Integrative analyses of the kinetochore and the spindle assembly checkpoint

动粒和纺锤体装配检查点的综合分析

• 批准号: 10188559
• 负责人: Ajit Joglekar
• 金额: $ 53.02万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188559
• 关键词: Aneuploidy; Architecture; Biochemical; Cancerous; Cell Death; Cell division; Cells; Chromosome Segregation; Chromosomes; Complex; Daughter; Defect; Diffuse; Ensure; Eukaryotic Cell; Fluorescence Microscopy; Generations; Genome; Goals; Human; Kinetochores; Knowledge; Measures; Mitosis; Modeling; Molecular; Output; Process; Proteins; Reaction; Research; Saccharomycetales; Shapes; Signal Transduction; Structure; System; Techniques; Thermodynamics; Work; Yeasts; base; chromosome movement; daughter cell; design; insight; mathematical model; nanoscale; protein complex; reconstitution; tumorigenesis

The primary goal of mitosis is to make two genetically identical copies of the dividing cell. To achieve this goal, the dividing cell must segregate exactly one copy of each chromosome into each daughter. Even a single error in chromosome segregation results in aneuploidy, which in turn leads to a plethora of defects, from cell death to tumorigenesis. Therefore, to accomplish accurate chromosome segregation, the eukaryotic cell uses two highly sophisticated systems: the kinetochore and the Spindle Assembly Checkpoint (SAC). The kinetochore is a multi- protein machine that moves and segregates each chromosome. If it is unable to do so, the kinetochore activates the SAC. The SAC is a signaling cascade that generates a diffusible checkpoint complex that arrests cell division. Extensive research has compiled a nearly complete list of proteins and activities necessary for the two systems. However, fundamental questions regarding each remain unanswered. How does the kinetochore seamlessly integrate the disparate molecular mechanisms that generate chromosome movement and activate the SAC? How does the cell calibrate SAC signaling output to maximize accurate chromosome segregation, but minimize the duration of mitosis? The most significant challenge in defining the molecular mechanisms of kinetochore function is its highly complex protein architecture. My lab reconstructed the nanoscale protein architecture of the kinetochore in budding yeast by developing an array of fluorescence microscopy techniques. We used this knowledge to undertake `architecture-function' analyses of the yeast kinetochore. Our work reveals how kinetochore architecture shapes functional mechanisms. Our next goal is to define how the architecture of the much more complex, human kinetochore shapes emergent mechanisms of force generation and SAC activation. The most significant challenge in studying the biochemical design of the SAC is our inability to measure the thermodynamic rate constants governing its signaling reactions. This is because these complex reactions are localized within the nanoscopic kinetochore. To circumvent this challenge, we designed the “eSAC”: an ectopic, quantifiable, and controllable, SAC activator. Preliminary characterization of the biochemical design of the SAC provides an elegant model to explain how the human cell optimizes the SAC signaling cascade. We will use the eSAC to quantify biochemical steps in the SAC cascade, reconstitute key steps to study them at the thermodynamic and structural level, and then synthesize a detailed mathematical model to completely establish the mechanistic platform describing the SAC. Our integrative analyses of the two systems will thus elucidate their respective functional designs, and reveal how they cooperate to ensure accurate chromosome segregation.

有丝分裂的主要目标是产生分裂细胞的两个遗传上相同的副本。为了实现这一目标， 分裂细胞必须将每条染色体的一个拷贝精确地分离到每个子代中。哪怕是一个小小的错误 染色体分离导致非整倍体，这反过来又导致了大量的缺陷，从细胞死亡到 肿瘤发生因此，为了实现精确的染色体分离，真核细胞使用两个高度分离的染色体。 复杂的系统：动粒和主轴组件检查点（SAC）。动粒是一个多- 移动和分离每个染色体的蛋白质机器。如果不能这样做，动粒就会激活 战略空军司令部SAC是一种信号级联反应，产生一种可扩散的检查点复合物，阻止细胞分裂。 广泛的研究已经汇编了这两个系统所需的蛋白质和活性的几乎完整的列表。 然而，关于每一个问题的根本问题仍然没有答案。动粒是如何无缝地 整合不同的分子机制，产生染色体运动和激活SAC？ 细胞如何校准SAC信号输出以最大化准确的染色体分离，但最小化 有丝分裂的持续时间在定义动粒的分子机制方面最重要的挑战是 其功能在于其高度复杂的蛋白质结构。我的实验室重建了 在芽殖酵母的动粒通过发展一系列的荧光显微镜技术。我们用这个 对酵母动粒进行"结构-功能"分析的知识。我们的工作揭示了 动粒结构塑造功能机制。我们的下一个目标是定义 更为复杂的是，人类动粒塑造了力产生和SAC激活的涌现机制。 在研究SAC的生化设计中，最重要的挑战是我们无法测量SAC的生物化学性质。 控制其信号传导反应的热力学速率常数。这是因为这些复杂的反应是 位于纳米级动粒内。为了规避这一挑战，我们设计了"eSAC"：一个异位的， 可量化和可控制的SAC激活剂。SAC生化设计的初步表征 提供了一个优雅的模型来解释人类细胞如何优化SAC信号级联。我们将使用 eSAC量化SAC级联中的生化步骤，重建关键步骤以在 热力学和结构水平，然后综合一个详细的数学模型， 描述SAC的机械平台。我们对这两个系统的综合分析将阐明 它们各自的功能设计，并揭示它们如何合作，以确保准确的染色体分离。