The total average was then computed over all cells (one region per cell). Spatial image correlation spectroscopy (ICS) [38] was used to measure the relative density (expression level) of fluorescent proteins. Spatial correlation functions are calculated for each region and the amplitude of the peak, after background noise correction, is inversely proportional to the fluorescent protein density per focal spot area. One uniform region was selected per cell, and the time average was obtained for each cell analyzed with STICS. FACS Cell sorting was done on a Becton Dickinson FACSVantage SE Turbo Sorter with DIVA Option at the Flow Cytometry Core Facility of UVA. A total of 2.06107 CHO.B2 cells were transfected and suspended in Basic Sorting Buffer (1x Ca/Mg2+ free PBS, 1 mM EDTA, 25 mM HEPES pH 7.0, 1% Heat- Inactivated FBS, filter sterile), then sorted into low, medium-low or high fluorescence groups, with at least 16106 cells for each condition. Cells were allowed to recover overnight in DMEM medium with 20% serum before analysis. FRAP and Bead Displacement Confocal images for FRAP (fluorescence recovery after photo- bleaching) analysis and bead displacements were acquired using an Olympus inverted microscope IX81 (1.40 (oil) PlanApo 60X objective) driven by FluoView software (Olympus). Bead displace- ments were measured using red fluorescent (580/605) Fluo- SpheresH carboxylate-modified microspheres (Invitrogen) imbed- ded into polyacrylamide gels at ,1 kPa stiffness [39,40] coated with the specified substratum [41,42]. CHO.B2 cells were transfected with the appropriate GFP integrin and 2-channel (GFP/beads) image time series of protrusion and retraction events were recorded. Bead displacements were determined using a particle tracking routine written in MATLAB (MathWorks) and interpolated onto a regular grid comprised of 32632 pixels subregions to find the gel displacement. The relative gel deformations were referenced to the beginning of the protrusions (or end of retraction) so that the measured gel deformation was caused by force differences during those events. For FRAP, a selected cellular area that contained GFP fusion protein was scanned five times, and then bleached using 15 scans at 100% laser power. To image the fluorescence recovery of fluorescence intensity after the photo-bleaching, we collected 200 scans in succession; 100 scans every 0.2 s followed by 50 scans every 0.5 s. Background subtraction and normalization were calculated for the averaged intensities from the bleach region, and normalized intensity vs. time were fitted by a single exponential equation. Data collected were processed using ImageJ, Excel (Microsoft) and SigmaPlot (SYSTAT) software. Adhesion Assay Affinities of different integrins were measured using a centrifu- gation assay [43]. Cells were transfected with a5, a6 or aL+ b2 construct, placed into in 96-well plates coated with FN, LN or ICAM-1, respectively, and incubated for 20 minutes at 37uC in a CO2 incubator. The plates were then sealed, inverted and centrifuged at 200 rpm three times for 5 minutes using the Beckman GH 3.8 rotor. Control plates were sealed and inverted for 15 min but not centrifuged. Cells in each well were counted and compared to its positive well. The adhesion strength for each integrin was presented as the fraction of cells remaining after centrifugation. Integrin-Ligand Interactions Control Migration Results Integrin Heterodimer Expression Dictates Migration, Protrusion and Adhesion To determine whether the migratory properties of cells depend on the integrin-ligand pairs utilized, we expressed either the a5 or a6 subunits or co-expressed the aL and b2 integrin subunits in CHO.B2 cells, a CHO cell variant that expresses the integrin b1 subunit but very little alpha subunit [15], and plated them onto fibronectin, laminin or an ICAM-1-Fc construct [44] (R&D Systems), respectively. a5-GFP expression mediated adhesion, spreading and migration on fibronectin but not on laminin or ICAM-1; a6-GFP promoted adhesion and spreading on laminin, but not on fibronectin or ICAM-1; simultaneous expression of aL- GFP and b2-GFP enabled adhesion and migration on ICAM-1 but not on fibronectin or laminin, and CHO.B2 cells expressing no ectopic integrin neither spread nor migrated on any substrate tested under comparable conditions (Figure S1, data not shown). Figure 1A shows the paths of seven typical cells expressing comparable levels of integrin-GFP for each condition plotted from a common origin traced over identical time periods. The cells on ICAM-1 migrate the fastest (larger displacement per unit time) and are the most directionally persistent (total net displacement from the origin), with cells on laminin intermediate and those on fibronectin slowest and least directionally persistent. The average speed of cells on fibronectin is about half that of cells on either laminin or ICAM-1 (Figure 1B, lower panel, P,161029). While the difference in speed between cells migrating on laminin and ICAM-1 is not as large, it is still significant (P = 0.0017). The differences in directional persistence were estimated as the ratio of the distance between the start and end points of the migration path to the total length of the path. The directionality of cells on fibronectin was much lower than that of cells on either laminin or ICAM-1 (P value of fibronectin vs. laminin = 0.00078); whereas the cells on fibronectin had significant movement but little net translation (Figure 1B, upper panel). We also analyzed the protrusions and adhesions of cells expressing a5-, a6-, or aLb2-GFP and paxillin-mCherry migrat- ing on fibronectin, laminin or ICAM-1, respectively. Cells migrating on laminin or ICAM-1 exhibited more numerous and rapid protrusions, as quantified by kymography, than those on fibronectin (Movie S1). Cells on laminin or ICAM-1 extended and retracted their protrusions more rapidly and frequently (,2.360.5 mm/min) than cells on fibronectin (1.660.5 mm/min, P,,0.001; Movie S1). This correlates well with the observed differences in migration. Adhesions, as visualized using paxillin- mCherry, were also different. Cells on fibronectin displayed prominent small, dynamic, nascent adhesions that actively turned over at the front of protrusions, as well as some larger, more stable, and slightly elongated adhesions in the more distal portions of protrusions (Figure 2A) [26,45]. In contrast, cells spread on laminin or ICAM-1 had few nascent adhesions; most adhesions in protrusions assembled and elongated quickly, and therefore, were highly elongated from very early time points (Figure 2A). To determine whether these observations are cell type- independent, we investigated the adhesion and migration of U2OS and HT1080 cells. These cells adhere to fibronectin through a5b1; but unlike CHO cells, they also spread and migrate spontaneously on laminin, likely due to their endogenous a3b1 and/or a6b1 [46–48]. Both the U2OS and HT1080 showed higher protrusiveness on laminin than on fibronectin (Figure S2). In addition, adhesions in cells on laminin elongated quickly (data not shown). Adhesion maturation was also observed in cells on fibronectin (data not shown). These results are similar to those PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40202