Mapping endogenous protein dynamics in living cells

项目摘要/摘要 后基因组时代的一个中心挑战是全面表征~20,000的细胞角色 人类基因组中编码的蛋白质。功能标记是描述细胞的一种强有力的策略 蛋白质的作用。特别是，标签允许访问蛋白质功能的两个关键特征：定位(使用 荧光标记)和相互作用伙伴(使用表位标记和免疫沉淀)。因此，通过标记 蛋白质在系统的方式下，生物体蛋白质组的全面功能描述可以是 已实现。为此，我们之前已经开发了基于自补分裂的FP11标签 荧光蛋白，与使用Cas9/sgRNA核糖核蛋白(RNPs)的基因编辑相结合， 能够快速、高效和高度可扩展地标记哺乳动物细胞系中的内源性蛋白质。而我们的 结果为大规模产生内源性标记的人类细胞系铺平了道路 在天然细胞环境中蛋白质定位和相互作用网络的蛋白质组学分析。然而， 对于大规模库的实际生成和分析，仍需要满足以下几个主要技术限制 解决：亮度、色彩可用性和低漂白的实时成像平台。在建议的 项目中，我们计划设计改进的FP11标签和新的分离蛋白片段标签来解决这些技术问题 挑战。我们还将通过开发一种新的选择性平面来演示强大的应用 照明显微镜(SPIM)系统用于筛选内源标记文库。

PROJECT SUMMARY/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. Functional tagging is a powerful strategy to characterize the cellular role of proteins. In particular, tags allow access to two key features of protein function: localization (using fluorescent tags) and interaction partners (using epitope tags and immuno-precipitation). Hence, by tagging proteins in a systematic manner, a comprehensive functional description of an organism’s proteome can be achieved. For this purpose, we have previously developed FP11 tags based on self-complementing split fluorescent proteins, which, in combination with gene editing using Cas9/sgRNA ribonucleoprotein (RNPs), enable rapid, efficient and highly scalable tagging of endogenous proteins in mammalian cell lines. While our results have paved the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. However, for practical generation and analysis of large-scale libraries, several major technical limitations still need to be addressed: brightness, color availability, and live imaging platforms with low photobleaching. In the proposed project, we plan to engineer improved FP11 tags and new split protein fragment tags to address these technical challenges. We will also demonstrate the powerful applications by developing a new selective plane illumination microscopy (SPIM) system to screen an endogenously tagged library.