Integrin-Ligand Interactions Control Migration Figure 1. Migration of CHO.B2 cells expressing different integrins. (A) CHO.B2 cells expressing a5-GFP, a6-GFP, or aL-GFP + b2-GFP migrate randomly on fibronectin (FN), laminin (LN) or ICAM-1, respectively. Typical cell paths are shown, with each individual cell track assigned a different colored line translated to a common origin. Experimental time: 6 hours. Scale Bar = 100 mm. (B) The speed and directionality, from at least three independent experiments, were calculated and plotted (cell number = 52, 36, 33, respectively). The speed was calculated from the total length of a cell path divided by the experimental time. Cells on FN migrate about half as fast as cells on either LN or ICAM-1 (P,161029), and the difference between cells on LN and ICAM-1 is small but still significant (P = 0.0017). Directionality was defined as the ratio of the length from the start to the end point and the length of the cell path. doi:10.1371/journal.pone.0040202.g001 presented above for the CHO cells and suggest that the observed behaviors on different substrates are due to intrinsic differences in the integrin-ECM interactions rather than different integrin expression levels or cell type-dependent differences. The Differences among Cells Migrating on Fibronectin, Laminin and ICAM-1 do not Originate from Major Alterations in Myosin II Activity Myosin II plays a pivotal role in the adhesion and migration of cells on fibronectin [49,50,21,23,12], and therefore, we asked whether the integrin heterodimer-dependent changes in migration and adhesion arise from differences in myosin activation or isoform expression. We first examined myosin II activation in CHO.B2 cells transfected with a5, a6, or aL and b2 that were plated onto fibronectin, laminin or ICAM-1, respectively. Myosin II activation was assessed by immunoblotting for phosphorylated RLC. CHO.B2 cells expressing the a5b1 or a6b1 integrins and plated on fibronectin or laminin, respectively, showed a substrate concentration-dependent RLC phosphorylation (Figure 3A). How- ever, the distribution of phosphoRLC (pRLC) was markedly different. In a5-expressing cells on fibronectin, pRLC localized robustly along thick actomyosin fibers that terminate in large adhesions. In a6-expressing cells on laminin, pRLC localized to thinner, strand-like actin structures in protrusions (Figure 3B). In PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40202