In Vitro Reconstitution of Dynamic Co-organization of Microtubules and Actin Filaments in Emulsion Droplets

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DOI http://dx.doi.org/10.1007/978-1-0716-0219-5_5
Reference K.J. A. Vendel, C. Alkemade, N. Andrea, G.H. Koenderink and A.M. Dogterom: In Vitro Reconstitution of Dynamic Co-organization of Microtubules and Actin Filaments in Emulsion Droplets In: Methods Mol. Biol., Springer Science, 2019. - pp. 53-75

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.