Self-organization of stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol.
Self-organization of stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol.
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DOI：
10.1091/mbc.e12-12-0850
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发表时间：
2013-05
影响因子：
3.3
通讯作者：
Groen AC
中科院分区：
文献类型：
--
作者：
Mitchison TJ;Nguyen P;Coughlin M;Groen AC
菠萝，或自组织的，紫杉醇稳定的微管组件，揭示了丰富的自组织机制，在细胞分裂过程中组装的微管上操作，并提供了一个生化易处理的系统，用于研究这些机制在减数分裂和胞质分裂。先前的研究表明，非洲爪蟾减数分裂提取物中紫杉醇稳定的微管自组织成星形，揭示了纺锤体中依赖于运动的组织机制。我们重新审视了这种方法，使用澄清的细胞溶质与糖原补充回供应能量和减少当量。我们添加了NUMA和Aurora B探针以揭示微管极性。紫杉醇和二甲基亚砜促进微管的快速聚合，微管缓慢地自组织成具有特征形态的组装体，所述特征形态由成对的线或平行束的开放圆组成。负端在外侧的包含NUMA的焦点中对齐，正端在内侧的包含Aurora B的焦点中对齐。组装体具有取决于初始组装条件的明确定义的宽度，但其中的微管具有宽的长度分布。电子显微镜显示，正端病灶被电子致密物质包裹，类似于细胞中单极中间区的类似病灶。功能测试表明，两个关键的纺锤体组装因子（动力蛋白和驱动蛋白-5）在组装过程中的作用与它们在纺锤体中的作用一样，而两个关键的中区组装因子（Aurora B和Kif 4）的作用与它们在中区的作用一样。这些数据揭示了丰富的自组织机制，操作微管后，他们在减数分裂细胞质中，并提供了一个生物化学上易于处理的系统，调查加端组织在中间区。
Pineapples, or self-organized, Taxol-stabilized microtubule assemblies, reveal the richness of self-organizing mechanisms that operate on assembled microtubules during cell division and provide a biochemically tractable system for investigating these mechanisms during meiosis and cytokinesis. 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.