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

ABSTRACT

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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    • "Another recent example of selective interactions with specific conformations of ab-tubulin comes from work in our laboratory showing that TOG domains from the microtubule polymerase Stu2p bind preferentially to the curved conformation of ab-tubulin (Ayaz et al., 2012;, and that this preference of TOG domains for curved ab-tubulin is probably what allows the polymerase to localize to the growing tip of the microtubule (Ayaz et al., 2014). These and numerous studies of other regulatory proteins (Desai et al., 1999;Gigant et al., 2000;Peters et al., 2010;Alushin et al., 2010;Bechstedt et al., 2014) contribute to the emerging view that selective binding to distinct conformations of ab-tubulin represents a common and important strategy for recognizing and controlling microtubules. How the ab-tubulin conformational cycle contributes to microtubule dynamics is less understood. "
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    ABSTRACT: Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking.
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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.
    Full-text · Article · Jun 2014 · The Journal of Cell Biology
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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.
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