Regulation of Chromosome Segregation in Human Cells

描述(由申请人提供)：准确的染色体分离对细胞增殖、组织动态平衡、胚胎发育和肿瘤抑制至关重要。一个关键的调控途径是纺锤体组装检查点(SAC)，它阻止姐妹染色单体的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两个纺锤体极点。即使是一个缺乏两极附着的单个染色体也足以触发SAC，因为它的动点招募和激活下游因子，这些下游因子不仅与核心细胞周期机制通信，而且还改变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Mps1参与了这两种输出，以及第三条独立于动粒的有丝分裂时钟或计时器的途径。这些见解是通过结合应用我们实验室首创的基因编辑和化学遗传学技术而产生的，通过这种技术，培养的人类细胞中的内源性Mps1从基因组中删除，并被对笨重的嘌呤类似物敏感的变异激酶等位基因所取代。使用这个系统，我们已经进行了全局和靶向的蛋白质组学筛选，并产生了一套广泛的磷酸特异性抗体，揭示了动粒-微管界面上Mps1依赖的磷酸化的图景。在目标1中，我们将挖掘这些信息来分析Mps1和反式磷酸酶如何调节动粒-微管连接，从而只有正确的双极连接才是稳定的。在目标2中，我们剖析了Mps1催化的磷酸化如何促进SAC效应器在动点的招募和激活，从而导致APC/C-CDC20泛素连接酶生化抑制剂的产生。在目标3中，我们使用化学遗传学来研究当SAC参与时，Mps1和其他激酶是如何相互作用来维持M期状态的。这些研究将阐明M期质量控制的分子和机制，并从长远来看，将促进针对非整倍体相关疾病(如癌症)的治疗剂的开发。

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.