αV-class integrins exert dual roles in α5β1 integrins to strengthen adhesion to fibronectin

Upon binding to the extracellular matrix protein, fibronectin, alpha V-class and alpha 5 beta 1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of alpha V-class and/or alpha 5 beta 1 integrins expressing fibroblasts initiating attachment to fibronectin (<= 120 s) by single-cell force spectroscopy. Our data reveals that alpha V-class integrins outcompete alpha 5 beta 1 integrins. Once engaged, alpha V-class integrins signal to alpha 5 beta 1 integrins to establish additional adhesion sites to fibronectin, away from those formed by alpha V-class integrins. This crosstalk, which strengthens cell adhesion, induces alpha 5 beta 1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix.