A minimal in vitro system for cell polarization

Polarity establishment is an evolutionary conserved mechanism for a cell to directionally redistribute proteins in order to divide, migrate, differentiate and grow. We know that yeast cell polarity is established by an accumulation of active Cdc42, a small Rho-type GTPase protein, on the membrane with enhancement of activating protein Cdc24 and scaffold protein Bem1. The interactions between these partially disordered proteins, membrane binding, crowding, and lipid dynamics drive the system towards polarization. Yet, we do not understand how these properties relate to the pattern formation that initiates polarity establishment. We have established all components to reconstitute Cdc42 based polarization: the three protein constructs with fluorescent tags and a 2D lipid bilayer. We are characterizing the membrane binding dynamics of our proteins through FRAP and TIRF microscopy and through a quartz crystal microbalance. We will induce binding of our proteins on the membrane through crowding agents. The combination of intrinsically disordered regions in the proteins and together with crowding effects should drive the system into a phase transition that could lead to polarization establishment. We also model these dynamics with a stochastic, reaction-diffusion based Gillespie simulation. Through a combination of experiments and modelling, we aim to get a better understanding of how single-molecule dynamics as well as collective dynamics lead to the pattern formation that initiates cell polarity.