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Title: The role of microtubule motors in membrane trafficking
Author: Hunt, Sylvie Dupont
ISNI:       0000 0004 5356 7289
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2014
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Little is known about the fundamental mechanisms required for cargo sorting into discrete intracellular trafficking pathways. A large body of evidence places opposing polarity microtubule motor proteins in the endosomal network. With specific roles for different family members, microtubule motors are suggested to play a central role in defining the high specificity of cargo sorting. The role of microtubule-based motor proteins in membrane trafficking pathways is first investigated in this thesis using total internal reflection fluorescence (TI RF) imaging techniques and high-resolution object tracking assays. Sorting nexins (SNX) are a group of cytoplasmic and membrane-associated proteins involved in trafficking, driving membrane deformation and coupling to microtubule-based motors hence their key role in endosomal dynamics. Coordination of opposite motors applies longitudinal tension to facilitate SNX-labelled endosome scission. The same cohort of motors ensures SNX-coated subdomain segregation and motility therefore participating in the maintenance of the functional architecture of these organelles. The cytoplasmic dynein interactome is subsequently established by stable-isotope by amino-acids labelling proteomics to identify novel dynein interacting proteins and allocate any prospective new adaptors within discrete intracellular trafficking pathways. A novel dynein adaptor, TIP47, required for lipid droplet biogenesis, is identified and characterised, driving the motility of these organelles; and crucial for their formation. Other proteins were identified as promising candidates to elucidate the variety of cargos transported by the unique minus-end direct motor cytoplasmic dynein. Microtubule-based motor protein functions in bacterial pathogen trafficking is later dissected using Salmonella enterica serovar Typhimurium infection of mammalian cells and confocal imaging. Opposing polarity motors dynein-l and kinesin-l are both required for efficient bacterial intracellular trafficking and replication. Recruited from the onset of bacterial invasion, dynein-l interaction with the Salmonella-containing vacuole (SCV) persists after its localisation near the Golgi apparatus indicating that at least one of the SCV positioning mechanisms requires dynein function. HOOK3, also identified as a dynein interacting protein in the proteomic assays undertaken in this thesis, is proposed to be the dynein adaptor at the SCV. Its Golgi -binding domain suggests its potential role in tethering the SCV to the Golgi apparatus, hence potentially mediating both dynein activity and Golgi tethering to explain SCV positioning. HOOK3 may also have a physiological function in docking endosomal subdomains destined to the Golgi to the microtubule network via interaction with dynein. This new body of evidence brings the scientific community a step closer to understanding the fundamental role of microtubule motors in membrane trafficking in physiological and pathological conditions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available