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首页> 外文学位 >Molecular dissection of Hec1 reveals novel roles in spindle assembly checkpoint, bipolar spindle assembly, and kinetochore targeting.
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Molecular dissection of Hec1 reveals novel roles in spindle assembly checkpoint, bipolar spindle assembly, and kinetochore targeting.

机译：Hec1的分子解剖揭示了纺锤体装配检查站，双极纺锤体装配和动粒靶向中的新作用。

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摘要

Hec1 is a critical structural component at the kinetochore outer layer, where it provides a major microtubule attachment site for establishing a stable bipolar spindle. Depletion of Hec1 results in defective spindle assembly checkpoint (SAC), unstable kinetochore-microtubule attachment, and multipolar mitotic spindles. Using siRNA technology and retrovirus infection, I am able to replace endogenous Hec1 with siRNA resistant Hec1 point mutants and deletion mutants. This allows me to precisely identify the amino acid residue or regions of Hec1 responsible for its role in spindle assembly checkpoint, bipolar spindle formation, and kinetochore targeting, and to elucidate the mechanism by which it is achieved.First, I discovered that Nek2-dependent phosphorylation of Hec1 S165 was required to recruit SAC proteins Mad1 and Mad2 to the kinetochores. Dephosphorylation mediated by protein phosphatase 1 is most likely responsible for dephosphorylation Hec1 S165, thus allowing for anaphase onset. In addition, I demonstrated that Hec1 and Hice1 contributes to centrosome-dependent microtubule growth during mitosis and their interaction at the centrosome is required for bipolar spindle formation. Lastly, Hec1's coiled-coil domain, which accounts for two-thirds of the encoded protein, functions as a scaffold for kinetochore-associated proteins. To identify novel Hec1 functions mediated by the coiled-coil domain, we performed a structure-function analysis of Hec1's first coiled-coil domain by generating a series of GFP-tagged deletion mutants spanning the entire first coiled-coil domain of Hec1 in which two consecutive leucine heptad repeats were removed at a time. Surprisingly, we identified two consecutive leucine heptad repeats that were required for Hec1 localization to the kinetochore. Consistently, this Hec1 deletion mutant has a dramatic reduction in its association with Nuf2, Spc24, and Spc25 compared to wildtype Hec1.
机译：Hec1是线粒体外层的关键结构组件，在这里它为建立稳定的双极纺锤体提供了主要的微管附着位点。 Hec1的耗尽会导致纺锤体装配检查点（SAC）损坏，动粒体-微管附着不稳定以及多极有丝分裂纺锤体。使用siRNA技术和逆转录病毒感染，我能够用抗siRNA的Hec1点突变体和缺失突变体替代内源性Hec1。这使我能够精确地识别Hec1在纺锤体装配检查点，双极纺锤体形成和线粒体靶向中起作用的氨基酸残基或区域，并阐明实现它的机理。首先，我发现了Nek2依赖性为了将SAC蛋白Mad1和Mad2募集到动植物体内，需要Hec1 S165的磷酸化。由蛋白磷酸酶1介导的去磷酸化最有可能引起Hec1 S165的去磷酸化，因此允许后期发生。此外，我证明了Hec1和Hice1在有丝分裂期间有助于中心体依赖的微管生长，并且双极纺锤体形成需要它们在中心体上的相互作用。最后，Hec1的卷曲螺旋结构域（占编码蛋白的三分之二）起着与线粒体相关蛋白的支架作用。为了鉴定由卷曲螺旋结构域介导的新的Hec1功能，我们通过产生一系列跨越整个Hec1的第一个卷曲螺旋结构域的GFP标记的缺失突变体，对Hec1的第一个卷曲螺旋结构域进行了结构功能分析。一次去除连续的亮氨酸七肽重复序列。出人意料的是，我们确定了两个连续的亮氨酸七肽重复序列，这些重复是Hec1定位到动粒体所需的。一致地，与野生型Hec1相比，此Hec1缺失突变体与Nuf2，Spc24和Spc25的结合显着减少。

著录项

  • 作者

    Wei, Randy Li-Hung.;

  • 作者单位

    University of California, Irvine.;

  • 授予单位 University of California, Irvine.;
  • 学科 Biology General.Biology Cell.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 180 p.
  • 总页数 180
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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