Motor Protein Dynamics and Mitotic Mechanisms

描述(申请人提供)：在细胞分裂过程中，有丝分裂纺锤体，一种多组件机器，准确地将细胞的复制DNA分离成两个子细胞。这一过程的失败与许多发育缺陷和肿瘤的发生有关。纺锤体的两极组织引导姐妹染色体进入每个子细胞。该项目的长期目标是了解两极纺锤体组装的分子机制。为此，我们将研究重点放在EG5上，这是一种进化上保守的动蛋白相关蛋白，是纺锤体形成所必需的。在细胞分裂过程中，EG5功能的丧失会导致单极纺锤体，并阻断细胞周期。有丝分裂特异的EG5保守序列的磷酸化(称为BIMC-box)被认为是调节EG5‘S纺锤体靶向的机制。EG5与Dynein一起组织纺锤体微管，控制纺锤体长度。Dynein是一种负端导向的微管马达，是一种基于微管的马达，它的调节因子是Dynactin。我们将使用EG5作为工具来剖析两极纺锤体形成和纺锤体内力产生的分子机制。具体地说，我们将：(1)利用荧光散斑显微镜和荧光光激活技术，检测微管动力学和组织对纺锤体组装过程中EG5动态行为的影响。(2)通过产生运动性降低和微管亲和力降低的突变体，测试这些突变体在纺锤体中的定位和动力学，以及它们在缺失EG5的细胞提取液中拯救纺锤体形成的能力，来确定EG5‘S运动功能在纺锤体组装中的作用。(3)通过比较在BIMC-box处磷酸化的EG5和不能磷酸化的突变体，分析EG5的磷酸化对其运动功能和结构的影响。(4)通过免疫沉淀和亲和层析检测dynactin和EG5之间的相互作用，以及通过荧光显微镜比较dynactin和EG5在纺锤体中的动态行为，来确定dynactin对纺锤体中EG5功能的影响。了解纺锤体中EG5功能的机制将有助于改进EG5的抑制剂，并可能产生更好的抗癌疗法。

DESCRIPTION (provided by applicant): During cell division the mitotic spindle, a multi-component machine, accurately segregates a cell's replicated DNA into two daughter cells. Failure of this process has been linked to numerous developmental defects and oncogenesis. The bipolar organization of the spindle directs movements of sister chromosomes into each daughter cell. The long-term goal of this project is to understand the molecular mechanisms of bipolar spindle assembly. To this end, we have focused our studies on Eg5, an evolutionarily conserved kinesin-related protein required for spindle formation. Loss of Eg5 function during cell division results in monopolar spindles and blocks the cell cycle. Mitosis-specific phosphorylation at a conserved sequence in Eg5 (called the bimC-box) has been proposed to regulate Eg5's spindle targeting. Together with dynein, a minus-end directed microtubule-based motor, and its regulator dynactin, Eg5 organizes spindle microtubules and controls spindle length. We will use Eg5 as a tool to dissect the molecular mechanisms of bipolar spindle formation and force generation in the spindle. Specifically, we will: (1) Examine the influence of microtubule dynamics and organization on the dynamic behavior of Eg5 during spindle assembly, by using fluorescent speckle microscopy and photoactivation of fluorescence. (2) Determine the role of Eg5's motor function in spindle assembly by generating Eg5 mutants with reduced motility and with reduced microtubule affinities, testing these mutants' localization and dynamics in spindles and their ability to rescue spindle formation in Eg5-depleted cell extracts. (3) Analyze the effect of Eg5 phosphorylation on its motor function and structure, by comparing Eg5 phosphorylated at the bimC-box to an Eg5 mutant that cannot be phosphorylated. (4) Determine how dynactin influences Eg5 function in spindles, by examining interactions between dynactin and Eg5 using immunoprecipitation and affinity chromatography, and by comparing the dynamic behavior of dynactin and Eg5 in spindles using fluorescence microscopy. Understanding the mechanism of Eg5 function in spindles should lead to improved inhibitors of Eg5 and could result in better anti-cancer therapeutics.