Chromosome dynamics and organizations necessary for faithful chromosome segregation

项目摘要 细胞分裂是一个保守的过程，通过这个过程，复制的染色体被均等地分成两个子细胞。 这个过程中的错误通常会导致染色体的获得或丢失，称为非整倍体，这可能导致和 促进肿瘤和发育疾病。在有丝分裂过程中，染色体动态地改变它们的 通过着丝粒产生的力以力依赖的方式定位，构建大分子蛋白质结构 作为微管组装平台的着丝粒染色质上。虽然染色体的领土，区域 优先被间期核中的特定染色体占据，已经建立，并且已知是 参与基因调控和基因组保护，有丝分裂中染色体组织的存在和功能 还没有得到充分的探索。我们的长期目标是表征“有丝分裂染色体领域”， 哺乳动物细胞，并揭示背后的染色体组织和时空调控功能， 动粒动力学确保忠实的染色体分离。在本提案中，我们将检验以下假设： 用超分辨率显微镜方法观察，有丝分裂中的染色体组织与间期核中的一样， 我们最近开发的，这将使我们能够识别完整的个体染色体，并确定它们的空间位置， 哺乳动物细胞中的组织。如果存在有丝分裂染色体区域，我们将探讨它们是如何以及何时发生的。 它们在有丝分裂过程中的进化。我们还假设，主要的有丝分裂缺陷（未对齐 染色体、落后染色体和染色体桥）与不正常染色体相关 organization.我们将通过以下方法来验证这一假设： 增加频率并确定其位置。有丝分裂细胞有两种主要的纠正有丝分裂的途径， 由Aurora A或Aurora B激酶介导的错误。这两种激酶都是空间调节的，并磷酸化高度依赖于蛋白质的激酶。 保守的微管结合动粒蛋白，Ndc 80/Hec 1，使不适当的微管结合不稳定， 促进错误纠正和SAC（纺锤体组装检查点）活性的调节。Aurora A介导错误 校正需要错误染色体接近纺锤体极点，极光A集中在那里。对 另一方面，极光B介导的错误校正依赖于动粒的动态变形。这些 这表明有丝分裂染色体定位，加上动粒动力学，协调合作 Aurora A和Aurora B介导的纠错机制之间的差异。我们将分析 染色体定位和动粒动力学对极光A和极光B错误校正使用力- 校准的微针和我们最近开发的半自动定量显微镜分析软件 3D Speckler（3D Speckler）我们提出的工作将为有丝分裂提供新的， 染色体组织及其对确保染色体分离完整性的贡献， 有助于为癌症和发育疾病制定更好的治疗和检测策略， 改善患者的预后。

PROJECT SUMMARY Cell division is a conserved process by which replicated chromosomes are equally partitioned into two daughter cells. Errors in this process often result in gains or losses of chromosomes, known as aneuploidy, which can cause and promote tumors and developmental diseases. During mitotic progression, chromosomes dynamically change their positions in a force-dependent manner via forces generated at kinetochores, macro-molecular protein structures built on centromeric chromatin that serves as platforms for microtubule assembly. While chromosome territories, regions preferentially occupied by specific chromosomes in interphase nuclei, have been established and are known to be involved in gene regulation and genomic protection, the presence and function of chromosome organization in mitosis have not been adequately explored. Our long-term goals are to characterize “mitotic chromosome territories” in mammalian cells and to uncover the function behind spatiotemporal regulation of both chromosome organization and kinetochore dynamics in ensuring faithful chromosome segregation. In this proposal, we will test the hypothesis that there exist chromosome organizations in mitosis as in interphase nuclei using a super-resolution microscopy method we recently developed, which will allow us to identify full sets of individual chromosomes and determine their spatial organization in mammalian cells. If there exist mitotic chromosome territories, we will explore how and when they are established and their evolution throughout mitosis. We also hypothesize that major mitotic defects (unaligned chromosomes, lagging chromosomes, and chromosome bridges) are associated with improper chromosome organization. We will examine this hypothesis by identifying which chromosomes are involved in each defect with increased frequency and determine their positionings. Mitotic cells have two major pathways for correcting mitotic errors, mediated by Aurora A or Aurora B kinases. Both kinases are spatially regulated and phosphorylate a highly conserved microtubule-binding kinetochore protein, Ndc80/Hec1, to destabilize improper microtubule bindings for promotion of error correction and regulation of SAC (spindle assembly checkpoint) activity. Aurora A-mediated error corrections require proximity of erroneous chromosomes to the spindle poles, where Aurora A is concentrated. On the other hand, Aurora B-mediated error corrections depend on dynamic deformations of kinetochores. These suggest that mitotic chromosome positioning, coupled with kinetochore dynamics, orchestrate the cooperation between Aurora A and Aurora B-mediated error correction machineries. We will dissect the contributions of chromosome positioning and kinetochore dynamics towards Aurora A and Aurora B error corrections using force- calibrated microneedles and a semi-automated, quantitative microscopy analysis software that we recently developed called the 3D speckle analyzer (3D-Speckler). Our proposed work will provide new, mechanistic insights into mitotic chromosome organization and its contribution toward ensuring the integrity of chromosome segregation, which will contribute towards developing better therapeutic and detection strategies for cancer and developmental diseases for improved patient outcomes.