Identification and functional characterisation of new ciliary base proteins and investigation of diffusion kinetics across the ciliary transition zone in Caenorhabditis elegans roundworms

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Title: Identification and functional characterisation of new ciliary base proteins and investigation of diffusion kinetics across the ciliary transition zone in Caenorhabditis elegans roundworms
Authors: Sanders, Anna Antoinette Wilhelmina Maria
Advisor: Blacque, Oliver E
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Date: 2014
Abstract: Cilia are evolutionarily conserved microtubule based organelles extending from the surface of most cells serving important sensory and signalling functions. Defects in cilia cause a variety of disorders with overlapping phenotypes, termed ciliopathies. The ciliary base, acting as a ciliary gate, plays a key role in regulating ciliary protein composition and cilia-related signal transduction pathways, forming membrane and cytosolic diffusion barriers that prevent exchange between ciliary and non-ciliary compartments. The transition zone at the proximal ~1 µm of the axoneme is part of the ciliary base and ciliopathy proteins localising specifically to the transition zone form two functional modules (MKS and NPHP). Together, these modules are required for ciliogenesis, and are implicated in membrane diffusion barrier function in <italic>Caenorhabditis elegans</italic> cilia. Active transport across the transition zone diffusion barrier is thought to be facilitated by intraflagellar transport. The protein composition of the ciliary base is not fully known and molecular mechanisms underlying the diffusion barriers are poorly understood.The work presented in this thesis is focussed on the ciliary base and the transition zone in <italic>C. elegans</italic> ciliated sensory neurons. Specifically, Chapter III focusses on characterisation of a candidate ciliary component, K04F10.2. Exhaustive phenotypic analysis indicates that K04F10.2 serves ciliary functions. Consistent with a ciliary role, subtle IFT defects were observed, as was a functional interaction with the ciliopathy protein, Joubert Syndrome-associated ARL-13. The interactome of human KIAA0556 (K04F10.2 ortholog) was identified and subsequent analysis in worms revealed novel ciliary localisations and transport properties for RAB-28 and F47G4.5/KATNBL1.Chapter IV describes the development of a Fluorescence Recovery After Photobleaching (FRAP)-based assay to validate the existence of a transition zone membrane diffusion barrier. The subsequent use of this assay to investigate the molecular mechanisms that establish and maintain this barrier reveals that various MKS- and NPHP-module components are differentially required for diffusion barrier integrity. Additionally, IFT components are required for active transport to overcome the transition zone membrane diffusion barrier. This FRAP assay is the first such described in a multicellular system and allows for in vivo investigation of exchange kinetics across the transition zone membrane diffusion barrier in real time.
Type of material: Doctoral Thesis
Publisher: University College Dublin. School of Biomolecular and Biomedical Science
Qualification Name: Ph.D.
Copyright (published version): 2014 the author
Keywords: Caenorhabditis elegansCiliaCiliopathyFRAPMicroscopyTransition zone
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Language: en
Status of Item: Peer reviewed
Appears in Collections:Biomolecular and Biomedical Science Theses

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