Organization of the Mammalian Mitotic Spindle

描述(申请人提供)：准确的染色体分离对于物种的繁殖和细胞的存活至关重要，它是由一种称为纺锤体的复杂的微管结构驱动的。密集的生化、遗传和蛋白质组学工作提供了广泛的蛋白质目录，参与纺锤体组织和纺锤体依赖的染色体运动。然而，这些努力并没有揭示确保哺乳动物细胞中忠实的染色体分离的分子机制。最近，我们发现人类肿瘤细胞中染色体错误分离的最常见原因是动粒微管(K-MT)附着错误的持续存在。然而，我们对如何监管K-MT附件以促进纠错的理解仍然完全不完整。我们不了解不同的分子成分如何在细胞周期转变过程中创造一致的输出来微调K-MT附着稳定性。我们也不了解非整倍体如何影响基因组稳定性和细胞存活。我们在即将到来的资助期的目标是结合生化方法和活细胞成像来测试模型并确定有丝分裂中K-MT连接的调节机制。了解这些机制可能会为癌症治疗带来新的治疗方法。这项建议的具体目标是：1.使用活细胞成像来确定从前中期到中期的潜在的生化变化；将活细胞成像与分子工具相结合，测试单基因拷贝数变化足以通过破坏K-MT附着动力学来破坏忠实的染色体分离的模型；3)量化持续的K-MT附着错误造成的遗传损害；以及4.在活体动物中测试抑制非整倍体细胞生长的机制。

DESCRIPTION (provided by applicant): Accurate chromosome segregation is essential for the propagation of species and the viability of cells, and is driven by a complex microtubule-based structure called the spindle. Intensive biochemical, genetic, and proteomic efforts provide an extensive catalogue of proteins that participate in spindle organization and spindle-dependent chromosome movement. However, these efforts don't reveal the molecular mechanisms that ensure faithful chromosome segregation in mammalian cells. Recently, we showed that the most common cause of chromosome mis-segregation in human tumor cells is the persistence of kinetochore-microtubule (K- MT) attachment errors. However, our understanding for how K-MT attachments are regulated to promote error correction remains starkly incomplete. We don't understand how different molecular components create a coherent output to fine-tune K-MT attachment stability during cell cycle transitions. We also don't understand how aneuploidy influences genome stability and cell survival. It is our goal in the forthcoming funding period to combine biochemical methods and live cell imaging to test models and determine the mechanisms of the regulation of K-MT attachments in mitosis. Understanding these mechanisms could lead to new therapeutic approaches for cancer treatment. The specific aims of this proposal are, 1.) To use live cell imaging to determine the biochemical changes underlying the transition from prometaphase to metaphase; 2.) To combine live cell imaging with molecular tools to test the model that changes in the copy number of single genes is sufficient to undermine faithful chromosome segregation by disrupting K-MT attachment dynamics; 3.) To quantify the genetic damage caused by persistent K-MT attachment errors; and 4.) To test mechanisms that suppress aneuploid cell growth in live animals.