How a cell cycle kinase, Aurora A, regulates polarity proteins organization

项目摘要 复杂生物体的发育需要细胞类型和组织的多样化。细胞如干 细胞和胚胎必须分裂，以分离细胞命运决定因素，然后不对称分裂， 产生功能不同的细胞。不能建立细胞极性是许多癌症的标志， 发育缺陷因此，了解细胞极性网络是如何耦合的具有重要意义 与细胞周期，以了解特定的细胞类型是如何形成在发展过程中。 PAR蛋白是许多生物体中控制细胞极性正确建立的特化蛋白。为 例如，小鼠胚胎干细胞通过将PAR蛋白定位于细胞的顶端或底部来建立顶端-基底极性。 基底结构域，以允许细胞命运决定子的分离。另一个例子是秀丽隐杆线虫 受精卵，通过形成相互排斥的PAR皮质区域建立前后极性。然而 PAR蛋白是如何调节的还不完全清楚。 最近的研究表明，细胞周期激酶，极光A，是需要启动极化在一个C。elegans 受精卵这些研究表明，极光A表现出动态定位，并在不同阶段发挥作用 的细胞周期，以控制皮质招聘PAR蛋白及时和精确的方式。但 Aurora A调节PAR蛋白质不对称性的确切机制仍然不清楚。 这项提案的目标是剖析不同的极光A池如何传输信号， 促进及时和精确的细胞极化，并确定极光A在极化过程中的作用是否 在其他极化细胞中是保守的。核心假设是极光A在不同的时间和地点起作用 调节极性蛋白组织，从而协调极性与细胞周期。这一假设将 使用独特的生物化学方法进行测试，用于在单细胞、单细胞和单细胞中定量蛋白质-蛋白质相互作用。 分子水平，结合高分辨率显微镜和靶向基因组编辑。实验目的 1将鉴定C.并测试极光A活性的丧失如何影响PAR蛋白质 本地化和复杂的组装。目标2将确定何时何地需要极光A活动， 协调PAR蛋白，使用光遗传学操纵Aurora A空间调控，并在商业上 可用的Aurora A抑制剂来控制Aurora A活性。目标3将评估极光A活动是否 在小鼠胚胎干细胞的顶端-基底极性建立过程中所需的。这些数据将 揭示极光A是如何定位，以协调极性蛋白质动力学和复杂的组装，并提供 极性如何建立并与细胞周期同步的基本知识。

Project Summary The development of complex organisms requires diversification of cell types and tissues. Cells such as stem cells and embryos must polarize in order to segregate cell fate determinants, and then divide asymmetrically to generate functionally distinct cells. Failure to establish cell polarity is a hallmark of many cancers and developmental defects. Therefore, it is of great importance to understand how cell polarity network is coupled with the cell cycle, to get insights into how specific cell types are formed during development. PAR proteins are specialized proteins that govern proper establishment of cell polarity in many organisms. For example, mouse embryonic stem cells establish apical-basal polarity by localizing PAR proteins to the apical or basal domains to allow the segregation of cell fate determinants. Another example is the Caenorhabditis elegans zygote, which establishes antero-posterior polarity by forming mutually exclusive PAR cortical domains. However how PAR proteins are regulated is not fully understood. Recent studies have revealed that a cell cycle kinase, Aurora A, is required to initiate polarization in a C. elegans zygote. These studies showed evidence that Aurora A exhibits dynamic localization and acts at different stages of the cell cycle to control cortical recruitment of PAR proteins in a timely and precise manner. However, the exact mechanisms by which Aurora A modulates PAR protein asymmetries remain obscure. The overreaching objectives of this proposal are to dissect how different pools of Aurora A transmit signals that promote timely and precise cell polarization, and to determine if the role of Aurora A during polarization is conserved in other polarizing cells. The central hypothesis is that Aurora A works at different times and places to modulate polarity proteins organizations, thereby coordinating polarity with the cell cycle. This hypothesis will be tested using a unique biochemical approach for quantifying protein-protein interactions at a single cell, single molecule level, in combination with high-resolution microscopy and targeted genome editing. Experiments in Aim 1 will identify Aurora A substrates in C. elegans and test how loss of Aurora A activity impacts PAR proteins localization and complex assembly. Aim 2 will determine where and when Aurora A activity is required to coordinate PAR proteins, using optogenetics to manipulate Aurora A spatial regulation and commercially available Aurora A inhibitors to control the Aurora A activity. Aim 3 will assess whether Aurora A activity is required during apical-basal polarity establishment of mouse embryonic stem cells. Collectively, these data will reveal how Aurora A is positioned to orchestrate polarity proteins dynamics and complex assembly and provide fundamental knowledge of how polarity is established and synchronized with the cell cycle.