Chmielewska, AE; (2015) Studying roles of the kinetochore component Ndc80 in spindle assembly checkpoint. Doctoral thesis , UCL (University College London).

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Abstract

Accurate chromosome segregation relies on the kinetochore, a multiprotein structure, which assembles around the centromeric region of the chromosome. Proper kinetochore-microtubule attachment is critical for the segregation of sister chromatids toward the opposite poles during mitosis. The outer kinetochore, notably the KMN network (KNL-1 complex/Mis12 complex/Ndc80 complex), links chromosomes to the mitotic spindle, in which the Ndc80 protein is a primary microtubule-binding site. Any improper attachments are recognised by the spindle assembly checkpoint (SAC), a surveillance pathway, which blocks premature segregation of chromosomes by preventing anaphase onset. Incorrect kinetochore-microtubule attachments lead to SAC activation, which is coupled with localisation of its components (Mph1/MPS1, Mad1, Mad2, Mad3/BubR1, Bub1 and Bub3) to the kinetochores. However, the mechanisms by which SAC components other than Bub1 and Bub3 are recruited to the kinetochore remain largely elusive. In this study, I show isolation and characterisation of an ndc80 mutant (ndc80-AK01) in fission yeast. The ndc80-AK01 mutant contains a single point mutation within an uncharacterised linker/hairpin region that connects a calponin-homology domain and a coiled-coil domain. This mutant is hypersensitive to microtubule poisons with no apparent growth defects in the absence of drugs. Subsequent analysis indicates that ndc80-AK01 is defective in SAC signalling. Localisation studies of SAC components have shown the absence of GFP-tagged Ark1, Mph1, Bub1, Bub3, Mad3, Mad2 and Mad1 from kinetochores under mitotic arrest conditions. Genetic and cell biological data indicate that the Ndc80 linker region may act as a structural platform for kinetochore recruitment of Mph1, which is one of the most upstream SAC components. Intriguingly, artificial tethering of Mph1 to the kinetochore restores checkpoint signalling in the ndc80-AK01 mutant, further confirming the function of Ndc80 as a kinetochore platform for Mph1. These data have unveiled a hitherto unknown function of the Ndc80 linker region in the SAC.

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