In vitro (or cell-free) reconstitution is a powerful tool to study the physical basis of cytoskeletal organization in eukaryotic cells. Cytoskeletal reconstitution studies have mostly been done for individual cytoskeleton systems in unconfined 3D or quasi-2D geometries, which lack complexity relative to a cellular environment. To increase the level of complexity, we present a method to study co-organization of two cytoskeletal components, namely microtubules and actin filaments, confined in cell-sized water-in-oil emulsion droplets. We show that centrosome-nucleated dynamic microtubules can be made to interact with actin filaments through a tip-tracking complex consisting of microtubule end-binding proteins and an actin-microtubule cytolinker. In addition to the protocols themselves, we discuss the optimization steps required in order to build these more complex in vitro model systems of cytoskeletal interactions.

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Every living organism consists of cells. Even for the simplest single-cell organism, this cell is extremely complex. Thousands of components (such asDNA, cytoskeletal filaments, proteins, lipids, nutrients and energy) are organized both spatially and temporally to ensure proper functioning of vital cellular processes. One of those processes is pattern formation, or cell polarity. Cell polarity is defined as the morphological and functional differentiation of cellular compartments in a directional manner. This directionality is crucial for processes like cell division, cell migration and cell growth, which require an asymmetric action of the cell. When polarity is inhibited by silencing of cell polarity proteins, cells become deformed and have trouble to function, if viable at all. It is well-known that cell polarity is the result of reaction-diffusion and cytoskeleton-based mechanisms, but the exact mechanisms remain unknown. In this thesis, we focus on the latter, and more specifically on one type of cytoskeletal filaments: microtubules (MTs). Our goal is to study the role of MTs in cell polarity establishment. @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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