Functional properties of centrosomes in somatic cells

描述(申请人提供)：这项提案的首要目标是建立负责在细胞分裂过程中适当分离染色体的机制。染色体分离的高保真度和低保真度是正常细胞和癌细胞之间的一个特征差异，了解导致这种差异的机制是开发新的抗癌疗法和治疗策略的关键一步。为了进行适当的分离，每条染色体都必须附着在有丝分裂装置的微管上(称为纺锤体)，这种附着是由染色体主体上的特殊大分子组装介导的，称为动点。最近的研究表明，微管/着丝粒相互作用的几何约束在确保有丝分裂纺锤体的正确形成方面发挥着至关重要的作用，而有丝分裂纺锤体的正确形成对于忠实的染色体分离是必不可少的。这些几何约束是由动粒内分子复合体的分布和取向建立的，这个项目旨在揭示动粒的结构以及该结构中单个分子复合体的取向如何适应有丝分裂纺锤体内快速变化的条件。我们的目标是剖析在有丝分裂的不同阶段和动粒的不同生理状态中发生在动粒内的结构重组的本质。为了实现这些目标，我们使用了一种独特的方法，即对运动中枢进行高分辨率的电子显微镜分析，这些运动中枢的行为和运动在活细胞中被跟踪到固定的时刻。这种被称为相关LM/EM的方法使我们可以评估处于已知功能状态的动心的结构，从而揭示在各种生理状态之间转换过程中发生的确切结构变化。我们还将在两种功能表征的实验条件下进行动点的比较结构分析：1)低浓度紫杉醇处理，已知这种条件允许在没有着丝粒张力的情况下满足有丝分裂检查点；2)处理 尽管着丝粒高度伸展，但低浓度的诺可达唑会阻碍SAC的满意。总之，这些研究将使我们能够为动粒的结构创建一个准确的模型，并确定动粒内分子成分的结构和分布如何适应各种类型的微管附着和各种类型的染色体运动。

DESCRIPTION (provided by applicant): The overarching goal of this proposal is to establish the mechanisms responsible for proper segregation of chromosomes during cell division. High vs. low fidelity of chromosome segregation is a characteristic difference between normal vs. cancer cells and understanding the mechanisms responsible for this difference is an essential step towards developing novel anti-cancer therapeutics and treatments strategies. For proper segregation, each chromosome must attach to the microtubules of mitotic apparatus (termed the 'spindle') and this attachment is mediated by special macromolecular assemblies on the chromosome's body known as the 'kinetochores'. The project is enabled by recent demonstrations that geometric constraints of microtubule/kinetochore interactions play a vital role in ensuring proper formation of the mitotic spindle essential for faithful chromosome segregation. These geometric constraints are established by the distribution and orientation of molecular complexes within the kinetochore and this project is designed to reveal how the architecture of the kinetochore, and the orientation of individual molecular complexes within that architecture, adapt to rapidly changing conditions within the mitotic spindle. We aim to dissect the nature of structural reorganizations known to occur within the kinetochore during different stages of mitosis and different physiological states of the kinetochore. To achieve these goals we use a unique approach in which high- resolution electron-microscopy analyses are conducted on the kinetochores whose behavior and movements were followed in live cells up to the moment of fixation. This approach, termed correlative LM/EM allows us evaluate the structure of kinetochores that are in known functional state thus revealing the exact structural changes that occur during transitions between various physiological states. We will also perform comparative structural analyses of kinetochores upon two functionally-characterized experimental conditions: 1) treatment with low concentration of taxol, a condition known to allow satisfaction of the mitotic checkpoint despite the lack of centromere tension vs. 2) treatment with low concentration of nocodazole, a condition known to prevent satisfaction of the SAC despite highly-stretched centromeres. Together, these studies will allow us to create an accurate model for the structure of the kinetochore and determine how the architecture and the distribution of molecular components within the kinetochore adapt in response to various types of microtubule attachment and various types of chromosome movements.