Ying Wang et al. (2008) C.elegans Neuronal Development Meeting "A glial DEG/ENaC channel functions with neuronal channel DEG-1 to mediate specific sensory functions in C. elegans"

Sun-Kyung Lee, Alfonso Apicella, Dan Slone, Shai Shaham, Marina Ezcurra, Ying Wang, Maya Goldmit, Laura Bianchi, William Schafer, Monica Driscoll

[C.elegans Neuronal Development Meeting, 2008]

DEG/ENaC channel subunits (named for founding members of the family C. elegans degenerin DEG-1 and the mammalian epithelial Na channels) are two-transmembrane domain proteins that trimerize to form Na or Na/Ca-selective ion channels. Across species, DEG/ENaC ion channels have been implicated in sensory transduction including mechanosensation (C. elegans MEC-4 and MEC-10), pain sensation, thermo-sensation, pheromone perception and proprioception (C. elegans UNC-8). Here we document the characterization of a novel C. elegans DEG/ENaC channel involved in sensory perception, ACD-1, which exhibits interesting interactions with the DEG-1 channel. DEG-1 is expressed in ASK chemosensory neurons (among other neurons), and is needed for normal avoidance of acidic solutions and attraction to lysine-acetate. We show that acd-1 deletion exacerbates these two sensory deficits of a deg-1 null mutant, such that acd-1; deg-1 double mutants are severely defective in responses. Unexpectedly, we find that ACD-1 is expressed in C. elegans glial amphid sheath cells, rather than in neurons. acd-1 mutants are not defective in overall glial structure or development, nor are they defective in aphid neuron development or viability. Thus glial-expressed acd-1 appears needed for sensory function. We characterized properties of the ACD-1 channel by expressing it in Xenopus oocytes and measuring its current properties. We report that ACD-1 is a Na-selective, constitutively open, and Ca-impermeable ion channel, and it is inhibited by both extracellular and intracellular acidification. We propose a mechanism for glial channel ACD-1 in controlling acid avoidance and lysine chemotaxis behaviors based on its acid sensitivity. Our findings indicate that C. elegans DEG/ENaC channel ACD-1 is expressed in glia but regulates sensory neuron function to influence behavior. Our findings suggest that channels with ACD-1 features may be expressed in mammalian glia to play important roles in controlling neuronal function.

Wang, Ying et al. (2009) International Worm Meeting "Glial DEG/ENaC channel ACD-1 functions in odor sensation in C. elegans."

Bianchi, Laura, Wang, Ying

[International Worm Meeting, 2009]

DEG/ENaC channel subunits are two-transmembrane domain proteins that trimerize to form voltage-independent Na+ or Na+/Ca2+-selective ion channels. Neuronally expressed DEG/ENaC channels have been implicated in sensory perception across species including touch sensation, pain sensation and proprioception. We recently reported that C. elegans DEG/ENaC channel ACD-1 participates in acid avoidance behavior and chemotaxis to lysine-acetate. Surprisingly, we found that ACD-1 is expressed in amphid glia rather than neurons where it acts to orchestrate these behaviors. More specifically, we found that acd-1 knock-out does not cause sensory deficits on its own but that it exacerbates mild defects in acid avoidance and chemotaxis to lysine-acetate caused by mutations in deg-1, another DEG/ENaC channel gene expressed sensory neurons. Because acidic solutions and lysine-acetate cause extracellular and intracellular acidification respectively, we tested if ACD-1 is sensitive to protons. In the heterologous expression studies, we found that ACD-1 is inhibited by both extracellular and intracellular acidification. We thus hypothesized that exacerbation of sensory defects by acd-1 knock-out may occur if ACD-1 channel is sensitive to the cue that acts on the specific sensory neuron whose function has been compromised by mutations in neuronal genes. To test this hypothesis, we assayed ACD-1 sensitivity to odors and tastants in Xenopus oocytes. We found that ACD-1 channels are inhibited by the odor isoamyl alcohol, which attracts C. elegans. We thus acquired a C. elegans mutant strain with no detectable abnormalities in sensory perception, but that is mutant for the gene tax-2 (a cyclic nucleotide gated channel) required for sensing isoamyl alcohol and expressed in AWC sensory neurons, among others. tax-2(p694) mutation deletes part of the promoter region of tax-2 reducing TAX-2 channel expression level. We built tax-2(p694);acd-1 double mutants and tested attraction to the odor isoamyl alcohol. Consistent with our hypothesis, we found that tax-2(p694);acd-1 double mutant animals are defective in attraction to this odor. Notably, acd-1 and tax-2(p694) single mutants detect this odor just like wild type animals. Functional imaging studies are under way to determine the consequences of acd-1 and tax-2 mutations on AWC neurons responses to odors.

Wang, Ying et al. (2013) International Worm Meeting "Neurotoxic unc-8 mutants encode constitutively active DEG/ENaC channels that are blocked by divalent cations."

Matthewman, Cristina, Wang, Ying, Bianchi, Laura, Miller, David, Han, Lu, Miller, Tyne

[International Worm Meeting, 2013]

Hyperactivation of ion channels of the DEG/ENaC family can induce swelling and degeneration of the cells in which they are expressed. A genetic screen for uncoordinated mutants in C. elegans identified dominant mutations in the DEG/ENaC channel subunit UNC-8 that cause neuronal swelling. Here we show that the movement defect of these unc-8d mutants is correlated with the selective death of DA and DB cholinergic motor neurons in the ventral nerve cord (VNC); GABAergic motor neurons are not affected. Our finding that < 25% of DA and DB motor neurons degenerate in unc-8(d) mutants may explain the original suggestion that neuronal swelling is transient and does not result in cell death (Shreffler et al, 1995). We performed electrophysiology experiments in Xenopus oocytes expressing the mutant proteins UNC-8 (G387E) and UNC-8(A586T) to confirm that these UNC-8d channels induce hyperactive cation transport. Our data show that currents are small due to blockage by extracellular divalent cations (Ca++, Mg++). Indeed, removal of extracellular divalent cations produces larger currents and results in the swelling and eventual death of oocytes expressing UNC-8(G387E). We suggest that the UNC-8 channel is intrinsically sensitive to extracellular divalent cations and that this property limits UNC-8(d) toxicity in vivo. Since the local concentration of extracellular calcium is likely to be decreased by the neurotransmitter release mechanism, regulation of external Ca2+ levels by neuronal activity may contribute to the selective death of neurons that express UNC-8d channels.

Wesley HUNG et al. (2008) C.elegans Neuronal Development Meeting "A Potential Gap Junction-Independent Role of a C. elegans Innexin in Neurons"

Edward YEH, Ying Wang, Mei ZHEN, Magali BOUHOURS, Wesley HUNG

[C.elegans Neuronal Development Meeting, 2008]

Connexins, pannexins and innexins are known for forming gap-junctions, intercellular channels between adjacent cells, allowing them to synchronize their activity through diffusion of ions or small molecules from cytoplasm to cytoplasm. In addition, some of these proteins were recently reported to play physiological and pathophysiological roles independently of gap-junctions, by forming hemichannels at the plasmic membrane and diffusing calcium and small signaling molecules to the extracellular space towards neighboring cells. Here we show that C. elegans animals lacking UNC-7 display irregularly spaced and sized active zones, and altered synaptic activity. This indicates that UNC-7 ultimately modulates synaptic morphology and synaptic activity. In C. elegans neurons, UNC-7 localizes not only to presumptive gap junctions but also to axonal regions devoid of gap junction structures. Our studies suggest that in addition to being structural components of gap junctions, innexins may also function as hemichannels to regulate membrane excitability, presumably by modulating the propagation of neuronal excitation along axons.

Wenying Zhang et al. (2007) International Worm Meeting "Specific interactions between MEC-4 and MEC-10 subunits change channel activity and induce neurotoxicity."

Wenying Zhang, Samuel Israel, Laura Bianchi, Ying Wang, Monica Driscoll

[International Worm Meeting, 2007]

The homologous DEG/ENaC channel subunits MEC-4 and MEC-10 are co-expressed in the 6 gentle touch neurons where they are thought to be co-assembled into heteromeric complexes that form the core of a mechanotransducing channel. A hyper-activated MEC-4 mutant channel (MEC-4(d)), specifying an A713V near the channel pore, induces touch neuron necrosis by enhancing calcium and sodium influx through the channel. Elevated ion influx appears to provoke ER calcium release. Increased cytoplasmic calcium concentration activates proteases that dismantle the neurons. Hyper-activation of the MEC-4 sequence-related channel ASIC1b can induce profound neuronal death in mouse brain ischemia models. Thus, channel hyperactivation induces a conserved neuronal necrosis response. We are identifying genes that both promote and protect against necrosis. To date, little work has been done to identify genetic enhancers of necrosis. We constructed lines that express mec-10 bearing an AA substitution analogous to MEC-4(A713V). In mec-10(d) transgenic lines, there is only a weak necrosis induction. We performed the first forward screen of the worm genome to identify mutations that enhance necrosis induced by mec-10(d). Among other enhancer mutations, we identified novel mec-4 allele bz301 as a potent enhancer of mec-10(d)-induced necrosis. The bz301 mutation encodes substitution MEC-4(A149V), which is situated in the extracellular loop, close to transmembrane domain I of MEC-4. This MEC-4 substitution causes strong enhancement of cell death induced by mec-10(d). While the A149V substitution (on its own) does not disrupt MEC-4 channel function and induces no cell death. MEC-4(A149V) requires mec-10(d) to be neurotoxic. We introduced MEC subunit combinations into Xenopus oocytes and found that MEC-4(A149V) increased MEC-10(d) sodium and calcium currents more than 10 times consistent with its potent neurotoxic effect in vivo when combined with mec-10(d). Conversely, when MEC-4(A149V) is expressed alone it produces much smaller currents and it is not permeable to calcium ion. This finding is in line with the lack of toxic activity of bz301 allele alone. We also found that MEC-4(A149V) changes amiloride sensitivity of the channel complex. These data suggest that MEC-4 and MEC-10 channel subunits interact in vivo in 3D to define the channel pore and the amiloride binding site. We will also report on other necrosis enhancers. This work was supported in part by a Predoctoral Fellowship from the NJ Commission on Spinal Cord Research to Wenying.

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.

Kyung Won Kim et al. (2005) International Worm Meeting "RNP-8 interacts with the GLD-2 cytoplasmic poly(A) polymerase and regulates its polyadenylation activity"

Kyung Won Kim, Judith Kimble, Liaoteng Wang

[International Worm Meeting, 2005]

Polyadenylation is critical for mRNA stability and translational activation. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs promotes their translation. The C. elegans gld-2 gene is required for multiple steps in germline development, including the mitosis/meiosis decision. GLD-2 is a cytoplasmic poly(A) polymerase (PAP) that lacks an RNA recognition motif (1); similar PAPs have been identified in virtually all eukaryotic organisms (2). A yeast two-hybrid screen using GLD-2 as bait isolated GLD-3, RNP-8 and LARP-1 (1; L. Wang and J. Kimble, unpublished). GLD-2 has little PAP activity on its own, but it is stimulated in vitro by GLD-3 (1). We have now focused on RNP-8 to ask whether this GLD-2 interactor also stimulates GLD-2 activity and to determine whether RNP-8 has a major role in germline development. Preliminary data reveal that rnp-8(RNAi) and gld-2 mutants have similar defects during oogenesis. Both gld-2 and rnp-8 mRNAs are abundant in embryos, L4 larvae, and adults, and rnp-8 mRNA is enriched in the germ line. To test whether RNP-8 stimulates the GLD-2 PAP activity, we performed assays for PAP activity in vitro. Preliminary data suggest that RNP-8 can indeed stimulate GLD-2 PAP activity. Taken together, we suggest that RNP-8 stimulates GLD-2 activity to control germline development.1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M., and Kimble J. (2002), A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble J. and Wickens, M. (2004), Mammalian GLD-2 homologs are poly(A) polymerases. PNAS 101: 4407-4412

Kendall Wu et al. (2004) West Coast Worm Meeting "Identification of Genetic Biomarkers and Regulators of Aging in C. elegans"

James Lund, John Wang, Yueyi Liu, Stuart Kim, Min Jiang, Kendall Wu

[West Coast Worm Meeting, 2004]

Despite the wealth of knowledge about conditions and mutations that cause worms to live longer, we still have an incomplete understanding of what varies from young to old worms -- especially at the molecular level. A long-term goal of our laboratory is to determine the molecular changes that occur as an organism ages in order to better understand the aging process and its regulation. Using the nematode Caenorhabditis elegans ( C. elegans ) as a genetic model for aging, we have used DNA microarrays to identify a common set of genes regulated throughout several age-related conditions: genes that change in old age (Lund et al. , 2002), genes that change in the exit from the dauer state (Wang and Kim, 2003), and genes that change expression in long-lived mutants of the insulin-like pathway such as the age-1 and daf-16 mutants (unpublished data). Analysis of the upstream regions of these age-regulated genes showed that they are heavily enriched for a GATA DNA consensus sequence suggesting that a GATA transcription factor may be involved in regulating the expression of these genes. This GATA motif may represent a novel regulatory pathway of the aging process that might act either together or separately from the daf-2 /insulin-like pathway. Using GFP reporters of these genes, we have begun to determine how these genes are age-regulated and to identify the key tissues regulating lifespan. We have also used RNAi to abrogate the expression of these genes to determine their role in longevity. The results of these studies will define a set of molecular biomarkers that will allow a more accurate description of the aging process. In addition, these biomarkers will allow identification of new aging mutants perhaps leading to identification of mutants with accelerated aging. Lund, J., Tedesco, P., Duke, K., Wang, J., Kim, S. K., and Johnson, T. E. (2002). Transcriptional profile of aging in C. elegans. Curr Biol 12, 1566-1573. Wang, J., and Kim, S. K. (2003). Global analysis of dauer gene expression in Caenorhabditis elegans. Development 130, 1621-1634.

Edward Yeh et al. (2007) International Worm Meeting "A novel putative calcium channel NCA-1 and its scaffold UNC-80 regulate synaptic activity."

Ying Wang, Kim Schuske, Katya Melnik-Martinez, William Schafer, Sharon Ng, Edward YEH, Mi Zhang, Mei ZHEN

[International Worm Meeting, 2007]

The entry of calcium (Ca2+) into cells is conducted through multiple Ca2+ channels that differ in composition, property and modulation. Influx of Ca2+ at synapses, typically through N-, P- and Q-type voltage-gated Ca2+ channels, is a prerequisite for vesicle fusion mediated synaptic transmission. In a screen to identify genes involved in synapse development and function, we isolated the novel putative Ca2+ channel-like protein NCA-1/UNC-77 and its accessory or scaffold protein UNC-80. Gain-of-function alleles of nca-1 lead to an abnormal distribution of an active zone marker at GABAergic synapses, constitutively activated Ca2+ influx at serotonergic synapses and hyperactivated locomotions. While nca-1 loss-of-function mutants exhibit wild type behavior, double mutants of nca-1 and nca-2 (a closely related homolog) display reduced Ca2+ influx frequency at synapses and unsustainable normal locomotion upon stimulation (fainter behavior). NCA-1 is expressed in the nervous system and is required in neurons to regulate synapse morphology and function. Unlike N/P/Q-type Ca2+ channels that closely associate with active zones, NCA-1 protein localizes to perisynaptic regions. The presence of NCA-1 perisynaptically regions requires UNC-80, a novel protein that also localizes to perisynaptic regions. Our studies thus reveal the regulation and function of a novel perisynaptic channel that determines the activation of synapses.

Alcantara, Alfredo Jr. et al. (2021) International Worm Meeting "Simulated Microgravity Impairs C. elegans Gut Immunity"

Alcantara, Alfredo Jr., Higashitani, Atsushi, Hashizume, Toko, Higashibata, Akira, Lee, Jin Il

[International Worm Meeting, 2021]

Humanity had been fascinated with space exploration and habitation. However, weak gravity force in outer space, termed as microgravity, has been known to alter normal human biology and induce health risks, such as muscle atrophy and compromised immunity. The underlying processes on how gravity is sensed by the body and how it affects immunity are still unclear. The nematode Caenorhabditis elegans, is a well-known model organism in the field of innate immunity and space research. Similar to humans, worm immune response signaling is regulating by MAPK and TGFbeta pathways. Previous microgravity experiments in C. elegans have shown an increase in p38 MAPK pmk-1 expression in simulated microgravity (Li, Wang, &amp; Wang, 2018) and decreased expression of TGFbeta ligand dbl-1 in space (Harada et al., 2016). In this study, we have utilized Enterobacter cloacae carrying tdTomato fluorescence reporter to observe gut colonization in C. elegans under simulated microgravity using a 3D clinostat. E. cloacae is a known commensal in C. elegans' gut but is invasive in immunocompromised dbl-1 mutant worms (Berg et al., 2019). Our preliminary results have shown that simulated microgravity can induce E. cloacae gut colonization in wild type C. elegans and aggravated colonization in pmk-1 mutants, while there is no difference observed in dbl-1 mutants which have robust colonization in both normal earth gravity and simulated microgravity, which demonstrates potential involvement of these pathways. Our study aims to discover the underlying mechanism of gravity sensing and how it affects the immune signaling pathways. In addition, spaceflights are already scheduled to send C. elegans aboard the International Space Station (ISS) for actual space gravity experiments.