CAREER: Mechanics of the Physical-Biological Interface: Mechanotransduction of Endothelial Cells

[238910]血液被心脏的泵送作用强行流过动脉，对血管内皮细胞（ECs）施加剪切应力。ECs对剪切应力的反应是产生影响血管直径、血液凝固、白细胞粘附血管壁以及动脉壁对水、蛋白质和胆固醇的渗漏的物质。因此，研究ec对剪切应力的敏感性将有助于了解ec如何向血管发出信号，以控制血流、免疫反应和向组织输送营养物质。在某些情况下，剪切应力会导致内皮细胞功能失调，从而导致高血压、动脉粥样硬化和其他血管疾病。用于研究ec对剪切应力敏感性的方法包括动物、组织、细胞、细胞成分和分子的研究。负责检测力的分子（脂质和蛋白质）存在于ec膜的特殊结构中，这些结构具有独特的物理特性（它们的脂质成分存在于液体有序（凝胶）相）。一个分子的活动在某种程度上是由它周围分子的动力学来调节的。因此，提出的研究旨在测量流体力对位于这些特殊结构中的分子动力学的影响。为了测量膜子域中单个分子的动力学（例如旋转速率，横向扩散），PI建议结合(i)以非常短的可控脉冲传递的偏振激光，（ii）某些荧光类脂分子对细胞膜特定区域的偏好，以及（iii）光学和快速响应光检测的使用。PI将结合强大的光谱和显微镜工具来研究受剪切应力的单细胞亚细胞膜域的分子动力学。PI假设剪切应力可能扰乱膜微域中的脂质，这反过来改变了在机械转导中重要的膜结合蛋白的动力学。为了验证这一假设，PI提出(i)测量剪切应力和膜修饰对通过三类膜受体的信号传导的影响：外周膜蛋白（g蛋白），细胞骨架连接的整体膜蛋白（整合素）和非细胞骨架连接的整体膜蛋白（受体酪氨酸激酶，flk-1），（ii）使用新型荧光探针测量剪切应力和膜修饰对这些分子旋转和横向动力学的影响，（iii）测量剪切应力对内皮细胞膜相亚结构域影响的时空方面。该提案的核心特征是将这些科学目标与教育目标相结合，将物理、应用、生命和临床科学以及生物伦理学纳入本科生和研究生的教育和培训中。作为整合研究和教育的详细计划的一部分，PI将担任宾夕法尼亚州立大学生物材料和生物纳米技术暑期研究所（PSSI）的联合主任和教育主任，该研究所由NIH和NSF共同资助。

0238910ButlerBlood forced through arteries by the pumping action of the heart exerts shear stresses on the endothelial cells (ECs) that line the arteries. ECs respond to shear stress by producing substances that affect blood vessel diameter, blood coagulation, the adhesion of white blood cells to the vessel wall, and the leakiness of the artery wall to water, proteins and cholesterol. Therefore,studies of EC-sensitivity to shear stress will lead to an understanding of how ECs signal the blood vessel to control blood flow, immune responses, and the delivery of nutrients to the tissues. In certain cases, shear stress can cause ECs to become dysfunctional, a situation which leads to hypertension, atherosclerosis and other vascular diseases. Methodologies used to study EC-sensitivity to shear stress range from studies in animals, tissues, cells, cell components to molecules. Molecules (lipids and proteins)responsible for detecting force reside in specialized structures in the EC-membrane and these structures have unique physical characteristics (their lipid components exist in a liquid-ordered (gel)phase). A molecule's activity is regulated, in part, by the dynamics of molecules around it. Therefore, the proposed research seeks to measure the effects of fluid force on the dynamics of molecules located in these specialized structures. To measure the dynamics (e.g. rotation rate, lateral diffusion) of single molecules in membrane subdomains, the PI proposes to combine (i) polarized laser light delivered at very short, controlled pulses, (ii) the preference of certain fluorescent lipid-like molecules for specialized domains in the cell membrane, and (iii) the use of optics and fast-response light detection. The PI will combine powerful spectroscopic and microscopy tools to investigate molecular dynamics in subcellular membrane domains in single cells subjected to shear stress. The PI hypothesizes that shear stress may perturb lipids in membrane microdomains, which, in turn, alters the dynamics of membrane bound proteins important in mechanotransduction. To test this hypothesis the PI proposes to (i) measure the effects of shear stress and membrane modifications on the signaling through three classes of membrane receptors: peripheral membrane proteins (G-proteins), cytoskeleton-linked integral membrane proteins (integrins) and non-cytoskeleton-linked integral membrane proteins (receptor tyrosine kinase, flk-1),(ii)to measure the effects of shear stress and membrane modification on the rotational and lateral dynamics of these molecules using novel fluorescent probes, and (iii)to measure the spatio-temporal aspects of the effects of shear stress on membrane phase sub-domains in endothelial cells. A central feature of this proposal is to integrate these scientific aims with educational aims to incorporate physical, applied, life, and clinical sciences and Bioethics in the education and training of undergraduate and graduate students. As part of a detailed plan to integrate research and education, the PI will act as co-director and educational director in a Penn State Summer Institute (PSSI)for Biomaterials and Bionanotechnology that is jointly funded by NIH and NSF.