In living cells, the architecture of the microtubule cytoskeleton is intimately linked to its function. The principles determining how microtubules arrange in space are, however, still not fully understood. Biochemical activities controlling microtubule nucleation and dynamics as well as mechanochemical activities exerted by molecular motors and the dynamic microtubules themselves are known to be critical for the correct spatial organization of the microtubule cytoskeleton. In vitro reconstitution approaches have revealed the morphogenetic properties of these activities in minimal systems. In most cases, such in vitro experiments were performed in experimental chambers of spatial dimensions that exceeded typical cell sizes by orders of magnitude. Here, we describe a method for the fluorescence microscopic study of the effects of spatial confinement on the self-organization of purified motors and microtubules that are encapsulated in micrometer-sized lipid-monolayered droplets emulsified in oil. In the future, this experimental setup can be extended in several ways. Additional proteins can be added, either to the lumen or to the boundary of the microcontainers, and the droplets can be transformed into liposomes. Such more complex in vitro reconstitutions would be another step closer to mimicking intracellular cytoskeleton organization.
