Mapping endogenous protein dynamics in living cells

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.