Project Summary This project aims to understand in molecular detail how fluid shear stress acting on endothelial cells triggers mechanical activation of signaling pathways at cell-cell junctions. Published data show that shear stress activates a PECAM1-dependent signaling pathway, Notch signaling and Alk1-Endoglin-Smad1/5 signaling, all of which occur at and depend on cell-cell contacts. These pathways play major roles in vascular embryonic development, postnatal physiology and adult disease. However, much remains to be learned about molecular mechanisms. The proposed work is based on two recent advances in our labs. First, we have recently identified latrophilins (LPHNs, also known as ADGRLs), members of the adhesion G protein coupled receptors family, as key upstream mediators of shear activation of all three of these pathways. Second, we have developed a new nanodevice that utilizes DNA origami to apply defined mechanical tension to proteins. Aim 1 will investigate (1) the molecular mechanisms by which LPHNs mediate the effects of shear stress on junctional signaling and (2) determine the role of LPHN2 in vascular development and function in vivo by doing endothelial-specific knockout in mice. Aim 2 will use the DNA origami device to apply defined tension to PECAM1 and visualize protein conformation change via cryoEM. These experiments will allow us to determine the effect of applied force on PECAM’s structural transitions. Together, the project will provide new understanding at unprecedented depth concerning how endothelial cell-cell junctional proteins respond to mechanical force generated by shear stress. .