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

对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，而且通常 患上了许多重要的人类疾病，包括癌症和一些免疫紊乱。 形态控制对细胞分化也很重要：促进不对称的机制 细胞命运决定因素的分离取决于细胞的基本结构。自病理性以来 状态(如浸润性转移性癌症)通常涉及不适当的运动性升高，化合物 调节控制细胞结构的通路的活性是候选的治疗剂。 发芽酵母RAM网络(ACE2和形态发生的调节)是一种新的信号通路 不对称基因表达和维持极化生长所必需的。这条路显然是 从酵母到人类的保守；在后生动物中，RAM网络蛋白的同源物参与了控制 细胞增殖、极化细胞的形态发生和树突域的组织。很少有 了解这些蛋白质的功能。 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 接近时，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。在未来的遗传学中 筛选这种基于选择的策略，很快就产生了400多个新的RAM网络功能丧失等位基因 基因。我们建议将这一策略应用于高通量筛选，以确定阻止 RAM网络。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，确保送往MLSCN的分析是可靠和便携的。在初步化验中， 相对于保持RAM网络功能的细胞的增长进行选择，得到Z‘约为0.72。

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 aboyt 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 approximately0.72.