TY - JOUR
T1 - Reconstitution of basic mitotic spindles in spherical emulsion droplets
AU - Vleugel, Mathijs
AU - Roth, Sophie
AU - Groenendijk, Celebrity F.
AU - Dogterom, Marileen
PY - 2016/8/13
Y1 - 2016/8/13
N2 - 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.
AB - 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.
KW - Ase1
KW - Cellular biology
KW - Centrosomes
KW - Dynein
KW - Issue 114
KW - Kinesin-5
KW - Microfluidics
KW - Microtubules
KW - Mitotic spindle formation
KW - Spindle positioning
UR - http://www.scopus.com/inward/record.url?scp=84983772213&partnerID=8YFLogxK
UR - http://resolver.tudelft.nl/uuid:399294ec-6e5e-4a77-8603-be0be5ba40d1
U2 - 10.3791/54278
DO - 10.3791/54278
M3 - Article
AN - SCOPUS:84983772213
SN - 1940-087X
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 114
M1 - e54278
ER -