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    Motility and regulation of kinesin-2

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

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    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.

    Metadata

    Item Type: Thesis
    Copyright Holders: The copyright of this thesis rests with the author, who asserts his/her right to be known as such according to the Copyright Designs and Patents Act 1988. No dealing with the thesis contrary to the copyright or moral rights of the author is permitted.
    Depositing User: Acquisitions And Metadata
    Date Deposited: 23 Sep 2022 13:46
    Last Modified: 01 Nov 2023 15:44
    URI: https://eprints.bbk.ac.uk/id/eprint/49184
    DOI: https://doi.org/10.18743/PUB.00049184

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