Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation
Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation
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DOI：
10.1038/ncb1702
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发表时间：
2008-04-01
影响因子：
21.3
通讯作者：
Davis, Trisha N.
中科院分区：
文献类型：
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作者：
Gestaut, Daniel R.;Graczyk, Beth;Davis, Trisha N.
在有丝分裂过程中，动粒与微管顶端形成持久的附着，并在Ipl 1（极光B）激酶磷酸化的作用下发生纠正性脱离（1）。Dam 1复合物是在体内建立和维持与微管顶端双向连接所必需的，它含有多个被Ipl 1磷酸化的位点（参考文献2-10）。此外，许多类似于运动舞蹈的功能可以在体外用纯Dam 1复合物重建（11-14）。这些功能被认为是来自于复合物自组装成环的能力（12，13，15 -17）。在这里，我们表明，环是没有必要的动态微管附着，Ipl 1依赖性的微管亲和力的调制或能力的Dam 1移动procectin与拆卸微管提示。使用两个基于荧光的测定，我们发现，该复合物表现出高亲和力的微管（Kd约为6 nM），这是减少在ser 20，一个单一的Ipl 1的目标残基Dam 1的磷酸化。此外，个别复合物进行一维扩散沿着微管和脱落后，Ipl 1磷酸化的2.5倍更频繁。由一到四个Dam 1复合物组成的颗粒-太少而不能包围微管-被捕获并通过拆卸尖端携带。因此，即使是少量的结合元件也可以提供动态的、磷酸调节的微管附着，从而促进准确的染色体分离。
During mitosis, kinetochores form persistent attachments to microtubule tips and undergo corrective detachment in response to phosphorylation by Ipl1 ( Aurora B) kinase(1). the Dam1 complex is required to establish and maintain bi-oriented attachment to microtubule tips in vivo, and it contains multiple sites phosphorylated by Ipl1 ( refs 2-10). Moreover, a number of kinetochore-like functions can be reconstituted in vitro with pure Dam1 complex(11-14). these functions are believed to derive from the ability of the complex to self-assemble into rings(12,13,15-17). Here we show that rings are not necessary for dynamic microtubule attachment, Ipl1-dependent modulation of microtubule affinity or the ability of Dam1 to move processively with disassembling microtubule tips. Using two fluorescence-based assays, we found that the complex exhibited a high affinity for microtubules ( K-d of approximately 6 nM) that was reduced by phosphorylation at ser 20, a single Ipl1 target residue in Dam1. Moreover, individual complexes underwent one-dimensional diffusion along microtubules and detached 2.5-fold more frequently after phosphorylation by Ipl1. Particles consisting of one to four Dam1 complexes - too few to surround a microtubule - were captured and carried by disassembling tips. thus, even a small number of binding elements could provide a dynamic, phosphoregulated microtubule attachment and thereby facilitate accurate chromosome segregation.