Title: Elucidating dynamic changes in the VE-cadherin interactome triggered by pro-angiogenic stimulation

Abstract: Vascular Endothelial Growth Factor (VEGF) is a pro-angiogenic factor and a strong modulator of cell-cell interactions. Under physiological conditions, VEGF mediates the formation of healthy and intact blood vessels. However, in disease states, dysregulation of VEGF signaling is responsible for developing disorganized and leaky vasculature, as observed in tumors. How VEGF leads to both the disruption and development of vasculature is unknown. VE-cadherin is the key component of adhering junctions, multiprotein structures controlling endothelial permeability. Although endothelial adherens junctions have been extensively studied, the dynamic changes in adherens junction protein complexes that follow VEGF stimulation have not been systemically characterized. Current technology makes examining dynamic interactions challenging. Immunoprecipitation is the most common method for dissecting protein interactions, yet it lacks the ability to discern weak interactions and heavily depends on the quality of the specific antibody. Biotin proximity labeling is a powerful technique that overcomes many limitations of immunoprecipitation and provides high sensitivity even for weak interactions. However, the existing proximity labeling tools lack temporal resolution and do not allow for the detection of dynamic changes in protein interactions. Thus, to examine the dynamic effects of VEGF stimulation on VE-cadherin interactions, traditional methods are not applicable. To address this issue, we have developed a light-regulated biotin ligase, LightR-TurboID, that provides high sensitivity and temporal resolution. We aim to use LightR-TurboID to examine VEGF-induced changes in the VE-cadherin interactome. Specifically, how does VEGF stimulation disrupt and amend barrier function, and how is this balance between leakiness and angiogenesis achieved? We hypothesize that there are previously unrecorded dynamic changes in the VE-cadherin interaction following VEGF stimulation occurring on a short time scale. Using our VE-cadherin-LightR-TurboID tool, we can determine what dynamic interactions are occurring and how they are affecting barrier integrity.