title: Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.
abstract: BACKGROUND
  Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades.
  
  
  RESULTS
  We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage.
  
  
  CONCLUSIONS
  These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.
authors.affiliation: Department of Fundamental Microbiology, University of Lausanne, CH-1015, Lausanne, Switzerland.
authors.forename: Eric
authors.initials: E
authors.lastname: Durandau
authors.affiliation: Department of Fundamental Microbiology, University of Lausanne, CH-1015, Lausanne, Switzerland.
authors.forename: Delphine
authors.initials: D
authors.lastname: Aymoz
authors.affiliation: Department of Fundamental Microbiology, University of Lausanne, CH-1015, Lausanne, Switzerland. serge.pelet@unil.ch.
authors.forename: Serge
authors.initials: S
authors.lastname: Pelet
mesh_terms: D020935:MAP Kinase Signaling System; D008856:Microscopy, Fluorescence / Q000379:methods; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D020928:Mitogen-Activated Protein Kinases