The structure of purified kinetochores reveals multiple microtubule attachment sites

1] Howard Hughes Medical Institute, Department of Biochemistry, University of Washington, Seattle, Washington, USA. [2] Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. [3].
Nature Structural & Molecular Biology (Impact Factor: 13.31). 08/2012; 19(9):925-9. DOI: 10.1038/nsmb.2358
Source: PubMed


Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an ~126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.

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Available from: Bungo Akiyoshi, Oct 05, 2015
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    • "In the budding yeast Saccharomyces cerevisiae, a single MT is captured by the kinetochore; the organization and composition of this site has been determined through high-resolution light and electron microscopy (Joglekar et al., 2009; Gonen et al., 2012). In vertebrate cells, the number of kinetochore-bound MTs (KMTs) increases during prometaphase, and by metaphase, the kinetochore MT fiber (K fiber) contains 20–30 MTs (McDonald et al., 1992; McEwen et al., 1997). "
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    ABSTRACT: Accurate chromosome segregation relies on dynamic interactions between microtubules (MTs) and the NDC80 complex, a major kinetochore MT-binding component. Phosphorylation at multiple residues of its Hec1 subunit may tune kinetochore-MT binding affinity for diverse mitotic functions, but molecular details of such phosphoregulation remain elusive. Using quantitative analyses of mitotic progression in mammalian cells, we show that Hec1 phosphorylation provides graded control of kinetochore-MT affinity. In contrast, modeling the kinetochore interface with repetitive MT binding sites predicts a switchlike response. To reconcile these findings, we hypothesize that interactions between NDC80 complexes and MTs are not constrained, i.e., the NDC80 complexes can alternate their binding between adjacent kinetochore MTs. Experiments using cells with phosphomimetic Hec1 mutants corroborate predictions of such a model but not of the repetitive sites model. We propose that accurate regulation of kinetochore-MT affinity is driven by incremental phosphorylation of an NDC80 molecular "lawn," in which the NDC80-MT bonds reorganize dynamically in response to the number and stability of MT attachments.
    The Journal of Cell Biology 06/2014; 206(1). DOI:10.1083/jcb.201312107 · 9.83 Impact Factor
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    • "The period leading up to blastulation is a period of intense cellular metabolic activity, gene activation, rapidly increasing cell division and differentiation. Cell division and the mitotic process is a series of complex structural rearrangements involving the kinetochore attachments to the microtubules (Gonen et al., 2012), cohesion molecules for the crucially precise separation of the chromosome to ensure correct alignment on the spindle (Clift and Marston, 2011), the spindle assembly complex and many highly specialized proteins subject to precise gene expression (Vogt et al., 2008). Although the cause of a temporal delay in aneuploid embryos compared with their euploid counterparts is not yet fully explained, there exist error detection and repair systems within the cell to prevent aneuploidy (Nasmyth and Haering, 2009). "
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    ABSTRACT: This study determined whether morphokinetic variables between aneuploid and euploid embryos differ as a potential aid to select euploid embryos for transfer. Following insemination, EmbryoScope time-lapse images from 98 blastocysts were collected and analysed blinded to ploidy. The morphokinetic variables were retrospectively compared with ploidy, which was determined following trophectoderm biopsy and analysis by array comparative genomic hybridization or single-nucleotide polymorphic array. Multiple aneuploid embryos were delayed at the initiation of compaction (tSC; median 85.1 hours post insemination (hpi); P = 0.02) and the time to reach full blastocyst stage (tB; median 110.9 hpi, P = 0.01) compared with euploid embryos (tSC median 79.7 hpi, tB median 105.9 hpi). Embryos having single or multiple aneuploidy (median 103.4 hpi, P = 0.004 and 101.9 hpi, P = 0.006, respectively) had delayed initiation of blastulation compared with euploid embryos (median 95.1 hpi). No significant differences were observed in first or second cell-cycle length, synchrony of the second or third cell cycles, duration of blastulation, multinucleation at the 2-cell stage and irregular division patterns between euploid and aneuploid embryos. This non-invasive model for ploidy classification may be used to avoid selecting embryos with high risk of aneuploidy while selecting those with reduced risk.
    Reproductive biomedicine online 02/2013; 26(5). DOI:10.1016/j.rbmo.2013.02.006 · 3.02 Impact Factor
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    • "Our biochemical experiments show that residues close to the CH-domains support the Ndc80–Dam1 interaction. This conclusion is independently supported by a recent EM analysis of isolated yeast kinetochores (Gonen et al., 2012). In this study the contact point between the Ndc80 complex and Dam1 rings appeared to be in close proximity to the CH-domains. "
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    ABSTRACT: Kinetochores are large protein complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore-microtubule interface is formed by the plus end-associated Dam1 complex and the kinetochore-resident Ndc80 complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80-Dam1 interaction and probe their function in vivo. A novel ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80-Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.
    The Journal of Cell Biology 12/2012; 200(1). DOI:10.1083/jcb.201210091 · 9.83 Impact Factor
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