Baker, Michael (2016) The assembly and disassembly of clathrin cages. PhD thesis, University of Warwick.

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Abstract

Clathrin mediated endocytosis (CME) is an integral process in eukaryotic cells and governs a wide range of processes in higher organisms including neurotransmitter release and cell signalling, to development and cell polarity. Due to its wide ranging roles this mechanism has been implicated in various disease states such as Huntington’s disease and various cancers as well as being a method of entry into cells for many viruses and bacteria. The process of CME involves the formation of a clathrin coat, primarily consisting of the protein clathrin, that drives uptake of cargo at the plasma membrane. These interactions are facilitated through a large family of proteins known as adaptor proteins, which drive the process of CME through specific interactions with clathrin, cargo, the plasma membrane and the cytoskeleton. Once the cargo has been endocytosed the process must be reversed through the actions of the disassembly proteins auxilin/GAK and Hsc70. A number of questions remain as to how adaptors promote assembly and how auxilin and Hsc70 drive disassembly through interaction with clathrin and potentially through interactions with the adaptor proteins.

By purifying adaptor proteins and clathrin I have used various biochemical and biophysical techniques to investigate these interactions in vitro. Using a novel assembly assay based on dynamic light scattering I have shown that it is possible to measure the effect of adaptors on clathrin cage size distribution during assembly. In disassembly I have shown how mutations in the disassembly protein auxilin affect its ability to catalyse the disassembly of clathrin cages and how the presence of various adaptor proteins alters the ability of auxilin and Hsc70 to disassemble these structures. Finally, I demonstrate an inhibitory effect on disassembly by the adaptor protein epsin and propose a mechanism of interaction with clathrin that can be disrupted through mutations to epsin clathrin-binding motifs and discuss the implications of this effect for the role of adaptors in vivo.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QH Natural history
Library of Congress Subject Headings (LCSH):	Endocytosis -- Research, Coated vesicles, Cell membranes -- Transport, Carrier proteins
Official Date:	September 2016
Dates:	Date Event September 2016 Submitted
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Smith, Corinne ; Dafforn, Tim
Sponsors:	Biotechnology and Biological Sciences Research Council (Great Britain)
Format of File:	pdf
Extent:	xxi, 209 leaves : illustrations, charts
Language:	eng

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