Project Summary Small secreted extracellular vesicles (EVs) play critical roles in communication between cells during physiological and pathological processes. The primary cilium, a sensory organelle protruding from cells, serves as a platform to transmit signals via shedding of microvesicles, a class of EVs that bud directly from the plasma membrane. In C. elegans, microvesicles shed from sensory neuron cilia are discharged into the environment where they play a role in animal-to-animal communication. We discovered that CLHM-1, the homolog of Illuminating the Druggable Genome (IDG) target FAM26 ion channels, is a cargo in EVs released from the ciliary base. tdTomato-tagged CLHM-1 and GFP-tagged PKD-2, a well described EV cargo and homolog of the IDG target PKD2L2 TRPP channel, colocalize in the ciliary base and cilium middle segment. However, these proteins are enriched in distinct EV subpopulations that are discharged in different quantities, with PKD-2 alone located in the cilium distal tip and EVs shed from this site. CLHM-1 versus PKD-2 containing EVs are differentially shed into the environment in response to the presence of mating partners, demonstrating that a single cilium can release distinct subpopulations of microvesicles, each with different cargo enrichment and functionalities. There is limited understanding of the mechanisms underlying microvesicle cargo loading, biogenesis and release. The membrane lipid phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 plays critical roles in protein localization and cytoskeletal remodeling. While a few studies suggest that PI(4,5)P2 levels affect EV cargo localization and biogenesis, it remains unclear whether PI(4,5)P2 promotes or inhibits microvesicle shedding. PI(4,5)P2 is predominantly generated by type I phosphatidylinositol 4-phosphate 5-kinases (PIP5K1s), the IDG target focus of this proposal. There is a single PIP5K1 in C. elegans, PPK-1. Our overarching goal is to draw upon the strengths of our genetic model system and cutting edge imaging approaches to define how PIP5K1- dependent generation of PI(4,5)P2 impacts EV cargo sorting and formation. The proposed research will use our unique transgenic animals that express multiple fluorescently tagged EV cargoes at single copy level and advanced imaging techniques to determine how overexpression of PPK-1 as well as inducible loss of this kinase affects shedding of cilia-derived microvesicle subpopulations and colocalization of cargoes in EVs. Using a a PI(4,5)P2 reporter consisting of the PH domain of PLCδ1 fused to GFP we will define the impact of PPK-1 on PI(4,5)P2 ciliary abundance and localization. Finally, we will determine if PPK-1 and PI(4,5)P2 subciliary localization underlies the differences in EV subpopulation abundance and release in response to mating partner presence. This work will lead to an understanding of how PIP5K1 activity impacts formation of heterogeneous EV populations with different physiological functions, impacting br...