Functional expression of mammalian bitter taste receptors in Caenorhabditis elegans

Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances appearing in the environment. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCR) called T2Rs and expressed at the surface of taste receptor cells. The functional characterisation of T2R receptors is far from been completed due to the difficulty to functionally express them in heterologous systems. Taking advantage of the parallelisms between the Caenorhabditis elegans (C. elegans) and mammalian GPCR signalling pathways, we developed a C. elegans-based expression system to express functional human and rodent GPCRs of the T2R family. We generated transgenic worms expressing T2Rs in ASI chemosensory neurons and performed behavioural assays using a variety of bitter tastants. As a proof of the concept, we generated transgenic worms expressing human T2R4 or its mouse ortholog T2R8 receptors, which respond to two bitter tastants previously characterised as their functional ligands, 6-n-propyl-2-thiouracil and denatoniun. As expected, expression of human T2R4 or its mouse ortholog T2R8 in ASI neurons counteracted the water-soluble avoidance to 6-n-propyl-2-thiouracil and denatoniun observed in control wild-type worms. The expression in ASI neurons of human T2R16, the ligand of which, phenyl-beta-d-glucopyranoside, belong to a chemically different group of bitter tastants, also counteracted the water-soluble avoidance to this compound observed in wild-type worms. These results indicate that C. elegans is a suitable heterologous expression system to express functional T2Rs providing a tool to efficiently search for specific taste receptor ligands and to extend our understanding of the molecular basis of gustation.     

The contribution of taste bud populations to bitter avoidance in mouse strains differentially sensitive to sucrose octa-acetate and quinine

Mice of the SWR/J (SW) strain avoid orally delivered sucrose octa-acetate (SOA), whereas the mice of the C3HeB/FeJ (C3) strain are insensitive to SOA. Mice of both strains and of a congenic strain (C3.SW) that shares more than 99% of the C3 genome, were tested in a taste-salient brief-access taste test for responses to SOA and quinine hydrochloride, before and after transection of the glossopharyngeal or chorda tympani nerve, or sham surgery. Prior to surgery, congenic SOA tasters (C3.SW(T)) were phenotypically identical to the SW strain in avoidance of SOA, but showed a greater reduction in sensitivity after nerve transection. For quinine avoidance, which is thought to be a polygenic trait, SW mice showed the greatest sensitivity to quinine, C3 the least and C3.SW(T) mice were different from both parental strains, showing intermediate sensitivity. Nerve transections had only a moderate effect on quinine sensitivity, suggesting that both anterior and posterior taste bud fields contribute to behavioral quinine avoidance. These findings are discussed with regard to the distribution in the oral cavity of putative taste receptors for quinine and SOA and the peripheral organization of bitter taste.     

Temperature- and touch-sensitive neurons couple CNG and TRPV channel activities to control heat avoidance in Caenorhabditis elegans

Background

Any organism depends on its ability to sense temperature and avoid noxious heat. The nematode Caenorhabditis elegans responds to noxious temperatures exceeding ∼35°C and also senses changes in its environmental temperature in the range between 15 and 25°C. The neural circuits and molecular mechanisms involved in thermotaxis have been successfully studied, whereas details of the thermal avoidance behavior remain elusive. In this work, we investigate neurological and molecular aspects of thermonociception using genetic, cell biological and physiological approaches.

Methodology/Principal Findings

We show here that the thermosensory neurons AFD, in addition to sensing temperature within the range within which the animals can thrive, also contribute to the sensation of noxious temperatures resulting in a reflex-like escape reaction. Distinct sets of interneurons are involved in transmitting thermonociception and thermotaxis, respectively. Loss of AFD is partially compensated by the activity of a pair of multidendritic, polymodal neurons, FLP, whereas laser ablation of both types of neurons abrogated the heat response in the head of the animals almost completely. A third pair of heat sensory neurons, PHC, is situated in the tail. We find that the thermal avoidance response requires the cell autonomous function of cGMP dependent Cyclic Nucleotide-Gated (CNG) channels in AFD, and the heat- and capsaicin-sensitive Transient Receptor Potential Vanilloid (TRPV) channels in the FLP and PHC sensory neurons.

Conclusions/Significance

Our results identify distinct thermal responses mediated by a single neuron, but also show that parallel nociceptor circuits and molecules may be used as back-up strategies to guarantee fast and efficient responses to potentially detrimental stimuli.     

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified C. elegans Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.     

Centriole translocation and degeneration during ciliogenesis in Caenorhabditis elegans neurons

Neuronal cilia that are formed at the dendritic endings of sensory neurons are essential for sensory perception. However, it remains unclear how the centriole‐derived basal body is positioned to form a template for cilium formation. Using fluorescence time‐lapse microscopy, we show that the centriole translocates from the cell body to the dendrite tip in the Caenorhabditis elegans sensory neurons. The centriolar protein SAS‐5 interacts with the dynein light‐chain LC8 and conditional mutations of cytoplasmic dynein‐1 block centriole translocation and ciliogenesis. The components of the central tube are essential for the biogenesis of centrioles, which later drive ciliogenesis in the dendrite; however, the centriole loses these components at the late stage of centriole translocation and subsequently recruits transition zone and intraflagellar transport proteins. Together, our results provide a comprehensive model of ciliogenesis in sensory neurons and reveal the importance of the dynein‐dependent centriole translocation in this process.     

Caenorhabditis elegans HOPS and CCZ-1 mediate trafficking to lysosome-related organelles independently of RAB-7 and SAND-1

As early endosomes mature, the SAND-1/CCZ-1 complex acts as a guanine nucleotide exchange factor (GEF) for RAB-7 to promote the activity of its effector, HOPS, which facilitates late endosome–lysosome fusion and the consumption of AP-3–containing vesicles. We show that CCZ-1 and the HOPS complex are essential for the biogenesis of gut granules, cell type–specific, lysosome-related organelles (LROs) that coexist with conventional lysosomes in Caenorhabditis elegans intestinal cells. The HOPS subunit VPS-18 promotes the trafficking of gut granule proteins away from lysosomes and functions downstream of or in parallel to the AP-3 adaptor. CCZ-1 also acts independently of AP-3, and ccz-1 mutants mistraffic gut granule proteins. Our results indicate that SAND-1 does not participate in the formation of gut granules. In the absence of RAB-7 activity, gut granules are generated; however, their size and protein composition are subtly altered. These observations suggest that CCZ-1 acts in partnership with a protein other than SAND-1 as a GEF for an alternate Rab to promote gut granule biogenesis. Point mutations in GLO-1, a Rab32/38-related protein, predicted to increase spontaneous guanine nucleotide exchange, specifically suppress the loss of gut granules by ccz-1 and glo-3 mutants. GLO-3 is known to be required for gut granule formation and has homology to SAND-1/Mon1–related proteins, suggesting that CCZ-1 functions with GLO-3 upstream of the GLO-1 Rab, possibly as a GLO-1 GEF. These results support LRO formation occurring via processes similar to conventional lysosome biogenesis, albeit with key molecular differences.     

Two antagonistic gustatory receptor neurons responding to sweet-salty and bitter taste in Drosophila

In Drosophila, gustatory receptor neurons (GRNs) occur within hair-like structures called sensilla. Most taste sensilla house four GRNs, which have been named according to their preferred sensitivity to basic stimuli: water (W cell), sugars (S cell), salt at low concentration (L1 cell), and salt at high concentration (L2 cell). Labellar taste sensilla are classified into three types, l-, s-, and i-type, according to their length and location. Of these, l- and s-type labellar sensilla possess these four cells, but most i-type sensilla house only two GRNs. In i-type sensilla, we demonstrate here that the first GRN responds to sugar and to low concentrations of salt (10-50 mM NaCl). The second GRN detects a range of bitter compounds, among which strychnine is the most potent; and also to salt at high concentrations (over 400 mM NaCl). Neither type of GRN responds to water. The detection of feeding stimulants in i-type sensilla appears to be performed by one GRN with the combined properties of S+L1 cells, while the other GRN detects feeding inhibitors in a similar manner to bitter-sensitive L2 cells on the legs. These sensilla thus house two GRNs having an antagonistic effect on behavior, suggesting that the expression of taste receptors is segregated across them accordingly.     

The neuropeptide receptor npr-38 regulates avoidance and stress-induced sleep in Caenorhabditis elegans

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Lifespan decrease in a Caenorhabditis elegans mutant lacking TRX-1, a thioredoxin expressed in ASJ sensory neurons

Thioredoxins are a class of small proteins that play a key role in regulating many cellular redox processes. We report here the characterization of the first member of the thioredoxin family in metazoans that is mainly associated with neurons. The Caenorhabditis elegans gene B0228.5 encodes a thioredoxin (TRX-1) that is expressed in ASJ ciliated sensory neurons, and to some extent also in the posterior-most intestinal cells. TRX-1 is active at reducing protein disulfides in the presence of a heterologous thioredoxin reductase. A mutant worm strain carrying a null allele of the trx-1 gene displays a reproducible decrease in both mean and maximum lifespan when compared to wild-type. The identification and characterization of TRX-1 paves the way to use C. elegans as an in vivo model to study the role of thioredoxins in lifespan and nervous system physiology and pathology.     

Target sequences of Tc1, Tc3 and Tc5 transposons of Caenorhabditis elegans

We report here the consensus target sequence of transposons Tc1, Tc3 and Tc5 of Caenorhabditis elegans. These sequences were obtained by molecular analysis of 1008 random new insertions which have not been exposed to natural selection. This analysis reveals consensus target sites slightly different from those previously reported, and confirms that the mariner elements Tc1 and Tc3 insert in sites which are not preferentially palindromic.     

SKN-1/Nrf,stress responses,and aging in Caenorhabditis elegans

The mammalian Nrf/CNC proteins (Nrf1, Nrf2, Nrf3, p45 NF-E2) perform a wide range of cellular protective and maintenance functions. The most thoroughly described of these proteins, Nrf2, is best known as a regulator of antioxidant and xenobiotic defense, but more recently has been implicated in additional functions that include proteostasis and metabolic regulation. In the nematode Caenorhabditis elegans, which offers many advantages for genetic analyses, the Nrf/CNC proteins are represented by their ortholog SKN-1. Although SKN-1 has diverged in aspects of how it binds DNA, it exhibits remarkable functional conservation with Nrf/CNC proteins in other species and regulates many of the same target gene families. C. elegans may therefore have considerable predictive value as a discovery model for understanding how mammalian Nrf/CNC proteins function and are regulated in vivo. Work in C. elegans indicates that SKN-1 regulation is surprisingly complex and is influenced by numerous growth, nutrient, and metabolic signals. SKN-1 is also involved in a wide range of homeostatic functions that extend well beyond the canonical Nrf2 function in responses to acute stress. Importantly, SKN-1 plays a central role in diverse genetic and pharmacologic interventions that promote C. elegans longevity, suggesting that mechanisms regulated by SKN-1 may be of conserved importance in aging. These C. elegans studies predict that mammalian Nrf/CNC protein functions and regulation may be similarly complex and that the proteins and processes that they regulate are likely to have a major influence on mammalian life- and healthspan.     

Oligomerisation of C. elegans Olfactory Receptors,ODR-10 and STR-112, in Yeast

It is widely accepted that vertebrate G-Protein Coupled Receptors (GPCRs) associate with each other as homo- or hetero-dimers or higher-order oligomers. The C. elegans genome encodes hundreds of olfactory GPCRs, which may be expressed in fewer than a dozen chemosensory neurons, suggesting an opportunity for oligomerisation. Here we show, using three independent lines of evidence: co-immunoprecipitation, bioluminescence resonance energy transfer and a yeast two-hybrid assay that nematode olfactory receptors (ORs) oligomerise when heterologously expressed in yeast. Specifically, the nematode receptor ODR-10 is able to homo-oligomerise and can also form heteromers with the related nematode receptor STR-112. ODR-10 also oligomerised with the rat I7 OR but did not oligomerise with the human somatostatin receptor 5, a neuropeptide receptor. In this study, the question of functional relevance was not addressed and remains to be investigated.     

Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans

The ability to optically excite or silence specific cells using optogenetics has become a powerful tool to interrogate the nervous system. Optogenetic experiments in small organisms have mostly been performed using whole-field illumination and genetic targeting, but these strategies do not always provide adequate cellular specificity. Targeted illumination can be a valuable alternative but it has only been shown in motionless animals without the ability to observe behavior output. We present a real-time, multimodal illumination technology that allows both tracking and recording the behavior of freely moving C. elegans while stimulating specific cells that express channelrhodopsin-2 or MAC. We used this system to optically manipulate nodes in the C. elegans touch circuit and study the roles of sensory and command neurons and the ultimate behavioral output. This technology enhances our ability to control, alter, observe and investigate how neurons, muscles and circuits ultimately produce behavior in animals using optogenetics.     

UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16’s inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.     

RNA interference in Caenorhabditis elegans: uptake, mechanism, and regulation

RNA interference (RNAi) is a powerful research tool that has enabled molecular insights into gene activity, pathway analysis, partial loss-of-function phenotypes, and large-scale genomic discovery of gene function. While RNAi works extremely well in the non-parasitic nematode C. elegans, it is also especially useful in organisms that lack facile genetic analysis. Extensive genetic analysis of the mechanisms, delivery and regulation of RNAi in C. elegans has provided mechanistic and phenomenological insights into why RNAi is so effective in this species. These insights are useful for the testing and development of RNAi in other nematodes, including parasitic nematodes where more effective RNAi would be extremely useful. Here, we review the current advances in C. elegans for RNA delivery methods, regulation of cell autonomous and systemic RNAi phenomena, and implications of enhanced RNAi mutants. These discussions, with a focus on mechanism and cross-species application, provide new perspectives for optimizing RNAi in other species.     

CLK-1 controls respiration, behavior and aging in the nematode Caenorhabditis elegans

Mutations in the clk-1 gene of the nematode Caenorhabditis elegans result in an average slowing of a variety of developmental and physiological processes, including the cell cycle, embryogenesis, post-embryonic growth, rhythmic behaviors and aging. In yeast, a CLK-1 homologue is absolutely required for ubiquinone biosynthesis and thus respiration. Here we show that CLK-1 is fully active when fused to green fluorescent protein and is found in the mitochondria of all somatic cells. The activity of mutant mitochondria, however, is only very slightly impaired, as measured in vivo by a dye-uptake assay, and in vitro by the activity of succinate cytochrome c reductase. Overexpression of CLK-1 activity in wild-type worms can increase mitochondrial activity, accelerate behavioral rates during aging and shorten life span, indicating that clk-1 regulates and controls these processes. These observations also provide strong genetic evidence that mitochondria are causally involved in aging. Furthermore, the reduced respiration of the long-lived clk-1 mutants suggests that longevity is promoted by the age-dependent decrease in mitochondrial function that is observed in most species.     

Gain-of-Function Mutations of fem-3, a Sex-Determination Gene in Caenorhabditis elegans

We have isolated nine gain-of-function (gf) alleles of the sex-determination gene fem-3 as suppressors of feminizing mutations in fem-1 and fem-2. The wild-type fem-3 gene is needed for spermatogenesis in XX self-fertilizing hermaphrodites and for male development in both soma and germ line of XO animals. Loss-of-function alleles of fem-3 transform XX and XO animals into females (spermless hermaphrodites). In contrast, fem-3(gf) alleles masculinize only one tissue, the hermaphrodite germ line. Thus, XX fem-3(gf) mutant animals have a normal hermaphrodite soma, but the germ line produces a vast excess of sperm and no oocytes. All nine fem-3(gf) alleles are temperature sensitive. The temperature-sensitive period is from late L4 to early adult, a period just preceding the first signs of oogenesis. The finding of gain-of-function alleles which confer a phenotype opposite to that of loss-of-function alleles supports the idea that fem-3 plays a critical role in germ-line sex determination. Furthermore, the germ-line specificity of the fem-3(gf) mutant phenotype and the late temperature-sensitive period suggest that, in the wild-type XX hermaphrodite, fem-3 is negatively regulated so that the hermaphrodite stops making sperm and starts making oocytes. Temperature shift experiments also show that, in the germ line, sexual commitment appears to be a continuing process. Spermatogenesis can resume even after oogenesis has begun, and oogenesis can be initiated much later than normal.