Chromosome dynamics and organizations necessary for faithful chromosome segregation

项目总结 细胞分裂是一个保守的过程，通过这个过程，复制的染色体被平等地分割成两个子细胞。 这一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，这可能会导致 促进肿瘤和发育性疾病。在有丝分裂过程中，染色体会动态地改变它们的 位置依赖于力的方式，通过在运动中心产生的力，即构建的大分子蛋白质结构 关于作为微管组装平台的着丝粒染色质。而染色体的区域、区域 在间期核中优先被特定染色体占据的基因已被建立，并已知是 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 还没有得到充分的勘探。我们的长期目标是确定“有丝分裂染色体区域”的特征。 并揭示染色体组织和染色体的时空调节背后的功能 动粒动力学在确保忠实的染色体分离中的作用。在这个提案中，我们将检验假设 用超分辨显微镜观察发现，有丝分裂过程中存在着与间期核一样的染色体组织。 我们最近开发的，这将使我们能够识别完整的个体染色体组并确定它们的空间 哺乳动物细胞中的组织。如果存在有丝分裂的染色体区域，我们将探索它们是如何以及何时存在的。 以及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、落后的染色体和染色体桥)与染色体不正确有关 组织。我们将通过确定哪些染色体与每个缺陷有关来检验这一假设 增加频率并确定它们的位置。有丝分裂细胞有两条纠正有丝分裂的主要途径 由Aurora A或Aurora B激酶介导的错误。这两种激酶都受空间调控，并高度磷酸化一种 保守的微管结合动粒蛋白Ndc80/Hec1，以破坏不正确的微管结合 促进纠错和规范SAC(主轴组件检查点)活动。Aurora A-中介的错误 纠正需要将错误的染色体靠近纺锤体极，那里是极光A的集中地。在……上面 另一方面，极光B介导的误差校正依赖于动心的动态变形。这些 建议有丝分裂染色体定位，加上着丝粒动态，协调合作 Aurora A和Aurora B之间的纠错机制。我们将仔细分析 Aurora A和Aurora B的染色体定位和动粒动力学使用力校正法 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 称为3D散斑分析器(3D-SPEKLER)。我们提议的工作将为有丝分裂提供新的、机械性的见解 染色体组织及其对确保染色体分离完整性的贡献，这将 有助于为癌症和发育性疾病制定更好的治疗和检测战略 改善了患者的预后。

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.