Single molecule force spectroscopy analysis of PECAM-1 mechanotransduction

描述（由申请人提供）：细胞通过称为机械转导的过程对环境中的物理力做出反应。 细胞表面上的机械力转导分子识别物理力并传递可影响细胞生长、基因表达等的内部生化信号。内皮细胞（EC）或衬在血管上的细胞可感知由血流诱导的剪切应力。在高剪切应力的区域中，细胞伸长并与流动方向对齐。然而，在低剪切应力或扰动流的区域中，EC不具有细长和定向的形态。这些低血流或血流紊乱的区域容易形成动脉粥样硬化病变。因此，对内皮细胞力学信号转导的研究将有助于我们对动脉粥样硬化和心血管疾病的认识。用暴露于流体流动或拉伸的EC的实验已经表明，血小板内皮细胞粘附分子-1（PECAM-1）的胞质结构域被蛋白激酶Fyn磷酸化。SHP-2是一种蛋白酪氨酸磷酸酶，沿着ERK/MAPK生化途径传播信号，最终改变EC的生长和排列。据推测，PECAM-1的物理拉伸解开了胞质结构域并暴露了磷酸化的区域。拟议的研究将通过三个目标建立对PECAM-1如何响应物理力的理解。在目标1中，将通过分子生物学和生物技术产生由PECAM-1的胞质结构域组成的构建体。在目标2中，将使用单分子力谱技术测量结构的物理特性。为了进行这些测量，将用原子力显微镜（AFM）伸长PECAM-1结构，并测量合力。最后，在目标3中，PECAM-1构建体将用AFM拉伸，同时信号传播事件将使用荧光以真实的时间测量。这将允许确定进行PECAM-1机械转导所需的力。 公共卫生相关性：动脉粥样硬化斑块形成于流体流动受到干扰的血管区域。该项目将解决细胞识别物理力（如流体流动）并将力作为生化信号传输的方式。这项研究将集中在血小板内皮细胞粘附分子-1（PECAM-1）上，这是一种参与血管细胞中机械信号传导并与斑块形成有关的分子。

DESCRIPTION (provided by applicant): Cells respond to physical forces in their environment through a process called mechanotransduction. Mechanotransduction molecules on the cell surface recognize physical forces and transmit an internal biochemical signal that can affect cell growth, gene expression, etc. Endothelial cells (ECs), or the cells lining the blood vessels, can sense shear stress induced by the blood flow. In regions of high shear stress, the cells elongate and align with the direction of the flow. However, in regions of low shear stress or disturbed flow, the ECs do not have an elongated and oriented morphology. These regions of low or disturbed flow are susceptible to the formation of atherosclerotic lesions. Therefore the study of mechanotransduction in ECs will aid in our understanding of atherosclerosis and cardiovascular disease. Experiments with ECs exposed to fluid flow or stretched ECs have shown that cytoplasmic domain of platelet endothelial cell adhesion molecule-1 (PECAM-1) is phosphorylated by the protein kinase Fyn. SHP-2, a protein tyrosine phosphatase, propagates the signal along the ERK/MAPK biochemical pathway, eventually altering the EC growth and alignment. It is hypothesized that physical stretching of PECAM-1 unravels the cytoplasmic domain and exposes the region that is phosphorylated. The proposed research will build an understanding of how PECAM-1 responds to physical forces through three aims. In Aim 1, a construct consisting of the cytoplasmic domain of PECAM-1 will be produced through molecular biology and biotechnology techniques. In Aim 2, the physical characteristics of the construct will be measured using single molecule force spectroscopy techniques. To perform these measurements, the PECAM-1 construct will be elongated with an atomic force microscope (AFM), and the resultant forces will be measured. Finally, in Aim 3, the PECAM-1 construct will be stretched with the AFM while the signal propagation event will be measured in real time using fluorescence. This will allow the determination of the forces required to perform PECAM-1 mechanotransduction. PUBLIC HEALTH RELEVANCE: Atherosclerotic plaques form in the region of blood vessels that experience disturbed fluid flow. This project will address the manner in which cells recognize physical forces, like fluid flow, and transmit the force as a biochemical signal. The research will focus on platelet endothelial cell adhesion molecule-1 (PECAM-1), a molecule that is involved in mechanical signaling in the cells that line the blood vessels and implicated in the formation of plaques.