Pitts, Lauren R et al. (2022) MicroPubl Biol

Pitts, Lauren R, Wehman, Ann M, Nguyen, Alexander T

[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.

Wang J et al. (2021) Curr Biol "Sensory cilia act as a specialized venue for regulated extracellular vesicle biogenesis and signaling."

Silva M, Akella JS, Tilton PE, Gu A, Walsh JD, Barr MM, Nikonorova IA, Wang J

[Curr Biol, 2021]

Ciliary extracellular vesicle (EV) shedding is evolutionarily conserved. In Chlamydomonas and C.elegans, ciliary EVs act as signaling devices.^1-3 In cultured mammalian cells, ciliary EVs regulate ciliary disposal but also receptor abundance and signaling, ciliary length, and ciliary membrane dynamics.^4-7 Mammalian cilia produce EVs from the tip and along the ciliary membrane.⁸^,⁹ 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.

Carmel L et al. (2009) Genome Biol Evol "A universal nonmonotonic relationship between gene compactness and expression levels in multicellular eukaryotes."

Koonin EV, Carmel L

[Genome Biol Evol, 2009]

Analysis of gene architecture and expression levels of four organisms, Homo sapiens, Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana, reveals a surprising, nonmonotonic, universal relationship between expression level and gene compactness. With increasing expression level, the genes tend at first to become longer but, from a certain level of expression, they become more and more compact, resulting in an approximate bell-shaped dependence. There are two leading hypotheses to explain the compactness of highly expressed genes. The selection hypothesis predicts that gene compactness is predominantly driven by the level of expression, whereas the genomic design hypothesis predicts that expression breadth across tissues is the driving force. We observed the connection between gene expression breadth in humans and gene compactness to be significantly weaker than the connection between expression level and compactness, a result that is compatible with the selection hypothesis but not the genome design hypothesis. The initial gene elongation with increasing expression level could be explained, at least in part, by accumulation of regulatory elements enhancing expression, in particular, in introns. This explanation is compatible with the observed positive correlation between intron density and expression level of a gene. Conversely, the trend toward increasing compactness for highly expressed genes could be caused by selection for minimization of energy and time expenditure during transcription and splicing and for increased fidelity of transcription, splicing, and/or translation that is likely to be particularly critical for highly expressed genes. Regardless of the exact nature of the forces that shape the gene architecture, we present evidence that, at least, in animals, coding and noncoding parts of genes show similar architectonic trends.

Aravind L et al. (2001) Genome Biol "The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases."

Koonin EV, Aravind L

[Genome Biol, 2001]

BACKGROUND: Protein fold recognition using sequence profile searches frequently allows prediction of the structure and biochemical mechanisms of proteins with an important biological function but unknown biochemical activity. Here we describe such predictions resulting from an analysis of the 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenases, a class of enzymes that are widespread in eukaryotes and bacteria and catalyze a variety of reactions typically involving the oxidation of an organic substrate using a dioxygen molecule. RESULTS: We employ sequence profile analysis to show that the DNA repair protein AlkB, the extracellular matrix protein leprecan, the disease-resistance-related protein EGL-9 and several uncharacterized proteins define novel families of enzymes of the 2OG-Fe(II) oxygenase superfamily. The identification of AlkB as a member of the 2OG-Fe(II) oxygenase superfamily suggests that this protein catalyzes oxidative detoxification of alkylated bases. More distant homologs of AlkB were detected in eukaryotes and in plant RNA viruses, leading to the hypothesis that these proteins might be involved in RNA demethylation. The EGL-9 protein from Caenorhabditis elegans is necessary for normal muscle function and its inactivation results in resistance against paralysis induced by the Pseudomonas aeruginosa toxin. EGL-9 and leprecan are predicted to be novel protein hydroxylases that might be involved in the generation of substrates for protein glycosylation. CONCLUSIONS: Here, using sequence profile searches, we show that several previously undetected protein families contain 2OG-Fe(II) oxygenase fold. This allows us to predict the catalytic activity for a wide range of biologically important, but biochemically uncharacterized proteins from eukaryotes and bacteria.

Pitts, Lauren R et al. (2023) MicroPubl Biol

Wehman, Ann M, Pitts, Lauren R, Nguyen, Alexander T, Frondoni, Julia

[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.

Wang J et al. (2024) Curr Biol "Ciliary intrinsic mechanisms regulate dynamic ciliary extracellular vesicle release from sensory neurons."

Nguyen KC, Cruz CN, Hall DH, Barr MM, Power KM, Nikonorova IA, Saul J, Wang J

[Curr Biol, 2024]

Extracellular vesicles (EVs) are submicron membranous structures and key mediators of intercellular communication.¹^,² Recent research has highlighted roles for cilia-derived EVs in signal transduction, underscoring their importance as bioactive extracellular organelles containing conserved ciliary signaling proteins.³^,⁴ Members of the transient receptor potential (TRP) channel polycystin-2 (PKD-2) family are found in ciliary EVs of the green algae Chlamydomonas and the nematode Caenorhabditis elegans⁵^,⁶ and in EVs in the mouse embryonic node and isolated from human urine.⁷^,⁸ In C. elegans, PKD-2 is expressed in male-specific EV-releasing sensory neurons, which extend ciliary tips to ciliary pore and directly release EVs into the environment.⁶^,⁹ Males release EVs in a mechanically stimulated manner, regulate EV cargo content in response to mating partners, and deposit PKD-2::GFP-labeled EVs on the vulval cuticle of hermaphrodites during mating.⁹^,¹⁰ Combined, our findings suggest that ciliary EV release is a dynamic process. Herein, we identify mechanisms controlling dynamic EV shedding using time-lapse imaging. Cilia can sustain the release of PKD-2-labeled EVs for 2 h. This extended release doesn't require neuronal transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The kinesin-3 motor kinesin-like protein 6 (KLP-6) is necessary for initial and extended EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dynamic replenishment of PKD-2 at the ciliary tip is key to sustained EV release. Our study provides a comprehensive portrait of real-time ciliary EV release and mechanisms supporting cilia as proficient EV release platforms.

Akella JS et al. (2020) Elife "Ciliary Rab28 and the BBSome negatively regulate extracellular vesicle shedding."

Hall DH, Tsiropoulou S, Silva M, Akella JS, Blacque OE, Carter SP, Kennedy BN, Nguyen K, Rizvi F, Moran AL, Barr MM

[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.

Wang J et al. (2015) Curr Biol "Cell-Specific Transcriptional Profiling of Ciliated Sensory Neurons Reveals Regulators of Behavior and Extracellular Vesicle Biogenesis."

Haas LA, Hodgkin J, Kaletsky R, Silva M, Wang J, Hall DH, Landis JN, Barr MM, Patrick C, Williams A, Murphy CT, Rashid A, Gravato-Nobre M, Santiago-Martinez D, Androwski RJ

[Curr Biol, 2015]

Cilia and extracellular vesicles (EVs) are signaling organelles [1]. Cilia act as cellular sensory antennae, with defects resulting in human ciliopathies. Cilia both release and bind to EVs [1]. EVs are sub-micron-sized particles released by cells and function in both short- and long-range intercellular communication. In C.elegans and mammals, the autosomal dominant polycystic kidney disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation [2]. A fundamental understanding of EV biology and the relationship between the polycystins, cilia, and EVs is lacking. To define properties of a ciliated EV-releasing cell, we performed RNA-seq on 27 GFP-labeled EV-releasing neurons (EVNs) isolated from adult C.elegans. We identified 335 significantly overrepresented genes, of which 61 were validated by GFP reporters. The EVN transcriptional profile uncovered new pathways controlling EV biogenesis and polycystin signaling and also identified EV cargo, which included an antimicrobial peptide and ASIC channel. Tumor-necrosis-associated factor (TRAF) homologs trf-1 and trf-2 and the p38 mitogen-activated protein kinase (MAPK) pmk-1 acted in polycystin-signaling pathways controlling male mating behaviors. pmk-1 was also required for EV biogenesis, independent of the innate immunity MAPK signaling cascade. This first high-resolution transcriptome profile of a subtype of ciliated sensory neurons isolated from adult animals reveals the functional components of an EVN.

Nikonorova IA et al. (2022) Curr Biol "Isolation, profiling, and tracking of extracellular vesicle cargo in Caenorhabditis elegans."

Shah P, Akella JS, Tilton PE, Power KM, Nikonorova IA, Wang J, Walsh JD, Cope AL, Krauchunas AR, Barr MM

[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 (&#8764;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.

Aravind L et al. (1998) Trends Biochem Sci "The catalytic domain of the P-type ATPase has the haloacid dehalogenase fold."

Koonin EV, Galperin MY, Aravind L

[Trends Biochem Sci, 1998]