The cytoskeletal mechanisms of cell-cell junction formation in endothelial cells

Intercellular adhesions are essential for compartmentalization and integrity of tissues in an organism, cell-cell communication, and morphogenesis. The actin cytoskeleton and associated proteins play a vital role in establishing and maintaining cell-cell adhesion. However, the procedure by which cells establish adherens junctions remains largely unclear. We investigated the dynamics of cell-cell junction formation and the corresponding architecture of the underlying cytoskeleton in cultured human umbilical vein endothelial cells (HUVECs). We show that the initial interaction between cells is mediated by protruding lamellipodia. Upon their retraction, cells maintain contact through thin bridges formed by filopodia-like protrusions connected by VE-cadherin-rich junctions. Bridges share multiple features with conventional filopodia, such as an internal actin bundle associated with fascin along the length and VASP at the tip. Strikingly, unlike conventional filopodia, transformation of actin organization from the lamellipodial network to filopodial bundle during bridge formation occurs in a proximal-to-distal direction and is accompanied by recruitment of fascin in the same direction. Subsequently, bridge bundles recruit nonmuscle myosin II and mature into stress fibers. Myosin II activity was important for bridge formation and accumulation of VE-cadherin in nascent adherens junctions. Our data reveal a mechanism of cell-cell junction formation in endothelial cells utilizing lamellipodia as the initial protrusive contact, subsequently transforming into filopodia-like bridges connected through adherens junctions. Moreover, a novel lamellipodia-to-filopodia transition is employed in this context