Franco, Cláudio AreiasCarvalho, Joana Cristina Reino2021-06-092021-06-092019-112017-12http://hdl.handle.net/10451/48450Collective cell migration is a widespread biologic process that is crucial not only during homeostasis, but also in the context of disease. Mechanistically, it is based on the establishment of a front-rear polarity axis, and on the maintenance and dynamic regulation of cell-cell adhesions, which are crucial for traction force generation and oriented cell movements. Sprouting angiogenesis, a biologic process that enables the expansion of the vascular network, relies on the collective migration of specialized strands of endothelial cells composed by tip and stalk cells. Wnt5a, a non-canonical Wnt ligand, has been described as a pro-angiogenic factor that promotes cell proliferation, migration and network formation. More recently, it was shown to be crucial during sprouting angiogenesis. Indeed, genetic deletion of Wnt5a in endothelial cells reduces the expansion of the vascular plexus in the mouse retina. However, it is still unclear how Wnt5a exerts these functions in the endothelium and which specific molecular components could be involved. Therefore, to investigate these questions, we used the in vitro wound-healing assay to study endothelial collective cell behavior. With this system we uncovered that loss of Wnt5a leads to randomized follower cell polarity and migration, dramatically impairing coordination of collective cell behavior. Moreover, we also showed that cells lacking Wnt5a had a significant decrease in cell-cell force transmission and displayed a specific downregulation of vinculin co-localization to VE-cadherin at cell junctions. Then, we showed that randomized cell polarity in Wnt5a-depleted cells could be rescued by forcing the association of vinculin to cell junctions by overexpressing the αCat-Vinc fusion protein, or a constitutive active form of vinculin (Vinc-T12). Mechanistically, we identified ROR2/Cdc42 as the most relevant transducers in the signaling cascade triggered by Wnt5a. Altogether, we propose a model where Wnt5a, via ROR2/Cdc42, promotes vinculin and Arp2/3 association to adherens junctions, enhancing actin polymerization and junction stabilization. These strengthened adherens junctions would then act as a platform to enable stable force transmission between adjacent cells and coordination of endothelial cell behaviors during collective migration. Given its implications in evolutionarily conserved morphogenetic processes that require cell-cell mechanocoupling, we believe the mechanotransduction function of Wnt5a we described here may be applicable to other tissues and contexts. In the context of vascular biology, our findings may be relevant to understand the mechanical and molecular mechanisms involved in the collective migration of vessel sprouts driven by tip cells in vivo. Moreover, as Wnt5a has been described as a potential key regulator of cancer, our results might be relevant to understand disease onset and progression and even contribute to the development of potential therapies.engAngiogenesisCollective cell migrationAdherens junctionsMechanotransductionNon-canonical Wnt signalingTeses de doutoramento - 2019doctoral thesis101460740