Motor Protein Dynamics and Mitotic Mechanisms

描述（由申请人提供）：在细胞分裂过程中，有丝分裂纺锤体（一种多组分机器）将细胞复制的DNA准确分离成两个子细胞。这一过程的失败与许多发育缺陷和肿瘤发生有关。纺锤体的双极结构引导姐妹染色体进入每个子细胞。本计画的长期目标是了解双极纺锤体组装的分子机制。为此，我们集中我们的研究Eg 5，一个进化上保守的驱动蛋白相关的蛋白质所需的纺锤体形成。在细胞分裂期间Eg 5功能的丧失导致单极纺锤体并阻断细胞周期。有丝分裂特异性磷酸化在Eg 5中的保守序列（称为bimC-box）已被提出来调节Eg 5的纺锤体靶向。连同动力蛋白，负端定向微管为基础的电机，其调节dynactin，Eg 5组织纺锤体微管和控制纺锤体长度。我们将使用Eg 5作为工具来剖析双极纺锤体形成和纺锤体中力产生的分子机制。具体而言，我们将：（1）利用荧光散斑显微镜和荧光的光活化，研究微管动力学和组织对Eg 5纺锤体组装过程中动力学行为的影响。(2)通过产生运动性降低和微管亲和力降低的Eg 5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Eg 5耗尽的细胞提取物中拯救纺锤体形成的能力，确定Eg 5的运动功能在纺锤体组装中的作用。(3)通过比较在bimC-box磷酸化的Eg 5与不能磷酸化的Eg 5突变体，分析Eg 5磷酸化对其运动功能和结构的影响。(4)确定如何dynactin影响Eg 5功能的纺锤体，通过检查dynactin和Eg 5之间的相互作用，使用免疫沉淀和亲和层析，并通过比较dynactin和Eg 5在纺锤体中的动态行为，使用荧光显微镜。了解Eg 5在纺锤体中的功能机制应该会导致Eg 5抑制剂的改进，并可能导致更好的抗癌治疗。

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.