Wts phosphorylates Ena and represses its activity to polarize F-actin and promote border cell migration. (A) Conserved Wts phosphorylation sites in Yki/YAP and Ena/VASP proteins from different species. (B) Recombinant Wts kinase can directly phosphorylate Ena on its conserved Wts site in vitro. (C) hpo^42-47, ena²¹⁰ double mutant clusters migrate almost normally. (D) Quantification of hpo^42-47 single mutant (n = 62), hpo^42-47, ena²¹⁰ double mutant (n = 50), and ena²¹⁰ single mutant (n = 35) cluster migration at stage 10. The double mutant clusters migrate as well as the ena²¹⁰ single mutant and do not show the strong delays observed in a hpo^42-47 mutant (see Fig 1). (E) Overexpression of Ena causes delayed border cell migration during stage 9, with prominent accumulation of F-actin throughout the border cell cluster (inset). (F) Ena-expressing clusters recover to achieve normal migration at stage 10. Inset shows increased levels of F-actin, but the cytoskeleton is still polarized. (G) Overexpression of EnaS187A phosphomutant causes delayed migration at stage 9 with F-actin accumulation throughout the cluster. (H) EnaS187A-expressing clusters can still be delayed at stage 10. (I) Overexpression of EnaS187D phosphomimic does not delay migration or depolarize F-actin at stage 9. (J) Overexpression of EnaS187D phosphomimic does not delay migration or depolarize F-actin at stage 10. (K) Quantification of migration delay at stage 10 for control (n = 69), UAS.Ena (n = 80), UAS.EnaS187A (n = 102), and UAS.EnaS187D (n = 63). Note all three transgenes are inserted in the same second-chromosomal attP landing site. Bars, 50 µm (5 µm for insets).