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Reconstitution of basic mitotic spindles in spherical emulsion droplets. / Vleugel, Mathijs; Roth, Sophie; Groenendijk, Celebrity F.; Dogterom, Marileen.

In: Journal of Visualized Experiments, No. 114, e54278, 13.08.2016.

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Vleugel, Mathijs ; Roth, Sophie ; Groenendijk, Celebrity F. ; Dogterom, Marileen. / Reconstitution of basic mitotic spindles in spherical emulsion droplets. In: Journal of Visualized Experiments. 2016 ; No. 114.

BibTeX

@article{399294ec6e5e4a778603be0be5ba40d1,
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.",
keywords = "Ase1, Cellular biology, Centrosomes, Dynein, Issue 114, Kinesin-5, Microfluidics, Microtubules, Mitotic spindle formation, Spindle positioning",
author = "Mathijs Vleugel and Sophie Roth and Groenendijk, {Celebrity F.} and Marileen Dogterom",
year = "2016",
month = "8",
day = "13",
doi = "10.3791/54278",
language = "English",
journal = "Journal of Visualized Experiments",
issn = "1940-087X",
publisher = "MYJoVE Corporation",
number = "114",

}

RIS

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

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UR - http://resolver.tudelft.nl/uuid:399294ec-6e5e-4a77-8603-be0be5ba40d1

U2 - 10.3791/54278

DO - 10.3791/54278

M3 - Article

JO - Journal of Visualized Experiments

T2 - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

IS - 114

M1 - e54278

ER -

ID: 7336143