ED DURANDAU¹, MM MA¹, DA AYMOZ¹, SP PELET<sup>1

1</sup>Département de Microbiologie Fondamentale

The phosphorylation of signaling cascade components is a key post-translational modification used in signal transduction. Although all kinases catalyze this bio-chemical reaction, each individual kinase possesses its specific pool of substrates. Moreover, the temporal activity of these enzymes varies from cell to cell depending on intra- or extra-cellular cues. Knowing that kinase mis-regulation is implicated in many diseases, such as cancer, it becomes crucial to quantify the heterogeneity of the dynamic kinase activity at the single cell level. Micros- copy is an ideal technique for such investigation. However, fluorescent assays have to be established to quantify this enzymatic activity in living cells.
Using a synthetic biology approach, we designed a fluorescent biosensor that undergoes nuclear-to-cytoplasmic relocation upon phosphorylation by the kinase of interest. Combination of time-lapse microscopy measurements and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. As proof of concept, we generated a Synthetic Kinase Activity Relocation Sensor (SKARS) for Mitogen Activated Protein Kinases (MAPK) of the mating pathway in S. cerevisiae. The kinetics of the MAPK acti- vation exhibits a large heterogeneity between single cells due to an inhibition of signal transduction at specific stages of the cell-cycle. The modularity of the architecture of the SKARS allows us to expand the range of kinases that we can monitor in yeast, providing biosensors for all MAPKs as well as cyclins. In addition, based on the same sensor structure, we also developped reporters for the mammalian MAPKs ERK, JNK and p38.