Pinder, Corinne; (2018) Exploring the roles of kinesin-8 in mitotic progression. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The mitotic spindle employs myriad proteins for chromosome segregation, in addition to monitoring the fidelity of this process and correcting mistakes before they become problematic for daughter cells. Key players in mitosis include the superfamily of kinesin motors, a variety of microtubule-associated proteins (MAPs) and members of the spindle assembly checkpoint; deregulation of these factors has been observed in multiple tumour types. Our interest lies in the conserved family of microtubule plus-end directed kinesin-8 motors, which have been implicated in some breast and colorectal cancers. Klp5/Klp6 constitute the S. pombe kinesin-8 complex, and like the human counterpart, Kif18A, localise dynamically on the spindle, interact with kinetochores and regulate microtubule dynamics to allow a timely and coordinated metaphaseanaphase transition. Although these functions have been assigned in multiple species, exact mechanisms of the diverse kinesin-8 functions have not been elucidated, particularly in fission yeast. In contrast to prior studies of Klp5/6, where mainly deletion or truncation mutants were used, we employ a targeted mutagenesis approach to look directly at kinesin-8 function and regulation. Though Klp5/6 are non-essential, we exploit their synthetic lethality with various MAPs to isolate temperature-sensitive alleles of klp5. We find that a specific mutation in the non-motor kinesin tail has a deletion-like phenotype, disrupting chromosome congression, bipolar kinetochore attachment, negative regulation of spindle length and overall control of microtubule dynamics. Additionally, the processivity of the mutant is greatly impaired in vitro. Using this loss-of-function mutant with the deletion of XMAP215/TOG family microtubule polymerase, dis1Δ, we find that both kinesin-8 and Dis1 synergistically negatively regulate pre-anaphase and anaphase A spindle elongation but promote bipolar kinetochore-microtubule attachments in distinct ways. The activity of both factors is required to prepare metaphase for a timely and concerted anaphase for high fidelity chromosome segregation.

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