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Insights from biochemical reconstitution into the architecture of human kinetochores

Nature volume 537, pages 249–253 (2016)Cite this article

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Abstract

Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle1. Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A)2. A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN)3,4,5,6, establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore7,8. Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described9,10,11, but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A:CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro. This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores.

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Figure 1: Reconstitution of the CHIKMLN complex.
Figure 2: Selective cooperative binding of CHIKMLN to CENP-A mononucleosomes.
Figure 3: KMN and CHIKMLN connect CENP-A to microtubules.
Figure 4: Centromere and kinetochore assembly and propagation.

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Acknowledgements

We are grateful to F. Martino and D. Rhodes for help in setting up NCP preparations, to A. F. Straight for providing plasmids for expression of CENP-A:H4, to K. A. Davey for plasmids to produce the ‘601’ 145-bp nucleosomal DNA, to C. Smith for setting up conditions for CENP-H depletion, to G. Ossolengo of the antibody facility at the European Institute of Oncology in Milan (Italy) for help with antibody production, and all other members of the Musacchio laboratory for discussions. A.C.F. is supported by an EMBO long-term fellowship (ALTF 1096-2012) and Marie Curie Intra-European Fellowship. A.M. acknowledges funding by the European Union’s 7th Framework Program Integrated Project MitoSys, the Horizon 2020 ERC agreement RECEPIANCE, and the DFG’s Collaborative Research Centre 1093. F.H. is supported by the European Research Council (MolStruKT StG number 638218) and by an LMU excellent junior grant.

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Author notes

  1. John R. Weir, Alex C. Faesen and Kerstin Klare: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, Dortmund, 44227, Germany

    John R. Weir, Alex C. Faesen, Kerstin Klare, Arsen Petrovic, Federica Basilico, Satyakrishna Pentakota, Jenny Keller, Marion E. Pesenti, Dongqing Pan, Doro Vogt, Sabine Wohlgemuth & Andrea Musacchio

  2. Gene Center Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, Munich, 81377, Germany

    Josef Fischböck & Franz Herzog

  3. Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstraße, Essen, 45141, Germany

    Andrea Musacchio

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Contributions

J.R.W., K.K., A.C.F. and A.M. designed the experiments. J.R.W, K.K., A.C.F., F.B., J.K., A.P., S.W., M.P. and S.P. purified proteins. D.V. and F.B. purified nucleosomes. D.P. created the engineered nucleosomes used in AUC experiments. J.R.W. performed gel filtration experiments. A.P. performed AUC experiments. K.K. performed cell biology experiments. A.C.F. performed microtubule binding experiments. J.F. and F.H. performed crosslinking and mass-spec experiments. A.M. coordinated the working team. J.R.W. and A.M. wrote the paper.

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Correspondence to John R. Weir or Andrea Musacchio.

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The authors declare no competing financial interests.

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Nature thanks A. Desai and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Building blocks of the kinetochore.

Schematic organization of protein and subcomplexes used in this study, with essential structural features. CENP-A is a histone H3 variant. Crucial to its function in kinetochore assembly are the so-called CATD box and the C-terminal region, which are believed to interact with CENP-N and CENP-C, respectively10,11,39. For our reconstitution studies, we reconstituted human CENP-A:H4 tetramers and combined them with X. laevis H2A:H2B dimers. Nucleosome core particles containing histone H3 were reconstituted with X. laevis H3, H4, H2A, and H2B (see Methods). CENP-C can be thought of as a blueprint for kinetochore assembly, with binding motifs for outer and inner kinetochore subunits ordered from the N to the C terminus. The N-terminal region starts with a binding site for the Mis12 complex40,41, followed by a binding site for the CENP-HIKM complex19. Two related nucleosome-binding motifs have been identified, in the so-called ‘central region’ and ‘CENP-C motif’11. The nucleosome-binding motifs interact with the H2A:H2B dimer and with the C-terminal region of CENP-A11. Finally, the dimerization motif has a cupin-like fold42. The C-terminal region also binds to M18BP1 (refs 43, 44), which is involved in CENP-A deposition. The two subunits of the CENP-LN complex have similar size and are structurally related, as revealed by the crystal structure of their S. cerevisiae homologues15. The four-subunit CENP-HIKM complex contains a tight subcomplex of the CENP-H and CENP-K subunits19. CENP-M is a pseudo-Ras-like small GTPase that has lost the ability to bind GTP19. It interacts with CENP-I and is required for its stability19, but no CENP-M orthologue has been identified in S. cerevisiae, whereas Ctf3 is the CENP-I orthologue in this organism (see Fig. 4a). Structurally, CENP-I may resemble the HEAT-repeat α-solenoid structure of Importin-β (ref. 19). The four-subunit NDC80 complex is crucial for microtubule-binding by kinetochores7,8. It is a dumbbell-shaped, elongated protein with large coiled-coil domains23,45. Calponin-homology (CH) domains near the N terminus of the NDC80 and NUF2 subunits have been implicated in microtubule-binding23,45. The RWD domains of the SPC24 and SPC25 subunits target the NDC80 complex to the kinetochore46,47 through interactions with the MIS12 complex. The four-subunit MIS12 complex remains structural uncharacterized, except for low-resolution negative-stain electron microscopy analyses47,48,49,50. It is a hub of interactions, including interactions with the CENP-C complex (discussed above), the NDC80 complex (also discussed above), and the Knl1 subunit of the Knl1 complex49. The two-subunit KNL1 complex plays a crucial role in spindle assembly checkpoint signalling51. The C-terminal region of KNL1, the largest known core kinetochore subunits, consists of tandem RWD domains and is sufficient with an interaction with the MIS12 complex47,49. A longer region, comprising approximately the last 300 residues, is also sufficient for tight binding to ZWINT. For our studies, we used a construct encompassing residues KNL12000–2311 that was endowed with the ability to bind the MIS12 complex and ZWINT.

Extended Data Figure 2 SEC analyses.

The indicated samples (at a concentration of 10 μM, 5 μM for nucleosome core particles) were loaded on the indicated SEC column and the resulting elution fractions were analysed by SDS–PAGE. a, CENP-LN complex. Note that CENP-L and CENP-N migrate identically in these gels because of their almost identical mass. They can be distinguished by selective addition of a tag, as shown in Fig. 1c. b, CENP-C1–544 complex. c, CENP-HIKM. d, KMN network. e, CENP-ANCP; the lower panel is a MidoriGreen-stained agarose gel of the same fractions analysed by SDS–PAGE in the upper panel. f, H3NCP; bottom panel as in e.

Extended Data Figure 3 Additional CENP-A binding experiments.

a, EMSAs were used to assess relative binding affinity of H3 or CENP-A NCPs to the CENP-LN complex. Quantification of binding data predicts the indicated dissociation constants. In quantifications of a–c, the mean ± s.d. from three independent experiments is shown. b, CENP:CHIKMLN was titrated against Alexa-647-labelled CENP-A NCPs (purple trace) or H3 NCPs (grey trace) in an EMSA assay. Experimental triplicates were performed, and the approximate dissociation constant determined. CENP:CHIKMLN binds with approximately sevenfold higher affinity to CENP-A NCPs than to H3 NCPs. c, EMSA assays were performed using Alexa-647-labelled DNA in free form (black trace), in complex with H3 containing octamers (grey trace), or in complex with CENP-A containing octamers (purple trace). CENP-HIKM complex was titrated against the DNA or NCPs. No binding preference emerged. d, Biotinylated CENP-A NCPs were used as bait to pull down CENP:CHIKMLN complex. Interactions were then competed for using an increasing concentration of free (non-biotylated) CENP-A NCPs (left) or H3 NCPs (right). The ratio of CENP-I to CENP-A was plotted in the lower graph. Free CENP-A NCPs effectively compete off biotinylated CENP-A NCPs from the CHIKMLN complex. Free H3 NCPs are unable to do so, even at concentrations 20-fold the biotinylated bait. The assay was performed in 200 mM NaCl, and used a shorter construct of CENP-C (189–544) owing to the greater stability of this construct at lower salt concentrations and to better separation of the CENP-C and CENP-I bands on SDS–PAGE for analysis. e, Biotinylated nucleosomes were used as bait to pull down CENP:CHIKMLN complex. Pull-downs were performed at increasing salt concentrations from 100–300 mM NaCl. CENP-A nucleosomes maintained a strong interaction with CENP:CHIKMLN in 300 mM salt. H3 NCPs lost the interaction with CENP:CHIKMLN at NaCl concentrations above 200 mM.

Extended Data Figure 4 Binding assays and analytical ultracentrifugation.

a, Normalized sedimentation coefficient (c(s)) distributions of the respective sedimentation velocity runs. The data were collected at 280 nm and the size distribution analysis of the sedimentation coefficient was performed with SEDFIT38 software using a continuous c(s) model. The rotor was spun at 42,000 rpm and equilibrated at 20 °C for 1 h before the start of the run. b, Normalized c(s) distributions of the indicated sedimentation velocity runs. The data were collected at 497 nm (thus analysing signals from CENP-HI57-CKM complex labelled with Alexa Fluor 488) and the size distribution analysis of the sedimentation coefficient was performed with SEDFIT using a continuous c(s) model. The rotor was spun at 42,000 rpm and equilibrated at 10 °C for 2 h before the start of the run. We were unable to carry out runs with isolated CENP-C1–544, CENP-C189–544, or CENP-LN complex, owing to sample instability during the centrifugation experiments. c, Normalized c(s) distributions of the indicated sedimentation velocity runs. The data were collected at 401 nm to monitor sedimentation of blue fluorescent protein (BFP) in chimaeric nucleosomes consisting of residues 2–75 of histone H3.1 and residues 75–140 of CENP-A (see Methods). The chimaeric nucleosomes were mixed with a threefold excess of CHIKMLN complex (containing CENP-C189–544: that is, a construct devoid of the binding domain for the MIS12 complex) to saturate binding.

Extended Data Figure 5 Kinetochore localization studies.

a, Representative images showing kinetochore levels in interphase cells of CENP-A, CENP-C, and CENP-HK (with an antibody raised against the CENP-HK complex) in Flp-In T-REx HeLa cells upon RNAi-based depletion of the indicated proteins. Kinetochores were visualized with anti-CENP-A sera. Scale bar, 10 μm. Magnification 630×. b, Western blots documenting protein depletion. RNAi-based depletion of CENP-C appears incomplete by western blotting, whereas it appears to be very penetrant in immunofluorescence experiments. We have described this phenomenon before20, and found that decreased CENP-C silencing correlates with the higher degree of cell confluence (~80%) for the relatively large-scale RNAi preparations required for western blotting compared with immunofluorescence (where we start with cells at ~30% confluence).

Extended Data Figure 6 Incorporation of CENP-TWSX.

In an in vitro pull-down assay, CENP-ANCP reconstituted with biotinylated DNA were incubated with streptavidin-coated beads and the other recombinant kinetochore species indicated in the INPUT (top) panel of the figure. Beads were recovered by centrifugation and washed, and proteins bound to the solid phase (PULLDOWN, bottom) were visualized by SDS–PAGE followed by Coomassie blue staining. Binding of CENP-TWSX tetramer (which contains only the histone fold domain of CENP-T) was contingent to binding of CENP-C1–544 and CENP-HI57-CKM.

Extended Data Figure 7 Topology of the kinetochore.

Using XL–MS, the inter-peptide interactions within the kinetochore sample were analysed. Intra-protein crosslinks are shown in red, intra-subcomplex crosslinks are shown in orange, inter-subcomplex crosslinks in black. Proteins are coloured according to their subcomplex: CENP-ANCP, purple; CENP-C1–544, red; CENP-HIKM, green; CENP-LN, blue; MIS12-C, peach; KNL1-C, orange; NDC80-C, yellow.

Extended Data Figure 8 Microtubule-binding experiments.

a, Description of reagents and buffer used in experiments in b and in Fig. 3b. b, Rhodamine-labelled microtubules (red channel) were tethered to glass coverslips and incubated in the presence of GFP–KMN (green), Alexa-405-labelled CHIKMLN (blue), or Alexa-647-labelled CENP-ANCP or H3NCP (purple), and combinations thereof. Only CENP-ANCP translocated to microtubules, whereas H3NCP did not. Single microtubules from these images have already been shown in Fig. 3b. c, Quantification of experiments, already shown in Fig. 3b, c. Shown for each channel is mean ± s.e.m. from at least 20 microtubules in at least 2 independent experiments.

Extended Data Figure 9 CENP-C545–943 does not interact with CCAN subunits.

a, SEC analysis of CENP-ANCP (purple), CENP-C631–943 (red trace), and their combination (green trace) shows a stoichiometric interaction. b, SEC analysis of CENP-LN (blue trace), CENP-HI57-CKM (green trace), CENP-C545–943 (red trace), and their combination (orange trace). No apparent shift of CENP-C545–943 was observed. c, Summary of known interactions at the centromere–kinetochore interface. The N-terminal region of CENP-C (exemplified by CENP-C1–544) binds the KMN, the CHIKMLN, and a CENP-A nucleosome. The C-terminal region of CENP-C (exemplified by CENP-C545–943) does not bind core kinetochore components (this study) but interacts with CENP-A loading machinery, including the Mis18 complex, which in turn recruits the CENP-A chaperone HJURP52,53. Each half of CENP-C contains a nucleosome-binding motif, and has therefore the potential to interact with two adjacent nucleosomes. After DNA replication, when CENP-A levels are halved, CENP-A is replaced with H3 (H3.3, refs 54, 55). After mitosis, the C-terminal region of CENP-C contributes to recruit machinery that replaces H3 with CENP-A.

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Weir, J., Faesen, A., Klare, K. et al. Insights from biochemical reconstitution into the architecture of human kinetochores. Nature 537, 249–253 (2016). https://doi.org/10.1038/nature19333

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