CAREER:The Development of Molecular Stress Biosensor to Study Mechanotransduction

[846429]动脉粥样硬化是一种以斑块形成导致动脉管腔狭窄为代表的疾病，是美国和大多数发达国家的主要死亡原因之一。动脉粥样硬化可能发生在血管分支部位，那里的血管壁暴露于血流紊乱，但不会发生在层流占主导地位的血管直部。有证据表明，不同的血流和随之而来的剪切应力在调节血管内皮细胞（ECs）以及随后的内皮通透性和动脉粥样硬化中起关键作用。然而，目前尚不清楚EC表面剪切应力的详细分布，以及EC如何感知这些机械刺激的时空特征来确定病理生理结果。在本提案中，PI将开发一种弹性分子生物传感器，利用荧光共振能量转移（FRET）来可视化活性EC表面的剪切应力分布。一种新的细胞内分子生物传感器也将被开发。因此，细胞外剪切应力和细胞内分子活动的信号都可以在单个活细胞中可视化。本研究提出了三个具体目标：(1)在体外开发和表征基于fret的剪切应力生物传感器；(2)可视化不同流动模式下活体ECs的剪应力分布；(3)同时观察不同流动模式下细胞外剪应力和细胞内分子活动。鉴于剪切应力在内皮通透性和随后的动脉粥样硬化中的重要性，该研究的意义可以为细胞感知机械刺激的时空特性并将其转化为生化/生理信号的分子机制提供新的信息。该提案的主要教育目标是激励高中生和本科生走向未来的职业目标，成为下一代生物工程领域的顶尖科学家。PI将把拟议的研究与教育结合起来，并为本科生组织以活细胞成像、生物纳米技术和机械生物学为特色的讲习班。将特别鼓励少数民族和妇女参加这些讲习班，以便她们了解这些跨学科领域的基本原则和令人兴奋的进展。还将投入大量的精力，把实验室中引人入胜的研究进展带给年轻一代，即高中生。研究实验将被录下来，并通过互联网分发给广大的高中教师和学生。重点将放在如何对细胞的机械和生化特性进行监测、测量、操纵和建模，以帮助我们理解复杂的生物系统。提出的研究的最终目的是帮助开发与机械力有关的疾病的早期诊断和改善治疗的新工具。教育影响是激励下一代学生，特别是代表性不足的群体，参与和促进新兴的生物工程领域，并为大学内外的社会带来广泛的影响。

0846429WangAtherosclerosis, a disease represented by the lumen narrowing of arteries due to plaque formation, is one of the leading causes of death in the United States and most other developed countries. Atherosclerosis occurs likely at vascular branch sites where the vessel walls are exposed to disturbed flow, but not at the straight parts of vessels where laminar flow dominates. Evidence has shown that different flows and the ensuing shear stresses play crucial roles in regulating vascular endothelial cells (ECs), and subsequently endothelium permeability and atherosclerosis. However, it is unclear about the detailed shear stress distribution on EC surface and how ECs sense the spatiotemporal features of these mechanical stimuli to determine patho-physiological outcomes. In this proposal, the PI will develop an elastic molecular biosensor to visualize the shear stress distribution on live EC surfaces, utilizing the fluorescence resonance energy transfer (FRET). A new intracellular molecular biosensor will also be developed. Hence, both the signals of extracellular shear stress and intracellular molecular activity can be visualized in a single live cell. Three specific aims for this career proposal have been proposed: (1) To develop and characterize a FRET-based shear stress biosensor in vitro; (2) To visualize the distribution of shear stress on live ECs under different flow patterns; (3) To visualize extracellular shear stress and intracellular molecular activities simultaneously under different flow patterns. Given the importance of shear stress in endothelium permeability, and subsequently atherosclerosis, the significance of the proposed study can provide new information on the molecular mechanism by which cells sense the spatiotemporal properties of mechanical stimuli and convert them into biochemical/physiological signals. The main educational objective of this proposal is to motivate the high school and undergraduate students toward future career goals as the next generation of leading scientists in bioengineering. The PI will combine the proposed research and education and organize workshops for undergraduate students, featuring live cell imaging, bio-nanotechnology, and mechanobiology. Minorities and women will be particularly encouraged to participate in these workshops so that they are exposed to the fundamental principles and exciting advancement in these interdisciplinary fields. A significant effort will also be invested to bring the fascinating research advancement in the laboratory to the younger generation, i.e. high-school students. Research experiments will be videotaped and distributed through the internet to reach a wide audience of high-school teachers and students. An emphasis will be placed on how the mechanical and biochemical properties of cell can be monitored, measured, manipulated, and modeled to help our understanding of the complex biological system.The ultimate goal of the proposed research is to help the development of new tools for the early diagnosis of diseases related to mechanical force and the improvement of the treatment. The educational impact is to motivate the next generation of students, particularly underrepresented groups, to participate and promote the emerging field of bioengineering, and to bring broad impact to the society within and beyond the university.