An Optogenetic Toolbox for the Interrogation of Dynamic Events During Angiogenesis

Characterization of highly dynamic processes such as the induction of angiogenesis has historically been limited by a lack of tools with sufficient spatiotemporal control. To address the spatiotemporal requirements for VEGF-induced tip cell development, we designed an optogenetic VEGFR2 utilizing the LightR regulatory system. We demonstrated that LightR regulation of RTKs provides spatiotemporal control over VEGFR2 signaling. Furthermore, we demonstrated that previous VVD modifications conserved their effect in RTK regulation. Despite these successes, LightR-VEGFR2 regulation was perturbed in endothelial cells. These findings highlight the challenges in the creation of LightR-RTKs but also provide a logical foundation for their creation. Another immediate consequence of angiogenic stimulation is rapid alteration of the endothelial barrier. VE-Cadherin is an essential junctional protein involved in the maintenance of the endothelial barrier. While many of the components involved in these processes have been characterized, the dynamic changes in VE cadherin interactome induced by angiogenic stimuli have not been fully established. To address this, we generated an optogenetic biotin proximity ligase termed LightR-TurboID. Fusion of LightR-TurboID to VE-Cadherin resulted in a construct capable of labelling known interacting partners with sub-minute resolution. Furthermore, the stability of biotinylation also enables the capture of weak or transient binders that may dissociate from the VE-Cadherin complex. Proteomics investigations have further supported the specificity of VE-Cadherin-LiTurboID towards endothelial and junctional complexes. We aim to utilize these characteristics to identify novel components of junctional machinery and further develop the short-timescale mechanism of barrier alteration following angiogenic stimulation. Together, these optogenetic tools have expanded our toolbox for the investigation of highly dynamic events with unparalleled spatiotemporal resolution.