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

R01 GM098389 NIGMS NIH HHS , R35 GM130302 NIGMS NIH HHS , R35 GM130365 NIGMS NIH HHS , R35 GM126930 NIGMS NIH HHS

             kinetochore microtubule
             kinetochore organization

	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).

Akiyoshi, The aurora B kinase promotes inner and outer kinetochore interactions in budding yeast. 2014, Pubmed

Akiyoshi, The aurora B kinase promotes inner and outer kinetochore interactions in budding yeast. 2014, Pubmed
Akiyoshi, Tension directly stabilizes reconstituted kinetochore-microtubule attachments. 2010, Pubmed
Allu, Structure of the Human Core Centromeric Nucleosome Complex. 2020, Pubmed
Barnhart, HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore. 2011, Pubmed
Bassett, Epigenetic centromere specification directs aurora B accumulation but is insufficient to efficiently correct mitotic errors. 2010, Pubmed
Bonner, Enrichment of Aurora B kinase at the inner kinetochore controls outer kinetochore assembly. 2020, Pubmed , Xenbase
Cai, Experimental and computational framework for a dynamic protein atlas of human cell division. 2019, Pubmed
Carroll, Dual recognition of CENP-A nucleosomes is required for centromere assembly. 2010, Pubmed
Chakraborty, In vitro reconstitution of lateral to end-on conversion of kinetochore-microtubule attachments. 2018, Pubmed
Chakraborty, Microtubule end conversion mediated by motors and diffusing proteins with no intrinsic microtubule end-binding activity. 2019, Pubmed , Xenbase
Cheeseman, The kinetochore. 2015, Pubmed
Cheeseman, The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. 2007, Pubmed
Coy, Kinesin's tail domain is an inhibitory regulator of the motor domain. 1999, Pubmed
De Wulf, Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes. 2004, Pubmed
Desai, A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts. 1997, Pubmed , Xenbase
Dimitrova, Structure of the MIND Complex Defines a Regulatory Focus for Yeast Kinetochore Assembly. 2017, Pubmed
Emanuele, Measuring the stoichiometry and physical interactions between components elucidates the architecture of the vertebrate kinetochore. 2006, Pubmed , Xenbase
Fachinetti, DNA Sequence-Specific Binding of CENP-B Enhances the Fidelity of Human Centromere Function. 2015, Pubmed
Falk, Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere. 2015, Pubmed
Falk, CENP-C directs a structural transition of CENP-A nucleosomes mainly through sliding of DNA gyres. 2016, Pubmed
Fukagawa, The centromere: chromatin foundation for the kinetochore machinery. 2014, Pubmed
Gascoigne, CDK-dependent phosphorylation and nuclear exclusion coordinately control kinetochore assembly state. 2013, Pubmed
Gascoigne, Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes. 2011, Pubmed
Grishchuk, Different assemblies of the DAM1 complex follow shortening microtubules by distinct mechanisms. 2008, Pubmed
Gudimchuk, Kinetochore kinesin CENP-E is a processive bi-directional tracker of dynamic microtubule tips. 2013, Pubmed
Guo, Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition. 2019, Pubmed
Gupta, Purification of kinetochores from the budding yeast Saccharomyces cerevisiae. 2018, Pubmed
Guse, In vitro centromere and kinetochore assembly on defined chromatin templates. 2011, Pubmed , Xenbase
Haase, Distinct Roles of the Chromosomal Passenger Complex in the Detection of and Response to Errors in Kinetochore-Microtubule Attachment. 2017, Pubmed , Xenbase
Hamilton, Reconstitution reveals two paths of force transmission through the kinetochore. 2021, Pubmed
Hara, Publisher Correction: Multiple phosphorylations control recruitment of the KMN network onto kinetochores. 2019, Pubmed
Hasson, The octamer is the major form of CENP-A nucleosomes at human centromeres. 2013, Pubmed
Hori, CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore. 2008, Pubmed
Hori, Artificial generation of centromeres and kinetochores to understand their structure and function. 2020, Pubmed
Hori, The CCAN recruits CENP-A to the centromere and forms the structural core for kinetochore assembly. 2013, Pubmed
Huis In 't Veld, Molecular basis of outer kinetochore assembly on CENP-T. 2017, Pubmed
Hyman, Preparation of modified tubulins. 1991, Pubmed
Itzhak, Global, quantitative and dynamic mapping of protein subcellular localization. 2017, Pubmed
Kato, A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C. 2013, Pubmed
Killinger, Auto-inhibition of Mif2/CENP-C ensures centromere-dependent kinetochore assembly in budding yeast. 2021, Pubmed
Kim, Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores. 2015, Pubmed
Krizaic, The distinct functions of CENP-C and CENP-T/W in centromere propagation and function in Xenopus egg extracts. 2016, Pubmed , Xenbase
Kudalkar, Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex. 2016, Pubmed
Lang, An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast. 2019, Pubmed
Logsdon, Both tails and the centromere targeting domain of CENP-A are required for centromere establishment. 2015, Pubmed
McIntosh, Fibrils connect microtubule tips with kinetochores: a mechanism to couple tubulin dynamics to chromosome motion. 2008, Pubmed
McKinley, The molecular basis for centromere identity and function. 2016, Pubmed
Miller, Preparation of microtubule protein and purified tubulin from bovine brain by cycles of assembly and disassembly and phosphocellulose chromatography. 2010, Pubmed
Musacchio, A Molecular View of Kinetochore Assembly and Function. 2020, Pubmed
Nagpal, Kinetochore assembly and function through the cell cycle. 2017, Pubmed
Nishino, CENP-T provides a structural platform for outer kinetochore assembly. 2013, Pubmed
Nishino, CENP-T-W-S-X forms a unique centromeric chromatin structure with a histone-like fold. 2012, Pubmed
Ohzeki, CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA. 2003, Pubmed
Pesenti, Reconstitution of a 26-Subunit Human Kinetochore Reveals Cooperative Microtubule Binding by CENP-OPQUR and NDC80. 2019, Pubmed
Petrovic, Structure of the MIS12 Complex and Molecular Basis of Its Interaction with CENP-C at Human Kinetochores. 2017, Pubmed
Petrovic, The MIS12 complex is a protein interaction hub for outer kinetochore assembly. 2010, Pubmed
Powers, The Ndc80 kinetochore complex forms load-bearing attachments to dynamic microtubule tips via biased diffusion. 2009, Pubmed
Rago, Distinct organization and regulation of the outer kinetochore KMN network downstream of CENP-C and CENP-T. 2016, Pubmed
Sandall, A Bir1-Sli15 complex connects centromeres to microtubules and is required to sense kinetochore tension. 2007, Pubmed
Scarborough, Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules. 2020, Pubmed
Schindelin, Fiji: an open-source platform for biological-image analysis. 2012, Pubmed
Schmidt, The kinetochore-bound Ska1 complex tracks depolymerizing microtubules and binds to curved protofilaments. 2013, Pubmed
Sekulic, Preparation of Recombinant Centromeric Nucleosomes and Formation of Complexes with Nonhistone Centromere Proteins. 2017, Pubmed
Sorger, Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro. 1994, Pubmed
Suzuki, A quantitative description of Ndc80 complex linkage to human kinetochores. 2016, Pubmed
Volkov, Preparation of segmented microtubules to study motions driven by the disassembling microtubule ends. 2014, Pubmed
Watanabe, CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization. 2020, Pubmed
Weir, Insights from biochemical reconstitution into the architecture of human kinetochores. 2017, Pubmed
Weissmann, biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes. 2016, Pubmed
Wimbish, The Hec1/Ndc80 tail domain is required for force generation at kinetochores, but is dispensable for kinetochore-microtubule attachment formation and Ska complex recruitment. 2021, Pubmed
Yan, Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome. 2019, Pubmed
Yang, Phosphorylation of HsMis13 by Aurora B kinase is essential for assembly of functional kinetochore. 2008, Pubmed
Zaytsev, Multisite phosphorylation of the NDC80 complex gradually tunes its microtubule-binding affinity. 2016, Pubmed
Zhang, Optogenetic control of kinetochore function. 2017, Pubmed
Zhang, Optogenetic control of mitosis with photocaged chemical dimerizers. 2018, Pubmed
Zou, Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. 1999, Pubmed , Xenbase