Extracellular vesicles (EVs) are membrane-wrapped particles that mediate cell-cell communication by transporting proteins, nucleic acids, and metabolites through biological fluids. EVs play roles in many physiological and pathophysiological processes, and their precise function is dependent on molecular cargo and parent cell type. A single cell can release distinct subpopulations of EVs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. In the nematode C. elegans, EVs bud from male sensory neuron cilia and are released into the hermaphrodite vulva during mating and the environment to mediate animal communication. We discovered that the calcium homeostasis modulator ion channel CLHM-1 localizes to cilia of EV-releasing neurons and is cargo in EVs released from males and hermaphrodites. Using total internal reflection fluorescence (TIRF) microscopy, we imaged EVs released from animals co-expressing tdTomato-tagged CLHM-1 and GFP-tagged PKD-2, a known EV cargo, in the same male sensory neurons, and observed that CLHM-1 and PKD-2 are significantly enriched in distinct subpopulations. Furthermore, release of these subpopulations is dependent on different sensory cues, as culturing males in the absence of hermaphrodites significantly increased the release of PKD-2::GFP EVs from adult males, but did not affect the number of CLHM-1::tdTomato EVs released. As CLHM-1::tdTomato and PKD-2::GFP do not completely colocalize in neuron cilia, we hypothesize that maintaining discrete separation of these proteins in the cilia is required for their enrichment into distinct EV subpopulations. Intraflagellar transport (IFT) is required for movement of proteins along the length of the cilia. Anterograde IFT is driven by kinesin motors, including homodimeric kinesin-II OSM-3, kinesin-III KLP-6, and a heterotrimeric kinesin-II complex containing KLP-11. In
osm-3 mutants, colocalization between CLHM-1::tdTomato and PKD-2::GFP in EVs significantly increases, indicating that EV protein cargo enrichment is dependent on OSM-3. Environmental release - but not biogenesis - of both CLHM-1 and PKD-2 EVs is dependent on KLP-6. Currently, we are investigating the role of KLP-11 and the heterotrimeric kinesin-II in EV subpopulation cargo enrichment. In conclusion, our results show that IFT is required for proper packaging and release of ciliary EVs.