title: A fluorescent reporter for mapping cellular protein-protein interactions in time and space.
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.
authors.affiliation: Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Moreno
authors.forename: Joachim
authors.initials: J
authors.lastname: Neller
authors.forename: Hans A
authors.initials: HA
authors.lastname: Kestler
authors.forename: Johann
authors.initials: J
authors.lastname: Kraus
authors.forename: Alexander
authors.initials: A
authors.lastname: Dünkler
authors.forename: Nils
authors.initials: N
authors.lastname: Johnsson
mesh_terms: D002453:Cell Cycle; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D056490:Nucleosome Assembly Protein 1 / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods; D059008:Time-Lapse Imaging / Q000379:methods*; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; C116847:HOF1 protein, S cerevisiae; C110443:KAR9 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; C533556:NAP1 protein, S cerevisiae; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D011993:Recombinant Fusion Proteins; C418484:SPC72 protein, S cerevisiae; C109414:STU2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D011494:Protein Kinases; C117190:KCC4 protein, S cerevisiae