Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis

Abstract

Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classes of molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsy samples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolate EVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C.elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on a scale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system. BACKGROUNDThe cellular secretion of small membrane-bound extracellular vesicles (EVs) into the external environment is an ancient capacity conserved throughout evolution 1,2,3 . EVs range in size from 30-1000 nm in diameter and can be internalized into recipient cells via endocytosis or membrane fusion. There is growing recognition that EVs may play important roles in facilitating intercellular communication through transferring protein, lipid, and genetic cargos 4,5 . The content of EVs are influenced by the physiological state of the cells and are thought to play critical roles in diverse cellular processes as well as multiple types of pathological conditions including cancer, immunity, and neurodegenerative diseases.Many studies have characterized the composition of mammalian EVs. Such EVs are highly enriched in the lipid raft species cholesterol, and sphingomyelin, and in proteins that associate with lipid rafts, including glycosylphosphatidylinositol-anchored (GPI) proteins 6,7,8 . The two main types of EVs studied so far are exosomes, which release from the endosomal network and microvesicles which bud directly from the plasma membrane 9,10 . Mammalian exosomes commonly contain membrane proteins such as CD9, CD63, and CD81, as well as lysosomal and endosomal-marking proteins, and various amounts of extracellular matrix proteins while are largely free of nuclear proteins 11 . Microvesicles have less defined protein markers but may contain proteins of mitochondria and endoplasmic reticulum origin 11 . However, the methods utilized to purify EVs do not separate these types of vesicles, so it is currently unclear how to definitively distinguish the cargos from different types of EVs 12 . EVs from diverse species, including humans, are known to carry RNA ...

Extracellular Vesicle-Contained microRNA of C. elegans as a Tool to Decipher the Molecular Basis of Nematode Parasitism

“…In line with the experimental design, 80/127 (63%) proteins assigned to a gene ontology cellular component (74% of all proteins) were associated with membrane (GO:0016020, GO:0016021, GO:0005886, GO:0045121), or the extracellular space (GO:0005615, GO:0005576). This is in agreement with a previous report (Russell et al, 2018).…”

The PMK-3 (p38) Mitochondrial Retrograde Response Functions in Intestinal Cells to Extend Life via the ESCRT Machinery

“…We predict that EVs emitted from the baso-lateral surface of intestinal cells into the coelomic cavity in response to PMK-3 activation will mediate signaling to the rest of the body to control longevity. However, formal testing of the role of EVs in PMK-3 retrograde response signaling is currently hampered by technical limitations which prevent isolation of EVs from specific tissues (26). Current, state-of-the-art isolation of EVs from C. elegans is restricted to extracorporeal populations from starved worms (26).…”

“…However, formal testing of the role of EVs in PMK-3 retrograde response signaling is currently hampered by technical limitations which prevent isolation of EVs from specific tissues (26). Current, state-of-the-art isolation of EVs from C. elegans is restricted to extracorporeal populations from starved worms (26). When non-stressed worms were treated with these EV populations from worms with activated PMK-3 signaling to see if they might mediate a broader retrograde signal, we find not unexpectedly that they do not ( Fig.…”

“…EV-containing fractions from adult worms was collected using size exclusion chromatography as described (26). Samples were utilized within a 72 hour window.…”

Purification and Analysis of <em>Caenorhabditis elegans</em> Extracellular Vesicles

“…This purification approach capitalizes on the active secretion of EVs outside the bodies of intact, living worms. This permits C. elegans EVs to be isolated at comparable purity and abundance as from cell culture, specifications that are sufficient for identifying hundreds of protein and RNA cargos via LC-MS-MS and RNAseq analysis 26 . Thus, the large library of reagents available for C. elegans research can be leveraged for investigating the influence of the genetic and physiological perturbation on EV cargo composition.…”