Akella, Jyothi et al. (2015) International Worm Meeting "C.elegans as a model to study extracellular vesicle biology, dynamics, and function."

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. .

Rizvi, F. et al. (2017) International Worm Meeting "C.elegans as a model to study extracellular vesicle biology."

Akella, J., Rizvi, F., Nguyen, K., Barr, M., Hall, D.

[International Worm Meeting, 2017]

Cells release extracellular vesicles (EVs) that serve as nano-sized packages allowing for exchange of protein and genetic content. Cilia are hair-like projections that play important roles in development and signaling. The cilium both releases and binds to EVs. EVs play a role in cell signaling in health and pathologies, and may carry beneficial or toxic cargo. An understanding of the biogenesis, release, uptake, and signaling of ciliary EVs is lacking. Using C. elegans as a model, we aim to identify the molecules and mechanisms involved in EV biology. A subset of the ciliated neurons of C. elegans release EVs containing cargo that include the polycystins LOV-1 and PKD-2 and a myristoylated protein CIL-7 (Wang et al. Current Biology 2014; Maguire et al. MBoC 2015). Transcriptional profiling of the EV releasing neurons (EVNs) revealed candidates that could play a role in EV biogenesis and/or release (Wang et al. Current Biology 2015). rab-28 is expressed in all ciliated neurons of C.elegans including the EVNs. rab-28 encodes a small RAB GTPase. RAB-28 is important for amphid ciliary ultrastructure, amphid glial sheath cell volumes, and amphid-mediated sensory behaviors (Jensen. et al. PLoS Genetics 2016). Here, we explore the role of RAB-28 in EVNs. We investigated the role of RAB-28 in EV biology. We examined shedding and release of GFP-tagged EV cargoes using live imaging of rab-28(tm2636) mutants. rab-28 mutants display altered localization of ciliary EV cargoes PKD-2 and CIL-7. Defects in the release of EV cargo from the tips of EVNs and/or alterations in the ciliary localization of GFP-tagged EV cargo in the EVNs may indicate defects in ciliary trafficking or defects in EV biogenesis and/or release. We are examining ciliary ultrastructure and EVs in rab-28 mutant males using transmission electron microscopy (TEM). The EV releasing CEM cilia have a unique ciliary ultrastructure and are housed within the cephalic sensillum (Silva et al. Current Biology 2017). The male cephalic sensillum is comprised of CEM and CEP neurons, and glial support and socket cells, the latter create a lumenal space that contains EVs. We are determining whether rab-28 regulates CEM ciliogenesis, the integrity of the cephalic sensillum, or EV biogenesis. Our work could shed light on the contribution of ciliary ultrastructure and cilia-glia interactions to EV biology.

Nikonorova, Inna et al. (2021) International Worm Meeting "Deciphering the ciliary extracellular vesicle (EV) proteome"

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.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Clupper, Michael et al. (2019) International Worm Meeting "C. elegans ciliated sensory neurons release distinct subpopulations of extracellular vesicles."

Touroutine, Denis, Clupper, Michael, Tanis, Jessica, Gill, Rachael

[International Worm Meeting, 2019]

Extracellular vesicles (EVs) are particles that transfer protein, miRNA, mRNA, lipid, and metabolite cargo between cells. They are released from all cell types and play important roles in both physiological and pathophysiological processes. The impact of EVs on recipient cells is dependent upon their molecular cargo. In C. elegans, EVs bud from the ciliary membrane of male-specific bilateral ray B type (RnB), hook B type (HOB), and cephalic male (CEM) sensory neurons and are released into the environment via cuticular pores, where they function in animal communication. We discovered that the ion channel CLHM-1 is expressed in the RnB, HOB, and CEM neurons, and that a functional GFP-tagged CLHM-1 fusion protein localizes to puncta in the cilia proper and cilia base at the site of EV formation. We found no difference in release of EVs containing GFP-tagged PKD-2, a known EV cargo protein, in a clhm-1 mutant, indicating that CLHM-1 is not required for EV biogenesis. Using high-resolution imaging, we observed GFP-tagged CLHM-1 in cilia-derived EVs, establishing CLHM-1 as EV cargo. In vitro studies have shown that a single cell type can release distinct subpopulations of EVs enriched in specific cargo. To determine if CLHM-1 and PKD-2 are in the same EV subpopulation, we analyzed transgenic animals expressing both CLHM-1::tdTomato and PKD-2::GFP and found that the two fluorescent proteins co-localized in very few EVs. In addition, release of PKD-2::GFP EVs, but not CLHM-1::GFP EVs, requires the kinesin KLP-6. Together, these results suggest that distinct subpopulations of EVs are released from the cilia of CEM, HOB, and RnB neurons. How cargo is specifically sorted into EV subpopulations is poorly understood. The ALG-2 interacting protein X (ALIX) can play a role in EV cargo sorting in vitro. Interestingly, we have found that loss of the C. elegans ALIX homolog alx-1 causes a significant increase in the number of CLHM-1 containing EVs. We are using high-resolution imaging to determine if CLHM-1::tdTomato is aberrantly packaged into vesicles that normally contain only PKD-2::GFP in alx-1 mutant animals. In conclusion, our results indicate that a unique cargo sorting mechanism exists that gives rise to unique subpopulations of ciliary EVs.

Beer, Katharina et al. (2017) International Worm Meeting "Retromer-associated proteins and PAD-1 control TAT-5 flippase activity to inhibit extracellular vesicle budding."

Nance, Jeremy, Stigloher, Christian, Wehman, Ann, Fazeli, Gholamreza, Beer, Katharina

[International Worm Meeting, 2017]

Cells release extracellular vesicles (EV) that can mediate intercellular communication to influence development and disease (Beer & Wehman, Cell Adh Migr 2017). Despite their pleiotropic functions, the molecular details of EV release are poorly understood, especially for plasma membrane budding (ectocytosis). Previously, we showed that TAT-5 phospholipid flippase activity inhibits ectocytosis and maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the inner leaflet of the plasma membrane (Wehman et al., Curr Biol 2011). In a screen for additional proteins that inhibit EV budding, we identified new TAT-5 regulators related to the retromer recycling pathway (PI3Kinase VPS-34, Beclin1 homolog BEC-1, and RME-8) together with the Dopey domain protein PAD-1. PI3K, RME-8, and sorting nexins are required for the localization of TAT-5 to the plasma membrane, which is important to maintain PE asymmetry. PAD-1 also localizes to the plasma membrane, but is not required for TAT-5 localization. Rather, PAD-1 is required for the lipid flipping activity of TAT-5, further supporting the model that PE asymmetry regulates plasma membrane budding. Our study identifies new proteins that regulate extracellular vesicle release and pinpoints TAT-5 and phosphatidylethanolamine as key regulators of plasma membrane budding. Understanding the mechanisms of EV release will enable us to determine the in vivo roles of EVs during development and homeostasis.

Borgonie G et al. (1995) International C. elegans Meeting "MISSORTING CAUSES RICIN RESISTANCE IN THE NEMATODE CAENORHABDITIS ELEGANS"

Coomans AV, Claeys M, Link CD, Driessche EV, Borgonie G

[International C. elegans Meeting, 1995]

The resistance of the nematode C. elegans towards the potent toxin ricin has been studied. Micro-injection of the ricin A-chain into the distal, syncitial gonad causes degeneration and sterility in test specimens, confirming that C. elegans ribosomes are sensitive to depurination. However, when nematodes are incubated in ricin no negative effect is observed on lifespan or progeny. Using TEM, it was observed that ricin is effectively internalized into the intestinal cells. When prelabelled with gold, the toxin reached only the lysosomes. When native toxin was used, the toxin was either routed to the lysosomes are undergoes transcytosis to the pseudocoelomatic cavity and into embryos. None of the ricin reached either the trans Golgi network or the Golgi apparatus, considered essential for toxicity. The observed oral non-toxicity is most likely due to missorting of the toxin. The data indicate that, although ricin is opportunistically binding and being internalized by several receptors, not all of these are suitable to elicit toxicity.

Clupper, Michael et al. (2021) International Worm Meeting "Intraflagellar transport is required for enrichment of CLHM-1 into a distinct subpopulation of extracellular vesicles released from ciliated sensory neurons"

Gill, Rachael, Elsayyid, Malek, Touroutine, Denis, Tanis, Jessica, Clupper, Michael

[International Worm Meeting, 2021]

Extracellular vesicles (EVs) are membrane-wrapped particles that mediate cell-cell communication by transporting proteins, nucleic acids, and metabolites through biological fluids. EVs play roles in many physiological and pathophysiological processes, and their precise function is dependent on molecular cargo and parent cell type. A single cell can release distinct subpopulations of EVs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. In the nematode C. elegans, EVs bud from male sensory neuron cilia and are released into the hermaphrodite vulva during mating and the environment to mediate animal communication. We discovered that the calcium homeostasis modulator ion channel CLHM-1 localizes to cilia of EV-releasing neurons and is cargo in EVs released from males and hermaphrodites. Using total internal reflection fluorescence (TIRF) microscopy, we imaged EVs released from animals co-expressing tdTomato-tagged CLHM-1 and GFP-tagged PKD-2, a known EV cargo, in the same male sensory neurons, and observed that CLHM-1 and PKD-2 are significantly enriched in distinct subpopulations. Furthermore, release of these subpopulations is dependent on different sensory cues, as culturing males in the absence of hermaphrodites significantly increased the release of PKD-2::GFP EVs from adult males, but did not affect the number of CLHM-1::tdTomato EVs released. As CLHM-1::tdTomato and PKD-2::GFP do not completely colocalize in neuron cilia, we hypothesize that maintaining discrete separation of these proteins in the cilia is required for their enrichment into distinct EV subpopulations. Intraflagellar transport (IFT) is required for movement of proteins along the length of the cilia. Anterograde IFT is driven by kinesin motors, including homodimeric kinesin-II OSM-3, kinesin-III KLP-6, and a heterotrimeric kinesin-II complex containing KLP-11. In osm-3 mutants, colocalization between CLHM-1::tdTomato and PKD-2::GFP in EVs significantly increases, indicating that EV protein cargo enrichment is dependent on OSM-3. Environmental release - but not biogenesis - of both CLHM-1 and PKD-2 EVs is dependent on KLP-6. Currently, we are investigating the role of KLP-11 and the heterotrimeric kinesin-II in EV subpopulation cargo enrichment. In conclusion, our results show that IFT is required for proper packaging and release of ciliary EVs.

Beer, Katharina et al. (2015) International Worm Meeting "PAD-1 prevents extracellular vesicle budding in C. elegans embryos."

Beer, Katharina, Wehman, Ann

[International Worm Meeting, 2015]

Cells release extracellular vesicles (EVs) that can mediate intercellular communication by the delivery of lipids, proteins and nucleic acids and are therefore able to influence development, immune responses or disease. In previous studies, we showed that the phospholipid flippase TAT-5 (a P4-ATPase) inhibits the budding of EVs in C. elegans embryos and maintains Phosphatidylethanolamine (PE) asymmetry in the plasma membrane (Wehman et al., Curr Biol, 2011). Loss of TAT-5 activity causes increased release of EVs, but it is not known how TAT-5 activity is regulated. To address this, we searched for potential TAT-5 interactors. The large and novel protein PAD-1 is a homolog of the yeast Dop1p, which interacts with the yeast TAT-5 homolog, Neo1p. Therefore, we hypothesized that PAD-1 could also regulate EV budding in C. elegans. We depleted PAD-1 by RNAi and tested for EVs using a fluorescent membrane marker and transmission electron microscopy (TEM). We found excess membrane between cells in pad-1 mutants and TEM analysis revealed excessive release of EVs similar to tat-5 mutants. Thus, PAD-1 also prevents EV budding in C. elegans. Whether PAD-1 regulates TAT-5 activity, localization or levels during EV budding remains unclear. To address this, we are currently creating a pad-1 deletion allele and GFP-tagged alleles using the CRISPR/Cas9 system. We have also generated antibodies against PAD-1 and TAT-5 and are analyzing their localization and expression levels to determine whether PAD-1 regulates TAT-5. We are further checking whether pad-1 mutants show defects in PE-asymmetry similar to tat-5 mutants by staining cells with lipid probes. In summary, this approach will give us a better understanding of TAT-5- and PAD-1-mediated EV release and will provide insight into the vital process of cell communication.

Gill, R. et al. (2019) International Worm Meeting "Identifying biogenesis mechanisms for discrete subpopulations of extracellular vesicles."

Tanis, J., Gill, R., Touroutine, D., Clupper, M.

[International Worm Meeting, 2019]

Extracellular vesicles (EVs) are membrane wrapped structures containing proteins, RNAs, lipids, and metabolites that are released from most if not all cell types to mediate intercellular communication in physiological and pathological conditions. EVs fall into subclasses based on their mode of biogenesis. Exosomes are released following fusion of multivesicular bodies (MVBs) with the plasma membrane, while microvesicles form directly from plasma membrane budding. Our goal is to use C. elegans to identify proteins required for EV biogenesis in vivo. EVs are released from the cilia of the male specific cephalic male (CEM) neurons in the head and hook B type (HOB) and bilateral ray B type neurons (RnB) in the tail. These EVs are likely microvesicles as there is no evidence of MVBs in these neurons1. We discovered that the ion channel CLHM-1 is cargo in EVs released from C. elegans ciliated male sensory neurons by performing high resolution imaging of animals expressing functional GFP-tagged CLHM-1 at endogenous levels. Remarkably, when we co-expressed tdTomato-tagged CLHM-1 with GFP-tagged PKD-2, a known EV cargo protein expressed in the same neurons1, we rarely observed colocalization of the fluorescent proteins in vesicles, suggesting that CLHM-1 and PKD-2 are in discrete EV subpopulations. We are using the power of genetics to manipulate candidate EV biogenesis pathways to identify factors required for release of CLHM-1 containing EVs. Lipid asymmetry between the inner and outer leaflets of the plasma membrane induces curvature which could drive microvesicle release. Type IV-ATPase flippases translocate phospholipids from the outer to the inner leaflet to maintain bilayer asymmetry, while scramblases disrupt membrane asymmetry. We are determining if the flippases TAT-1, TAT-3, and TAT-6 as well as the scramblases ANOH-1 and SCRM-4 play a role in the biogenesis of one or both fluorescently marked EV subpopulations derived from male ciliated neurons. 1. Wang J, Silva M, Haas LA, Morsci NS, Nguyen KC, Hall DH, Barr MM. (2014) C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr Biol. 24(5):519-25.