Single molecule force spectroscopy analysis of PECAM-1 mechanotransduction

PECAM-1 机械力转导的单分子力谱分析

基本信息

批准号：
8282428
负责人：
ROBERT L CLARK
金额：
$ 18.22万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-16 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8282428
关键词：
Actins Address Affect Arterial Fatty Streak Atherosclerosis Atomic Force Microscopy Binding Biochemical Biochemical Pathway Biotechnology Biotin Blood Vessels Blood flow CD31 Antigens Cardiovascular Diseases Cattle Cell Line Cell Surface Receptors Cell surface Cells Characteristics Chemicals Coupled Coupling Cysteine Cytoplasmic Tail Cytoskeleton Endothelial Cells Energy Transfer Environment Escherichia coli Event Extracellular Domain Fluorescence Gene Expression Imagery In Vitro Ligase Link Liquid substance MAP Kinase Gene Maleimides Measurement Measures Mechanics Molecular Biology Molecular Conformation Monitor Morphology Nickel PTPN11 gene Phosphorylation Phosphotyrosine Plasmids Preparation Process Protein Kinase Protein Tyrosine Phosphatase Proteins Recombinants Research Scanning Probe Microscopes Signal Pathway Signal Transduction Site Spectrum Analysis Stimulus Streptavidin Stretching Structure System Techniques Translating Tyrosine cantilever cell growth design experience fluid flow fluorophore in vivo interest receptor research study shear stress single molecule src Homology Region 2 Domain time use

项目摘要

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.

描述（由申请人提供）：细胞通过称为机械转移的过程对环境中的物理力响应。细胞表面上的机械传导分子识别物理力并传递内部生化信号，该信号可能影响细胞生长，基因表达等。内皮细胞（ECS）或衬有血管的细胞，可以感觉到血液流动引起的剪切应力。在高剪切应力的区域中，细胞拉长并与流动方向排列。但是，在低剪切应力或流动干扰的区域中，EC没有伸长且定向的形态。这些低或干扰流的区域易受动脉粥样硬化病变的形成。因此，对EC的机械转导的研究将有助于我们理解动脉粥样硬化和心血管疾病。暴露于流体流或拉伸EC的EC进行的实验表明，血小板内皮细胞粘附分子1（PECAM-1）的细胞质结构域被蛋白激酶FYN磷酸化。 SHP-2是一种蛋白质酪氨酸磷酸酶，沿ERK/MAPK生化途径传播信号，最终改变了EC的生长和比对。假设PECAM-1的物理拉伸会散发细胞质结构域，并暴露于磷酸化的区域。拟议的研究将对PECAM-1通过三个目标对身体力量做出反应的反应有所了解。在AIM 1中，将通过分子生物学和生物技术技术产生由PECAM-1的细胞质结构域组成的构造。在AIM 2中，将使用单分子力光谱技术测量构建体的物理特性。为了执行这些测量，PECAM-1构建体将用原子力显微镜（AFM）伸长，并将测量所得的力。最后，在AIM 3中，PECAM-1构建体将用AFM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。公共卫生相关性：在流体流动障碍的血管区域的动脉粥样硬化斑块。该项目将解决细胞识别物理力（如流体流量）的方式，并将其作为生化信号传递。该研究将重点放在血小板内皮细胞粘附分子-1（PECAM-1）上，这是一种参与血管上的细胞机械信号传导并与形成斑块相关的细胞中的分子。