Regulation of Chromosome Segregation in Human Cells

描述（由申请方提供）：准确的染色体分离对于细胞增殖、组织稳态、胚胎发育和肿瘤抑制至关重要。一个关键的调节途径是纺锤体组装检查点（SAC），其防止姐妹染色单体分离并退出有丝分裂，直到所有染色体通过微管纤维连接到纺锤体两极。即使是一条缺乏双极附着的染色体也足以触发SAC，因为它的动粒招募并激活下游因子，这些因子不仅与核心细胞周期机制沟通，而且还改变了动粒本身的微管结合特性，从而使不正确的微管附着不稳定。我们实验室最近的工作表明，蛋白激酶Mps 1参与了这两种输出，以及第三种途径，该途径作为有丝分裂的“时钟”或“计时器”独立于动粒。这些见解是通过我们实验室开创的基因编辑和化学遗传学技术的结合应用而出现的，其中培养的人类细胞中的内源性Mps 1从基因组中删除，并被对大嘌呤类似物敏感的变体激酶等位基因所取代。使用这个系统，我们已经进行了全球和有针对性的蛋白质组学筛选，并产生了广泛的一套磷酸化抗体，揭示了Mps 1依赖的磷酸化在kinetochore-microtubule接口的景观。在目标1中，我们将挖掘这些信息来分析Mps 1和抵消磷酸酶如何调节着丝粒微管附着，这样只有适当的双极附着才能稳定。在目标2中，我们剖析了Mps 1催化的磷酸化如何在着丝粒上促进SAC效应子的募集和激活，从而产生APC/C-Cdc 20泛素连接酶的生化抑制剂。在目标3中，我们使用化学遗传学来研究Mps 1和其他激酶如何相互作用，以维持SAC参与时的M期状态。这些研究将阐明M期质量控制的分子和机制，从长远来看，将有助于开发针对非整倍体相关疾病（如癌症）的治疗药物。

DESCRIPTION (provided by applicant): Accurate chromosome segregation is vital for cell proliferation, tissue homeostasis, embryonic development, and tumor suppression. One key regulatory pathway is the spindle assembly checkpoint (SAC), which prevents the separation of sister chromatids and exit from mitosis until all chromosomes are linked to both spindle poles by microtubule fibers. Even a single chromosome lacking bipolar attachment is sufficient to trigger the SAC, as its kinetochores recruit and activate downstream factors that not only communicate with the core cell-cycle machinery, but also alter the microtubule-binding properties of the kinetochore itself, so that incorrect microtubule attachments are destabilized. Recent work from our lab has implicated the protein kinase Mps1 in both of these outputs, as well as in a third pathway that acts as a mitotic 'clock' or 'timer' independently of kinetochores. These insights emerged through combined application of gene editing and chemical genetics techniques pioneered in our lab, whereby endogenous Mps1 in cultured human cells was deleted from the genome and replaced by a variant kinase allele sensitized to bulky purine analogs. Using this system, we have performed global and targeted proteomics screens and generated an extensive suite of phosphospecific antibodies, revealing the landscape of Mps1-dependent phosphorylation at the kinetochore-microtubule interface. In Aim 1, we will mine this information to analyze how Mps1 and counteracting phosphatases regulate kinetochore-microtubule attachments, such that only proper bipolar attachments are stabilized. In Aim 2, we dissect how Mps1-catalyzed phosphorylation fuels the recruitment and activation of SAC effectors at kinetochores, resulting in the production of biochemical inhibitors of the APC/C-Cdc20 ubiquitin ligase. In Aim 3, we use chemical genetics to ask how Mps1 and other kinases interact to maintain the M phase state when the SAC is engaged. These studies will illuminate the molecules and mechanisms underlying M phase quality control, and in the long term will empower development of therapeutic agents that target aneuploidy-associated diseases such as cancer.