NEDD9 Stabilization of Focal Adhesions Figure 4. NEDD9 increases 3D migration speed. A. Western blot analysis of cell extracts following treatment with siRNA targeting NEDD9 or control siRNA sequences. Blots were probed with antibodies to NEDD9 which detects 105 kD and 115 kD NEDD9 phospho-forms and cross-reacted with p130Cas as indicated. Probing with anti-tubulin antibodies confirmed equal loading. B. MSD calculated from trajectories of WT MEFs (black squares, average of 3 independent experiments), NEDD92/2 MEFs (white squares, average of 3 independent experiments n.22 cells per experiment), MEFs treated with control siRNA (black circles, average of n = 38 cells) and NEDD9 siRNA (white circles, average of n = 25 cells). Note that identical values were obtained for the WT MEFs and WT MEFs treated with control siRNA; these data points are therefore super-imposed on the graph. C. Cell speed is significantly reduced in NEDD92/2 MEFs and in NEDD9 siRNA treated cells relative to the matched controls. Horizontal bars indicate the average speed for all cells tracked. D. Cell persistence is significantly reduced in NEDD92/2 and NEDD9 siRNA treated MEFs. Histogram shows the average persistence for all cells tracked. *p,0.05, ** p,0.001, *** p,0.0001, Students’ t-test. doi:10.1371/journal.pone.0035058.g004 be secreted by the MEFs. By immunofluorescence we confirmed the presence of fibronectin in the 3D gels (Figure 7C). Therefore, we analysed adhesion force to fibronectin by incubating cells with fibronectin-coated magnetic beads and exposing them to increas- ing, lateral pulling force in the range of 0 to 40 nN. The force at which a bead detached, if at all, was recorded. Importantly, significantly less force was required to detach the fibronectin- coated beads from the NEDD92/2 cells than from the control cells (Figure 7D). Given that there was reduced adhesion force despite high levels of a5b1 expression, we questioned whether this reflects decreased inside-out activation of b1 integrin receptors. Indeed we observe a striking suppression of activated b1 integrin receptor (clone 9EG7) on the NEDD92/2 MEFs relative to the total levels of a5b1 expression (Figure 7E). Thus, despite elevated a5b1 integrin receptor surface expression, receptor activation was inhibited. We therefore next determined the sub-cellular distribu- tion of activated b1 integrin. As the antibodies to the a5b1 integrin and activated b1 integrin are from the same species it was not possible to co-stain for these two populations. However, in agreement with the surface expression data (Figure 7E), compar- ison of the staining patterns for these two antibodies reveals that while there is greater organization of a5b1 in focal adhesions in the NEDD92/2 MEFs (Figure 7B), there is no corresponding increase in the organization of activated b1 integrin (Figure 8A). In 3D collagen gels the WT MEFs display robust stress fibre formation and activated b1 integrin co-localizes with stress fibres (Figure 8B). In contrast, the majority of the NEDD92/2MEF cells were rounded and lacked obvious stress fibres. In those NEDD92/2 MEFs in which stress fibres were evident, there is no corresponding organization of activated b1 integrin (Figure 8B). Thus, together these data suggest that lack of NEDD9 may decrease 3D cell speed by reducing integrin receptor activation and adhesion force to the ECM. Discussion Previous studies have used MEFs derived from mice in which focal adhesion molecules have been genetically deleted to investigate the function of these molecules in focal adhesion dynamics [15]. In the present study, we have used the same approach to investigate the role of NEDD9 and show that the lack of this protein appears to increase focal adhesion turnover in these cells, accompanied by increased cell migration speed in 2D. Thus, in contrast to most other molecules that have been investigated to date, it appears that NEDD9 may suppress focal adhesion disassembly. Further, despite NEDD9 depletion causing increased 2D migration speed, this conversely decreased 3D migration speed. Our data suggesting that NEDD9 suppresses focal adhesion disassembly is intriguing in light of reports that p130Cas conversely stimulates adhesion turnover [15]. A key difference between these two proteins is the presence of a poly-proline domain in p130Cas C-terminus that is not found in NEDD9. Both proteins contain a consensus Src SH2 binding site in their C- termini, but in p130Cas this site together with the poly-proline domain creates a bipartite binding site for the Src SH2 and SH3 domains respectively [33]. Through the bipartite Src binding motif, p130Cas is thought to amplify Src kinase activity [34], which is a major regulator of focal adhesion disassembly [35]. Additionally, the poly-proline domain of p130Cas has been directly implicated in focal adhesion disassembly [6]. Therefore, the absence of the poly-proline motif in NEDD9 may be one explanation for the differences of these two highly similar proteins on focal adhesion stability in MEFs. In the present study we show that while there is no change in the total levels of Src or FAK phosphorylation in the absence of NEDD9, phosphorylation of the Src/FAK substrate paxillin is increased at focal adhesions, PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35058