title: Self-organization of stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol.
abstract: Previous study of self-organization of Taxol-stabilized microtubules into asters in Xenopus meiotic extracts revealed motor-dependent organizational mechanisms in the spindle. We revisit this approach using clarified cytosol with glycogen added back to supply energy and reducing equivalents. We added probes for NUMA and Aurora B to reveal microtubule polarity. Taxol and dimethyl sulfoxide promote rapid polymerization of microtubules that slowly self-organize into assemblies with a characteristic morphology consisting of paired lines or open circles of parallel bundles. Minus ends align in NUMA-containing foci on the outside, and plus ends in Aurora B-containing foci on the inside. Assemblies have a well-defined width that depends on initial assembly conditions, but microtubules within them have a broad length distribution. Electron microscopy shows that plus-end foci are coated with electron-dense material and resemble similar foci in monopolar midzones in cells. Functional tests show that two key spindle assembly factors, dynein and kinesin-5, act during assembly as they do in spindles, whereas two key midzone assembly factors, Aurora B and Kif4, act 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.
authors.affiliation: Marine Biological Laboratory, Woods Hole, MA 02543, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Mitchison
authors.forename: Phuong
authors.initials: P
authors.lastname: Nguyen
authors.forename: Margaret
authors.initials: M
authors.lastname: Coughlin
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Groen
mesh_terms: D000818:Animals; D064107:Aurora Kinase B / Q000378:metabolism; D002474:Cell-Free System; D048749:Cytokinesis; D003600:Cytosol / Q000378:metabolism*; D004398:Dyneins / Q000378:metabolism; D016547:Kinesins / Q000378:metabolism; D008540:Meiosis; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D009865:Oocytes / Q000378:metabolism; D055550:Protein Stability; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D059008:Time-Lapse Imaging; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D029867:Xenopus Proteins; D064107:Aurora Kinase B; C576938:aurkb protein, Xenopus; D004398:Dyneins; D016547:Kinesins