Development of a robust high throughput assay for the discovery of RAM network in

描述(由申请人提供)：细胞结构的控制对于功能组织的形成和维持是必不可少的，并且经常在包括癌症和一些免疫紊乱在内的许多重要的人类疾病中错乱。形态控制对细胞分化也很重要：促进细胞命运决定因素不对称分离的机制依赖于细胞的基本结构来做到这一点。由于病理状态(如浸润性转移癌)通常涉及不适当的运动性升高，因此调节控制细胞结构的通路的活性的化合物是候选的治疗剂。发芽酵母RAM网络(调控ACE2和形态发生)是基因不对称表达和维持极化生长所必需的一条新的信号通路。这条途径从酵母到人类明显是保守的；在后生动物中，RAM网络蛋白的同源物参与控制细胞增殖、极化细胞的形态发生和树突域的组织。人们对这些蛋白质的功能知之甚少。我们已经开发出一种简单而强大的方法来寻找阻断RAM网络的治疗方法。在这种方法中，保留通路功能的细胞被杀死，而失去通路功能的细胞可以存活。在正向遗传筛查中，这种基于选择的策略迅速产生了400多个新的RAM网络基因功能丧失的等位基因。我们建议将这一策略用于高通量筛选，以确定阻止RAM网络的化合物。我们将使用西北大学先进的NTS设备来开发这种方法并进行中试筛选，以确保送往MLSCN的分析是可靠和便携的。在初步分析中，对照保留RAM网络功能的细胞的生长进行选择，得到Z‘约为0.72。

DESCRIPTION (provided by applicant): Control of cell architecture is essential for formation and maintenance of functional tissues, and is often deranged in a number of important human diseases including cancer and some immune disorders. Morphology control is also important for cell differentiation: mechanisms that promote asymmetric segregation of cell fate determinants rely upon the cell's underlying structure to do so. Since pathological states (such as invasive metastatic cancer) often involve inappropriate elevation of motility, compounds that modulate the activity of pathways that control cell architecture are candidate therapeutic agents. The budding yeast RAM network (Regulation of Ace2 and Morphogenesis) is a novel signaling pathway required for asymmetric gene expression and maintenance of polarized growth. This pathway is evidently conserved from yeast to humans; in metazoans, homologs of RAM network proteins are involved in control of cell proliferation, morphogenesis of polarized cells, and organization of dendritic fields. Very little is understood about these proteins' functions. We have developed a simple and powerful way to find treatments that block the RAM network. In this approach, cells retaining pathway function are killed, while those that lose it can live. In a forward genetic screen this selection-based strategy quickly yielded over 400 new loss-of-function alleles of RAM network genes. We propose to adapt this strategy for high-throughput screening to identify compounds that block the RAM network. We will use advanced NTS equipment at Northwestern to develop this approach and conduct pilot screens, ensuring that the assay sent to the MLSCN is robust and portable. In preliminary assays, selection against growth of cells that retain RAM network function gives a Z' of approximately 0.72.