Syx, a RhoA Guanine Exchange Factor, Is Essential for Angiogenesis In Vivo

Abstract

Rho GTPases play an important and versatile role in several biological processes. In this study, we identified the zebrafish ortholog of the mammalian Rho A guanine exchange factor (GEF), Synectin-binding GEF (Syx), and determined its in vivo function in the zebrafish and the mouse. We found that Syx is expressed specifically in the vasculature of these organisms. Loss-of-function studies in the zebrafish and mouse point to a specific role for Syx in angiogenic sprouting in the developing vascular bed. Importantly, vasculogenesis and angioblast differentiation steps were unaffected in syx-knockdown (KD) zebrafish embryos, and the vascular sprouting defects were partially rescued by the mouse ortholog. Syx KD in vitro impairs vascular endothelial growth factor-A (VEGF-A) induced endothelial cell migration and angiogenesis. We have also uncovered a potential mechanism of endothelial sprout guidance in which Angiomotin (Amot), a component of endothelial cell junctions, plays an additive role with Syx in directing endothelial sprouts. These results identify Syx as an essential contributor to angiogenesis in vivo.

Rab13-dependent Trafficking of RhoA Is Required for Directional Migration and Angiogenesis

“…Both cell types migrated in response to a uniform VEGF-A 164 concentration, although the distances traveled by syx Ϫ/Ϫ ECs were shorter than those traveled by syx ϩ/ϩ ECs (Fig. 1B), similar to our previous observations (19). The responses of the two cell types to a VEGF-A 164 gradient were drastically different from each other: syx ϩ/ϩ EC followed the direction of the gradient, whereas syx Ϫ/Ϫ ECs failed to respond in a directional manner (Fig.…”

“…The lengths of the ISVs and the shape of their tips were variable (right-hand panels in Fig. 6A), unlike the growth-arrested ISVs in syx and amot morphants that ended uniformly in a round tip (19,47). These observations conform to a scenario in which the loss of directional migration caused by Rab13 depletion randomizes the shape of the tips cells of sprouting ISVs.…”

“…However, the functional versatility of these proteins and their alternate localization at cell junctions and at the leading edge are probably essential for conferring the dynamic cell remodeling inherent to growing vessels, where the same cell regions may alternate between being a leading edge and forming junctions. Amot and Syx had already been shown to be required for vascular development in the mouse and zebrafish (19,47).…”

“…6 We had found earlier that Syx was localized to the leading edge of migrating ECs and that it was required for EC migration (17,19). Syx associates through the PDZ adaptor protein synectin with myosin VI (17), an actin-based molecular motor involved in membrane traffic (30).…”

“…Syx binds via this motif the PDZ domain of the adaptor protein synectin (named alternatively Gipc1) (17) and PDZ domains 10 and 13 of multi-PDZ domain protein 1 (Mupp1) (18). Syx is required for cell migration (17), for angiogenesis in the zebrafish and the mouse (19), and for the regulation of cell junctions. 6 Here we report that the functional duality of Syx and RhoA as regulators of both cell junctions and migration is conferred by their cotrafficking from EC junctions to the leading edge.…”