Genomic Structure and Mutational Analysis of the<i>Dα7</i> Gene

<div><p>(A) The<i>Dα7</i> gene consists of 16 exons. The 5′ and 3′UTRs are drawn in blue, and the ORF is colored red. The insertion sites of the three P-elements are shown above the gene, while the extent of each deletion generated by imprecise excision of the P-elements is depicted below.</p> <p>(B) The structural domains of the Dα7 protein are shown in a schematic representation, and the minimum extent of the lesion associated with the two deletions that remove part of the ORF are indicated by brackets, (), below. Abbreviations: LBD, ligand-binding domain; M1–M4, transmembrane domains 1–4; SP, signal peptide.</p> <p>(C) Immunostaining using an anti-Dα7 antibody is absent in<i>PΔEY6</i>. Here representative staining in the medulla is shown. The precise excision<i>PΔEY5</i> (left image) was used as a control, and whole-mount of the mutant and control brains were processed together. Scale bar, 20 μm.</p></div

gfA¹ is a Mutant Allele of<i>Dα7</i>

<div><p>(A) The response of transheterozygous animals to giant fiber stimulation at 1, 10, and 100 Hz is shown as a histogram using Canton-S (<i>CS</i>) flies as controls.<i>p</i>-Values (1 Hz):<i>CS</i> (<i>n</i> = 6);<i>gfA¹,</i> 0.004 (<i>n</i> = 6);<i>gfA¹/PΔD5,</i> 0.0009 (<i>n</i> = 4);<i>gfA¹/PΔEY6,</i> 0.02 (<i>n</i> = 5).<i>p</i>-Values for 10 Hz and 100 Hz were 2.8 × 10⁻⁵ for all mutant genotypes.</p> <p>(B)<i>gfA¹</i> flies do not show loss of Dα7 protein. Representative staining in the medulla of<i>CS</i> and<i>gfA¹</i> flies is shown. Control and mutant flies were processed together for immunohistochemistry. Scale bar: 20 μm.</p> <p>(C)<i>gfA¹</i> and<i>PΔEY6</i> show dominant phenotype at the PSI-DLM synapse. Comparison of the response to 100 Hz giant fiber stimulation of<i>PΔD5, PΔEY6,</i> and<i>gfA¹</i> in transheterozygous combinations with<i>CS</i> flies as controls is shown as a histogram.<i>p</i>-Values:<i>CS</i> (<i>n</i> = 6),<i>PΔD5</i>/+<i>,</i> 0.99 (<i>n</i> = 6);<i>PΔEY6</i>/+<i>,</i> 0.0002 (<i>n</i> = 7);<i>gfA¹</i>/+<i>,</i> 0.0001 (<i>n</i> = 5).</p> <p>(D)<i>gfA¹</i> flies are dominant in jump behavior. Comparison of jump behavior in<i>bw; st, PΔEY6/+; bw; st,</i> and<i>gfA¹/+; bw; st</i> is shown in a histogram.<i>gfA¹/+; bw; st</i> flies fail to jump while<i>bw; st</i> and<i>PΔEY6/+; bw; st</i> show no significant difference.<i>p</i>-Values:<i>bw; st</i> (<i>n</i> = 10),<i>PΔEY6/+; bw; st</i> (<i>n</i> = 10), 0.23,<i>gfA¹ /+; bw; st</i>, (<i>n</i> = 10), 0.00001.</p> <p>(E) Electropherogram showing basepair change that results in amino acid substitution in<i>gfA¹</i> mutant relative to the<i>CS</i> (wt) sequence.</p> <p>(F) Location of mutated residue in<i>gfA¹</i> (shown in red) indicated on the structure of the nAChR fromTorpedo marmorata [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040063#pbio-0040063-b035" target="_blank">35</a>]. This protein consists of five subunits, one of which is depicted in blue, while the others are in cyan. The residue is drawn in van der Waals representation to emphasize its position. This figure was generated using VMD [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040063#pbio-0040063-b056" target="_blank">56</a>].</p></div

Intracellular Recordings from DLMmn

<div><p>(A) mCD8-GFP driven by<i>Dα7</i> GAL4 labels the DLMmns including the dorsal MN5 (arrows). Scale bar: 50 μm.</p> <p>(B) Experimental method for recording from MN5. The fly is injected in DLMs with Texas red dextran, which is then allowed to be transported back to the cell body (left). Scale bar: 50 μm. Whole cell patch clamp recordings are made from the cell body after dissection to remove the asynchronous flight muscles and the gut. The giant fiber was stimulated through insulated tungsten electrodes placed in the eyes.</p> <p>(C) The backfilled MN5 is shown on the left. The organization of the branch of the giant fiber circuit supplying DLMs is depicted on the right. Synapses between the giant fiber and PSI and between the PSI and DLMmn are circled and indicated with arrowheads. Abbreviations: ADMN, anterior dorsal motor nerve; GF, giant fiber; PDMN, posterior dorsal motor nerve.</p> <p>(D) Response of DLMmn to current injection is shown. Current pulses in steps of 200 pA were applied starting at −500 pA.</p> <p>(E) The top tracings show recordings from MN5 in response to 1-Hz giant fiber stimulation. The middle tracings show stimulation at 10 Hz. The bottom tracings show several overlaid EPSPs at higher magnification.</p></div

Defective Neurotransmission at the PSI-DLMmn Synapse in<i>Dα7</i> Mutants

<div><p>The response of the DLMs to direct activation of the giant fiber is shown. The allele<i>PΔL1</i> served as control for<i>PΔ14G, PΔ41,</i> and<i>PΔD5,</i> while<i>PΔEY5</i> served as control for<i>PΔEY6</i>. For rescue of the<i>PΔD5</i> and<i>PΔEY6</i> mutant alleles, experiments were performed in males with genotype<i>PΔD5/Y; UAS Dα7/OK307 GAL4</i> and<i>PΔEY6/Y; UAS Dα7/OK307 GAL4,</i> respectively.</p> <p>(A) Schematic representation of the giant fiber circuit. Visual and mechanosensory input is transmitted via mixed electrical and chemical synapses to the giant fiber, which carries it to the thoracic ganglion. The TTMmn and PSI neurons are connected via electrical synapses to the giant fiber, and the PSI makes a chemical synapse onto the axon of the DLMmns (encircled).</p> <p>(B) Representative traces of intracellular recordings from DLM muscles for<i>PΔEY5, PΔEY6,</i> and<i>PΔEY6/Y; UAS Dα7/OK307 GAL4</i>.</p> <p>(C) The response of the mutant and control alleles to giant fiber stimulation is summarized in histograms. The top bar graph shows the average latency of the responses at 1 Hz. The second, third, and fourth bar graphs show the number of responses to ten stimuli at 1 Hz, 10 Hz, and 100 Hz respectively. The<i>p</i>-values are as follows. For 1 Hz:<i>PΔL1, n</i> = 6;<i>PΔ14G,</i> 1 (<i>n</i> = 6);<i>PΔ41,</i> 0.04 (<i>n</i> = 7);<i>PΔD5,</i> 2 × 10⁻⁶ (<i>n</i> = 5);<i>PΔD5/Y; UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 4);<i>PΔEY5, n</i> = 5;<i>PΔEY6,</i> 2 × 10⁻⁶ (<i>n</i> = 9);<i>PΔEY6/Y</i>;<i>UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 5). For 10 Hz:<i>PΔL1, n</i> = 6;<i>PΔ14G,</i> 1 (<i>n</i> = 6);<i>PΔ41,</i> 2 × 10⁻⁶ (<i>n</i> = 7);<i>PΔD5,</i> 2 × 10⁻⁶ (<i>n</i> = 5);<i>PΔD5/Y; UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 4);<i>PΔEY5, n</i> = 5;<i>PΔEY6,</i> 2 × 10⁻⁶ (<i>n</i> = 9);<i>PΔEY6/Y; UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 5). For 100 Hz:<i>PΔL1, n</i> = 6;<i>PΔ14G,</i> 4 × 10⁻⁶ (<i>n</i> = 6);<i>PΔ41,</i> 2 × 10⁻⁶ (<i>n</i> = 7);<i>PΔD5,</i> 2 × 10⁻⁶ (<i>n</i> = 5);<i>PΔD5/Y; UAS Dα7/OK307 GAL4,</i> 0.9 (<i>n</i> = 4);<i>PΔEY5, n</i> = 5;<i>PΔEY6,</i> 2 × 10⁻⁶ (<i>n</i> = 9);<i>PΔEY6/Y; UAS Dα7/OK307 GAL4,</i> 0.7 (<i>n</i> = 5). All error bars represent SEM. The<i>p</i>-values for latency measurements at 1 Hz are:<i>PΔL1,</i> (<i>n</i> = 5);<i>PΔ14G,</i> 1 (<i>n</i> = 6);<i>PΔ41,</i> 8.6 × 10⁻⁵ (<i>n</i> = 5);<i>PΔD5,</i> 3.2 × 10⁻⁵ (<i>n</i> = 2);<i>PΔD5/Y</i>;<i>UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 4);<i>PΔEY5,</i> (<i>n</i> = 5);<i>PΔEY6,</i> 0.02 (<i>n</i> = 3);<i>PΔEY6/Y</i>;<i>UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 5).</p> <p>(D) The top panel shows representative intracellular recording from the DLM with direct stimulation of its motor neuron at 1, 10, and 100 Hz. The bottom traces show representative recording from TTM with activation of the giant fiber.</p></div

Behavioral Characterization of<i>Dα7</i> Mutations

<div><p>The response of flies in a variety of behavioral assays is shown in this figure. The error bars represent SEM. In all assays, the precise excision of P-element line KG3295,<i>PΔL1,</i> served as a control for the mutant lines<i>PΔ14G, PΔ41,</i> and<i>PΔD5</i>. The precise excision of line EY10801,<i>PΔEY5,</i> served as a control for<i>PΔEY6</i>.</p> <p>(A) Flight test. Flies were individually dropped in a plastic cylinder, and the height at which they landed was recorded. Ten flies were tested for each genotype.</p> <p>(B) Olfactory response. Ten flies were placed in a Petri dish containing traps baited with food, and the number of trapped flies was recorded. Averages were calculated from between eight and ten trials for each genotype.</p> <p>(C) Visually mediated jump assay. Flies were placed in a Petri dish illuminated with green LEDs. A lights-off stimulus was presented by turning off the LEDs for 20 ms. For this assay it was necessary to use white-eyed flies. Since<i>PΔEY5</i> and<i>PΔEY6</i> are red-eyed, we placed them in a<i>bw; st</i> background, which produces white-eyed flies.<i>p</i>-Values:<i>PΔ14G,</i> 1.9 × 10⁻⁶ (<i>n</i> = 9);<i>PΔ41,</i> 1.9 × 10⁻⁶ (<i>n</i> = 11);<i>PΔD5,</i> 1.9 × 10⁻⁶ (<i>n</i> = 9);<i>PΔEY6,</i> 1.9 × 10⁻⁶ (<i>n</i> = 7).</p> <p>(D) Representative ERGs are shown for control and mutant alleles. We recorded extracellular responses from the eye to 1-s pulses of white light. The timing of the light pulse is shown below each voltage trace.</p> <p>(E) Visual performance was tested using the counter-current assay of Benzer [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040063#pbio-0040063-b029" target="_blank">29</a>].<i>p</i>-Values:<i>PΔ14G,</i> 0.22 (<i>n</i> = 5);<i>PΔ41,</i> 0.02 (<i>n</i> = 5);<i>PΔD5,</i> 0.003 (<i>n</i> = 5);<i>PΔEY6,</i> 1 (<i>n</i> = 5).</p></div

Long-Latency Response of TTM to Giant Fiber Stimulation

<div><p>(A) Recording configuration. The giant fiber was stimulated via tungsten electrodes in the eyes, and extracellular potentials were recorded from the TTM.</p> <p>(B) Average long- and short-latency response of TTM to giant fiber stimulation in different genotypes.<i>PΔD5 rescue</i> and<i>PΔEY6 rescue</i> had genotypes<i>PΔD5/Y; UAS Dα7/c17 GAL4</i> and<i>PΔEY6/Y; UAS Dα7/c17 GAL4,</i> respectively. The success rate of long-latency stimulation was as follows:<i>PΔL1</i> 8/10,<i>PΔ14G</i> 2/4,<i>PΔ41</i> 0/5,<i>PΔD5</i> 0/6,<i>PΔD5/Y; UAS Dα7/c17 GAL4</i> 2/2,<i>PΔEY5</i> 4/6,<i>PΔEY6</i> 0/4,<i>PΔEY6/Y; UAS Dα7/c17 GAL4</i> 3/3,<i>gfA¹</i> 0/3.</p> <p>(C) Representative traces showing the presence of short- and long-latency responses.<i>PΔEY5</i> and<i>PΔEY6/Y; UAS Dα7/c17 GAL4</i> showed both short- and long-latency TTM responses, but the long-latency response was absent in<i>PΔEY6</i>.</p></div

Similarities of Rab GTPases based on subcellular localization features.

<p>Pair-wise analyses for all 27 <i>rabs</i> were performed, and their similarity was determined as described in Materials and Methods. Bold <i>rabs</i> indicate the closest homologous Rabs in protein sequence alignments based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040912#pone-0040912-g001" target="_blank">Figure 1</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040912#pone.0040912-Zhang1" target="_blank">[18]</a> and Figure S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040912#pone.0040912-Chan1" target="_blank">[17]</a>. In cases of two or three pone.0040912.g006.tifclose homologs.</p

Cellular Expression Analysis of <i>rab30</i>-Gal4, <i>rab40</i>-Gal4, <i>rabX5</i>-gal4 and <i>rabX6</i>-Gal4.

<p>All rab-Gal4 lines were crossed to UAS-CD8-GFP. (A) Larval tissues showing GFP expression in green, 3×P3-RFP (positive marker of the Gal4 knock-in cassette) and nuclear Toto3 in blue. (B) Pupal (P+30% +/−5%) and 1-day adult brains (top panel: anterior; bottom panel: posterior. All scale bars represent 100 µm.</p

Analysis of Cellular Expression in Developing and Adult Tissues.

<p>Qualitative analysis of <i>rab</i>-Gal4>UAS-CD8-GFP expression (columns) for specific cells and tissues (rows). If expression was found to be particularly strong compared to other cells and tissues it was designated ‘YS’ for ‘Yes Strong’. If expression was found to be present but very weak it was designed ‘YW’ for ‘Yes Weak’.</p

Subcellular Localization Features of YFP-Rab30, YFP-Rab40, YFP-RabX5, and YFP-RabX6.

<p>(A) Double immunolabelings of the posterior larval brain ventral ganglion at high resolution are shown for the four YFP-Rabs driven by their respective rab-Gal4 lines. Left column: YFP-Rab (green), anti-Rab11 (red, recycling endosomes), anti-Rab5 (blue, early endosomes); right column: YFP-Rab (green), anti-CSP (red, synaptic vesicles), anti-Rab7 (blue, late endosomes). Cell bodies are peripherally and synaptic neuropils centrally located. Scale bar for all panels represents 20 µm. (B) Corresponding Gal4-lines drive the expression of wild type YFP-tagged Rabs in the left column, constitutively active (GTP-bound) YFP-tagged Rabs in the middle column and dominant negative (GDP-bound) YFP-tagged Rabs in the right column. Toto-3 labels nuclei (blue). Scale bar for all panels represents 20 µm.</p