Studying the subcellular localization of proteins using microscopy has been instrumental in understanding cellular processes. In particular, changes in protein localization induced by perturbations such as environmental stress, drug treatment or gene knockout can reveal protein functions and help researchers understand cellular responses. However, testing large collections of antibodies or fluorescently labelled reporter cell lines against many perturbations remains challenging and time-consuming.

To improve throughput and scalability of live-cell imaging-based monitoring of subcellular protein localization, we have developed a tool called visual proteomics cells (vpCells), which facilitates two key advances. The first advance lies in the development of a pooled multicolour endogenous protein-tagging strategy, which enables the rapid labelling of many proteins in a single experiment. The result is a cell pool in which every cell carries multiple tagged proteins, each labelled with a fluorophore of a different colour. Using genome-scale single-guide RNA libraries enabled us to target specific proteins, leading to the isolation of thousands of clonal cell lines with a total of 1,158 unique tagged proteins, which can be accessed in our interactive web atlas.