Mechanisms of kinetochore-microtubule attachment and regulation

项目摘要 细胞分裂的目标是可靠地产生两个相同的子细胞，每个子细胞都有一个完全相同的 原始遗传物质。当这个过程出错时，一个常见的结果是非整倍体，这是一种领先的 出生缺陷的原因和癌症的一个关键特征。有丝分裂细胞的分裂在很大程度上取决于动粒， 协调染色体分离并整合有丝分裂机制的所有方面的结构 确保高保真地进行有丝分裂。着丝点在物理上将染色体连接到纺锤体。 微管(MT)，它们调节这些连接的强度，从而使错误附着的MT 释放并正确连接的MT已稳定下来。重要的是，运动控制确保细胞不会退出 如果染色体不能或错误地附着在线粒体上，就会有丝分裂。在以下方面取得了很大进展 确定参与这些过程的动粒蛋白；然而，这些分子是如何发挥作用的 以确保染色体附着和分离的准确性，并确保及时的有丝分裂 进展情况仍然知之甚少。我们的实验室在定义动觉节律如何调节方面发挥了作用 MT附着，以及有丝分裂的这一基本方面是如何整合到控制细胞周期进程中的。 此外，我们的实验室已经开始在了解致癌转化如何 导致动粒功能失调，以及这些缺陷如何导致癌细胞特有的脆弱性 这可能会被用于癌症治疗。我们在研究动粒功能方面的专业知识 与我们新开发的实验方法相结合-特别是那些提供更多 动粒蛋白的空间和时间分辨率-使我们处于一个战略位置来解决如何 着丝点确保准确的染色体分离，并推动有丝分裂进程。在接下来的五年， 我们的研究将集中在四个方面。我们将：(1)研究动粒-MT附着的机制 使用生化和细胞生物学工具进行监管，以及跟踪特定物种动态的新检测方法 在高时间分辨率的细胞中的磷酸化事件；(2)研究Aurora B激酶，“主人”是如何 依附调节因子被招募到离散的着丝粒和着丝粒区域，具有精确的时间 使用细胞内诱变方法、邻近相互作用/质谱分析进行对照， 和磷酸化修饰示踪技术；(3)检测动粒-MT附着状态 通信到主轴组件检查点，部分是通过使用超分辨率成像来映射 在稳定的MT附着时发生的动粒结构的变化；(4)决定如何致癌 信号导致动粒-MT连接解除调控使用从 原生细胞。总而言之，我们的研究将提供对监管的基本机制的批判性洞察 动粒-MT连接，并将这一关键的有丝分裂功能与其他有丝分裂过程整合在一起 包括染色体结构、纺锤体MT动力学和检查点信号。 好了！

Project Summary The goal of cell division is to reliably produce two identical daughter cells, each with an exact copy of the original genetic material. When this process goes awry, a common result is aneuploidy, which is a leading cause of birth defects and a key characteristic of cancer. Mitotic cell division critically depends on kinetochores, structures that orchestrate chromosome segregation and integrate all aspects of the mitotic machinery to ensure mitosis is executed with high fidelity. Kinetochores physically connect chromosomes to spindle microtubules (MTs), and they regulate the strength of these connections so that erroneously-attached MTs are released, and correctly-attached MTs are stabilized. Importantly, kinetochores ensure that cells do not exit mitosis if chromosomes fail to attach, or are incorrectly attached, to MTs. Much progress has been made in identifying the kinetochore proteins that participate in these processes; however, how these molecules function in concert to ensure the accuracy of chromosome attachment and segregation, and to ensure timely mitotic progression remains poorly understood. Our lab has been instrumental in defining how kinetochores regulate MT attachment, and how this fundamental aspect of mitosis is integrated into controlling cell cycle progression. Additionally, our lab has begun to make significant inroads to understanding how oncogenic transformation leads to deregulated kinetochore function, and how these defects lead to cancer cell-specific vulnerabilities that can potentially be exploited for cancer therapies. Our expertise in studying kinetochore function in combination with our newly developed experimental approaches – especially those that provide increased spatial and temporal resolution of kinetochore proteins – puts us in a strategic position to resolve how kinetochores ensure accurate chromosome segregation and drive mitotic progression. In the next five years, our research will focus on four areas. We will: (1) Examine the mechanisms of kinetochore-MT attachment regulation using biochemical and cell biological tools, as well as new assays to track the dynamics of specific phosphorylation events in cells with high temporal resolution; (2) Investigate how Aurora B kinase, the “master” regulator of attachment, is recruited to discrete centromere and kinetochore regions with precise temporal control using in-cell mutagenesis approaches, proximity-dependent interaction/mass spectrometry analysis, and phospho-modification tracking techniques; (3) Probe how kinetochore-MT attachment status is communicated to the spindle assembly checkpoint, in part by using super-resolution imaging to map the changes in kinetochore architecture that occur upon stable MT attachment; (4) Determine how oncogenic signaling leads to kinetochore-MT attachment deregulation using a tumor progression model system built from primary cells. In sum, our studies will provide critical insight into the fundamental mechanisms that regulate kinetochore-MT attachment, and that integrate this critical mitotic function with other mitotic processes including chromosome architecture, spindle MT dynamics, and checkpoint signaling. !