New methods for monitoring the immune system, in individual cells and in vivo

描述（由申请人提供）：了解单细胞先天免疫信号网络的动力学是免疫学的基本目标。隐蔽实验室采用一种结合了活细胞成像、高通量图像分析、微流体细胞培养和计算网络建模等最新技术的方法，通过测量NFkB对刺激和时间依赖性刺激组合的单细胞反应，研究细胞如何解码复杂的环境信息（Nature, 2010; Science Signaling 2009）。尽管这些和类似的方法在描述细胞群体内的表型异质性方面非常有用（例如，在研究p53方面也是如此），但由于可测量输出的数量相对较少，以及到目前为止，几乎所有这类研究都是在培养细胞中进行的，因此可以从中得出的结论受到限制。我们建议大幅扩大免疫系统活细胞动态成像的范围，开发新技术以大幅增加可测量输出的数量，并使体内测量成为可能。我们的具体目标是：(1)创建一个构建物和细胞库，能够同时在单个细胞中监测多种因素，包括多种平行信号通路和内源性表达水平。(2)为了了解网络动力学如何控制基因表达，我们建议通过将最近开发的RNA FISH技术与我们的活细胞成像技术相结合，开发将转录因子动力学与单细胞内源基因表达动力学相关联的方法。这将是第一次将动态转录因子活性与单个细胞中的基因表达直接比较。(3)隐蔽实验室将与加州大学圣地亚哥分校的Tannishtha Reya合作，将我们在单细胞中成像和定量蛋白质定位的方法与她在体内监测细胞运动的开创性工具结合起来。通过结合这些方法，我们将首次观察到单个细胞中转录因子在完整动物骨髓中移动时的动态。在实现这些目标的过程中，我们期望对环境信息如何在信号网络动态中编码有更详细和系统级的理解，并为科学界提供一些首创的技术。

DESCRIPTION (provided by applicant): To understand the dynamics of the innate immune signaling network in single cells is a fundamental goal of immunology. Using an approach that combines the latest technologies for live-cell imaging, high-throughput image analysis, microfluidic cell culture and computational network modeling, the Covert Lab studies how cells decode complex environmental information by measuring the single-cell responses of NFkB to combinations of stimuli and time-dependent stimuli (Nature, 2010; Science Signaling 2009). Although these and similar approaches have been extremely useful in characterizing phenotypic heterogeneity within a population of cells (also in studying p53, for example), the conclusions that can be drawn from them are limited by the relatively low number of measureable outputs as well as the fact that until now, virtually all of this kind of research has been performed in cultued cells. We propose to dramatically expand the scope of live-cell dynamic imaging of the immune system, developing new technologies to dramatically increase the number of measureable outputs, and enable in vivo measurements. Our Specific Aims are: (1) to create a library of constructs and cells that will enable monitoring of a variety of factors, encompassing multiple parallel signaling pathways and at endogenous expression levels, simultaneously in individual cells. (2) To understand how network dynamics control gene expression, we propose to develop methods to correlate the dynamics of transcription factors with the dynamics of endogenous gene expression in single cells, by integrating recently developed techniques for RNA FISH with our live cell imaging technology. This will be the first time that dynamic transcription factor activity has ever been directly compared with gene expression in individual cells. (3) The Covert Lab will partner with Tannishtha Reya at UCSD to integrate our methods for imaging and quantifying protein localization in single cells with her pioneering tools for monitoring cellular movement in vivo. By combining these approaches, we will be the first to observe the dynamics of transcription factors in individual cells as they move through the bone marrow of intact animals. In achieving these goals, we expect to achieve a significantly more detailed and system-level understanding of how environmental information is encoded in signaling network dynamics, and to have produced some first-of-its-kind technology for the scientific community. PUBLIC HEALTH RELEVANCE: New methods for monitoring the immune system, in individual cells and in vivo Project Narrative In recent years, scientists have realized how important each individual cell in our bodies are, and how even two neighboring cells of the same kind can behave quite differently. There are some ways to observe individual cells, and specifically the activation of key proteins, in real- time, but there are many limitations of the technology. We are proposing to develop some new technology that will make it much easier to track and the immune response as it occurs in single cells -- even as the cells move around in a living animal.