Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1

Animals in complex environments must discriminate between salient and uninformative sensory cues. Caenorhabditis elegans uses one pair of olfactory neurons called AWC to sense many different odorants, yet the animal can distinguish each odorant from the others in discrimination assays. We demonstrate that the transmembrane guanylyl cyclase ODR-1 is essential for responses to all AWC-sensed odorants. ODR-1 appears to be a shared signaling component downstream of odorant receptors. Overexpression of ODR-1 protein indicates that ODR-1 can influence odor discrimination and adaptation as well as olfaction. Adaptation to one odorant, butanone, is disrupted by ODR-1 overexpression. Olfactory discrimination is also disrupted by ODR-1 overexpression, probably by overproduction of the shared second messenger cGMP. We propose that AWC odorant signaling pathways are insulated to permit odor discrimination.     

A network of stimulatory and inhibitory Galpha-subunits regulates olfaction in Caenorhabditis elegans

The two pairs of sensory neurons of C. elegans, AWA and AWC, that mediate odorant attraction, express six Galpha-subunits, suggesting that olfaction is regulated by a complex signaling network. Here, we describe the cellular localization and functions of the six olfactory Galpha-subunits: GPA-2, GPA-3, GPA-5, GPA-6, GPA-13, and ODR-3. All except GPA-6 localize to sensory cilia, suggesting a direct role in sensory transduction. GPA-2, GPA-3, GPA-5, and GPA-6 are also present in cell bodies and axons and GPA-5 specifically localizes to synaptic sites. Analysis of animals with single- to sixfold loss-of-function mutations shows that olfaction involves a balance between multiple stimulatory and inhibitory signals. ODR-3 constitutes the main stimulatory signal and is sufficient for the detection of odorants. GPA-3 forms a second stimulatory signal in the AWA and AWC neurons, also sufficient for odorant detection. In AWA, signaling is suppressed by GPA-5. In AWC, GPA-2 and GPA-13 negatively and positively regulate signaling, respectively. Finally, we show that only ODR-3 plays a role in cilia morphogenesis. Defects in this process are, however, independent of olfactory behavior. Our findings reveal the existence of a complex signaling network that controls odorant detection by C. elegans.     

An ER Complex of ODR-4 and ODR-8/Ufm1 Specific Protease 2 Promotes GPCR Maturation by a Ufm1-Independent Mechanism

Despite the importance of G-protein coupled receptors (GPCRs) their biogenesis is poorly understood. Like vertebrates, C. elegans uses a large family of GPCRs as chemoreceptors. A subset of these receptors, such as ODR-10, requires the odr-4 and odr-8 genes to be appropriately localized to sensory cilia. The odr-4 gene encodes a conserved tail-anchored transmembrane protein; the molecular identity of odr-8 is unknown. Here, we show that odr-8 encodes the C. elegans ortholog of Ufm1-specific protease 2 (UfSP2). UfSPs are cysteine proteases identified biochemically by their ability to liberate the ubiquitin-like modifier Ufm1 from its pro-form and protein conjugates. ODR-8/UfSP2 and ODR-4 are expressed in the same set of twelve chemosensory neurons, and physically interact at the ER membrane. ODR-4 also binds ODR-10, suggesting that an ODR-4/ODR-8 complex promotes GPCR folding, maturation, or export from the ER. The physical interaction between human ODR4 and UfSP2 suggests that this complex''s role in GPCR biogenesis may be evolutionarily conserved. Unexpectedly, mutant versions of ODR-8/UfSP2 lacking catalytic residues required for protease activity can rescue all odr-8 mutant phenotypes tested. Moreover, deleting C. elegans ufm-1 does not alter chemoreceptor traffic to cilia, either in wild type or in odr-8 mutants. Thus, UfSP2 proteins have protease- and Ufm1-independent functions in GPCR biogenesis.     

Polarized dendritic transport and the AP-1 mu1 clathrin adaptor UNC-101 localize odorant receptors to olfactory cilia

Odorant receptors and signaling proteins are localized to sensory cilia on olfactory dendrites. Using a GFP-tagged odorant receptor protein, Caenorhabditis elegans ODR-10, we characterized protein sorting and transport in olfactory neurons in vivo. ODR-10 is transported in rapidly moving dendritic vesicles that shuttle between the cell body and the cilia. Anterograde and retrograde vesicles move at different speeds, suggesting that dendrites have polarized transport mechanisms. Residues immediately after the seventh membrane-spanning domain of ODR-10 are required for localization; these residues are conserved in many G protein-coupled receptors. UNC-101 encodes a mu1 subunit of the AP-1 clathrin adaptor complex. In unc-101 mutants, dendritic vesicles are absent, ODR-10 receptor is evenly distributed over the plasma membrane, and other cilia membrane proteins are also mislocalized, implicating AP-1 in protein sorting to olfactory cilia.     

The divergent orphan nuclear receptor ODR-7 regulates olfactory neuron gene expression via multiple mechanisms in Caenorhabditis elegans

Nuclear receptors regulate numerous critical biological processes. The C. elegans genome is predicted to encode approximately 270 nuclear receptors of which >250 are unique to nematodes. ODR-7 is the only member of this large divergent family whose functions have been defined genetically. ODR-7 is expressed in the AWA olfactory neurons and specifies AWA sensory identity by promoting the expression of AWA-specific signaling genes and repressing the expression of an AWC-specific olfactory receptor gene. To elucidate the molecular mechanisms of action of a divergent nuclear receptor, we have identified residues and domains required for different aspects of ODR-7 function in vivo. ODR-7 utilizes an unexpected diversity of mechanisms to regulate the expression of different sets of target genes. Moreover, these mechanisms are distinct in normal and heterologous cellular contexts. The odr-7 ortholog in the closely related nematode C. briggsae can fully substitute for all ODR-7-mediated functions, indicating conservation of function across 25-120 million years of divergence.     

Noncell- and cell-autonomous G-protein-signaling converges with Ca2+/mitogen-activated protein kinase signaling to regulate str-2 receptor gene expression in Caenorhabditis elegans

In the sensory system of C. elegans, the candidate odorant receptor gene str-2 is strongly expressed in one of the two AWC neurons and weakly in both ASI neurons. Asymmetric AWC expression results from suppression of str-2 expression by a Ca2+/MAPK signaling pathway in one of the AWC neurons early in development. Here we show that the same Ca2+/MAPK pathway promotes str-2 expression in the AWC and ASI neurons together with multiple cell-autonomous and noncell-autonomous G-protein-signaling pathways. In first-stage larvae and adult animals, signals mediated by the Galpha subunits ODR-3, GPA-2, GPA-5, and GPA-6 and a Ca2+/MAPK pathway involving the Ca2+ channel subunit UNC-36, the CaMKII UNC-43, and the MAPKK kinase NSY-1 induce strong str-2 expression. Cell-specific rescue experiments suggest that ODR-3 and the Ca2+/MAPK genes function in the AWC neurons, but that GPA-5 and GPA-6 function in the AWA and ADL neurons, respectively. In Dauer larvae, the same network of genes promotes strong str-2 expression in the ASI neurons, but ODR-3 functions in AWB and ASH and GPA-6 in AWB. Our results reveal a complex signaling network, encompassing signals from multiple cells, that controls the level of receptor gene expression at different developmental stages.     

The claudin superfamily protein nsy-4 biases lateral signaling to generate left-right asymmetry in C. elegans olfactory neurons

Early in C. elegans development, signaling between bilaterally symmetric AWC olfactory neurons causes them to express different odorant receptor genes. AWC left-right asymmetry is stochastic: in each animal, either the left or the right neuron randomly becomes AWC(ON), and the other neuron becomes AWC(OFF). Here we show that the nsy-4 gene coordinates the lateral signaling that diversifies AWC(ON) and AWC(OFF) neurons. nsy-4 mutants generate 2 AWC(OFF) neurons, as expected if communication between the AWC neurons is lost, whereas overexpression of nsy-4 results in 2 AWC(ON) neurons. nsy-4 encodes a transmembrane protein related to the gamma subunits of voltage-activated calcium channels and the claudin superfamily; it interacts genetically with calcium channels and antagonizes a calcium-to-MAP kinase cascade in the neuron that becomes AWC(ON). Genetic mosaic analysis indicates that nsy-4 functions both cell-autonomously and nonautonomously in signaling between AWC neurons, providing evidence for lateral signaling and feedback that coordinate asymmetric receptor choice.     

Changes in cGMP levels affect the localization of EGL-4 in AWC in Caenorhabditis elegans

The Protein Kinase G, EGL-4, is required within the C. elegans AWC sensory neurons to promote olfactory adaptation. After prolonged stimulation of these neurons, EGL-4 translocates from the cytosol to the nuclei of the AWC. This nuclear translocation event is both necessary and sufficient for adaptation of the AWC neuron to odor. A cGMP binding motif within EGL-4 and the Gα protein ODR-3 are both required for this translocation event, while loss of the guanylyl cyclase ODR-1 was shown to result in constitutively nuclear localization of EGL-4. However, the molecular changes that are integrated over time to produce a stably adapted response in the AWC are unknown. Here we show that odor-induced fluctuations in cGMP levels in the adult cilia may be responsible in part for sending EGL-4 into the AWC nucleus to produce long-term adaptation. We found that reductions in cGMP that result from mutations in the genes encoding the cilia-localized guanylyl cyclases ODR-1 and DAF-11 result in constitutively nuclear EGL-4 even in naive animals. Conversely, increases in cGMP levels that result from mutations in cGMP phosphodiesterases block EGL-4 nuclear entry even after prolonged odor exposure. Expression of a single phosphodiesterase in adult, naive animals was sufficient to modestly increase the number of animals with nuclear EGL-4. Further, coincident acute treatment of animals with odor and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased the number of animals with nuclear EGL-4. These data suggest that reducing cGMP levels in AWC is necessary and even partially sufficient for nuclear translocation of EGL-4 and adaptation as a result of prolonged odor exposure. Our genetic analysis and chemical treatment of C. elegans further indicate that cilia morphology, as defined by fluorescent microscopic observation of the sensory endings, may allow for odor-induced fluctuations in cGMP levels and this fluctuation may be responsible for sending EGL-4 into the AWC nucleus.     

Microtubule-based localization of a synaptic calcium-signaling complex is required for left-right neuronal asymmetry in C. elegans

The axons of C. elegans left and right AWC olfactory neurons communicate at synapses through a calcium-signaling complex to regulate stochastic asymmetric cell identities called AWC(ON) and AWC(OFF). However, it is not known how the calcium-signaling complex, which consists of UNC-43/CaMKII, TIR-1/SARM adaptor protein and NSY-1/ASK1 MAPKKK, is localized to postsynaptic sites in the AWC axons for this lateral interaction. Here, we show that microtubule-based localization of the TIR-1 signaling complex to the synapses regulates AWC asymmetry. Similar to unc-43, tir-1 and nsy-1 loss-of-function mutants, specific disruption of microtubules in AWC by nocodazole generates two AWC(ON) neurons. Reduced localization of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons strongly correlates with the 2AWC(ON) phenotype in nocodazole-treated animals. We identified kinesin motor unc-104/kif1a mutants for enhancement of the 2AWC(ON) phenotype of a hypomorphic tir-1 mutant. Mutations in unc-104, like microtubule depolymerization, lead to a reduced level of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons. In addition, dynamic transport of TIR-1 in the AWC axons is dependent on unc-104, the primary motor required for the transport of presynaptic vesicles. Furthermore, unc-104 acts non-cell autonomously in the AWC(ON) neuron to regulate the AWC(OFF) identity. Together, these results suggest a model in which UNC-104 may transport some unknown presynaptic factor(s) in the future AWC(ON) cell that non-cell autonomously control the trafficking of the TIR-1 signaling complex to postsynaptic regions of the AWC axons to regulate the AWC(OFF) identity.     

A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans

Innate chemosensory preferences are often encoded by sensory neurons that are specialized for attractive or avoidance behaviors. Here, we show that one olfactory neuron in Caenorhabditis elegans, AWC(ON), has the potential to direct both attraction and repulsion. Attraction, the typical AWC(ON) behavior, requires a receptor-like guanylate cyclase GCY-28 that acts in adults and localizes to AWC(ON) axons. gcy-28 mutants avoid AWC(ON)-sensed odors; they have normal odor-evoked calcium responses in AWC(ON) but reversed turning biases in odor gradients. In addition to gcy-28, a diacylglycerol/protein kinase C pathway that regulates neurotransmission switches AWC(ON) odor preferences. A behavioral switch in AWC(ON) may be part of normal olfactory plasticity, as odor conditioning can induce odor avoidance in wild-type animals. Genetic interactions, acute rescue, and calcium imaging suggest that the behavioral reversal results from presynaptic changes in AWC(ON). These results suggest that alternative modes of neurotransmission can couple one sensory neuron to opposite behavioral outputs.     

The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans

The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWC(ON) and AWC(OFF), by inhibiting a calcium-mediated signaling pathway in the future AWC(ON) cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWC(ON) fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWC(ON) cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWC(ON) identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWC(ON) neurons. tir-1 expression is downregulated through its 3' UTR in AWC(ON), in which mir-71 is expressed at a higher level than in AWC(OFF). In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3' UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWC(ON) identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.     

The cyclic GMP-dependent protein kinase EGL-4 regulates olfactory adaptation in C. elegans

Prolonged odor exposure causes a specific, reversible adaptation of olfactory responses. A genetic screen for negative regulators of olfaction uncovered mutations in the cGMP-dependent protein kinase EGL-4 that disrupt olfactory adaptation in C. elegans. G protein-coupled olfactory receptors within the AWC olfactory neuron signal through cGMP and a cGMP-gated channel. The cGMP-dependent kinase functions in AWC neurons during odor exposure to direct adaptation to AWC-sensed odors, suggesting that adaptation is a cell intrinsic process initiated by cGMP. A predicted phosphorylation site on the beta subunit of the cGMP-gated channel is required for adaptation after short odor exposure, suggesting that phosphorylation of signaling molecules generates adaptation at early time points. A predicted nuclear localization signal within EGL-4 is required for adaptation after longer odor exposure, suggesting that nuclear translocation of EGL-4 triggers late forms of adaptation.     

G protein-coupled receptor kinase function is essential for chemosensation in C. elegans

G protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca(2+) imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Galpha subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation.     

Diverse regulation of sensory signaling by C. elegans nPKC-epsilon/eta TTX-4

Molecular and pharmacological studies in vitro suggest that protein kinase C (PKC) family members play important roles in intracellular signal transduction. Nevertheless, the in vivo roles of PKC are poorly understood. We show here that nPKC-epsilon/eta TTX-4 in the nematode Caenorhabditis elegans is required for the regulation of signal transduction in various sensory neurons for temperature, odor, taste, and high osmolality. Interestingly, the requirement for TTX-4 differs in different sensory neurons. In AFD thermosensory neurons, gain or loss of TTX-4 function inactivates or hyperactivates the neural activity, respectively, suggesting negative regulation of temperature sensation by TTX-4. In contrast, TTX-4 positively regulates the signal sensation of ASH nociceptive neurons. Moreover, in AWA and AWC olfactory neurons, TTX-4 plays a partially redundant role with another nPKC, TPA-1, to regulate olfactory signaling. These results suggest that C. elegans nPKCs regulate different sensory signaling in various sensory neurons. Thus, C. elegans provides an ideal model to reveal genetically novel components of nPKC-mediated molecular pathways in sensory signaling.     

An innexin-dependent cell network establishes left-right neuronal asymmetry in C. elegans

Gap junctions are widespread in immature neuronal circuits, but their functional significance is poorly understood. We show here that a transient network formed by the innexin gap-junction protein NSY-5 coordinates left-right asymmetry in the developing nervous system of Caenorhabditis elegans. nsy-5 is required for the left and right AWC olfactory neurons to establish stochastic, asymmetric patterns of gene expression during embryogenesis. nsy-5-dependent gap junctions in the embryo transiently connect the AWC cell bodies with those of numerous other neurons. Both AWCs and several other classes of nsy-5-expressing neurons participate in signaling that coordinates left-right AWC asymmetry. The right AWC can respond to nsy-5 directly, but the left AWC requires nsy-5 function in multiple cells of the network. NSY-5 forms hemichannels and intercellular gap-junction channels in Xenopus oocytes, consistent with a combination of cell-intrinsic and network functions. These results provide insight into gap-junction activity in developing circuits.     

Molecular evolution and quantitative variation for chemosensory behaviour in the nematode genus Caenorhabditis

Caenorhabditis elegans is a model organism in biology, yet despite the tremendous information generated from genetic, genomic and functional analyses, C. elegans has rarely been used to address questions in ecological genetics. Here, we analyse genetic variation for chemosensory behaviour, an ecologically important trait that is also genetically well characterized, at both the phenotypic and molecular levels within three species of the genus Caenorhabditis. We show that the G-protein ODR-3 plays an important role in chemosensory avoidance behaviour and identify orthologues of odr-3 in C. briggsae and C. remanei. Both quantitative genetic analysis of chemosensory behaviour and molecular population genetic analysis of odr-3 show that there is little genetic variation among a worldwide collection of isolates of the primarily selfing C. elegans, whereas there is substantially more variation within a single population of the outcrossing C. remanei. Although there are a large number of substitutions at silent sites within odr-3 among the three species, molecular evolution at the protein level is extremely conserved, suggesting that odr-3 plays an important role in cell signalling during chemosensation and/or neuronal cilia development in C. remanei and in C. briggsae as it does in C. elegans. Our results suggest that C. remanei may be a more suitable subject for ecological and evolutionary genetic studies than C. elegans.     

Lateral signaling mediated by axon contact and calcium entry regulates asymmetric odorant receptor expression in C. elegans

C. elegans detects several odorants with the bilaterally symmetric pair of AWC olfactory neurons. A stochastic, coordinated decision ensures that the candidate odorant receptor gene str-2 is expressed in only one AWC neuron in each animal--either the left or the right neuron, but never both. An interaction between the two AWC neurons generates asymmetric str-2 expression in a process that requires normal axon guidance and probably AWC axon contact. This interaction induces str-2 expression by reducing calcium signaling through a voltage-dependent Ca2+ channel and the CaM kinase II UNC-43. CaMKII activity acts as a switch in the initial decision to express str-2; thus, calcium signals can define distinct cell types during neuronal development. A cGMP signaling pathway that is used in olfaction maintains str-2 expression after the initial decision has been made.