Project summary Spatiotemporal regulation of signal molecules is critical for development and adult tissue homeostasis. Indeed, misactivation or mislocalization of signaling events can cause congenital disorders and cancers, and understanding how information transfer is regulated in cells is essential for understanding and improving human health. The Hedgehog pathway is conserved across metazoan animals where it controls cell proliferation, differentiation, migration, and homeostasis. In vertebrates, Hedgehog signals are transduced through primary cilia that project from the surface of most cells, including cells in cancers driven by misactivation of the Hedgehog pathway. Cilia are required for vertebrate Hedgehog signaling for reasons that remain unknown. Our central hypothesis is that the ciliary microenvironment enables protein and lipid interactions that are necessary for Hedgehog signal transduction. Our lab has identified an important role for cilia-associated lipids that bind to Smoothened, accumulate Smoothened to cilia, and activate the Hedgehog pathway. When the Hedgehog pathway is off, Smoothened accumulation and activity in cilia is inhibited by Patched1. How Patched1 and Smoothened accumulate and function in cilia, and how Patched1 inhibits Smoothened, are poorly understood despite increasing recognition that these activities of the Hedgehog pathway are critical in health and disease. To address these key gaps in our understanding of human biology, the objectives of this proposal are to define the protein interactions that are necessary for Patched1 and Smoothened accumulation and activity in primary cilia, and to determine if Patched1 inhibited Smoothened through compartmentalization of ciliary lipids. Recently, we have begun to appreciate that the Hedgehog pathway can gather information directly from the ciliary microenvironment. Our discovery of ciliary lipids that specify Hedgehog pathway output establishes a new paradigm for information input onto the Hedgehog transcriptional program, one in which changes in the ciliary microenvironment reprogram the Hedgehog response. In this proposal, we will systematically examine the dynamic repertoire of protein and lipid interactions in cilia that specify the Hedgehog pathway across normal and tumor cells. We will leverage recent technical advances in proteomic proximity-labeling mass spectrometry, lipidomic mass spectrometry, and functional genomics to interpret high-dimensional landscapes of cell states induced by Hedgehog pathway activation. The long-term goal of this proposal is to understanding mechanisms underlying Hedgehog signal transduction well enough to develop new treatments for Hedgehog-associated cancers. To do so, the data generated by this proposal will establish a rationale to interrogate these mechanisms in vivo and in samples from human patients with Hedgehog-associated cancers. More broadly, this proposal will elucidate why cilia are required for Hedgehog signaling a...