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American Society for Cell Biology, Molecular Biology of the Cell, 10(24), p. 1559-1573

DOI: 10.1091/mbc.e12-12-0850

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Self-organization of stabilized microtubules by both spindle and midzone mechanisms inXenopusegg cytosol

Journal article published in 2013 by Timothy J. Mitchison, Phuong Nguyen, Margaret Coughlin, Aaron C. Groen ORCID
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Abstract

Self-organization of taxol-stabilized microtubules into asters in Xenopus meiotic extracts revealed motor-dependent organizational mechanisms in the spindle (Verde et al. 1991). We revisited this approach using clarified cytosol with glycogen added back to supply energy and reducing equivalents. Probes for NUMA and Aurora B were added to reveal microtubule polarity. Taxol and DMSO promoted rapid polymerization of microtubules that slowly self-organized into assemblies with a characteristic morphology consisting of paired lines or open circles of parallel bundles. Minus ends aligned in NUMA-containing foci on the outside, and plus ends in Aurora-B containing foci on the inside. Assemblies had a well-defined width that depended on initial assembly conditions, but microtubules within them had a broad length distribution. Electron microscopy showed that plus end foci were coated with electron dense material and resembled similar foci in monopolar midzones in cells (Hu et al. 2008). Functional tests showed that two key spindle assembly factors, Dynein and Kinesin-5, acted during assembly as they do in spindles, while two key midzone assembly factors, Aurora B and Kif4, acted as they do in midzones. These data reveal the richness of self-organizing mechanisms that operate on microtubules after they polymerize in meiotic cytoplasm and provide a biochemically tractable system for investigating plus end organization in midzones.