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How to control microtubule dynamics: Function and regulation of kinesin -13s in Drosophila S2 cells.

机译：如何控制微管动力学：果蝇S2细胞中驱动蛋白-13s的功能和调控。

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摘要

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here we report that two proteins of the Drosophila melanogaster Kinesin-13 family, Klp10a and Klp59c, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; Klp10a stimulates catastrophe (a switch from growth to shrinkage) whereas Klp59c suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live cell analysis of cells expressing tagged kinesins reveal that Klp10a and Klp59c target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that Klp10a is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the Kinesin-13 family divide the labor of microtubule depolymerization.;To investigate regulation of Klp10a in vivo, peptide mapping by ESI-MALDI-TOF mass spectrometry was performed. These experiments identified a novel in vivo site of serine phosphorylation (s573) in the motor domain of Klp10a. To assess the role of serine 573 phosphorylation on the cellular activity of Klp10a, double transgenic Drosophila S2 cell lines expressing mRFP tubulin and GFP-Klp10a bearing phospho-mimic (S573E) or dephospho-mimic (S573A) were developed. These studies show that phosphorylated Klp10a is locked in a low depolymerizing activity state compared to the wild type. In vitro biochemical analysis of the mutants suggest that Klp10a phosphorylation directly affects the motor's ability to depolymerize microtubules, while not affecting its ability to efficiently bind microtubules.;To gain structural insights into how phosphorylation affects depolymerization activity, we also performed electron microscopy studies with purified recombinant proteins. In our assays we show that the phospo-mimic form of Klp10a is unable to form ring and spiral structures on the microtubules, while the wild type form can.*.;*This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: Windows MediaPlayer or RealPlayer.
机译：微管聚合和解聚的调节对于适当的细胞发育是必需的。在这里我们报告果蝇果蝇Kinesin-13家族的两个蛋白质，Klp10a和Klp59c，协同驱动相间细胞中的微管解聚。对这些蛋白质消耗掉的S2细胞中微管动力学的分析表明，这两种蛋白均能刺激解聚反应，但会改变动态不稳定的不同参数。 Klp10a刺激了灾难（从增长到收缩的转变），而Klp59c抑制了救援（从收缩到增长的转变）。此外，对表达标记的驱动蛋白的细胞进行免疫荧光和活细胞分析表明，Klp10a和Klp59c分别靶向于微管正末端的聚合和解聚。我们的数据还表明，Klp10a被正向追踪蛋白EB1沉积在微管上。我们的发现支持了其中Kinesin-13家族的这两个成员分担微管解聚工作的模型。为了研究Klp10a在体内的调控，通过ESI-MALDI-TOF质谱进行了肽图分析。这些实验在Klp10a的运动域中发现了一个新的丝氨酸磷酸化（s573）体内位点。为了评估丝氨酸573磷酸化对Klp10a细胞活性的作用，开发了表达mRFP微管蛋白和带有磷酸化模拟物（S573E）或脱磷酸化模拟物（S573A）的双转基因果蝇S2细胞系。这些研究表明，与野生型相比，磷酸化的Klp10a处于低解聚活性状态。突变体的体外生化分析表明，Klp10a磷酸化直接影响电机解聚微管的能力，而不影响其有效结合微管的能力。;为了获得结构化见解，了解磷酸化如何影响解聚活性，我们还进行了纯化的电子显微镜研究重组蛋白。在我们的测定中，我们证明了拟态形式的Klp10a不能在微管上形成环和螺旋结构，而野生型则可以。*。*本文是复合文件（包括纸质副本和纸质副本）。 CD作为论文的一部分）。该CD需要满足以下系统要求：Windows MediaPlayer或RealPlayer。

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  • 作者

    Mennella, Vito.;

  • 作者单位

    Yeshiva University.;

  • 授予单位 Yeshiva University.;
  • 学科 Biology Cell.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 132 p.
  • 总页数 132
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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