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Title: Kinetochore dynamics and their attachment to the mitotic spindle
Author: Smith, Christopher
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2016
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Chromosome segregation is a mechanical process that requires assembly of the mitotic spindle – a dynamic microtubule-based force-generating machine (Dumont & Mitchison, 2009). Connections between chromosomes and the spindle are mediated by kinetochores, pairs of multi-protein complexes assembled on centromeric chromatin that harness the pushing and pulling forces associated with microtubule dynamics to power chromosome movement (Rago & Cheeseman, 2013). This structure is reported to be compliant (Wan et al., 2009; Maresca & Salmon, 2009; Uchida et al., 2009; Dumont et al., 2012; Drpic et al., 2015; Etemad et al., 2015; Tauchman et al., 2015; Magidson et al., 2016), which is the basis upon which it can transduce forces, and satisfy mitotic checkpoints. However recent studies have suggested that the kinetochore is a non-compliant linkage (Suzuki et al., 2014), while others negate the requirement for intra-kinetochore stretch to allow onset of anaphase (Etemad et al., 2015; Tauchman et al., 2015; Magidson et al., 2016). It is therefore crucial that the reasons for these differences are elucidated, and a new model developed to explain regulatory pathways sensitive to intra-kinetochore structure. I have developed a novel semi-automated imaging assay to track kinetochore proteins tagged with two different coloured fluorescent proteins in 3D, coupled with a detailed assay for measurement of the required correction for 3D chromatic aberration directly from cells. I reveal using three dimensional tracking that the kinetochore forms a non-compliant linkage between centromeric chromatin and kintetochore spindle fibres. Instead the outer kinetochore layer can swivel around the inner kinetochore/centromere, which results in a reduction in intra-kinetochore distance when viewed in lower dimensions, the only 3D measurement thus far being by Etemad et al. (2015). I show that swivel provides a mechanical flexibility that enables kinetochores at the periphery of the spindle to engage microtubules. Swivel reduces as cells approach anaphase, explaining previous suggestions that the kinetochore needs to be stretched upon bi-orientation prior to anaphase onset (Maresca & Salmon, 2009; Uchida et al., 2009). Reduction in swivel suggests there is an organisational change linked to checkpoint satisfaction, and/or changes required in kinetochore mechanochemistry before sister chromatid dysjunction. I show that Cdk1 inactivation is required to suppress kinetochore-microtubule dynamics in anaphase, and therefore may mediate required changes in kinetochore state. Finally, my work opens up the possibility to map the 3D architecture of the kinetochore, and between different domains within proteins and complexes. With such an architectural understanding, it will now be possible to investigate the mechanisms of organisational changes that occur during cycles of microtubule attachment and the regulatory processes of error-correction and SAC activation/silencing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology