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.

Springer
doi.org/10.1007/978-1-0716-0219-5_5
Methods Mol. Biol.
Biological Soft Matter-Former Group

Vendel, K.J. A, Alkemade, C, Andrea, N, Koenderink, G.H, & Dogterom, M. (2019). In Vitro Reconstitution of Dynamic Co-organization of Microtubules and Actin Filaments in Emulsion Droplets. Methods Mol. Biol., 2101, 53–75. doi:10.1007/978-1-0716-0219-5_5