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 interaction profiles of the investigated proteins in single cells.
*These authors contributed equally.
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Acknowledgement
This work was supported by the BMBF Initiative SysTec (0315690B) and by the BMBF Initiative GerontoSys2 (SyStaR, 03158 94A).
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Dünkler, A., Rösler, R., Kestler, H.A., Moreno-Andrés, D., Johnsson, N. (2015). SPLIFF: A Single-Cell Method to Map Protein-Protein Interactions in Time and Space. In: Singh, A., Chandrasekaran, A. (eds) Single Cell Protein Analysis. Methods in Molecular Biology, vol 1346. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2987-0_11
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DOI: https://doi.org/10.1007/978-1-4939-2987-0_11
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