Elucidating the role of Scribble in angiogenic symmetry breaking and adherens junction remodeling - PROJECT SUMMARY/ABSTRACT Angiogenesis is crucial for tissue homeostasis and growth. Despite extensive studies, it remains unclear how angiogenesis is spatially and temporally regulated to drive therapeutic neovascularization or conversely to block vascularization of tumors. Initiation of angiogenesis is a symmetry breaking process across scales: at tissue level, a blood vessel must asymmetrically generate a new vessel; at cellular level, a polarized endothelial “tip” cell must break symmetry by remodeling its cell-cell adhesions to initiate migration towards an angiogenic stimulus. This symmetry break is carefully regulated, as a tip cell must maintain sufficient vascular endothelial (VE)-cadherin-based adherens junctions with follower “stalk” cells to generate a continuous neovessel. VE- cadherin remodeling is largely mediated by (1) VE-cadherin endocytosis and recycling and (2) remodeling of junction-associated actomyosin. As these two processes closely regulate each other, it remains unclear what initiates and regulates the break in symmetry that permits tip cell migration. To identify new molecular regulators of VE-cadherin remodeling, VE-cadherin interactors were profiled using unbiased proximal biotinylation (BioID) and mass spectrometry in Preliminary Studies, revealing Scribble (Scrib). Mice with germline Scrib loss in literature exhibit developmental vascular defects; however, the subcellular endothelial role of Scrib in human vascular development remains unclear. Preliminary studies show Scrib localizes at adherens junctions in primary human microvascular endothelial cells, and this localization is regulated by angiogenic stimuli. Scrib knockout 3D organotypic microvessels exhibit aberrantly increased sprouting. Mechanistically, Scrib regulates VE- cadherin turnover and junctional actomyosin. The central hypothesis of this proposal is that Scrib regulates angiogenic symmetry breaking through limiting VE-cadherin remodeling, and this regulation is exerted through stabilization of junctional actomyosin. This hypothesis will be tested by two Aims: (1) Determine how Scrib regulates angiogenesis across endothelial tissue and cell scales, and (2) Elucidate molecular mechanisms by which Scrib regulates VE-cadherin remodeling and endothelial symmetry breaking during angiogenesis. These Aims leverage engineered vascular models, CRISPR-Cas9 genome editing of primary human endothelial cells, and advanced live microscopy to capture unappreciated mechanisms underlying angiogenic initiation. These research goals will be conducted alongside a comprehensive training plan at the University of California, San Francisco, including structured conceptual and technical mentorship from primary sponsor Dr. Matthew Kutys, an expert in endothelial cell-cell adhesions, morphogenesis, and engineered vascular models. Co-sponsor and physician-scientist Dr. Dean Sheppard will provide further conceptual, translational, and career mentorship. The applicant will gain additional training by participation in the Cardiovascular Research Institute at UCSF and structured shadowing experiences with physician-scientists in vascular biology. Altogether this work will provide invaluable training for an aspiring physician-vascular biologist.
