Scribble regulates angiogenic symmetry breaking through control of cortical actomyosin

Introduction: Angiogenesis, the growth of new blood vessels from previously existing vessels, is essential for tissue growth and injury repair. Despite extensive studies, it remains unclear how angiogenesis is spatially and temporally regulated to drive proper neovascularization therapeutically in settings such as tissue ischemia and tissue-engineered organs, or to block vascularization of tumors. Initiation of angiogenic sprouting is a symmetry breaking process across scales: a vascular tube asymmetrically generates a new tube; a polarized endothelial “tip” cell breaks symmetrical adhesive and cytoskeletal organization to migrate towards an angiogenic stimulus. Technical limitations have prevented an understanding of the molecular control systems regulating dynamics of vascular endothelial (VE)-cadherin and associated actomyosin during tip cell symmetry breaking. Herein we describe a novel mechanism by which the polarity protein Scribble (Scrib) controls adherens junction stability through cortical actomyosin mechanics, ultimately gating the emergent multicellular behaviors required for angiogenic symmetry breaking.

Materials and

Methods: To identify unappreciated regulators of adherens junction stability, VE-cadherin interactors were profiled using unbiased proximal biotinylation (BioID) and mass spectrometry (Fig. 1a), revealing significant interaction with the multifunctional scaffold Scrib. Scrib is a critical regulator of epithelial polarity and morphogenesis, typically through stabilizing cadherin-catenin coupling and planar cell polarity. We sought to understand the role of Scrib in endothelial cell (EC) organization and the multicellular process of angiogenesis using 2D and 3D engineered models of primary human vasculature.

We generated primary human microvascular endothelial cells (hMVECs, derived from neonatal dermis) with CRISPR-Cas9-mediated deletion of Scrib (SCRIBKO). The impact of SCRIBKO on angiogenesis was studied using an engineered 3D angiogenesis model (Fig. 1c-e). Mechanistically, we studied the effect of SCRIBKO on adherens junctions and associated actomyosin using immunofluorescence of hMVECs on compliant 2D polyacrylamide gels, which more closely mimic the soft substrate of human tissue. To determine whether Scrib regulates adherens junctions through cadherin-catenin coupling to actin, we generated hMVECs with CRISPR deletion of ɑ catenin (CTNNA1KO) to destabilize coupling. We also generated a VE cadherin-ɑ catenin fusion protein to stabilize VE cadherin – actin coupling in both knockout cell lines. The resulting adherens junction and actomyosin morphologies from these perturbations were compared to that of SCRIBKO. We also examined actin dynamics of SCRIBKO at cell-cell adhesions using a fluorescent actin probe. To study the functional consequence of disrupted actomyosin tension in cells, we employed tension force microscopy (TFM).

Results, Conclusions, and Discussions: We find that Scrib localizes at endothelial adherens junctions (Fig. 1b), and SCRIBKO microvessels demonstrate aberrantly increased angiogenic sprouting (Fig. 1f-g). Upon examining adherens junctions components in SCRIBKO hMVECs, we find decreased phosphorylated myosin light chain (pMLC) and non-muscle myosin IIA (NMIIA) and IIB (NMIIB) at junctions, suggesting a role for Scrib in adherens junction stability (Fig 2a-b). Surprisingly, SCRIBKO hMVECs display disrupted cortical NMIIIB that is not phenocopied in CTNNA1KO (Fig. 2c-d), or rescued by constitutively coupling VE cadherin to actin via a VE cadherin-ɑ catenin fusion protein. This demonstrates Scrib regulates adherens junctions and cortical actomyosin independently of cadherin-catenin coupling to actin. In addition to junctional localization, Scrib localizes to punctate, contractile regions of actomyosin (Fig. 2e), which are disrupted in SCRIBKO and may serve to stabilize retrograde actin flow from cell-cell contacts (Fig. 2f). As a putative consequence of disrupted actomyosin contractility, SCRIBKO cells exert significantly less force across the cell-ECM interface (Fig. 2g-h). Altogether, these results suggest that Scrib maintains mechanical homeostasis within the cell through regulation of cortical actomyosin.

Importantly, Scrib localization shifts from adherens junctions to cytoplasmic actomyosin puncta in response to angiogenic cues, suggesting actomyosin regulation by Scrib is altered in the angiogenic context to permit sprouting. Ongoing work is decoding Scrib molecular interactors and examining this regulatory mechanism during live 3D angiogenic sprouting (Fig 1h). Altogether, we reveal a novel role for Scrib in regulating adherens junctions and endothelial cell mechanics via cortical actomyosin rather than direct effects on the cadherin-catenin complex. This mechanism illustrates important distinctions between endothelial and epithelial junctional architectures and regulatory mechanisms. Scrib regulation of cortical actomyosin is ultimately necessary for endothelial cell organization and coordination required for the earliest stage of angiogenesis, and a deeper understanding of this process may present an opportunity for more precise therapeutic control of vascularization.

Acknowledgements (Optional): This work was generously supported by NHLBI F31HL172606, NIGMS R35GM150987, and the UCSF-UCB Joint Bioengineering Program. The authors would like to thank the members of the Kutys, Barber, and Wittmann laboratories for critical input.