Investigating late mitotic events using Xenopus egg extracts

描述（由申请人提供）：在有丝分裂期间，一套完整的染色体分布到每个子细胞对生物体的生存能力至关重要，该过程中的缺陷被认为是导致癌症的原因。不可逆的和最重要的步骤发生在后期。在分裂后期，A复制的姐妹染色体分离并移动到基于微管的有丝分裂纺锤体的相反两极，而分裂后期B被定义为纺锤体极分离，其发生为在分离的染色体之间形成称为中心纺锤体的微管结构，其也有助于胞质分裂（将细胞捏成两个）。目前尚不清楚中央纺锤体是如何组装的，以及它在后期染色体分离中起什么作用。建议的实验是阐明后期事件使用爪蟾卵提取物，在体外重建细胞周期，并开放生化操作和高分辨率显微镜。中心纺锤体的组件将被检查，涉及在晚期有丝分裂事件，但其作用和相互作用还不清楚。这些成分中的许多在有丝分裂的其他方面（包括胞质分裂）具有重叠的功能。非洲爪蟾卵提取物系统的一个优点是胞质分裂不发生，允许在后期参与的中央纺锤体组件的具体研究。具体的目的是检查三个潜在的球员的中央主轴的作用，在周围的事件后期没有明确界定。化学抑制剂将与免疫荧光分析结合使用，以检查有丝分裂激酶Aurora B在后期事件中的作用。将开发试剂来破坏微管捆绑蛋白PRC1，并测量其对中央纺锤体形成和后期进展的影响。实验将进行，以确定和定义高尔基体衍生的膜在中央纺锤体组装和组织的作用。这些实验将揭示驱动后期的分子途径，并可能有助于开发化疗药物的新靶点。

DESCRIPTION (provided by applicant): Distribution of a complete set of chromosomes to each daughter cell during mitosis is essential for organism viability, and defects in this process are thought to contribute to cancer. The irreversible and most important steps occur during anaphase. At anaphase A duplicated sister chromosomes separate and move to opposite poles of the microtubule-based mitotic spindle, while anaphase B is defined as spindle pole separation, which occurs as a microtubule structure called the central spindle forms between the segregating chromosomes that also contributes to cytokinesis (pinching of the cell into two). It is unknown how the central spindle assembles and what role it plays in anaphase chromosome separation. The experiments proposed are to elucidate anaphase events using Xenopus egg extracts, which reconstitute the cell cycle in vitro, and are open to biochemical manipulation and high resolution microscopy. Components of the central spindle will be examined that are implicated in late mitotic events, but whose roles and interactions are not understood. Many of these components have overlapping functions in other aspects of mitosis including cytokinesis. An advantage of the Xenopus egg extract system is that cytokinesis does not occur, allowing for the specific study of central spindle components involvement at anaphase. The specific aims are to examine three potential players of the central spindle whose role in the events surrounding anaphase are not clearly defined. Chemical inhibitors will be used in conjunction with immunofluorscence analysis to examine the role of the mitotic kinase Aurora B in anaphase events. Reagents will be developed to disrupt the microtubule bundling protein PRC1 and measure its effects on central spindle formation and anaphase progression. Experiments will be performed to identify and define the role of Golgi-derived membrane in central spindle assembly and organization. These experiments will reveal the molecular pathways that drive anaphase and may be useful developing novel targets for chemotherapeutic agents.