Exploration and stabilization of Ras1 mating zone: A mechanism with positive and negative feedbacks

In mating fission yeast cells, sensing and response to extracellular pheromone concentrations occurs through an exploratory Cdc42 patch that stochastically samples the cell cortex before stabilizing towards a mating partner. Active Ras1 (Ras1-GTP), an upstream regulator of Cdc42, and Gap1, the GTPase-activating protein for Ras1, localize at the patch. We developed a reaction-diffusion model of Ras1 patch appearance and disappearance with a positive feedback by a Guanine nucleotide Exchange Factor (GEF) and Gap1 inhibition. The model is based on new estimates of Ras1-GDP, Ras1-GTP and Gap1 diffusion coefficients and rates of cytoplasmic exchange studied by FRAP. The model reproduces exploratory patch behavior and lack of Ras1 patch in cells lacking Gap1. Transition to a stable patch can occur by change of Gap1 rates constants or local increase of the positive feedback rate constants. The model predicts that the patch size and number of patches depend on the strength of positive and negative feedbacks. Measurements of Ras1 patch size and number in cells overexpressing the Ras1 GEF or Gap1 are consistent with the model.
Author summary Unicellular fission yeasts mate by fusing with partners of the opposite mating type. Each pair member grows towards its selected partner that signals its presence through secreted pheromone. The process of partner selection occurs through an exploratory patch (containing activated signaling protein Cdc42 and upstream regulator Ras1) that assembles and disassembles on the cell cortex, stabilizing in regions of higher opposite pheromone concentration. We present a computational model of the molecular mechanisms driving the dynamical pattern of patch exploration and stabilization. The model is based on reaction and diffusion along the curved cell membrane, with diffusion coefficients measured experimentally. In the model, a positive Ras1 activation feedback loop generates a patch containing most of the activating protein (Ras1 GEF). The fast diffusing inhibitor Gap1 that is recruited locally from the cytoplasm spreads on the cell membrane, limiting patch size and causing its decay. Spontaneous reinitiation of Ras1 activation elsewhere on the cortex provides a mechanism for exploration. Transition of the system’s behavior to that of a single stable patch is possible upon simulated pheromone sensing. The computational model provides predictions for the number of patches and patch size dependence on parameters that we tested experimentally.