NEDD9 Stabilization of Focal Adhesions Figure 1. Focal adhesion turnover is more rapid in the absence of NEDD9 expression. A. Western blot analysis of extracts of wild-type (WT) and NEDD9 knockout (2/2) MEFs. Blots were probed with antibodies to NEDD9 which detects the 105 kD and 115 kD phospho-forms of NEDD9 and p130Cas antibodies as indicated. Blots were then probed with anti-tubulin or anti-HSP70 antibodies to confirm equal loading. B. WT and NEDD9 2/2 MEFs immunostained with antibodies to paxillin (pax) to detect focal adhesions (white arrows) and TRITC-phalloidin to detect actin stress fibres. C. Examples of YFP-paxillin positive focal adhesions in transfected WT (72 minute lifetime) and NEDD92/2 MEFs (52 minute lifetime) are shown. D. Focal adhesion assembly rate constants (k) for cells transfected with YFP-paxillin (n = 32 focal adhesions per cell line). Horizontal bars show the average value per cell line. E. As for D, except data showing the disassembly rate constants (k). (WT n = 26, 2/2 n = 33). *p,0.01, N.S. = not significant. doi:10.1371/journal.pone.0035058.g001 conditions, both Src and FAK displayed reduced phosphorylation in 3D cultures (Figure 5A and B). However, global activation of these molecules was not affected by the presence of NEDD9 as there was no difference in Src and FAK phosphorylation between WT MEF and NEDD92/2 cells in either 2D or 3D cultures. In contrast, there is a striking differential effect on MLCK activity. Under 2D culture conditions, MLC-phosphorylation appears to be significantly reduced in NEDD2/2 MEFs. Conversely MLC- phosphorylation is significantly increased in NEDD92/2 MEFs grown in 3D collagen gels (Figure 5A and B). The observed increased MLC phosphorylation in the NEDD92/2 MEFs in 3D collagen gels agrees with earlier reports showing that NEDD9 depletion is accompanied by activation of MLCK [9]. Following the differential signalling effects in 2D versus 3D, we questioned whether NEDD9 is also differentially regulated in 2D versus 3D. NEDD9 is detected as two distinct phospho-forms of 115 kDa and 105 kDa apparent molecular mass [36]. We have previously shown that 115 kDa NEDD9 is formed in response to integrin receptor stimulation [11]. Analysis of the NEDD9 phospho-forms in WT MEFs revealed that 115 kDa NEDD9 is significantly decreased in 3D cell cultures (Figure 5A and B). During observation of the time-lapse imaging series, it was noted that the majority of the NEDD92/2 MEFs had a rounded appearance in 3D collagen gels and quantification of the percentage of rounded cells confirmed this (Figure 6A and 6B). We considered whether the previously described mitotic defects associated with altered NEDD9 expression levels [31,32] might account for cell rounding. However this was not the case, as we observe fewer mitotic events in NEDD92/2 cells in 3D collagen gels when compared with WT controls (Figure 6C). Based on the increased rounding and increased levels of MLC phosphorylation, we questioned whether NEDD9 depletion stimulated a switch to amoeboid invasion in the MEFs, as has previously been shown in other cell types [9]. However, the reduced speed of NEDD92/2 MEFs suggests that the observed rounding does not reflect a switch to an amoeboid mode of migration. The rounded shape of the NEDD92/2 MEFs, suggested that these cells may have reduced interaction with the surrounding matrix. NEDD9 increases adhesion strength and b1 integrin activation Following the demonstration that NEDD9 2/2 MEFs have reduced speed and persistence and are more rounded in 3D PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35058