Extracellular vesicles (EVs) are particles that transfer protein, miRNA, mRNA, lipid, and metabolite cargo between cells. They are released from all cell types and play important roles in both physiological and pathophysiological processes. The impact of EVs on recipient cells is dependent upon their molecular cargo. In C. elegans, EVs bud from the ciliary membrane of male-specific bilateral ray B type (RnB), hook B type (HOB), and cephalic male (CEM) sensory neurons and are released into the environment via cuticular pores, where they function in animal communication. We discovered that the ion channel CLHM-1 is expressed in the RnB, HOB, and CEM neurons, and that a functional GFP-tagged CLHM-1 fusion protein localizes to puncta in the cilia proper and cilia base at the site of EV formation. We found no difference in release of EVs containing GFP-tagged PKD-2, a known EV cargo protein, in a
clhm-1 mutant, indicating that CLHM-1 is not required for EV biogenesis. Using high-resolution imaging, we observed GFP-tagged CLHM-1 in cilia-derived EVs, establishing CLHM-1 as EV cargo. In vitro studies have shown that a single cell type can release distinct subpopulations of EVs enriched in specific cargo. To determine if CLHM-1 and PKD-2 are in the same EV subpopulation, we analyzed transgenic animals expressing both CLHM-1::tdTomato and PKD-2::GFP and found that the two fluorescent proteins co-localized in very few EVs. In addition, release of PKD-2::GFP EVs, but not CLHM-1::GFP EVs, requires the kinesin KLP-6. Together, these results suggest that distinct subpopulations of EVs are released from the cilia of CEM, HOB, and RnB neurons. How cargo is specifically sorted into EV subpopulations is poorly understood. The ALG-2 interacting protein X (ALIX) can play a role in EV cargo sorting in vitro. Interestingly, we have found that loss of the C. elegans ALIX homolog
alx-1 causes a significant increase in the number of CLHM-1 containing EVs. We are using high-resolution imaging to determine if CLHM-1::tdTomato is aberrantly packaged into vesicles that normally contain only PKD-2::GFP in
alx-1 mutant animals. In conclusion, our results indicate that a unique cargo sorting mechanism exists that gives rise to unique subpopulations of ciliary EVs.