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.
