Best fluorescence discrimination metrics and their accuracy landscapes when used in combination.

<p><b>A)</b> Histograms of the <i>Max 5% Ratio</i> metric for GFP females (light blue, n = 270 flies), non-GFP females (red, n = 267 flies), GFP males (dark blue, n = 270 flies), non-GFP males (dark red, n = 268 flies). Here the mean of the maximum 5% pixel values in the Front ROI is divided by the mean of the maximum 5% pixel values in the Rear ROI. Data is taken from homogeneous group experiments. <b>B)</b> Histograms of the <i>Skewness</i> metric for the same dataset. Here the skewness of pixel value distributions in each Total ROI is measured. <b>C)</b> Fluorescence discrimination accuracy of heterogeneous groups of female flies (n = 14 experiments; GFP females, n = 123; non-GFP females, n = 125) or <b>D)</b> male flies (n = 15 experiments; GFP males, n = 142; non-GFP males, n = 136). Here the number of flies expected in each genotype is incorporated into the genotype discrimination algorithm. X-axes show the weighting of each metric. Y-axes show the cumulative number of images averaged for metric measurements. Color bars indicate the discrimination accuracy.</p

Processing of video images to yield genetic identities in heterogeneous groups of <i>Drosophila</i>. A)

<p>Infrared backlit image of a genetically heterogeneous group of flies. Black box indicates the inset to the right of the image. <b>B)</b> Fluorescence image of the same group of flies. Grey box indicates inset. Note that extraneous autofluorescence from the experimental arena (grey arrowhead) does not impede subsequent analysis. <b>C)</b> Infrared image overlaid with size, position and orientation tracking data acquired using Ctrax <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048381#pone.0048381-Branson1" target="_blank">[2]</a>. Each colored triangle corresponds to a single fly. <b>D)</b> Fluorescence image overlaid with Regions of interest (ROIs). Green boxes encompass Front ROIs and blue boxes Rear ROIs for each fly. <b>E)</b> Infrared image overlaid with FBI data identifying each fly as GFP (blue) or non-GFP (red).</p

Workflow and tools for Fluorescence Behavioral Imaging (FBI). A)

<p>Workflow of FBI experiments. Experiments are performed using an FBI hardware system, <i>sQuid</i> software for multi-camera acquisition/LED control, and Actin88F:eGFP transgenic <i>Drosophila melanogaster</i>. Subsequently, infrared movies are processed using Ctrax <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048381#pone.0048381-Branson1" target="_blank">[2]</a> tracking software. FBI post-processing scripts then employ tracking data and fluorescence images to determine the genetic identity of behaving flies in an automated fashion. <b>B)</b> Bright-field (top) and fluorescence (bottom) images of an Actin88F:eGFP female fly. <b>C)</b> FBI hardware system used in this paper. <b>D)</b> Illumination during a single FBI experiment. FBI requires infrared backlight and blue fluorescence illumination following each experiment (solid blocks). Infrared and blue fluorescence illumination prior to each experiment and/or green illumination for vision-dependent behaviors are optional (hatched blocks).</p

FBI analysis of courtship interactions among mixed groups of <i>fruitless</i> (<i>fru^−/−</i>) and wild-type males. A)

<p>Raw (left) and Ctrax/FBI (right) images of courtship interactions between wild-type GFP males (blue) and <i>fru^−/−</i> non-GFP males (red) (flies are trailed by colored dots indicating their position in the previous 50 images/2.5 s). Three flies at the bottom-right form a courtship chain, as <i>fru^−/−</i> mutants court other males. <b>B)</b> Encounter density heat maps for wild-type reference flies with respect to other wild-type neighbors (top-left), <i>fru^−/−</i> reference flies with respect to wild-type neighbors (top-right), wild-type reference flies with respect to <i>fru^−/−</i> neighbors (bottom-left), and <i>fru^−/−</i> reference flies with respect to <i>fru^−/−</i> neighbors (bottom-right); n = 10 experiments; 60 <i>fru^−/−</i> flies and 60 GFP flies. Color bar indicates the percent of encounters observed at a given pixel. White triangles denote the orientation and approximate size of reference flies. <b>C)</b> Bar plots indicating the proportion of <i>fru^−/−</i> courtship events and courtship time towards wild-type flies (n = 10 experiments, mean and s.e.m.; chance based on the proportion of wild-type flies indicated in grey dashed line). <b>D)</b> The average duration of each courtship event of <i>fru^−/−</i> flies (n = 10 experiments) towards <i>fru^−/−</i> males (red) or wild-type males (blue).</p

The N-Terminus of <i>Drosophila</i> ORs Is Intracellular

<div><p>(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 <i>Drosophila</i> RH1 fusion proteins.</p> <p>(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:</p> <p>(B<i>) Or83b-Gal4/+;UAS-YFP(1):zip/UAS-YFP(2):zip</i></p> <p>(C) <i>Or83b-Gal4/UAS-YFP(2):zip:Or83b;UAS-YFP(1):zip/+</i></p> <p>(D) <i>Or83b-Gal4/+;UAS-YFP(1):zip/UAS-YFP(2):zip:Or43a</i></p></div

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

<div><p>(A) Immunostaining for GFP:OR43a (α-GFP, green) and OR83b (red) when misexpressed singly or in combination in <i>Gr21a</i> neurons. These animals are homozygous mutant for <i>Or83b</i>, which allows visualization of protein distributions specifically in <i>Gr21a</i> neurons. The arrowhead marks very weak localization of OR83b to cilia when expressed alone. Genotypes: Top row: <i>Gr21a-Gal4/UAS-GFP:Or43a;Or83b¹/Or83b²</i>. Middle row: <i>Gr21a-Gal4/+;UAS-Or83b,Or83b¹/Or83b²</i>. Bottom row: <i>Gr21a-Gal4/UAS-GFP:Or43a;UAS-Or83b,Or83b¹/Or83b²</i>.</p> <p>(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 <i>oseg2-Gal4</i> 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: <i>oseg2-Gal4/UAS-GFP:Or43a</i>. Middle row: <i>oseg2-Gal4/+;UAS-Or83b/+</i>. Bottom row: <i>oseg2-Gal4/UAS-GFP:Or43a;UAS-Or83b/+</i>.</p></div

Bioinformatic Analysis Defines <i>Drosophila</i> ORs As a Novel Family of TM Proteins

<div><p>(A) Unrooted neighbor-joining tree of selected <i>Drosophila</i> ORs [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040020#pbio-0040020-b037" target="_blank">37</a>], Class A GPCRs [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040020#pbio-0040020-b084" target="_blank">84</a>]<i>,</i> Methuselah family receptors [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040020#pbio-0040020-b085" target="_blank">85</a>], Frizzled receptors [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040020#pbio-0040020-b086" target="_blank">86</a>], potassium channels, and mouse ORs [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040020#pbio-0040020-b087" target="_blank">87</a>]. Sequences were aligned in ClustalX with 1000 bootstrap iterations.</p> <p>(B) TM domain predictions of <i>Drosophila</i> ORs (<i>n</i> = 61) and a representative subset of mouse ORs (<i>n</i> = 61) by the HMMTOP version 2.0 algorithm, including all those ORs depicted in (A).</p> <p>(C) Membrane-insertion orientation predictions of <i>Drosophila</i> 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 <i>Drosophila</i> 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.</p></div

Probing OR83b Topology by Antibody Epitope Staining

<div><p>(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: <i>AB1-Gal4/+;UAS-GFP:Or83b/+</i>. 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.</p> <p>(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 (<i>AB1-Gal4/+</i>) (bottom). Images are single confocal sections of cells in a plane through or just above the cell nuclei (visualized with DAPI staining, blue).</p> <p>(C) Salivary glands expressing GFP:OR83b (<i>AB1-Gal4/+;UAS-GFP:Or83b/+</i>) 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).</p> <p>(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.</p> <p>(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).</p> <p>(F) Quantification of gold particle distribution scored from four sections obtained in two independent experiments.</p></div

Pheromone-dependent conformational changes in LUSH.

<p>(A) Schematic of a pheromone-sensing trichoid sensillum illustrating the major ultrastructural features and proteins involved in detection of cVA. The OSN bears a single sensory cilium where the heteromeric pheromone receptor OR67d/ORCO and the CD36-related SNMP are localized. Auxiliary cells secrete at least three OBPs, including LUSH, and ODEs, including the carboxylesterase Est-6 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001546#pbio.1001546-Chertemps1" target="_blank">[49]</a>, into the lymph that bathes the OSN cilium within the sensillar hair lumen. (B) cVA-dependent conformational changes in LUSH. Ribbon view of the superimposed backbones of apo LUSH (grey) and cVA/LUSH (green, monomer A, conformation A only is represented; see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001546#pbio.1001546.s002" target="_blank">Figure S2</a>) (PDB IDs 1T14 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001546#pbio.1001546-Thode1" target="_blank">[27]</a> and 2GTE <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001546#pbio.1001546-Laughlin1" target="_blank">[29]</a>, respectively). The ligand, cVA, is depicted in stick form (yellow, carbon atoms; red, oxygen atoms). The most prominent conformational differences between the structures are within the C-terminal tail (Ct). (C) Close-up of the regions of LUSH corresponding to residues 83–87 and 115–123 for the structures shown in (B). The side chains of K87, D118, and F121 are represented by sticks. In apo LUSH (grey)—but not in this conformation of cVA/LUSH (green)—K87 and D118 can form a salt bridge (dotted line) (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001546#pbio-1001546-t001" target="_blank">Table 1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001546#pbio.1001546.s002" target="_blank">Figure S2</a>).</p

OR83b and ORs Associate via Conserved C-Terminal Domains

<div><p>(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): <i>Or83b^+/−</i> neuron [<i>Or83b-Gal4/UAS-GFP:Or83b(1–170):Or43a(159–376);Or83b¹/+</i>]; <i>Or83b^−/−</i> neuron [<i>Or83b-Gal4/UAS-GFP:Or83b(1–170):Or43a(159–376);Or83b¹/Or83b²</i>]; <i>Gr21a</i> neuron + OR83b [<i>Gr21a-Gal4/UAS-GFP:Or83b(1–170):Or43a(159–376);UAS-Or83b/+</i>]; <i>Gr21a</i> neuron + OR43a [<i>Gr21a-Gal4/UAS-GFP:Or83b(1–170):Or43a(159–376);UAS-Rho:Or43a/+</i>]. Bottom table: summary of localization (+) or no localization (−) to cilia of the chimera and, for comparison, OR83b and OR43a.</p> <p>(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 <i>HIS3</i> and <i>ADE2</i> reporters.</p></div