PROJECT SUMMARY/ABSTRACT We will investigate how cytoskeletal organelles organize into patterns in the cell. The fields of motile cilia in epithelial cells and the rows of stereocilia in the inner hair cells are examples of physiologically- important organelle patterns. The known pathways do not satisfactorily explain how organelle patterns form. The ciliated protists (ciliates) assemble unusually complex surface (cortical) patterns. Ciliates faithfully duplicate their cortical pattern during cell division and, despite its complexity, the cortical pattern remains nearly invariable in a population. Thus, even subtle deviations from the normal pattern (e.g. due to a mutation or trauma) can be detected and analyzed over many generations. In the genetic model ciliate Tetrahymena thermophila, hundreds of cortical organelles, including cilia, are arranged along the anteroposterior and circumferential (left-right) axes. We recently identified causal mutations in several classical Tetrahymena pattern mutants, in which organelles assemble at incorrect positions. We found that orthologs of the Hippo pathway kinases and cyclin E are critical for patterning on the anteroposterior axis. We identified a strong candidate for Hpo1, a protein that regulates the positions of organelles on the cell’s circumferential axis. The molecular activities through which these pattern-regulating proteins act, and the basis of their restricted cortical localizations, remain unknown. We will explore the molecular composition of the pathways that drive pattern formation in Tetrahymena, study the properties of the pattern-regulating proteins and develop genetic and biochemical screens to systematically unravel the principles of intracellular patterning through unbiased approaches. The overarching goal for Aim 1 is to uncover the molecular circuitry of “early” Hippo signaling that controls the initial positions of organelles that form during cell division on the anteroposterior cell axis in Tetrahymena. Aim 2 will investigate how the “late” Hippo signaling circuit and cyclin E interact (in a mutually antagonistic manner) to induce and maintain cortical structures that form at the division plane. Aim 3 will investigate factors that regulate the circumferential pattern, including Hpo1, a protein that localizes to the right side of the cell.