Periodic Reporting for period 2 - FocAd (Structural Studies on Focal Adhesions)
Periodo di rendicontazione: 2019-01-01 al 2020-06-30
The aim of the project FocAd is to understand a comprehensive molecular view of focal adhesion (FA) growth using interdisciplinary cutting-edge techniques, such as cryo-EM, X-ray crystallography, biochemistry and biophysical approaches.
The aim is divided into 4 sub-categories described below:
A. To biochemically characterize individual recombinantly produced proteins involved in the FA initiation. To understand the regulation of the interaction partners in binary as well as in complex modes.
B. To reconstitute a stable complex and test the interaction of the complex with integrin tail - signaling master controller and analyze its molecular architecture by cryo-EM.
C. To understand the interactions of the FA proteins with actin cytoskeleton.
D. To extend the reconstitution from integrin receptor to the actin layer and understand the effect of the tension and elucidate the molecular mechanism of the FA growth.
The aim is divided into 4 sub-categories described below:
A. To biochemically characterize individual recombinantly produced proteins involved in the FA initiation. To understand the regulation of the interaction partners in binary as well as in complex modes.
B. To reconstitute a stable complex and test the interaction of the complex with integrin tail - signaling master controller and analyze its molecular architecture by cryo-EM.
C. To understand the interactions of the FA proteins with actin cytoskeleton.
D. To extend the reconstitution from integrin receptor to the actin layer and understand the effect of the tension and elucidate the molecular mechanism of the FA growth.
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).
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).
We have used a wide range of cutting-edge techniques for the stabilization and characterization of complexes. We used various synthetic platforms like membrane mimics, DNA origami and we incorporated the FA machinery into the inner lumen of GUVs to facilitate a biophysical and biochemical analysis. Furthermore, we have established the variation of ionic strength to sample different activity states of talin for a molecular characterization. Using the techniques established during this period of the projects, we expect to produce more results leading to publications.