Our initial goal is to prepare key components of focal adhesion machinery recombinantly and to optimize the protein purification to a preparative amount for their characterization. We have completed this step with talin and vinculin. Other components are well established for purification and the efforts of characterization continue.
We have put extensive efforts in characterizing the master regulator talin. It is critical to understand the regulation of talin, as it is 1) an integrin activator, initiating the adhesion process 2) tension sensor, sensing the tension given by the stretching of cells and 3) platform for binding of more than 10 signaling protein components, directly connecting between membrane surface and actomyosin cytoskeleton. Talin is a large protein with 270 kDa in size. We are the first group that purified this large protein recombinantly, and this facilitated us to biophysically characterize them. By changing the surrounding ionic strength, we successfully activated the protein, changing the size from 15 nm to 80 nm by untangling its string-like folding. The biochemical analysis facilitated for the optimization of talin to be structurally stabilized for cryo-EM analysis. We obtained the structure of talin in autoinhibited form, which is now published to Cell (Dedden et al., 2019). Understanding the structure of talin and its activation machinery to a molecular level moved us forward, and we moved forward and tested the protein interactions of talin and vinculin, in the presence of membrane as well as cytoskeleton components actin. We found that the activation of talin occurs in the presence of PIP2 enriched membrane surface and vinculin is only recruited to the site and get activated in the presence of both talin and PIP2-rich membrane. Finally the complex formation of talin-vinculin at the membrane is necessary for the recruitment of actin cytoskeleton to the inner lumen of the membrane. This part of study is recently published in Elife (Kelley et al., 2020).
