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

描述(申请人提供)：我们建议的研究涉及细胞的全球“系统水平”行为，特别是随着细胞分化而在单个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。我们专注于一个明确的模型系统，在这个系统中，酵母对交配信息素的反应改变了形态，我们打算绘制信息素诱导的大多数酵母蛋白质和RNA的空间重组图。为了构建这一图谱并定义影响重组的基因，我们提出了一系列基于“细胞芯片”的实验，这是一种基于微阵列的新平台，用于自动化、高通量、高分辨率的细胞显微成像。细胞芯片提供了一种方法，通过在单细胞水平上全面地测量蛋白质和RNA的表达和定位变化来表征酵母细胞。在每个芯片上，可以测量给定目标蛋白质/RNA在一组全面的遗传背景中的定位和表达，也可以测量在单一遗传背景中的一组全面的蛋白质/RNA的定位和表达。由于每一次采集单个菌种可以生产100个细胞芯片，而且每个芯片都可以用针对不同目标的荧光探针进行探测，因此细胞芯片提供了传统方法实际上无法获得的新型高通量遗传相互作用屏幕。我们建议发展细胞芯片技术，大规模地测量单细胞水平的蛋白质/RNA的功能、表达和定位，重点关注伴随交配信息素反应的全球变化。除了描述蛋白质组/转录组的空间动态外，这项工作还将讨论RNA在酵母中的定位程度，以及比较酵母蛋白质组中大部分单细胞的转录和翻译水平。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理。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中有一些在人类细胞中保守的重要元件，因此，这项研究的结果直接影响到我们对人类细胞生物学和信号转导的理解，以及人类细胞与环境的相互作用。

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.