Andrei Karginov, PhD

2023-24

Mentors: Andrei Karginov, PhD and Andrius Kazlauskas, PhD 

VEGFR2 is an essential endothelial receptor involved in the induction of angiogenic responses. Deregulation of this receptor results in many diseases and its total knockout is embryonic lethal. The current model indicates that upon VEGFR2 activation a single endothelial cell outcompetes the others to form a tip cell which begins to extend towards the gradient of VEGF. Additional tip cell formation is stopped through lateral inhibition of surrounding cells, which subsequently develop into stalk cells that form the initial structure of the new vessel. This tip cell selection is the first essential step in angiogenesis, but how a particular cell is selected to become a tip cell remains unknown. To address this question, a tool exhibiting tight spatiotemporal regulation of VEGFR2 is required. However, current tools used in the study of VEGFR2 function do not provide sufficient spatiotemporal control of its signaling. Thus, we have developed an optogenetically controlled VEGFR2 which provides spatiotemporal control over its activation. This was accomplished through the insertion of a light-regulatable (LightR) clamp created from two photosensitive VVD proteins connected by a linker. In the dark, the VVD monomers remain far apart and upon illumination with blue light they homodimerize which brings the ends of the clamp together. The LightR clamp was inserted into the catalytic domain of VEGFR2 at a specific allosteric site. In the dark, catalytic activity is disrupted due to distortion imparted by the clamp. Upon illumination with blue light the structure is reestablished, restoring catalytic activity. This approach enables control over VEGFR2 signaling in living cells. Light-induced stimulation of LightR-VEGFR2 leads to trans-phosphorylation of the receptor which is a hallmark of VEGFR2 activation and essential to its further activity. Furthermore, LightR-VEGFR2 induces downstream signaling through canonical VEGFR2 pathways. The kinetics of this signaling mimic those seen in the wild-type enzyme and can be further modulated by mutations within the LightR domain. Activation of LightR-VEGFR2 in endothelial cells stimulates their invasion into three-dimensional collagen matrix, closely mimicking physiologically relevant angiogenic sprouting mediated by wild type VEGFR2. These data indicate that LightR-VEGFR2 can be used as a direct analogue to VEGFR2 to probe the molecular events that occur when endothelial cells are defined as tip cells. These findings will help to further our understanding of the early stages of angiogenesis.