ligand pairs, we perturbed myosin II expression and activity in cells on different substrates. Overexpression of MHC IIA, but not MHC IIB, decreased the protrusion rates of a5 expressing CHO.B2 cells (Figure 3C), as previously shown in CHO.K1 cells [21]. However, overexpression of MHC IIA did not affect protrusion in the a6b1-expressing CHO.B2 cells (Figure 3C) and in aLb2-expressing cells (data not shown). Similarly, myosin II activation by overexpression of phospho-mimetic RLC mutants (RLC-A,D and RLC-D,D) [23,24], or inhibition by addition of ML7 and Y27632 to inhibit MLCK and ROCK, respectively, which are upstream of RLC phosphorylation in these cells [23,24], did not show significant differences on protrusion rates (Figure S3). Thus, enhancing myosin II expression or activity has little effect on protrusion and adhesion in cells using a6b1 or aLb2 for migration, and therefore, differences in migration properties between CHO.B2 cells with a6b1 or aLb2 integrins do not appear to result primarily from alterations in myosin II activity. To ensure that possible variations in integrin expression do not produce the observed phenotypes, we used Fluorescence-Activated Cell Sorting (FACS) to sort cells by the expression level of integrin- GFP in CHO.B2 cells. The cells were binned into three populations: low, medium-low, and high fluorescence; this range of expression includes the endogenous level in wild type cells. Immunoblotting and kymography were performed on the three populations. Although pRLC levels increased somewhat in the cells expressing high a5-GFP, the protrusion rates remained similar for each integrin expressed (Figure S4A). This result suggests that the observed behaviors on different substrates are due primarily to intrinsic differences in the integrin-ECM binding rather than differences in integrin expression levels. Similar results were observed in cells expressing a6-GFP or aL-GFP (Figure S4A, data not shown). The Integrin-ligand Interactions of the a6b1 and aLb2 Integrins Differ from those of a5b1 The absence of a clear relation between myosin II activity and migration, adhesion and protrusion on the different substrates suggests that the myosin II activity is not coupled efficiently to signaling and adhesion in cells using some integrins. Therefore, we queried whether there are differences in the efficiency of the linkage, or molecular clutch, that couples actin and adhesion [9,51,52]. To do this, we measured the retrograde movement, or fluxing, of adhesion components in protrusions, since these adhesions are the traction points through which cellular forces are shunted to the substratum and thereby inhibit retrograde flow. Interestingly, in cells expressing either a6 or aLb2, most of the elongated adhesions in protrusions exhibited a rapid retrograde, flux of paxillin, which is seen as parallel downhill slopes in the kymographs (Figure 2B). Conversely, in cells on fibronectin, we did not observe robust centripetal fluxing of paxillin in adhesions 26], except in adhesions located within regions that were retracting actively. Previous studies have identified a ‘‘slip’’ point within the adhesion-actin linkage. That is, in some adhesions, the integrins remain fixed while other adhesion molecules flux; a-actinin, which is bound to actin, moves the fastest [9,10]. To localize the ‘‘slip’’ point, in the linkage with integrins, we assayed the movement of adhesion components including the a6 and aL integrins on laminin and ICAM-1 using their GFP derivatives at expression levels low enough to observe ‘‘speckles’’ [53]. Surprisingly, all of the components (data not shown) including the integrins displayed robust retrograde movement even when the protrusions were stationary; however a comparable, retro- grade fluxing of a5 in cells on fibronectin was seldom seen Integrin-Ligand Interactions Control Migration (Figure 4) [9,51]. The integrin fluxing does not appear to arise from a weak interaction between the matrix ligand and the glass coverslip, since laminin covalently cross-linked to the coverslip or adsorbed onto pre-bound poly-L-lysine, which improves laminin binding [33,34], did not have a significant effect on the flux and protrusion rates of a6 or paxillin (Figure 5A, B). In addition variations in integrin expression levels do not correlate well with the level of fluxing (Figure S4B). Also, unlike cells expressing a5 on fibronectin, the protrusion rate of a6 or aLb2 does not appear to arise from the density of laminin (Figure 5C) or ICAM-1 (data not shown). While few cells attached to very low concentrations of ICAM-1, those that did went on to migrate (data not shown). In contrast, increasing the density of fibronectin decreased the protrusion rate; however, lowering the fibronectin concentration to the threshold for adhesion of these cells, 0.5–1 mg/ml, did not increase protrusion to a rate comparable to that seen for cells on LM or ICAM-1 (data not shown). These observations further suggest that the differences among different integrin-ligand pairs are intrinsic. Finally, we altered the strength of the integrin-substrate interaction by adding Mn2+ [54], which increases integrin affinity, to CHO.B2 cells expressing paxillin-Cherry and a6- or aLb2- GFP. Mn2+ inhibited the retrograde fluxing and the protrusion rates of both a6 and aLb2 (Figure 4D, Figure 5D). We quantified these differences using spatio-temporal image correlation spec- troscopy (STICS) [37,9]. In the presence of Mn2+ the rate of retrograde flux for aLb2 decreased ,3-fold in cells on ICAM-1 (Figure 4C, from 2.860.3 to 1.060.1 mm/min, P = 0.0016); while the rate of paxillin slowed from 4.160.4 to 1.760.1 mm/min (P = 0.0016). A similar effect was observed with a6-expressing cells migrating on laminin (Figure 4B); the average retrograde flux of a6 slowed from 1.6860.09 to 1.060.1 mm/min (P = 0.0051), which is similar to that of paxillin (from 1.6460.09 to 1.060.2 mm/min, P = 0.0051). In the absence of Mn2+, cells plated on laminin exhibited faster flux for both proteins than those plated on fibronectin (P,0.01). The dramatic and novel differences in retrograde integrin fluxing suggest that the adhesion strength, i.e., the apparent affinity, avidity or ligand-integrin-actin linkage, of a5b1 for fibronectin is higher than that for either a6b1or aLb2 interacting with its respective ligand. To test this, we used fluorescence recovery after photobleaching (FRAP). This measures the diffusion of the integrins in the membrane plane, a parameter that would be affected by the affinity of integrin for its ligand and alterations in the integrin-actin linkage. CHO.B2 cells expressing a5-GFP, a6- GFP or aLb2-GFP were plated on fibronectin, laminin or ICAM- 1, respectively, and the mobility of the fluorescently-labelled integrin was measured by FRAP. The data in Figure 6A, B shows that the recovery of a6 or aLb2 after photobleaching is significantly faster, and the fractional recovery higher, than that of a5. Since diffusion in the membrane is largely insensitive to the size of the integrin [55,56], the data support an altered ligand- integrin-actin linkage that is stronger between a5 and fibronectin than between a6 and laminin, or aLb2 and ICAM-1. Finally, we measured the adhesion strength directly using a centrifugation assay. In this assay, cells are plated and allowed to adhere, and then the plates are inverted and centrifuged [43]. The fraction of cells remaining on the dish is a measure of the relative adhesion strength. The fraction of cells on remaining on fibronectin is significantly higher than that on laminin or ICAM-1 (P,0.04) (Figure 6C). This shows that the adhesion strength of a5b1 to fibronectin is stronger than that of a6b1 to laminin or aLb2 to ICAM-1 under these conditions. PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40202