Accurate regulation of kinetochore–microtubule affinity is driven by incremental phosphorylation of an NDC80 molecular “lawn,” in which NDC80–microtubule bonds reorganize dynamically in response to the number and stability of microtubule attachments.

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment.

The NDC80 complex is known to function in kinetochore-microtubule attachment during mitosis. We analyzed the mitotic roles of three separate structural motifs within the complex and found that the Nuf2 CH domain, the Hec1 CH domain, and the Hec1 tail domain each make distinct contributions at the kinetochore-microtubule interface.

Kinetochore-associated NDC80 complexes serve as the primary binding site for the plus-ends of spindle microtubules in mitosis. A recent study proposes a novel mechanism for regulating kinetochore-microtubule binding involving NDC80 complex oligomerization, which could be mediated by Aurora B kinase.

Best macular dystrophy (BMD) is an autosomal dominant form of macular degeneration, linked to mutations in the hBEST1 gene, which encodes the calcium activated chloride channel (CACC) bestrophin-1. The bestrophin family includes three additional members: hBEST2, 3 and 4. Because mutations in hBEST1 cause BMD, but a knockout does not, hBEST1 mutants have been suggested to exert a dominant negative effect through interaction with other CACCs. Using single molecule subunit counting and co-localization we find that each hBEST forms a homotetrameric channel. Despite considerable conservation among hBESTs, hBEST1 has little or no interaction with other hBESTs. Deletion and chimera analysis are being used to identify the portions of hBEST1 that allow assembly of like subunits and prevent assembly with other hBESTs. Our results suggest that the pathology caused by hBEST1 mutations is not due to assembly with other CACCs.

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