Nikonorova, Inna, Cope, Alexander, Barr, Maureen, Power, Kaiden, Walsh, Jonathon, Wang, Juan, Shah, Premal, Akella, Jyothi
[
International Worm Meeting,
2021]
Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies. However, the EV field lacks a fundamental understanding of biogenesis, cargo content, signaling, and target interactions. EVs that are transmitted by cilia represent a particular challenge due to small volume of the organelle. Here, we used our established C. elegans system to determine the composition and explore the function of ciliary EVs. We took advantage of the fact that C. elegans releases ciliary EVs from 21 male-specific neurons and 6 core IL2 neurons into environment and thus provides a great platform for discovery of evolutionarily conserved ciliary EV cargo. To collect ciliary EVs we developed a biochemical enrichment procedure based on buoyant density centrifugation and high-resolution fractionation. Using fluorescent-tagged EV cargo PKD-2::GFP and superresolution microscopy we tracked ciliary EVs in the collected fractions and identified two populations of PKD-2 carrying EVs that differ in their densities. Proteomic analysis of the PKD-2 EV-enriched fractions revealed 2,888 proteins of C. elegans EVome that likely originate from multiple tissues. Top candidates were validated via generation of transgenic or CRISPR reporters and visualization of EV release using super-resolution microscopy. This strategy revealed that the male reproductive system is a major source of non-ciliary EVs. To extract ciliary EV cargoes, we integrated our dataset with published transcriptomic data. We identified new ciliary EV cargo involved in nucleotide binding and RNA interference, suggesting that environmentally-released ciliary EVs may also carry nucleic acids. Our work serves as a springboard for discoveries in the EV field and will help shed light on the contribution of ciliary EVs to the pathophysiology of abnormal EV signaling, including ciliopathies, cancer, and neurodegenerative diseases.