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制作两个分裂细胞的两个遗传相同副本。为了实现这一目标， 分隔的细胞必须将每个染色体的一个副本恰好隔离到每个女儿中。即使是一个错误 在染色体分离中，导致非整倍性，从而导致了很多缺陷，从细胞死亡到 肿瘤发生。因此，为了完成准确的染色体分离，真核细胞使用两个高度 软化系统：动力学和主轴组件检查点（SAC）。动力学是一个多 移动和分离每个染色体的蛋白质机。如果不能这样做，则动力学会激活 囊。 SAC是一个信号级联，可产生可扩散的检查点复合物，以阻止细胞分裂。 广泛的研究已汇总了两种系统所需的蛋白质和活动的几乎完整列表。 但是，关于每个人的基本问题仍然没有得到答复。动力学如何无缝接缝 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？定义动力学分子机制的最重大挑战 功能是其高度复杂的蛋白质结构。我的实验室重建了 通过开发一系列荧光显微镜技术，在发芽酵母中的动力学。我们使用了这个 进行酵母动物学的“建筑功能”分析的知识。我们的工作揭示了如何 动力学结构塑造功能机制。我们的下一个目标是定义如何 更复杂的人类动力学形状会形成力产生和SAC激活的紧急机理。 研究囊的生化设计最大的挑战是我们无法衡量 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是 局部位于纳米镜内运动学中。为了应对这一挑战，我们设计了“ ESAC”：异位， 可量化和受控的SAC激活剂。 SAC生化设计的初步表征 提供了一个优雅的模型，以解释人类细胞如何优化SAC信号级联。我们将使用 ESAC量化SAC级联的生化步骤，重构在研究中研究它们的关键步骤 热力学和结构水平，然后合成一个详细的数学模型以完全建立 描述SAC的机械平台。因此，我们对这两个系统的集成分析将阐明 它们各自的功能设计，并揭示它们如何协调以确保准确的染色体分离。