[15,16]. We expressed the a5 or a6 integrin subunit in CHO.B2 cells and measured their migration on fibronectin and laminin, respectively. We also transfected a leukocyte-specific integrin, aLb2, into CHO.B2 cells and measured their migration on ICAM-1, an inflammation-related substrate [17]. To parse contributions arising from integrin-substrate interactions and cell type, we investigated cell types that naturally express the integrins studied in the CHO model. For a6b1 we used the osteosarcoma cell U2OS, which expresses both a5b1 and a6b1 [18] and therefore migrates on both fibronectin and laminin. For aLb2, we used HL60 cells, which migrate robustly on ICAM-1. We found that myosin II plays a greatly reduced role in adhesion and migration of cells on laminin and ICAM-1 compared to that on fibronectin. This difference appears to arise from a novel retrograde fluxing of a6b1 and aLb2 integrins that results in reduced adhesion strength and force transmission to adhesions. Leukocyte migration (using the HL-60 cells) was also largely myosin II-independent; but the differences in morphology of the cells and their adhesions from that of the CHO cells using the same integrins seem to reflect intrinsic cell type differences in actin organization. Materials and Methods Plasmids a5-GFP has been described previously [16]; a6-GFP was made by excising the a6 cDNA from the a6-pRSVneo plasmid [19] and inserting into the 59 of the pEGFP-N1 vector (Clontech) using the flanking Apa I and Kpn I sites; aL-GFP and b2-GFP were generated by in-frame replacement of YFP with GFP in aL-YFP and b2-YFP, which were kind gifts from T. Springer [17]. The low expression, ‘‘speckle’’ versions of a5-GFP, a6-GFP and aL-GFP were made by replacement of the CMV promoter with a truncated version that enables very low expression [20]. mCherry-MIIA and mCherry-MIIB were made from their GFP version as described [21], which were gifts from R. Adelstein [22]; wild type RLC-mCherry, and its mutant versions: RLC-AA- mCherry, RLC-AD-mCherry, RLC-DA-mCherry, RLC-DD- mCherry were prepared from RLC-GFP and RLC-DD (from Kathleen Kelly, NCI/NIH) as described previously [23,24]. The mCherry plasmid was a gift from R. Tsien [25]. Paxillin-GFP and Paxillin-mCherry were also described previously [16,26,27]. Low expression, ‘‘speckle’’ mGFP-dSH2 and mCherry-dSH2 were generated from the YFP-SH2 construct donated by Benjamin Geiger [28] by replacement of the YFP with mGFP or mCherry and of the CMV promoter with a truncated version that enables very low expression [20]. Use of the mGFP version has been recently described [12]. Cell Culture and Transfection CHO.B2 cells (from Rudi Juliano) [15] and HL60 cells (from Orion Weiner) [29] were cultured in DMEM medium and RPMI medium 1640, respectively, from Invitrogen. CHO.B2 cells were transfected using Lipofectamine or Lipofectamine 2000 (Invitro- gen) according to the manufacturer’s instructions. HL60 cells were differentiated and transfected with the Amaxa nucleofection system from Lonza [30]. U2OS cells and HT1080 cells were obtained from ATCC and transfected using Lipofectamine 2000 according to the manufac- turer’s instructions. Antibodies and Reagents Fibronectin and laminin were from Sigma-Aldrich. Recombi- nant human ICAM-1 (Fc fragment) was from R&D Systems. Integrin-Ligand Interactions Control Migration Phospho-RLC (pRLC) and total RLC antibodies were from Rockland Inc. and Sigma, respectively. MIIA and MIIB antibod- ies were from Covance. Immunoblots Cells (,106) were incubated on the indicated substrates for one hour, washed using PBS (Invitrogen), and lysed in RIPA buffer (Pierce). The resulting lysates were separated by 4–20% SDS/ PAGE (BioRad). Proteins were transferred onto PVDF mem- branes, blocked using SuperBlock blocking buffer (Thermo Scientific) and immunoassayed for pRLC, MIIA, MIIB or total RLC by Western blot using Amersham ECL system (GE healthcare). When indicated, densities were quantified using ImageJ. Migration Assays Cell migration was assayed under 10X phase microscopy (Nikon TE300). The cells were plated in CMM1 medium (Hyclone from Thermo Scientific), and allowed to migrate for the desired time. Images were captured using a CCD camera (Hamamatsu) with Metamorph software (Molecular Devices) and then analyzed by ImageJ as previously described [31]. TIRF Microscopy TIRF images were acquired on an Olympus IX70 inverted microscope (1.45 (oil) PlanApo 60X TIRFM objective), fitted with a Ludl modular automation controller (Ludl Electronic Products) and controlled by Metamorph (Molecular Devices). GFP and mCherry were excited using the 488 nm laser line of an Argon ion laser and the 543 nm laser line of a He-Ne laser (Melles Griot), respectively. A dichroic mirror (HQ485/30) was used for GFP- labeled cells. For dual GFP- mCherry/mOrange, a dual emission filter (z488/543) was used. Images were acquired with a charge- coupled device (CCD) camera (Retiga Exi; Qimaging) controlled by Metamorph software. To confirm the co-localization of adhesion components, some images were acquired using Olympus inverted microscope IX71 (1.45 (oil) PlanApo 100X TIRFM objective) fitted with a Dual- View (Photometrics) to simultaneously acquire both colors. When indicated, substrates were covalently cross-linked to the coverslip using GMBS as previously reported [32]. For other experiments, we adsorbed the substrates to coverslips pre-coated with 1 mg/mL poly-L-lysine, which has been described to improve adsorption [33,34]. All images were analyzed using ImageJ. For kymography, a single line is drawn from the edge of the protrusion toward the cell center, using images captured every 2 seconds, then the line intensity is plotted with the x-axis representing total time (4 minutes), and y-axis representing the movement of the cell edge [35,36]. Image Correlation Spectroscopy STICS (spatio-temporal image correlation spectroscopy) [37] was used to quantify (magnitude and direction) transport of adhesion components during fluxing. This method measures the peak displacement of the spatio-temporal correlation function calculated from fluorescence intensity fluctuations recorded in a time series of images in order to compute the average velocity of the labeled species in a small region of interest. It was used previously in the same context to characterize the relative transport of different adhesion components [9]. The average velocities were computed from only the top 20% of measured velocities to remove non-fluxing vectors in the region of analysis. PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40202