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

描述(由申请人提供)：要了解后生动物细胞如何在它们所在的组织中发挥作用，需要了解在细胞内和细胞之间运行的无数信号转导途径。经典的生化技术已经揭示了信号通路中的许多信号元件及其作用方式；然而，这些技术无法在体内可视化信号通路的激活状态。最近，先进的光学技术与工程荧光蛋白探针的结合使用，使直接可视化信号通路的激活状态成为可能。我们建议开发一个普遍适用的工具包，用于在完整组织或生物体的背景下研究Rho家族GTP酶途径的激活。这些工具包括一组优化的荧光探针，用于可视化GTP酶何时激活，何时与同源效应器结合，以及如何形成信号复合体。通过荧光寿命分析用多光子显微镜光学检测结合，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。使用极化各向异性衰减测量的新技术将被开发用于检测激活时GTP酶的关联以及大信号复合体的形成。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复性。公共卫生相关性：基于FRET的先进成像方法的发展可以极大地提高我们对细胞间和细胞内信号传递的了解，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生和发展背后的潜在机制，并有助于确定未来的治疗目标。

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.