Molecular requirements for kinetochore-associated microtubule formation in mammalian cells
Molecular requirements for kinetochore-associated microtubule formation in mammalian cells
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DOI：
10.1016/j.cub.2006.01.060
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发表时间：
2006-03-07
期刊：
影响因子：
9.2
通讯作者：
Wadsworth, P
中科院分区：
文献类型：
--
作者：
Tulu, US;Fagerstrom, C;Wadsworth, P
在含有中心体的细胞中，在中心体处成核的微管被认为在纺锤体组装中起主要作用[1]。此外，还观察到着丝粒处的微管形成[2-5]，最近在活细胞中的生理条件下[6]。微管在着丝粒和中心体的形成对纺锤体组装的相对贡献，以及它们的分子要求，仍然不完全清楚。使用哺乳动物细胞释放nocodazole诱导拆卸，我们观察到微管形成的中心体和Bub 1阳性位点的染色体上。动力蛋白依赖的方式Kinetochorse相关的微管迅速合并成极样结构。显微注射过量的importin-β或耗尽的RAN依赖的纺锤体组装因子，TPX 2，阻断了kinetochorse相关微管的形成，增强了中心体相关微管的形成，但并没有阻止中心体微管的染色体捕获。耗尽的染色体乘客蛋白，生存素，减少微管形成在着丝粒中的MCAK依赖性的方式。微管形成的细胞中耗尽的Bub 1或Nuf 2是无法区分的，在控制。我们的数据表明，微管组装在中心体和动粒是动力学上不同的和差异调节。微管在动粒的存在提供了一种机制，以调和所需的时间在体内纺锤体组装与观察到的计算机模拟搜索和捕获。
In centrosome-containing cells, microtubules nucleated at centrosomes are thought to play a major role in spindle assembly [1]. In addition, microtubule formation at kinetochores has also been observed [2-5], most recently under physiological conditions in live cells [6]. The relative contributions of microtubule formation at kinetochores and centrosomes to spindle assembly, and their molecular requirements, remain incompletely understood. Using mammalian cells released from nocodazole-induced disassembly, we observed microtubule formation at centrosomes and at Bub1-positive sites on chromosomes. Kinetochore-associated microtubules rapidly coalesced into pole-like structures in a dynein-dependent manner. Microinjection of excess importin-beta or depletion of the Ran-dependent spindle assembly factor, TPX2, blocked kinetochore-associated microtubule formation, enhanced centrosome-associated microtubule formation, but did not prevent chromosome capture by centrosomal microtubules. Depletion of the chromosome passenger protein, survivin, reduced microtubule formation at kinetochores in an MCAK-dependent manner. Microtubule formation in cells depleted of Bub1 or Nuf2 was indistinguishable from that in controls. Our data demonstrate that microtubule assembly at centrosomes and kinetochores is kinetically distinct and differentially regulated. The presence of microtubules at kinetochores provides a mechanism to reconcile the time required for spindle assembly in vivo with that observed in computer simulations of search and capture.