Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.800096
Title: Integration of microtubule-kinetochore attachment formation and spindle assembly checkpoint signalling
Author: Barnard, Christina H. Y.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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Abstract:
This study focuses on mitosis and the regulation of microtubule-kinetochore attachment formation by phosphorylation. Kinetochores act as platforms for both the attachment of bi-polar spindle microtubules to chromosomes and the generation of the surveillance systems which regulate this process. Both the error-correction pathway and the spindle assembly checkpoint are activated in the presence of non-attached or non-tension bearing kinetochores. These mechanisms rely on dynamic phosphorylation and dephosphorylation for the transduction of signals allowing the regulation of protein:protein interactions. Many kinetochore proteins have been found to be highly phosphorylated in mitosis, but less is known about the temporal regulation of these phospho-sites. In addition, only a small proportion of sites and their biological function have been investigated in detail. Here, I have developed a mass spectrometry protocol combined with a synchronisation step to isolate the outer kinetochore and compare the phosphorylation state at unattached and attached kinetochore states. Our analysis showed that multiple phosphorylation sites on the outer kinetochore are dephosphorylated upon attachment. As key examples we identified the six active MELT motifs on Knl1, which regulate the recruitment of SAC proteins. Intriguingly, we also identified two Ndc80 phosphorylation sites, pThr61/Ser62 and pSer69, which are up-regulated upon kinetochore-microtubule attachment. This suggests that the regulation of microtubule attachment to the outer kinetochore by different kinases may be more complex than hitherto thought, as up-regulation upon attachment has so far not been reported. As an example of a specific kinase-regulated protein complex involved in the generation of microtubule-kinetochore attachment formation we investigated the role of Plk1 associating with the Astrin-Kinastrin complex. Previous results in our lab showed depletion of Astrin or Kinastrin +results in delayed chromosome alignment and loss of spindle bipolarity. In this study we demonstrate that Plk1 binds directly to Astrin via a Cdk1-phosphorylated sequence in the N-terminus of Astrin. Using mass spectrometry we identify four Plk1-dependent phosphorylation sites on Astrin at residues, pSer157, pSer159, pSer353 and pSer411 and demonstrate that phosphorylation of these sites is dependent on Plk1 activity in vivo. Interestingly, we can demonstrate that the interaction of the Astrin-Kinastrin complex with Plk1 is dispensable for bipolar spindle formation but does play a role in timely chromosome congression and anaphase onset, thus separating the different aspect of the Astrin/Kinastrin depletion phenotype.
Supervisor: Not available Sponsor: Sir William Dunn School of Pathology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.800096  DOI: Not available
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