Osmotic Avoidance Research Articles

Crotamiton derivative JM03 extends lifespan and improves oxidative and hypertonic stress resistance in Caenorhabditis elegans via inhibiting OSM-9.

While screening our in-house 1072 marketed drugs for their ability to extend the lifespan using Caenorhabditis elegans (C. elegans) as an animal model, crotamiton (N-ethyl-o-crotonotoluidide) showed anti-aging activity and was selected for further structural optimization. After replacing the ortho-methyl of crotamiton with ortho-fluoro, crotamiton derivative JM03 was obtained and showed better activity in terms of lifespan-extension and stress resistance than crotamiton. It was further explored that JM03 extended the lifespan of C. elegans through osmotic avoidance abnormal-9 (OSM-9). Besides, JM03 improves the ability of nematode to resist oxidative stress and hypertonic stress through OSM-9, but not osm-9/capsaicin receptor related-2 (OCR-2). Then the inhibition of OSM-9 by JM03 reduces the aggregation of Q35 in C. elegans via upregulating the genes associated with proteostasis. SKN-1 signaling was also found to be activated after JM03 treatment, which might contribute to proteostasis, stress resistance and lifespan extension. In summary, this study explored a new small molecule derived from crotamiton, which has efficient anti-oxidative, anti-hypertonic, and anti-aging effects, and could further lead to promising application prospects.

Contributions of Adaptive Plant Architecture to Transgressive Salinity Tolerance in Recombinant Inbred Lines of Rice: Molecular Mechanisms Based on Transcriptional Networks.

Genetic novelties are important nucleators of adaptive speciation. Transgressive segregation is a major mechanism that creates genetic novelties with morphological and developmental attributes that confer adaptive advantages in certain environments. This study examined the morpho-developmental and physiological profiles of recombinant inbred lines (RILs) from the salt-sensitive IR29 and salt-tolerant Pokkali rice, representing the total range of salt tolerance including the outliers at both ends of the spectrum. Morpho-developmental and physiological profiles were integrated with a hypothesis-driven interrogation of mRNA and miRNA transcriptomes to uncover the critical genetic networks that have been rewired for novel adaptive architecture. The transgressive super-tolerant FL510 had a characteristic small tiller angle and wider, more erect, sturdier, and darker green leaves. This unique morphology resulted in lower transpiration rate, which also conferred a special ability to retain water more efficiently for osmotic avoidance. The unique ability for water retention conferred by such adaptive morphology appeared to enhance the efficacy of defenses mediated by Na+ exclusion mechanism (SalTol-effects) inherited from Pokkali. The super-tolerant FL510 and super-sensitive FL499 had the smallest proportions of differentially expressed genes with little overlaps. Genes that were steadily upregulated in FL510 comprised a putative cytokinin-regulated genetic network that appeared to maintain robust growth under salt stress through well-orchestrated cell wall biogenesis and cell expansion, likely through major regulatory (OsRR23, OsHK5) and biosynthetic (OsIPT9) genes in the cytokinin signaling pathway. Meanwhile, a constitutively expressed cluster in FL510 prominently featured two transcription factors (OsIBH1, TAC3) that control tiller angle and growth habit through the brassinosteroid signaling pathway. Both the putative cytokinin-mediated and brassinosteroid-mediated clusters appeared to function as highly coordinated network synergies in FL510. In contrast, both networks appeared to be sub-optimal and inferior in the other RILs and parents as they were disjointed and highly fragmented. Transgressively expressed miRNAs (miR169, miR397, miR827) were also identified as prominent signatures of FL510, with functional implications to mechanisms that support robust growth, homeostasis, and osmotic stress avoidance. Results of this study demonstrate how genetic recombination creates novel morphology that complements inducible defenses hence transgressive adaptive phenotypes.

Signal Decoding for Glutamate Modulating Egg Laying Oppositely in Caenorhabditis elegans under Varied Environmental Conditions.

SummaryAnimals' ability to sense environmental cues and to integrate this information to control fecundity is vital for continuing the species lineage. In this study, we observed that the sensory neurons Amphid neuron (ASHs and ADLs) differentially regulate egg-laying behavior in Caenorhabditis elegans under varied environmental conditions via distinct neuronal circuits. Under standard culture conditions, ASHs tonically release a small amount of glutamate and inhibit Hermaphrodite specific motor neuron (HSN) activities and egg laying via a highly sensitive Glutamate receptor (GLR)-5 receptor. In contrast, under Cu2+ stimulation, ASHs and ADLs may release a large amount of glutamate and inhibit Amphid interneuron (AIA) interneurons via low-sensitivity Glutamate-gated chloride channel (GLC)-3 receptor, thus removing the inhibitory roles of AIAs on HSN activity and egg laying. However, directly measuring the amount of glutamate released by sensory neurons under different conditions and assaying the binding kinetics of receptors with the neurotransmitter are still required to support this study directly.

Chemically induced oxidative stress affects ASH neuronal function and behavior in C. elegans.

Oxidative stress (OS) impact on a single neuron’s function in vivo remains obscure. Using C. elegans as a model organism, we report the effect of paraquat (PQ)-induced OS on wild type worms on the function of the ASH polymodal neuron. By calcium (Ca2+) imaging, we quantified ASH activation upon stimulus delivery. PQ-treated worms displayed higher maximum depolarization (peak of the Ca2+ transients) compared to untreated animals. PQ had a similar effect on the ASH neuron response time (rising slope of the Ca2+ transients), except in very young worms. OS effect on ASH was partially abolished in vitamin C-treated worms. We performed octanol and osmotic avoidance tests, to investigate the OS effect on ASH-dependent behaviors. PQ-treated worms have enhanced avoidance behavior compared to untreated ones, suggesting that elevated ASH Ca2+ transients result in enhanced ASH-mediated behavior. The above findings suggest a possible hormetic effect of PQ, as a factor inducing mild oxidative stress. We also quantified locomotion parameters (velocity, bending amplitude), which are not mediated by ASH activation. Bending amplitude did not differ significantly between treated and untreated worms; velocity in older adults decreased. The differential effect of OS on behavioral patterns may mirror a selective impact on the organism’s neurons.

Caenorhabditis elegans glutamylating enzymes function redundantly in male mating.

ABSTRACTMicrotubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans. We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons.

Abnormal Osmotic Avoidance Behavior in C. elegans Is Associated with Increased Hypertonic Stress Resistance and Improved Proteostasis.

Protein function is controlled by the cellular proteostasis network. Proteostasis is energetically costly and those costs must be balanced with the energy needs of other physiological functions. Hypertonic stress causes widespread protein damage in C. elegans. Suppression and management of protein damage is essential for optimal survival under hypertonic conditions. ASH chemosensory neurons allow C. elegans to detect and avoid strongly hypertonic environments. We demonstrate that mutations in osm-9 and osm-12 that disrupt ASH mediated hypertonic avoidance behavior or genetic ablation of ASH neurons are associated with enhanced survival during hypertonic stress. Improved survival is not due to altered systemic volume homeostasis or organic osmolyte accumulation. Instead, we find that osm-9(ok1677) mutant and osm-9(RNAi) worms exhibit reductions in hypertonicity induced protein damage in non-neuronal cells suggesting that enhanced proteostasis capacity may account for improved hypertonic stress resistance in worms with defects in osmotic avoidance behavior. RNA-seq analysis revealed that genes that play roles in managing protein damage are upregulated in osm-9(ok1677) worms. Our findings are consistent with a growing body of work demonstrating that intercellular communication between neuronal and non-neuronal cells plays a critical role in integrating cellular stress resistance with other organismal physiological demands and associated energy costs.

A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4.

Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit, NPHP-4 influences localization and function of a distal ciliary kinesin. Moreover, data suggest human OSM-3 homolog (Kif17) could act as a modifying locus affecting disease penetrance or expressivity in NPHP patients.

Kinesin family 17 (osmotic avoidance abnormal-3) is dispensable for photoreceptor morphology and function.

In Caenorhabditis elegans, homodimeric [kinesin family (KIF) 17, osmotic avoidance abnormal-3 (OSM-3)] and heterotrimeric (KIF3) kinesin-2 motors are required to establish sensory cilia by intraflagellar transport (IFT) where KIF3 and KIF17 cooperate to build the axoneme core and KIF17 builds the distal segments. However, the function of KIF17 in vertebrates is unresolved. We expressed full-length and motorless KIF17 constructs in mouse rod photoreceptors using adeno-associated virus in Xenopus laevis rod photoreceptors using a transgene and in ciliated IMCD3 cells. We found that tagged KIF17 localized along the rod outer segment axoneme when expressed in mouse and X. laevis photoreceptors, whereas KIF3A was restricted to the proximal axoneme. Motorless KIF3A and KIF17 mutants caused photoreceptor degeneration, likely through dominant negative effects on IFT. KIF17 mutant lacking the motor domain translocated to nuclei after exposure of a C-terminal nuclear localization signal. Germ-line deletion of Kif17 in mouse did not affect photoreceptor function. A rod-specific Kif3/Kif17 double knockout mouse demonstrated that KIF17 and KIF3 do not act synergistically and did not prevent rhodopsin trafficking to rod outer segments. In summary, the nematode model of KIF3/KIF17 cooperation apparently does not apply to mouse photoreceptors in which the photosensory cilium is built exclusively by KIF3.

Identification of C. elegans RAB-5-dependent downstream effectors as regulators of ciliogenesis and ciliary membrane homeostasis

Cilia are microtubule based organelles enveloped by a specialised extension of plasma membrane. Serving as cellular antennae, cilia are essential for perceiving extracellular cues and mediating downstream signal transduction crucial for normal cellular physiology and development. These functions are mediated in large part by the specialised ciliary membrane, which differs in composition from the plasma membrane, housing many of the receptors, channels and effectors that mediate cilium-based signalling. However, the molecular mechanisms underlying the establishment, maintenance and regulation of the ciliary membrane remain poorly understood. In this project we are seeking to identify and functionally characterize genes with ciliogenic roles in ciliary membrane transport and organisation. Specifically, we are taking a candidate gene approach using the Caenorhabditis elegans ciliated sensory neuronal model, employing assays for cilium integrity (dye filling assay) and functionality (osmotic avoidance assay; foraging). Using predicted loss-of-function alleles, we screened 55 mutants of evolutionarily conserved membrane organising and trafficking genes and identified 4 rab-5-related endocytic gene mutants with defects in cilium integrity and function. One of these, rabs-5, encodes a RAB-5 effector, which we found is expressed mainly in ciliated cells. rabs-5 mutants possess short cilia and an expanded periciliary membrane compartment, indicative of defects in ciliary membrane homeostasis. Transgenic expression of a rabs-5(WT) construct rescues the cilium integrity and foraging defects of rabs-5 mutants, confirming the association of rabs-5 with cilia-related functions. By taking a reverse genetics candidate gene approach in C. elegans, a number of RAB-5-dependent downstream effectors have been identified as potential regulators of ciliogenesis and ciliary membrane homeostasis.

TRPV channel-mediated calcium transients in nociceptor neurons are dispensable for avoidance behaviour.

Animals need to sense and react to potentially dangerous environments. TRP ion channels participate in nociception, presumably via Ca2+ influx, in most animal species. However, the relationship between ion permeation and animals’ nocifensive behaviour is unknown. Here we use an invertebrate animal model with relevance for mammalian pain. We analyse the putative selectivity filter of OSM-9, a TRPV channel, in osmotic avoidance behaviour of Caenorhabditis elegans. Using mutagenized OSM-9 expressed in the head nociceptor neuron, ASH, we study nocifensive behaviour and Ca2+ influx. Within the selectivity filter, M601-F609, Y604G strongly reduces avoidance behaviour and eliminates Ca2+ transients. Y604F also abolishes Ca2+ transients in ASH, while sustaining avoidance behaviour, yet it disrupts behavioral plasticity. Homology modelling of the OSM-9 pore suggests that Y604 may assume a scaffolding role. Thus, aromatic residues in the OSM-9 selectivity filter are critical for pain behaviour and ion permeation. These findings have relevance for understanding evolutionary roots of mammalian nociception.

The SOL-2/Neto Auxiliary Protein Modulates the Function of AMPA-Subtype Ionotropic Glutamate Receptors

The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents.

Functional Phenotypic Rescue of Caenorhabditis elegans Neuroligin-Deficient Mutants by the Human and Rat NLGN1 Genes

Neuroligins are cell adhesion proteins that interact with neurexins at the synapse. This interaction may contribute to differentiation, plasticity and specificity of synapses. In humans, single mutations in neuroligin encoding genes lead to autism spectrum disorder and/or mental retardation. Caenorhabditis elegans mutants deficient in nlg-1, an orthologue of human neuroligin genes, have defects in different behaviors. Here we show that the expression of human NLGN1 or rat Nlgn1 cDNAs in C. elegans nlg-1 mutants rescues the fructose osmotic strength avoidance and gentle touch response phenotypes. Two specific point mutations in NLGN3 and NLGN4 genes, involved in autistic spectrum disorder, were further characterized in this experimental system. The R451C allele described in NLGN3, was analyzed with both human NLGN1 (R453C) and worm NLG-1 (R437C) proteins, and both were not functional in rescuing the osmotic avoidance behavior and the gentle touch response phenotype. The D396X allele described in NLGN4, which produces a truncated protein, was studied with human NLGN1 (D432X) and they did not rescue any of the behavioral phenotypes analyzed. In addition, RNAi feeding experiments measuring gentle touch response in wild type strain and worms expressing SID-1 in neurons (which increases the response to dsRNA), both fed with bacteria expressing dsRNA for nlg-1, provided evidence for a postsynaptic in vivo function of neuroligins both in muscle cells and neurons, equivalent to that proposed in mammals. This finding was further confirmed generating transgenic nlg-1 deficient mutants expressing NLG-1 under pan-neuronal (nrx-1) or pan-muscular (myo-3) specific promoters. All these results suggest that the nematode could be used as an in vivo model for studying particular synaptic mechanisms with proteins orthologues of humans involved in pervasive developmental disorders.

The kinesin superfamily protein KIF17: one protein with many functions

Kinesins are ATP-dependent molecular motors that carry cargos along microtubules, generally in an anterograde direction. They are classified into 14 distinct families with varying structural and functional characteristics. KIF17 is a member of the kinesin-2 family that is plus end-directed. It is a homodimer with a pair of head motor domains that bind microtubules, a coiled-coil stalk, and a tail domain that binds cargos. In neurons, KIF17 transports N-methyl-D-aspartate receptor NR2B subunit, kainate receptor GluR5, and potassium Kv4.2 channels from cell bodies exclusively to dendrites. These cargos are necessary for synaptic transmission, learning, memory, and other functions. KIF17's interaction with NXF2 enables the transport of mRNA bidirectionally in dendrites. KIF17 or its homolog OSM-3 also mediates intraflagellar transport of cargos to the distal tips of flagella or cilia, thereby aiding in ciliogenesis. In many invertebrate and vertebrate sensory cells, KIF17 delivers cargos that contribute to chemosensory perception and signal transduction. In vertebrate photoreceptors, KIF17 is necessary for outer segment development and disc morphogenesis. In the testis, KIF17 (KIF17b) mediates microtubule-independent delivery of ACT from the nucleus to the cytoplasm and microtubule-dependent transport of Spatial-ε, both are presumably involved in spermatogenesis. KIF17 is also implicated in epithelial polarity and morphogenesis, placental transport and development, and the development of specific brain regions. The transcriptional regulation of KIF17 has recently been found to be mediated by nuclear respiratory factor 1 (NRF-1), which also regulates NR2B as well as energy metabolism in neurons. Dysfunctions of KIF17 are linked to a number of pathologies.

Microfluidic Devices for Analysis of Spatial Orientation Behaviors in Semi-Restrained Caenorhabditis elegans

This article describes the fabrication and use of microfluidic devices for investigating spatial orientation behaviors in nematode worms (Caenorhabditis elegans). Until now, spatial orientation has been studied in freely moving nematodes in which the frequency and nature of encounters with the gradient are uncontrolled experimental variables. In the new devices, the nematode is held in place by a restraint that aligns the longitudinal axis of the body with the border between two laminar fluid streams, leaving the animal's head and tail free to move. The content of the fluid streams can be manipulated to deliver step gradients in space or time. We demonstrate the utility of the device by identifying previously uncharacterized aspects of the behavioral mechanisms underlying chemotaxis, osmotic avoidance, and thermotaxis in this organism. The new devices are readily adaptable to behavioral and imaging studies involving fluid borne stimuli in a wide range of sensory modalities.

Roles for class IIA phosphatidylinositol transfer protein in neurotransmission and behavioral plasticity at the sensory neuron synapses of Caenorhabditis elegans

Growing evidence suggests that sensory neuron synapses not merely pass, but actively encode sensory information and convey it to the central nervous system. The chemosensory preferences of Caenorhabditis elegans, as manifested in the direction of chemotaxis, are reversibly regulated by prior experience at the level of sensory neurons; the attractive drive is promoted by diacylglycerol (DAG) signaling, whereas the counteracting repulsive drive requires PtdIns(3,4,5)P(3) signaling. Here we report that the two opposing drives require a class IIA phosphatidylinositol transfer protein (PITP), PITP-1, which localizes to the sensory neuron synapses. In pitp-1 mutants, attraction behavior to salt is reduced, whereas conditioned repulsion from salt is eliminated: the mutants inflexibly show weak attraction behavior to salt, irrespective of prior experience. To generate flexible behavioral outputs, attraction and repulsion, PITP-1 acts in the gustatory neuron ASER and likely regulates neurotransmission from ASER, as pitp-1 mutations do not affect the ASER Ca(2+) response to sensory stimulus. Furthermore, full attraction to salt is restored in pitp-1 mutants by expression of the phosphatidylinositol transfer domain alone, and also by mutations of a DGK gene that cause accumulation of DAG, suggesting that PITP-1 serves for DAG production via phosphatidylinositol transport and, hence, regulates synaptic transmission. In addition to gustatory behavior, olfactory behaviors and osmotic avoidance are also regulated by PITP-1 in the sensory neurons that detect each sensory stimulus. Thus, PITP-1-dependent phosphatidylinositol transport is essential for sensory neuron synapses to couple sensory inputs to effective behavioral responses.

Osmotic Avoidance in <em>Caenorhabditis elegans</em>: Synaptic Function of Two Genes, Orthologues of Human <em>NRXN1</em> and <em>NLGN1</em>, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function. These proteins are found at the presynaptic and postsynaptic membranes. Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders. Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegans nrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains. The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response. To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring. The behavioral phenotypes were confirmed using specific RNAi clones. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.

Evolutionary Conserved Role for TARPs in the Gating of Glutamate Receptors and Tuning of Synaptic Function

Neurotransmission in the brain is critically dependent on excitatory synaptic signaling mediated by AMPA-class ionotropic glutamate receptors (AMPARs). AMPARs are known to be associated with Transmembrane AMPA receptor Regulatory Proteins (TARPs). In vertebrates, at least four TARPs appear to have redundant roles as obligate chaperones for AMPARs, thus greatly complicating analysis of TARP participation in synaptic function. We have overcome this limitation by identifying and mutating the essential set of TARPs in C. elegans (STG-1 and STG-2). In TARP mutants, AMPAR-mediated currents and worm behaviors are selectively disrupted despite apparently normal surface expression and clustering of the receptors. Reconstitution experiments indicate that both STG-1 and STG-2 can functionally substitute for vertebrate TARPs to modify receptor function. Thus, we show that TARPs are obligate auxiliary subunits for AMPARs with a primary, evolutionarily conserved functional role in the modification of current kinetics.

Selective Role for TRPV4 Ion Channels in Visceral Sensory Pathways

Although there are many candidates as molecular mechanotransducers, so far there has been no evidence for molecular specialization of visceral afferents. Here, we show that colonic afferents express a specific molecular transducer that underlies their specialized mechanosensory function: the transient receptor potential channel, vanilloid 4 (TRPV4). We found TRPV4 mRNA is highly enriched in colonic sensory neurons compared with other visceral and somatic sensory neurons. TRPV4 protein was found in colonic nerve fibers from patients with inflammatory bowel disease, and it colocalized in a subset of fibers with the sensory neuropeptide CGRP in mice. We characterized the responses of 8 subtypes of vagal, splanchnic, and pelvic mechanoreceptors. Mechanosensory responses of colonic serosal and mesenteric afferents were enhanced by a TRPV4 agonist and dramatically reduced by targeted deletion of TRPV4 or by a TRP antagonist. Other subtypes of vagal and pelvic afferents, by contrast, were unaffected by these interventions. The behavioral responses to noxious colonic distention were also substantially reduced in mice lacking TRPV4. These data indicate that TRPV4 contributes to mechanically evoked visceral pain, with relevance to human disease. In view of its distribution in favor of specific populations of visceral afferents, we propose that TRPV4 may present a selective novel target for the reduction of visceral pain, which is an important opportunity in the absence of current treatments.

IFTA-2 is a conserved cilia protein involved in pathways regulating longevity and dauer formation inCaenorhabditis elegans

Defects in cilia are associated with diseases and developmental abnormalities. Proper cilia function is required for sonic hedgehog and PDGFRalpha signaling in mammals and for insulin-like growth factor (IGF) signaling in Caenorhabditis elegans. However, the role of cilia in these pathways remains unknown. To begin addressing this issue, we are characterizing putative cilia proteins in C. elegans that are predicted to have regulatory rather than structural functions. In this report, we characterized the novel cilia protein T28F3.6 (IFTA-2, intraflagellar transport associated protein 2), which is homologous to the mammalian Rab-like 5 protein. We found that, unlike the intraflagellar transport (IFT) genes, disruption of ifta-2 does not result in overt cilia assembly abnormalities, nor did it cause chemotaxis or osmotic avoidance defects typical of cilia mutants. Rather, ifta-2 null mutants have an extended lifespan phenotype and are defective in dauer formation. Our analysis indicates that these phenotypes result from defects in the DAF-2 (insulin-IGF-1-like) receptor signaling pathway in ciliated sensory neurons. We conclude that IFTA-2 is not a ciliogenic protein but rather is a regulator of specific cilia signaling activities. Interestingly, a mammalian IFTA-2 homolog is also found in cilia, raising the possibility that its function has been conserved during evolution.

TRPV4 plays an evolutionary conserved role in the transduction of osmotic and mechanical stimuli in live animals

The TRPV4 ion channel, previously named vanilloid receptor-related osmotically activated channel (VR-OAC), functions in vivo in the transduction of osmotic and mechanical stimuli. In trpv4 null mice, TRPV4 was found to be necessary for the maintenance of systemic osmotic equilibrium, and for normal thresholds in response to noxious mechanical stimuli. In a Caenorhabditis elegans TRPV mutant transgenic for mammalian TRPV4, the mammalian transgene was directing the osmotic and mechanical avoidance response in the context of the ASH 'nociceptive' neurone. Molecular mechanisms of gating of TRPV4 in vivo are not known at this point and have to be determined.