Vias Moleculares Involucradas en la Migración e Invasión Celular Dependiente de Integrinas

COLO G.P.; ALONSO, E.N.; CURINO A. C.; GANDINI N.A.; MASCARO M.; HAGA R.B.; GUEVARA J.A.; FASSLER R

Congreso; LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica; 2018

Mechanotransduction is mediated by the integrinfamily of cell adhesion receptors. Integrins bind cell extracellular matrixproteins an connect to the F-actin cytoskeleton and non-muscle-myosin inside thecells. Using genetically engineered cells, biochemical assays, in combinationwith mass spectroscopy (MS), traction force microscopy and micropatterns, weobserved that ¦Á5¦Â1-integrin expressing cells promotethe formation of small nascent adhesions, low RhoA activation  and high  force,  while  ¦ÁV¦Â3-integrin expressing  cells showed largefocal adhesions connected to contractile stress fibers (SFs), resulting in highRhoA but low force. To further analyze pKOcells phenotypes, we looked for specificRhoA activators (GEFs). We performed a MS-proteomic analysis and amongst theinteresting hits was GEF-H1, together with biochemical assays we observed that  GEF-H1 activation  is  dependent on  a  specific integrin-class suggesting that integrins may activate specific GEFsduring adhesion, migration and invasion. Recent studies have also shown that anincrease in GEF-H1 expression correlates with an increase in tumor progressionand met¨¢stasis. In addition, GEF-H1 is involved in the cross-talk betweenmicrotubules and the actin cytoskeleton. Our data shows that GEF-H1 islocalized in the cytoplasm and more active in ¦ÁV¦Â3-cells when compared to ¦Á5¦Â1-cells, whereGEF-H1 is in an inactive state bounded to the microtubules. Similar results weobserved in breast cancer cells depending their invasiveness and the integrin-class-expression.These results could explain the increase in SFs formation in ¦ÁV¦Â3-cells and RhoA activation. GEF-H1 can be releasedto the cytoplasm either by microtubule depolymerization or by proteinphosphorylation. A phosphoenrichment-label-free MS analysis revealed thatGEF-H1 is highly phosphorylated in ¦ÁV¦Â3-cells.Furthermore, using integrin-tail pull-down and MS assay, we observed thatGEF-H1 binds to ¦Â3-integrintail. These results show for the first time that GEF-H1-RhoA activation is ¦ÁV¦Â3-integrin dependent and it can mediate thesignaling involved in controlling cell structure, force generation, migrationand invasion.