N. MIRA¹, S. PELET<sup>1

1</sup>Quantitative Signaling Group, Department of Fundamental Microbiology, University of Lausanne, UNIL-Sorge District, Biophore Building CH-1015 Lausanne, Switzerland

Studies of signaling pathways have focused on the quantification of global changes of MAPK activity. However, localized activity of MAPK may play a crucial role in many signal transduction cascades. For instance, in the yeast mating process, Fus3 MAPK and other signaling components are enriched at the tip of the mating projection. To quantify this local activity, we sought to develop a Bimolecular Synthetic Kinase Activity Relocation Sensor (Bi-SKARS), which consists of two synthetic modules: the docking module includes a MAPK docking site, a phosphorylatable sequence and a membrane targeting sequence, while the translocating module bears a phospho-binding domain as well as a fluorescent protein to track its sub-cellular location. The MAPK binds to the docking site upon pheromone stimulation and phosphorylates the Ser/Thr-containing motif. The recruitment of the translocating module via the phospho-Ser/Thr binding domain results in a change in fluorescence distribution.
Using this sensor, we observed a robust membrane enrichment of the sensor at the plasma membrane 4 min after stimulation of cells with pheromone. The specificity of the system to Fus3 MAPK was confirmed by the absence of recruitment with a non-docking version of the biosensor. Our next experiment will be to study the dynamic response of the biosensor using a fus3-as allele, which can be inhibited by an ATP analog. We will also verify the specificity of interaction between the two modules using a non-phosphorylatable and a phosphomimetic Bi-SKARS. The quantification of MAPK local activity will allow us to gain new insights into the conversion of Fus3 MAPK activation from an isotropic response to a polarized signal at the shmoo tip and its contribution to the establishment of a stable polarity site.