The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly
The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly
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DOI：
10.1091/mbc.e06-08-0724
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发表时间：
2007-01-01
影响因子：
3.3
通讯作者：
Walczak, Claire E.
中科院分区：
文献类型：
--
作者：
Ems-McClung, Stephanie C.;Hertzer, Kathleen M.;Walczak, Claire E.
纺锤体组装和准确的染色体分离需要对微管动力学进行适当的调节。 MCAK 是一种驱动蛋白 13，可催化解聚微管，调节生理微管动力学，是鸡蛋提取物中的主要灾难因子。对纯化的 GFP 标记的 MCAK 结构域突变体进行分析，以了解不同的 MCAK 结构域如何促进鸡蛋提取物中的体外微管解聚活性和生理纺锤体组装活性。我们的生化结果表明，颈部和 C 末端结构域对于强大的体外微管解聚活性都是必需的。特别是，颈部对于微管末端结合至关重要，而 C 末端结构域对于在过量微管蛋白异二聚体存在的情况下紧密微管结合至关重要。我们的生理学结果表明，N 端结构域对于调节微管动力学、刺激纺锤体双极性和动粒靶向至关重要；而C端结构域对于强大的微管解聚活性、限制纺锤体双极性和增强着丝粒靶向是必需的。出乎意料的是，精子诱导的纺锤体组装不需要强大的 MCAK 微管 (MT) 解聚活性。然而，正如 Ran 诱导的 aster 组装所测定的那样，高活性对于适当的生理 MT 动力学是必需的。我们认为 MCAK 活性在空间上受纺锤体组装过程中 N 端和 C 端结构域之间的相互作用控制。
Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.