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• W848483837 abstract "Focal adhesions (FAs) are dynamic, macromolecular complexes that regulate cell adhesion and migration. I aimed to elucidate how FA protein turnover correlates with FA stability and cell motility. To measure protein dynamics I established protocols for fluorescence recovery after photobleaching (FRAP) and photoactivation (PA). Custom MATLAB-based scripts and functions were used to analyse intensity measurements and quantify time-lapse experiments of FRAP and PA in great detail. Using these protocols and analytical tools, I demonstrated that the differential turnover of key FA proteins correlates with their presumed endogenous function. It became evident that adaptor proteins known to play a structural role in linking integrins to the actin cytoskeleton (e.g. vinculin) were slow, whereas proteins that have an indirect regulatory role (e.g. paxillin) were fast in their turnover within FAs. In an attempt to test how mutations that influence protein activation may affect protein turnover, I showed that vinculin dynamics are highly dependent on its activation state. Conversely, paxillin turnover remained unchanged irrespective of mutations that affect their phosphorylation states at two prominent regulatory sites. The lab had previously shown that constitutively active vinculin constructs decrease talin dynamics, which I have confirmed in this study using photoactivatable talin constructs. The decrease in talin dynamics correlated with the overall stability of FAs, which upon co-expression of activated vinculin remained stable. To search for the essential domains in talin that mediate this effect, I measured the dynamics of photoactivatable talin deletion mutants lacking 9 of 11 vinculin binding sites (VBSs). The data revealed that talin lacking domains R1-10 (?R1-10) displayed a significantly higher turnover rate and was unable to stabilise FAs in the presence of constitutively active vinculin. Expression of a talin ?R4-10 mutant (lacking 4 VBSs) only partially restored this defect suggesting that VBSs in both regions R1-3 and R4-10 contribute to vinculin-mediated FA stabilisation. Surprisingly, both talin deletion constructs triggered integrin activation. These data suggest that effective FA stabilisation is dependent on both integrin activation and the efficient connection with vinculin and the tensile actin cytoskeleton. Since vinculin activity is able to regulate the dissociation of critical proteins from FAs, such as talin, I hypothesised that it may have a subsequent effect on cell migration. Individual migration patterns of fibroblasts expressing constitutively active vinculin constructs were tracked. The data revealed dramatic defects in migration speed. Furthermore, it was clear that cell polarity was also perturbed. These data show that vinculin activity is critical for efficient cell migration.Overall, this study reveals distinct subdivisions of FA proteins based on differential protein dynamics, which correlate to their endogenous functions. We found that the activation state of vinculin is vital for regulating talin turnover but not paxillin or zyxin. Specifically, I showed that talin R1-10 domains contain essential binding sites for interacting partners that may infer FA stability. By dissecting the dynamic cross talk of FA proteins we can further our understanding of the spatial and temporal molecular mechanisms in the regulation of cell migration." @default.
• W848483837 created "2016-06-24" @default.
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• W848483837 date "2012-11-22" @default.
• W848483837 modified "2023-09-26" @default.
• W848483837 title "Investigating the differential dynamics of focal adhesion proteins : a key role for vinculin in their coordination" @default.
• W848483837 hasPublicationYear "2012" @default.
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