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

心脏的泵送作用迫使血液通过动脉，对排列在动脉上的内皮细胞(ECs)施加剪应力。内皮细胞通过产生影响血管直径、血液凝固、白血球与血管壁的粘连以及动脉壁对水、蛋白质和胆固醇的渗漏的物质来响应剪切力。因此，研究内皮细胞对切应力的敏感性将有助于理解内皮细胞如何向血管发出信号来控制血流、免疫反应和营养物质向组织的输送。在某些情况下，剪切力会导致内皮细胞功能障碍，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(脂类和蛋白质)驻留在EC膜的特殊结构中，这些结构具有独特的物理特性(它们的脂类成分以液体有序(凝胶)相存在)。分子的活动在一定程度上受到其周围分子动态的调节。因此，这项拟议的研究试图测量流体力对位于这些特殊结构中的分子动力学的影响。为了测量膜亚区中单分子的动力学(例如，旋转速度、横向扩散)，PI建议结合(I)在非常短的受控脉冲下发射的偏振激光，(Ii)某些荧光类脂分子对细胞膜中特定区域的偏好，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单细胞在剪应力作用下亚细胞膜域的分子动力学。PI假设剪切力可能扰乱膜微区中的脂类，进而改变在机械转导中重要的膜结合蛋白的动力学。为了验证这一假说，PI建议：(I)测量剪切力和膜修饰对通过三类膜受体：外周膜蛋白(G蛋白)、细胞骨架连接的整膜蛋白(整合素)和非细胞骨架连接的整膜蛋白(受体酪氨酸激酶，Flk-1)对信号的影响；(Ii)使用新型荧光探针测量剪切力和膜修饰对这些分子旋转和侧向动力学的影响；以及(Iii)测量剪切力对内皮细胞膜相亚结构域影响的时空方面。这项建议的一个主要特点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。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夏季研究所(PSSI)的联席主任和教育主任，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.