FLIM and FRET for the Visualization of Flow-induced Molecular Hierarchies

FLIM 和 FRET 用于流诱导分子层次结构的可视化

• 批准号: 8840314
• 负责人: Yingxiao Wang
• 金额: $ 10.98万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8840314
• 关键词: Affect; Atherosclerosis; Biology; Biosensor; Blood Vessels; Books; Cardiovascular Diseases; Cardiovascular Physiology; Cell membrane; Cell physiology; Cells; Color; Coupling; Educational workshop; Endothelial Cells; Equipment; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Goals; Health; Illinois; Image; Image Analysis; Imagery; In Vitro; Knowledge; Laboratories; Learning; Life; Mammalian Cell; Maps; Marine Biology; Measurement; Measures; Mechanical Stimulation; Mechanics; Membrane; Membrane Microdomains; Microscopy; Molecular; Monitor; Morphologic artifacts; National Heart, Lung, and Blood Institute; Optics; PTK2 gene; Pattern; Personal Satisfaction; Physics; Physiological; Physiological Processes; Play; Problem Solving; Public Health; Reagent; Research; Research Activity; Resolution; Role; Signal Transduction; Specificity; Spectrum Analysis; Structure; Technology; Text; Thrombosis; Training; Training Programs; Treatment Efficacy; Tyrosine; Universities; Virginia; Wood material; base; career development; cellular imaging; design; experience; fluorescence imaging; improved; inhibitor/antagonist; insight; instrument; journal article; light microscopy; meetings; new technology; novel; optical imaging; programs; reconstruction; restenosis; shear stress; spatiotemporal; success; symposium; technology development; tool

DESCRIPTION (provided by applicant): The Integration of FLIM and FRET for the Visualization of Flow-induced Molecular Hierarchies The research activity of the PI's laboratory is focused on the integration of cutting edge technologies for the elucidation of molecular mechanism in vascular biology, with the ultimate goal of enhancing our knowledge on cardiovascular functions and contributing to the health and well-being of humankind. In particular, novel imaging biosensors and technologies will be developed and employed to visualize the signaling transduction with high spatiotemporal resolutions in live cells under different mechanical forces, e.g. shear stress. In the long run, we aim to apply the knowledge and insights gained via these novel technologies for the development of new strategies and reagents to treat cardiovascular diseases, including thrombosis and atherosclerosis. Along this line, we proposed to apply biosensors based on fluorescence resonance energy transfer (FRET) to visualize the Src activity and its related molecular hierarchy at sub-cellular levels during mechanotransduction, which is funded by NHLBI (R01HL098472). Since FAK activity and its autophosphorylation at tyrosine 397 are critical for the Src activation, we plan to investigate the spatiotemporal coupling between FAK and Src activations in endothelial cells under mechanical stimulation. Recently, we have developed a new FRET pair, mOrange2 and mCherry, which contains colors distinct from the popular FRET pair, CFP and YFP. As such, two different molecular events, such as the activities of FAK and Src, can be simultaneously monitored in the same live cell with biosensors based on mOrange2/mCherry and CFP/YFP. However, there is a certain overlap between the excitation spectra of mOrange2 and mCherry to cause a non-specific cross- excitation of mCherry, which can cause artifacts and lower the sensitivity of FRET biosensors. Fluorescence lifetime imaging microscopy (FLIM) could help to solve this problem because the emission lifetime measurement of the donor mOrange2 alone is sufficient to deduce the FRET efficiency, without the need to measure the emission lifetime of the acceptor mCherry. The main objective of the current proposal is hence for the PI to obtain in-depth training in optics and fluorescence, particularly FLIM and its associated instruments and imaging analysis. Part of the training requires the PI to attend formal courses and training programs whereas the other aspects of training involve more self-studying and interactions with collaborators. Accordingly, three specific aims are proposed for the research plan: (1) develop a mOrange2/mCherry-based FAK biosensor and characterize it by FLIM; (2) visualize the spatiotemporal map of FAK activity under different flows with FLIM; (3) simultaneously monitor FAK and Src activities under different flows with FLIM. The information obtained will significantly advance our systematic understanding of the molecular mechanism by which different flows affect cardiovascular diseases, such as atherosclerosis and restenosis. The newly developed biosensors will also provide powerful tools for detecting cardiovascular diseases and assessing the efficacy of therapeutic inhibitors.

描述（由申请人提供）：PI实验室的研究活动集中在整合尖端技术来阐明血管生物学中的分子机制，最终目标是提高我们对心血管功能的认识，为人类的健康和福祉做出贡献。特别是，新的成像生物传感器和技术将被开发和应用，以高时空分辨率可视化信号转导在不同的机械力，如剪切应力下的活细胞。从长远来看，我们的目标是将通过这些新技术获得的知识和见解应用于开发治疗心血管疾病（包括血栓和动脉粥样硬化）的新策略和试剂。在此基础上，我们提出应用基于荧光共振能量转移（FRET）的生物传感器在机械转导过程中观察Src活性及其相关分子层次，该研究由NHLBI资助（R01HL098472）。由于FAK活性及其酪氨酸397位点的自磷酸化对Src激活至关重要，我们计划研究机械刺激下内皮细胞中FAK和Src激活之间的时空耦合。最近，我们开发了一种新的FRET对，mOrange2和mCherry，其中包含不同于流行的FRET对，CFP和YFP。因此，基于mOrange2/mCherry和CFP/YFP的生物传感器可以在同一个活细胞中同时监测两种不同的分子事件，如FAK和Src的活性。然而，mOrange2和mCherry的激发光谱之间存在一定的重叠，导致mCherry的非特异性交叉激发，这可能导致伪影，降低FRET生物传感器的灵敏度。荧光寿命成像显微镜（FLIM）可以帮助解决这一问题，因为仅测量供体mOrange2的发射寿命就足以推断FRET效率，而无需测量受体mCherry的发射寿命。因此，目前提案的主要目标是让PI获得光学和荧光方面的深入培训，特别是FLIM及其相关仪器和成像分析方面的培训。部分培训要求PI参加正式课程和培训项目，而培训的其他方面则涉及更多的自学和与合作者的互动。为此，提出了研究计划的三个具体目标：(1)开发基于mOrange2/ mcherry的FAK生物传感器并通过FLIM对其进行表征；(2)利用FLIM可视化不同流量下FAK活动的时空图；(3)利用FLIM同时监测不同流量下FAK和Src的活动。所获得的信息将大大促进我们对不同血流影响心血管疾病（如动脉粥样硬化和再狭窄）的分子机制的系统理解。新开发的生物传感器还将为检测心血管疾病和评估治疗抑制剂的功效提供强大的工具。