Cilia are microtubule-based cellular extensions that play key roles in sensing the extracellular environment, processing developmental signals and generating propulsive force and fluid flow. They also act as secretory organelles releasing bioactive vesicular ectosomes involved in cell-cell communication and other processes. Cilia are ancient and complex; in humans, more than 5% of all genes are involved in their assembly/function. Defects in cilia result in a wide array of ciliopathies which have broad and often devastating multi-systemic consequences. Although much has been learned about how cilia are assembled, many key aspects of their assembly and signaling are unknown. Recently, we demonstrated that a neuroendocrine enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), which acts on glycine-extended peptide precursors to generate amidated bioactive peptides such as oxytocin, vasopressin and neuropeptide Y, is present in Chlamydomonas. We found that PAM, a transmembrane protein, is localized to both the Golgi and cilia (flagella) in Chlamydomonas and is present in both motile and non-motile (primary) cilia in mammals. Furthermore, PAM is required for normal ciliary assembly in Chlamydomonas, planaria and vertebrates (mice and zebrafish). Ciliary PAM, with its active site exposed to extracellular fluid, is tightly associated with the axoneme. Although localized to the cilium in both vegetative cells and gametes, PAM is present in ectosomes released by mating gametes, but not in ectosomes released by vegetative cells. We found that mating ectosomes contain multiple amidated protein products and demonstrated that an amidated peptide derived from one of these acts as a chemoattractant, thereby revealing a novel cilia-based bioactive peptide signaling process. In this application, we will investigate these unexplored properties of cilia using the genetic and biochemical approaches employed so successfully in Chlamydomonas to understand intraflagellar transport and ciliary motility. The first Aim will directly address the role(s) of PAM protein and amidating activity in ciliogenesis and dissect the function of the catalytic cores and the transmembrane/cytosolic domains. Aim 2 will define the signaling mechanism that leads to the controlled release of PAM in ciliary ectosomes specifically from mating gametes. This will enable us to assess how cells control cilia-based secretion and how this might impact downstream cell-cell communication. The final Aim will examine the function of amidated products secreted in ciliary ectosomes, and how the trafficking and processing of the amidated product precursors are coordinated as they pass from the Golgi, through cilia to ultimately be secreted in ectosomes. This will define a completely novel cilia-based signaling pathway and thereby provide new concepts in understanding cilia-based deficiencies and ciliopathies.