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. Rutgers University researchers 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 their dataset. By integrating their EV proteomic dataset with single-cell transcriptomic data, the researchers 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.

Identified EV proteome is enriched in nucleic acid binding proteins and ciliary EV cargo

(A) Consolidated groups of InterPro domains enriched in the identified EV proteome (for the full list, see Data S2). Bars represent fold enrichment in the EVome over the whole proteome. (B) Identified EVome contained proteins encoded by previously identified transcripts specific to EV-releasing neurons (EVNs). The Venn diagram shows EV cargo candidates that overlap with two transcriptomic datasets.8,19 (C) GFP::KLP-6 EV shedding from the IL2 cilia of a wild-type hermaphrodite, orthogonal projection. GFP::KLP-6 was observed in EVs released outside the animal. (D) The transcriptome of EV-releasing neurons is most similar to the cholinergic cluster 15 from Cao et al.,14 likely representing the IL2 neurons. (E) EV cargo candidates were categorized into three groups based on their relative enrichment in the IL2 sex-shared EV-releasing neurons (x axis) and in the ciliated neurons (y axis). Three categories are indicated by colors. Previously validated ciliary EV cargoes are bolded.