0.8. From the single exponential fits, the fractional recoveries for the three typical curves are 0.39, 0.49, 0.56, respectively; estimated half- times (T1/2, sec) are 19, 6.3, 4.1, respectively. (B) From the single exponential fits, recovery half-times (T1/2) were plotted as mean 6 SD. The T1/2 for a5-GFP is significantly larger than for either a6-GFP or aLb2- GFP (n = 3 cells for each condition, P,0.05). (C) Adhesion strength assay. Cells expressing a5, a6 or aL+b2 integrins were plated onto substrates coated with FN, LN, or ICAM-1, then either simply inverted (positive control), or inverted and centrifuged at low speed (200 RPM). The number of remaining cells was counted. The relative adhesion strength was estimated by the fraction of cells remained after centrifugation divided by the positive control. More cells remained on FN than on LN or ICAM-1: P,0.04. doi:10.1371/journal.pone.0040202.g006 leukemia (HL-60) cells, which become highly migratory following differentiation into neutrophil-like cells with DMSO [29]. HL-60 cells use aLb2 to migrate on ICAM-1 and a4b1/a5b1 to migrate on fibronectin. When plated on fibronectin or ICAM-1, HL-60 cells migrate roughly 10 times faster than CHO.B2 cells expressing either a5 or aLb2 and migrating on fibronectin or ICAM-1, respectively (Figure 8A and 8B). In addition, their adhesions are small in the periphery with a focal subventral area of contact with the substrate [58] (movie S2). This rapid migration is consistent with previous reports of high speeds and a polarized morphology consisting of a small leading protrusion [14] (Figure S5B). We also analyzed the phosphorylation of RLC in response to adhesion. RLC phosphorylation is generally higher than that observed in CHO.B2 cells but largely ligand concentration- independent (Figure 9A). Adhesion and migration of the HL-60 cells were not significantly altered by the RLC mutants, RLC-A,D and RLC-D,D, regardless of whether the cells were migrating on fibronectin or ICAM-1(Figure 9B, data not shown). In addition, both RLC mutants localized to the rear of the cell as does wild type and endogenous RLC, as reported previously [59,14]. Interestingly, the adhesions in the HL-60 cells, as visualized using paxillin-mCherry, were present as small dot-like structures at the side of cell, the uropod, and along the retraction path. The ‘‘touch, hold, and release’’ motion of the adhesions is clearly visible at the side edge and the rear of the cell, especially when the cell is changing direction (movie S2). Therefore, HL-60 adhesion and migration differs from that of the CHO cells using the same integrin-ligand pairs and is largely refractory to increases in myosin II activity. Since their adhesions are small and do not appear to flux retrograde, it appears that the major difference between HL-60 and CHO cells resides in fundamental differences in cytoskeleton organization (e.g. actin organization) rather than to large differences in myosin II mediated contractile forces. Discussion We have addressed the effects of different integrins on migration and adhesion using a common cell type expressing different integrins and different cell types expressing the same integrins. In contrast to cells migrating on fibronectin using the a5b1 integrin, the same cells migrating on laminin or ICAM-1 using the a6b1 and aLb2 integrins, respectively, show the following phenotypes: increased migration rates and directional persistence, a rapid fluxing of integrins and other adhesion components in protrusions, a reduced effect of myosin II activation on migration, increased tyrosine phosphorylation in large adhesions, decreased adhesion strength, and inhibited force transmission from actomyosin to the substratum. In contrast, HL-60 cells, a leukocyte model, migrating on either fibronectin or ICAM-1 exhibit rapid migration and only small transient adhesions that do not flux and are largely Integrin-Ligand Interactions Control Migration Figure 7. Bead displacements for CHO.B2 cells migrating on FN or LN. Cells were plated onto bead embedded polyacrylamide substrates (E = 1 kPa) coated with adhesive ligand. The relative bead displacements were measured in regions of protrusion, retraction and the cell body. Maximum deformation was measured in gel sub-regions (see Methods) by interpolating bead displacements onto a regular grid during events of protrusion and retraction. Independent protrusion and retraction events show no significant difference (P = 0.61 and P = 0.18, for FN and LN respectively, in a cell paired t-test), indicating that in each case we measured the maximum deformation of the gel. Cells plated on FN-coated gels deform them significantly more than LN-coated gels (P = 0.028 and P = 0.016 for protrusion and retractions respectively), suggesting that they exert stronger forces. Data are expressed as the mean 6 SEM (n = 13 cells). doi:10.1371/journal.pone.0040202.g007 independent of the myosin II activity. These observations indicate that intrinsic cellular differences, e.g., actin organization, regulate their adhesion and migration. The striking differences in adhesion and migration between cells expressing a5b1 and a6b1 or aLb2 and their decreased dependence on myosin II activity were unexpected. Both a5 and a6 dimerize with the b1 subunit in these cells and therefore potentially share similar signaling properties through the b1 cytoplasmic domain. While functional differences between a5b1 and a6b1 have been reported previously, their specific role in the signaling that controls cell migration is not understood [19,60,61]. One possibility is the tail of the alpha chain modulates signaling by the beta chain. It is also possible that unique binding proteins may modulate signaling triggered by a given integrin; for example, tetraspanins associate with a6b1 but not a5b1 [62]. Retrograde fluxing of adhesion components has been reported previously for cells using avb3 and a5b1 integrins and arises primarily from membrane resistance and actomyosin driven retrograde forces [9,10]. Our studies show that a6b1 and aLb2 expressing cells on laminin and ICAM-1, respectively, show an unusually prominent and rapid retrograde fluxing of adhesion components when protrusions pause, and a novel retrograde fluxing of the integrins, themselves, that is not commonly seen with a5b1 on fibronectin. This fluxing will inhibit the transfer of cellular forces to the substratum and therefore is likely responsible, at least in part, for the reduced forces sensed by the substratum. The enhanced fluxing, in turn, appears to arise from altered adhesion strength. Taken together, these differences indicate that differential mechanotransduction underlies the different migration properties. Several recent studies propose tension as a regulator of both adhesion maturation and the signals that adhesions produce [10,63–65,12]. In this regard, it is particularly interesting that CHO cells expressing a6b1 and aLb2 have very large adhesions PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40202