levels of myosin II activity by knockdown, overexpression, inhibition, or altering RLC phosphorylation using mutants or RLC phosphatase or kinase inhibitors, all produce dramatic effects. In contrast, the migration of the same cells on laminin or ICAM-1 is more rapid, less dependent on either RLC phosphor- ylation or substrate density, and the adhesions in protrusions are large and highly elongated with high relative levels of phosphotyr- osine. Based on fibroblast studies on fibronectin, large adhesions arise from the high tension generated by rigid substrata and tend to have reduced phosphotyrosine [12,70]. However, laminin- or ICAM-1-based adhesions undergo retrograde flow and are under less tension. It is interesting in this context that the large adhesions in the a6b1 and aLb2 expressing cells still contain tyrosine phosphorylated molecules. This reinforces the notion that force transmission through myosin II, rather than adhesion size, per se, regulates phosphotyrosine phosphorylation. The difference in migration and response to RLC activation among different cell types using the same integrins is surprising. The CHO.B2 cells migrate faster on ICAM-1 or laminin than on fibronectin; yet HL-60 cells migrate faster on fibronectin, via a4b1 or a5b1, than on ICAM-1. In addition, the adhesions in the protrusions of migrating HL-60 cells are small despite the high level of RLC phosphorylation. Myosin IIB, which regulates polarity in CHO cell migration [23,12], is not present in the HL-60 cells and provides a partial explanation for the differences in actin and myosin organization and their polarity. Dictyostelium also expresses a single myosin II [71] and shows migration properties closer to that of neutrophils [72]. Therefore, it appears that in more contractile cells, myosin II regulates the production of large actomyosin bundles and their associated adhesions. But for some highly migratory cells, the role of actomyosin is different and results in fundamentally different structures and associated adhesions. In these cells, the integrin-substrate linkage may largely serve as a brake at the back of the cell or while cells change direction, resulting in a more fluid movement due to the force provided by actin polymerization at the leading edge. Supporting Information Figure S1 Phase contrast images of CHO.B2 cells transfected with or without its appropriate integrins and plated onto FN, LN or ICAM-1. Cells do not spread without the necessary integrins. (TIF) Figure S2 Protrusion, RLC distribution and phosphor- ylation depend on integrin engagement in U2OS and HT1080 cells. (A) Protrusion speed of HT-1080 (top) and U2OS (bottom) cells migrating on FN (1 mg/ml) or LN (5 mg/ml). Data are the mean 6 SD of 17 independent measurements per condition. (B) Differential phosphorylation of RLC in response to increasing amounts of FN (left) and LN (right) in U2OS. Cells were plated for 60 min on either substrate, in the presence of RLC phosphorylation inhibitors (Y/M stands for 20 mM Y27632+10 mM ML7) or a phosphatase inhibitor (calyculinA, 10 nM), and blotted for phosphorylated (p) or total (t) RLC. Quantification (bottom) represents the mean 6 SD of three independent experiments. (C) Subcellular distribution of the adhesion marker vinculin, actin and pRLC in U2OS plated on FN (1 mg/ml) or LN (5 mg/ml) for 60 min. Representative cells are shown. Scale Bar = 10 mm. (TIF) Figure S3 (A) Inhibiting MII activity does not change protrusion rates. Upper panel: CHO.K1 cells, or CHO.B2 cells transfected Integrin-Ligand Interactions Control Migration cells with a6, were plated onto fibronectin or laminin, respectively, then treated with ML7, Y27632, both or Control (blebbistatin) for half hour. The level of pRLC was clearly reduced. Typical immunoblots of pRLC, with quantification of 3 blots, are shown. Lower panel: CHO.B2 cells transfected cells with integrin-GFP were plated on fibronectin or laminin and treated with ML7 or Y27632. Protrusion rates were calculated from kymographs. No significant difference was observed for cells on same substrate. (B) CHO.B2 cells expressing the appropriate GFP-coupled integrin were co-transfected with the indicated mCherry mutants, allowed to adhere to the corresponding substrate, and protrusion was assayed by kymography. Data are the mean 6 SD of at least 3 independent experiments with 8–19 measurements per condition. There were no significant differences caused by expression of the RLC mutants. (TIF) Figure S4 (A) pRLC levels and protrusion rates on cells with different integrin expression levels. CHO.B2 cells were co- transfected with paxillin-mCherry and the appropriate integrin- GFP, sorted into three population by FACS: very low, low- medium, and high fluorescence, and then plated on FN or LN. Immunoblots for pRLC (upper panel) and protrusion rates are shown. High a5-GFP expressing cells show a small increase in pRLC level. Despite the integrin-GFP expression level, the protrusion rates remained similar on same substrate. (B) Relation between retrograde flow velocities, as determined by STICS, and average fluorescence protein expression level as estimated by ICS. Each point corresponds to a cell in a specific condition. Little, if any, influence of the expression level is observed on the retrograde fluxing. Correlation coefficients were computed for each condition (average of 0.24) and none was significant (P..0.05). (TIF) Figure S5 (A) Adhesions in protrusions of CHO.B2 cells expressing a6-GFP or aL+b2-GFP express SH2 domain binding sites. CHO.B2 cells were double transfected with SH2-mCherry and a5-GFP, a6-GFP or aL+b2-GFP and plated on FN, LN or ICAM-1, respectively (Upper, middle or lower panel, respectively). For the merged channel, SH2-mCherry is in purple and integrins are in green. Scale Bar = 10 mm. (B) T lymphocytes migrating on fibronectin display small, nascent adhesions. Jurkat E6.1 cells were transfected with GFP-vinculin, plated on FN (10 mg/ml) and allowed to migrate. The adhesions were imaged using TIRF microscopy after 30 min plating. Images were captured every 10 seconds for 1 hour. Representative time points are shown. Arrows indicate small adhesions that can be visualized as the cell extends new protrusions and retracts the rear. Scale Bar = 5 mm. (TIF) Movie S1 CHO.B2 cells expressing different integrins show differential integrin engagement and paxillin and integrin dynamics. CHO.B2 cells expressing paxillin-mCherry and a5, a6-GFP or aL+b2-GFP (left, center, or right, respectively) were imaged using a 60X TIRF microscope as they migrate on FN, LN or ICAM-1, respectively. Paxillin-mCherry is in magenta and GFP-integrins are in green. Images were captured every 2 seconds, and the total imaging time was 4 minutes. (AVI) Movie S2 HL60 cells expressing paxillin-mCherry and migrat- ing on FN (left and center) or ICAM-1 (right). Notice the touch, hold and release motion of paxillin (black dots and patches). Images were captured every 2 seconds and total imaging time was 4 minutes. (AVI) PLoS ONE | www.plosone.org 13 July 2012 | Volume 7 | Issue 7 | e40202