Arginylation Regulates Neural Crest Migration Figure 6. Ate1 knockout affects cell attachment to the substrate via intracellular and not extracellular mechanisms. (A) Left, extracellular fibronectin staining in a dense monolayer of wild-type (WT) and Ate1 knockout (KO) cultured fibroblasts shows no difference between the two cultures. Right, quantification of the fibronectin level in the two cultures measured as average gray value in the entire field of view confirms that there is no difference between WT and KO cells in the amount of extracellular fibronectin. Bars show the ratio between WT and KO and error bars represent the average of the measurements in 10 different fields of view in each culture. (B) Left, an overlay of the fluorescence staining of the edge of the cell monolayer moving into the wound co-stained with rhodamine-phalloidin (red) to visualize the actin filaments and anti-paxillin (green) to visualize the focal adhesions. Right, quantification of the number of focal adhesions per mm of the wound edge shows that the number of prominent focal adhesion in wild-type exceeds that in the knockout by over 5-fold. Error bars represent the measurements in 21 and 18 different images in WT and KO, respectively. Bar, 20 mm. doi:10.1371/journal.pgen.1000878.g006 monolayer migrating into the wound in wild-type and Ate1 knockout cells by staining these cells with three different focal adhesion markers, paxillin, talin, and focal adhesion kinase (FAK) (Figure 6B and Figure S11). While in wild-type cultures cells moving into the wound developed prominent focal adhesions that appeared firmly anchored to the substrate (Figure 6B, top panel, and Figure S11, left panels WT), in Ate1 knockout cells focal adhesions appeared smaller and scarcer, sometimes difficult to detect (Figure 6B, bottom panel, and Figure S11, right panels). This decrease in focal adhesion size and number was accompanied by a reduced level of paxillin but did not correlate with the changes in talin and FAK protein level (Figure S11, middle panels KO), suggesting that the decrease in focal adhesion constituted a genuine structural defect rather than a secondary effect of down- regulation of any of these markers. Manual counting of the number of prominent elongated paxillin-containing structures per mm of the wound edge, or the area of talin-containing structures per leading edge in each cell, revealed an almost 10-fold reduction in the focal adhesion area and number in Ate1 knockout cells compared to wild-type (Figure 6B and Figure S11, right panels). Such a prominent difference is highly likely to severely affect both the cell’s ability to attach to the substrate and the resulting cell migration. Thus, Ate1 knockout-dependent impairment in cell migration likely originates at the intracellular level. To further study the focal adhesion defects in Ate1 knockout cells we quantified the total area of talin and paxillin-containing focal adhesions in single wild-type and Ate1 knockout cells, and found that, consistent with the results obtained in the moving cell monolayer, the focal adhesion area in single cells is significantly reduced (Figure S12). This result suggests that the focal adhesion defects in Ate1 knockout cells are the intrinsic property of these cells and not the secondary result of the impaired movement of these cells along the substrate. Finally, to corroborate this observation with the situation in situ, we stained embryo sections for talin and quantified the staining intensity in the migrating subpopulations of neural crest cells at the sides of the neural tube (Figure S13). While this assay measures not only talin localized to the adhesion sites but the total talin level in the tissue (which is similar in wild-type and knockout, as seen in Figure S11), the un- localized talin is expected to show a reduced, more diffuse signal compared to talin bound at the focal adhesion sites. Consistent with this hypothesis and the results of focal adhesion quantification in culture, the talin staining in situ was reduced in Wnt1-Ate1 embryos by a small but statistically significant number (Figure S13), suggesting that migrating neural crest cells in these embryos have focal adhesion defects that may prevent them from proper attachment and movement through the tissues during embryo- genesis. Unlike in the complete knockout, when cells migrate in situ in a conditional mouse model, in many cases knockout cells are found in the immediate vicinity of the wild-type and are expected to co- migrate during different developmental events. Therefore, the straight comparison between wild-type and Ate1 knockout cell movements does not necessarily reflect the complex situation in which these cells co-exist in situ, potentially affecting each others’ migration. To test the motility of Ate1 knockout cells in the environment of the surrounding wild-type cells, we performed wound healing migration assays of wild-type cells co-cultured with Ate1 knockout cells labeled with stably transfected GFP. Control experiments showed that GFP-expressing cells moved at rates PLoS Genetics | www.plosgenetics.org 8 March 2010 | Volume 6 | Issue 3 | e1000878