Project Summary of Parent Grant Autosomal dominant polycystic kidney disease (ADPKD) is a common, life threatening disease that affects 1/400-1/1000. ADPKD is caused by mutations in PKD1 and PKD2, which encode polycystin-1 and polycystin-2 (PC1 and PC2). Remarkably, the function of the polycystins remains enigmatic almost 30 years after their cloning and 20 years after their discovery on renal primary cilia. Besides cilia, PC1 and PC2 are also found in other subcellular locations including extracellular vesicles (EVs). Urinary EVs can be used as biomarkers of renal disease including ADPKD. Whether these polycystin-carrying EVs are of ciliary origin and what role EVs play in healthy and diseased kidneys remains unknown. In the model organism C. elegans and mammals, the polycystins LOV-1/PC1 and PKD-2/PC2 are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to primary/sensory cilia, and are shed in EVs, suggesting ancient conservation. We will use our established C. elegans model and fluorescently labeled ciliary EV cargoes PC1 (LOV-1) and PC2 (PKD-2) to study ciliary EV shedding, bioactivity, and targeting in living animals. Super resolution, real time imaging reveals that LOV-1 and PKD-2 co-localize on ciliary EVs and that cilia shed polycystin-carrying EVs from two distinct sites - the ciliary tip and the ciliary base. Ciliary tip EVs shedding is triggered by mechanical stimulation and functions in animal-to-animal communication. For example, males deposit PKD-2-carrying EVs onto the vulva of the hermaphrodite during mating. We also used PKD-2::GFP to enrich and profile the C. elegans EV proteome, and, more specifically, the polycystin EV signalosome. We hypothesize that polycystin-carrying EVs act as discrete signaling units and carry specific cargo for biogenesis, cargo sorting, signaling, and targeting. In Aim 1, we will define the relationships between polycystins and the components of polycystin- carrying EVs. In Aim 2, we will ascertain the function of polycystin-associated EV cargo and test the hypotheses that the polycystins and associated proteins function as a signalosome in ciliary EVs and in ciliated sensory neurons. This competing renewal application will uncover the fundamentals of ciliary EV biology and the functions of the polycystins on cilia and EVs. Our studies will provide insight about the biology of normal and pathological processes that require or are modulated by EVs including ciliopathies.