Print Email Facebook Twitter Reconstitution of basic mitotic spindles in spherical emulsion droplets Title Reconstitution of basic mitotic spindles in spherical emulsion droplets Author Vleugel, M. (TU Delft BN/Marileen Dogterom Lab) Roth, S.C.A. (TU Delft BN/Marileen Dogterom Lab) Groenendijk, Celebrity F. Dogterom, A.M. (TU Delft BN/Bionanoscience) Department BN/Bionanoscience Date 2016-08-13 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. Subject Ase1Cellular biologyCentrosomesDyneinIssue 114Kinesin-5MicrofluidicsMicrotubulesMitotic spindle formationSpindle positioning To reference this document use: http://resolver.tudelft.nl/uuid:399294ec-6e5e-4a77-8603-be0be5ba40d1 DOI https://doi.org/10.3791/54278 ISSN 1940-087X Source Journal of Visualized Experiments (114) Part of collection Institutional Repository Document type journal article Rights © 2016 M. Vleugel, S.C.A. Roth, Celebrity F. Groenendijk, A.M. Dogterom Files PDF jove_protocol_54278_recon ... ulsion.pdf 1.19 MB Close viewer /islandora/object/uuid:399294ec-6e5e-4a77-8603-be0be5ba40d1/datastream/OBJ/view