Abstract
Multiple angiogenic cues modulate phosphotyrosine signaling to promote vasculogenesis and angiogenesis. Despite its functional and clinical importance, how vascular cells integrate phosphotyrosine-dependent signaling to elicit cytoskeletal changes required for endothelial morphogenesis remains poorly understood. The family of Nck adaptors couples phosphotyrosine signals with actin dynamics and therefore is well positioned to orchestrate cellular processes required in vascular formation and remodeling. Culture of endothelial cells in three-dimensional collagen matrices in the presence of VEGF stimulation was combined with molecular genetics, optical imaging, and biochemistry to show that Nck-dependent actin remodeling promotes endothelial cell elongation and proper organization of VE-cadherin intercellular junctions. Major morphogenetic defects caused by abrogation of Nck signaling included loss of endothelial apical-basal polarity and impaired lumenization. Time-lapse imaging using a Förster resonance energy transfer biosensor, immunostaining with phospho-specific antibodies, and GST pull-down assays showed that Nck determines spatiotemporal patterns of Cdc42/aPKC activation during endothelial morphogenesis. Our results demonstrate that Nck acts as an important hub integrating angiogenic cues with cytoskeletal changes that enable endothelial apical-basal polarization and lumen formation. These findings point to Nck as an emergent target for effective antiangiogenic therapy.