The Amot/Patj/Syx signalling complex spatially controls RhoA GTPase activity in migrating endothelial cells

Controlled regulation of Rho GTPase activity is an essential component mediating growth factor stimulated migration. We have previously shown that angiomotin (Amot), a membrane associated scaffold protein, plays a critical role during vascular patterning and endothelial migration during embryogenesis. However, the signaling pathways by which Amot controls directional migration are not known. Here we have employed peptide pull-down and yeast two-hybrid (Y2H) screening to identify proteins that interact with the C-terminal PDZ-binding motifs of Amot and its related proteins AmotL1 and 2. We report that Amot and its related proteins bind to the RhoA GTPase Exchange Factor (RhoGEF) protein Syx. We show that Amot forms a ternary complex together with Patj (or its paralogue Mupp1) and Syx. By using FRET analysis we provide evidence that Amot controls targeting of RhoA activity to lamellipodia in vitro. We also report that, similar to Amot, morpholino knock down of Syx in zebrafish results in inhibition of migration of inter-segmental arteries. Taken together, our results indicate that the directional migration of capillaries in the embryo is governed by the Amot:Patj/Mupp1:Syx signaling that control local GTPase activity.