Fluid shear stress mechanotransduction at endothelial cell-cell junctions

项目摘要 本项目旨在从分子水平详细了解流体剪切应力如何作用于内皮细胞， 细胞在细胞-细胞连接处触发信号传导途径的机械活化。公布数据 显示剪切应力激活PECAM 1依赖性信号传导途径，Notch信号传导， Alk 1-Endoglin-Smad 1/5信号传导，所有这些都发生在并依赖于细胞-细胞接触。这些 血管通路在胚胎发育、出生后生理和成年中起重要作用 疾病然而，还有很多关于分子机制的知识有待了解。拟议 这项工作是基于我们实验室的两项最新进展。首先，我们最近发现了亲latrophilins （LPHN，也称为ADGRL），粘附G蛋白偶联受体的成员 家族，作为剪切激活所有这三种途径的关键上游介质。第二、 我们已经开发出一种新的纳米装置，利用DNA折纸， 蛋白质的张力目的1：研究（1）LPHNs的分子机制 介导剪切应力对连接信号传导的影响;（2）确定LPHN 2的作用 在血管发育和功能在体内通过做内皮特异性敲除小鼠。 目标2将使用DNA折纸装置对PECAM 1施加规定的张力， 通过cryoEM观察蛋白质构象变化。这些实验将使我们能够确定 施加力对PECAM结构转变的影响。该项目将提供新的 对内皮细胞-细胞连接蛋白 响应于由剪切应力产生的机械力。 .

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. .