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Title: Analysis of the function of the cytoskeletal proteins vinculin and talin using gene disruption
Author: Sutton, Deborah Helen
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2001
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Talin, a 270 kDa cytoskeletal protein is thought to be a key player in the formation of focal adhesions. Talin (-/-) embryonic stem (ES) cells expressing no intact talin spread less efficiently on gelatin, laminin and fibronectin compared to wild-type ES cells, exhibited extensive membrane blebbing, showed a decrease in cell polarity, and were unable to form focal adhesions when planted on fibronectin. They also showed reduced adhesion to laminin (but not fibronectin), probably due to reduced expression of 1-integrins. The phenotypic characteristics of the talin (-/-) ES cells could be revealed to wild-type by expression of the full length mouse talin cDNA. Indeed, adhesion to laminin and fibronectin was greater than seen with the wild-type cells. These results provide strong support to the conclusion that talin is essential for integrin-mediated cell spreading and the assembly of focal adhesions. However, expression of a talin polypeptide lacking the C-terminal actin-binding site failed to rescue the mutant phenotype demonstrating the importance of this site in talin function. The talin (-/-) ES cells formed normal embryoid bodies, and when these were plated on gelatin, a population of spread cells slowly emerged from the central cell mass. Interestingly, these cells formed small vinculin and paxillin-containing focal adhesions, raising the possibility that talin is not essential for focal adhesion formation in all cell types. The talin-binding protein vinculin is also localised to focal adhesions. Although vinculin (-/-) null mouse embryo fibroblasts (MEFs) spread less efficiently on fibronectin, they were more motile compared to vinculin (+/-) MEFs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available