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Title: Microtubule organisation in normal and colon cancer cells
Author: Gadsby, Jonathan
ISNI:       0000 0004 5347 5114
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2014
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ApicoNbasal polarisation of epithelial cells is essential for normal function, with the rearrangement of the microtubule cytoskeleton on differentiation critical for the establishment of polarity. During this process, microtubules reorganise to form a stabilised array of apicoNbasal bundles via a release and capture mechanism. Disruption of this array can cause a loss of epithelial polarity, which can contribute to diseases such as cancer. Microtubule rearrangements are driven by their inherent dynamics, which are regulated by +TIP proteins. This project aims to examine the mechanisms behind apicoNbasal array formation, and how they may be compromised in colorectal cancer. It has focussed on two +TIPs; APC (which is mutated in most colorectal cancers), and EB2 (which has also been observed upregulated in cancer). The role of APC at the cortex was examining using cell lines containing wild type or APC truncation mutations. Microtubule orientation at junctional sites was determined, with Nocodazole regrowth assays used to determine cortical approach. Additionally, the angle of the mitotic spindles was analysed in the different cell lines. The impact of EB2 loss on microtubule organisation was determined by immunolabelling following siRNA treatment. A fluorescently tagged EB2 construct was created and used to rescue depleted phenotypes. The effect of EB2 depletion on microtubule stability was assessed by analysing live microtubule dynamics. The impact of EB2 overexpression on cell migration was assessed by creating a stable EB2 overexpressing cell line and live imaging sparsely seeded cells and scratch assays. Overexpressing cells were grown in 3D culture to study any effects on polarisation. The TC7 cell line containing truncated APC possessed fewer microtubules orientated perpendicular to the cortex than wildNtype expressing cells. This could be rescued with full length GFPNAPC. Nocodazole regrowth showed microtubules in TC7 cells would approach the cortex perpendicularly, and then curl away. TC7 cells also had an increased proportion of misorientated spindles. EB2 depletion caused a dramatic increase in cell size, and redeployment of EB1 and the microtubuleNactin crosslinking protein ACF7 along the microtubule lattice. These effects could be rescued by transfection with mCherryNEB2. Analysis revealed EB2 depleted cells possessed less dynamic microtubules. The large depleted cells were found capable of division, although some appeared to fail during the process, with cell cycle analysis revealing suggesting the potential induction of a cell death pathway. Overexpressed EB2 could be located at the migratory leading edge, and was found to promote cell migration, with cells moving at an increased rate and wounds healing more quickly. There was also evidence of compromised tissue integrity in overexpressing cells grown in 3D culture The results gathered identified a potential role for full length APC in stabilising cortical microtubule contacts, and helped identify EB2 as a central regulator of microtubule dynamics and organisation during polarisation. EB2 overexpression was found to increase cell migration, and may be able to induce EMT.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available