The NDC80 complex forma oligomeric arrays along microtubules

Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.
Nature (Impact Factor: 41.46). 10/2010; 467(7317):805-10. DOI: 10.1038/nature09423
Source: PubMed


The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment (a process required for cell division), but the molecular mechanism underlying its function remains unknown. Here we present a subnanometre-resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that the Ndc80 complex binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-tubulin dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments through interactions mediated by the amino-terminal tail of the NDC80 protein, which is the site of phospho-regulation by Aurora B kinase. The complex's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing kinetochore-microtubule attachments.

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Available from: Nikolaus Grigorieff, Oct 03, 2015
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    • "For example, it is possible that one MT end may interact only with a defined set of " dedicated " MAPs within the molecular lawn (Powers et al., 2009; Tooley and Stukenberg, 2011), such that the resulting interface remains similar to the traditionally described interface with repetitive sites, despite different structural organization. Such restricted interactions, for instance, can occur if the kinetochore MAPs interact with each other on the MT surface, so their binding may become biased to the same MT (Alushin et al., 2010; Tooley and Stukenberg, 2011). Alternatively, the kinetochore MAPs may retain their independence, and their interactions with MTs may be unconstrained throughout mitosis. "
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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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    • "Thus, upon initial contact with the motor domain, the unstructured C-terminal tail of MKlp2 may induce conformation adaptation to promote intimate complex formation in a headto-tail geometry, thus clustering. Similarly, the disordered N-terminal tail of Ndc80 interacts with the E-hook of tubulin and induces Ndc80 clustering (Alushin et al., 2010, 2012), suggesting that microtubule binding and cluster formation are intimately connected. We also show that Cdk1/cyclin B1 phosphorylation inhibits oligomerization/clustering and microtubule bundling of MKlp2 in order to avoid premature stabilization and bundling of microtubules that causes defects in chromosome congression in early mitosis. "
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    ABSTRACT: The chromosome passenger complex (CPC) must relocate from anaphase chromosomes to the cell equator for successful cytokinesis. Although this landmark event requires the mitotic kinesin MKlp2, the spatiotemporal mechanistic basis remains elusive. Here, we show that phosphoregulation of MKlp2 by the mitotic kinase Cdk1/cyclin B1 coordinates proper mitotic transition with CPC relocation. We identified multiple Cdk1/cyclin B1 phosphorylation sites within the stalk and C-terminal tail that inhibit microtubule binding and bundling, oligomerization/clustering, and chromosome targeting of MKlp2. Specifically, inhibition of these abilities by Cdk1/cyclin B1 phosphorylation is essential for proper early mitotic progression. Upon anaphase onset, however, reversal of Cdk1/cyclin B1 phosphorylation promotes MKlp2-CPC complex formation and relocates the CPC from anaphase chromosomes for successful cytokinesis. Thus, we propose that phosphoregulation of MKlp2 by Cdk1/cyclin B1 ensures that activation of MKlp2 kinesin and relocation of the CPC occur at the appropriate time and space for proper mitotic progression and genomic stability.
    Cell Reports 03/2014; 7(1). DOI:10.1016/j.celrep.2014.02.034 · 8.36 Impact Factor
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    • "Three lysine residues in the CH domain (Lys 33 , Lys 41 and Lys 115 ) were shown to be critical for this phenotype. However, structural studies showed that these are not directly involved in microtubule binding, since the Nuf2 domain does not directly interact with microtubules (Alushin et al., 2010). The current model is that the CH domain is involved in either stabilizing and/or positioning the Ndc80 complex on the microtubules (Sundin et al., 2011). "
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    ABSTRACT: The kinetochore is a multi-protein structure assembled on eukaryotic centromeres mediating chromosome attachment to spindle microtubules. Here we identified the kinetochore proteins Nuf2 and Ndc80 in the apicomplexan parasite Toxoplasma gondii. Localization revealed that kinetochores remain clustered throughout the cell cycle and colocalize with clustered centromeres at the centrocone, a structure containing the spindle pole embedded in the nuclear envelope. Pharmacological disruption of microtubules resulted in partial loss of some kinetochore and centromere clustering, indicating microtubules are necessary but not strictly required for kinetochore clustering. Generation of a TgNuf2 conditional knock-down strain revealed it is essential for chromosome segregation, but dispensable for centromere clustering. The centromeres actually remained associated with the centrocone suggesting microtubule binding is not required for their interaction with the spindle pole. The most striking observation upon TgNuf2 depletion was that the centrosome behaved normally, but that it lost its association with the centrocone. This suggests that microtubules are essential to maintain contact between the centrosome and chromosomes, and this interaction is critical for the partitioning of the nuclei into the two daughter parasites. Finally, genetic complementation experiments with mutated TgNuf2 constructs highlighted an apicomplexan-specific motif with a putative role in nuclear localization.
    Cellular Microbiology 08/2013; 16(1). DOI:10.1111/cmi.12185 · 4.92 Impact Factor
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