Investigating late mitotic events using Xenopus egg extracts

描述(由申请人提供)：在有丝分裂过程中，将一套完整的染色体分配给每个子细胞是生物体生存所必需的，这一过程中的缺陷被认为是导致癌症的原因。不可逆转的和最重要的步骤发生在后期。在后期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，而后期B被定义为纺锤体极分离，它发生在分离染色体之间的称为中心纺锤体的微管结构中，这也有助于细胞质分裂(将细胞挤压成两个)。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扮演了什么角色。提出的实验是为了阐明后期事件使用非洲爪哇卵子提取物，在体外重建细胞周期，并开放的生化操作和高分辨率显微镜。我们将研究与晚期有丝分裂事件有关，但其作用和相互作用尚不清楚的中央纺锤体的组成部分。这些成分中的许多在有丝分裂的其他方面有重叠的功能，包括胞质分裂。非洲爪哇卵子提取系统的一个优点是不发生细胞质分裂，允许对中央纺锤体组件参与后期的具体研究。具体目的是考察三个可能的中心纺锤体的参与者，他们在后期周围事件中的作用没有明确的定义。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绑定蛋白PRC1，并测量其对中心纺锤体形成和后期进展的影响。将进行实验，以确定和确定高尔基体衍生膜在中心纺锤体组装和组织中的作用。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.