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基因相同的副本。 分隔的细胞必须完全将每个染色体隔离到每个女儿中。 在染色体隔离中，导致非整倍性，从而导致从细胞死亡到到。 因此，为了完成精确的染色体隔离，真核细胞用于高度 复杂的系统：Kinetchore和主轴组件检查点（SAC）。 蛋白质机，将每个染色体移动并分离。 SAC。 广泛的研究已经编译了这两个系统的蛋白质和活动的几乎完整列表。 但是，基本问题提出的问题仍然没有得到答复。 整合产生染色体运动并激活SAC的不同分子机制？ 精确的染色体隔离如何，但如何最小化，但如何最小化 有丝分裂的持续时间？ 功能是高度comprex蛋白结构。 通过开发一系列荧光显微镜技术来发展的Kinetchore 进行年度Kinetchore的“建筑 - 功能”分析的知识。 Kinetchore架构塑造功能机制。 更复杂的人类Kinetchore塑造了力产生和SAC激活的新兴机制。 研究囊的生化设计最大的挑战是我们的能力来测量 控制其信号反应的热力学速率常数是因为这些复杂的反应是 局部纳米镜子的挑战。 SACC的生物化学设计的量化和可控的SAC激活剂 为人类细胞提供了优雅的模型，优化了SAC信号级联。 ESAC量化SAC级联的生化步骤 热力学和结构水平，这些详细的数学模型可以综合建立 因此，描述SAC的机械平台。 它们各自的功能设计，并揭示如何合作以确保准确的染色体分离。