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[
MicroPubl Biol,
2022]
Cells release extracellular vesicles (EVs) carrying cargos that can influence development and disease, but the mechanisms that govern EV release by plasma membrane budding are poorly understood. We previously showed that the Dopey protein PAD-1 inhibits EV release from the plasma membrane in C. elegans . However, PAD-1 is large, and the domains required to regulate EV release were unknown. Here, we reveal that the conserved N-terminal EWAD motif and C-terminal leucine zippers are required to inhibit EV release from the plasma membrane. Revealing a role for these domains is an important first step to identifying how EV release is regulated.
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[
Cell Mol Neurobiol,
2016]
Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV-cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and Caenorhabditis elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. Caenorhabditis elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport-dependent manner. Caenorhabditis elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia-EV interactions. Until the 21st century, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies.
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[
Curr Biol,
2021]
Ciliary extracellular vesicle (EV) shedding is evolutionarily conserved. In Chlamydomonas and C.elegans, ciliary EVs act as signaling devices.<sup>1-3</sup> In cultured mammalian cells, ciliary EVs regulate ciliary disposal but also receptor abundance and signaling, ciliary length, and ciliary membrane dynamics.<sup>4-7</sup> Mammalian cilia produce EVs from the tip and along the ciliary membrane.<sup>8</sup><sup>,</sup><sup>9</sup> This study aimed to determine the functional significance of shedding at distinct locations and to explore ciliary EV biogenesis mechanisms. Using Airyscan super-resolution imaging in living C.elegans animals, we find that neuronal sensory cilia shed TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites: the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. A big unanswered question in the EV field is how cells sort EV cargo. Here, we show that two EV cargoes- CIL-7 and PKD-2-localized and trafficked differently along cilia and were sorted to different environmentally released EVs. In response to mating partners, C.elegans males modulate EV cargo composition by increasing the ratio of PKD-2 to CIL-7 EVs. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.
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[
MicroPubl Biol,
2023]
Cells release extracellular vesicles (EVs) from their surface, but the mechanisms that govern EV release by plasma membrane budding are poorly understood. The lipid flippase TAT-5 inhibits EV release from the plasma membrane in C. elegans , but how the level of flippase activity regulates EV release was unknown. We generated point mutations in the DGET motif of TAT-5 predicted to lead to a partial or complete loss of ATPase activity. We discovered that
tat-5(E246Q) mutants were sterile, while
tat-5(D244T) mutants produced embryos that arrested during development. Using degron-based reporters, we found that EV release was increased in
tat-5(D244T) mutant embryos and that phagocytosis was also disrupted. These data suggest that a low level of flippase activity can promote fertility, while a higher level of flippase activity is required to inhibit EV release, allow phagocytosis, and carry out embryonic development.
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[
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.
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Power, Kaiden, Akella, Jyothi, Nikonorova, Inna, Cope, Alexander, Shah, Premal, Wang, Juan, Walsh, Jonathon, Barr, Maureen
[
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.
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Akella, Jyothi, Silva, Malan, Maguire, Julie, Barr, Maureen, Hall, David, Wang, Juan
[
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. .
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Rizvi F, Tsiropoulou S, Kennedy BN, Carter SP, Moran AL, Barr MM, Hall DH, Nguyen K, Silva M, Blacque OE, Akella JS
[
Elife,
2020]
Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis are almost entirely unknown. Here, we show that the ciliary G-protein Rab28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of <i>Caenorhabditis elegans</i> in a cilia specific manner. Sequential targeting of lipidated Rab28 to periciliary and ciliary membranes is highly dependent on the BBSome and the prenyl-binding protein phosphodiesterase 6 subunit delta (PDE6D), respectively, and BBSome loss causes excessive and ectopic EV production. We also find that EV defective mutants display abnormalities in sensory compartment morphogenesis. Together, these findings reveal that Rab28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane and suggest that EVs may mediate signaling between cilia and glia to shape sensory organ compartments. Our data also suggest that defects in the biogenesis of cilia-related EVs may contribute to human ciliopathies.
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[
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.
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[
Curr Biol,
2022]
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.