Mapping endogenous protein dynamics in living cells

摘要 后基因组时代的一个核心挑战是全面表征约20，000个基因的细胞作用。 人类基因组中编码的蛋白质。为此，我们开发了FP 11标签和高- 高效的CRISPR/Cas9基因编辑管道，能够大规模生成内源性标记的人类基因。 细胞系，并支付的方式，蛋白质定位和相互作用网络的蛋白质组范围内的分析， 原生细胞环境在前期资助的项目中，我们建立了细胞工程平台， 并证明了这种方法在创建OpenCell文库（约1300种靶向蛋白质）中的作用 及其成像和蛋白质组学表征。尽管如此，技术挑战仍然存在于长期的现场成像， 蛋白质动力学和这些电影的定量解释。在拟议的更新中，我们计划进一步 发展epi-illumination选择性平面照明显微镜（eSPIM）技术，使其成为一种广泛的 适用的活细胞显微镜平台，用于低分辨率的亚细胞动力学的长期成像， 光漂白和光毒性。我们还将开发相应的仪器软件包 控制和图像处理，使其易于被其他实验室采用。最后，我们将演示 通过使用细胞周期筛选不寻常的蛋白质动力学， OpenCell图书馆

ABSTRACT A central challenge of the post-genomic era is to comprehensively characterize the cellular role of the ~20,000 proteins encoded in the human genome. For this purpose, we have developed the FP11 tags and a high- efficiency CRISPR/Cas9 gene editing pipeline, enabling large-scale generation of endogenously tagged human cell lines, and paying the way to proteome-wide analysis of protein localization and interaction networks in a native cellular context. In the previously funded preject, we have established the cellular engineering platform and demonstrated the power of such an approach in creating the OpenCell library (~1300 targeted proteins) and its imaging and proteomics characterization. Still, technical challenges remain in long-term live imaging of protein dynamics and quantitative interpretation of these movies. In the proposed renewal, we plan to further develop the epi-illumination selective plane illumination microscopy (eSPIM) technique, turning it into a widely applicable live cell microscopy platform for high-resolution, long-term imaging of subcellular dynamics with low photobleaching and phototoxicity. We will also develop the corresponding software packages for instrument control and image processing to enable its easy adoption by other labs. Finally, we will demonstrate the applicability of this technological platform by screening for unusual protein dynamics through cell cycle using the OpenCell library.