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Alghamdi, Hanan (2023) Characterisation of the cilia-associated Kinesin-16, KIF12. PhD thesis, University of Nottingham.

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Abstract

Kinesin-16 is a family of kinesins that are only found in organisms that possess cilia or flagella. The human Kinesin-16, KIF12, is implicated as a polycystic kidney disease (PKD) modifier. Dysfunction of KIF12 may also be involved in the progression of certain types of diabetes and mutations to KIF12 have been shown to be strongly correlated with paediatric cholestatic liver disease.

KIF12 localises to the primary cilia and may affect the function of cilia by playing a role in either transport within the cilia or controlling microtubule dynamics and the assembly and length of cilia. The activity of KIF12 in relation to microtubules and the coupled ATP turnover have not yet been defined. Therefore, my research interest is to characterise KIF12 microtubule interaction and ATPase cycle. This information will aid in elucidating a molecular understanding of the cellular role of KIF12.

Using a truncated version of KIF12 which comprised the first 434 amino acids and included the motor domain and a stretch of coiled-coil, I studied KIF12 in a microtubule “gliding assay” and a single molecule “stepping assay” to characterise microtubule motility. The results of these assays show that KIF12 does not exhibit any ability to translocate in a directional manner along microtubules. A microtubule depolymerisation assay showed that KIF12 has no depolymerisation activity but may stabilise microtubules. Observing the impact of KIF12 in a dynamic microtubule assay, shows that it causes a small but significant increase in microtubule growth length but no significant increase in growth rate. I did not observe KIF12 interacting with microtubules in a dynamic microtubule assay as in the presence of unpolymerized tubulin KIF12 is sequestered away from the microtubules by binding to unpolymerized tubulin. An attempt to quantify the affinity of KIF12 for unpolymerized tubulin and microtubules suggest that KIF12 has higher affinity for unpolymerized tubulin than microtubules. ATP turnover by KIF12 is not accelerated by the presence of unpolymerized tubulin resulting in KIF12 remaining in a tightly bound ATP containing state when interacting with unpolymerized tubulin. Microtubules accelerate the ATPase of KIF12 ~20-fold, suggesting that KIF12 is driven into a nucleotide state that binds less tightly to microtubules by interaction with microtubules.

Due to solubility issues for the full-length protein, I worked with a truncated version of KIF12. Expression and purification of the remaining tail region of KIF12 was carried out but it proved difficult to obtain a soluble protein of sufficient purity. Nevertheless, studying partially purified KIF12 tail suggests that this region does not interact with microtubules and lack of this region likely does not impact the affinity of KIF12 for microtubules.

A mutant form of KIF12, KIF12∆PPGGG, results in a less severe disease phenotype in a PKD mouse model. This deletion was found to decrease the affinity of KIF12 for microtubules. Neither wild type KIF12-434 nor KIF12-434∆PPGGG could distinguish GTP-lattice (GMPCPP microtubules) from GDP lattice (Taxol microtubules). However, both recognise and concentrate at the ends of Taxol- stabilised microtubules indicating that KIF12 can distinguish a conformational difference between the end and the lattice of Taxol-stabilised microtubules.

Finally, as an attempt to obtain and study full-length KIF12, I expressed full length GFP tagged KIF12 in a HEK293 cell line. It did not prove possible to purify the protein from these cells. However, observation of the localisation of KIF12 in cells shows that KIF12 decorates the microtubule network. No evidence for translocating activity was observed for KIF12 expressed in HEK293 cells.

The data collected on KIF12-434 and full length KIF12, show that KIF12 interacts with microtubules both in vitro and in cells in a diffusive fashion and does not display translocating activity on microtubules. This suggests that KIF12 does not play a transport role in cilia. KIF12 has a small but significant stabilising effect on both GMPCPP-stabilised microtubules and dynamic microtubules and likely acts in cilia as a microtubule regulating kinesin. It is possible that KIF12 is involved in regulation of cilia assembly or disassembly. Further investigations are required for a complete characterisation of this KIF12 and its function in cilia and involvement in disease progression.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Friel, Claire Wickstead, Bill
Keywords:	Kinesins; KIF12; Microtubules; ATPase cycle; Cilia; Disease progression
Subjects:	Q Science > QP Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	72423
Depositing User:	Alghamdi, Hanan
Date Deposited:	31 Jul 2023 04:40
Last Modified:	31 Jul 2023 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/72423

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