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

描述（由申请人提供）：为了理解后生动物细胞如何在它们所处的组织中起作用，需要理解在细胞内和细胞之间起作用的无数信号转导途径。经典的生物化学技术已经揭示了许多信号传导元件及其在通路中的作用模式;然而，这些技术无法可视化体内信号传导通路的活化状态。最近，结合使用先进的光学技术，与工程荧光蛋白探针，开辟了直接可视化的信号通路的激活状态的可能性。我们建议开发一种普遍适用的工具包，用于研究在完整组织或生物体的背景下Rho家族GTdR通路的激活。该工具由一组优化的荧光探针组成，用于可视化何时GTdR被激活，何时它与同源效应物结合以及如何形成信号复合物。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。将生成和完善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的新技术，用于检测激活后GTP酶的关联以及大信号复合物的形成。我们预计，我们提出的方法将促进FRET测量的应用和重现性，许多在体内信号研究。公共卫生相关性：强大的基于FRET的先进成像方法的发展可以大大提高我们对细胞间和细胞内信号传导的认识，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。

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.