MECHANISMS OF CELL POLARITY ESTABLISHMENT

描述（由申请人提供）：细胞极化是生命中最基本的组织方面之一，从单细胞生物如大肠杆菌和出芽酵母到人类大脑的线路。细胞极化是干细胞分化为多种细胞类型、形态发生、定向运动、抗原呈递和轴突引导所必需的；而细胞极性的丧失是癌症发展的关键一步。对细胞极性的广泛兴趣推动了对极化机制的范围和复杂性的理解取得了快速进展。然而，极性蛋白如何组织细胞结构的基本问题在很大程度上仍未得到解答。这项建议是在上一个供资期间为解决这些问题所取得进展的基础上提出的。我们的目标是了解PAR极性蛋白和小gtpase之间的联系。我们将重点关注3D培养中的MDCK上皮细胞极化和海马神经元中的树突棘形态发生- PAR蛋白在两个可处理的系统中具有明确但不同的作用。具体目的如下：1。确定在MDCK细胞极化囊肿形成过程中，PAR-3、PAR-6和aPKC组织顶端结构域（我们称之为“斑块”）的分子机制。使用活细胞成像，我们将研究极化的最早步骤，这发生在3D培养中生长的细胞的初始细胞分裂期间。我们将测试一个新的模型，在这个模型中，细胞动力学事件被提议产生根尖斑块组装的里程碑。2. 确定CDC42在根尖斑块组装中的作用，并确定细胞极化所必需的CDC42 GEF和GAP。RNAi筛选被用于识别GEF和GAP。基于GEF RNAi筛选的初步数据，我们提出一个名为Tuba的CDC42-GEF驱动PAR-6/aPKC的招募和资本化。3. 确定PAR-3控制神经元中TIAM1功能的机制；并确定PAR- 6如何独立于PAR-3，通过Rho GTPase调节树突棘的形态发生。我们将确定PAR-6/aPKC活化p190 RhoGAP的分子基础。PAR-6调节海马神经元突触活动的机制也将被研究。总之，这些研究将为细胞极化的基本重要过程提供新的见解，并为PAR极性蛋白如何表现出环境特异性行为来控制细胞形态的不同方面提供新的见解。公共卫生相关性：超过90%的人类癌症起源于上皮细胞。上皮细胞的一个关键特性是它们相互粘附形成薄片，其中上表面（面向环境）与下表面在功能上不同。癌症的进展包括这种细胞-细胞粘附和上下极性的暂时丧失。因此，为了了解人类癌症，并确定新的化疗靶点，了解上皮细胞如何极化是很重要的。值得注意的是，帮助这些细胞相互粘在一起的过程对于大脑中神经细胞之间的联系也是必不可少的，这使得我们能够思考和存储记忆。这项资助研究了使上皮细胞和神经细胞极化并形成连接的详细分子机制，并将为这些机制提供新的见解。

DESCRIPTION (provided by applicant): Cell polarization is one of the most fundamental organizational aspects of life, from single-celled organisms such as E. coli and budding yeast up to the wiring of the human brain. Cell polarization is required for the generation of diverse cell types from stem cells, for morphogenesis, directional motility, antigen presentation, and axon guidance; and the loss of cell polarity is a critical step in cancer progression. The wide interest in cell polarity has pushed rapid progress in understanding the scope and complexity of the polarization machinery. However, the fundamental question of how the polarity proteins operate to organize cell structure remains largely unanswered. This proposal builds on progress over the last funding period to address these questions. Our objective is to understand the connections between the PAR polarity proteins and small GTPases. We will focus on MDCK epithelial cell polarization in 3D cultures, and on dendritic spine morphogenesis in hippocampal neurons - two tractable systems in which the PAR proteins have defined but distinct roles. The specific aims are as follows: 1. Identify the molecular mechanisms through which PAR-3, PAR-6, and aPKC organize the apical domain (which we term a "patch") during polarized cyst formation of MDCK cells. Using live cell imaging, we will investigate the earliest steps in polarization, which occur during the initial cell division of cells grown in 3D cultures. We will test a new model in which cytokinetic events are proposed to generate the landmark for apical patch assembly. 2. Determine the role of CDC42 in assembly of the apical patch, and identify the CDC42 GEF and GAP that are necessary for cell polarization. RNAi screens are being used to identify the GEF and GAP. Based on preliminary data from a GEF RNAi screen, we propose that a CDC42-GEF called Tuba drives recruitment of PAR-6/aPKC and apicalization. 3. Identify the mechanisms by which PAR-3 controls TIAM1 function in neurons; and determine how PAR- 6, independently of PAR-3, regulates dendritic spine morphogenesis via the Rho GTPase. We will determine the molecular basis for activation of p190 RhoGAP by PAR-6/aPKC. The mechanism by which PAR-6 regulates synaptic activity in hippocampal neurons will also be investigated. Together, these studies will provide new insights into the fundamentally important process of cell polarization, and into the ways by which the PAR polarity proteins exhibit context-specific behavior to control different aspects of cell morphology. PUBLIC HEALTH RELEVANCE: Over 90% of all human cancers originate from epithelial cells. A key property of epithelial cells is that they adhere to one another to form sheets in which the upper surface (which faces the environment) is functionally different from the bottom surface. Cancer progression involves the temporary loss of this cell-cell adhesion, and of up-down polarity. Thus, to understand human cancer, and to identify new chemotherapeutic targets, it is important to find out how epithelial cells polarize. Remarkably, the same process that helps these cells stick to one another is also essential for making the contacts between nerve cells in the brain, which enable us to think and store memories. This grant investigates the detailed molecular mechanisms that enable epithelial cells and nerve cells to polarize and form junctions, and will provide new insights into these mechanisms.