In different cellular activities such as signal transduction, cell division, and intracellular transportation, small guanosine triphosphatases (GTPases) take on a vital role. Their function involves hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article, we explain the application of a commercially available GTPase assay—the GTPase Glo assay by Promega—for investigation of GTPase-effector interactions. We provide experimental protocols together with an analysis model and software to obtain GTPase cycling rates of GTPases and GTPase:effector mixtures. GTPase cycling rates refer to the rates by which a GTPase completes an entire GTPase cycle. These rates enable quantification of the strength of GTPase effectors in a concentration-dependent fashion, as well as quantification of the combined effect of two effectors, independent of which GTPase cycle step they are affecting.

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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.@en

Cell polarity - the morphological and functional differentiation of cellular compartments in a directional manner - is required for processes such as orientation of cell division, directed cellular growth and motility. How the interplay of components within the complexity of a cell leads to cell polarity is still heavily debated. In this Review, we focus on one specific aspect of cell polarity: the non-uniform accumulation of proteins on the cell membrane. In cells, this is achieved through reaction-diffusion and/or cytoskeleton-based mechanisms. In reaction-diffusion systems, components are transformed into each other by chemical reactions and are moving through space by diffusion. In cytoskeleton-based processes, cellular components (i.e. proteins) are actively transported by microtubules (MTs) and actin filaments to specific locations in the cell. We examine how minimal systems - in vitro reconstitutions of a particular cellular function with a minimal number of components - are designed, how they contribute to our understanding of cell polarity (i.e. protein accumulation), and how they complement in vivo investigations. We start by discussing the Min protein system from Escherichia coli, which represents a reaction-diffusion system with a well-established minimal system. This is followed by a discussion of MT-based directed transport for cell polarity markers as an example of a cytoskeleton-based mechanism. To conclude, we discuss, as an example, the interplay of reaction-diffusion and cytoskeleton-based mechanisms during polarity establishment in budding yeast.

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