Cell chips for genome-wide protein and RNA localization in single cells

描述（由申请人提供）：我们提出的研究解决了细胞的全局“系统水平”行为，特别是随着细胞分化在单细胞中发生的蛋白质和RNA的动态定位和表达变化，以及控制这些过程的基因。我们专注于一个定义的模型系统中，酵母改变形态交配信息素的反应，我们打算映射信息素诱导的空间重组的大多数酵母蛋白质和RNA。为了构建这个地图和定义影响重组的基因，我们提出了一组实验的基础上围绕“细胞芯片”，一个新的基于微阵列的平台，自动化，高通量，高分辨率的显微成像细胞。细胞芯片提供了一种通过在单细胞水平上全面测量蛋白质和RNA表达和定位变化来表征酵母细胞的方法。在每个芯片上，定位和表达可以针对给定的靶蛋白/RNA跨一组全面的遗传背景或针对一组全面的蛋白质/RNA在单一遗传背景中进行测量。由于每个阶段可以从单个菌株收集中制备>100个细胞芯片，并且每个芯片可以用不同靶标的荧光探针进行探测，因此细胞芯片提供了传统方法无法有效获得的新型高通量遗传相互作用筛选。我们建议开发细胞芯片技术，以测量单细胞水平的蛋白质/RNA的功能，表达和大规模定位，专注于全球变化伴随着交配信息素的反应。除了描述蛋白质组/转录组的空间动力学，这项工作将解决RNA在酵母中定位的程度，以及比较大部分酵母蛋白质组在单细胞中的转录与翻译水平。这些实验将告诉我们真核细胞分化，RNA定位和转录后调控的一般原则。我们提出的实验和计算技术应该很容易扩展到其他系统，包括人类细胞，使我们更接近真核细胞行为的“系统水平”描述。我们研究的模型系统具有人类细胞中保守的重要元素，因此这项研究的结果直接影响我们对人类细胞生物学和信号转导以及人类细胞与环境相互作用的理解。

DESCRIPTION (provided by applicant): Our proposed research addresses the global "systems level" behavior of cells, especially dynamic localization and expression changes of proteins and RNA occurring in single cells as the cells differentiate, as well as the genes controlling these processes. We focus on a defined model system in which yeast change morphology in response to mating pheromone, and we intend to map the pheromone-induced spatial reorganization of the majority of yeast proteins and RNAs. To construct this map and define genes affecting the reorganization, we propose a set of experiments based around "cell chips", a new microarray-based platform for automated, high-throughput, high-resolution microscopic imaging of cells. Cell chips offer an approach for characterizing yeast cells by measuring, comprehensively and at the single cell level, protein and RNA expression and localization changes. On each chip, localization and expression can be measured either for a given target protein/RNA across a comprehensive set of genetic backgrounds or for a comprehensive set of proteins/RNAs in a single genetic background. Because >100 cell chips can be made per session from a single strain collection and each chip can be probed with fluorescent probes for a different target, cell chips offer new types of high-throughput genetic interaction screens effectively inaccessible by conventional approaches. We propose to develop cell chip technology to measure single-cell level protein/RNA function, expression and localization on a large scale, focusing on global changes accompanying the response to mating pheromone. Beyond describing the spatial dynamics of the proteome/transcriptome, this work will address the extent to which RNAs are localized in yeast, as well as to compare transcription versus translation levels in single cells for a large fraction of the yeast proteome. These experiments will inform us about general principles of eukaryotic cell differentiation, RNA localization, and post-transcriptional regulation. The experimental and computational technologies we propose should readily extend to other systems, including human cells, moving us closer to "systems level" descriptions of eukaryotic cell behavior. The model system we study has important elements conserved in human cells, and therefore results of this study directly impact our understanding of human cell biology and signal transduction, and the interaction of human cells with the environment.