Studying the role of Aurora A and Protein Phosphatase 6 in regulation of NDC80 during mitosis

准确的染色体分离对新细胞和组织的产生至关重要。在有丝分裂纺锤体形成过程中，极光激酶及其调控因子通过调控微管动力学和微管与着丝点的结合，在精确的染色体分离中发挥关键作用。然而，许多机械细节仍不清楚。我们最近发现，Aurora A通过磷酸化微管结合着丝点蛋白NDC80，是微管-着丝点界面的主要调节剂。Aurora A的活性被蛋白磷酸酶6 （PP6）抵消，缺乏PP6的细胞表现出NDC80磷酸化升高和染色体分离缺陷。在这个项目中，我们计划研究Aurora A-PP6轴和Aurora B在细胞分裂过程中调控微管细胞骨架的作用。我们的假设是，极光A途径促进微管附着到着丝点上，而极光B通过其在纺锤体组装检查点中的作用，充当这些缺陷的传感器。我们之前的工作主要集中在Aurora A-PP6通路在人类癌细胞中的作用，并产生了许多所需的试剂和工具。这个新项目将利用基因组工程、显微镜和光学方法在细胞分裂过程中操纵NDC80，以更好地了解Aurora A调控的机制。我们方法的基石是蛋白质动力学的定量成像和单分子成像，以及细胞周期系统动力学的计算建模。利用这些方法，我们旨在描述细胞分裂机制的动力学和功能以及极光激酶的作用。哲学博士将在以下方面提供广泛的培训：细胞周期生物学与体外生化超分辨率microscopy3。光学方法和化学/光活化降解技术利用CRISPR/CAS95进行基因组工程。计算模型

Accurate chromosome segregation is crucial for the generation of new cells and tissues. Aurora kinases and their regulators play a key role in accurate chromosome segregation through regulation of microtubule dynamics and microtubule binding to kinetochores during mitotic spindle formation. However, many of the mechanistic details remain unclear. We have recently found that Aurora A is the primary regulator of the microtubule-kinetochore interface through phosphorylation of the microtubule binding kinetochore protein NDC80. Aurora A activity is counteracted by protein phosphatase 6 (PP6), and cells lacking PP6 show elevated NDC80 phosphorylation and chromosome segregation defects.In this project we plan to study the role of the Aurora A-PP6 axis and Aurora B in regulating the microtubule cytoskeleton during cell division. Our hypothesis is that the Aurora A pathway promotes microtubule attachments to kinetochores and that Aurora B acts as the sensor for these defects through its role in the spindle assembly checkpoint. Our previous work has focussed on the role of the Aurora A-PP6 pathway in human cancer cells and has generated many of the reagents and tools required. This new project will take advantage of genome engineering, microscopy and optical methods to manipulate NDC80 during cell division to better understand the mechanism of Aurora A regulation. A cornerstone of our approach is quantitative imaging and single molecule imaging of protein dynamics, and computational modelling of the dynamics of the cell cycle system. Using these approaches, we aim to describe the dynamics and function of the cell division machinery and roles of Aurora kinases. The DPhil will provide extensive training in:1. Cell-cycle biology and in vitro biochemistry2. Super-resolution microscopy3. Optical methods and chemical/light-activated degron technology4. Genome engineering using CRISPR/CAS95. Computational modelling