Arginylation Regulates Neural Crest Migration similar to the regular Ate1 knockout cells when cultured separately from wild-type (data not shown). After the scratching of the wound the wild-type cells recovered first; even though areas with GFP-expressing cells at the edge were chosen for observation, shortly after the start of the time lapse, wild-type cells moved to the front and continued leading the way for the entire observation period (Figure 7A and Video S5). Ate1 knockout cells moved prominently slower than wild-type, however, unlike in individual cultures, co-cultured Ate1 knockout cells showed a less significant difference in migration speed from the wild-type cells (around 2-fold, Figure 7B) and were able to cover larger distances. This observation suggests that wild-type cells are capable of aiding the knockout cells along to cover larger distances over a period of time. Observations of the time-lapse images (Video S5) suggested that rather than moving, Ate1 knockout cells, when possible, ‘rode’ on the expanding wild-type monolayer, possibly due to their poorer substrate attachment, and that their ability to cover greater distances was likely due to passive, rather than active movement. Reciprocal assay, with GFP-labeled wild- type cells co-cultured with unlabeled knockout cells, confirmed that, independently of the GFP transfection, wild-type cells indeed migrated faster and filled the wound sooner than the knockout (Figure 7C). It should be noted that in the co-culture assay the wild-type cells on average moved somewhat slower than when cultured individually. A possible explanation for this effect could be that in this assay we did not wait for the edge of the monolayer to recover after the scratch wound and the delay may be simply due to the additional time the dislodged cells at the edge needed to re-attach and polarize. Thus, Ate1 knockout in the mesenchymal cells results in greatly delayed migration speeds that originate at the intracellular level and are likely to result in severe morphogenic defects in vivo. Figure 7. Ate1 knockout cells co-cultured with wild-type move with speeds closer to the wild-type cells than when cultured individually. (A) Phase contrast (top), fluorescence (middle), and overlayed (bottom) images of the first and last frame of a 12-hour time lapse series of GFP-labeled Ate1 knockout cells co-cultured with wild-type moving into the wound. Wild-type cells quickly bypass the Ate1 knockout cells initially found at the wound edge and ‘lead’ for the rest of the time lapse, consistent with their faster migration speeds. Ate1 knockout cells lag behind, often ‘riding’ on the wild-type cells rather than moving on their own, however they are able to cover greater distance over the 12 hours than in individual cultures (Figure 5). See Video S5 for the merged time lapse. Bar, 200 mm. (B) Average speeds of the wild-type (WT, unlabeled) and Ate1 knockout (KO, GFP-labeled) cells moving in co-culture show that the difference between the speeds of the two cell types is much less than in individual cultures. Average migration speed of wild-type cells was 24.12+/24.00(SEM), and the knockout 11.29+/22.78(SEM), n = 4. (C) Last frame of a 12-hour time lapse series of GFP-labeled wild-type cells co-cultured with Ate1 knockout cells moving into the wound, overlayed similarly to that shown in (A). doi:10.1371/journal.pgen.1000878.g007 PLoS Genetics | www.plosgenetics.org 9 March 2010 | Volume 6 | Issue 3 | e1000878