Mouse embryo fibroblasts (MEFs) derived from genetically depleted mouse models of the focal adhesion molecules paxillin, FAK and p130Cas were used to establish that each of these molecules stimulates focal adhesion disassembly [6,14,15] and these cells exhibit reduced speed [16–18]. By contrast, vinculin stabilizes focal adhesions [19,20] and fibroblasts lacking vinculin expression have more rapid locomotion in 2D migration assays [21]. We have previously shown that short peptides of NEDD9 dominantly interfere with focal adhesions, causing adhesion disassembly and cell rounding [11] and more recently NEDD9 depletion has been associated with increased 2D migration in breast epithelial cells [22] and granulocytes [23]. In contrast, in other cell backgrounds NEDD9 depletion inhibits cell migration [24–29]. Thus it appears that NEDD9 may differentially regulate 2D migration speed and focal adhesion dynamics depending on cell type. Therefore, it is important to establish the role of NEDD9 in focal adhesion dynamics in MEFs, to allow comparison with reported effects of other focal adhesion proteins, without the potentially confounding influence of cell-type specific effects. In contrast to the well-developed models of migration mechanisms in 2D, the understanding of cell migration in 3D and the role of focal adhesion molecules in this process is comparatively less well understood. Indeed, while fibroblasts with genetically deleted vinculin have increased 2D cell speed, conversely these cells have decreased speed in 3D collagen gels [21] and other adhesion proteins similarly give different 2D versus 3D migration outcomes [30]. In 3D environments migrating cells must overcome a different set of physical constraints than those in a 2D environment. Notably, not only does vinculin appear to stabilize focal adhesions, it also regulates cell adhesion force to the ECM and increases traction forces to the ECM [21]. Together, these functions of vinculin are likely to regulate the ability of the cell to negotiate the physical barriers of the 3D environment. The role of NEDD9 as a key signalling intermediary in mesenchymal cell invasion that relies on adhesion to the ECM suggests the possibility that NEDD9 may also regulate adhesion force to the ECM. In the present study we employ NEDD92/2 MEFs to show that NEDD9 stabilizes focal adhesions and increases b1 integrin receptor activation and adhesion force to the ECM. We show that NEDD9 depletion from fibroblasts increases 2D migration speed and conversely, significantly decreases 3D speed and migration persistence. Together our data reveal an important new role for NEDD9 in the stabilization of adhesions and adhesion force to the ECM. Results NEDD9 suppresses focal adhesion disassembly To address whether NEDD9 plays an active role in regulating focal adhesion turnover we used mouse embryo fibroblasts (MEFs) expressing endogenous wild-type NEDD9 (WT) and NEDD92/2 MEFs (Figure 1A). Due to the high degree of similarity between NEDD9 and p130Cas and the known role of p130Cas in stimulating focal adhesion disassembly in MEFs, we checked for changes in p130Cas expression the NEDD92/2 MEFs. Impor- tantly, there was no difference in either the expression level or the phospho-forms of p130Cas (Figure 1A), thus suggesting there is no compensatory changes in p130Cas in NEDD92/2 MEFs. Immunofluorescence analysis of fixed cells revealed robust focal adhesion and stress fibre formation in both WT and NEDD9 2/2 MEFs (Figure 1B), indicating that NEDD9 is not essential for the formation of focal adhesions and stress fibres. We next directly compared the rates of focal adhesion formation and disassembly in NEDD9 Stabilization of Focal Adhesions cells transfected with YFP-tagged paxillin. Rapid focal adhesion disassembly was observed in regions of protruding membrane in NEDD9 2/2 MEFs. By contrast in WT MEFs adhesion disassembly at leading edge appeared slower, with a period of focal adhesion stability preceding membrane extension. Quantification of the assembly and disassembly rates revealed that while assembly rates were essentially equivalent between the two cell lines (Figure 1C and 1D), disassembly rates were significantly faster in the NEDD9 2/2 MEFs (Figure 1C and 1E, p,0.001). We confirmed that there was no difference in the peak YFP-paxillin fluorescence intensity at focal adhesions in the WT versus NEDD9 2/2 MEFs (Figure S1) thus differences in adhesion disassembly rates were not due to differences in exogenous YFP-paxillin expression between the cell lines. Based on the increased rate of paxillin-positive adhesion disassembly in the NEDD92/2 MEFs we questioned whether paxillin phosphorylation was increased in NEDD92/2 MEFs, as this is associated with increased focal adhesion turnover [4,14]. Fluorescence ratio imaging of fixed WT and 2/2 MEFs co- immunostained with paxillin and phospho-paxillin antibodies revealed increased paxillin phosphorylation at focal adhesions in NEDD92/2 MEFs (Figure 2A). Calculation of the ratio of phoshorylated paxillin at individual adhesions confirmed signifi- cantly increased paxillin phosphorylation in the NEDD92/2 MEFs (Figure 2B, p,0.0001). These data are therefore consistent with the idea that the paxillin focal adhesion disassembly switch is more active in NEDD92/2 MEFs. NEDD9 differentially regulates 2D versus 3D cell migration Increased rates of focal adhesion disassembly and paxillin phosphorylation in NEDD92/2 MEFs together suggested that these cells might migrate more rapidly in 2D cell migration assays. Indeed, tracking of individual cells and quantification of the Mean Square Displacement (MSD) revealed that the NEDD92/2 MEFs have significantly faster 2D migration speed (Figure 3A and 3B). To confirm that these effects are directly due to the absence of NEDD9 we performed a rescue experiment with exogenously expressed GFP-tagged NEDD9. Importantly, exogenous GFP.NEDD9 significantly reduced 2D cell migration speed when compared with GFP control transfected NEDD92/2 MEFs (Figure 3C and 3D). We next determined the role of NEDD9 in the 3D migration of MEFs, comparing NEDD9 WT MEFs, NEDD92/2 MEFs, and MEFs either exposed to scrambled siRNA controls (control) or following treatment with siRNA targeting NEDD9 expression (NEDD9 siRNA). Control western blot analysis confirmed depletion of NEDD9 protein following NEDD9 siRNA treatment and no effect on p130Cas (Figure 4A). Analysis of the MSD demonstrated that both genetic deletion of NEDD9 (2/2) and NEDD9 depletion with siRNA (NEDD9 siRNA) significantly reduced the migration of MEFs in 3D collagen gels (Figure 4B). The wild-type MEFs and control siRNA-treated MEFs migrated through the 3D collagen gels with greater cell speed (p,0.0001, Figure 4C) and tended to have increased migration persistence (Figure 4D). Thus, in contrast to the effects seen in 2D, we find that 3D cultures cells lacking NEDD9 expression have reduced cell migration speed. Previous reports have demonstrated that Src, FAK, ERK and MLCK are important regulators of focal adhesion dynamics [15]. Due to the striking difference in motility effects in 2D versus 3D culture conditions, we therefore employed phospho-antibodies to analyse the activity of these kinases. This revealed distinct profiles of kinase activity between cells cultured in 2D and 3D. While ERK phosphorylation was equivalent between both 2D and 3D culture PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35058