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

项目摘要 复杂生物体的发展需要细胞类型和组织的多样化。干细胞等细胞 细胞和胚胎必须极化，以分离细胞命运决定因素，然后不对称地分裂到 生成功能不同的单元格。未能确定细胞极性是许多癌症和 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 与细胞周期有关，以深入了解特定细胞类型在发育过程中是如何形成的。 PAR蛋白是一种特殊的蛋白质，它控制着许多生物体中细胞极性的正确建立。为 例如，小鼠胚胎干细胞通过将PAR蛋白定位到顶端或顶端来建立顶端-基底端的极性 基本结构域允许分离细胞命运决定因素。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皮质结构域来建立前后极性。然而， PAR蛋白是如何被调控的，目前还不完全清楚。 最近的研究表明，细胞周期激酶Aurora A是启动线虫极化所必需的 受精卵。这些研究表明，极光A在不同的阶段表现出动态的定位和行为 控制细胞周期，以及时和精确地控制PAR蛋白的皮质募集。然而， Aurora A调节PAR蛋白不对称性的确切机制仍不清楚。 这项提案的过分目标是剖析不同的极光A池是如何传递信号的 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 在其他极化细胞中保守。中心假设是极光A在不同的时间和地点工作 调节极性蛋白质组织，从而使极性与细胞周期相协调。这一假说将 使用独特的生化方法进行测试，以量化单个细胞、单个细胞和单个细胞中的蛋白质相互作用 分子水平，结合高分辨率显微镜和定向基因组编辑。AIM中的实验 1将在线虫中鉴定Aurora A底物，并测试Aurora A活性丧失对PAR蛋白的影响 本地化和复杂组装。目标2将确定何时何地需要Aurora A活动 协调PAR蛋白，利用光遗传学操纵Aurora A空间调节和商业 可用Aurora A抑制剂控制Aurora A活性。目标3将评估Aurora A活动是否 在建立小鼠胚胎干细胞的顶端-基底极时所需的。总而言之，这些数据将 揭示Aurora A如何定位于协调极性蛋白质的动态和复杂组装，并提供 关于极性如何建立以及如何与细胞周期同步的基础知识。

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.