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Structural Basis for the Molecular Mechanisms Involving the Ska Complex in Establishing Stable Kinetochore - Microtubule Attachments.

Final Report Summary - STRUCTMITO (Structural Basis for the Molecular Mechanisms Involving the Ska Complex in Establishing Stable Kinetochore - Microtubule Attachments.)

Chromosome segregation during cell division requires the physical attachment of chromosomes to spindle microtubules mediated by the kinetochore, a large proteinaceous structure assembled on a specialized region of the chromosome called the centromeres. Inappropriate kinetochore-microtubule attachments often lead to chromosome segregation errors implicated in cancer. The four subunit Ndc80 complex and the three subunit Ska complex are the major microtubule components of the kinetochore. While the Ndc80 complex initiates kinetochore-microtubule attachments, the Ska complex further stabilizes the attachments and by doing so facilitate the spindle driven chromosome segregation. Incorrect attachments are eliminated by the centromere associated Chromosomal Passenger Complex containing the Aurora B kinase. My work during the funding period mainly aimed to understand the structural basis for the role of the Ska complex in achieving stable yet dynamic kinetochore-microtubule attachments and on understanding the mechanisms regulating the centromere localization of the CPC.

We had previously shown that the Ska complex (Ska1-Ska2-Ska3) forms a ‘W’-shaped dimer made of triple helical bundles with microtubule-binding domains flanking at the ends of each bundle and that this architecture is critical for stabilizing kinetochore-microtubule attachments (Jeyaprakash et al., 2012, Mol Cell). We further demonstrated that unlike the Ndc80 complex, the Ska complex binds surface-exposed regions of tubulin-monomers that are not affected by microtubule-polymerisation/depolymerisation, thus allowing it to bind dynamic MTs, a key requirement for chromosome segregation (Abad et al., 2014, Nat Commun). Recently, we identified the Ska complex subunit Ska3 as a direct binder of MTs whose activity is required for timely mitotic progression (Abad et al., 2016, Sci Rep).

Error-correction mediated by the CPC depends on its timely inner centromere localisation of during prometaphase mediated by Histone-H3 (HH3) and hSgo1. We had previously demonstrated that the N-terminal tails of both HH3 and hSgo1 are capable of binding Survivin (Jeyaprakash et al., 2011, Structure). Our recent work unprecedentedly showed that CPC-HH3 and CPC-hSgo1 interactions are mutually exclusive in-vitro and might happen in a temporally and/or spatially restricted manner (Gupta et al., MS in prep).

Correct kinetochore assembly and function rely on the maintenance of the centromeric chromatin underlying the kinetochore. Mis18 is a central player regulating this process. We showed that Mis18 centromere localisation and function requires its ‘Yippee-like’ domain mediated oligomerisation (Subramanian et al., 2016, EMBO Rep).

To explore RNA-mediated centromere regulation, we structurally characterised CENP-32, originally identified as a kinetochore-associated RNA-methyltransferase. While CENP-32 kinetochore role is unclear, it is required to integrate centrosomes into mitotic spindle. Ongoing CENP-32 substrate identification and functional characterization will provide novel-insights into RNA-mediated regulation of chromosome segregation.

Over all our work during the funding period provided key mechanistic insights into the regulation of kinetochore function essential for chromosome segregation.