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.@en

Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.@en