Regulation of Chromosome Segregation in Human Cells

项目总结/摘要 为了确保基因组的稳定性，纺锤体组装检查点（SAC）延迟后期 直到所有的动粒对都从相反的方向连接到微管纤维的末端， 纺锤杆此外，不适当的染色体-纺锤体连锁是选择性的， 在后期之前被摧毁。通过基因组编辑和化学遗传学， SAC激酶Mps 1控制这两种反应。通过大规模 磷酸蛋白质组学，我们发现了新的Mps 1调节底物在动粒， 包括Ska复合物（这有助于Ndc 80复合物抓住动态 微管末端）和与外套体相关的RZZ复合物（其整合Mad 1-Mad 2 依赖于SAC信号传导和动力蛋白依赖性转运）。在目标1中，我们研究如何 Mps 1影响Ska和Ndc 80配合物的协同作用 微管，无论是在细胞和体外系统重建。在目标2中，我们剖析了 Mps 1激活RZZ复合物，用于Mad 1-Mad 2招募，长期SAC停滞， 独立动粒的结构扩张。在目标3中，我们开发了一种新的化学物质- Aurora A的遗传系统，一种有丝分裂激酶，仍然很难研究， 目前的抑制剂在体外具有Aurora A选择性，但在体外交叉抑制Aurora B， 体内生物活性浓度。这些研究将揭示动粒如何 结构、微管附着和SAC信号在有丝分裂过程中进化， 最大化无错误染色体分离的概率。最终这 这些信息将促进靶向非整倍体的治疗剂的开发- 相关疾病，如癌症。

PROJECT SUMMARY/ABSTRACT To ensure genome stability, the spindle assembly checkpoint (SAC) delays anaphase until all pairs of kinetochores are attached to the ends of microtubule fibers from opposite spindle poles. In addition, improper chromosome-spindle linkages are selectively destroyed before anaphase. Through genome editing and chemical genetics, we found that the SAC kinase Mps1 controls both responses. Through large-scale phosphoproteomics, we discovered novel Mps1-regulated substrates at the kinetochore, including the Ska complex (which helps the Ndc80 complex grip onto dynamic microtubule ends) and the coatomer-related RZZ complex (which integrates Mad1-Mad2 dependent SAC signaling and dynein-dependent transport). In Aim 1, we investigate how Mps1 affects the synergistic interactions of the Ska and Ndc80 complexes on dynamic microtubules, both in cells and in reconstituted in vitro systems. In Aim 2, we dissect how Mps1 activates the RZZ complex for Mad1-Mad2 recruitment, long-term SAC arrest, and structural expansion of unattached kinetochores. In Aim 3, we develop a new chemical- genetic system for Aurora A, a mitotic kinase that remains difficult to study because current inhibitors with Aurora A selectivity in vitro nonetheless cross-inhibit Aurora B at bioactive concentrations in vivo. Together these studies will reveal how kinetochore structure, microtubule attachment, and SAC signaling evolve during mitosis, in order to maximize the probability of error-free chromosome segregation. Ultimately this information will empower development of therapeutic agents that target aneuploidy- associated diseases such as cancer.