Complex Simplicity: Towards reconstituting Cdc42-based polarity establishment

Saccharomyces cerevisiae proliferates through budding, where a daughter cell grows by budding off one side of the mother. The first step towards budding is polarity establishment: here Cdc42 accumulates in one spot on the membrane, marking the site of bud-emergence. Cdc42 accumulation arises through at least two interconnected regulatory feedback loops, based on I) a reaction-diffusion mechanism and II) the actin cytoskeleton. Cdc42 is a highly regulated protein and dissecting the molecular mechanisms and coupling between the different feedback loops has turned out to be controversial, because of both the parameter sensitivity and the high level of observed redundancy and interdependence within and between the feedback loops. This calls for the development of a minimal in vitro system. In this thesis I show our progress towards reconstituting Cdc42-based polarity establishment. Our system is based on, due to theoretical predictions, the three proteins: the GTPase Cdc42, its GDP/GTP Exchange Factor Cdc24, and the scaffold protein Bem1. Such a minimal system, where proteins can be added and removed at will, will not only facilitate mechanistic studies, but also help to understand how molecular functions necessary for pattern formation are distributed within the polarity network.