Unsworth, A.; (2007) The mitotic role and regulation of Kinesin-8 Klp5 and Klp6 in fission yeast. Doctoral thesis , University of London.

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Abstract

Chromosome segregation in mitosis is mediated by the spindle, a structure consisting of microtubule fibres. Microtubules are inherently dynamic that is, constantly growing and shrinking and this property is utilised in the cell to create pushing and pulling forces. Cells contain an array of stabilising and destabilising factors to achieve precise spatial and temporal control of microtubule dynamics. During mitosis, this enables spindle elongation and shortening to be co-ordinated with chromosome segregation. It also facilitates the capture of kinetochores by microtubules. Several kinesin subfamilies regulate microtubule dynamics, namely Kinesin-8, -13 and -14. Klp5 and Klp6 in fission yeast belong to the Kinesin-8 family, which are thought to function both as plus-end motors and as microtubule depolymerisers. Klp5 and Klp6 form a heterocomplex. They localise to cytoplasmic microtubules during interphase, and to kinetochores, the spindle, and the spindle midzone during mitosis. Deletion mutants are viable but show defects in chromosome congression and segregation, and activation of the spindle checkpoint. We show that mitotic chromosome movements are abnormal in Aklp5/Aklp6 cells, and that in the absence of spindle checkpoint function, chromosome missegregation may occur. We also show that Klp5/6 are required for maintenance of constant metaphase spindle length. We constructed rigor (ATPase) mutants and show their phenotype. We explore the significance of Klp5/6 heterocomplex formation for subcellular localisation and regulation of activity. We find that Klp5 and Kp6 are co-dependent for their localisation to the nuclear mitotic spindle, and that monomers are able to enter the nucleus, but cannot be retained. We identify a conserved NLS in Klp5 and Klp6. We consider whether heterocomplex formation is required solely for nuclear retention of KJp5 and Klp6, or if they contribute differing properties to the complex. We made N- and C-terminal deletions, and constructed Klp5 and Klp6 chimeric molecules. We found that the C-termini are dispensable, but that both Klp5 and Klp6 N-termini contribute essential and distinct functions to the Klp5/Klp6 heterocomplex.

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