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Title: Role of fascin in regulating microtubule and focal adhesion dynamics in carcinoma cells
Author: Villari, Giulia
ISNI:       0000 0004 5364 1549
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2014
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Fascin is an actin-bundling protein whose upregulation is correlated with poor prognosis in cancer. Previous studies have shown a role for fascin in regulating assembly of actin bundles and stability of adhesion structures. Fascin has not previously been localised at focal adhesions and the mechanisms by which it contributes to adhesion dynamics and assembly is still unknown. The aim of this thesis is to investigate whether fascin regulates focal adhesion (FA) dynamics and tumour cell invasion through control of actin and microtubule (MT) crosstalk. To analyse this, MT network integrity and focal adhesion area were quantified in control, fascin knockdown (KD) and fascin mutant rescued breast carcinoma cells treated with the MT-depolymerising agent nocodazole or following washout of this drug. Fascin KD cells showed a delay in the recovery of MT re-growth, increased MT stability and an increase in FA area following drug washout, compared to control cells. Both events were rescued by the re-expression of WT and S39A fascin (which constitutively associates with F-actin), but also by S39D fascin (is unable to bind F-actin). Interestingly, MT and FA assembly were increased compared to controls in cells re-expressing fascin that is mutated within a recently identified additional c-terminal actin-binding domain (S274D). To investigate whether there is a direct binding between MT and fascin that may explain these altered MT dynamics, I performed in vitro MT polymerisation/binding assays, in absence and presence of actin. WT and all fascin mutants showed association with tubulin in biochemical co- sedimentation assays. Interestingly, S274D fascin soluble fraction was not increased in the presence of actin, suggesting mutations in this actin-binding site are preferentially involved in controlling association with MT. To elucidate the mechanism regulating fascin binding to MT and its role in FA dynamics, I considered the Focal Adhesion Kinase (FAK) as a possible downstream effector. I found fascin to form a complex with FAK. In addition I showed fascin-FAK complex formation to be dependent on MT cytoskeleton integrity/stability and fascin-binding to F-actin and MT. Thus, fascin regulates MT and adhesion dynamics coordinating the actin-MT crosstalk and contributing towards carcinoma cell invasion.
Supervisor: Parsons, Madeline ; Jones, Gareth E. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available