A fluorescent reporter for mapping cellular protein-protein interactions in time and space

Daniel Moreno; Joachim Neller; Hans A Kestler; Johann Kraus; Alexander Dünkler; Nils Johnsson

doi:10.1038/msb.2013.3

A fluorescent reporter for mapping cellular protein-protein interactions in time and space

Mol Syst Biol. 2013:9:647. doi: 10.1038/msb.2013.3.

Authors

Daniel Moreno¹, Joachim Neller, Hans A Kestler, Johann Kraus, Alexander Dünkler, Nils Johnsson

Affiliation

¹ Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany.

Abstract

We introduce a fluorescent reporter for monitoring protein-protein interactions in living cells. The method is based on the Split-Ubiquitin method and uses the ratio of two auto-fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two-channel time-lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio-temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran-GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein-protein interactions identified from large-scale screens can be effectively followed up by high-resolution single-cell analysis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Cycle
Cell Nucleus / genetics
Cell Nucleus / metabolism
Fluorescent Dyes / analysis*
Fluorescent Dyes / metabolism
Microscopy, Confocal / methods
Microtubule-Associated Proteins / genetics
Microtubule-Associated Proteins / metabolism
Microtubules / metabolism
Nuclear Proteins / genetics
Nuclear Proteins / metabolism
Nucleosome Assembly Protein 1 / genetics
Nucleosome Assembly Protein 1 / metabolism
Protein Interaction Mapping / methods*
Protein Kinases / genetics
Protein Kinases / metabolism
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism*
Saccharomyces cerevisiae Proteins / genetics
Saccharomyces cerevisiae Proteins / metabolism
Single-Cell Analysis / methods
Time-Lapse Imaging / methods*
Ubiquitin / metabolism

Substances

Fluorescent Dyes
HOF1 protein, S cerevisiae
KAR9 protein, S cerevisiae
Microtubule-Associated Proteins
NAP1 protein, S cerevisiae
Nuclear Proteins
Nucleosome Assembly Protein 1
Recombinant Fusion Proteins
SPC72 protein, S cerevisiae
STU2 protein, S cerevisiae
Saccharomyces cerevisiae Proteins
Ubiquitin
Protein Kinases
KCC4 protein, S cerevisiae