Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Dana L. Miller, Mark B. Roth

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

HIROSHI SUZUKI et al. (2006) East Asia C. elegans Meeting "In vivo neuroimaging and activity-manipulation of ASE salt-sensory neurons"

Shawn Lockery, William Schafer, HIROSHI SUZUKI, Tod Thiele

[East Asia C. elegans Meeting, 2006]

Since the first successful application in mechanosensory neurons, we have been extensively using in vivo calcium imaging to monitor activity in various individual neurons in C.elegans (1, 2). The genetically encoded calcium sensor cameleon enables us to express the GFP-based probe in specific neurons and optically monitor calcium transients in living animals. One of the advantages of this imaging technique is that we can monitor multiple neurons at the same time, provided they are in the same focal plane. Simultaneous in vivo imaging from ASE-left and -right neurons has revealed an unpredicted asymmetric nature in the salt-sensory neurons: ASE-left responds to salt-increase with a calcium increase while ASE-right is activated by a salt-decrease. Thus, ASE-left is a salt-increase sensor and ASE-right is a salt-decrease sensor. Despite their divergent sensory nature, laser ablation studies showed that each of the two ASEs alone is capable of mediating salt-chemotaxis. How does the neuronal circuit process the opposite sensory cues to yield the same behavior? To address this question, we segregated the two sensory pathways by using newly emerging molecular tools to manipulate neuronal activity: a light-gated cation channel, channelrhodopsin-2(ChR2), and a capsaicin-gated cation channel, TRP-V1. We can activate the neurons expressing these channels by light-illumination or application of capsaicin. Transgenic worms expressing ChR2 or TRP-V1 in unilateral ASEs are generated by using gcy-7 and gcy-5 promoters. We found that specific activation of ASE-left decreased the probability of turning behavior while ASE-right activation increased the turning probability. Based on the Pirouette/ biased random-walk model proposed for chemotaxis and thermotaxis in C.elegans, this data suggests that opposite sensory cues induce opposite motor programs to navigate the worms in the proper direction. We are currently exploring how the apparently symmetric circuit processes the asymmetric sensory cues. We are also characterizing the sensory nature of ASEs to salts and other taste compounds in further detail. 1. Suzuki, H., Kerr, R., et al. Neuron (2003) 39, 1005-17. 2. Schafer, W.R., Curr. Biol. (2005) 15, R723-9.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zelmanovich, V., Livshits, L., Zuckerman, B., Smith, Y., Gross, E., Abergel, R., Romero, L., Abergel, Z.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Hiroshi Suzuki et al. (2004) West Coast Worm Meeting "Bilaterally asymmetric nature of the ASE chemosensory neuron"

Shawn Lockery, William R Schafer, Christian Froekjaer-Jensen, Tod R Thiele, HIROSHI SUZUKI

[West Coast Worm Meeting, 2004]

Most animals have a body plan of bilateral symmetry, which extends to the positioning of the sensors of various modalities (e.g., vision, acoustics). The difference of the inputs from left and right sensors are processed in the brain to enrich perceptions. In C.elegans , most bilateral pairs of neurons are thought to be functionally symmetric. Exceptions are the amphid neurons ASE and AWC, which are bilaterally asymmetric at the level of gene expression and/or function (1-2). We are interested in addressing how this asymmetry contributes to information processing and behavioral control by the C. elegans neural circuitry. ASE is the major chemosensory neuron mediating chemotaxis toward water-soluble attractants like NaCl. Using cameleon, a genetically encoded calcium sensor, we can optically monitor in vivo neuronal calcium transients in intact animals (3). When we challenged worms expressing cameleon in ASE with a pulsed increase of NaCl from 0 to 40mM for 10sec, the calcium level increased reversibly in ASE-Left, while simultaneous imaging recorded a decrease in ASE-Right. Such transients were not observed with the mutant cameleon that has mutations in all the calcium binding sites. No significant difference was observed in unc-13 background, ruling out the contribution of the synaptic inputs from other cells to the opposite changes of the calcium transient in ASE-Left and -Right. The transients are abolished in tax-4(p678) , suggesting the TAX-4 cGMP-gated channel is involved in the calcium mobilizations. In order to see the response to a decrease in chemoattractant concentration, we kept the worms in 40mM NaCl and challenged with 80mM (40mM up-step) and 0mM (40mM down-step) NaCl. Interestingly, ASE-Left showed a calcium increase in response to the NaCl upstep (40 to 80mM in upstep protocol and 0 to 40mM in down-step protocol), while ASE-Right showed a calcium increase in response to the downstep (80 to 40mM and 40 to 0mM). Such opposite responses to chemoattractant stimuli, reminiscent of the ON- and OFF-center cells in mammalian retina, suggest that activation of ASE-Left and ASE-Right have opposite effects on navigation behavior during chemotaxis. To address this hypothesis, we are expressing vanilloid receptor in hemilateral ASE to artificially stimulate the cells with capsaicin and determine the effect on locomotion behavior. 1.Pierce-Shimomura-JT, et al. Nature (2001) 2. Wes-PD & Bargmann CI. Nature (2001) 3. Suzuki-H, Kerr-R, et al. Neuron (2003)

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Rasika Kalamegham, Wendy Hanna-Rose

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Kotera, I. et al. (2011) International Worm Meeting "In vivo calcium imaging of the pharyngeal muscles in freely moving animals."

Suzuki, H., Truong, K., Wong, S., Su, L., Kotera, I.

[International Worm Meeting, 2011]

Imaging Ca2+ concentrations in live cells provides temporospatial dynamics of cellular activities, which is of vital importance in understanding how multi-cellular systems function. The first in vivo Ca2+ imaging in C. elegans has been established in the pharynx using a genetically encoded Ca2+ probe, cameleon; the pharynx is an attractive model for its neuronal regulation because the pharyngeal nervous system has few neurons, and functions semi-autonomously. However, previously reported imaging methods require immobilization of the worms and exogenously added serotonin just to observe pharyngeal activity. We have developed a new imaging system, which combines two independent imaging units to enable two simultaneous imaging, along with "worm tracker" functionality to perform imaging on a freely moving worm. We have also developed a pattern matching algorithm, which recognizes the expression pattern of the probe, and automatically extracts Ca2+ dynamics of the pharynx. Pharyngeal Ca2+ dynamics of freely-moving worms was measured in the presence of serotonin. Both the rise time (0.14s) and firing rate (3.14 Hz) of the transients in the pharynx are in good agreement with the previous report, confirming the validity of our new system. The free worms showed pharyngeal activity even in the absence of exogenous serotonin, albeit a reduced firing rate (0.24 Hz). The rise slope of the transients stayed relatively unchanged with or without serotonin (106%/s and 65%/s), but the rise duration has increased from 0.14s with serotonin to 0.39s without it. With an incorporation of new GCaMP, our result has demonstrated over 10-fold improvement in both the temporal resolution and dynamic range when used on our system. By employing all the techniques developed, an intriguing relation was revealed between the pharyngeal firing rate and locomotion direction. When worms are moving forward, the firing rate was similar to what we have measured with the serotonin-stimulated worms. However, when the worms autonomously reversed its direction, the firings quickly ceased; this pharyngeal firing was restored when the worm resumed forward movement. Thus we have developed a novel imaging system which is capable of concurrently performing Ca2+ imaging and transmission image acquisition on a freely moving worm. The ability to record multiple behaviors of the worm provides a unique opportunity to investigate how multiple systems are interacted and coordinated; further investigation of the seemingly isolated pharyngeal and somatic nervous systems should yield a compelling model for the inter-systems relation.

Schwartz, Hillel et al. (2013) International Worm Meeting "Developing Heterorhabditis nematodes as an experimental system for the study of mutualistic symbiosis."

Sternberg, Paul, Schwartz, Hillel

[International Worm Meeting, 2013]

Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora. Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility. In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.

Schwartz H et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Towards a SNP map for Heterorhabditis bacteriophora"

Sternberg P, Schwartz H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Heterorhabditis bacteriophora is a species of insect-parasitic nematode that lives in mutually beneficial symbiosis with pathogenic Photorhabdus luminescens bacteria. P. luminescens bacteria are lethal to insects and to other nematodes, including the soil nematode Caenorhabditis elegans, but are required for H. bacteriophora growth. The symbiosis between H. bacteriophora and P. luminescens therefore offers the potential to study the molecular genetic basis of their cooperative relationship. We are interested in developing tools to make such studies more feasible; in particular, we propose to generate tools for genetic mapping in H. bacteriophora. We will test independent isolates of H. bacteriophora to ensure that the isolates are cross-fertile. We will then examine the abilities of these isolates to grow on and to become infected with different wild-type and mutant Photorhabdus bacteria. From these tests we will select an isolate on which we will use next-generation high-throughput sequencing technology for the purpose of refining the existing draft genome sequence and for the creation of a SNP map. We anticipate that this SNP map will enable us and the wider insect-parasitic nematode community to identify induced mutations and natural variations affecting the interactions between H. bacteriophora nematodes and pathogenic Photorhabdus bacteria.

Carmela Bergamasco et al. (2006) European Worm Meeting "Signalling in quinine avoidance"

Paolo Bazzicalupo, Giovanni Esposito, Salvatore Arbucci, Carmela Bergamasco

[European Worm Meeting, 2006]

Chemical sensitivity allows animals to identify and respond appropriately to the chemical composition of the environment. Noxious water-soluble compounds that are avoided by C. elegans are generally sensed as bitter by humans and are discarded in double choice test by mice. We have used C. elegans to focus on the molecular mechanisms involved in primary sensing of quinine, a molecule detected as bitter by humans. ASH is the main sensory neuron involved in sensing quinine. Two G? subunits, GPA-3 and ODR-3 are necessary for the response of ASH to repellent stimuli (Hilliard et al., 2004 and 2005). In addition the TRPV channel proteins, OSM-9 and OCR-2, are also necessary for the ASH avoidance responses (Colbert et al 1997, Tobin et al 2002). Finally we identified a novel protein, QUI-1, as an essential components of the response to quinine (Hilliard et al., 2004). With regard to the molecular function of QUI-1, we demonstrate that QUI-1 function is required in ASH for the response to quinine and, using specific antibodies, that the protein is localized to the sensory cilia. These results, together with the discovery that QUI-1 contains an RGS (Regulator of G protein Signaling) domain, strongly suggest that this novel protein might be involved in quinine signaling.. Are there other components of the quinine signal transduction pathway?. We are using a best candidate approach and a variety of behavioral assays to identify new molecules involved in sensing repellent chemicals and in particular quinine. We analyzed behaviorally loss of function and overexpression mutants in several molecules known to act in the G protein signaling pathways (G? subunits, G? subunits, RGS proteins, etc.). The results obtained will be discussed.. Colbert, H. A., Smith, T. L. and Bargmann, C. I. (1997). OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J Neurosci 17, 8259-69.. Hilliard, M. A., Apicella, A. J., Kerr, R., Suzuki, H., Bazzicalupo, P. and Schafer, W. R. (2005). In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. Embo J 24, 63-72.. Hilliard, M. A., Bergamasco, C., Arbucci, S., Plasterk, R. H. and Bazzicalupo, P. (2004). Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans. Embo J 23, 1101-11.. Tobin, D., Madsen, D., Kahn-Kirby, A., Peckol, E., Moulder, G., Barstead, R., Maricq, A. and Bargmann, C. (2002). Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron 35, 307-18.

Chu, Yu-De et al. (2013) International Worm Meeting "An RNAi-based screen for the DExD/H-box RNA helicases involved in Caenorhabditis elegans microRNA function."

Chan, Shih-Peng, Chen, Shin-Kai, Chu, Yu-De, Chen, Guan-Rong, Huang, Tao

[International Worm Meeting, 2013]

MicroRNAs (miRNAs) are small non-coding regulatory RNAs that regulate gene expression at the post-transcriptional level and are involved in a broad spectrum of biological processes. Rearrangements of inter- or intra-molecular RNA structures and conformational changes of ribonucleoprotein complexes in the multiple processes of the miRNA pathway have led to the involvement of RNA helicase activities but little is known so far. In eukaryotes, RNA helicases generally belong to the superfamily 2 (SF2) in helicase classification, especially the DExD/H-box helicase family. The DExD/H-box proteins have been shown in association with many cellular processes involving RNA. To better understand the possible roles of DExD/H-box RNA helicases in miRNA function, we employed RNAi screen to identify genetic interaction between C. elegans DExD/H-box RNA helicases and the let-7 miRNA, which controls the timing of cell cycle exit and terminal differentiation. In addition to the RNA helicase p72, a component of Drosha Microprocessor complex, and CGH-1 that has been reported to facilitate the function of miRNA-induced silencing complex (miRISC), we found several DExD/H-box RNA helicases, which are involved in ribosomal RNA processing, pre-mRNA splicing and mRNA surveillance, may also take part in miRNA biogenesis and/or function. (Support: National Science Council, Taiwan. NSC 100-2311-B-002-006-MY3).