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Title: Analysis of the gating structures at the cilia base
Author: Hazime, Khodor
ISNI:       0000 0004 7230 4687
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Cilia are microscopic microtubule-based projections that emanate from basal bodies and protrude from the cell membrane of most eukaryotic cells. The base of the cilium is an evolutionary conserved sub-domain that acts as a gate to regulate and modulate trafficking of proteins to and from cilia. All proteins destined for the cilium cross the gating barriers at the base of the cilium in a tightly regulated manner preventing small polypeptides from entering while permitting the entrance of large protein complexes, such as the intraflagellar transport (IFT). Where exactly these gating structures are positioned, where IFT assembly takes place, and how this process is regulated is still ambiguous. Based on STORM imaging, I present the first detailed 3D-model that shows the position of over 30 epitope tags in the IFT, transition zone (TZ), and basal body. We established Tetrahymena thermophila transgenic lines with Cand/or N- termini epitope tags of over 18 different proteins. I show that IFT, TZ, and other ciliary proteins form 9-fold symmetrical rings at the cilia base. I reveal for the first time the architecture of IFT particles in their docking sites. By using particle averaging of the radial and axial locations at the N- and C- termini of the proteins, I was able to determine the orientation of these proteins. I show that in the radial dimension, IFT-A complex lies outside IFT-B complex. I also reveal the localisation of the binding sites of cargoes, such as tubulin and outer dynein arms, at the cilia base. These studies made it possible to generate a 3D model of the cilia base encompassing crucial components in unprecedented details. Moreover, I examined the relationship between the apico-basal polarity determinants, crumbs genes, and the gating mechanisms at the cilia base. I investigated mutant phenotypes of zebrafish crumbs genes. These mutations affect cilia length in a subset of tissues, and in some mutants, this is accompanied by accumulation of other Crumbs proteins and IFT particle components in the ciliary shaft. These findings reveal crumbs-dependent mechanisms that regulate the localisation of ciliary proteins at the cilia base, including Crumbs proteins themselves, and show that crumbs genes modulate intraflagellar transport and cilia elongation possibly acting as a component of a gating mechanism.
Supervisor: Malicki, Jarema Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available