Integrin-Ligand Interactions Control Migration Figure 8. HL-60 migration on FN or ICAM-1. (A) Migration tracks of HL-60 cells on the indicated substrate over 1 hour were translated to a common origin and marked with a different color. Scale Bar = 100 mm. (B) The speed and directionality were calculated and plotted (n = 36, 39 cell tracks, respectively). At least three independent experiments were quantified. P value = 361027. doi:10.1371/journal.pone.0040202.g008 with prominent tyrosine phosphorylation in their protrusions, despite the reduced tension on the substratum. Presumably this reflects the residual cross-linking activity that sustains the large adhesions and actin bundles even in the presence of reduced force. Recently, Oakes, et al. also showed a lack of correlation between adhesion size and force [66]. Several reports document the retrograde translation of adhe- sions and the fluxing of molecules within them [54,9,52]. The translation appears to arise from the release of complexes at the rear of the adhesion and addition of new components in the front, i.e., direction of movement [67,54,27] presumably along actin filaments; although a net physical movement has not been ruled out. The retrograde movement of adhesion components presum- ably also arises from a treadmilling mechanism and reveals a clutch-like effect in which components closely associated with actin, like a-actinin, flux rapidly [9,51]; whereas other components more closely associated with the substratum flux more slowly or not at all. Most studies have focused on the avb3 and a5b1 integrins in cells adhering to fibronectin. In these previous studies, the integrins are largely immobile suggesting that the force sensitive interaction lies in the associations among cytoplasmic components that comprise the integrin-actin linkage [9,51]. Changes in the efficiency of the interaction can affect cell signaling by adhesions, since contractile forces couple to the substratum through this linkage. The fluxing of integrins that we observe for a6b1 and aLb2 is novel and reveals a new force sensitive locus. The fluxing itself, inhibition by Mn2+ and the FRAP data suggest that the effective adhesion strength, e.g., apparent affinity, avidity or linkage to actin, is reduced. The adhesion, migration, and polarity of fibroblast-like cells migrating on fibronectin depend on the activation status of myosin II via a poorly understood signaling loop [11,21,23,12,68,69]. As RLC phosphorylation status increases, migration rates show a biphasic dependence, and small nascent adhesions in the lamellipodium tend to undergo rapid maturation into larger adhesions tethered to actin bundles. The adhesions in the protrusions show high relative phosphotyrosine levels; whereas larger adhesions outside the protrusions do not. Manipulating the PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e40202