Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo methods
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ORs and OR83b Adopt a Novel Membrane Topology
(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 None of the antibodies stain control salivary glands under permeabilized conditions (unpublished data).
OR83b Is Continuously Required for OR Localization
To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system to achieve temporal control of OR83b expression, specifically in Or22a/b neurons [ ]. This technique combines cell-type specific induction of a UAS-Or83b transgene by an Or22a-Gal4 driver line with temporal regulation through use of a ubiquitously expressed temperature-sensitive Gal80 (Gal80ts) transgene. At the permissive temperature (18 °C), the GAL80 protein is active and represses induction of OR83b expression by inhibiting GAL4 ( A, top row). At the restrictive temperature (29 °C), GAL80 is inactivated, permitting expression of OR83b in OR22a/b neurons ( A, bottom row).
Histology
Antennae: 14-µm frozen sections of antennae were collected and stained with primary and secondary antibodies (see below) as described [ ]. Salivary glands: the anterior quarter of the third instar larvae was separated from the rest of the body, inverted to expose the salivary glands, and fixed in 4% formaldehyde in PBS for 20 min. These were then stained as for antennal sections, either in the presence of 0.25% Triton X-100 (Fisher Scientific, Springfield, New Jersey, United States) (permeabilized) or without detergent (non-permeabilized). After mounting in Vectashield (Vector Labs, Burlingame, California, United States), salivary glands were dissected away from other tissues. Images were collected with a Zeiss LSM510 confocal microscope (Zeiss, Oberkochen, Germany).
Optical imaging
In vivo preparation of flies (3- to 8-d-old animals) and optical imaging of odor-evoked calcium responses in the antennal lobe were essentially as described [ ] using a modified TILL Photonics imaging system (TILL Photonics, Ludwig Maximilians University, Munich, Germany). For each measurement, a series of 40 frames was taken at 4 Hz. A constant air stream (2,000 ml/min) produced by an aquarium pump was guided through a 1-ml syringe with the tip placed 1 cm from the antennae. Pure odorants were diluted in paraffin oil (see figure legends for concentrations), and 10 µl of this solution was placed on a filter paper in a second syringe that was laterally inserted into this air flow. Odorant stimuli (1 s, i.e., frames 12-16) were puffed into the constant airstream using a custom-made electronic valve under computer control with a 1-min interstimulus interval. Imaging data were...
Topology-mapping with β-gal fusion proteins
lacZ and TM:lacZ (encoding β-gal with an upstream synthetic TM domain) were amplified from pPD16.43 and pPD34.110 [ ], respectively, and cloned downstream of OR and Rh1 cDNA fragments in pMT-V5-His A (Invitrogen). Codons of test genes fused to lacZ and TM:lacZ: OR83b (1-46, 1-72), OR9a (1-41, 1-68), RH1 (1-49, 1-82). Plasmids were transfected into S2-R+ cultured Drosophila cells [ ] with Fugene (Qiagen, Valencia, California, United States). Expression of fusion proteins was induced for 24 h with 5 mM Cu 2 S0 4 and verified by Western blotting of cell extracts using antibodies against β-gal (Cappel from MP Biomedicals, Irvine, California, United States). To assess β-gal activity, cells were rinsed in PBS, fixed in 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 min, rinsed twice in PBS, and incubated in staining solution [5 mM K 3 Fe(...
Electron microscopy
Prelabeling immunoelectron microscopy: 6-µm cryostat sections of fresh-frozen adult antennae were collected on an Aclar film (EMS) treated with Alcian blue. Sections were fixed for 10 min in 4% paraformaldehyde, washed in PBS, and incubated with α-EC2 (diluted 1:5,000) overnight in the cold. After washing with PBS, the sections were incubated with goat-α-rabbit conjugated to 5 nm gold particles (Amersham Biosciences, Little Chalfont, United Kingdom). Incubations were stopped by buffer washes followed by fixation with 2.5% glutaraldehyde. Sections were further processed as described above except embedding was in EPON. Silver sections were collected on copper grids and viewed unstained in a JEOL 100 CX electron microscope operated at 80 kV. Distributions of gold particles were scored blindly by an impartial observer.
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Evidence quotes (6)
(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).
To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system to achieve temporal control of OR83b expression, specifically in Or22a/b neurons [ ]. This technique combines cell-type specific induction of a UAS-Or83b transgene by an Or22a-Gal4 driver line with temporal regulation through use of a ubiquitously expressed temperature-sensitive Gal80 (Gal80ts) transgene. At the permissive temperature (18 °C), the GAL80 protein is active and represses induction of OR83b expression by inhibiting GAL4 ( A, top row). At the restrictive temperature (29 °C), GAL80 is inactivated, permitting expression of OR83b in OR22a/b neurons ( A, bottom row).
Antennae: 14-µm frozen sections of antennae were collected and stained with primary and secondary antibodies (see below) as described [ ]. Salivary glands: the anterior quarter of the third instar larvae was separated from the rest of the body, inverted to expose the salivary glands, and fixed in 4% formaldehyde in PBS for 20 min. These were then stained as for antennal sections, either in the presence of 0.25% Triton X-100 (Fisher Scientific, Springfield, New Jersey, United States) (permeabilized) or without detergent (non-permeabilized). After mounting in Vectashield (Vector Labs, Burlingame, California, United States), salivary glands were dissected away from other tissues. Images were collected with a Zeiss LSM510 confocal microscope (Zeiss, Oberkochen, Germany).
In vivo preparation of flies (3- to 8-d-old animals) and optical imaging of odor-evoked calcium responses in the antennal lobe were essentially as described [ ] using a modified TILL Photonics imaging system (TILL Photonics, Ludwig Maximilians University, Munich, Germany). For each measurement, a series of 40 frames was taken at 4 Hz. A constant air stream (2,000 ml/min) produced by an aquarium pump was guided through a 1-ml syringe with the tip placed 1 cm from the antennae. Pure odorants were diluted in paraffin oil (see figure legends for concentrations), and 10 µl of this solution was placed on a filter paper in a second syringe that was laterally inserted into this air flow. Odorant stimuli (1 s, i.e., frames 12-16) were puffed into the constant airstream using a custom-made electronic valve under computer control with a 1-min interstimulus interval. Imaging data were analyzed with custom-written IDL software (Research Systems, Boulder, Colorado, United States) including noise filtering, movement, and bleaching correction. Relative fluorescence changes (ΔF/F) were calculated by subtracting the averaged fluorescence intensities from frames 4-6 from the...
lacZ and TM:lacZ (encoding β-gal with an upstream synthetic TM domain) were amplified from pPD16.43 and pPD34.110 [ ], respectively, and cloned downstream of OR and Rh1 cDNA fragments in pMT-V5-His A (Invitrogen). Codons of test genes fused to lacZ and TM:lacZ: OR83b (1-46, 1-72), OR9a (1-41, 1-68), RH1 (1-49, 1-82). Plasmids were transfected into S2-R+ cultured Drosophila cells [ ] with Fugene (Qiagen, Valencia, California, United States). Expression of fusion proteins was induced for 24 h with 5 mM Cu 2 S0 4 and verified by Western blotting of cell extracts using antibodies against β-gal (Cappel from MP Biomedicals, Irvine, California, United States). To assess β-gal activity, cells were rinsed in PBS, fixed in 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 min, rinsed twice in PBS, and incubated in staining solution [5 mM K 3 Fe(CN) 6, 5 mM K 4 Fe(CN) 6.6H 2 0, 2 mM MgCl 2, 1 mg/ml X-gal, in PBS] for 30 min at 37 °C. After staining, cells were rinsed in PBS and viewed on an inverted microscope.
Prelabeling immunoelectron microscopy: 6-µm cryostat sections of fresh-frozen adult antennae were collected on an Aclar film (EMS) treated with Alcian blue. Sections were fixed for 10 min in 4% paraformaldehyde, washed in PBS, and incubated with α-EC2 (diluted 1:5,000) overnight in the cold. After washing with PBS, the sections were incubated with goat-α-rabbit conjugated to 5 nm gold particles (Amersham Biosciences, Little Chalfont, United Kingdom). Incubations were stopped by buffer washes followed by fixation with 2.5% glutaraldehyde. Sections were further processed as described above except embedding was in EPON. Silver sections were collected on copper grids and viewed unstained in a JEOL 100 CX electron microscope operated at 80 kV. Distributions of gold particles were scored blindly by an impartial observer.
Machine-readable layer
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