Integrative analyses of the kinetochore and the spindle assembly checkpoint

有丝分裂的主要目标是为分裂的细胞制造两个基因上相同的副本。为了实现这一目标， 分裂细胞必须准确地将每个染色体的一个拷贝分离到每个子代中。哪怕是一个小错误 染色体分离导致非整倍体，进而导致过多的缺陷，从细胞死亡到 肿瘤发生学。因此，为了实现准确的染色体分离，真核细胞使用两个高度 复杂的系统：动粒和主轴组件检查点(SAC)。动毛虫是一种多- 移动和分离每条染色体的蛋白质机器。如果它不能这样做，则动粒被激活 SAC。SAC是一个信号级联，它产生一个可扩散的检查点复合体，阻止细胞分裂。 广泛的研究已经汇编了一份几乎完整的两个系统所需的蛋白质和活动的清单。 然而，关于这两个问题的根本问题仍然没有得到回答。动粒是如何无缝连接的 整合产生染色体运动和激活SAC的不同分子机制？ 细胞如何校准SAC信号输出，以最大限度地实现准确的染色体分离，但最小化 有丝分裂的持续时间？定义动粒分子机制的最大挑战 其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尺度的蛋白质结构 通过发展一系列荧光显微镜技术，在萌芽中的酵母中发现动粒。我们用了这个 具备对酵母着丝粒进行“结构-功能”分析的知识。我们的工作揭示了 动粒结构塑造了功能机制。我们的下一个目标是定义 更为复杂的是，人类动粒形成了力量产生和SAC激活的紧急机制。 研究SAC的生化设计最大的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 定位于纳米动粒内。为了绕过这一挑战，我们设计了“ESAC”：一个异类， 可量化、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将量化SAC级联中的生化步骤，重建关键步骤以在 热力学层次和结构层次，然后综合出详细的数学模型，完全建立 描述SAC的机械平台。因此，我们对这两个系统的综合分析将阐明 他们各自的功能设计，并揭示他们如何合作，以确保准确的染色体分离。

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.