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: 10439662
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10439662
• 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; 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基因相同的副本。为了实现这一目标， 分裂细胞必须将每条染色体的一份拷贝准确地分离到每个子细胞中。哪怕是一个错误 染色体分离会导致非整倍体，进而导致大量缺陷，从细胞死亡到 肿瘤发生。因此，为了实现准确的染色体分离，真核细胞使用两种高度 复杂的系统：动粒和主轴装配检查点（SAC）。着丝粒是一个多 移动和分离每条染色体的蛋白质机器。如果无法做到这一点，动粒就会激活 国家安全委员会。 SAC 是一种信号级联，可产生可阻止细胞分裂的扩散性检查点复合物。 广泛的研究已经编制了这两个系统所需的蛋白质和活性的近乎完整的列表。 然而，关于每个问题的基本问题仍未得到解答。着丝粒如何无缝衔接 整合产生染色体运动并激活 SAC 的不同分子机制？ 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？定义着丝粒分子机制中最重大的挑战 功能在于其高度复杂的蛋白质结构。我的实验室重建了纳米级蛋白质结构 通过开发一系列荧光显微镜技术来研究出芽酵母中的动粒。我们用这个 对酵母动粒进行“结构功能”分析的知识。我们的工作揭示了如何 着丝粒结构塑造了功能机制。我们的下一个目标是定义如何构建 更为复杂的是，人类动粒塑造了力产生和 SAC 激活的新兴机制。 研究 SAC 生化设计的最大挑战是我们无法测量 控制其信号反应的热力学速率常数。这是因为这些复杂的反应 位于纳米级着丝粒内。为了规避这一挑战，我们设计了“eSAC”：一个异位、 可量化、可控的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，重构关键步骤以在 热力学和结构层面，然后综合详细的数学模型，彻底建立 描述 SAC 的机械平台。因此，我们对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。