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.

Original languageEnglish
Title of host publicationCytoskeleton Dynamics
Subtitle of host publicationMethods and Protocols
EditorsHelder Maiato
Number of pages23
ISBN (Electronic)978-1-0716-0219-55
Publication statusPublished - 2020

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

    Research areas

  • Confinement, Cytolinkers, Cytoskeleton, In vitro reconstitution, Microtubule dynamic instability, Self-organization

ID: 68595049