It is still unknown how exactly cells respond to their extracellular matrix (ECM) and how they react to changes in it. For example a stiffer ECM environment in breast tissue heightens the likelihood to develop breast cancer, but how theses cells detect and react to changed stiffness is not clear. In order gain a better insight into the link between ECM and the control of cell behaviour it is essential to understand the complexity of focal adhesions (FAs), the complex of proteins linking cells to the ECM and interpreting information about their extracellular environment. To identify novel regulatory components of FAs, we have used in vivo biotinylation techniques to measure changes in protein interactions by quantitative mass spectrometry. BirA is a modified bacterial enzyme capable of biotinylating proteins in close proximity while it has access to biotin. It can therefore be expressed in fusion with proteins of interest and used to identify interactions in situ. We have set out to use integrin Î±5 and focal adhesion kinase (FAK) as BirA fusions, and then stably integrated these into U2OS cells. With this approach our goal was to label proteins interacting with either integrin Î±5 or FAK, and to identify potential novel regulatory proteins. Biotin labeled proteins were purified by affinity purification and then analyzed via SILAC mass spectroscopy. The integrin and FAK interactomes thus identified were compared with those of other focal adhesion proteins studied in our lab, allowing further dissection of the network within the adhesome. To understand how these cells react to different environments, the U2OS BirA FAK cells were grown on a stiff and soft matrix. Therein a new potential role of FAK was discovered in response to ECM stiffness. Further more using the BioID approach we now identified a possibly new interactor of the adhesome, ARHGAP1. Together, these results indicate that proximity biotin labeling can be a useful tool to identify potential new transient interacting proteins in FAs.
|Date of Award||1 Aug 2019|
- The University of Manchester
|Supervisor||Charles Streuli (Supervisor) & Andrew Gilmore (Supervisor)|
- Focal adhesion
Characterization of the focal adhesion interactome
Hohensee, T. (Author). 1 Aug 2019
Student thesis: Phd