expression was achieved following trypsinization to detach cells. Cells were immediately washed (0.5% BSA/0.1% NaN3/PBS), incubated with primary antibodies, washed, incubated with fluorescently labelled secondary antibodies and after a final wash stained cells were analysed using a FACSCalibur (Becton Dickinson). The isotype control corrected geometric mean fluorescence of at least 3 separate experiments was compared. Magnetic tweezers 1 mg of carboxylated superparamagnetic beads (Microparticles, Berlin) with 560.21 mm diameter were coated with 50 mg/ml human fibronectin (Roche, Basel, Switzerland) in 1 ml of carbonate buffer (pH 9.4) for overnight or longer at 4uC. After coating, the beads were washed and stored in PBS. 10,000 cells were plated in 35 mm dishes for 24 hours, and fibronectin-coated beads were then added to the cells and incubated for 30 min at 37uC and 5% CO2. Prior to measurements, the dishes were gently washed with fresh medium to remove unbound beads and then placed on a heated microscope stage. With a magnetic tweezer device, a force that was linearly increasing from 0 to 40 nN over 10 seconds was then applied to the beads. The speed of the force ramp was 4 nN/s. Bright-field images of the cell, the bead, and the needle tip were taken by a CCD camera (ORCA ER Hamamatsu) at a rate of 40 frames per second at 406 magnification. Bead positions were tracked in real time using an intensity-weighted centre-of-mass algorithm. The force value at which a bead detached from the cell surface was recorded. Focal adhesion dynamics YFP-paxillin positive focal adhesions were imaged with an Olympus IX81 inverted microscope, with a 606 (NA 1.35) oil objective, using fluorescence filters of BP 460–495/BP510–550 (GFP) and BP 490–500/BP 515–560 (YFP). Images were captured every 2 minutes for 90 minutes total, 250 ms exposure times. Focal adhesions at the protruding edge of the membrane were analysed by measuring the temporal changes in integrated pixel intensity of an individual focal adhesion. Using MetaMorph V6.1r0 (Molecular Devices, USA), a box was drawn close to the focal adhesion to be analysed (in the cell cytoplasm) and the background corrected by subtracting the average pixel intensity of References 1. DiMilla PA, Stone JA, Quinn JA, Albelda SM, Lauffenburger DA (1993) Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength. J Cell Biol 122: 729–737. 2. Harms BD, Bassi GM, Horwitz AR, Lauffenburger DA (2005) Directional persistence of EGF-induced cell migration is associated with stabilization of lamellipodial protrusions. Biophys J 88: 1479–1488. 3. Vicente-Manzanares M, Choi CK, Horwitz AR (2009) Integrins in cell migration–the actin connection. J Cell Sci 122: 199–206. 4. Bach CT, Creed S, Zhong J, Mahmassani M, Schevzov G, et al. (2009) Tropomyosin isoform expression regulates the transition of adhesions to determine cell speed and direction. Mol Cell Biol 29: 1506–1514. 5. O’Neill GM (2009) The coordination between actin filaments and adhesion in mesenchymal migration. Cell Adh Migr 3: 355–357. 6. Meenderink LM, Ryzhova LM, Donato DM, Gochberg DF, Kaverina I, et al. (2010) P130Cas Src-binding and substrate domains have distinct roles in sustaining focal adhesion disassembly and promoting cell migration. PloS ONE 5: e13412. 7. Sanz-Moreno V, Marshall CJ (2010) The plasticity of cytoskeletal dynamics underlying neoplastic cell migration. Curr Opin Cell Biol 22: 690–696. 8. Friedl P (2004) Prespecification and plasticity: shifting mechanisms of cell migration. Curr Opin Cell Biol 16: 14–23. 9. Sanz-Moreno V, Gadea G, Ahn J, Paterson H, Marra P, et al. (2008) Rac activation and inactivation control plasticity of tumor cell movement. Cell 135: 510–523. 10. Law SF, Estojak J, Wang B, Mysliwiec T, Kruh G, et al. (1996) Human Enhancer of Filamentation 1, a novel p130cas-like docking protein, associates NEDD9 Stabilization of Focal Adhesions the boxed region from the entire image as previously described [45]. A polygon was drawn around the focal adhesion at time of maximum intensity and the average pixel intensity of the polygon recorded for all time points. Intensity values (I) were expressed relative to the minimum intensity value (Io) for calculating adhesion assembly rates, or were expressed relative to the maximal intensity value (Imax) for calculating adhesion disassembly rates. Assembly and disassembly rate constants (k) were then calculated from the slope of the lines generated by plotting the data as either ln(I/Io) or ln(I/Imax), versus time. Fluorescence ratio imaging of phosphorylated paxillin and semi-quantitative analysis of paxillin phosphorylation at focal adhesions was performed as previously described [4], with the addition of a masking step using Metamorph software, whereby images were first subject to a high-pass filter (sensitivity 1) to emphasize the peripheral cell adhesions. The filtered images were then used as a mask for background subtracted images to high-light the peripheral focal adhesions. Note that the high level of phospho-paxillin antibody reactivity in the nucleus means that the nuclear staining is not filtered out and hence gives a strong signal in the final ratio images. Image preparation Final micrograph images and grey level adjustments were prepared in Adobe Photoshop. Supporting Information Figure S1 Equivalent expression of fluorescently-tagged fusion proteins at focal adhesions. Comparison of the peak fluorescence intensity (FI) in arbitrary units (a.u.) for YFP-paxillin positive focal adhesions in either wild-type (WT) or NEDD9 2/2 transfected MEFs. (TIF) Author Contributions Conceived and designed the experiments: GO JZ JBB BF. Performed the experiments: JZ JBB NB JL YWC. Analyzed the data: GO JZ JBB BF. Contributed reagents/materials/analysis tools: EP. Wrote the paper: GO. with focal adhesion kinase and induces pseudohyphal growth in Saccharomyces cerevisiae. Mol Cell Biol 16: 3327–3337. 11. O’Neill GM, Golemis EA (2001) Proteolysis of the docking protein HEF1 and implications for focal adhesion dynamics. Mol Cell Biol 21: 5094–5108. 12. Singh M, Cowell L, Seo S, O’Neill G, Golemis E (2007) Molecular basis for HEF1/NEDD9/Cas-L action as a multifunctional co-ordinator of invasion, apoptosis and cell cycle. Cell Biochem Biophys 48: 54–72. 13. 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