2 In vitro olfactory receptor assay
2.5 Discussion
Olfaction plays a major role for insect survival. Over the past 20 years much effort has been taken by the researchers to understand various aspects of olfaction. But still the mechanism of insect olfactory signal transduction is unclear and the proposed models are controversial. Using Drosophila Or22a and Or92a receptors as a model of insect OR, we developed an in vitro assay as a new tool to study the role of G proteins in insect olfactory signaling. Or22a and Or92a were activated by well described ligands of the receptors. Addition of odorants to dORs transfected HEK293T cells elicited a dose-dependent and significant calcium increase in most of the cells. About 60% of the transfected cells responded to the odorant, which is higher than reported in literature (Kiely et al., 2007; Neuhaus et al., 2005). One of these studies used insect cell lines for OR expression (dOr22a) and only ~4% of cells responded to the odor (Kiely et al., 2007), whereas other study used mammalian cells lines (HEK293 cells) and about 8-10%
(dOr22a and Orco) or 10-15% (dOr43a and Orco) of cells responded to the odor (Neuhaus et al., 2005). We used HEK293T cells for dOR (dOr22a) expression together with Orco, similar to the expression system used by the latter study, but in our assay more number of cells responded to the odor (~6 times higher number of cells). This difference may arise from the transfection efficiency, in our assay ~50% of cells were transfected but in the other study the transfection efficiency was much lower - ~20%
(Neuhaus et al., 2005). Hence the expression system and the greater transfection efficiency in our experiments is the reason for the higher number of cells responded in our assay than reported before.
For expression of Or22a in the mammalian cells, expression of the co-receptor Orco was required. The co-co-receptor Orco forms the integral part of the functional odorant receptor and is also shown to be involved in dendritic localization of the ORs in vivo (Benton et al., 2006). Insect ORs expressed in Xenopus oocytes did not respond to the odorants when expressed alone, whereas they responded to odorants after co-expression of Orco or a promiscuous Gα subunit (Wetzel, 2001). However insect ORs responded to odorants without the co-expression of Orco in mammalian cell lines; in this cell system co-expression of Orco was shown to enhance the sensitivity of the receptor (Neuhaus et al., 2005; Wicher et al., 2008). In our studies we found that Orco is required for the trafficking /stabilization of Or22a in cell membranes (Fig 2.3), which may indicate the necessity of co-expression of Orco in HEK cells for odor response as shown previously for odor responses in Xenopus oocytes and in vivo (Benton et al., 2006).
Odor responses were dose-dependent, strong (about 8-fold higher than in the control group of cells) and reproducible. With increase in odor concentration the response increased in dOr22a and Orco cells (Fig 2.6A), whereas in dOr92a and Orco
Chapter 2. Discussion
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cells the response increased till the 10-4 dilution and at the 10-3 dilution the response decreased (Fig 2.6B). We speculate that very high odor concentrations lead to the deactivation of the receptor. Although the odorant was present for 150 s, the response was transient. Individual cells elicited peak calcium response at different time points (typically 30 s after odor addition; Fig 2.4D). Non-automated odorant addition could have contributed to the difference observed. But the time window used to image the cells after odor addition (30 frames, 150 s) is large enough to record the response of most cells in the view.
Calcium response mediated by a particular concentration of the odorant differed within an experiment. Based on the response, responding cells can be categorized into 4 different groups: high responders (Ro/Bo value greater than 4), intermediate responders (Ro/Bo value greater than 2 and less than 4) and weak responders (Ro/Bo value greater than 1.5 and less than 2). A cell with a high copy number of receptors at the plasma membrane will elicit a larger increase in second messengers (leading to a high response) than a similar cell with lower amounts of receptor molecules exposed to the ligand (leading to an intermediate or weak response) (Clyne et al., 2003). Although all the transfected cells express dORs, the number of receptors that a cell expresses in their plasma membrane is unknown. Hence we speculate that different levels of expression of ORs on the cell surface lead to different levels of activation of second messengers that result in variations of response to the same concentration of the odorant. About 70% of cells didn’t respond to the odor; for 50% of them the reason for this might be that they were not transfected as we may conclude from our transfection efficiency estimation;
however, this leaves 20% of nonresponders that were transfected with dORs. Several possibilities could explain why this phenomenon takes place. These are: misfolding of ORs which results in their degradation in the cytosol; mistargeting of dORs to cell membranes; expression of one of the receptor proteins at very low quantities, insufficient for second messenger activation.
EtBE is one of the best ligands for the receptor Or22a, as shown in vivo by single sensillum recordings (Hallem et al., 2004) and also by in vitro studies (through expression of Or22a in Sf9 cells (Kiely et al., 2007)). Stimulation of flies with varied dilutions of EtBE elicited significant responses at the 10-2-10-4 but not at 10-6-10-8 dilutions (Hallem et al., 2004). In vitro, stimulation of the receptor (Or22a) elicited a significant response to low concentrations of the odorant as well (EtBE; EC50 value is 1.58±0.82 x 10-11 M) (Kiely et al., 2007; Wicher et al., 2008). In our experiments the sensitivity of Or22a was much lower (the significant response was seen only at 10-2 M; Fig 2.6A) than it was reported before. Odor concentrations between in vivo and in vitro experiments cannot be compared directly. Odorants are delivered in the vapor phase for in vivo assays and the quantification of the substrate in its initial mixture does not necessarily reflect the final
Chapter 2. Discussion
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concentration in the antennae (it is impossible to detect the actual concentration of the odorant that reaches the sensilla). Hence the sensitivity observed in the in vivo assays cannot be directly compared with our results, but our results can be compared with other in vitro studies.
The great discrepancy in the sensitivity of Or22a in our study and the results obtained in the insect cell line (Sf9 cells; (Kiely et al., 2007)) can be explained by the differences in the model cells. Since we co-expressed Orco in all our experiments, the difference is unlikely to be due to its endogenous expression in Sf9 cells. Rather an unknown endogenous protein in Sf9 cells enhanced the sensitivity of the receptor or affected its folding and targeting to the membrane. Sensitivity of in vitro pheromone transduction was shown to be enhanced by the co-expression of additional proteins (SNMPs and OBPs; (Grosse-Wilde, 2006; Grosse-Wilde et al., 2007; Nolte et al., 2013)).
But for general odor detection till now the requirement of additional proteins are not known. In another study Or22a was expressed in a mammalian cell line, however the odor induced whole cell currents were used as the readout (Wicher et al., 2008). The background noise is higher in calcium imaging experiments when compared to the electrophysiological measurements. This might explain why our responses at low concentrations were insignificant.
Stimulation of Or92a with BDOL elicited a significant response also at low concentrations (Fig 2.6B). BDOL is one of the best ligands for the receptor Or92a as shown in vivo (calcium imaging (Galizia et al., 2010)). Stimulation of flies with varied dilutions of BDOL elicited significant responses at the 10-2-10-4 dilution but not at the 10-6 dilution (unpublished data; for reference see Chapter 4). Sensitivity of the Or92a receptor was similar to the one reported before for in vivo studies. Using the same method we observed differences in the sensitivity of the receptors tested; Or92a was more sensitive than Or22a (Fig 2.6A, B). The only difference in the methodology when studying these receptors was the solvent used for odor dilution. For EtBE the assay buffer was used as a solvent (EtBE is partially water soluble; EtBE was diluted in water and functional studies were described before by using water as a solvent (Kiely et al., 2007)), while for BDOL, DMSO was used as a solvent. We chose DMSO as a solvent even though the odorant is soluble in water due to further plans to evaluate the sensitivity of the receptor to more ligands, most of which are insoluble in water.
Dissolving the odorants in DMSO, especially for hydrophobic compounds (pheromones) was shown to increase the sensitivity of the receptors in vitro (Grosse-Wilde, 2006). Odorant binding proteins (OBPs) in sensillar lymph enhances the solubility of hydrophobic compounds and transport the odor to the ORs. DMSO in our studies might play the similar role in vitro.
Chapter 2. Discussion
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Addition of increasing odor concentrations during single experiment resulted in a significant but weak response at the higher concentrations (10-2-10-3 dilution); repeated stimulation elicited a significant response at the 10-3 dilution, whereas single-time stimulation elicited insignificant responses. About 50% of cells responded to higher odor concentrations (10-2-10-3 M) and not to lower odor concentration (10-4 M; Fig 2.7B). We speculate that these cells expressed very low quantities of ORs at the cell surface that leads to an insufficient activation of second messengers and thus no observable response. Single-time stimulation of dORs transfected cells with EtBE leads to an about eight-fold increase in calcium levels as compared to the control cells (Figs 2.4E and 2.6B), whereas at the same concentration the cells that had already responded to other concentrations before elicited a weak response (~ 1.7-fold, Fig 2.8C, D). These data may reflect the phenomenon of adaptation.
Sensory systems adapt i.e. they adjust their sensitivities to external stimuli according to the ambient level. In the context of sensory processing, odor adaptation refers to the ability of the olfactory system to adjust its sensitivity at different stimulus intensities. It is essential for preventing saturation of the cellular transduction machinery and allows the retention of high sensitivity during continuous or repetitive odor stimulation. Odor adaptation occurs in peripheral (i.e. in ORNs) and central parts of the brain. In vertebrate ORNs co-existence of three different forms of odor adaptation was described: short term adaptation, desensitization and long-lasting adaptation (Zufall and Leinders-Zufall, 2000). Calcium-meditated feedback signaling was shown to play a major role in odor adaptation (Kurahashi and Menini, 1997). Also several calcium-independent mechanisms have been proposed for odor adaptation, such as OR phosphorylation by protein kinase A (Boekhoff and Breer, 1992) and G protein-coupled receptor kinase 3 (Dawson et al., 1993; Peppel et al., 1997; Schleicher et al., 1993). Acute attenuation of signaling of various receptors (including GPCRs and insect ORs) can be accomplished either via ligand-induced internalization of receptors (endocytic downregulation) or via ligand-induced receptor desensitization (Backer et al., 1991; Ferguson, 2001; Ferguson and Caron, 1998; Flores-Morales et al., 2006; Freedman and Lefkowitz, 1996;
Gainetdinov et al., 2004; Law et al., 1983; Quick and Lester, 2002; Wiley et al., 1991). We contemplate that any of the odor adaptation mechanisms described above could explain the transient and weak response observed in our experiments.
Our assay can be used in future to characterize molecules involved in olfactory signal transduction. Downregulation or overexpression of proteins or manipulations with second messengers that are presumably involved in the signal transduction cascade can be used to study their involvement in the signal transduction. These molecules can be then tested in vivo. With this assay screening of the molecules involved in signal transduction can be performed faster because it is less time-consuming when compared
Chapter 2. Discussion
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to in vivo studies. The reductionist principle of this system allows inferring a direct involvement of the identified molecules in signaling. Apart from the molecules involved in general odor detection, those involved in pheromone transduction can be studied with this assay; we plan to study the Drosophila odorant receptor 67d and the role of G proteins in signaling by this receptor in this system. For this purpose the gene encoding for this receptor was cloned in mammalian expression vector pCDNA3.1(+).
Using this assay with some modifications we can also characterize the molecules involved in gustatory (taste) signaling of insects. Three of the gustatory receptors (GRs) were cloned in the mammalian expression vector pCDNA3.1(+): Gr64c, d and Gr64e.
The assay can be validated with these receptors, and the role of G proteins in gustation can be studied in future. Also this assay can be used to de-orphanize ORs from various insects. The mechanisms of odor adaptation known to date are mostly from vertebrate ORNs and very little is known in this regard for insect ORNs. Although this assay may not reveal all the mechanisms of odor adaptation as it is only suitable to those linked to ORs it can give useful insights about odor adaption in insects. In the present work we have made the first step in application of this assay and studied the role of the Go/i subgroup of G proteins in dOr22a signal transduction. The results are described in the next chapter.
Acknowledgements
I thank Prof Dr Eva M Neuhaus, Charite - Universitatsmedizin Berlin, Germany for the kind gift of pcDNA3-Or22a-GFP and pcDNA3-Or83b-GFP constructs and Prof Dr Dean Smith, Department of Pharmacology and Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Texas, USA for the kind gift of Or67d construct. Also I thank the group of Prof. Marcel Leist, Department of biology, University of Konstanz, Germany for the kind gift of HEK293T cells and Martin Horn, University of Konstanz, Germany for helping with the image analysis in KNIME (http://knime.org/). All Images were recorded from the Bioimaging Center (BIC) of University of Konstanz, Germany.
38
CHAPTER 3
Role of G
o/isubgroup of G proteins in olfactory signaling of Drosophila melanogaster
3.1 Abstract
Intracellular signaling in insect olfactory receptor neurons remains unclear, with both metabotropic and ionotropic components being discussed. Here, we investigated the role of heterotrimeric Go and Gi proteins using a combined behavioral, in vivo and in vitro approach. Specifically, we show that inhibiting Go in sensory neurons by pertussis toxin leads to behavioral deficits. We heterologously expressed the olfactory receptor dOr22a in human embryonic kidney cells. Stimulation with an odor led to calcium influx, which was amplified via calcium release from intracellular stores. Subsequent experiments indicate that the signaling is mediated by the Gβγ subunits of the heterotrimeric Go/i proteins. Finally, using in vivo calcium imaging, we show that Go and Gi contribute to odor responses both for the fast (phasic) as for the slow (tonic) response component. We propose a transduction cascade model involving several parallel processes, in which the metabotropic component is activated by Go and Gi, and uses Gβγ.
3.2 Introduction
The sense of smell - olfaction - plays a major role for all animals and mediates behavioral and physiological responses. Odor molecules bind to the odorant receptors (ORs) present at the dendrites of the olfactory receptor neurons (ORNs) located at the peripheral olfactory organs, which send information to the central parts of the brain for further processing. Even though chemical senses are the most ancient in evolution, ORs have evolved creating several evolutionary distinct and independent gene families, which differ in structure and in intracellular signaling. All OR families in vertebrates are G-protein-coupled receptors (GPCRs) (Bargmann, 2006; Buck and Axel, 1991; Mombaerts, 1999). They activate metabotropic G protein-dependent signaling cascades, but different OR families activate different cascades (Berghard and Buck, 1996; Berghard et al., 1996;
Jones and Reed, 1989; Kaupp, 2010).
Chapter 3. Introduction
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Insects have more than one family of receptors for olfaction. One family consists of ionotropic receptors (IRs) related to glutamate channels, which respond to odor binding by opening an ion channel (Benton et al., 2009). The other family consists of ORs with a predicted seven transmembrane topology reminiscent of classical GPCRs, but with an inverted membrane topology and low sequence homology to all known GPCRs (Benton et al., 2006; Clyne et al., 1999; Gao and Chess, 1999; Lundin et al., 2007;
Vosshall et al., 1999). Insect ORs form heteromeric complexes with a conserved ortholog protein called Orco (Benton et al., 2006; Larsson et al., 2004). It remains unclear whether and if so how insect ORs depend on G proteins for olfactory signaling.
Two different hypotheses have been proposed for insect olfactory signal transduction:
either insect ORs may act as ligand gated ion channels (ionotropic signaling pathway) or combine an ionotropic and a G protein-dependent pathway for olfactory signaling (Sato et al., 2008; Smart et al., 2008; Wicher et al., 2008). However, the involvements of different G proteins in insect olfactory signaling are still enigmatic.
Insect ORNs express several G proteins that could be involved in signal transduction, in particular the Go/i subgroup of G proteins (Boto et al., 2010; Kang et al., 2011; Miura et al., 2005; Rützler et al., 2006). Therefore, in this study we tested whether Go/i are required for olfaction in behavior, for odor responses in the native tissue (antenna; in vivo) or when expressed in a heterologous cell-culture system (HEK293T cells: Human Embryonic Kidney 293T cells; in vitro). We found that in vivo disruption of Gαo/i subunits in the ORNs of Drosophila leads to olfactory behavioral deficits and reduced the amplitude of the odor responses regardless of odor identity and intensity. In vitro inhibition and over-expression of Gαo/i subunits indicated that the Gβγ heterodimer is the key player in the transduction mechanisms. Altogether our results indicate a role of Go/i subgroup of G proteins in olfactory signaling of Drosophila.
3.3 Materials and Methods
3.3.1 In vivo experiments
Flies
Flies were reared on standard corn meal medium containing yeast and were kept at 25ºC and a humidity of 50 % on a 12 h:12 h light:dark cycle. We used 1–3 days old flies for behavioral experiments and 7–14 days old female flies of F1 progeny for in vivo calcium imaging experiments. The following lines were used: UAS-PTX (Katanaev et al., 2005), UAS-RNAi-Gαi (Kopein and Katanaev, 2009) (Vienna Drosophila RNAi Center),
Chapter 3. Materials and Methods
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UAS-GCaMP;Or22a-Gal4/Cyo (crossed from UAS-GCaMP;Cyo/Sp;+ flies provided by Jing Wang, University of California, San Diego, La Jolla, CA (Nakai et al., 2001; Wang et al., 2003)), UAS-GCaMP;Or22a-Gal4/UAS-PTX (crossed from UAS-PTX (Katanaev et al., 2005) and UAS-GCaMP;Or22a-Gal4/Cyo) and UAS-GCaMP;Or22a-Gal4;UAS-RNAi-Gαi (crossed from UAS-RNAi-Gαi (Kopein and Katanaev, 2009) and UAS-GCaMP;Or22a-Gal4/Cyo).
Behavior
Ca. 150 young flies, with equal representation of males and females, were flipped into a large cylindrical bottle 8 cm in diameter and 14 cm in height, without anesthesia by CO2 or cold. Inside the bottles were two trap containers made of blue pipette tips, one with ca. 0.3 ml of mineral oil and one with equal volume of kitchen apple vinegar. Flies were kept in bottles for 1 h at 25°С, followed by counting the number of flies trapped in each container and those remaining in the bottle. Results were shown as mean ± standard error of mean (s.e.m.), where n represents number of experiments. The evaluation of statistical significance of differences was tested with Student’s t-test.
In vivo preparation of flies
Flies were immobilized on ice for 15 min and then slipped with their neck into a horizontal slit in a plastic recording chamber. The head was fixed to the chamber using dental glue. Antennae were prevented from moving by an electron-microscopy grid placed on top of the proximal part of the third antennal segment. The method of preparation leaves the animal surgically intact.
In vivo calcium imaging
Intact fly antennae were recorded as described before (Pelz et al., 2006). The calcium sensor GCaMP1.3 was expressed in the ORNs expressing the odorant receptor Or22a and the odor evoked calcium changes were measured at the receptor neuron dendrites and somata through the intact antennal cuticle. The setup consists of an upright microscope (Olympus BX50WI, Tokyo, Japan) equipped with a 50x air objective (NA = 0.5) and a CCD/monochromator based imaging system (Till Photonics, Gräfelfing, Germany). A monochromator (Polychrome II, TILL Photonics) produced excitation light of 470nm wavelength that was directed onto the antenna via a 500nm low-pass filter and a 495nm dichroic mirror, emission light was filtered through a 505nm high-pass emission filter. Images were acquired with a TILL imago CCD camera with a binning of 8x8 on the chip. We varied the exposures time between 180 and 220 ms to
Chapter 3. Materials and Methods
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adjust for different basal fluorescence values across preparations. Twenty-second films were recorded with an acquisition rate of 4 Hz.
Odorant preparation and application
Odorants [ethyl butyrate (EtBE), ethyl hexanoate, 1-heptanol, 4-methoxybenzene and 1-butanol] were greater than 99.5% pure or of the highest purity
Odorants [ethyl butyrate (EtBE), ethyl hexanoate, 1-heptanol, 4-methoxybenzene and 1-butanol] were greater than 99.5% pure or of the highest purity