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，复制的染色体被平均分割成两个子代 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 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 通过在运动中心产生的力，大分子以依赖于力的方式改变它们的位置 蛋白质结构建立在着丝粒染色质上，作为微管组装的平台。而当 染色体区域，即间期核中特定染色体优先占据的区域，有 已被证实并已知参与基因调控和基因组保护，存在和 染色体组织在有丝分裂中的作用还没有得到充分的研究。我们的长期目标是 描述哺乳动物细胞中的“有丝分裂染色体区域”并揭示其背后的功能 确保忠诚的染色体组织和动粒动力学的时空调节 染色体分离。在这个方案中，我们将检验存在染色体组织的假设 在有丝分裂中，就像在间期核中一样，使用我们最近开发的超分辨率显微镜方法，这将 使我们能够识别哺乳动物的全套个体染色体并确定它们的空间组织 细胞。如果存在有丝分裂染色体区域，我们将探索它们是如何以及何时建立的，以及它们的 在整个有丝分裂过程中的进化。我们还假设主要的有丝分裂缺陷(染色体不对齐，滞后 染色体和染色体桥)与不正确的染色体组织有关。我们会 通过以更高的频率确定哪些染色体与每个缺陷有关来检验这一假设 并确定它们的位置。有丝分裂细胞有两条主要途径来纠正有丝分裂错误 由Aurora A或Aurora B激酶激活。这两个激酶都是空间调节的，并磷酸化一个高度保守的 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。极光A介体 纠错需要错误的染色体接近纺锤体极点，Aurora A在纺锤体极点 集中精力。另一方面，Aurora B中介的误差校正依赖于 动感家务。这些表明，有丝分裂染色体的定位，加上动粒动力学， 协调Aurora A和Aurora B之间的合作--由Aurora B调解的纠错机制。我们会 剖析染色体定位和着丝粒动力学对极光A和极光B的贡献 使用力校准微针和半自动定量显微镜分析进行误差校正 我们最近开发的软件称为3D散斑分析器(3D-SPEKLER)。我们提议的工作将 提供了对有丝分裂染色体组织及其对确保 染色体分离的完整性，这将有助于开发更好的治疗和检测 癌症和发育疾病的战略，以改善患者的预后。

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.