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性组织的形成和维持是必不可少的，并且在许多重要的人类疾病中经常被打乱，包括癌症和一些免疫疾病。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底层结构。由于病理状态（如侵袭性转移性癌症）通常涉及不适当的运动性升高，因此调节控制细胞结构的途径活性的化合物是候选治疗剂。出芽酵母RAM网络（Regulation of Ace2 and Morphogenesis）是酵母非对称基因表达和维持细胞极化生长所需的一种新的信号通路。这一途径显然是保守的，从酵母到人类；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状场的组织。人们对这些蛋白质的功能了解甚少。我们已经开发了一种简单而有效的方法来寻找阻断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，而那些失去通路功能的细胞可以存活。在正向遗传筛选中，这种基于选择的策略迅速产生了400多个新的RAM网络基因丧失功能等位基因。我们建议将这一策略用于高通量筛选，以识别阻断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，并进行试点筛选，确保发送到MLSCN的分析是可靠和便携的。在初步分析中，保留RAM网络功能的细胞的生长选择的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.