Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo
(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:
(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.