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Title: A role for giantin in primary ciliogenesis through control of the localisation of the cytoplasmic dynein-2 intermediate chains
Author: Asante, David
ISNI:       0000 0004 5356 7086
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2014
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Properly assembled primary cilia play fundamental roles in the development and function of nearly all cells. The cilium is assembled by extension of a microtubule bundle, the axoneme from the mother centriole ensheathed by a specialised membrane, the ciliary membrane which is continuous with the plasma membrane. Intraflagellar transport (IFT) traffics ciliary components along the length of the nascent cilium and is required for the assembly, maintenance and function of the cilium. The microtubule motor, cytoplasmic dynein-2 mediates retrograde IFT from the axoneme tip to base and has been shown to be necessary for cilia assembly. Vital cilia machinery constituents localise to the centrosome. The Golgi is positioned adjacent to the centrosome in most cells and both organelles polarise towards the apical plasma membrane during ciliogenesis. Furthermore, some Golgi-localised proteins have been shown to execute cilia-related functions. Here, the transmembrane Golgi protein giantin (GOLG81) is shown to be required for cilia formation. Giantin is not required for the RABll-RABIN8-RAB8 pathway that is implicated in the early stages of ciliary membrane formation. Instead, giantin depletion caused mislocalisation of the intermediate chains of dynein-2, WDR34 and WDR60. Highly effective suppression of giantin, WDR34 or WDR60 in cells completely inhibited primary cilia formation; while, partial depletion of giantin, WDR34 or WDR60 permitted the assembly of cilia, albeit of abnormally increased lengths. These data implicate giantin in ciliogenesis through control of dynein-2 localisation. Unlike the cytoplasmic dynein-l complex, existing knowledge of cytoplasmic dynein-2 composition is limited. This work details the subunit composition of the human cytoplasmic dynein-2 motor complex. WDR34 and WDR60 are genuine dynein-2 intermediate chains and associate with the known heavy (DYNC2Hl) and light intermediate (LlC3) chains of dynein-2. Dynein-2 possesses all the' known dynein-l light chains in addition to exclusive possession of the light chain, TCTEX-2. The only dynein-l accessory factor found to be shared by dynein-2 was NUDCD3, whose depletion resulted in mislocalisation of WDR34, failure of cilia assembly and assembly of abnormally long cilia in partial knockdowns. No interactions were found between dynein-2 and dynactin, LlSl, NUDE/NUDEL or BICD2. NUDCD3, thus, presents a common avenue for regulating dynein-l and dynein-2 functions, indicating a functional interplay between the two motors. This novel biochemical characterisation of the human cytoplasmic dynein-2 complex could potentially advance research into 1FT and dynein-2 function in ciliopathies
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available