Figure 5. Adhesion signalling pathways are differentially activated in 2D versus 3D. A. Western blot analysis of lysates extracted from WT MEF and NEDD92/2 MEF grown on tissue culture plastic dishes (2D) or in collagen gels (3D). Blots were probed with the indicated antibodies to total proteins and phosphorylated proteins (-p-). Note that total Src was immunoprecipitated (IP) prior to probing with anti-p-Src antibodies. Blots were probed with Hsp70 or tubulin as a loading control. B. Histograms showing densitometric measurements of the level of phosphorylated protein divided by the matching total protein amount and expressed relative to the level in WT MEFs under 2D culture conditions. Data represent the average of triplicate repeats on separate days and error bars show the S.E.M. *p,0.05, ** p,0.001, NS = not significant, Students’ t-test. doi:10.1371/journal.pone.0035058.g005 suggesting small increases in localized Src or FAK activity specifically at focal adhesion in these cells. However, reports that Src and FAK activity are reduced in mouse mammary tumours derived from a NEDD92/2 mouse [26] suggest that NEDD9 regulation of these kinases is complex and may depend on cell-type and tumorigenic status. Similar to our results for NEDD92/2 MEFs, NEDD9 depletion from MCF10a breast epithelial cells and granulocytes also led to increased 2D cell migration [22,23]. The granulocyte data is particularly interesting, given that lymphocytes isolated from the same NEDD2/2 mice conversely display decreased 2D cell migration [23], in keeping with studies in a variety of cancer cell types showing that NEDD9 depletion inhibits migration in scratch wound healing assays [24–27] and transwell migration assays [28,29]. It was suggested that the differential effects of NEDD9 knock-out on granulocyte versus lymphocyte populations NEDD9 Stabilization of Focal Adhesions Figure 6. NEDD9 2/2 MEFs adopt a rounded morphology. A. Time-lapse series of WT and NEDD92/2 MEFs showing cellular morphologies in 3D collagen gels. B. Percentage of cells exhibiting a rounded phenotype. ** p,0.001, Students’ t-test. C. Mitotic events are decreased in NEDD92/2 fibroblasts. The number of cells observed to undergo mitosis in 3D collagen gels during 6 hours of filming were quantified and expressed as a percentage of the total cells analysed. Data shown are the average of two independent experiments. doi:10.1371/journal.pone.0035058.g006 may be due to differing expression of the two discrete NEDD9 phospho-forms [23]. Related to this, we have recently established that the NEDD9 phospho-forms have discrete functions in cell spreading [36,37]. In the present study we demonstrate reduced levels of 115 kDa NEDD9 in cells grown in 3D collagen gels therefore reflecting differential control of NEDD9 phosphorylation in 2D versus 3D conditions. Thus NEDD9 phospho-form expression may play a role in determining the function of NEDD9 as a regulator of focal adhesion turnover. Alternatively, the biphasic response between cell adhesion and migration in 2D [38] means that the consequence of altered focal adhesion turnover rates for final cell speed will depend on whether cells initially had optimal adhesion for the fastest rate of migration. As focal adhesion dynamics are ‘‘tuneable’’ the expression levels of an adhesion stabilizer such as NEDD9 may therefore give different speed readouts in a 2D environment. In contrast to the results seen in 2D migration assays, we find that NEDD9 depletion significantly reduced migration speeds of MEFs in 3D collagen gels. The critical role of integrin-mediated interaction with the ECM in 3D migration is indicated by studies showing decreased b1 integrin receptor clustering accompanying the switch to amoeboid migration [39]. In the present study we show that cells with genetically depleted NEDD9 have reduced cell adhesion force to fibronectin and the inside-out activation of b1 integrin receptors. These data extend previous findings that NEDD9 regulates Rac-mediated induction of mesenchymal migration and invasion [9], by suggesting that NEDD9 may also determine interaction with the ECM. Further, our data have confirmed earlier reports that MLCK activity is increased following NEDD9 depletion in 3D collagen gels, but in addition, we now show that MLCK activity is conversely decreased under 2D culture conditions. Notably, the data reported here for NEDD9 are highly similar to those previously reported for vinculin. Vinculin has also been suggested to be a negative regulator of adhesion turnover [19] and vinculin knockout fibroblasts have increased 2D migration speed, but decreased 3D migration and invasion [21]. It has previously been suggested that vinculin activation leads to an open conformation with tight PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35058