Molecular Dissection of Cytokinesis

描述(申请人提供)：我们试图了解动物细胞中细胞分裂的分子基础，即胞质分裂。中心纺锤体是纺锤体后期的一组反平行的束状微管，调节肌动球蛋白收缩环的形成，是完成胞质分裂所必需的。我们认为，中心纺锤体的组织和功能是中心纺锤体复合体的结构组织和生化性质的结果。Centralspindlin是一个进化保守的多聚体复合体，它含有一个激动素样蛋白(Zen-4/MKLP1)和一个Rho家族间隙(Cyk-4/MgcracGAP)，分别在晚期和末期高度集中在中央纺锤体和中体上。我们建议将线虫胚胎和哺乳动物细胞的体外生物化学和体内挽救分析结合起来，以解决三个特定目标：(1)剖析中心纺锤体复合体的分子结构，并表征非典型的动蛋白Zen-4。通过在相对分离的情况下表征中央纺锤体复合体中的关键蛋白质结构域，我们将建立必要的生化框架，以了解这些分子在更复杂的反应中以及在体内的功能。(2)破译中心纺锤体组装的分子机制。中心主轴组件将在体外由纯化的组件重组，以确定这组动态组件组装成高度有序和稳定结构的原则。(3)确定中央纺锤体如何调节胞质分裂的完成。Centralspindlin包含两个不同的蛋白质结构域，被认为用于控制胞质分裂的后期步骤。因此，将建立这些结构域的分子功能。这项研究将提供对细胞增殖至关重要的细胞结构的分子洞察力。因此，我们的研究有助于开发治疗癌症的新型抗有丝分裂药物。此外，对Zen-4的分子解剖将加深我们对微管马达作用机制的理解。

DESCRIPTION (provided by applicant): We seek to understand the molecular basis of cell division, cytokinesis, in animal cells. The central spindle, a set of antiparallel bundled microtubules in the anaphase spindle, regulates formation of the actomyosin- based contractile ring and is essential for completion of cytokinesis. We propose that the organization and function of the central spindle is a consequence of the structural organization and biochemical properties of the centralspindlin complex. Centralspindlin is an evolutionary conserved, multimeric complex containing a kinesin-like protein (ZEN-4/MKLP1) and a Rho family GAP (CYK-4/MgcRacGAP) that is highly concentrated on the central spindle and midbody during anaphase and telophase, respectively. We propose to combine in vitro biochemistry and in vivo rescue assays in C. elegans embryos and in mammalian cells to address three specific aims: (1) To dissect the molecular organization of the centralspindlin complex and to characterize the atypical kinesin protein, ZEN-4. By characterizing the critical protein domains in the centralspindlin complex in relative isolation, we will develop the biochemical framework necessary to understand the function of these molecules in more complex reactions as well as in vivo. (2) To decipher the molecular mechanism of central spindle assembly. Central spindle assembly will be reconstituted in vitro from purified components in order to define the principles by which this set of dynamic components assemble into a highly ordered and stable structure. (3) To determine how the central spindle mediates completion of cytokinesis. Centralspindlin contains two distinct protein domains that are proposed to control late steps in cytokinesis. Therefore, the molecular function of these domains will be established. This research will provide molecular insights into a cellular structure that is critical for cell multiplication. Therefore our research could contribute to the development of novel anti-mitotic agents for the treatment of cancer. Furthermore, molecular dissection of ZEN-4 will enhance our understanding of the mechanism of action of microtubule motors.