Spectral/FLIM strategies for FRET based analysis of signal transduction pathways

基于 FRET 的信号转导途径分析的光谱/FLIM 策略

• 批准号: 7817157
• 负责人: Patricia J Keely
• 金额: $ 21.67万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7817157
• 关键词: Algorithms; Anisotropy; Behavior; Binding; Biochemical; Cell Polarity; Cell membrane; Cell physiology; Cells; Complex; Computer software; Data Analyses; Data Set; Development; Diffusion; Dominant-Negative Mutation; Elements; Energy Transfer; Engineering; Family; Fluorescence; Fluorescence Polarization; Fluorescent Probes; Freedom; Future; Guanosine Triphosphate Phosphohydrolases; Guidelines; Homo; Image; Knowledge; Label; Length; Life; Location; Malignant Neoplasms; Maps; Measurement; Measures; Methods; Microscopy; Mitosis; Modeling; Molecular; Monomeric GTP-Binding Proteins; Multiphoton Fluorescence Microscopy; Nuclear Import; Optics; Organelles; Organism; Pathway interactions; Proteins; Regulation; Reproducibility; Research Project Grants; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Simulate; Spatial Distribution; Stimulus; Techniques; Testing; Tissues; base; cell motility; fluorophore; in vivo; malignant breast neoplasm; novel; public health relevance; response; rho; rho GTP-Binding Proteins; tool; trafficking

DESCRIPTION (provided by applicant): To understand how metazoan cells function in the context of the tissue in which they reside requires an understanding of the myriad signal transduction pathways that operate both within and between cells. Classical biochemical techniques have revealed many of the signaling elements and their mode of action within a pathway; however, such techniques are unable to visualize the activation state of a signaling pathway in vivo. Recently, the combined use of advanced optical techniques, used in conjunction with engineered fluorescent protein probes, have opened up the possibility of directly visualizing the activation state of a signaling pathway. We propose to develop a generally applicable toolkit for studying the activation of Rho-family GTPase pathways in the context of intact tissues or organisms. The tools consist of a set of optimized fluorescent probes for visualizing when a GTPase becomes activated, when it binds to a cognate effector and how a signaling complex may be formed. Binding is detected optically with multiphoton microscopy by fluorescence lifetime analysis to reveal the presence of Fvrster resonant energy transfer (FRET). Analysis algorithms for obtaining spatial lifetime maps indicating localized regions of signal activation will be generated and refined. Novel techniques using polarization anisotropy decay measurements for detecting associations of GTPases upon activation and also the formation of large signaling complexes will be developed. We anticipate that the methods we are proposing will facilitate the application and reproducibility of FRET measurements for many in vivo signaling studies. PUBLIC HEALTH RELEVANCE: The development of robust FRET-based advanced imaging approaches could enhance considerably our knowledge of inter- and intra-cellular signaling particularly in regards to cancers such as breast cancer. FRET-based methods could help elucidate underlying mechanisms behind cancer onset and progression and help identify future targets for treatment.

描述（由申请人提供）：为了了解后生动物细胞在其所在组织环境中如何发挥作用，需要了解在细胞内和细胞间运作的无数信号转导途径。经典生化技术已经揭示了许多信号传导元件及其在通路内的作用模式；然而，此类技术无法可视化体内信号通路的激活状态。最近，先进光学技术与工程荧光蛋白探针的结合使用，开辟了直接可视化信号通路激活状态的可能性。我们建议开发一个普遍适用的工具包，用于研究完整组织或生物体中 Rho 家族 GTP 酶途径的激活。这些工具由一组优化的荧光探针组成，用于可视化 GTP 酶何时被激活、何时与同源效应子结合以及如何形成信号复合物。使用多光子显微镜通过荧光寿命分析对结合进行光学检测，以揭示 Fvrster 共振能量转移 (FRET) 的存在。将生成并改进用于获得指示信号激活的局部区域的空间寿命图的分析算法。将开发使用偏振各向异性衰减测量来检测 GTP 酶在激活时的关联以及大型信号复合物的形成的新技术。我们预计我们提出的方法将促进 FRET 测量在许多体内信号研究中的应用和重现性。公共健康相关性：基于 FRET 的先进成像方法的开发可以大大增强我们对细胞间和细胞内信号传导的了解，特别是在乳腺癌等癌症方面。基于 FRET 的方法可以帮助阐明癌症发病和进展背后的潜在机制，并帮助确定未来的治疗目标。