Isolation, profiling, and tracking of extracellular vesicle cargo in Caenorhabditis elegans

Extracellular vesicles (EVs) may mediate intercellular communication by carrying protein and RNA cargo. The composition, biology, and roles of EVs in physiology and pathology have been primarily studied in the context of biofluids and in cultured mammalian cells. The experimental tractability of C. elegans makes for a powerful in vivo animal system to identify and study EV cargo from its cellular source. We developed an innovative method to label, track, and profile EVs using genetically encoded, fluorescent-tagged EV cargo and conducted a large-scale isolation and proteomic profiling. Nucleic acid binding proteins (∼200) are overrepresented in our dataset. By integrating our EV proteomic dataset with single-cell transcriptomic data, we identified and validated ciliary EV cargo: CD9-like tetraspanin (TSP-6), ectonucleotide pyrophosphatase/phosphodiesterase (ENPP-1), minichromosome maintenance protein (MCM-3), and double-stranded RNA transporter SID-2. C. elegans EVs also harbor RNA, suggesting that EVs may play a role in extracellular RNA-based communication. •Large-scale identification and a pipeline for validation of EV cargo in C. elegans•Multiple tissues shed EVs, and different neuronal cilia shed distinct EV cargo•Ciliary EVs carry nucleic acid-binding SID-2 and MCM-3 and phosphodiesterase ENPP-1•The male vas deferens transfers ENPP-1 to the hermaphrodite uterus during mating Extracellular vesicles (EVs) are an ancient and conserved form of intercellular and interorganismal communication. Nikonorova et al. used the conserved ciliary EV cargo PKD-2::GFP to enrich and profile the EV proteome of Caenorhabditis elegans. They identified and validated new EV cargo using a bioinformatic mining app MyEVome and live imaging of fluorescent reporters. Nucleic acid binding proteins SID-2 and MCM-3 are bona fide ciliary EV cargo. C. elegans EVs also carry RNA, suggesting that EVs may mediate extracellular RNA-based communication.