Quantitative Characterization of Budding Yeast Polarization at the Mesoscale

Cellular symmetry breaking or polarization is a key process that underlies many types of cellular behavior like division, migration and differentiation. In budding yeast, polarization is required for bud site selection. Polarization of key regulator Cdc42 has been molecularly dissected for decades, yet systematic quantification of protein localization dynamics during symmetry breaking has been lacking. Here, we present a phenomenological description of polarization in terms of physical characteristics using live cell microscopy and quantitative analysis. We identify multiple stages of Cdc42 polarization and derive mesoscale observables that describe each stage, such as establishment timescales and spot morphology indicators. We then apply this description to two case studies in which the wildtype polarity network is perturbed: a small perturbation after eliminating spatial cue communication and a larger perturbation involving removal of near-essential scaffold Bem1. This confirms the applicability of the framework in non-wildtype contexts while systematically revealing differences between genetic backgrounds. Finally, we study the dynamics of other polarizing components by extending our analysis to the Gic2 PBD domain, a Cdc42-GTP biosensor, and to polarisome component Spa2, highlighting variation in spot dynamics between protein species while also showing similarities to Cdc42. This approach enables standardized comparison between diverse molecular backgrounds at the mesoscale and helps constrain future modeling efforts.