Hall, David, Wang, Juan, Barr, Maureen, Silva, Malan, Akella, Jyothi, Maguire, Julie
[
International Worm Meeting,
2015]
Extracellular vesicles (EVs) are membrane bound vesicles released by most cells in the body. EVs aid the exchange of cargo such as proteins, lipids, and nucleic acids between cells without requiring direct contact. EVs are proposed to play important roles in the nervous system in vitro. Under healthy conditions, EVs are neuroprotective but, may propagate and promote neurodegeneration under conditions such as injury and infection. The functions of EVs, and the factors that affect EV dynamics and composition in vivo are unknown. A subset of ciliated neurons of C. elegans release GFP-tagged EVs containing select cargo into the environment. We use the environmentally released EVs of C.elegans as a model to identify the components and conditions that affect EV dynamics in vivo i.e. cause a change in total EV content and EV composition. Our strategy includes the identification of genes and mechanisms that regulate EV biogenesis and release under normal conditions, as well as determining the functions of EVs.Using in vivo imaging of fluorescently tagged EV cargo and transmission electron microscopy, we identified proteins that regulate EV biogenesis and release including a kinesin and a myristoylated novel protein. We previously identified that purified EVs from C.elegans trigger male tail chasing behavior, which is the first example of EVs mediating animal-animal communication (Wang et al; 2014). We also found that C.elegans EVs are bactericidal. Our future studies are aimed at identifying the components important for bactericidal activity and for identifying conditions that affect the bactericidal properties of EVs. Furthermore, we will determine whether EVs purified from mutants of the known regulators of EV biogenesis and release demonstrate differences in behavioral and bactericidal assays. Our studies are expected to provide insights into the factors that regulate EV biogenesis and release, and identify factors that affect the composition of EVs under normal conditions, and under other environmental stress. This knowledge is important for our understanding of the functions of EVs in health and disease, and the factors that modulate EV properties in disease. .
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.