We have made outstanding progress on the proposed research as summarised below:
WP1 Establishment of inner centromere signalling platform: One of the key protein assemblies that associates with the inner centromere is the Chromosomal Passenger Complex (consisting of four different proteins, Borealin, Survivin, INCENP and Aurora B (a protein kinase)). The CPC interacts with two other major regulators knows as Shugoshin 1 and HP1. Towards understanding how these proteins interact with each other to establish the inner centromere signalling platform, we first focused on determining the structure of CPC bound to nucleosome. We have been successful in determining a high-resolution cryo-EM structure of the core inner centromere component of CPC (consisting of Borealin, Survivin and INCENP) bound to nucleosome at 2.8 angstrom resolution. The molecular details of their interactions reveal that CPC-nucleosome binding stabilises the centromeric chromatin (nucleosomes forming the microtubule attachment sites). We are now working towards characterising the interaction of CPC, Shugoshin 1 (Sgo1) and HP1 with nucleosomes. Structural and biochemical characterisation these interactions are in progress.
WP2 Mechanisms of recruitment of enzymatic activities by the inner centromere interaction network: Protein phosphatase 2A (PP2A), which exists in different forms due to the presence of different regulatory protein subunits (B55 and B56) plays critical roles in regulating centromere functions such as maintaining centromere cohesion, error-correction (selective stabilisation of bi-polar attachments) and timely onset of anaphase (cell division stage when sister chromatids move towards the opposite poles of the mitotic spindle). However, the mechanistic basis of how different Shugoshin isoforms (Sgo1 and Sgo2) preferentially recruit PP2A to different sub-centromeric locations remains an open question. We have now reconstituted these protein complexes for further structural characterisation.
WP3. Mechanisms of centromere maintenance: As outlined above, the centromere is defined by the enrichment of a Histone H3 Variant, CENP-A, containing nucleosomes, which is critical for assembling the microtubule-capturing protein hub called the kinetochore. Importantly, the CENP-A levels undergo dilution during S-phase (a specific phase of the cell cycle, where the genetic information is duplicated for subsequent segregation to the newly formed daughter cells), as existing CENP-A is shared among the new and old DNA. To ensure stable maintenance of the centromere, the original CENP-A levels must be restored through active CENP-A deposition. By characterising the architecture of the CENP-A deposition machinery and its interaction with a protein modifying PLK1 kinase, we unravelled molecular details of how PLK1 allows CENP-A loading machinery to deposit CENP-A at the right time during the cell cycle, a question that remained unanswered for more than a decade.
During this grant period, through a collaborative effort, we have also characterised CENP-32 (an RNA modifying enzyme critical for the assembly of functional chromosome segregation apparatus, the mitotic spindle) using biochemical, structural, cell biological and clinical approaches. Our work showed that CENP-32 mutations found in patients with developmental defects affect CENP-32 enzymatic activity. We also showed that mutants compromising CENP-32 activity lead to spindle defects, causing chromosome segregation errors and altered cell proliferation.
