Cell polarity is a fundamental characteristic of many cell types and essential for their function. Several protein complexes mediate cell polarisation one of these being the Scrib/Dlg/Lgl-polarity module. We hypothesized that the polarity protein Scrib also controls directed processes in endothelial cells and is therefore involved in the angiogenic process.

Scrib siRNA blocked directed migration in chemotaxis and transwell assays, whereas random migration in the scratched wound assay was not affected. This effect was accompanied by an increased number and a disturbed orientation of cellular lamellipodia shown by Rac-1 staining. Co-Immunoprecipitation and protein identification by LTQ-Orbitrap-Mass spectroscopy identified an interaction of Scrib with integrin a5. Duolink analysis, a new method detecting single-molecule protein interaction events in situ and TIRF microscopy confirmed an interaction of Scrib and integrin a5 which colocalize in the basal membrane. Western blot and FACS analysis showed that Scrib siRNA reduced protein amount and surface expression of integrin a5 as well as RGD-binding capacity. This is the consequence of reduced integrin a5 recycling and protein stability after Scrib siRNA treatment as determined by a cycloheximide time course. By re-inducing the wild type protein of Scrib using a siRNA-resistant expression plasmid we could also re-induce integrin a5 protein expression, but overexpression of a Scrib mutant missing the LRR domain failed to re-induce integrin a5 expression. Consistent with this finding re-induction of wild type Scrib rescued directed cell migration in the transwell assay whereas the LRR domain-missing mutant did not. Showing that the regulation of integrin a5 signalling by Scrib is involved in the Scrib-mediated effect on directed cell migration, directed cell migration was only inhibited on fibronectin, the ligand for integrin a5 but not collagen, the ligand for other a integrins. In flkGFP zebrafish mutants, Scrib morphants showed a delayed sprouting of the intersegmental vessels and malformations of vessels in the brain.

In conclusion, Scrib represents a new endothelial protein mediating directed migration by modulating integrin a5 turnover and is therefore required for sprouting angiogenesis in vitro and in vivo.