substrates. Average velocity of protein fluxing was quantified by STICS. Data are expressed as the mean 6 SEM (n = 28 cells). Two-way ANOVA reveals that neither poly-L lysine attachment nor crosslinking have a significant effect on the flux velocity (P = 0.48). (B) Covalent cross-linking (Upper) or poly-L-lysine attachment (Lower) of the indicated integrin ligands did not significantly affect protrusion. Data are expressed as the mean 6 SD of at least 3 independent experiments (n = 7, 7, 12, 15, 10, 7 for cross-linking, respectively; n = 8, 9, 8, 9, 8, 7 protrusions for poly- lysine, respectively.) Protrusion rates were obtained from kymographs. (C) Protrusion rates of cells expressing a5 or a6 integrins and plated on FN or LN, respectively. On FN, the protrusion rate decreases with increasing concentration (P,0.006). No significant difference was detected with LN. (D) Protrusion of CHO.B2 cells was inhibited by activating integrins with Mn2+ (P,161026). Data are expressed as the mean 6 SD of at least 3 independent experiments. (n = 9, 9, 10, 12, 9, 10 protrusions respectively.). doi:10.1371/journal.pone.0040202.g005 a6b1 Adhesions Show Reduced Traction Forces but Still Signal Together, our data suggest a weakened ligand-integrin-actin linkage in cells migrating using a6b1 or aLb2 integrins. In this interpretation, the traction forces exerted by migrating cells on the substratum would be lower. To test this, we plated CHO.B2 cells expressing a5-GFP or a6-GFP on fluorescent bead embedded polyacrylamide gels coated with covalently-bound fibronectin or laminin, respectively [41,42]. The maximum gel deformation was measured for protrusion and retraction events; however, the relative displacements for different events were similar on a given substrate (P = 0.61 and P = 0.18, for fibronectin and laminin respectively, in a cell paired t-test), indicating that in each case we measured the maximum deformation of the gel. Overall, the gel deformation during protrusions and retractions with a5-expressing cells was significantly higher than those in a6-expressing cells (Figure 7), suggesting that the a5-expressing cells generate higher traction on fibronectin than the a6-expressing cells do on laminin (P = 0.028 and P = 0.016 for protrusion and retractions respec- tively). Reduced forces suggest that the signaling by adhesions might also be altered since adhesive signaling is thought to be force dependent [57,8]. Therefore, we determined whether the differ- ences in fluxing and traction force affected the signals produced by the adhesions in protrusions using a mGFP-dSH2 probe that binds to a Src-like kinase mediated phosphorylation of tyrosine on adhesion proteins [28,12]. Cells migrating on fibronectin display small adhesions at the front of protrusions, and large adhesions at the rear, which result from myosin II activation [12]. The small adhesions exhibit high levels of mGFP-dSH2, indicating that they signal actively, whereas large adhesions display relatively low levels of mGFP-dSH2 [12]. Conversely, a6b1-expressing cells on laminin and aLb2-expressing cells on ICAM-1 contain large adhesions even in their protrusions. They are comparable in size to those observed at the back of a5b1-expressing cells. However, these adhesions displayed an accumulation of the mCherry-dSH2 probe (Figure S5A). This indicates that large adhesions can signal robustly when cell adhesion occurs via a6b1or aLb2, in contrast to adhesions of similar size forming in cells on fibronectin. Rapid Leukocyte Migration Arises from Cell Type Differences Rather than Alterations in Myosin II Activity or the Ligand-integrin-actin Linkage We next sought to assess the contribution of cell type to the differences in adhesion and migration reported above in the CHO cells. We first compared the migration of CHO.B2 cells ectopically expressing aLb2, with the spontaneous migration of promyelocytic Integrin-Ligand Interactions Control Migration Figure 6. Fluorescence Recovery After Photobleaching (FRAP) and adhesion assays for CHO.B2 cells expressing a5, a6 or aL+b2 integrins and migrating on FN, LN and ICAM-1, respectively. (A) Typical FRAP curve for each condition. Data points are in blue, and a single exponential fit is in black. Notice the time scale differences. The R2 for a single and double exponential fit were both PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e40202