Integrin-mechanosignaling role in small GTPases activation

COLÓ, GEORGINA P.; FERNÁNDEZ CHÁVEZ, L.; FACCHINETTI, M.M.; FÄSSLER, R.; COLÓ, GEORGINA P.; FERNÁNDEZ CHÁVEZ, L.; FACCHINETTI, M.M.; FÄSSLER, R.; HAGA, R.; CURINO, A.C.; HAGA, R.; CURINO, A.C.

Bariloche

Simposio; The Fourth American Spring Symposium in Signal Transduction and Molecular Medicine (SISTAM 2018); 2018

The ability of cells to adhere and simultaneously probe their mechanical environment is central to many developmental, homeostatic as well as pathological processes. We determined the importance of studying integrins in genetically modified cell models that only express fibronectina (FN)-binding integrins. Using biochemical assays, in combination with mass spectroscopy (MS), traction force microscopy and micropatterns, we observed that α5β1-integrin expressing cells promote the formation of small nascent adhesions, low RhoA activation and high force, while αVβ3 integrin expressing cells showed large focal adhesions connected to contractile stress fibers, resulting in high RhoA but low force. To further analyze these phenotypes, we looked for specific RhoA activators (GEFs) or inhibitors (GAPs). We performed a proteomic analysis and amongst the interesting hits was GEF-H1, together with other less abundant GEFs (11 proteins) and GAPs (10 proteins). It has been described that GEF-H1 is involved in the cross-talk between microtubules and the actin cytoskeleton. Our data shows that GEF-H1 is localized in the cytoplasm and more active in αV expressing cells when compared to β1-cells, where GEF-H1 is in an inactive state bounded to the microtubules. These results could explain the increase in stress fibers formation in αV-cells and RhoA activation. GEF-H1 can be released to the cytoplasm either by microtubule depolymerization or by protein phosphorylation. A phosphoenrichment label free MS analysis revealed that GEF-H1 is highly phosphorylated in αV-cells. Furthermore, using integrin-tail pull-down and MS assay, we observed that GEF-H1 binds to β3-integrin tail. The results show for the first time that GEF-H1-RhoA activation is αVβ3-integrin dependent and it can mediate the signaling involved in controlling cell structure and force generation.