Integrin-mechanosignaling role in small GTPases activation

COLO, GEORGINA P.; CURINO, A.C.; TYANOVA, S.; HAGA, R.; FÄSSLER R

Buenos Aires

Simposio; Fronteras en biociencia 3; 2018

Sociedad Planck de Alemania y de la Argentina

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 fibronectin (FN)-binding integrins. Using biochemical assays, in combination with mass spectroscopy (MS), traction force microscopy and micropatterns, we observed that α5β1-integrin expressing cells (pKO-β1) promote the formation of small nascent adhesions, low RhoA activation and high force, while αVβ3 integrin expressing cells (pKO-αV) 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). In addition, 37 GAPs and 38 GEFs were identified as being differentially expressed in pKO-αV cells when compared to pKO-β1 cells using mRNASeq data and bioinformatics analysis. 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 pKO-αV cells when compared to pKO-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 pKO-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 pKO-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.