Single molecule force spectroscopy analysis of PECAM-1 mechanotransduction

描述(由申请人提供)：细胞通过一种称为机械转导的过程对其环境中的物理力量做出反应。细胞表面的机械转导分子识别物理力，并传递内部生化信号，影响细胞生长、基因表达等。内皮细胞(ECs)或血管衬里的细胞可以感知血流诱导的剪应力。在高剪应力区域，细胞伸长，并与流动的方向一致。然而，在低剪应力或扰动流动的区域，内皮细胞不具有拉长和取向的形态。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，研究内皮细胞的机械转导有助于我们对动脉粥样硬化和心血管疾病的理解。对暴露于流体或拉伸内皮细胞的内皮细胞的实验表明，血小板内皮细胞黏附分子-1(PECAM-1)的细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，它沿着ERK/MAPK生化途径传递信号，最终改变EC的生长和排列。据推测，PECAM-1的物理拉伸解开了细胞质结构域，暴露了被磷酸化的区域。这项拟议的研究将建立对PECAM-1如何通过三个目标响应物理力量的理解。在目标1中，将通过分子生物学和生物技术产生由PECAM-1的细胞质结构域组成的结构。在目标2中，将使用单分子力谱技术来测量该结构的物理特性。为了进行这些测量，将用原子力显微镜(AFM)拉长PECAM-1结构，并测量合力。最后，在目标3中，PECAM-1结构将用原子力显微镜拉伸，而信号传播事件将使用荧光实时测量。这将允许确定执行PECAM-1机械转导所需的力。

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.