title: Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets.
abstract: Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.
authors: Department of Bionanoscience, Delft University of Technology.
authors: Mathijs
authors: M
authors: Vleugel
authors: Department of Bionanoscience, Delft University of Technology.
authors: Sophie
authors: S
authors: Roth
authors: Department of Bionanoscience, Delft University of Technology.
authors: Celebrity F
authors: CF
authors: Groenendijk
authors: Department of Bionanoscience, Delft University of Technology; m.dogterom@tudelft.nl.
authors: Marileen
authors: M
authors: Dogterom
mesh_terms: D018385:Centrosome; D008870:Microtubules; D008938:Mitosis; D008941:Spindle Apparatus; D014404:Tubulin
chemical_list: D014404:Tubulin