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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    • "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.
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    • "Another important development brought about by our studies is the availability of the first 3D structure of a KMN network complex . Previously, 2D negative-stain EM analyses provided insight into the overall shape of the Ndc80 and Mis12 complexes (Gonen et al., 2012; Hornung et al., 2011; Maskell et al., 2010; Petrovic et al., 2010; Screpanti et al., 2011; Wang et al., 2008; Wei et al., 2005), while hybrid methods have shed light on the interaction of the Ndc80 complex with microtubules (Alushin et al., 2012, 2010). By fitting the crystal structures of the Ndc80-C Bonsai complex (Ciferri et al., 2008) and of C-terminal region of Knl1 (this study) into the 3D structure of Complex 4, we unequivocally identify the relative position of the RWD-containing subunits that interact with the Mis12 complex. "
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    ABSTRACT: Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore.
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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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