Single molecule force spectroscopy analysis of PECAM-1 mechanotransduction

PECAM-1 机械力转导的单分子力谱分析

• 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
• 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

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导的过程对其环境中的物理力做出反应。 细胞表面的力转导分子识别物理力并传递内部生化信号，从而影响细胞生长、基因表达等。内皮细胞 (EC) 或血管内壁细胞可以感知血流引起的剪切应力。在高剪切应力区域，细胞伸长并与流动方向对齐。然而，在低剪切应力或扰动流动的区域，EC 不具有拉长和定向的形态。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，EC 中机械传导的研究将有助于我们了解动脉粥样硬化和心血管疾病。对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 的物理拉伸会解开细胞质结构域并暴露磷酸化区域。拟议的研究将通过三个目标加深对 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生产由 PECAM-1 细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构建体的物理特性。为了进行这些测量，将使用原子力显微镜 (AFM) 拉长 PECAM-1 结构，并测量合力。最后，在目标 3 中，将使用 AFM 拉伸 PECAM-1 结构，同时使用荧光实时测量信号传播事件。这将允许确定执行 PECAM-1 机械转导所需的力。 公众健康相关性：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）的方式，并将力作为生化信号传输。该研究将重点关注血小板内皮细胞粘附分子-1 (PECAM-1)，这种分子参与血管内壁细胞的机械信号传导，并与斑块的形成有关。