Indeed, these fibroblasts are mesenchymal cells derived from the back portion of the embryos where the majority of the migrating neural crest cells are also found, and they are morphologically indistinguishable from the cells composing the neural crest explants in culture. Therefore, such fibroblasts can be expected to behave in the same manner as the neural crest cells in terms of their motile properties, and using these cells as a model in culture makes it possible to perform much more detailed tests than possible with neural crest explants. Ate1 knockout fibroblasts have been previously shown to have a defective lamella [24], however no changes in the speed or directionality of their migration have been reported. To test whether these cells move slower than wild-type, we performed scratch wound assays by removing an area of a dense cell monolayer and taking time lapse images of cells moving from the dense area to the scarce. The observations were performed over long periods of time similar to the estimated duration of many neural crest-dependent migratory events in development (10– 15 hours). While individual Ate1 knockout cells during shorter stretches of time were capable of moving at speeds similar to wild-type (not shown), the migration speed of the Ate1 knockout cell monolayers over continuous periods of time was nearly 4 times slower than that of wild-type cells (average speed of 11.5 mm/h compared to 41.3 mm/h in wild-type) (Figure 5, Video S3 and Video S4). This Arginylation Regulates Neural Crest Migration difference constitutes a significant delay in the overall migration, and, when transferred to an in vivo environment of a developing embryo would be likely to create significant morphogenic defects. Cell migration in culture and in situ is mediated by attachment to the substrate and forming a connection between the intracellular actin cytoskeleton and the extracellular matrix via focal adhesions. To test whether the slow migration speeds in Ate1 knockout cells were due to their impaired adhesion on the intra- or extracellular side, we first tested whether these cells are capable of creating a local extracellular environment that favors migration. To do this, we stained wild-type and Ate1 knockout non- permeabilized cell monolayers for fibronectin, a major extracel- lular matrix component that directs the migration and adhesion of the mesenchymal cells and is secreted by these cells in culture and in situ (reviewed in [46]). No differences were found in the amount or distribution of fibronectin per area in each culture (Figure 6A), suggesting that Ate1 knockout cells are capable of creating and utilizing the same local extracellular environment as wild-type. Thus, the migration defects in Ate1 knockout cells do not originate at the local extracellular level. We next tested whether the intracellular defects in Ate1 knockout cells could be responsible for slower migration speeds. To do this, we compared the number of focal adhesions (the structures that connect cells to the extracellular matrix and thus are responsible for cell movement) at the edge of the cell Figure 5. Knockout of the arginyltransferase Ate1 in the mesenchymal cells results in a significant reduction in their migration speeds. (A) The first and the last frame of a time lapse series of a wild-type (WT) and Ate1 knockout (KO) fibroblast monolayer moving into the wound. See Video S3 and Video S4 for the corresponding time lapse series. Bar, 200 mm. (B) Quantification of the average migration speed in wild- type (WT) and Ate1 knockout (KO) cultures calculated from the time lapse series similar to the one shown in (A). In wild-type average migration speed was 39.97+/24.90(SEM), n = 2; in the knockout the speed was almost 4 times slower at 11.34+/21.32(SEM), n = 4. doi:10.1371/journal.pgen.1000878.g005 PLoS Genetics | www.plosgenetics.org 7 March 2010 | Volume 6 | Issue 3 | e1000878