Wood, Katherine M. (2021) The role of adaptor proteins in the dynamics of clathrin coat disassembly. PhD thesis, University of Warwick.
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Abstract
Clathrin mediated endocytosis (CME) allows selective uptake of extracellular molecules into cells using cell surface receptors. CME is associated with different diseases (e.g., Parkinson’s disease) where mutations and differing expression levels of CME adaptor proteins are observed. The current understanding of clathrin-adaptor interactions is limited to low-resolution cryo-EM structures and crystal structures of the clathrin terminal domain with bound peptides derived from adaptor proteins, despite the wealth of data on the role of adaptor proteins within endocytosis. There are also still many questions with regards to how multiple adaptor proteins interact and bind to clathrin cages to enable controlled clathrin coat assembly and disassembly. This project aimed to gain a greater understanding of how adaptor proteins interact with clathrin, and the impact these interactions have on clathrin cage disassembly.
Initially cryo-EM was conducted to determine the structure of a clathrin cage hub with no adaptor proteins bound and terminal domains resolved, providing new insights into clathrin-clathrin interactions in the absence of adaptor proteins. This was followed by cryo-EM of clathrin cages with the disassembly adaptor protein auxilin bound, to acquire higher resolution details on auxilin binding to clathrin cages. Finally, effects of clathrin-adaptor interactions on clathrin disassembly were further analysed using fluorescence anisotropy (FA) to assess adaptor protein competition for clathrin binding sites. Overall, structures of the clathrin cage complex and the clathrin auxilin complex were successfully produced providing information on how auxilin interacts with clathrin during clathrin disassembly. The FA technique has been developed for measuring clathrin-adaptor interactions and clathrin disassembly rates, enabling competition between adaptor proteins auxilin, AP180, epsin, ARH and β2-adaptin to be identified. Further use of FA will be beneficial for assessing clathrin adaptor interactions and adaptor competition to understand how adaptor protein mutations and expression levels may cause or contribute to disease states.
| Item Type: | Thesis [via Doctoral College] (PhD) |
|---|---|
| Subjects: | Q Science > QH Natural history Q Science > QP Physiology Q Science > QR Microbiology |
| Library of Congress Subject Headings (LCSH): | Endocytosis, Cell membranes, Cell receptors, Signal transducing adaptor proteins, Electron microscopy |
| Official Date: | November 2021 |
| Dates: | Date Event November 2021 UNSPECIFIED |
| Institution: | University of Warwick |
| Theses Department: | Warwick Medical School |
| Thesis Type: | PhD |
| Publication Status: | Unpublished |
| Supervisor(s)/Advisor: | Smith, Corinne J. ; Chmel, Nikola Paul |
| Sponsors: | Medical Research Council (Great Britain) |
| Format of File: | |
| Extent: | xxvi, 275 leaves : illustrations |
| Language: | eng |
| URI: | https://wrap.warwick.ac.uk/166648/ |
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