huis In ‘t Veld et al. (2016).