Motility and regulation of kinesin-2

Webb, Stephanie (2022) Motility and regulation of kinesin-2. PhD thesis, Birkbeck, University of London.

Abstract

This thesis investigates the intrinsic and extrinsic regulation of kinesin-2 motor proteins, using the human family member Kif3 as a paradigm. Kif3 is the essential motor for anterograde transport along the microtubules of cilia and flagella. Unusually among kinesins, Kif3 is a heterotrimer of two different force-generating heavy chains, Kif3A and Kif3B, and an accessory subunit, Kap3. To date, how kinesin-2 activity is spatiotemporally controlled to elicit proper transport within cilia is not fully understood. This work uses biochemical methods, single-particle electron microscopy, structural modelling, and single-molecule TIRF microscopy to investigate the mechanism of Kif3 regulation. The first results chapter uncovers a novel auto-inhibitory motif specific to kinesin-2. This motif, named a charged b-hairpin, was found in the tail domains of Kif3A and Kif3B. The data lead to a model in which the charged b-hairpins of Kif3 interact with its motor domains, facilitating the occlusion of the microtubule-binding sites, with potentially additional inhibitory effects from the disordered regions of the tail. This mechanism of auto-inhibition is likely to be conserved across the kinesin-2 family. The second results chapter investigates how Kap3 interacts with Kif3A and Kif3B. These experiments revealed that both the Kif3A and Kif3B tails interact with Kap3, with a region in Kif3A being the most important for Kap3 binding. Congruently, I show that Kap3 can also bind to another motor in the kinesin-2 family, Kif3A/C, which shares the Kif3A subunit with Kif3A/B. Single-particle electron microscopy shows that the Kif3 heterotrimer has a compact, inhibited conformation. The final results chapter probes the regulation of Kif3 by extrinsic factors. I reconstitute the interaction between Kif3 and its partner in anterograde intraflagellar transport, the IFT-B1/B2 complex. Finally, I show that Kif3 motility is inhibited by kinesin binding protein (KBP) and present two-dimensional cryo-electron microscopy analysis of the Kif3A motor domain complexed with KBP. Together, the work in this thesis sheds light on the multiple layers of regulation that enable kinein-2 motors to perform their essential functions in living cells.

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