Odorant receptors of Drosophila are sensitive to the molecular volume of odorants.
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ABSTRACT:
SUBMITTER: Saberi M
PROVIDER: S-EPMC4844992 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Odorants are thought to activate sets of odorant receptors in vivo, but capturing sets of responsive receptors in vivo has never been accomplished. GeneChip microarrays were used to identify the odorant receptor mRNAs enriched in samples from activated olfactory neurons
2014-12-02 | GSE59336 | GEO
Project description:Flying insects have developed a remarkably sensitive olfactory system to detect faint and turbulent odor traces. This ability is linked to the olfactory receptors class of odorant receptors (ORs), occurring exclusively in winged insects. ORs form heteromeric complexes of an odorant specific receptor protein (OrX) and a highly conserved co-receptor protein (Orco). The ORs form ligand gated ion channels that are tuned by intracellular signaling systems. Repetitive subthreshold odor stimulation of olfactory sensory neurons sensitizes insect ORs. This OR sensitization process requires Orco activity. In the present study we first asked whether OR sensitization can be monitored with heterologously expressed OR proteins. Using electrophysiological and calcium imaging methods we demonstrate that D. melanogaster OR proteins expressed in CHO cells show sensitization upon repeated weak stimulation. This was found for OR channels formed by Orco as well as by Or22a or Or56a and Orco. Moreover, we show that inhibition of calmodulin (CaM) action on OR proteins, expressed in CHO cells, abolishes any sensitization. Finally, we investigated the sensitization phenomenon using an ex vivo preparation of olfactory sensory neurons (OSNs) expressing Or22a inside the fly's antenna. Using calcium imaging, we observed sensitization in the dendrites as well as in the soma. Inhibition of calmodulin with W7 disrupted the sensitization within the outer dendritic shaft, whereas the sensitization remained in the other OSN compartments. Taken together, our results suggest that CaM action is involved in sensitizing the OR complex and that this mechanisms accounts for the sensitization in the outer dendrites, whereas further mechanisms contribute to the sensitization observed in the other OSN compartments. The use of heterologously expressed OR proteins appears to be suitable for further investigations on the mechanistic basis of OR sensitization, while investigations on native neurons are required to study the presently unknown additional mechanisms involved in OSN sensitization.
| S-EPMC4751262 | biostudies-literature
Project description:Odorant degrading enzymes (ODEs) are thought to be responsible, at least in part, for olfactory signal termination in the chemosensory system by rapid degradation of odorants in the vicinity of the receptors. A carboxylesterase, specifically expressed in Drosophila antennae, called "juvenile hormone esterase duplication (JHEdup)" has been previously reported to hydrolyse different fruit esters in vitro. Here we functionally characterize JHEdup in vivo. We show that the jhedup gene is highly expressed in large basiconic sensilla that have been reported to detect several food esters. An electrophysiological analysis demonstrates that ab1A olfactory neurons of jhedup mutant flies exhibit an increased response to certain food acetates. Furthermore, mutant flies show a higher sensitivity towards the same odorants in behavioural assays. A phylogenetic analysis reveals that jhedup arose as a duplication of the juvenile hormone esterase gene during the evolution of Diptera, most likely in the ancestor of Schizophora, and has been conserved in all the 12 sequenced Drosophila species. Jhedup exhibits also an olfactory-predominant expression pattern in other Drosophila species. Our results support the implication of JHEdup in the degradation of food odorants in D. melanogaster and propose a neofunctionalization of this enzyme as a bona fide ODE in Drosophilids.
| S-EPMC5626784 | biostudies-literature
Project description:Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles.
| S-EPMC4633494 | biostudies-literature
Project description:BackgroundPost-translational transport is a vital process which ensures that each protein reaches its site of function. Though most do so via an ordered ER-to-Golgi route, an increasing number of proteins are now shown to bypass this conventional secretory pathway.ResultsIn the Drosophila olfactory sensory neurons (OSNs), odorant receptors (ORs) are trafficked from the ER towards the cilia. Here, we show that Or22a, a receptor of various esters and alcoholic compounds, reaches the cilia partially through unconventional means. Or22a frequently present as puncta at the somatic cell body exit and within the dendrite prior to the cilia base. These rarely coincide with markers of either the intermediary ER-Golgi-intermediate-compartment (ERGIC) or Golgi structures. ERGIC and Golgi also displayed axonal localization biases, a further indication that at least some measure of OR transport may occur independently of their involvement. Additionally, neither the loss of several COPII genes involved in anterograde trafficking nor ERGIC itself affected puncta formation or Or22a transport to the cilium. Instead, we observed the consistent colocalization of Or22a puncta with Grasp65, the sole Drosophila homolog of mammalian GRASP55/Grh1, a marker of the unconventional pathway. The numbers of both Or22a and Grasp65-positive puncta were furthermore increased upon nutritional starvation, a condition known to enhance Golgi-bypassing secretory activity.ConclusionsOur results demonstrate an alternative route of Or22a transport, thus expanding the repertoire of unconventional secretion mechanisms in neurons.
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Project description:Mosquitoes rely heavily on the olfactory system to find a host for a bloodmeal, plants for a source of energy and suitable sites for oviposition. Here, we examined a cluster of eight odorant receptors (ORs), which includes one OR, CquiOR1, previously identified to be sensitive to plant-derived compounds. We cloned 5 ORs from Culex quinquefasciatus and two ORs from Aedes aegypti, ie, CquiOR2, CquiOR4, CquiOR5, CquiOR84, CquiOR85, AaegOR14, and AaegOR15 and then deorphanized these receptors using the Xenopus oocyte recording system and a large panel of odorants. 2-Phenylethanol, phenethyl formate, and phenethyl propionate were the best ligands for CquiOR4 somewhat resembling the profile of AaegOR15, which gave the strongest responses to phenethyl propionate, phenethyl formate, and acetophenone. In contrast, the best ligands for CquiOR5 were linalool, PMD, and linalool oxide. CquiOR4 was predominantly expressed in antennae of nonblood fed female mosquitoes, with transcript levels significantly reduced after a blood meal. 2-Phenylethanol showed repellency activity comparable to that of DEET at 1%. RNAi experiments suggest that at least in part 2-phenylethanol-elicited repellency is mediated by CquiOR4 activation.
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Project description:Drosophila olfactory sensory neurons (OSNs) each express two odorant receptors (ORs): a divergent member of the OR family and the highly conserved, broadly expressed receptor OR83b. OR83b is essential for olfaction in vivo and enhances OR function in vitro, but the molecular mechanism by which it acts is unknown. Here we demonstrate that OR83b heterodimerizes with conventional ORs early in the endomembrane system in OSNs, couples these complexes to the conserved ciliary trafficking pathway, and is essential to maintain the OR/OR83b complex within the sensory cilia, where odor signal transduction occurs. The OR/OR83b complex is necessary and sufficient to promote functional reconstitution of odor-evoked signaling in sensory neurons that normally respond only to carbon dioxide. Unexpectedly, unlike all known vertebrate and nematode chemosensory receptors, we find that Drosophila ORs and OR83b adopt a novel membrane topology with their N-termini and the most conserved loops in the cytoplasm. These loops mediate direct association of ORs with OR83b. Our results reveal that OR83b is a universal and integral part of the functional OR in Drosophila. This atypical heteromeric and topological design appears to be an insect-specific solution for odor recognition, making the OR/OR83b complex an attractive target for the development of highly selective insect repellents to disrupt olfactory-mediated host-seeking behaviors of insect disease vectors.
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Project description:Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-β-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.
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Project description:This paper describes a highly sensitive and selective chemical sensor using living cells (Xenopus laevis oocytes) within a portable fluidic device. We constructed an odorant sensor whose sensitivity is a few parts per billion in solution and can simultaneously distinguish different types of chemicals that have only a slight difference in double bond isomerism or functional group such as -OH, -CHO and -C(=O)-. We developed a semiautomatic method to install cells to the fluidic device and achieved stable and reproducible odorant sensing. In addition, we found that the sensor worked for multiple-target chemicals and can be integrated with a robotic system without any noise reduction systems. Our developed sensor is compact and easy to replace in the system. We believe that the sensor can potentially be incorporated into a portable system for monitoring environmental and physical conditions.
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Project description:The Southern house mosquito Culex quinquefasciatus has the largest repertoire of odorant receptors (ORs) of all mosquitoes and dipteran species whose genomes have been sequenced to date. Previously, we have identified and de-orphanized two ORs expressed in female antennae, CquiOR2 and CquiOR10, which are sensitive to oviposition attractants. In view of a new nomenclature for the Culex genome (VectorBase) we renamed these ORs as CquiOR21 (formerly CquiOR10) and CquiOR121 (CquiOR2). In addition, we selected ORs from six different phylogenetic groups for deorphanization. We cloned four of them by using cDNA from female antennae as a template. Attempts to clone CquiOR87 and CquiOR110 were unsuccessful either because they are pseudogenes or are not expressed in adult female antennae, the main olfactory tissue. By contrast, CquiOR1, CquiOR44, CquiOR73, and CquiOR161 were highly expressed in female antennae. To de-orphanize these ORs, we employed the Xenopus oocyte recording system. CquiORx-CquiOrco-expressed oocytes were challenged with a panel of 90 compounds, including known oviposition attractants, human and vertebrate host odorants, plant kairomones, and naturally occurring repellents. While CquiOR161 did not respond to any test compound in two different laboratories, CquiOR1 showed the features of a generic OR, with strong responses to 1-octen-3-ol and other ligands. CquiOR44 and CquiOR73 showed preference to plant-derived terpenoids and phenolic compounds, respectively. While fenchone was the best ligand for the former, 3,5-dimethylphenol elicited the strongest responses in the latter. The newly de-orphanized ORs may be involved in reception of plant kairomones and/or natural repellents.
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