Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo

Abstract

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.

Figure 1. OR83b Is Essential for OR Membrane Trafficking

(A) The Drosophila olfactory system. Left panel: false-colored scanning electron micrograph image of a Drosophila head illustrating the major olfactory (antenna) and gustatory (proboscis) organs (photo: Jürgen Berger, Max Planck Institute for Developmental Biology, Tübingen, Germany). Center panel: schematic of olfactory sensilla distribution on the third antennal segment. Right panel: schematic of OSN anatomy. (B) Immunostaining for OR83b (green) and OR22a/b (red) in antennal sections of a wild-type (yw) animal. Arrowheads mark enrichment of perinuclear OR83b that co-localize with the trace amount of OR22a/b present in the cell body. (C) Immunostaining for OR22a/b (red) and mCD8:GFP (α-GFP, green) in antennal sections of control heterozygous (Or83b¹/+, top row) and homozygous (Or83b¹/Or83b², bottom row) Or83b null-mutant animals. The position of the field of view, in this and subsequent figures, is illustrated by the blue square in the antennal schematic (inset in upper left panel). Images of control and mutant samples were taken at identical confocal settings to permit comparison of signal intensities. Therefore, levels of OR22a/b are lower in (C) than in (D and E), in which confocal settings have been adjusted to permit visualization of OR22a/b in Or83b mutants. (D) Immunostaining for OR22a/b (red) and Golgi (α-Golgi, green) in antennal sections of control heterozygous (top panel) and homozygous (bottom panel) Or83b null-mutant animals. (E) Immunostaining for OR22a/b (red) and ER (α-KDEL, green) in antennal sections of heterozygous control (top panel) and homozygous (bottom panel) Or83b null-mutant animals. Mislocalized OR22a/b and KDEL accumulations overlap in the mutant (arrowheads in lower left panel). Autofluorescence of the antennal cuticle is visible in the green channel at the base of the sensilla. In (D and E), confocal settings of the red channel were adjusted to permit clear visualization of the weaker OR22a/b signal in Or83b mutants.

Figure 2. OR83b Is Required to Maintain OR Localization in Adults and Has No Essential Developmental Function

(A) Schematic of the TARGET system. (B) Immunostaining for OR83b (green) and OR22a/b (red) in Or83b null-mutant flies rescued using a UAS-Or83b transgene under the control of the TARGET system (Or22a-Gal4,tubP-Gal80ts/tubP-Gal80ts;UAS-Or83b,Or83b¹/tubP-Gal80ts,Or83b²). Three copies of the tubP-Gal80ts transgene were used to ensure efficient suppression of GAL4 activity by GAL80 at 18 °C. Flies were cultured and aged as adults for 10 d at 18 °C and then incubated for a further 2 d either at 18 °C (top row) or at 29 °C (bottom row) to induce late expression of Or83b. (C) Immunostaining for OR83b (green) and OR22a/b (red) in flies [genotype as in (B)] that were cultured and aged as adults for 3 d at 29 °C, transferred to 18 °C to switch off Or83b expression, and stained at the time points indicated.

Figure 3. Spatial Requirements for OR83b in OR Localization

(A) Immunostaining of GFP:OR43a (α-GFP, green) expressed in all Or83b neurons and OR83b (red) in antennal sections of control heterozygous (Or83b-Gal4/UAS-GFP:Or43a;Or83b¹/+, left panels) and homozygous (Or83b-Gal4/UAS-GFP:Or43a;Or83b¹/Or83b², right panel) Or83b null-mutant animals. (B) Immunostaining for GFP:OR47b (α-GFP, green) expressed in Or47b neurons and OR83b (red) in antennal sections of control heterozygous (Or47b-Gal4/UAS-GFP:Or47b;Or83b¹/+, left panels) and homozygous (Or47b-Gal4/UAS-GFP:Or47b;Or83b¹/Or83b², right panel) Or83b null-mutant animals. Arrowheads illustrate low level of OR83b expression in Or47b neurons. (C) Immunostaining for GFP:GR21a (α-GFP, green) expressed in Gr21a neurons and OR83b (red) in antennal sections of control heterozygous (Gr21a-Gal4/UAS-GFP:Gr21a;Or83b¹/+, left panel) and homozygous (Gr21a-Gal4/UAS-GFP:Gr21a;Or83b¹/Or83b², right panel) Or83b null-mutant animals. The OR83b signal in this sensillum reflects expression in the non-Gr21a-expressing neurons ab1A, ab1B, and ab1D, whose olfactory responses are Or83b-dependent [22,32]. (D) Immunostaining for GFP:GR21a (α-GFP, green) expressed in all Or83b neurons and OR83b (red) in antennal sections of wild-type animals (Or83b-Gal4/+;UAS-GFP:Gr21a/+).

Figure 4. OR-Independent Localization of OR83b to OSN Ciliated Dendrites

Immunostaining for OR22a/b (red) and GFP:OR83b (α-GFP, green) in antennal sections of control heterozygous (Or22a/b^Δhalo/+;Or22a-Gal4/UAS-GFP:Or83b, top panels) and homozygous (Or22a/b^Δhalo/Or22a/b^Δhalo;Or22a-Gal4/UAS-GFP:Or83b, bottom panels) Or22a/b null-mutant animals. Images of control and mutant samples were taken at identical confocal settings to permit comparison of signal intensities.

Figure 5. OR83b Is Necessary and Sufficient to Mediate OR Localization to Ciliated Dendrites in Other Sensory Neurons

(A) Immunostaining for GFP:OR43a (α-GFP, green) and OR83b (red) when misexpressed singly or in combination in Gr21a neurons. These animals are homozygous mutant for Or83b, which allows visualization of protein distributions specifically in Gr21a neurons. The arrowhead marks very weak localization of OR83b to cilia when expressed alone. Genotypes: Top row: Gr21a-Gal4/UAS-GFP:Or43a;Or83b¹/Or83b². Middle row: Gr21a-Gal4/+;UAS-Or83b,Or83b¹/Or83b². Bottom row: Gr21a-Gal4/UAS-GFP:Or43a;UAS-Or83b,Or83b¹/Or83b². (B) Immunostaining for GFP:OR43a (α-GFP, green) and OR83b (red) when misexpressed singly or in combination in second antennal segment mechanosensory neurons using the ciliated cell-specific oseg2-Gal4 driver. OR83b shows extremely weak localization to cilia that is not visible under these imaging conditions. Merged images are overlaid on a bright-field image to visualize tissue morphology. Genotypes: Top row: oseg2-Gal4/UAS-GFP:Or43a. Middle row: oseg2-Gal4/+;UAS-Or83b/+. Bottom row: oseg2-Gal4/UAS-GFP:Or43a;UAS-Or83b/+.

Figure 6. Expression of ORs and OR83b Reconstitutes a Functional OR in Gr21a Neurons

(A) Representative stimulus-evoked calcium signals recorded from the axon terminals of Gr21a neurons in the antennal lobe V glomerulus of a control animal (UAS-G-CaMP/UAS-G-CaMP;Gr21a-Gal4/Gr21a-Gal4; left column) and an animal misexpressing OR83b and GFP:OR43a in Gr21a neurons (UAS-G-CaMP/+;Gr21a-Gal4/UAS-GFP:Or43a;UAS-G-CaMP/UAS-Or83b; right column). Top row: intrinsic G-CaMP fluorescence of the V glomerulus. Dotted lines mark the antennal lobe border, and the black squares mark the area of the V glomerulus evaluated for stimulus-evoked changes in fluorescence. Middle row: false-color–coded images during stimulation with CO₂ (5%) or cyclohexanol (10⁻²) represent ΔF/F (%) according to the scales on the right panel. Bottom row: time traces of stimulus-evoked signals of the V glomerulus. Black bars indicate odor stimulation time. The diminished responses to CO₂ in animals expressing GFP:OR43a/OR83b may reflect competition between the resident and ectopic receptors in engaging the ciliary trafficking pathway or downstream signaling components. (B) Normalized odor-evoked calcium responses of control (blue) and GFP:OR43a/OR83b-misexpressing (red) animals [genotypes as in (A)] expressed as a percentage of the CO₂ response in each genotype. GFP:OR43a/OR83b-misexpressing animals show stronger responses than control animals for the odor stimuli (all at 10⁻²) marked with an asterisk (p < 0.05; two-tailed unpaired t-test; n = 4 animals per genotype and stimulus). Chemical Abstracts Service (CAS) registry numbers: cyclohexanol (108–93–0), cyclohexanone (108–94–1), hexanol (111–27–3), benzaldehyde (100–52–7), isoamyl acetate (123–92–2), geranyl acetate (105–87–3), octanol (111–87–5), linalool (126–91–0), caproic acid (142–62–1).

Figure 7. In Vivo Formation and Distribution of OR/OR83b Complexes

(A) Immunostaining for OR83b (blue), OR22a/b (red), and intrinsic YFP fluorescence (green) in antennal sections of Or83b null-mutant animals expressing YFP fragment:OR83b fusions, singly or in combination, as illustrated in the snake plots on the left. We note that the snake plots in this and subsequent figures are generated by computational analysis of OR sequences and the exact number and precise placement of the TM domains has not been experimentally verified. Genotypes: Or83b-Gal4/UAS-YFP(1):Or83b;Or83b¹/Or83b² (top row); Or83b-Gal4/+;UAS YFP(2):Or83b,Or83b¹/Or83b² (middle row); Or83b-Gal4/UAS-YFP(1):Or83b;UAS-YFP(2):Or83b,Or83b¹/Or83b² (bottom row). (B–E) Intrinsic YFP fluorescence (green) in antennal sections of animals expressing the indicated combinations of complementary YFP fragment fusions with these genotypes: (B) Or83b-Gal4/UAS-YFP(1):Or83b;UAS-YFP(2):Or83b,Or83b¹/Or83b² (C) Gr21a-Gal4/UAS-YFP(1):Or83b;UAS-YFP(2):Or83b/+ (D) Or83b-Gal4/UAS-YFP(1):Or43a;UAS-YFP(2):Or83b,Or83b¹/Or83b² (E) Or83b-Gal4/UAS-YFP(1):Gr21a;UAS-YFP(2):Or83b,Or83b¹/Or83b² (F) Left column: representative stimulus-evoked calcium signals recorded from the axon terminals of Or83b neurons in the antennal lobe of an Or83b mutant animal expressing YFP(2):OR83b alone (UAS-G-CaMP/UAS-G-CaMP;Or83b-Gal4/+;UAS-YFP(2):Or83b,Or83b¹/Or83b²) or YFP(1):OR43a and YFP(2):OR83b (UAS-G-CaMP/UAS-G-CaMP;Or83b-Gal4/UAS-YFP(1):Or43a;UAS-YFP(2):Or83b,Or83b¹/Or83b²) as indicated. Top row: intrinsic G-CaMP fluorescence in glomeruli innervated by Or83b neurons. Dotted lines mark the antennal lobe border and the black squares mark the area of the three selected glomeruli (DM2, DM3, and DM5) evaluated for stimulus-evoked changes in fluorescence. Below are false-color-–coded images during stimulation with two characterized OR43a ligands (cyclohexanol and benzaldehyde, both at 10⁻³ dilution) and a control odor that does not activate OR43a (ethyl-3-hydroxybutyrate, 10⁻⁵ dilution) representing ΔF/F (%) according to the scale at the bottom. Right column: quantification of odor-evoked calcium responses in the three indicated glomeruli of animals expressing YFP(2):OR83b alone (blue) or YFP(1):OR43a (red), and YFP(2):OR83b YFP(1):OR43a/YFP(2):OR83b-expressing animals show stronger responses for the known OR43a ligand stimuli (** p < 0.01; *** p < 0.001). DM2 and DM3 glomeruli show reduced responses to the control odor (* p < 0.05) similar to the effects of ectopic OR expression on the endogenous CO₂ responses of Gr21a neurons (Figure 6), while DM5 activity does not differ significantly between genotypes (marked N.S. on the bar graph). Significance was assessed with a two-tailed unpaired t-test; n = 4 animals per genotype and stimulus. CAS Registry Numbers: cyclohexanol (108–93–0), benzaldehyde (100–52–7), ethyl-3-hydroxybutyrate (54058–41–4). (G) Intrinsic YFP fluorescence (green) and immunostaining for YFP (red) in antennal sections of animals expressing YFP(1):OR43a and YFP(2):OR43a in control heterozygous (Or83b-GAL4/UAS-YFP(1):Or43a;UAS-YFP(2):Or43a,Or83b¹/+ [top panel]) and homozygous (Or83b-GAL4/UAS-YFP(1):Or43a;UAS-YFP(2):Or43a,Or83b¹/Or83b² [bottom panel]) Or83b null-mutant animals.

Figure 8. Bioinformatic Analysis Defines Drosophila ORs As a Novel Family of TM Proteins

(A) Unrooted neighbor-joining tree of selected Drosophila ORs [37], Class A GPCRs [84], Methuselah family receptors [85], Frizzled receptors [86], potassium channels, and mouse ORs [87]. Sequences were aligned in ClustalX with 1000 bootstrap iterations. (B) TM domain predictions of Drosophila ORs (n = 61) and a representative subset of mouse ORs (n = 61) by the HMMTOP version 2.0 algorithm, including all those ORs depicted in (A). (C) Membrane-insertion orientation predictions of Drosophila and mouse ORs (same sets as in (B)) by HMMTOP version 2.0. All mouse ORs mispredicted to have an intracellular N-terminus (8/8) and most Drosophila ORs predicted to have an extracellular N-terminus (8/12) are not predicted to have seven TM domains, suggesting that this algorithm may have difficulty in analyzing these particular sequences.

Figure 9. The N-Terminus of Drosophila ORs Is Intracellular

(A) Determination of OR N-terminus membrane insertion orientation by the β-gal fusion technique in S2 cells. Top left bars: schematic of fusion constructs and predictions of β-gal activity for proteins with an intracellular or extracellular N-terminus. Top right panels: sample field of view of S2 cells expressing OR83b N-term: β-gal (top panel) and OR83b N-term:artificial TM domain: β-gal (bottom panel) stained with X-gal to reveal active and inactive β-gal. Bottom table: active (+) and inactive (−) β-gal in the indicated OR83b, OR9a, and Drosophila RH1 fusion proteins. (B–D) Intrinsic YFP fluorescence (green) in antennal sections of animals expressing the indicated combinations of complementary YFP fragment and ZIP dimerization domain fusions with these genotypes: (B) Or83b-Gal4/+;UAS-YFP(1):zip/UAS-YFP(2):zip (C) Or83b-Gal4/UAS-YFP(2):zip:Or83b;UAS-YFP(1):zip/+ (D) Or83b-Gal4/+;UAS-YFP(1):zip/UAS-YFP(2):zip:Or43a

Figure 10. Probing OR83b Topology by Antibody Epitope Staining

(A) Left panel: whole-mount view of a third instar larval salivary gland expressing GFP:OR83b (green) counterstained with DAPI (blue) to visualize the cell nuclei. Genotype in this and subsequent panels: AB1-Gal4/+;UAS-GFP:Or83b/+. The white box marks the approximate field of view of this tissue shown in all subsequent panels. Right bar graphic: snake plot of OR83b showing the predicted topological location of the N-terminal GFP epitope and the OR83b α-EC2 antibody epitope. (B) Immunostaining of GFP:OR83b (intrinsic fluorescence in green) in larval salivary gland cells with α-EC2 (red) and α-GFP (purple) when permeabilized (0.25% Triton X-100 detergent, top row) or unpermeabilized (no detergent, middle row). The cell membrane staining of OR83b α-EC2 under unpermeabilized conditions is not detected in control salivary glands (AB1-Gal4/+) (bottom). Images are single confocal sections of cells in a plane through or just above the cell nuclei (visualized with DAPI staining, blue). (C) Salivary glands expressing GFP:OR83b (AB1-Gal4/+;UAS-GFP:Or83b/+) were stained with antibodies against the epitopes, illustrated in red in the snake plots on the left, under permeabilized or unpermeabilized conditions. For clarity, only the red channel is shown. None of the antibodies stain control salivary glands under permeabilized conditions (unpublished data). (D) Horizontal section of an antennal sensillum viewed by conventional EM reveals cross-sections of dendritic membranes (scale bar = 1 μm). C, cuticle; P, pore; D, dendrite; SL, sensillum lymph. (E) ImmunoEM on a horizontal section of an antennal sensillum using OR83b α-EC2 and a secondary antibody conjugated to 5 nm colloidal gold reveals distribution of the EC2 epitope on the extracellular face of the dendritic membranes (scale bar = 200 nm). (F) Quantification of gold particle distribution scored from four sections obtained in two independent experiments.

Figure 11. OR83b and ORs Associate via Conserved C-Terminal Domains

(A) Top rows: Immunostaining of the GFP:OR83b(1–170):OR43a(159–376) chimera (α-GFP, green) in antennal sections of animals of the following genotypes (left to right): Or83b^+/− neuron [Or83b-Gal4/UAS-GFP:Or83b(1–170):Or43a(159–376);Or83b¹/+]; Or83b^−/− neuron [Or83b-Gal4/UAS-GFP:Or83b(1–170):Or43a(159–376);Or83b¹/Or83b²]; Gr21a neuron + OR83b [Gr21a-Gal4/UAS-GFP:Or83b(1–170):Or43a(159–376);UAS-Or83b/+]; Gr21a neuron + OR43a [Gr21a-Gal4/UAS-GFP:Or83b(1–170):Or43a(159–376);UAS-Rho:Or43a/+]. Bottom table: summary of localization (+) or no localization (−) to cilia of the chimera and, for comparison, OR83b and OR43a. (B) Interactions between OR83b and OR cytoplasmic domains detected by the yeast two-hybrid assay by observation of growth (+) or no growth (−) of yeast co-transformed with the indicated bait/prey combinations on media selecting for expression of HIS3 and ADE2 reporters.