Spliff: A single-cell method to map protein-protein interactions in time and space

Abstract

Protein interactions occur at certain times and at specific cellular places. The past years have seen a massive accumulation of binary protein-protein interaction data. The rapid increase of this context-free information necessitates robust methods to monitor protein interactions with temporal and spatial resolution in single cells. We have developed a simple split-ubiquitin-based method (SPLIFF) that uses the ratio of two fluorescent reporters as a signal for protein-protein interactions. One protein of the pair of interest is attached to the linear fusion of mCherry, the C-terminal half of ubiquitin, and GFP (mCherry-Cub-GFP). The other potential binding partner is expressed as a C-terminal fusion to the N-terminal half of ubiquitin (Nub). Upon co-expression the interaction between the two proteins of interest induces the reassociation of Nub and Cub to the native-like ubiquitin. GFP is subsequently cleaved from the C-terminus of Cub and degraded whereas the red-fluorescent mCherry stays attached to the Cub-fusion protein. We first implemented this method in the model yeast Saccharomyces cerevisiae. One fusion protein is expressed in cells of the a-mating type and the complementary fusion protein in cells of the α-mating type. Upon mixing, both cell types fuse and the Nub- and Cub-fusion proteins are free to interact. The red and green fluorescence is monitored by two-channel fluorescence time-lapse microcopy. The moment of cell fusion defines the start of the analysis. The calculated ratio of green to red fluorescence allows mapping the spatiotemporal inter- action profiles of the investigated proteins in single cells.